Ilya Serebriiskii

Publications

• Interaction trap/two-hybrid system to identify interacting proteins.

  Erica A Golemis, Ilya Serebriiskii, Russell L Finley, Mikhail G Kolonin, Jeno Gyuris, Roger Brent

  Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]. 12/2011; Chapter 17:Unit 17.3..

  The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate pro... [more] The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins.
• 12.58

  Impact points

  Protein-intrinsic and signaling network-based sources of resistance to EGFR- and ErbB family-targeted therapies in head and neck cancer.

  Ranee Mehra, Ilya G Serebriiskii, Roland L Dunbrack, Matthew K Robinson, Barbara Burtness, Erica A Golemis

  Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy. 09/2011; 14(6):260-79.

  Agents targeting EGFR and related ErbB family proteins are valuable therapies for the treatment of many cancers. For some tumor types, including squamous cell carcinomas of the head and neck (SCCHN), antibodies targeting EGFR were the first protein-directed agents to show clinical benefit, and remai... [more] Agents targeting EGFR and related ErbB family proteins are valuable therapies for the treatment of many cancers. For some tumor types, including squamous cell carcinomas of the head and neck (SCCHN), antibodies targeting EGFR were the first protein-directed agents to show clinical benefit, and remain a standard component of clinical strategies for management of the disease. Nevertheless, many patients display either intrinsic or acquired resistance to these drugs; hence, major research goals are to better understand the underlying causes of resistance, and to develop new therapeutic strategies that boost the impact of EGFR/ErbB inhibitors. In this review, we first summarize current standard use of EGFR inhibitors in the context of SCCHN, and described new agents targeting EGFR currently moving through pre-clinical and clinical development. We then discuss how changes in other transmembrane receptors, including IGF1R, c-Met, and TGF-β, can confer resistance to EGFR-targeted inhibitors, and discuss new agents targeting these proteins. Moving downstream, we discuss critical EGFR-dependent effectors, including PLC-γ; PI3K and PTEN; SHC, GRB2, and RAS and the STAT proteins, as factors in resistance to EGFR-directed inhibitors and as alternative targets of therapeutic inhibition. We summarize alternative sources of resistance among cellular changes that target EGFR itself, through regulation of ligand availability, post-translational modification of EGFR, availability of EGFR partners for hetero-dimerization and control of EGFR intracellular trafficking for recycling versus degradation. Finally, we discuss new strategies to identify effective therapeutic combinations involving EGFR-targeted inhibitors, in the context of new system level data becoming available for analysis of individual tumors.

• Interaction trap/two-hybrid system to identify interacting proteins.

  Erica A Golemis, Ilya Serebriiskii, Russell L Finley, Mikhail G Kolonin, Jeno Gyuris, Roger Brent

  Current protocols in neuroscience / editorial board, Jacqueline N. Crawley ... [et al.]. 04/2011; Chapter 4:Unit 4.4.

  The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate pro... [more] The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins.
• 2.71

  Impact points

  Chemotherapy and signaling: How can targeted therapies supercharge cytotoxic agents?

  Tetyana V Bagnyukova, Ilya G Serebriiskii, Yan Zhou, Elizabeth A Hopper-Borge, Erica A Golemis, Igor Astsaturov

  Cancer biology & therapy. 11/2010; 10(9):839-53.

  In recent years, oncologists have begun to conclude that chemotherapy has reached a plateau of efficacy as a primary treatment modality, even if toxicity can be effectively controlled. Emerging specific inhibitors of signaling and metabolic pathways (i.e., targeted agents) contrast with traditional ... [more] In recent years, oncologists have begun to conclude that chemotherapy has reached a plateau of efficacy as a primary treatment modality, even if toxicity can be effectively controlled. Emerging specific inhibitors of signaling and metabolic pathways (i.e., targeted agents) contrast with traditional chemotherapy drugs in that the latter primarily interfere with the DNA biosynthesis and the cell replication machinery. In an attempt to improve on the efficacy, combination of targeted drugs with conventional chemotherapeutics has become a routine way of testing multiple new agents in early phase clinical trials. This review discusses the recent advances including integrative systematic biology and RNAi approaches to counteract the chemotherapy resistance and to buttress the selectivity, efficacy and personalization of anti-cancer drug therapy.
• 4.63

  Impact points

  Yeast two-hybrid system for studying protein-protein interactions--stage 1: Construction and characterization of a bait protein.

