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Crystal Structure of Human Survivin Reveals a Bow Tie–Shaped Dimer with Two Unusual α-Helical Extensions
Abstract
Survivin is a mitotic spindle-associated protein involved in linking mitotic spindle function to activation of apoptosis in mammalian cells. The structure of the full-length human survivin has been determined by X-ray crystallography to 2.7 A. Strikingly, the structure forms a very unusual bow tie-shaped dimer. It does not dimerize through a C-terminal coiled-coil, contrary to sequence analysis prediction. The C-terminal helices contain hydrophobic clusters with the potential for protein-protein interactions. The unusual shape and dimensions of survivin suggest it serves an adaptor function through its alpha-helical extensions.
... Unlike other IAPs, it lacks the RING finger domain carboxy-terminal and instead it has a long alpha-helix coil of 40 amino acids and 65Å involved in the association of the protein with the microtubules during the G2/M phase of the cell cycle. This domain is key to the interaction with the components of the chromosomal passage complex, during late mitosis [4,5]. Regarding its behavior, crystallographic studies have shown that it forms homodimers across the interface of dimerization, which includes the residues 6-10 and 89-102. ...
... Regarding its behavior, crystallographic studies have shown that it forms homodimers across the interface of dimerization, which includes the residues 6-10 and 89-102. The dimeric structure is stabilized by the formation of non-polar contacts between residues, mostly hydrophobic, with a total contact area between monomers of around 500Å 2 [4,6]. It is expressed mainly in embryonic tissues [2] and has a significant role in regulation of cell division and cell survival [4,7,8]. ...
... The dimeric structure is stabilized by the formation of non-polar contacts between residues, mostly hydrophobic, with a total contact area between monomers of around 500Å 2 [4,6]. It is expressed mainly in embryonic tissues [2] and has a significant role in regulation of cell division and cell survival [4,7,8]. However, its expression levels decrease during development, being undetectable in terminally differentiated normal tissues [2,5]. ...
Survivin protein is a metalloprotein member of the inhibitors of apoptosis proteins family, involved in the regulation of programmed cell death. Due to the recent development of antitumor therapies having survivin as molecular target, several strategies to interfere with the expression or function of survivin have emerged. This work describes the discovery of a new potential inhibitor of survivin function using a computer-aided drug design approach. Structure-based virtual screening and molecular dynamic simulations were carried out to select two compounds as possible inhibitors. According to the binding energy, possible ligand localization is in a cavity, close to dimerization interface. Next, cell-based assays were performed on three cell lines: two with tumor phenotype and over-expression of survivin, and another with normal phenotype and low expression of survivin. One of the selected compounds shows a selectively antitumor effect on panel cell lines suggesting that the compound effect could be correlated with the survivin expression.
... The survivin protein structure in the form of a homodimer has been determined by both crystallography [114][115][116] and nuclear magnetic resonance (NMR) [117]. The N-terminal BIR domain consists of a three-stranded β-sheet and four α-helices, with a zinc-binding fold, and the survivin protein forms a bow-tie-shaped dimer via part of the N-terminal region and the linker region between the BIR domain and the C-terminal helix [114,115]. ...
... The survivin protein structure in the form of a homodimer has been determined by both crystallography [114][115][116] and nuclear magnetic resonance (NMR) [117]. The N-terminal BIR domain consists of a three-stranded β-sheet and four α-helices, with a zinc-binding fold, and the survivin protein forms a bow-tie-shaped dimer via part of the N-terminal region and the linker region between the BIR domain and the C-terminal helix [114,115]. Notably, the ubiquitination of survivin on several lysine residues within the BIR domain is implicated in playing a role in modulating its localization to the centromere [118]. ...
Simple Summary
Survivin is overexpressed in a wide variety of human cancers and is associated with increased chemotherapy resistance, recurrence, and shorter patient survival. Although survivin was first identified as an inhibitor of apoptosis based on its sequence homology, recent studies show that survivin primarily plays a role in the regulation of cell division, as a component of the chromosome passenger complex, which can be localized to the centromere, the spindle midzone, or midbody in a cell cycle-dependent manner. Disruption of survivin function generally leads to mitotic catastrophe and is associated with elevated levels of aneuploidy. In contrast, ectopic expression of survivin can promote cell survival under certain conditions and is implicated in mediating resistance to various cancer drugs, possibly by interactions with molecules that modulate apoptotic pathways. A potential role of survivin in the regulation of mitochondrial functions and processes of autophagy has emerged. Because of its prevalent overexpression in cancer and very limited expression in normal tissues, survivin has been proposed as an ideal therapeutic target, and various approaches have been investigated for survivin inhibition. Here we provide a critical review of our current understanding of the role of survivin in promoting malignancy and strategies for the development of survivin-targeted therapy for cancer.
Abstract
Survivin was initially identified as a member of the inhibitor apoptosis (IAP) protein family and has been shown to play a critical role in the regulation of apoptosis. More recent studies showed that survivin is a component of the chromosome passenger complex and acts as an essential mediator of mitotic progression. Other potential functions of survivin, such as mitochondrial function and autophagy, have also been proposed. Survivin has emerged as an attractive target for cancer therapy because its overexpression has been found in most human cancers and is frequently associated with chemotherapy resistance, recurrence, and poor survival rates in cancer patients. In this review, we discuss our current understanding of how survivin mediates various aspects of malignant transformation and drug resistance, as well as the efforts that have been made to develop therapeutics targeting survivin for the treatment of cancer.
... The survivin protein structure in the form of a homodimer has been determined by both crystallography [107][108][109] and by nuclear magnetic resonance (NMR) [110]. The N-terminal BIR domain consists of a three-stranded β-sheet and four α-helices, with a zinc-binding fold, and the survivin protein forms a bow-tie shaped dimer via part of the N-terminal region and the linker region between the BIR domain and the C-terminal helix [107,108]. ...
... The survivin protein structure in the form of a homodimer has been determined by both crystallography [107][108][109] and by nuclear magnetic resonance (NMR) [110]. The N-terminal BIR domain consists of a three-stranded β-sheet and four α-helices, with a zinc-binding fold, and the survivin protein forms a bow-tie shaped dimer via part of the N-terminal region and the linker region between the BIR domain and the C-terminal helix [107,108]. Notably, ubiquitination of survivin on several lysine residues within the BIR domain are implicated in playing a role in modulating localization to the centromere [111]. ...
Survivin was initially identified as a member of the inhibitor apoptosis (IAP) protein family and has been shown to play a critical role in regulation of both apoptosis and mitosis. Survivin has emerged as an attractive target for cancer therapy because its overexpression has been found in most human cancers and is frequently associated with chemotherapy resistance, recurrence, and poor survival rate in cancer patients. In this review, we discuss our current understanding of how survivin mediates various aspects of malignant transformation and drug resistance, as well as efforts that have been made to develop therapeutics targeting survivin for the treatment of cancer.
... Accordingly, the SUR D70A/D71A mutant was the one selected for further studies. Finally, another interesting thing that was hypothesized, once the Survivin structure was revisited, and considered that the Survivin protein dimerizes, is that the SUR D70A/D71A mutant might act as a dominant-negative protein since the residues Asp70 and Asp71 appear on the surface of the Survivin dimer, and do not contribute to the monomers' interface (32). ...
... The Survivin peptide spanning the Cdk1-binding region (Figs. S3A and C) comprises several negatively-charged residues that reside on the Survivin dimer's acidic surface (32). In fact, even though I used the SUR D70A/D71A mutant as a Survivin antagonist due to this protein not being able to bind Cdk1, both the GST-SUR 71-142 truncated protein, which could not bind the kinase (Fig. S2C), and the alanine point mutant SUR D71, which slightly did (Fig. S5, bottom), suggested that only the Asp70 is crucial for binding to Cdk1. ...
Survivin has roles in repairing incorrect microtubule-kinetochore attachments at prometaphase, and the execution of cytokinesis, both as part of the chromosomal passenger complex. Here, errors lead to aneuploidy, polyploidy and cancer. Adding to these roles of Survivin, this work shows that Survivin is also required for cancer cells to enter mitosis, and that, in its absence, HeLa cells accumulate at early prophase. This blockage is demonstrated by the presence of an intact nuclear lamina and low Cdk1 activity. Interestingly, Survivin and Cdk1 form a complex at mitosis, and its molecular targeting indicates that Survivin is needed for Cdk1 to activate. Subsequently, escaping the blockage induced by Survivin abrogation leads to mitotic defects and apoptosis. Mechanistically, recombinant Survivin induces the activation of Cdk1 via Cdc25 in vitro. Coincidentally, Cdk1 mislocalizes at the centrosome when Survivin is not expressed. Moreover, Survivin interacts with Cdc25B in vitro and in vivo, and when absent, an inactive cytosolic Cdc25B-Cdk1-Cyclin B1 complex accumulates. Finally, the increase in prophase cells when Survivin was absent, could be bypassed by a gain-of-function Cdc25B mutant.
... Crystallographic studies have revealed that survivin forms homodimers across the dimerization interface that includes the amino acid residues 6-10 and 89-102. This dimeric construction is stabilized through non-polar contacts, mainly hydrophobic interactions, between residues [17,32,33]. By analysis of the dimerization interface, Leu6, Pro7, Pro8, Ala9, Trp10, Phe93, Glu94, Glu95, Leu96, Thr97, Leu98, Gly99, Phe101, and Leu102 were found to interact between the two subunits, forming a dimerization core. ...
... Here, a molecular docking study was carried out to predict and score the poses of the protein-ligand binding using survivin monomer (PDB ID: 1E31) [32] by the MOE program [37]. The results are displayed in Table 5 and Figures 4 and 5. Results analysis revealed that the top poses of compounds 5c and 5e are located mainly in the dimerization core. ...
The overexpression of survivin is usually accompanied by an increased resistance of cancer cells to chemotherapeutic agents in addition to cancer aggressiveness. Consequently, survivin is considered as an attractive target to develop new promising anticancer candidates. A series of novel 3-cyanopyridine derivatives was synthesized and assessed for their cytotoxic activity against three human cancer cell lines: prostate carcinoma (PC-3), breast cancer (MDA-MB-231) and hepatocellular carcinoma (HepG2). In addition, their activities were evaluated in comparison with a standard anticancer drug 5-FU. Compounds 5c and 5e both exhibited promising cytotoxicity against all the tested cell lines; especially, 5e showed better cytotoxic effect than the reference drug 5-FU. In order to evaluate the safety of these compounds, they were tested on the normal cell line WI-38, revealing their toxic selectivity toward cancer cells over normal ones. Further studies were performed in order to understand their mechanism of action; we examined the ability of our promising compounds 5c and 5e to induce cell cycle arrest. Both resulted in a notable induction of cell cycle arrest at the G2/M phase, along with an increase in the DNA content in the pre-G1 phase, giving us an indication of the incidence of apoptosis. 5c and 5e were further subjected to additional study using Annexin V-FITC assay in order to evaluate their ability to induce apoptosis. The results showed a marked increase in the early and late apoptotic cells, as well as an increase in the percentage of necrosis. Furthermore, Western blotting assay was accomplished using different concentrations of 5c and 5e. The results revealed a striking reduction in survivin expression through proteasome-dependent survivin degradation in addition to a decrease in the expression of some other inhibitor of apoptosis proteins (IAP) family proteins: Livin, XIAP, and C-IAP1 in a concentration-dependent manner. A docking study of 5c and 5e compounds in the dimerization site of survivin was also performed, showing agreement with the in vitro anti-survivin activity.
... The wild type survivin splice WT S (16.5 kDa) consists of four exons and three introns (Fig. 1) and forms a chain of 142 amino acids (Ambrosini et al., 1997). In solution, it forms a bow tie-shaped homodimer (Altieri, 2008a;Chantalat et al., 2000;Muchmore et al., 2000;Verdecia et al., 2000), as illustrated in Fig. 1A. The role of Sur in cancer biology and the methods of Sur gene detection have recently been evaluated in a series of excellent reviews (Andersen et al., 2007;Chen et al., 2016;Chiou et al., 2003;Dallaglio et al., 2012;Fangusaro et al., 2006;Johnson and Howerth, 2004;Li et al., 2018;Sah et al., 2006;Wheatley and Altieri, 2019). ...
... Here, we describe and evaluate different approaches based on the detection of Sur protein and Sur mRNA biomarkers which are by far the most common signature of the malignant tumors. In comparison with the current molecular biology techniques and methods based on sophisticated instrumental (Chantalat et al., 2000); (B) Five splice variants of survivin; (C) Schematic exhibition of survivin roles in apoptosis and cellular proliferation. Reprinted with permission from (Ebrahimiyan et al., 2018). ...
