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Research Item (81)
- Jun 2017
Purpose: Crystallin is a major protein present in eye lens. Peptide fragment αA(66-80) derived from αA-crystallin possesses high aggregation propensity and forms amyloid-like structures. αA(66-80) aggregates are known to interact with soluble crystallins and destabilize native structures that subsequently undergo aggregation. Crystallin aggregation in eye lens leads to reduction in lens opacity, the condition generally referred to as a cataract. Thus, αA(66-80) aggregation appears to be an important event during cataract development, and therefore, inhibition of αA(66-80) aggregation may be an attractive strategy to intervene in cataract development. Materials and Methods: αA(66-80) peptide derived from αA-crystallin possesses high aggregation potential and has a crucial role in cataract development. In order to inhibit the aggregation of αA(66-80) peptide, epigallocatechin-3-gallate (EGCG), a major active constituent of green tea, was employed. The inhibitory effect was assessed by Congo Red (CR) spectral shift assay, Thioflavin-T binding assay, transmission electron microscopy and fluorescence microscopy. Results: The inhibitory potential of EGCG toward αA-crystallin was clearly observed as in the presence of EGCG, the αA(66-80) aggregation was considerably inhibited and the pre-formed fibrillary aggregates of αA(66-80) were found to be disassembled. Conclusion: In the present study, we are able to successfully demonstrate that EGCG efficiently blocks the aggregation of αA(66-80) peptide in a concentration-dependent manner. Furthermore, it is also evident that EGCG is able to disaggregate pre-formed αA(66-80) aggregates. The study suggests that EGCG can be a potential molecule that can prevent the initiation of cataract as well as be helpful in the disease reversal.
Gefitinib, an EGFR tyrosine kinase inhibitor, is used as FDA approved drug in breast cancer and non-small cell lung cancer treatment. However, this drug has certain side effects and complications for which the underlying molecular mechanisms are not well understood. By systems biology based in silico analysis, we identified off-targets of gefitinib that might explain side effects of this drugs. The crystal structure of EGFR-gefitinib complex was used for binding pocket similarity searches on a druggable proteome database (Sc-PDB) by using IsoMIF Finder. The top 128 hits of putative off-targets were validated by reverse docking approach. The results showed that identified off-targets have efficient binding with gefitinib. The identified human specific off-targets were confirmed and further analyzed for their links with biological process and clinical disease pathways using retrospective studies and literature mining, respectively. Noticeably, many of the identified off-targets in this study were reported in previous high-throughput screenings. Interestingly, the present study reveals that gefitinib may have positive effects in reducing brain and bone metastasis, and may be useful in defining novel gefitinib based treatment regime. We propose that a system wide approach could be useful during new drug development and to minimize side effect of the prospective drug.
- Aug 2015
Both villous and extravillous trophoblast (EVT) cells produce a wide range of cytokines and also respond to them in autocrine and paracrine manner. Deregulation of cytokine secretion may lead to various pathologic conditions including preeclampsia. IL-8, a pro-inflammatory cytokine, regulates various cellular functions such as neutrophil trafficking, cell adhesion, tumor growth and has a role in placental development. IL-8 also promotes trophoblast cell migration and invasion, and stimulates the secretion of progesterone. The induction and mechanism of IL-8 secretion by EVT is still unknown. IL-8 mRNA expression and secretion was determined using real-time PCR and ELISA respectively. To identify the mechanism of IL-8 expression and secretion, selective antagonists and agonist of S1P receptor subtypes, Rac1 and Rho-kinase inhibitors were used. We found that S1P induces IL-8 gene expression and protein secretion in EVT derived HTR-8/SVneo cells but not in BeWo cells. SEW2781, the selective agonist of S1PR1, induced IL-8 gene expression but not protein secretion. The specific S1PR2 inhibitor JTE-013 could drastically inhibit IL-8 secretion. Furthermore, pre-treatment of cells with the selective S1PR1/S1PR3 antagonist VPC23019 inhibited IL-8 secretion by ∼45%. Selective Rho-kinase inhibitor Y27632 and Rac1 inhibitor NSC23766 could block IL-8 secretion in these cells. In this study, we could show for the first time that S1P induces IL-8 mRNA expression and protein secretion in EVT cell line. S1P-induced IL-8 gene expression is mainly regulated via S1PR1 and its secretion is regulated through S1PR2 receptor subtype. Rho GTPases signaling is essential for S1P-induced IL-8 secretion. Copyright © 2015 Elsevier Ltd. All rights reserved.
Glycoprotein VI (GPVI) is the essential platelet collagen receptor in atherothrombosis, but its inhibition causes only a mild bleeding tendency. Thus, targeting this receptor has selective antithrombotic potential. This study sought to compare compounds interfering with platelet GPVI-atherosclerotic plaque interaction to improve current antiatherothrombotic therapy. Human atherosclerotic plaque-induced platelet aggregation was measured in anticoagulated blood under static and arterial flow conditions (550/s, 1,100/s, and 1,500/s). Inhibition by dimeric GPVI fragment crystallizable region of IgG (Fc) masking GPVI binding sites on collagen was compared with that of 3 anti-GPVI antibodies: BLO8-1, a human domain antibody; 5C4, a fragment antigen-binding (Fab fragment) of monoclonal rat immunoglobulin G; and m-Fab-F, a human recombinant sFab against GPVI dimers. GPVI-Fc reduced plaque-triggered platelet aggregation in static blood by 51%, BLO8-1 by 88%, and 5C4 by 93%. Under arterial flow conditions, BLO8-1 and 5C4 almost completely inhibited platelet aggregation while preserving platelet adhesion on plaque. Inhibition by GPVI-Fc, even at high concentrations, was less marked but increased with shear rate. Advanced optical imaging revealed rapid persistent GPVI-Fc binding to collagen under low and high shear flow, upstream and downstream of plaque fragments. At low shear particularly, platelets adhered in plaque flow niches to GPVI-Fc-free segments of collagen fibers and recruited other platelets onto aggregates via ADP and TxA2 release. Anti-GPVI antibodies inhibit atherosclerotic plaque-induced platelet aggregation under static and flow conditions more effectively than GPVI-Fc. However, potent platelet inhibition by GPVI-Fc at a higher shear rate (1,500/s) suggests localized antithrombotic efficacy at denuded or fissured stenotic high-risk lesions without systemic bleeding. The compound-specific differences have relevance for clinical trials targeting GPVI-collagen interaction combined with established antiplatelet therapies in patients with spontaneous plaque rupture or intervention-associated plaque injury. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.