  Ilya Serebriiskii

  Cold Spring Harbor protocols. 05/2010; 2010(5):pdb.prot5429.

  An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the yeast two-hybrid system, uses transcriptional reporters in yeast to indirectly reflect the interaction b... [more] An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the yeast two-hybrid system, uses transcriptional reporters in yeast to indirectly reflect the interaction between two proteins. The term two-hybrid derives from the two classes of chimeric, or "hybrid," proteins used in each screen. The first, commonly referred to as the "bait," is a fusion of a protein of interest "x" with a DNA-binding domain (DBD-x). The second, sometimes called the "prey," is a fusion of a cDNA library "y" to a transcriptional activation domain (AD-y). Together, DBD-x and AD-y provide the basis of the detection system. The two-hybrid approach has gained wide popularity because of the relative ease and speed with which it can be used to identify novel protein-protein interactions and to analyze known interactions. In stage 1 of the method, detailed in this protocol, characterization of a novel bait is described, with attention to controls that increase the chance of the bait functioning in a two-hybrid screen.
• 4.63

  Impact points

  Yeast two-hybrid system for studying protein-protein interactions--stage 2: Transforming and characterizing the library.

  Ilya Serebriiskii

  Cold Spring Harbor protocols. 05/2010; 2010(5):pdb.prot5430.

  An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the "yeast two-hybrid system," uses transcriptional reporters in yeast to indirectly reflect the i... [more] An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the "yeast two-hybrid system," uses transcriptional reporters in yeast to indirectly reflect the interaction between two proteins. The term "two-hybrid" derives from the two classes of chimeric, or "hybrid," proteins used in each screen. The first, commonly referred to as the "bait," is a fusion of a protein of interest "x" with a DNA-binding domain (DBD-x). The second, sometimes called the "prey," is a fusion of a cDNA library "y" to a transcriptional activation domain (AD-y). Together, DBD-x and AD-y provide the basis of the detection system. The two-hybrid approach has gained wide popularity because of the relative ease and speed with which it can be used to identify novel protein-protein interactions and to analyze known interactions. The second stage of the method, described in this protocol, includes the transformation of yeast with a cDNA library, followed by library characterization. It can be performed in parallel with construction of a bait protein (stage 1).
• 4.63

  Impact points

  Yeast two-hybrid system for studying protein-protein interactions--stage 3: Screen for interacting proteins.

  Ilya Serebriiskii

  Cold Spring Harbor protocols. 05/2010; 2010(5):pdb.prot5431.

  An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the "yeast two-hybrid system," uses transcriptional reporters in yeast to indirectly reflect the i... [more] An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the "yeast two-hybrid system," uses transcriptional reporters in yeast to indirectly reflect the interaction between two proteins. The term "two-hybrid" derives from the two classes of chimeric, or "hybrid," proteins used in each screen. The first, commonly referred to as the "bait," is a fusion of a protein of interest "x" with a DNA-binding domain (DBD-x). The second, sometimes called the "prey," is a fusion of a cDNA library "y" to a transcriptional activation domain (AD-y). Together, DBD-x and AD-y provide the basis of the detection system. The two-hybrid approach has gained wide popularity because of the relative ease and speed with which it can be used to identify novel protein-protein interactions and to analyze known interactions. Once the bait strain has been made and characterized (stage 1) and the library strain has been transformed and frozen in aliquots (stage 2), the next step is to mate the two strains. Stage 3 of the method, described here, details the mating and screening of the cDNA library for positive interactors.
• 4.63

  Impact points

  Yeast two-hybrid system for studying protein-protein interactions--stage 4: Isolation of library plasmid insert and second confirmation of positive interactions.

  Ilya Serebriiskii

  Cold Spring Harbor protocols. 05/2010; 2010(5):pdb.prot5432.

  An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the "yeast two-hybrid system," uses transcriptional reporters in yeast to indirectly reflect the i... [more] An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the "yeast two-hybrid system," uses transcriptional reporters in yeast to indirectly reflect the interaction between two proteins. The term "two-hybrid" derives from the two classes of chimeric, or "hybrid," proteins used in each screen. The first, commonly referred to as the "bait," is a fusion of a protein of interest "x" with a DNA-binding domain (DBD-x). The second, sometimes called the "prey," is a fusion of a cDNA library "y" to a transcriptional activation domain (AD-y). Together, DBD-x and AD-y provide the basis of the detection system. The two-hybrid approach has gained wide popularity because of the relative ease and speed with which it can be used to identify novel protein-protein interactions and to analyze known interactions. Stage 4, described here, outlines a series of first-order and subsequent control experiments designed to establish whether an interacting protein is likely to be biologically significant. The number of positive interactions obtained will vary drastically from bait to bait. Subsequent processing methods will depend on the number initially obtained and on the preference of the individual investigator.
• 5.98

  Impact points

  Numb independently antagonizes Sanpodo membrane targeting and Notch signaling in Drosophila sensory organ precursor cells.