The development of biosensors for cancer biomarkers has recently been expanding rapidly, offering promising biomedical applications of these sensors as highly sensitive, selective, and inexpensive bioanalytical tools that can provide alternative methodology to that afforded by the advanced hyphenated-instrumental techniques. In this review, we focus particularly on the detection of a member of the inhibitor of apoptosis proteins (IAP) family, protein survivin (Sur), a ubiquitous re-organizer of the cell life cycle with the ability to inhibit the apoptosis and induce an enhanced proliferation leading to the unimpeded cancer growth and metastasis. Herein, we critically evaluate the progress in the development of novel biosensing systems and biosensors for the detection of two survivin (Sur) biomarkers: the Sur protein and its messenger RNA (Sur mRNA), including immunosensors, electrochemical piezo- and impedance-sensors, electrochemi-luminescence biosensors, genosensors based on oligonucleotide molecular beacons (MBs) with fluorescent or electrochemical transduction, as well as the microfluidic and related analytical platforms based on solution chemistry. The in-situ applications of survivin biomarkers' detection technologies to equip nanocarriers of the controlled drug delivery systems with MB-based fluorescence imaging capability, apoptosis control, and mitigation of the acquired drug resistance are also presented and critically evaluated. Finally, we turn the attention to the application of biosensors for the analysis of Sur biomarkers in exosomes and circulating tumor cells for a non-invasive liquid biopsy. The prospect of a widespread screening for early cancers, based on inexpensive point-of-care testing using biosensors and multiplex biosensor arrays, as a means of reducing the high cancer fatality rate, is discussed.
... Survivin is the best studied and the smallest member of the IAP family, with a weight of 16.5kda. It contains a single BIR and lacks a carboxy-terminal RING finger unlike other proteins of the family ( Figure 1A) (9). It is encoded by the BIRC5 gene on chromosome chr17q25 (3). ...
Background and Objective
A versatile biomarker, survivin, is highly expressed in proliferating cells of multiple cancers in humans and animals. It is an apoptosis-regulating protein, engaging in a cascade of reactions that involve several other genes and protein interactions. Currently, researchers are investigating its therapeutic potential due to the evidence linking its overexpression to advanced-stage lung cancer. This review is centered around examining survivin-related molecular mechanisms and its therapeutic role specifically in lung cancer. Our objective is to discuss the role of survivin in prognosis and treatment response, shedding light on immune-targeted therapies, as well as outlining future directions for survivin-based vaccines in lung cancer.
Methods
The PubMed database and the United States National Library of Medicine search engine at the National Institutes of Health were searched on 24 August 2023 to identify published research studies. Searching “((((((airway [Title/Abstract]) OR (lung [Title/Abstract])) OR (pulm[Title/Abstract])) OR (bronch[Title/Abstract])) OR (nslc[Title/Abstract])) AND (((cancer[Title/Abstract]) OR (carcino[Title/Abstract])) OR (oncol[Title/Abstract]))) AND (survivin[Title/Abstract])” gave 728 results. After screening the title and abstracts and excluding the review articles 168 titles were shortlisted and full text studied. The discussions are added to relevant sections.
Key Content and Findings
Survivin is a cell cycle-dependent, inhibitor of apoptosis protein that contributes to carcinogenesis, tumor vascularization, metastasis, and treatment resistance. Several treatments that impact survivin either directly or indirectly have been reported as effective in treating lung cancer. Immunity-based therapy, a novel approach known for its targeted nature and minimal side effects, is currently under investigation for lung cancer treatment. Emerging survivin-centered vaccines exhibit promising attributes in terms of safety, effectiveness, and ability to stimulate an immune response. These factors point towards a significant potential for advancing the future of lung cancer prevention and enhancing overall survival rates.
Conclusions
Nuclear survivin is a potential biomarker for advanced non-small cell lung cancer. It plays a role in determining drug responsiveness and is found to be significantly elevated in cases of resistance to chemotherapy. Multiple compounds and immunization strategies have been identified to impact lung cancer cells; however, they are currently in the early stages of phase I or phase II clinical trials. The substantial promise of survivin-based immunogenicity-focused treatments warrants in-depth investigation and exploration.
... These exosomes with survivin are secreted by the tumor cell, hence, these extracellular survivin can be used as a tool for the early diagnosis of malignancies. 12 Survivin expression has been noteworthy in the occurrence of OSCC when compared with other epithelial tumors of the oral cavity. Also, there are many studies which had documented the salivary expression of survivin in other potentially malignant disorders such as oral lichen planus, and oral leukoplakia, but its expression in OSMF has been highly correlated to malignant transformation of the condition. ...
... Due to its key role in apoptosis, proliferation, and angiogenesis, survivin has received increasing attention as a potential oncotherapeutic target [8,9]. However, survivin is an intracellular protein, does not have intrinsic enzymatic activity, and has few potential druggable sites, making it a relatively complex and difficult therapeutic target [10]. ...
Most cancer cells overexpress the anti-apoptotic protein survivin and display redox dysregulation originating from genotypic and phenotypic alterations. These disturbances contribute to the uncontrolled proliferation, invasion , and chemoresistance of cancer cells, yet they also represent a specific vulnerability that could be exploited therapeutically in selected tumors. YM155 (sepantronium bromide) is a naphthoquinone-containing imidazole-based compound that selectively inhibits survivin expression at the transcriptional and post-transcriptional levels. Here, we performed a systematic review and meta-analysis of clinical studies in which YM155 was administered as monotherapy or combination therapy for patients with cancer. We assessed fully or partially reported clinical outcomes and pharmacological parameters, and further performed subgroup analysis based on tumor type and treatment regimen. Our comprehensive analysis, which included patients of many ethnicities, demonstrated that YM155 was effective as a monotherapy or combination therapy. Clinical benefits, including regression and/or stabilization of tumor progression and prolonged survival, were observed within a reasonable time after treatment initiation, and YM155 displayed good synergistic effects with combination drugs. YM155 appears to be effective against a wide range of tumor types and has an acceptable safety profile, with the main toxicities being decreased blood cell counts; fatigue/weakness; renal, hepatic, and/or cardiac issues; and electrolyte disturbance. KEYWORDS YM155 (sepantronium bromide); systematic review; safety; anticancer effect; meta-synthesis; Naphthoquinone This work is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
... Due to its key role in apoptosis, proliferation, and angiogenesis, survivin has received increasing attention as a potential oncotherapeutic target [8,9]. However, survivin is an intracellular protein, does not have intrinsic enzymatic activity, and has few potential druggable sites, making it a relatively complex and difficult therapeutic target [10]. ...
... To find candidate drugs for survivin inhibition property, we have carried out a computational study to screen for effective drugs, endoxifen (PubChem CID:10090750), tamoxifen (PubChem CID: 2733526), and olanzapine (PubChem CID: 135398745) which may work as an inhibitor for apoptosis. [36] The structure of survivin (PDB ID: 1E31) was obtained from the Protein Data Bank (PDB) database, and molecular docking was performed with AutoDock Vina ver4.0 and as in literature. [37][38][39] ...
Background: Epidemiologic findings revealed approximately one-Third of patients with breast cancer develop brain metastases. Recent research has found that schizophrenia patients who take antipsychotic medications on a long-Term basis have a decreased risk of cancers than normal individuals. This serendipitous anticancer action of antipsychotic medications is now being investigated by many studies. The ability of these drugs to penetrate the blood-brain barrier may target brain metastases. We investigated antiproliferative activity of antipsychotic drug. The present study aimed to determine the antiproliferative effects of olanzapine against MCF-7 cells and also to examine its molecular interactions with survivin. Methods: The antiproliferative effects of olanzapine were demonstrated using MTT assay and molecular interactions were analyzed using AutoDock Vina ver4.0 between olanzapine (PubChem CID-135398745) and survivin (PDB ID-1E31). These molecular interactions were also compared with tamoxifen (PubChem CID: 2733526). Results: We found that olanzapine has extensive antiproliferative effects against MCF-7 human breast cancer cells, with an IC 50 of 10.9 g/mL. We also discovered that olanzapine had possible interactions with the survivin protein at Lys15, Phe86, and Val89 amino acid residues, which could be related to effects of olanzapine on MCF-7 cell viability. Conclusion: Our research establishes that olanzapine has promising anticancer properties against breast tumors, with prospective application to target brain metastases in patients with breast cancer. © 2022 Wolters Kluwer Medknow Publications. All rights reserved.
... Survivin (BIRC5), a homo-dimeric protein of 16.5-kDa, 1 is a member of the Inhibitor of Apoptosis Protein (IAP) family with a single BIR domain, a zinc-finger fold, and an extended C-terminal helical coiled coil. [2][3][4] Ectopic overexpression of survivin inhibits both the intrinsic and extrinsic apoptosis pathways in cell lines and animal models and has been suggested to contribute to resistance in cancer treatments. [5][6][7][8] Interestingly, survivin is expressed at undetectable or low levels in normal adult tissues while it has been shown to be overexpressed in almost all solid tumors. ...
Survivin, a member of the inhibitor of apoptosis protein family, exists as a homodimer and is aberrantly upregulated in a wide spectrum of cancers. It was thought to be an ideal target due to its lack of expression in most adult normal tissues and importance in cancer cell survival. However, it has been challenging to target survivin due to its “undruggable” nature. We previously attempted to target its dimerization domain with a hypothesis that inhibiting survivin dimerization would promote its degradation in proteasome, which led to identification of a lead small-molecule inhibitor, LQZ-7F. LQZ-7F consists of a flat tetracyclic aromatic core with labile hydrazone linking a 1,2,5-oxadiazole moiety. In this study, we tested the hypothesis that LQZ-7F could be developed as a prodrug because the labile hydrazone linker could be hydrolyzed, releasing the tetracyclic aromatic core. To this end, we synthesized the tetracyclic aromatic core (LQZ-7F1) using reported procedure and tested LQZ-7F1 for its biological activities. Here we show that LQZ-7F1 has a significantly improved potency with submicromolar IC50’s and induces spontaneous apoptosis in prostate cancer cells. It also more effectively inhibits survivin dimerization and induces survivin degradation in a proteasome-dependent manner than LQZ-7F. We also show that the combination of LQZ-7F1 and docetaxel have strong synergism in inhibiting prostate cancer cell survival. Together, we conclude that the hydrazone linker with the oxadiazole tail is dispensable for survivin inhibition and the survivin dimerization inhibitor, LQZ-7F, may be developed as a prodrug for prostate cancer treatment and to overcome docetaxel resistance.
... Survivin forms a homodimer by a symmetrical interaction between two survivin monomers across the dimerization interface, which consists of amino acid residues 6-10 and 89-102. This dimeric structure is essential for survivin protein stabilization and functionality by establishing non-polar interactions between residues (16,17). Mechanistically, the single zinc finger folds BIR domain is implicated in the anti-apoptotic activities of survivin. ...
It has been well established that the etiopathogenesis of diverse autoimmune diseases is rooted in the autoreactive immune cells’ excessively proliferative state and impaired apoptotic machinery. Survivin is an anti-apoptotic and mitotic factor that has sparked a considerable research interest in this field. Survivin overexpression has been shown to contribute significantly to the development of autoimmune diseases via autoreactive immune cell overproliferation and apoptotic dysregulation. Several microRNAs (miRNAs/miRs) have been discovered to be involved in survivin regulation, rendering the survivin-miRNA axis a perspective target for autoimmune disease therapy. In this review, we discuss the role of survivin as an immune regulator and a highly implicated protein in the pathogenesis of autoimmune diseases, the significance of survivin-targeting miRNAs in autoimmunity, and the feasibility of targeting the survivin-miRNA axis as a promising therapeutic option for autoimmune diseases.
... The Survivin gene named baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) is translated to a protein (16.5 kDa), including an N-terminal Zn2 +-binding baculoviral inhibitor of apoptosis repeat (BIR) domain bound to a 65 A o amphipathic C-terminal α-helix [15]. It is considered a positive mitosis regulator and a negative apoptosis regulator. ...
Survivin is one of the novel members of the apoptosis inhibitor protein family in humans. The main activity of the Survivin protein is to suppress caspases activity resulting in negative regulation of apoptosis. Survivin protein can be a potential target for the treatment of cancers between cancerous and normal cells. In the present research, the synthetic Survivin gene with PelB secretion signal peptide was cloned into a prokaryotic expression vector pET21a. The recombinant plasmid pET21a-PelB-Surv was expressed in Escherichia coli (E.coli) BL21, and the relative molecular mass of expressed protein was calculated 34,000 g/mol, approximately. The recombinant protein was purified through chromatography column and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Response surface methodology (RSM) was used to design 20 experiments for optimization of IPTG concentration, post-induction period, and cell density of induction (OD600). The optimum levels of the selected parameters were successfully determined to be 0.28 mM for IPTG concentration, 10 h for post-induction period, and 3.40768 for cell density (OD600). These findings resulted in 4.14-fold increases in the Survivin production rate of optimum expression conditions (93.6363 mg/ml).
... 11 Exists as a homodimer which is functional. 12 Is implicated in controlling cell survival and regulating mitosis in cancers. 13 Survivin is a substrate of CK2. ...
CK2 enzyme is up regulated in several cancers. It has many substratesincluding survivin which is up regulated in cancers. Objectives: To find out the correlationbetween expression of CK2α and survivin and evaluate it as a prospective prognostic marker inpathogenesis of the breast cancer and to find if positive correlation between CK2 and survivinwas associated with advancing disease. Study Design: Cross Sectional Analytical type of study.Setting: Department of Biochemistry & Molecular Biology, Army Medical College, Rawalpindiand Armed Forces Institute of Pathology, Rawalpindi. Duration of study: January 2013-December 2014. Methods: The research protocol was approved by Armed Forces Institute ofPathology Ethical Committee. Paraffin embedded tissue sections of diagnosed breast cancer,obtained from AFIP, were used. Immunohistochemistry was performed to determine nuclearand cytoplasm expression of survivin, and CK2 .Scoring done by three histopathologists,independently. Results: Total CK2 expression was high in invasive as compared to noninvasivecases (p =0.209). Cytoplasm and nuclear localization of CK2 in invasive group was alittle higher too (p = 0.092) and (p=0.286) respectively. Total survivin expression was high ininvasive as compared to non-invasive cases (p= 0.449). Cytoplasm and nuclear localizationof survivin in invasive group was higher as compared to noninvasive group with no significantdifferent (p=0.472) and (p=0.367) respectively. A positive and strong correlation was found inCK2 and survivin expression and localization in both non-invasive as well as invasive groups.Conclusion: CK2 and survivin correlation in cancers can be used in predicting the cancerphenotype and aggression at early stages.
... 33 In solution, purified survivin exists in a bow tie-shaped dimerization form. 33,34 Both dimer and monomer survivin have been implicated in the inhibition of apoptosis, although the detailed dimer-monomer balance regulation has not yet been elucidated. 35 ...
Survivin belongs to the inhibitor of apoptosis protein (IAP) family, which is consistently overexpressed in most cancer cells, whereas it is rarely expressed in normal adult tissues. Therefore, the detection and inhibition of survivin are regarded as attractive strategies for cancer-specific treatment. In this study, we designed and synthesized 7-19 residues of INCENP-derived small peptides (INC peptides) as novel survivin targeting agents. The INC peptides demonstrated binding affinity for the human survivin protein (Kd = 91.4-255 nM); INC16-22 , which contains residues 16-22 of INCENP, showed the highest affinity (91.4 nM). Confocal fluorescence imaging demonstrated consistent colocalization of FITC-INC16-22 and survivin in cell lines. Nonaarginine-linked INC16-22 (r9-INC16-22 ) rendered INC16-22 cell penetrable and strongly inhibited cell growth of MIA PaCa-2 cells (52% inhibition at 1.0 µM) and MDA-MB-231 cells (60% inhibition at 10 µM) as determined by MTT assays. On one hand, the exposure of MIA PaCa-2 cells to 40 µM r9-INC16-22 apparently reduced the intracellular protein expression levels of survivin. On the other hand, cleaved caspase-3 was significantly increased in the cells treated with r9-INC16-22 even at 10 µM compared to those in the untreated cells. Flow cytometry revealed that r9-INC16-22 strongly induced apoptosis in MIA PaCa-2 cells. These results indicate that the cytotoxic effects of r9-INC16-22 may be mediated mainly through the disruption of survivin-dependent anti-apoptotic functions and partly because of the direct degradation of the survivin protein. Our findings suggest that INC peptides can act as useful scaffolds for novel cancer imaging and anticancer agents.
... Crystal structure analysis of both human and mouse survivin revealed that survivin forms a homodimer through a symmetric interaction of two survivin monomers along the molecular dyad axis [24][25][26], which is required for survivin protein stabilization for its function. This finding lays a foundation for designing compounds to disrupt survivin homodimerization for possible cancer therapeutics. ...
Abstract Survivin (also named BIRC5) is a well-known cancer therapeutic target. Since its discovery more than two decades ago, the use of survivin as a target for cancer therapeutics has remained a central goal of survivin studies in the cancer field. Many studies have provided intriguing insight into survivin’s functional role in cancers, thus providing promise for survivin as a cancer therapeutic target. Despite this, moving survivin-targeting agents into and through the clinic remains a challenge. In order to address this challenge, we may need to rethink current strategies in order to develop a new mindset for targeting survivin. In this Review, we will first summarize the current survivin mechanistic studies, and then review the status of survivin cancer therapeutics, which is classified into five categories: (i) survivin-partner protein interaction inhibitors, (ii) survivin homodimerization inhibitors, (iii) survivin gene transcription inhibitors, (iv) survivin mRNA inhibitors and (v) survivin immunotherapy. We will then provide our opinions on cancer therapeutics using survivin as a target, with the goal of stimulating discussion that might facilitate translational research for discovering improved strategies and/or more effective anticancer agents that target survivin for cancer therapy.
... It is not fully understood, however, how these modifications regulate the activity of survivin during mitosis. X-ray crystallography data have shown that survivin forms a stable homodimer in solution (Chantalat et al., 2000;Sun et al., 2005;Verdecia et al., 2000), but definitive evidence that this structure is needed for survivin function(s) in vivo is still lacking. By contrast, there is evidence that survivin interacts as a monomer with other CPC components (Bourhis et al., 2007;Jeyaprakash et al., 2007). ...
An acto-myosin contractile ring, which forms after anaphase onset and is highly regulated in time and space, mediates cytokinesis, the final step of mitosis. The chromosomal passenger complex (CPC), composed of Aurora-B kinase, INCENP, borealin and survivin (also known as BIRC5), regulates various processes during mitosis, including cytokinesis. It is not understood, however, how CPC regulates cytokinesis. We show that survivin binds to non-muscle myosin II (NMII), regulating its filament assembly. Survivin and NMII interact mainly in telophase, and Cdk1 regulates their interaction in a mitotic-phase-specific manner, revealing the mechanism for the specific timing of survivin–NMII interaction during mitosis. The survivin–NMII interaction is indispensable for cytokinesis, and its disruption leads to multiple mitotic defects. We further show that only the survivin homodimer binds to NMII, attesting to the biological importance for survivin homodimerization. We suggest a novel function for survivin in regulating the spatio-temporal formation of the acto-NMII contractile ring during cytokinesis and we elucidate the role of Cdk1 in regulating this process.
This article has an associated First Person interview with the first author of the paper.
... [8][9][10] Survivin is the smallestm ember of the inhibitoro fa poptosis protein (IAP) family and exists as as table homodimer.T his protein is composed of 142 amino acids and contains ab aculovirus repeat( BIR) domain, ad imerizationd omain and aC -terminal a-helicald omain. [11] One of antiapoptotic processes responsible for survivin is itss uppression of the function of the proapoptotic protein Smac (second mitochondria-derived activator of caspases, also referred to as DIABLO). [12] In the normal state, Smac is located exclusively in mitochondria in cells. ...
Herein we report the first small molecule that disrupts the survivin‐Smac interaction taking place in mitochondria. The inhibitor, PZ‐6‐QN, was identified by initially screening a phenothiazine library using a fluorescence anisotropy assay and then conducting a structure‐activity relationship study. Mutagenesis and molecular docking studies suggest that PZ‐6‐QN binds to survivin similarly to the known Smac peptide, AVPI. The results of the effort also show that PZ‐6‐QN exhibits good anticancer activity against various cancer cells. Moreover, cell‐based mechanistic studies provide evidence for the proposal that PZ‐6‐QN enters mitochondria to inhibit the survivin‐Smac interaction and promotes release of Smac and cytochrome c from mitochondria into the cytosol, a process that induces apoptosis in cancer cells. Overall, the present study suggests that PZ‐6‐QN can serve as a novel chemical probe for study of processes associated with the mitochondrial survivin‐Smac interaction and it will aid the discovery of novel anticancer agents.
... This gene with 15 kb length is on the long arm of chromosome 17 (17q25; Chiou et al., 2003). This gene encodes a protein with 142 residues including a baculovirus IAP repeat (BIR) domain in the N-terminal and long alpha-helical region in the C-terminal ( Chantalat et al., 2000;T Shamsabadi et al., 2016). The survivin gene has six exons which prevent apoptosis by its four mRNA isoforms including survivin-ΔEx3, survivin-2α, survivin-2β, and survivin-3β due to alternative splicing (Consortium, 2013;Noton et al., 2006). ...
Survivin is a member of the family of apoptosis inhibitory proteins with increased expression level in most cancerous tissues. Evidence shows that survivin plays regulatory roles in proliferation or survival of normal adult cells, principally vascular endothelial cells, T lymphocytes, primitive hematopoietic cells, and polymorphonuclear neutrophils. Survivin antiapoptotic role is, directly and indirectly, related to caspase proteins and shows its role in cell division through the chromosomal passenger complex. Survivin contains many genetic polymorphisms that the role of some variations has been proven in several cancers. The −31G/C polymorphism is one of the most important survivin mutations which is located in the promoter region on a CDE/CHR motif. This polymorphism can upregulate the survivin messenger RNA. In addition, its allele C can increase the risk of cancers in 1.27‐fold than allele G. Considering the fundamental role of survivin in different cancers, this protein could be considered as a new therapeutic target in cancer treatment. For this purpose, various strategies have been designed including the prevention of survivin expression through inhibition of mRNA translation using antagonistic molecules, inhibition of survivin gene function through small inhibitory molecules, gene therapy, and immunotherapy. In this study, we describe the structure, played roles in physiological and pathological states and genetic polymorphisms of survivin. Finally, the role of survivin as a potential target in cancer therapy given challenges ahead has been discussed. Survivin plays regulatory and pathologic roles in cell function depending on its expression level. Considering the fundamental role of survivin in different cancers, this protein could be considered as a new therapeutic target in cancer treatment.
... Survivin is a ubiquitously expressed in tumor cells where it inhibits apoptosis and promotes mitosis, leading to cell proliferation. It belongs to IAP (inhibitor of apoptosis) family of proteins and is dependent on Hsp90 for maintaining its structure and function, ie., client of Hsp90 [35]. ...
The ninety kilo Dalton molecular weight heat shock protein (Hsp90) is an attractive target for the discovery of novel anticancer agents. Several strategies have been employed for the development of inhibitors against this polypeptide. The most successful strategy is targeting the N-terminal ATP binding region of the chaperone. However, till date not a single molecule reached Phase-IV of clinical trials from this class of Hsp90 inhibitors. The other approach is to target the Cterminal region of the protein. The success with this approach has been limited due to lack of well-defined ligand binding pocket in this terminal. The other promising strategy is to prevent the interaction of client proteins/co-chaperones with Hsp90 protein, i.e., protein-protein interaction inhibitors of Hsp90. The review focuses on advantage of this approach along with the recent advances in the discovery of inhibitors by following this strategy. Additionally, the biology of the client protein/co-chaperone binding site of Hsp90 is also discussed.
... At the molecular level, the survivin gene (BIRC5) encodes a 16.5 kDa protein consisting of an N-terminal Zn 2+ -binding baculoviral inhibitor-of-apoptosis repeat (BIR) domain linked to a 65 A o amphipathic C-terminal α-helix [15]. Functionally, it acts as a positive regulator of mitosis and also as a negative regulator of apoptosis. ...
Survivin is a well-known inhibitor-of-apoptosis proteins family member and a promising molecular target for anti-cancer treatment. However, it is widely accepted that survivin is only a “semi-druggable” target and development of survivin-specific small molecule inhibitors has shown to be difficult. In this study, we demonstrated that a histidine-tagged survivin T34A-C84A mutated protein (T34A-C84A-dNSur-His) can be produced using a bacterial recombinant protein expression system [E. coli ArcticExpress (DE3) cells] and solubilized using 1% (w/v) Sarkosyl. In addition, we showed that the purified T34A-C84A-dNSur-His protein formed dimers as predicted by in silico protein structure and molecular dynamics analysis. Importantly, results of the MTT assay revealed that the purified recombinant protein was biologically active in decreasing the viability of the human MDA-MB-231 breast adenocarcinoma and MIA-PaCa pancreatic carcinoma cells in vitro. Furthermore, the purified T34A-C84A-dNSur-His protein, but not of the histidine-peptide, induced apoptosis (i.e. caspase-9 activation and DNA fragmentation) in MDA-MB-231 cells at concentrations from 50 to 400 nM. In conclusion, our study provides a protocol of producing a biologically active survivin-targeting macromolecule, T34A-C84A-dNSur-His, which can be used as a tool for studying the molecular and cellular roles of survivin in cells. T34A-C84A-dNSur-His is also a potential therapeutic agent for augmenting cancer therapy
... This protein is produced by the BIRC5 gene in human [10]. In contrast to the structure of other IAP family proteins this protein is lack of C-terminal RING finger domain, though it possesses a BIR domain [11,12]. The survivin protein functions to inhibit apoptotic pathway and negatively regulates apoptosis of the cells [13][14][15]. ...