  Xin Tong, Diana Zitserman, Ilya Serebriiskii, Mark Andrake, Roland Dunbrack, Fabrice Roegiers

  Molecular biology of the cell. 03/2010; 21(5):802-10.

  In Drosophila, mitotic neural progenitor cells asymmetrically segregate the cell fate determinant Numb in order to block Notch signaling in only one of the two daughter cells. Sanpodo, a membrane protein required for Notch signaling in asymmetrically dividing cells, is sequestered from the plasma me... [more] In Drosophila, mitotic neural progenitor cells asymmetrically segregate the cell fate determinant Numb in order to block Notch signaling in only one of the two daughter cells. Sanpodo, a membrane protein required for Notch signaling in asymmetrically dividing cells, is sequestered from the plasma membrane to intracellular vesicles in a Numb-dependent way after neural progenitor cell mitosis. However, the significance of Numb-dependent Sanpodo regulation is unclear. In this study, we conducted a structure-function analysis to identify the determinants of Sanpodo targeting in vivo. We identified an NPAF motif in the amino-terminal cytoplasmic tail of Sanpodo, which is conserved among insect Sanpodo homologues. The Sanpodo NPAF motif is predicted to bind directly to the Numb phosphotyrosine-binding domain and is critical for Numb binding in vitro. Deletion or mutation of the NPAF motif results in accumulation of Sanpodo at the plasma membrane in Numb-positive cells in vivo. Genetic analysis of Sanpodo NPAF mutants shows that Numb-dependent Sanpodo endocytic targeting can be uncoupled from Notch signaling regulation. Our findings demonstrate that Sanpodo contains an evolutionarily conserved motif that has been linked to Numb-dependent regulation in vertebrates and further support the model that Numb regulates Notch signaling independently of Sanpodo membrane trafficking in neural progenitor cells.

• Synthetic lethal screen of an EGFR-centered network to improve targeted therapies.

  Igor Astsaturov, Vladimir Ratushny, Anna Sukhanova, Margret B Einarson, Tetyana Bagnyukova, Yan Zhou, Karthik Devarajan, Joshua S Silverman, Nadezhda Tikhmyanova, Natalya Skobeleva, Anna Pecherskaya, Rochelle E Nasto, Catherine Sharma, Sandra A Jablonski, Ilya G Serebriiskii, Louis M Weiner, Erica A Golemis

  Science signaling. 01/2010; 3(140):ra67.

  Intrinsic and acquired cellular resistance factors limit the efficacy of most targeted cancer therapeutics. Synthetic lethal screens in lower eukaryotes suggest that networks of genes closely linked to therapeutic targets would be enriched for determinants of drug resistance. We developed a protein ... [more] Intrinsic and acquired cellular resistance factors limit the efficacy of most targeted cancer therapeutics. Synthetic lethal screens in lower eukaryotes suggest that networks of genes closely linked to therapeutic targets would be enriched for determinants of drug resistance. We developed a protein network centered on the epidermal growth factor receptor (EGFR), which is a validated cancer therapeutic target, and used small interfering RNA screening to comparatively probe this network for proteins that regulate the effectiveness of both EGFR-targeted agents and nonspecific cytotoxic agents. We identified subnetworks of proteins influencing resistance, with putative resistance determinants enriched among proteins that interacted with proteins at the core of the network. We found that clinically relevant drugs targeting proteins connected in the EGFR network, such as protein kinase C or Aurora kinase A, or the transcriptional regulator signal transducer and activator of transcription 3 (STAT3), synergized with EGFR antagonists to reduce cell viability and tumor size, suggesting the potential for a direct path to clinical exploitation. Such a focused approach can potentially improve the coherent design of combination cancer therapies.
• 7.54

  Impact points

  NEDD9 Promotes Oncogenic Signaling in Mammary Tumor Development.