... Both domains are essential for its functions; the former binds the target proteins involved in the regulation of apoptosis and mitosis, the latter contains a microtubule binding site that allows interactions between survivin and the cytoskeleton (15,16). Analysis of crystal structure of human survivin demonstrates its bow tie-shaped dimer containing two unusual C-terminal α-helical extensions interacting with several proteins (18). ...
Head and neck squamous cell carcinoma (HNSCC) is the sixth most commonly diagnosed cancer worldwide. It has poor clinical outcome due to intrinsic or acquired drug resistance. Deregulation of both apoptosis and autophagy contributes to chemotherapy resistance and disease progression. A new member of the inhibitors of apoptosis protein (IAP) family, namely survivin, is selectively overexpressed in tumors, including HNSCC, but not in normal tissues. Thus, it is considered a tumor biomarker. Here, we reviewed survivin expression and function in tumor progression focusing on its nodal role in the regulation of cell apoptosis and autophagy. Based on literature data, survivin targeting may be envisaged as a novel therapeutic strategy.
... The αhelix on the other hand, is involved in extensive protein-protein interactions essential for SVV's function during mitosis; the domain binds polymerized tubulin and chromosomal passenger complex (CPC) proteins borealin and INCENP (Figure 6a and see section 3.3). X-ray crystallography shows that SVV dimerizes in solution, generating an elongated, bowtie shaped dimer (Figure 6b) (118,119). Dimerization occurs via two distinct interfaces, encompassing residues 6-10 and 89-102. Both monomeric as dimeric SVV seem to appear in vivo (120), but their individual roles long have been a matter of debate. ...
... Survivin has a single BIR domain, which discriminates it from other IAPs. In addition, it has been found that the interaction between two hydrophobic positions in survivin structure forms a bow tie-shaped dimer in protein product [5] (Figure 1). ...
Survivin, an antiapoptotic molecule from inhibitor of apoptosis protein (IAP) family, is most known for its implication in cancer as there are some efforts to apply it for diagnostic as well as therapeutic purposes in oncology. On the other hand, it could be a useful molecule to be positively targeted when trying to save tissue and promote cells viability. Since protecting the allograft from ischemia reperfusion injury and inflammation-induced damage is a considerable objective in transplantation, it is reasonable to take advantage from antiapoptotic agents like survivin in order to achieve this goal. However, survivin’s potential ability to induce malignancies makes some concerns about its use in clinic. The other barrier is this molecule’s involvement in lymphocytes development and proliferation which might increase the risk of graft rejection due to adaptive immune system overactivation. In this review we summarize the few studies carried out about survivin’s effect on graft survival and probable advantages and disadvantages of its overexpression in transplantation.
... There is also one zinc finger in its structure formed by four Zn 2 + -binding residues including Cys-60, Cys-57, Cys-84, and His-77 that retain survivin integrity (Chantalat et al., 2000). Survivin is also distinguished from the other IAP family members by the lack of caspase activation and recruitment domain and RING finger (really interesting gene) motifs (Deveraux and Reed, 1999). ...
Survivin is one of the most cancer-specific proteins overexpressed in almost all malignancies, but is nearly undetectable in most normal tissues in adults. Functionally, as a member of the inhibitor of apoptosis family, survivin has been shown to inhibit apoptosis and increase proliferation. The antiapoptotic function of survivin seems to be related to its ability to inhibit caspases directly or indirectly. Furthermore, the role of survivin in cell cycle division control is related to its role in the chromosomal passenger complex. Consistent with its determining role in these processes, survivin plays a crucial role in cancer progression and cancer cell resistance to anticancer drugs and ionizing radiation. On the basis of these findings, recently survivin has been investigated intensively as an ideal tumor biomarker. Thus, multiple molecular approaches such as use of the RNA interfering technique, antisense oligonucleotides, ribozyme, and small molecule inhibitors have been used to downregulate survivin regulation and inhibit its biological function consequently. In this review, all these approaches are explained and other compounds that induced apoptosis in different cell lines through survivin inhibition are also reported.
... Survivin is a homodimer of a 16.5-kDa protein (7). Located at the tip of chromosome 17 in humans (17q25), the survivin gene has four dominant (1, 2, 3, and 4) and two hidden (2B and 3B) exons. ...
Survivin, which is highly expressed in the majority of tumors, but not in most normal adult tissues, has been identified to have significant clinical applications. In the present study, using survivin‑specific monoclonal antibodies (mAbs), we aimed to establish methods for detecting the expression of survivin in cancer cell lines, serum samples, urine samples and cancer tissues from patients with bladder cancer (BCa) and renal cell carcinoma (RCC), and to evaluate the efficacy of survivin as a tumor marker in the surveillance of BCa and RCC. First, mAbs were labeled with horseradish peroxidase (HRP), and a sandwich enzyme‑linked immunosorbent assay (ELISA) with mAbs and HRP‑conjugated mAbs was developed to detect survivin expression in serum and urine samples from BCa and RCC patients, with samples from healthy controls (HCs) used for comparison. The HRP‑conjugated mAbs were also used to detect survivin expression in cancer cell lines by western blotting. Survivin expression in cancer tissues from BCa patients was also evaluated by immunohistochemistry. The results showed that the sandwich ELISA was successfully established, and significantly higher expression of survivin was subsequently detected in BCa and RCC patients as compared with HCs in both urinary and serum samples (P<0.05), and was more pronounced in urine. The HRP‑mAbs could recognize survivin in cancer cell lines. Western blotting and immunohistochemistry results confirmed survivin expression in the 5637 BCa cell line, as well as BCa tissues. In addition, the expressions of survivin in BCa tissues, urine and serum were consistent in our study. In conclusion, the sandwich ELISA successfully established in the present study was of high sensitivity and specificity in the detection of survivin expression. The results also indicated that survivin is a potential tumor marker for the surveillance of BCa and RCC.
... Survivin, also called baculoviral inhibitor of apoptosis repeat-containing 5, is a member of the inhibitor of apoptosis protein family (IAP), which also includes X-linked inhibitor of apoptosis (XIAP), cIAP1, cIAP2, NOD-like receptor family apoptosis inhibitory protein, livin, IAP-like protein 2 and baculovirus inhibitor of apoptosis protein repeat (BIR) containing ubiquitin-conjugating enzyme, isoform C (7,8). Survivin is a 142-amino acid, 16.5-kDa protein encoded by a single gene located on human chromosome 17q25, consisting of an N-terminal Zn 2+ -binding BIR domain linked to a 65A˚ amphipathic C-terminal α-helix, as well as 3 introns and 4 exons (3,(9)(10)(11). Heat shock protein 90 (HSP90) maintains the stability and folding of multiple bioenergetic effectors of survivin (12). Unlike other IAP members, survivin is highly expressed in the majority of neoplasms, whereas it is rarely expressed in normal adult tissues (13). ...
Survivin, also known as baculoviral inhibitor of apoptosis repeat-containing 5, is a novel member of the inhibitor of apoptosis protein family. Survivin is highly expressed in tumors and embryonic tissues and is associated with tumor cell differentiation, proliferation, invasion and metastasis; however, survivin is expressed at low levels in normal terminally differentiated adult tissues. Meanwhile, the expression level of survivin is also a negative prognostic factor for patients with cancer. These unique characteristics of survivin make it an exciting potential therapeutic target for cancer treatment. This review will discuss the biological characteristics of survivin and its potential use as a treatment target to reduce cancer cell proliferation.
... These strategies include but are not limited to introducing recombinant cell-permeable dominant-negative survivin protein [7,8], obstructing protein translation using antisense oligonucleotides [9], and developing small-molecule based survivin antagonists [10,11]. The crystal structure of human survivin was obtained in the early of this century, revealing its unusual bow tie-shape dimer structure [12]. However, targeting survivin using small-molecule survivin inhibitors has been proven to be challenging because no experimentally validated binding pocket in survivin has been identified yet. ...
The anti-apoptotic protein survivin is highly expressed in cancer cells but has a very low expression in fully differentiated adult cells. Overexpression of survivin is positively correlated with cancer cell resistance to chemotherapy and radiotherapy, cancer cell metastasis, and poor patient prognosis. Therefore, selective targeting survivin represents an attractive strategy for the development of anticancer therapeutics. Herein, we reported the extensive structural modification of our recently discovered selective survivin inhibitor UC-112 and the synthesis of thirty-three new analogs. The structure-activity relationship (SAR) study indicated that replacement of the benzyloxy moeity in UC-112 with an indole moiety was preferred to other moieties. Among these UC-112 analogs, 10f, 10h, 10k, 10n showed the most potent antiproliferative activities. Interestingly, they were more potent against the P-glycoprotein overexpressing cancer cell lines compared with the parental cancer cell lines. Mechanistic studies confirmed that new analogs maintained their unique selectivity against survivin among the IAP family members. In vivo study using 10f in a human A375 melanoma xenograft model revealed that it effectively inhibited melanoma tumor growth without observable acute toxicity. Collectively, this study strongly supports the further preclinical development of selective survivin inhibitors based on the UC-112 scaffold.
... In this complex, Aurora B acts as the enzymatic core, while survivin dictates chromosomal passenger complex localisation (10). X-ray crystallography has revealed an unusual bow tie-shaped dimer with two α-helical extensions interacting with the microtubules through these α-helical extensions at the carboxyl termini (11). Survivin is uniquely placed at the border of both the cell-death machinery and mechanisms of cell cycle progression/ microtubule stability linking mitotic spindle functions to apoptotic pathways (12). ...
Chemotherapy drugs usually inflict a lethal dose to tumour cells with the consequence that these cells are being killed by cell death. However, each round of chemotherapy also causes damage to normal somatic cells. The DNA cross-linking agent oxaliplatin (OXP), which causes DNA double-strand breaks, and vinflunine (VFN), which disrupts the mitotic spindle, are two of these chemotherapy drugs which were evaluated in vitro using peripheral lymphocytes from colorectal cancer patients and healthy individuals to determine any differential response. Endpoints examined included micronucleus (MN) induction using the cytokinesis-blocked micronucleus (CBMN) assay and pancentromeric fluorescence in situ hybridisation. Also, survivin expression was monitored since it regulates the mitotic spindle checkpoint and inhibits apoptosis. OXP produced cytogenetic damage (micronuclei in binucleated cells) via its clastogenic but also previously unknown aneugenic action, possibly through interfering with topoisomerase II, whilst VFN produced micronuclei in mononucleated cells because of incomplete karyokinesis. Survivin expression was found to be significantly reduced in a concentration-dependent manner by not only OXP but surprisingly also VFN. This resulted in large numbers of multinucleated cells found with the CBMN assay. As survivin is upregulated in cancers, eliminating apoptosis inhibition might provide a more targeted chemotherapy approach; particularly, when considering VFN, which only affects cycling cells by inhibiting their mitotic spindle, and alongside possibly other pro-apoptotic compounds. Hence, these newly found properties of VFN -the inhibition of survivin expression-might demonstrate a promising chemotherapeutic approach as VFN induces less DNA damage in normal somatic cells compared to other chemotherapeutic compounds.
... Survivin is the smallest member of the mammalian IAP family containing only a single N-terminal baculovirus IAP repeat (BIR) domain combined with long C-terminal α -helix coiled region [20]. In solution it is present in dimeric form. ...
Squamous cell carcinoma (SCC) is the most common cancer worldwide. The treatment of locally advanced disease generally requires various combinations of radiotherapy, surgery, and systemic therapy. Despite aggressive multimodal treatment, most of the patients relapse. Identification of molecules that sustain cancer cell growth and survival has made molecular targeting a feasible therapeutic strategy. Survivin is a member of the Inhibitor of Apoptosis Protein (IAP) family, which is overexpressed in most of the malignancies including SCC and totally absent in most of the normal tissues. This feature makes survivin an ideal target for cancer therapy. It orchestrates several important mechanisms to support cancer cell survival including inhibition of apoptosis and regulation of cell division. Overexpression of survivin in tumors is also associated with poor prognosis, aggressive tumor behavior, resistance to therapy, and high tumor recurrence. Various strategies have been developed to target survivin expression in cancer cells, and their effects on apoptosis induction and tumor growth attenuation have been demonstrated. In this review, we discuss recent advances in therapeutic potential of survivin in cancer treatment.
Tumor-related mRNA detection is significant and interesting. The current mRNA detection method has the challenge of quantifying long mRNA sequences. Herein, a Y-shaped DNA probe with three target-binding segments was developed to detect tumor-related mRNA. This Y-shaped DNA probe (Y-probe) was assembled by six single DNA strands. Among these DNA strands, two DNA strands contained the split G-quadruplex sequence, and two DNA strands were modified with a pair of fluorophore and quencher, which were used to produce the detectable signal. In the presence of a long target mRNA sequence, target mRNA was hybridized with the three target-binding segments of the Y-probe, resulting in the increased fluorescence of G-quadruplex specific dye Thioflavin T and the decreased fluorescence of fluorophore, which could achieve the ratio detection of target mRNA. The Y-probe exhibited a low detection limit of 17.53 nM. Moreover, this probe showed high accuracy due to the benefits of three target-binding segments.
Survivin is overexpressed in most cancer cells but is rarely expressed in normal adult tissues. It is associated with poor prognosis and resistance to radiation therapy and chemotherapy. In this study, we designed and synthesized borealin-derived small peptides (Bor peptides) to function as survivin-targeting agents for the diagnosis and treatment of cancers. These peptides exhibited binding affinities for recombinant human survivin (Kd = 49.6-193 nM), with Bor65-75 showing the highest affinity (Kd = 49.6 nM). Fluorescence images of fluorescein isothiocyanate-labeled Bor65-75 showed its co-localization with survivin expression in the human pancreatic cancer cell line, MIA PaCa-2. In the WST-1 assay, cell penetrable nona-d-arginine-conjugated Bor65-75 (r9-Bor65-75) inhibited the growth of MIA PaCa-2 cells and MDA-MB-231 cells (89 and 88% inhibition at 10 μM, respectively), whereas it had almost no effect on the human mammary epithelial cell line, MCF-10A, that inherently does not have high survivin expression. Flow cytometry with annexin V and propidium iodide staining revealed that r9-Bor65-75 induced apoptosis in MIA PaCa-2 cells in a dose-dependent manner. An increase in cleaved poly ADP-ribose polymerase protein expression was observed in MIA PaCa-2 cells exposed to r9-Bor65-75 by western blotting, suggesting that r9-Bor65-75 inhibits cell proliferation by inducing apoptosis. In vivo, r9-Bor65-75 significantly suppressed tumor growth in MIA PaCa-2 xenograft mice, without any marked weight loss. Hence, Bor peptides are promising candidates for the development of cancer imaging and anticancer agents targeting survivin.
The Chromosomal Passenger Complex (CPC), composed of Aurora B, Borealin, INCENP, and Survivin, contributes to a sequence of critical mitotic processes to ensure proper chromosome inheritance in daughter cells. Early in mitosis, the CPC concentrates at centromeres to monitor kinetochore-spindle attachments, destabilizing those which could cause chromosome mis-segregation. Next, the CPC associates with the spindle midzone to help coordinate cell division. During cytokinesis, the CPC localizes to the intercellular bridge to monitor for chromosomes trapped in the cleavage furrow and block abscission if such is the case. Through these activities, the CPC makes critical contributions to mitosis and cell division.
Context: Malnutrition outbreak in 2018 caused increased morbidity and mortality of Asmat children. Many studies indicated that malnourished children should receive adequate nutrients. Aim: The study aims to analyze food consumption among under-five children in Asmat. Settings and design: The cross-sectional study was conducted in Agats subdistrict, Asmat district, and Papua province, in July 2018 and included 62 under-five children. Children were selected using purposive sampling from five villages. Materials and methods: The primary data were collected by interview, direct measurement, questionnaire fulfillment consisting of general, anthropometric, and food consumption data. Statistical analysis used: descriptive data, composed of the characteristics of children, children’s nutritional status, and food consumption. Results: The average energy intake was 561.7 ± 335.3 kcal/day. The mean carbohydrate and protein were 93.3 ± 52.9 and 18.2 ± 11.2 g, respectively. The median fat intake was 7.53 (2.6, 16.9) g and fiber was 1.63 (1.0, 2.9) g/day. The median iron and zinc were 1.5 (0.8, 2.6) g and 1.5 (0.8, 2.3) g. The average folic acid intake was 36.4 ± 25.2 g. Conclusion: Compared to the Indonesian Recommended Dietary Allowance (RDA), the percentage of children’s energy (93.5%), protein (75.8%), fat (96.8%), carbohydrate (83.9%), fiber (91.9%), iron (88.7%), folic acid (98.4%), and zinc (88.7%) intakes was included as below of Indonesian RDA category. These results can be used to make appropriate dietary recommendations, which will be used as a substantial improvement in community service programs
High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division. The complex contains 4 protein components: Aurora B kinase, survivin, borealin and INCENP. The CPC acts as a node to dynamically organize other partnering subcomplexes to build multiple functional structures during mitotic progression. Using phage display mutagenesis, a cohort of synthetic antibodies (sABs) were generated against different domains of survivin, borealin and INCENP. Immunofluorescence established that a set of these sABs can discriminate between the form of the CPC complex in the midbody versus the spindle. Others localize to targets, which appear to be less organized, in the nucleus or cytoplasm. This differentiation suggests that different CPC epitopes have dynamic accessibility depending upon the mitotic state of the cell. An IP/mass spec analysis was performed using sABs that bound specifically to the CPC in either the midbody or MT spindle macromolecular assemblies. Thus, sABs can be exploited as high performance reagents to profile the accessibility of different components of the CPC within macromolecular assemblies during different stages of mitosis suggesting this high throughput approach will be applicable to other complex macromolecular systems.
Survivin’s dual function as apoptosis inhibitor and regulator of cell proliferation is mediated via its interaction with the export receptor CRM1. This protein–protein interaction represents an attractive target in cancer research and therapy. Here, we report a sophisticated strategy addressing Survivin’s nuclear export signal (NES), the binding site of CRM1, with advanced supramolecular tweezers for lysine and arginine. These were covalently connected to small peptides resembling the natural, self-complementary dimer interface which largely overlaps with the NES. Several biochemical methods demonstrated sequence-selective NES recognition and interference with the critical receptor interaction. These data were strongly supported by molecular dynamics simulations and multiscale computational studies. Rational design of lysine tweezers equipped with a peptidic recognition element thus allowed to address a previously unapproachable protein surface area. As an experimental proof-of-principle for specific transport signal interference, this concept should be transferable to any protein epitope with a flanking well-accessible lysine. Survivin’s dual function as apoptosis inhibitor and regulator of cell proliferation is mediated via its interaction with the export receptor CRM1. Here authors report a strategy addressing its dimer interface overlapping with the nuclear export signal, the binding site of CRM1, with advanced supramolecular tweezers for lysine and arginine.
Survivin, a homodimeric member of the Inhibitor of Apoptosis Protein (IAP) family, is required for cancer cell survival and overexpressed in almost all solid tumors. However, targeting survivin has been challenging due to its “undruggable” nature. Recently, we used a novel approach to target the dimerization interface and identified inhibitors of two scaffolds that can directly bind to and inhibit survivin dimerization. One of the scaffolds, represented by the compound LQZ-7, con-tain an undesirable labile hydrazone linker and a potentially non-functional furazanopyrazine ring that we attempted to eliminate in this study. We found one compound, 7I, that is more active than the parent compound, LQZ-7, and when giv-en orally effectively inhibits xenograft tumor growth and induces survivin loss in tumors. These findings indicate that 7I with a stable linker and a quinoxaline ring can be used as a lead for further optimization of this novel class of survivin inhibitors.
Viral myocarditis (VMC) is a widely studied but poorly understood inflammatory cardiomyopathy which mainly affects children and young adults and results in adverse outcomes. Cardiomyocyte apoptosis was reported important in the progress of coxsackievirus B3 (CVB3)-induced VMC and the blocking of this process may contribute to the therapeutic effect towards VMC. Therefore, this study was designed to explore whether survivin, one of the strongest antiapoptotic proteins, can protect cardiomyocytes from apoptosis in VMC and to discover its related mechanisms. Here, the cultured neonatal mouse cardiomyocytes (NMCs) were exposed to CVB3 to establish the cell model of VMC and the results of Western Blot showed that the protein expression of survivin in CVB3-infected NMCs varied at different post-infection time. Lentivirus was next used to examine the function of survivin in CVB3-infected NMCs. TUNEL assay demonstrated that the overexpression of survivin interrupted CVB3-induced apoptosis. It was next examined whether caspase-3 and -9 were involved in the antiapoptotic pathway initiated by survivin via Western Blot. The results showed a reverse relationship between the protein expression of survivin and that of cleaved caspase-3 and cleaved caspase-9, suggesting that survivin may attenuate apoptosis through restraining the activity of caspase-3 and -9. Moreover, the supernatant fluid of cultured NMCs was extracted to detect the quantitation of released lactate dehydrogenase (LDH) and a sharp decrease was discovered in the survivin-overexpressed group compared to the CVB3-infected group, indicating a protective role of survivin in the cell model of CVB3-induced myocarditis. This study demonstrated that survivin was triggered by CVB3 infection in NMCs and survivin executed its antiapoptotic effects via caspase-3- and caspase-9-dependent signaling pathway.
Ugi reaction was a reliable procedure for the synthesis of new coumarin-quinoline frameworks. Excellent yields, mild reaction conditions and easily available and inexpensive starting materials are advantages of this protocol. Cytotoxic effects of fourteen products were investigated in A2780 human ovarian cancer cells. Two synthesized compounds (L11 and L12) exhibited more anti-cancer activity than other derivatives with IC50 values of 0.042 mmol/L and 0.102 mmol/L, respectively and were thus selected for further studies. Apoptosis was induced through the intrinsic pathway by activating caspase 9 and ended at the executioner pathway of caspase 3.
Measurement of intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were also carried out for both of them. Further studies on a mechanism by Real Time-PCR and Western blot analysis were performed for anti-apoptotic proteins Bcl-2 and survivin both in mRNA and protein level relating to the
untreated A2780 cells. The treatment of A2780 cells with compound L11 significantly (P-value≤0.05) induced apoptosis by down-regulation of Bcl-2 and survivin both in mRNA and protein level via a single dose (0.042 mmol/L), as well as activation of caspase 9 and 3, loss of MMP, and high ROS. Accordingly, findings supported the first report under which the pro-apoptotic activity of compound L11 as an apoptosis-inducing agent was related to mitochondrial-mediated dysfunction signaling pathways. Molecular docking supports experimental
outcomes. Evidently, coumarin-quinoline scaffolds are potentially favorable options for further assessment as influential chemotherapeutic agents for the future.
Baculoviral IAP repeat containing 5 (BIRC5) gene encodes the important protein as survivin, a multifunctional protein, which is involved in cellular and molecular networks, progression of cell cycle, homeostasis, developmental morphogenesis, and apoptosis. The proximal BIRC5 promoter possesses specific binding sites for key transcription factors such as nuclear factor κB and signal transducer and activator of transcription 3. Upregulation of survivin exacerbates the autoimmune diseases (AIDs) including multiple sclerosis and myasthenia gravis by reducing the activity threshold of survivin‐specific cytotoxic T cells. DNA damage along with upregulation or downregulation of survivin have been demonstrated in initiation and pathogenesis of cancers and AIDs. However, detailed mechanism of survivin function in pathogenesis of AIDs is not well understood. This review focuses on the structure, specificity, regulation, and function of survivin in physiologic conditions and pathogenesis of AIDs.
Background:
Gastric cancer is one of the most common and deadly malignancies worldwide. Despite recent medical progress, the 5-year survival rate of gastric cancer is still unsatisfactory. 5-fluorouracil (5-Fu) is one of the first-line antineoplastic treatments for gastric cancer, as it can effectively induce cancer cell apoptosis. However, the effect of 5-Fu is limited due to drug resistance of the malignant tumor. Previous studies have reported that Sotetsuflavone from Cycas revoluta Thunb. can markedly suppress lung cancer cell proliferation by apoptosis, though its effect on gastric cancer remains unknown.
Aim:
To investigate the inhibitory effect of Cycas revoluta Thunb. and to determine whether it can overcome gastric cancer cell drug resistance to 5-Fu.
Methods:
Cell viability was examined to determine whether the natural extract of Cycas revoluta Thunb. induced gastric cancer cell death. The half-maximal effective concentration and the half-maximal lethal concentration were calculatede. Wound-healing and transwell assays were performed to examine gastric cancer cell motility. Clonogenic assays were performed to investigate the synergistic effects of Cycas revoluta Thunb. with 5-Fu, and apoptotic bodies were detected by Hoechst staining. Western blotting was performed to examine the expression of related proteins and to investigate the molecular mechanism of Cycas revoluta Thunb.-induced cancer cell apoptosis. The expressions of proteins, including mammalian target of rapamycin (mTOR) and p-AKT, were detected in different combinations of treatments for 48 h, then analyzed by ECL detection.
Results:
Gastric cancer cells were more sensitive to the natural extract of Cycas revoluta Thunb. compared to normal gastric epithelial cells, and the extract effectively inhibited gastric cancer cell migration and invasion. The extract improved the anti-cancer effect of 5-Fu by enhancing the chemosensitization of gastric cancer cells. Extract plus 5-Fu further reduced the expression of the drug-resistance-related proteins p-AKT and mTOR after 48 h compared to 5-Fu alone. Compared to 5-Fu treatment alone, mTOR and p-AKT expression was significantly reduced by about 50% and 75%, respectively. We also found that the natural extract of Cycas revoluta Thunb. further increased 5-Fu-induced gastric cancer cell apoptosis. Expression of apoptosis-related protein X-linked inhibitor of apoptosis protein and apoptosis inducing factor were significantly reduced and increased, respectively, in the 5-Fu-resistant gastric cancer line SGC-7901/R treated with extract plus 5-Fu, while the expression of survivin did not change.