  Eugene Izumchenko, Mahendra K Singh, Olga V Plotnikova, Nadezhda Tikhmyanova, Joy L Little, Ilya G Serebriiskii, Sachiko Seo, Mineo Kurokawa, Brian L Egleston, Andres Klein-Szanto, Elena N Pugacheva, Richard R Hardy, Marina Wolfson, Denise C Connolly, Erica A Golemis

  Cancer research. 10/2009;

  In the past 3 years, altered expression of the HEF1/CAS-L/NEDD9 scaffolding protein has emerged as contributing to cancer metastasis in multiple cancer types. However, whereas some studies have identified elevated NEDD9 expression as prometastatic, other work has suggested a negative role in tumor p... [more] In the past 3 years, altered expression of the HEF1/CAS-L/NEDD9 scaffolding protein has emerged as contributing to cancer metastasis in multiple cancer types. However, whereas some studies have identified elevated NEDD9 expression as prometastatic, other work has suggested a negative role in tumor progression. We here show that the Nedd9-null genetic background significantly limits mammary tumor initiation in the MMTV-polyoma virus middle T genetic model. Action of NEDD9 is tumor cell intrinsic, with immune cell infiltration, stroma, and angiogenesis unaffected. The majority of the late-appearing mammary tumors of MMTV-polyoma virus middle T;Nedd9(-/-) mice are characterized by depressed activation of proteins including AKT, Src, FAK, and extracellular signal-regulated kinase, emphasizing an important role of NEDD9 as a scaffolding protein for these prooncogenic proteins. Analysis of cells derived from primary Nedd9(+/+) and Nedd9(-/-) tumors showed persistently reduced FAK activation, attachment, and migration, consistent with a role for NEDD9 activation of FAK in promoting tumor aggressiveness. This study provides the first in vivo evidence of a role for NEDD9 in breast cancer progression and suggests that NEDD9 expression may provide a biomarker for tumor aggressiveness. [Cancer Res 2009;69(18):7198-206].

• Interaction trap/two-hybrid system to identify interacting proteins.

  Erica A Golemis, Ilya Serebriiskii, Russell L Finley, Mikhail G Kolonin, Jeno Gyuris, Roger Brent

  Current protocols in protein science / editorial board, John E. Coligan ... [et al.]. 09/2009; Chapter 19:Unit19.2.

  The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate pro... [more] The yeast two-hybrid method (or interaction trap) is a powerful technique for detecting protein interactions. The procedure is performed using transcriptional activation of a dual reporter system in yeast to identify interactions between a protein of interest (the bait protein) and the candidate proteins for interaction. The method can be used to screen a protein library for interactions with a bait protein or to test for association between proteins that are expected to interact based on prior evidence. Interaction mating facilitates the screening of a library with multiple bait proteins.

• Two-hybrid dual bait system.

  Elena Kotova, Thomas Coleman, Ilya Serebriiskii

  Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]. 05/2009; Chapter 20:Unit 20.7.

  The yeast two-hybrid system, or interaction trap, is one of the most versatile methods available with which to identify and establish protein-protein interactions. The dual bait system is one adaptation of the classic approach. This system facilitates the simultaneous comparison of two distinct bait... [more] The yeast two-hybrid system, or interaction trap, is one of the most versatile methods available with which to identify and establish protein-protein interactions. The dual bait system is one adaptation of the classic approach. This system facilitates the simultaneous comparison of two distinct baits with one prey. One protein of interest is expressed as a fusion to the DNA-binding protein LexA (bait 1), while a second protein of interest is expressed as a fusion to the DNA-binding protein cI (bait 2). Strains of yeast engineered for screening of these dual baits possess four separate reporter genes. A plasmid expressing an activation domain-fused protein (prey), which can be either a defined protein interactor or a cDNA library, is also expressed to allow dual hybrid-mediated transcriptional activation.
• 5.98

  Impact points

  A Novel Cas Family Member, HEPL, Regulates FAK and Cell Spreading.

  Mahendra K Singh, Disha Dadke, Emmanuelle Nicolas, Ilya G Serebriiskii, Sinoula Apostolou, Adrian Canutescu, Brian L Egleston, Erica A Golemis

  Molecular biology of the cell. 05/2008; 19(4):1627-36.