Conclusion:
The natural extract of Cycas revoluta Thunb. effectively inhibited gastric cancer cell growth and enhanced the anti-cancer effect of 5-Fu through the AKT-mTOR pathway.
Survivin is a small protein that belongs to the inhibitor of apoptosis protein family. It is abundantly expressed in tumors compared with adult differentiated tissues, being associated with poor prognosis in many human neoplasms. This apoptotic inhibitor has a relevant role in both the promotion of cancer cell survival and in the inhibition of cell death. Consequently, aberrant survivin expression stimulates tumor progression and confers resistance to several therapeutic strategies in a variety of tumors. In fact, efficient survivin downregulation or inhibition results in spontaneous apoptosis or sensitization to chemotherapy and radiotherapy. Therefore, all these features make survivin an attractive therapeutic target to treat cancer. Currently, there are several survivin inhibitors under clinical evaluation, although more specific and efficient survivin inhibitors are being developed. Moreover, novel combination regimens targeting survivin together with other therapeutic approaches are currently being designed and assessed. In this review, recent progress in the therapeutic options targeting survivin for cancer treatment is analyzed. Direct survivin inhibitors and their current development status are explored. Besides, the major signaling pathways implicated in survivin regulation are described and different therapeutic approaches involving survivin indirect inhibition are evaluated. Finally, promising novel inhibitors under preclinical or clinical evaluation as well as challenges of developing survivin inhibitors as a new therapy for cancer treatment are discussed.
Survivin, the smallest member of the inhibitor of apoptosis protein (IAP) family, is overexpressed in cells of almost all cancers but not in most normal tissues in adults. Survivin expression is required for cancer cell survival and knocking down its expression or inhibiting its function using molecular approaches results in spontaneous apoptosis. Thus, survivin is an attractive and perhaps ideal target for cancer drug discovery. However, a US Food and Drug Administration (FDA)-approved drug targeting survivin has yet to emerge. In this Foundation Review, we examine and evaluate various strategies that have been used to target survivin and the stages of each survivin inhibitor to help understand this lack of success. We also provide future perspectives moving forward in targeting survivin for drug discovery.
Survivin is the smallest member of the inhibitors of apoptosis (IAP) family. However, participation in inhibition of apoptosis is not the only function of this molecule. Survivin can also affect the proper process of mitosis and even promoting of angiogenesis or DNA repair. High levels of survivin expression are connected with foetal tissues during intrauterine development. In the overwhelming majority of healthy, differentiated adult tissues, amounts of survivin are markedly reduced. On the other hand, survivin is also often abundantly expressed in cases of various types of cancer. Generally, high expression levels of survivin are associated with a poor prognosis, an increased rate of tumour recurrence and high resistance to chemo- as well as radiotherapy, hence survivin can be considered a factor in the initiation and progression of many types of cancer with great significance and potential for cancer therapy. Nonetheless, progress in development of survivin inhibitors or primarily, in survivin-related molecular therapies, is surprisingly not very fast and indeed remains a challenge for the future. The objective of this article is to summarize known facts about survivin, its contribution to inhibition of apoptosis and cell division and its implication in the development of gynaecological tumours. At the end, known survivin inhibitors and their effect on regulation of tumour growth will be referenced.
BACKGROUND: Inhibitor of apoptosis proteins (IAPs) suppress apoptotic cell death in several model systems and are highly conserved between insects and mammals. All IAPs contain at least one copy of the approximately 70 amino-acid baculovirus IAP repeat (BIR), and this domain is essential for the anti-apoptotic activity of the IAPs. Both the marked structural diversity of IAPs and the identification of BIR-containing proteins (BIRPs) in yeast, however, have led to the suggestion that BIRPs might play roles in other, as yet unidentified, cellular processes besides apoptosis. Survivin, a human BIRP, is upregulated 40-fold at G2-M phase and binds to mitotic spindles, although its role at the spindle is still unclear. RESULTS: We have identified and characterised two Caenorhabditis elegans BIRPs,BIR-1 and BIR-2; these proteins are the only BIRPs in C. elegans. The bir-1 gene is highly expressed during embryogenesis with detectable expression throughout other stages of development; bir-2 expression is detectable only in adults and embryos. Overexpression of bir-1 was unable to inhibit developmentally occurring cell death in C. elegans and inhibition of bir-1 expression did not increase cell death. Instead, embryos lacking bir-1 were unable to complete cytokinesis and they became multinucleate. This cytokinesis defect could be partially suppressed by transgenic expression of survivin, the mammalian BIRP most structurally related to BIR-1, suggesting a conserved role for BIRPs in the regulation of cytokinesis. CONCLUSIONS: BIR-1, a C. elegans BIRP, is probably not involved in the general regulation of apoptosis but is required for embryonic cytokinesis. We suggest that BIRPs may regulate cytoskeletal changes in diverse biological processes including cytokinesis and apoptosis.
Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ 2 ) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
Inhibitors of programmed cell death (apoptosis) aberrantly prolonging cell viability may contribute to cancer by facilitating the insurgence of mutations and by promoting resistance to therapy. Despite the identification of several new apoptosis inhibitors related to bcl-2 or to the baculovirus IAP gene, it is not clear whether apoptosis inhibition plays a general role in neoplasia. Here, we describe a new human gene encoding a structurally unique IAP apoptosis inhibitor, designated survivin. Survivin contains a single baculovirus IAP repeat and lacks a carboxyl-terminal RING finger. Present during fetal development, survivin is undetectable in terminally differentiated adult tissues. However, survivin becomes prominently expressed in transformed cell lines and in all the most common human cancers of lung, colon, pancreas, prostate and breast, in vivo. Survivin is also found in approximately 50% of high-grade non-Hodgkin's lymphomas (centroblastic, immunoblastic), but not in low-grade lymphomas (lymphocytic). Recombinant expression of survivin counteracts apoptosis of B lymphocyte precursors deprived of interleukin 3 (IL-3). These findings suggest that apoptosis inhibition may be a general feature of neoplasia and identify survivin as a potential new target for apoptosis-based therapy in cancer and lymphoma.
Survivin is a new IAP apoptosis inhibitor expressed during development and in human cancer in vivo. The coding strand of the survivin gene was extensively complementary to that of effector cell protease receptor-1 (EPR-1), prompting the present investigation
on the origin and functional relationship of these two transcripts. Southern blots of genomic DNA were consistent with the
presence of multiple, evolutionarily conserved, EPR-1/Survivin-related genes. By pulsed field gel electrophoresis and single-
and two-color fluorescence in situ hybridization, these were contained within a contiguous physical interval of 75–130 kilobases (kb) on chromosome 17q25. In
Northern blots, a single strand-specific probe identified a 1.3-kb EPR-1 mRNA broadly distributed in normal adult and fetal
tissues, structurally distinct from the 1.9-kb Survivin transcript expressed in transformed cell lines. Transient co-transfection
of an EPR-1 cDNA potentially acting as a Survivin antisense with a lacZ reporter plasmid resulted in loss of viability of HeLa cells. In contrast, co-transfection of an antisense cDNA of intercellular
adhesion molecule-1 or a sense-oriented Survivin cDNA was without effect. In stably transfected HeLa cells, ZnSO4 induction of an EPR-1 mRNA under the control of a metallothionein promoter suppressed the expression of endogenous survivin.
This resulted in (i) increased apoptosis as detected by analysis of DNA content and in situ internucleosomal DNA fragmentation and (ii) inhibition of cell proliferation as compared with induced vector control transfectants.
These findings suggest the existence of a potential EPR-1/survivin gene cluster and identify survivin as a new target for disrupting cell viability pathways in cancer.
A novel inhibitor of apoptosis designated survivin has recently been found in many common human cancers but not in normal tissues. A potential distribution of survivin in gastric cancer and its implication for apoptosis inhibition have been investigated. Recombinant survivin expressed in Escherichia coli as a glutathione S-transferase fusion protein was used to raise a novel panel of mouse monoclonal antibodies. In an immunohistochemical analysis of 174 cases of gastric carcinomas (stages I-III), anti-survivin monoclonal antibody 8E2 (IgG1) reacted with 34.5% of cases (60 of 174 cases) with a variable number of tumor cells stained (20-100%). In contrast, no expression of survivin in neighboring normal tissues was observed. When stratified for p53 and bcl-2 expression and apoptotic index, the expression of survivin significantly segregated with p53- and bcl-2-positive cases [56.1 versus 15.2% (P = 0.001) and 69.2 versus 31.6% (P = 0.006), respectively] and with a decreased apoptotic index as compared with that of survivin-negative tumors (0.97 +/- 0.64 versus 0.62 +/- 0.39%, P < 0.001). These data identify a role for survivin in promoting aberrantly increased cell viability in gastric cancer and suggest a potential correlation between accumulated p53 and survivin expression in neoplasia.
A new software suite, called Crystallography & NMR System (CNS), has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. In contrast to existing structure-determination programs, the architecture of CNS is highly flexible, allowing for extension to other structure-determination methods, such as electron microscopy and solid-state NMR spectroscopy. CNS has a hierarchical structure: a high-level hypertext markup language (HTML) user interface, task-oriented user input files, module files, a symbolic structure-determination language (CNS language), and low-level source code. Each layer is accessible to the user. The novice user may just use the HTML interface, while the more advanced user may use any of the other layers. The source code will be distributed, thus source-code modification is possible. The CNS language is sufficiently powerful and flexible that many new algorithms can be easily implemented in the CNS language without changes to the source code. The CNS language allows the user to perform operations on data structures, such as structure factors, electron-density maps, and atomic properties. The power of the CNS language has been demonstrated by the implementation of a comprehensive set of crystallographic procedures for phasing, density modification and refinement. User-friendly task-oriented input files are available for nearly all aspects of macromolecular structure determination by X-ray crystallography and solution NMR.
Progression of the cell cycle and control of apoptosis (programmed cell death) are thought to be intimately linked processes, acting to preserve homeostasis and developmental morphogenesis. Although proteins that regulate apoptosis have been implicated in restraining cell-cycle entry and controlling ploidy (chromosome number), the effector molecules at the interface between cell proliferation and cell survival have remained elusive. Here we show that a new inhibitor of apoptosis (IAP) protein, survivin, is expressed in the G2/M phase of the cell cycle in a cycle-regulated manner. At the beginning of mitosis, survivin associates with microtubules of the mitotic spindle in a specific and saturable reaction that is regulated by microtubule dynamics. Disruption of survivin-microtubule interactions results in loss of survivin's anti-apoptosis function and increased caspase-3 activity, a mechanism involved in cell death, during mitosis. These results indicate that survivin may counteract a default induction of apoptosis in G2/M phase. The overexpression of survivin in cancer may overcome this apoptotic checkpoint and favour aberrant progression of transformed cells through mitosis.
IAP family proteins are conserved throughout animal evolution and can block apoptosis when expressed in cells derived from multiple species. In many instances IAP family proteins can suppress apoptosis across species barriers (for review, see Clem and Duckett 1998), implying that although the details of their regulation may vary, these proteins evidently target a common mechanism involved in programmed cell death. Although all IAP family proteins require at least one BIR domain for their anti-apoptotic function, it should be emphasized that not all BIR-containing proteins are necessarily involved in apoptosis regulation as indicated by the failure of the Ac-IAP protein to suppress apoptosis despite harboring a BIR domain. Until proven otherwise, the most likely explanation for how IAPs prevent apoptosis is by binding to and inhibiting caspases, as indicated by recent studies (Deveraux et al. 1997 1998; Roy et al. 1997; Takahashi et al. 1998; Tamm et al. 1998). Assuming that this biased viewpoint is correct, how important are IAP family proteins likely to be for ensuring cell survival in vivo? The answer to that question probably depends on the type of cell under investigation and the specific cell death stimulus involved. I IAPs function primarily as inhibitors of caspases, then we can anticipate from other experiments where artificial means were used to suppress these proteases that APs will be capable of rescuing cells from some cell death signals but not others (Green and Reed 1998). Mitochondrial involvement appears to be one of the key variables that determines whether caspase inhibitors are sufficient to provide long-term protection and preservation of clonigenic potential, versus merely delaying death by converting an apoptotic stimulus into a necrotic one (Reed 1997; Green and Reed 1998). In many types of cells, loss of cytochrome c from mitochondria, for example, has two ways of killing cells: (1) activation of caspases through Apaf-1; or (2) cessation of mitochondrial electron chain transport with subsequent ATP depletion, generation of reactive oxygen species, and related sequelae. If the role of IAPs is relegated to caspase suppression, this may prevent cytochrome c-induced apoptosis, but not necessarily stop cell death induced by caspase-independent mechanisms. Recent studies in which release of cytochrome c was found to be a potentially reversible event suggest that whether cytochrome c loss from mitochondria defines a cell death commitment point will likely vary among cell types and depending on a variety environmental factors (Q. Chen et al. 1998). These factors may include the extent to which cells are able to produce sufficient ATP from anaerobic glycolysis in the cytosol and the method by which mitochondrial membrane barrier function was altered to allow for exodus of cytochrome c (i.e., reversible versus irreversible/rupture). Defining the in vivo requirements for IAPs in the maintenance of cell survival may be difficult because of potential redundancy. Humans have at least five and possibly more IAP family genes and even lower organisms, such as Drosophila, appear to contain at least two IAP genes, implying evolutionary pressure to ensure adequate back-up if one of these genes were to become inactivated. If IAPs do indeed function predominantly as caspase inhibitors, then one could imagine a very important role for these endogenous protease inhibitors in ensuring that the small amounts of adventitial caspase activation, which must surely occur on a routine basis, do not amplify out of control, resulting in inappropriate cell death. In this regard, virtually every other protease system studied to date contains molecules whose sole function is directed toward dampening proteolysis through the cascade (for review, see Colman et al. 1994), thus ensuring that biologically appropriate triggering of the pathway only occurs when certain thresholds are surpassed. By analogy, it is attractive to consider IAP family proteins in the same way, as proteins that set thresholds for how much caspase activation is necessary to successfully trigger apoptosis. Through alterations in the levels of IAP family gene expression and interactions of IAPs with other proteins, this IAP-dependent threshold for caspase-induced apoptosis could be varied to suit various physiological needs. Dysregulation of these normal control mechanisms then could be a contributor to various diseases characterized by excessive (ischemia, AIDS, SMA) or inadequate (cancer, autoimmunity) cell death.
Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely alpha-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among beta subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.
Inhibitors of apoptosis (IAPs) are a family of proteins that bear baculoviral IAP repeats (BIRs) and regulate apoptosis in vertebrates and Drosophila melanogaster. The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both encode a single IAP, designated BIR1 and bir1, respectively, each of which bears two BIRs. In rich medium, BIR1 mutant S. cerevisiae underwent normal vegetative growth and mitosis. Under starvation conditions, however, BIR1 mutant diploids formed spores inefficiently, instead undergoing pseudohyphal differentiation. Most spores that did form failed to survive beyond two divisions after germination. bir1 mutant S. pombe spores also died in the early divisions after spore germination and became blocked at the metaphase/anaphase transition because of an inability to elongate their mitotic spindle. Rather than inhibiting caspase-mediated cell death, yeast IAP proteins have roles in cell division and appear to act in a similar way to the IAPs from Caenorhabditis elegans and the mammalian IAP Survivin.
The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murine survivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin(140), similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin(121) that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin(40), lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin(140) and survivin(121) inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin(140) mRNA, whereas survivin(121)-specific transcripts were detected in all tissues, while those representing survivin(40) were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis. (Blood. 2000;95:1435-1442)
The inhibitor of apoptosis (IAP) gene family comprises molecules that block the activity of pro-apoptotic caspase proteases. Paradoxically, yeasts contain IAP proteins but no caspases and no apoptotic program. To determine the function of these proteins in vivo, we disrupted the BIR1 gene, encoding the only known IAP in yeast Saccharomyces cerevisiae. Sporulation of heterozygous diploids yielded no viable mutant haploids, indicating that BIR1 is an essential gene. By flow cytometry, some heterozygous mutants were polyploid accumulating >4 N DNA content. These cells exhibited a 20-40% reduction in growth rate, which was rescued by plasmid-borne over-expression of BIR1 but not by its human counterpart, survivin. Deletion analysis revealed that the N-terminal domain of Bir1, containing the conserved baculovirus IAP repeat, was able to partially complement the cell growth defect caused by BIR1 deletion. Moreover, the full-length and truncated forms of Bir1 accelerated cell division in wild-type cells. Finally, BIR1 heterozygous mutants exhibited grossly altered cell morphology with misshapen or abnormally long buds connected to an unusually large mother cell. These findings identify a novel function of IAP proteins in the pleiotropic control of cell division, in addition to their role in the suppression of apoptosis.
Deterin, a new apoptosis inhibitor fromDrosophila melanogaster, possesses an unusual structure of only a single baculovirus inhibitor of apoptosis (IAP)-type repeat and no RING finger motif.
The biochemical actions of deterin are demonstrated in SF9 and S2 cell transfection assays, in which the expressed protein
acts in the cytoplasm to inhibit or deter cells from apoptosis otherwise induced by the caspase-dependent apoptosis activator
reaper or by cytotoxicants. A loss of function phenotype for deterin of cell death was indicated by transfections with either
a dominant negative deterin mutant or with inhibitory RNA (RNAi) for deterin. The dominant negative C-terminal fragment that
antagonized antiapoptotic activity of deterin did not affect antiapoptotic activity of DIAP1 or p35. Both the baculovirus
IAP-type repeat (BIR) domain and the α-helical C-terminal domain are necessary in both SF9 and S2 cells for deterin to manifest
its activity to prevent cell death. The approximately 650-base deterin transcript is present in embryos, third instar larvae,
and late stage nurse cells of adult females. The deterin transcript is distributed throughout early stage embryos, whereas
in later stage embryos it becomes progressively restricted to the central nervous system and gonads. Whereas the nematode
survivin-type IAP has thus far been implicated only as a mitotic regulator,Drosophila deterin constitutes the first invertebrate member of the survivin-type IAP group to exhibit apoptosis-inhibitory activity.
Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murinesurvivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin140, similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin121 that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin40, lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin140 and survivin121 inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin140 mRNA, whereas survivin121-specific transcripts were detected in all tissues, while those representing survivin40 were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis.
The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The programs are written mainly in standard Fortran77. They are from a wide variety of sources but are connected by standard data file formats. The package has been ported to all the major platforms under both Unix and VMS. The suite is distributed by anonymous ftp from Daresbury Laboratory and is widely used throughout the world.
The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource.
The inhibitor-of-apoptosis (IAP) family of proteins, originally identified in baculoviruses, regulate programmed cell death in a variety of organisms. IAPs inhibit specific enzymes (caspases) in the death cascade and contain one to three modules of a common 70-amino-acid motif called the BIR domain. Here we describe the nuclear magnetic resonance structure of a region encompassing the second BIR domain (BIR2) of a human IAP family member, XIAP (also called hILP or MIHA). The structure of the BIR domain consists of a three-stranded antiparallel β-sheet and four α-helices and resembles a classical zinc finger. Unexpectedly, conserved amino acids within the linker region between the BIR1 and BIR2 domains were found to be critical for inhibiting caspase-3. The absence or presence of these residues may explain the differences in caspase inhibition observed for different truncated and full-length IAPs. Our data further indicate that these residues may bind to the active site and that the BIR domain may interact with an adjacent site on the enzyme.
Here we investigate the role of the control of apoptosis in normal cell division. We show that interference with the expression or function of the apoptosis inhibitor survivin causes caspase-dependent cell death in the G2/M phase of the cell cycle, and a cell-division defect characterized by centrosome dysregulation, multipolar mitotic spindles and multinucleated, polyploid cells. Use of a dominant-negative survivin mutant or antisense survivin complementary DNA disrupts a supramolecular assembly of survivin, caspase-3 and the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 within centrosomes, and results in caspase-dependent cleavage of p21. Polyploidy induced by survivin antagonists is accentuated in p21-deficient cells, and corrected by exogenous expression of p21. These findings show that control of apoptosis and preservation of p21 integrity within centrosomes by survivin are required for normal mitotic progression.
The Crystallography and NMR System (CNS) software suite is the result of an international collaborative effort amongst several research groups. It has been developed for macromolecular structure determination by X-ray crystallography or solution nuclear magnetic resonance (NMR) spectroscopy. CNS is available to academic institutions under a licence agreement. The CNS website provides information on the software and its use, an application form for downloading the software, and a bibliography.
Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
An expression system has been established for the incorporation of selenomethionine into recombinant proteins produced from plasmids in Escherichia coli. Replacement of methionine by selenomethionine is demonstrated at the level of 100% for both T4 and E. coli thioredoxins. The natural recombinant proteins and the selenomethionyl variants of both thioredoxins crystallize isomorphously. Anomalous scattering factors were deduced from synchrotron X-ray absorption measurements of crystals of the selenomethionyl proteins. Taken with reference to experience in the structural analysis of selenobiotinyl streptavidin by the method of multiwavelength anomalous diffraction (MAD), these data indicate that recombinant selenomethionyl proteins analyzed by MAD phasing offer a rather general means for the elucidation of atomic structures.
The solvent-accessible surface area (As) of 23 oligomeric proteins is calculated using atomic co-ordinates from high-resolution and well-refined crystal structures. As is correlated with the protein molecular weight, and a power law predicts its value to within 5% on average. The accessible surface of the average oligomer is similar to that of monomeric proteins in its hydropathy and amino acid composition. The distribution of the 20 amino acid types between the protein surface and its interior is also the same as in monomers. Interfaces, i.e. surfaces involved in subunit contacts, differ from the rest of the subunit surface. They are enriched in hydrophobic side-chains, yet they contain a number of charged groups, especially from Arg residues, which are the most abundant residues at interfaces except for Leu. Buried Arg residues are involved in H-bonds between subunits. We counted H-bonds at interfaces and found that several have none, others have one H-bond per 200 A2 of interface area on average (1 A = 0.1 nm). A majority of interface H-bonds involve charged donor or acceptor groups, which should make their contribution to the free energy of dissociation significant, even when they are few. The smaller interfaces cover about 700 A2 of the subunit surface. The larger ones cover 3000 to 10,000 A2, up to 40% of the subunit surface area in catalase. The lower value corresponds to an estimate of the accessible surface area loss required for stabilizing subunit association through the hydrophobic effect alone. Oligomers with small interfaces have globular subunits with accessible surface areas similar to those of monomeric proteins. We suggest that these oligomers assemble from preformed monomers with little change in conformation. In oligomers with large interfaces, isolated subunits should be unstable given their excessively large accessible surface, and assembly is expected to require major structural changes.
An analysis has been made, from the data which are currently available, of the solvent content of 116 different crystal forms of globular proteins. The fraction of the crystal volume occupied by solvent is most commonly near 43 %, but has been observed to have values from about 27 to 65%. In many cases this range will be sufficiently restrictive to enable the probable number of molecules in the crystallographic asymmetric unit to be determined directly from the molecular weight of the protein and the space group and unit cell dimensions of the crystal.
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Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
Various algorithms are described, developed for the dm density modification package, which have not been described elsewhere. Methods are described for the following problems: determination of the absolute scale and overall temperature factor of a data set, by a method which is less dependent on data resolution than Wilson statistics; an efficient interpolation algorithm for averaging and its application to refinement of averaging operators; a method for the automatic determination of averaging masks.
Survivin is a member of the inhibitor of apoptosis protein (IAP) family. We investigated the antiapoptotic mechanism of Survivin, as well as its expression in 60 human tumor cell lines used for the National Cancer Institute's anticancer drug screening program. In cotransfection experiments, cell death induced by Bax or Fas (CD 95) was partially inhibited (mean +/- SD, 65% +/- 8%) by Survivin, whereas XIAP, another IAP family member, almost completely blocked cell death (93% +/- 4%) under the same conditions. Survivin and XIAP also protected 293 cells from apoptosis induced by overexpression of procaspase-3 and -7 and inhibited the processing of these zymogens into active caspases. In vitro binding experiments indicated that, like other IAP-family proteins, Survivin binds specifically to the terminal effector cell death proteases, caspase-3 and -7, but not to the proximal initiator protease caspase-8. Using a cell-free system in which cytosolic extracts were derived from control- or Survivin-transfected cells and where caspases were activated either by addition of cytochrome c and dATP or by adding recombinant active caspase-8, Survivin was able to substantially reduce caspase activity, as measured by cleavage of a tetrapeptide substrate, AspGluValAsp-aminofluorocoumarin. Similar results were obtained in intact cells when Survivin was overexpressed by gene transfection and caspase activation was induced by the anticancer drug etoposide. Survivin was expressed in all 60 cancer cell lines analyzed, with highest levels in breast and lung cancers and lowest levels in renal cancers. These findings indicate that Survivin, which is commonly expressed in human tumor cell lines, can bind the effector cell death proteases caspase-3 and -7 in vitro and inhibits caspase activity and cell death in cells exposed to diverse apoptotic stimuli. Although quantitative differences may exist, these observations suggest commonality in the mechanisms used by IAP-family proteins to suppress apoptosis.