  For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. ... [more] For over a decade, p130Cas/BCAR1, HEF1/NEDD9/Cas-L, and Efs/Sin have defined the Cas (Crk-associated substrate) scaffolding protein family. Cas proteins mediate integrin-dependent signals at focal adhesions, regulating cell invasion and survival; at least one family member, HEF1, regulates mitosis. We here report a previously undescribed novel branch of the Cas protein family, designated HEPL (for HEF1-Efs-p130Cas-like). The HEPL branch is evolutionarily conserved through jawed vertebrates, and HEPL is found in some species lacking other members of the Cas family. The human HEPL mRNA and protein are selectively expressed in specific primary tissues and cancer cell lines, and HEPL maintains Cas family function in localization to focal adhesions, as well as regulation of FAK activity, focal adhesion integrity, and cell spreading. It has recently been demonstrated that upregulation of HEF1 expression marks and induces metastasis, whereas high endogenous levels of p130Cas are associated with poor prognosis in breast cancer, emphasizing the clinical relevance of Cas proteins. Better understanding of the complete protein family should help inform prediction of cancer incidence and prognosis.
• 3.56

  Impact points

  Fibroblast-derived 3D matrix differentially regulates the growth and drug-responsiveness of human cancer cells.

  Ilya Serebriiskii, Remedios Castelló-Cros, Acacia Lamb, Erica A Golemis, Edna Cukierman

  Matrix biology : journal of the International Society for Matrix Biology. 04/2008;

  Recent studies have emphasized the importance of cellular microenvironment in modulating cell growth and signaling. In vitro, collagen matrices, Matrigel, and other synthetic support systems have been used to simulate in vivo microenvironments, and epithelial cells grown in these matrices manifest s... [more] Recent studies have emphasized the importance of cellular microenvironment in modulating cell growth and signaling. In vitro, collagen matrices, Matrigel, and other synthetic support systems have been used to simulate in vivo microenvironments, and epithelial cells grown in these matrices manifest significant differences in proliferation, differentiation, response to drugs, and other parameters. However, these substrates do not closely resemble the mesenchymal microenvironment that is typically associated with advanced carcinomas in vivo, which is produced to a large extent by fibroblasts. In this study, we have evaluated the ability of a fibroblast-derived three-dimensional matrix to regulate the growth of a panel of 11 human tumor epithelial cell lines. Although proliferative and morphological responses to three-dimensional cues segregated independently, general responsiveness to the matrix correlated with the ability of matrix to influence drug responses. Fibroblast-derived three-dimensional matrix increased beta1-integrin-dependent survival of a subset of human cancer cell lines during taxol treatment, while it sensitized or minimally influenced survival of other cells. beta1-integrin-dependent changes in cell resistance to taxol did not correlate with the degree of modulation of FAK and Akt, implying that additional signaling factors are involved. Based on these results, we propose that these matrices potentially have value as in vitro drug screening platforms.
• 3.71

  Impact points

  Selective Raf inhibition in cancer therapy.

  Vladimir Khazak, Igor Astsaturov, Ilya G Serebriiskii, Erica A Golemis

  Expert opinion on therapeutic targets. 01/2008; 11(12):1587-609.

  Over the past 5 years, the Raf kinase family has emerged as a promising target for protein-directed cancer therapy development. The goal of this review is to first provide a concise summary of the data validating Raf proteins as high-interest therapeutic targets. The authors then outline the mode of... [more] Over the past 5 years, the Raf kinase family has emerged as a promising target for protein-directed cancer therapy development. The goal of this review is to first provide a concise summary of the data validating Raf proteins as high-interest therapeutic targets. The authors then outline the mode of action of Raf kinases, emphasizing how Raf activities and protein interactions suggest specific approaches to inhibiting Raf. The authors then summarize the set of drugs, antisense reagents and antibodies available or in development for therapeutically targeting Raf or Raf-related proteins, as well as existing strategies combining these and other therapeutic agents. Finally, the authors discuss recent results from systems biology analyses that have the potential to increasingly guide the intelligent selection of combination therapies involving Raf-targeting agents and other therapeutics.
• 7.54

  Impact points

  A new central scaffold for metastasis: parsing HEF1/Cas-L/NEDD9.

  Geraldine M O'Neill, Sachiko Seo, Ilya G Serebriiskii, Stuart R Lessin, Erica A Golemis

  Cancer research. 11/2007; 67(19):8975-9.

  Greater understanding of metastasis is required to improve cancer treatment outcomes. Recently, changes in expression of the scaffold protein HEF1/CAS-L/NEDD9 were found to be a potent prometastatic stimulus in melanoma and other cancers. Mechanistic studies suggest diverse cellular roles of HEF1 an... [more] Greater understanding of metastasis is required to improve cancer treatment outcomes. Recently, changes in expression of the scaffold protein HEF1/CAS-L/NEDD9 were found to be a potent prometastatic stimulus in melanoma and other cancers. Mechanistic studies suggest diverse cellular roles of HEF1 and highlight its importance in the response to extracellular cues that drive invasion and metastasis. As a metastatic "hub" for signaling in cancer, HEF1 may provide a useful target for drug discovery efforts.
• 3.09

  Impact points

  A 1.55 A resolution X-ray crystal structure of HEF2/ERH and insights into its transcriptional and cell-cycle interaction networks.

  Tengchuan Jin, Feng Guo, Ilya G Serebriiskii, Andrew Howard, Yu-Zhu Zhang

  Proteins. 09/2007; 68(2):427-37.

  Functional complementation screens can identify known or novel proteins with important intracellular activities. We have isolated human enhancer of filamentation 2 (HEF2) in a screen to find human genes that promote pseudohyphal growth in budding yeast. HEF2 is identical to enhancer of rudimentary h... [more] Functional complementation screens can identify known or novel proteins with important intracellular activities. We have isolated human enhancer of filamentation 2 (HEF2) in a screen to find human genes that promote pseudohyphal growth in budding yeast. HEF2 is identical to enhancer of rudimentary homolog (ERH), a highly conserved protein of 104 amino acids. In silico protein-interaction mapping implies that HEF2/ERH interacts with transcription factors, cell-cycle regulators, and other proteins shown to enhance filamentous growth in S. cerevisiae, suggesting a context for studies of HEF2/ERH function. To provide a mechanistic basis to study of HEF2/ERH, we have determined the crystal structure of HEF2/ERH at 1.55 A. The crystal asymmetric unit contains a HEF2/ERH monomer. The two monomers of the physiological dimer are related by the y, x, -z crystal symmetric operation. The HEF2/ERH structure is characterized by a novel alpha + beta fold, a four-strand antiparallel beta-sheet with three alpha-helixes on one side of the sheet. The beta-sheets from the two monomers together constitute a pseudo-beta-barrel, and form the center of the functional HEF2/ERH dimer, with a cavity channel at the dimer interface. Docking of this structure to the HEF2/ERH partner protein DCOH/PCD suggests that HEF2/ERH may regulate the oligomeric state of this protein. These data suggest that HEF2/ERH may be an important transcription regulator that also functions in the control of cell-cycle progression.

• A bacterial/yeast merged two-hybrid system: protocol for bacterial screening.

  Ilya G Serebriiskii, Nadia Milech, Erica A Golemis

  Methods in molecular biology (Clifton, N.J.). 02/2007; 408:291-315.

  Yeast two-hybrid systems are artificial genetic systems that allow identification and characterization of protein-protein interactions. One common limit to the use of these techniques is when the intrinsic property of "bait" proteins of interest transcriptionally autoactivates reporters, e... [more] Yeast two-hybrid systems are artificial genetic systems that allow identification and characterization of protein-protein interactions. One common limit to the use of these techniques is when the intrinsic property of "bait" proteins of interest transcriptionally autoactivates reporters, eliminating the basis for interaction detection. To circumvent this problem, autoactivating baits can be alternatively used in bacteria wherein such activation does not occur. A single-vector system has been developed, which can be used either in yeast or in bacteria, streamlining and expanding capacity for protein-protein interaction screens. A concise proposal is provided for use of this system in bacteria; a companion article, chapter 15, describes use of the system in yeast.

• A bacterial/yeast merged two-hybrid system: protocol for yeast screening with single or parallel baits.

  Nadezhda Y Tikhmyanova, Eugene A Izumchenko, Ilya G Serebriiskii, Erica A Golemis

  Methods in molecular biology (Clifton, N.J.). 02/2007; 408:257-90.

  The yeast two-hybrid system is a useful tool for identifying new protein-protein interactions, and for the dissection of previously identified interactions. An important issue in protein-interaction studies is frequently that of determining whether a protein associates specifically with one protein ... [more] The yeast two-hybrid system is a useful tool for identifying new protein-protein interactions, and for the dissection of previously identified interactions. An important issue in protein-interaction studies is frequently that of determining whether a protein associates specifically with one protein or domain of interest, or has a more promiscuous interaction profile. To help address this issue, the authors have created a new two-hybrid system, which can be used either in bacteria or in yeast to counterscreen against "decoy" baits in parallel with a primary screen, hence improving the power and specificity of the method. Protocols of this system for use in yeast are provided; a companion article, Serebriiski et al., describes alternative use of this system in bacteria.