The inhibitor of apoptosis (IAP) proteins form a highly conserved gene family that prevents cell death in response to a variety of stimuli. Herein we describe a newly defined murine IAP, designated Tiap, that proved to be a murine homologue of human survivin based on sequence comparison. TIAP has one baculovirus IAP repeat and lacks a C-terminal RING finger motif. TIAP interacted with the processed form of caspase 3 and inhibited caspase-induced cell death. Histological examinations revealed that TIAP is expressed in growing tissues such as thymus, testis, and intestine of adult mice and many tissues of embryos. In in vitro studies, TIAP was induced in splenic T cells activated with anti-CD3 antibody or Con A, and the expression of TIAP was up-regulated in synchronized NIH 3T3 cells at S to G2/M phase of the cell cycle. We propose that during cell proliferation, cellular protective activity may be augmented with inducible IAPs such as TIAP.
Inhibitor of apoptosis proteins (IAPs) suppress apoptotic cell death in several model systems and are highly conserved between insects and mammals. All IAPs contain at least one copy of the approximately 70 amino-acid baculovirus IAP repeat (BIR), and this domain is essential for the anti-apoptotic activity of the IAPs. Both the marked structural diversity of IAPs and the identification of BIR-containing proteins (BIRPs) in yeast, however, have led to the suggestion that BIRPs might play roles in other, as yet unidentified, cellular processes besides apoptosis. Survivin, a human BIRP, is upregulated 40-fold at G2-M phase and binds to mitotic spindles, although its role at the spindle is still unclear.
We have identified and characterised two Caenorhabditis elegans BIRPs,BIR-1 and BIR-2; these proteins are the only BIRPs in C. elegans. The bir-1 gene is highly expressed during embryogenesis with detectable expression throughout other stages of development; bir-2 expression is detectable only in adults and embryos. Overexpression of bir-1 was unable to inhibit developmentally occurring cell death in C. elegans and inhibition of bir-1 expression did not increase cell death. Instead, embryos lacking bir-1 were unable to complete cytokinesis and they became multinucleate. This cytokinesis defect could be partially suppressed by transgenic expression of survivin, the mammalian BIRP most structurally related to BIR-1, suggesting a conserved role for BIRPs in the regulation of cytokinesis.
BIR-1, a C. elegans BIRP, is probably not involved in the general regulation of apoptosis but is required for embryonic cytokinesis. We suggest that BIRPs may regulate cytoskeletal changes in diverse biological processes including cytokinesis and apoptosis.
Caspase-9-mediated apoptosis (programmed cell death) plays a central role in the development and homeostasis of all multicellular organisms. Mature caspase-9 is derived from its procaspase precursor as a result of recruitment by the activating factor Apaf-1. The crystal structures of the caspase-recruitment domain of Apaf-1 by itself and in complex with the prodomain of procaspase-9 have been determined at 1.6 and 2.5 A resolution, respectively. These structures and other evidence reveal that each molecule of Apaf-1 interacts with a molecule of procaspase-9 through two highly charged and complementary surfaces formed by non-conserved residues; these surfaces determine recognition specificity through networks of intermolecular hydrogen bonds and van der Waals interactions. Mutation of the important interface residues in procaspase-9 or Apaf-1 prevents or reduces activation of procaspase-9 in a cell-free system. Wild-type, but not mutant, prodomains of caspase-9 completely inhibit catalytic processing of procaspase-9. Furthermore, analysis of homologues from Caenorhabditis elegans indicates that recruitment of CED-3 by CED-4 is probably mediated by the same set of conserved structural motifs, with a corresponding change in the specificity-determining residues.
Members of the inhibitor of apoptosis (IAP) family of proteins are able to inhibit cell death following viral infection, during development or in cell lines in vitro. All IAP proteins bear one or more baculoviral IAP repeats (BIRs). Here we describe the solution structure of the third BIR domain from the mammalian IAP homolog B (MIHB/c-IAP-1). The BIR domain has a novel fold that is stabilized by zinc tetrahedrally coordinated by one histidine and three cysteine residues. The structure consists of a series of short alpha-helices and turns with the zinc packed in an unusually hydrophobic environment created by residues that are highly conserved among all BIRs.
The caspase recruitment domain (CARD) of Apaf-1 binds to the CARD of caspase-9 to trigger a proteolytic cascade that leads to apoptotic cell death. We report the crystal structure of the Apaf-1 CARD at 1. 3 A resolution, solved in a two-element multiwavelength anomalous dispersion (MAD) X-ray diffraction experiment. This CARD adopts a six-helix bundle fold with Greek key topology surrounding an extensive hydrophobic core. This fold, which we call the "death fold", is found in other domains that mediate interactions in apoptotic signaling despite very low sequence identity. From a structure-based alignment, we identify conserved patterns that characterize the death fold and its subclasses. Like the Ig-fold, it provides a rigid structural scaffold upon which diverse recognition surfaces are assembled.
Yeast two-hybrid and genetic interaction screens indicate that Bir1p, a yeast protein containing phylogenetically conserved antiapoptotic repeat domains called baculovirus inhibitor of apoptosis repeats (BIRs), is involved in chromosome segregation events. In the two-hybrid screen, Bir1p specifically interacts with Ndc10p, an essential component of the yeast kinetochore. Although Bir1p carries two BIR motifs in the N-terminal region, the C-terminal third of the protein is sufficient to provide strong interaction with Ndc10p and moderate interaction with Skp1p, another essential component of the yeast kinetochore. In addition, deletion of BIR1 is synthetically lethal with deletion of CBF1 or CTF19, genes specifying two other components of the yeast kinetochore. Yeast cells deleted of BIR1 have a chromosome-loss phenotype, which can be completely rescued by elevating NDC10 dosage. Furthermore, overexpression of either full-length or the C-terminal region of Bir1p can efficiently suppress the chromosome-loss phenotype of both bir1Delta null and skp1-4 mutants. Our data suggest that Bir1p participates in chromosome segregation events, either directly or via interaction with kinetochore proteins, and these effects are apparently not mediated by the BIR domains of Bir1p.
The coupling of apoptosis (programmed cell death) to the cell division cycle is essential for homeostasis and genomic integrity. Here, we report the crystal structure of survivin, an inhibitor of apoptosis, which has been implicated in both control of cell death and regulation of cell division. In addition to a conserved N-terminal Zn finger baculovirus IAP repeat, survivin forms a dimer through a symmetric interaction with an intermolecularly bound Zn atom located along the molecular dyad axis. The interaction of the dimer-related C-terminal alpha helices forms an extended surface of approximately 70 A in length. Mutagenesis analysis revealed that survivin dimerization and an extended negatively charged surface surrounding Asp-71 are required to counteract apoptosis and preserve ploidy. These findings may provide a structural basis for a dual role of survivin in inhibition of apoptosis and regulation of cell division.
The determination of macromolecular structure by crystallography involves fitting atomic models to the observed diffraction data. The traditional measure of the quality of this fit, and presumably the accuracy of the model, is the R value. Despite stereochemical restraints, it is possible to overfit or 'misfit' the diffraction data: an incorrect model can be refined to fairly good R values as several recent examples have shown. Here I propose a reliable and unbiased indicator of the accuracy of such models. By analogy with the cross-validation method of testing statistical models I define a statistical quantity (R(free) (T) that measures the agreement between observed and computed structure factor amplitudes for a 'test' set of reflections that is omitted in the modelling and refinement process. As examples show, there is a high correlation between R(free) (T) and the accuracy of the atomic model phases. This is useful because experimental phase information is usually inaccurate, incomplete or unavailable. I expect that R(free) (T) will provide a measure of the information content of recently proposed models of thermal motion and disorder, time-averaging and bulk solvent.
Crystal structure and mutagenic analysis of the inhibitor-of-apoptosis pro-Brü nger Free R value: a novel statistical quantity for assessing the accuracy of crystal structures
- W Wu
- H Zhang
- F Li
- S.-C Ng
- D C Altieri
Wu, W., Zhang, H., Li, F., Ng, S.-C., and Altieri, D.C. (2000). Crystal structure and mutagenic analysis of the inhibitor-of-apoptosis pro-Brü nger, A.T. (1992). Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475.
The CCP4 Sun, Chen, suite: programs for protein crystallography
- C Cai
- M Gunasekera
- A H Meadows
- R P Wang
CCP4 (Collaborative Computational Project 4) (1994). The CCP4 Sun, C., Cai, M., Gunasekera, A.H., Meadows, R.P., Wang, H., Chen, suite: programs for protein crystallography. Acta Crystallogr. D 50, J., Zhang, H., Wu, W., Xu, N., Ng, S.C., et al. (1999). NMR structure 760–763.
The protein Muchmore
- H Weissig
- I N Shindyalov
- P E S W Bourne
- J Chen
- C Jakob
- D Zakula
- E D Matayoshi
Weissig, H., Shindyalov, I.N., and Bourne, P.E. (2000). The protein
Muchmore, S.W., Chen, J., Jakob, C., Zakula, D., Matayoshi, E.D.,
data bank. Nucleic Acids Res. 28, 235-242.
Control of apoptosis and mitotic spindle supported by an EMBO fellowship
- La Li
- F Ambrosini
- G Chu
- E Y Plescia
- J Tognin
- S Marchisio
- Le Recherche
- La Cancer
- Ligue
This work was supported by grants from the Association pour la Li, F., Ambrosini, G., Chu, E.Y., Plescia, J., Tognin, S., Marchisio, Recherche sur le Cancer and La Ligue (R. L. M.). D. A. S. was P.C., and Altieri, D.C. (1998). Control of apoptosis and mitotic spindle supported by an EMBO fellowship. We thank Drs. G. Sainz and E. checkpoint by survivin. Nature 396, 580–584.
ESRF-Grenoble) for assistance in data collection and Drs
- Mitchell
Mitchell (ESRF-Grenoble) for assistance in data collection and Drs.
Role for yeast inhibitor of apo-Three differentially expressed survivin cDNA variants encode proptosis (IAP)-like proteins in cell division
- D L Vaux
- T Lithgow
Vaux, D.L., and Lithgow, T. (1999). Role for yeast inhibitor of apo-Three differentially expressed survivin cDNA variants encode proptosis (IAP)-like proteins in cell division. Proc. Natl. Acad. Sci. USA
teins with distinct antiapoptotic functions. Blood 95, 1435-1442.
96, 10170-10175.
The CCP4 Sun, Chen, suite: programs for protein crystallography NMR structure 760–763. and mutagenesis of the inhibitor-of-apoptosis protein XIAP
- C Cai
- M Gunasekera
- A H Meadows
- R P Wang
- H J Zhang
- H Wu
- W Xu
- N Ng
CCP4 (Collaborative Computational Project 4) (1994). The CCP4
Sun, C., Cai, M., Gunasekera, A.H., Meadows, R.P., Wang, H., Chen,
suite: programs for protein crystallography. Acta Crystallogr. D 50,
J., Zhang, H., Wu, W., Xu, N., Ng, S.C., et al. (1999). NMR structure
760–763.
and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature
401, 818–822.
Miscellaneous algorithms for den sity modification Crystal structure of Apaf-1 caspase recruitment domain: an alpha- Deveraux IAP family proteins— helical Greek key fold for apoptotic signaling 293, suppressors of apoptosis
- K Cowtan
- P Main
- D E Vaughn
- J Rodriguez
- Y Lazebnik
Cowtan, K., and Main, P. (1998). Miscellaneous algorithms for den-
Vaughn, D.E., Rodriguez, J., Lazebnik, Y., and Joshua-Tor, L. (1999).
sity modification. Acta Crystallogr. D 54, 487–493.
Crystal structure of Apaf-1 caspase recruitment domain: an alpha-
Deveraux, Q.L., and Reed, J.C. (1999). IAP family proteins—
helical Greek key fold for apoptotic signaling. J. Mol. Biol. 293,
suppressors of apoptosis. Genes Dev. 13, 239–252.
439–447.
Zinc fingers are sticking of apoptosis and inhibition of cell proliferation by survivin gene together
- G Ambrosini
- C Adida
- G Sirugo
- D C Mackay
- J P Crossley
Ambrosini, G., Adida, C., Sirugo, G., and Altieri, D.C. (1998). Induction
Mackay, J.P., and Crossley, M. (1998). Zinc fingers are sticking
of apoptosis and inhibition of cell proliferation by survivin gene
together. Trends Biochem. Sci. 23, 1-4.
targeting. J. Biol. Chem. 273, 11177-11182.
Free R value: a novel statistical quantity for assessing the accuracy of crystal structures
- W Wu
- H Zhang
- F Li
- S.-C Ng
Wu, W., Zhang, H., Li, F., Ng, S.-C., and Altieri, D.C. (2000). Crystal
structure and mutagenic analysis of the inhibitor-of-apoptosis pro-Brü nger, A.T. (1992). Free R value: a novel statistical quantity for
assessing the accuracy of crystal structures. Nature 355, 472-475.
tein survivin. Mol. Cell 6, this issue, 173-182.
Crystal structure of Apaf-1 caspase recruitment domain.
- Vaughn D.E.
- Rodriguez J.
- Lazebnik Y.
- Joshua-Tor L.
A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.
- Ambrosini G.
- Adida C.
- Altieri D.C.