This is the author accepted manuscript. It is currently under an infinite embargo pending publication of the final version.
Question - Can I use -20°C incubation temperature, when permeabilization of PBMCs is recommended to done on ice?
The best way is to incubate on ice or refrigerate (4oC). -20oC is not allowed as it may damage the cells. The time is also very critical and it is dependent on the application.
Ovarian cancer still represents a challenge in gynecological oncology. Most patients are diagnosed in an advanced tumor stage. No specific screening or prevention strategies for ovarian cancer exist as of yet. Interleukin 8 (IL-8) is a pro-inflammatory chemokine known for its angiogenetic activity, and is supposedly responsible for tumor-associated angiogenesis in several malignant tumors. The aim of the study was to investigate the susceptibility of patients with an IL-8 gene polymorphism to developing ovarian cancer. Four single nucleotide polymorphisms (SNPs) (IL-8 −251, IL-8 +781, IL-8 +1633 and IL-8 +2767) of the IL-8 gene were screened, using the PCR method in 268 patients with ovarian cancer and 426 healthy women as a control group. Significant associations were noted in patients with the IL-8 +781 (T/T) genotype (p = 0.0048) with increased frequencies of ovarian cancer, while women with the IL-8 +781 (C/C) allele suffer from ovarian cancer significantly less frequently (p = 0.0003). Furthermore, the IL-8 +2767 (T/T) genotype is also associated with a higher risk of ovarian cancer (p = 0.0177). Our results indicate, for the first time, that IL-8 polymorphism is associated with ovarian cancer.
Angiogenesis and vascular development are essential for foetal development and growth, whereby early pregnancy loss and other pregnancy-related pathologies have been linked to aberrant vascular development. As Wnt signalling has been suggested to play a role in the vascularization of chorionic villi, we investigated the expression of Wnt family members in trophoblasts and stromal cells isolated from chorionic villi of early placenta and the influence of Wnt signalling on CD133+CD34+-hematopoietic progenitor (CD133+CD34+) cells. Wnt-5a was expressed in human placental stromal cells and to a lesser extent in human trophoblast cells. rWnt-5a impeded migration and induced adhesion of CD133+CD34+ cells, in accordance with the expression of adhesion proteins, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). rWnt-5a-related regulation of the ICAM-1 and VCAM-1 expression were dependent on the release of Ca2+ and the activation of transcription factor – nuclear factor of activated T-cells (NF-AT). We propose that Wnt-5a is required during early placenta development to mediate adhesion and homing of CD133+CD34+ cells.
In the luteal phase, human endometrial stromal cells (HESCs) undergo proliferation, migration and differentiation during the decidualization process under the control of the ovarian steroids progesterone and estrogen. Proper decidualization of stromal cells is required for blastocyst implantation and the development of pregnancy. The proliferation, migration and differentiation of HESCs in decidualization do not require the presence of a blastocyst but are greatly accelerated during implantation. Lysophosphatidic acid (LPA) and Sphingosine-1-phosphate (S1P) are potent bioactive lysophospholipids that have critical roles in various physiological and pathophysiological processes including inflammation, angiogenesis and cancer. The expression of the enzymes involved in LPA and S1P turnover and their receptors in HESCs during decidualization has not been characterized yet. We found that the LPAR1 and LPAR6 and S1PR3 receptors are highly expressed in HESCs. LPAR1, autotaxin (ATX), an LPA producing enzyme and lipid phosphate phosphatase 3 (LPP3) were upregulated during decidualization. Interestingly, the expression of all S1P receptor subtypes and LPA receptors (LPAR2-6) mRNA was downregulated after decidualization. We found that SPHK1 is highly expressed in HESCs, and is upregulated during decidualization. S1P phosphatase SGPP1 and S1P lyase SGPL1 are highly expressed in HESCs. SGPP1 mRNA expression was significantly upregulated in decidualized HESCs. In conclusion, this study shows the first time that specific LPA and S1P receptors and their metabolizing enzymes are highly regulated in HESCs during decidualization. Furthermore, we suggest that LPAR1 receptor-mediated signaling in HESCs may be crucial in decidualization process. SPHK1 activity and high turnover of S1P and LPA might be essential for precise regulation of their signaling during decidualization of human endometrium and implantation.
Background: ADF/cofilin proteins are key regulators of actin dynamics. Their function is inhibited by LIMK-mediated phosphorylation at Ser-3. Previous in vitro studies have shown that dependent on its concentration, cofilin either depolymerizes F-actin (at low cofilin concentrations) or promotes actin polymerization (at high cofilin concentrations). Methodology/Principal Findings: We found that after in vivo cross-linking with different probes, a cofilin oligomer (65 kDa) could be detected in platelets and endothelial cells. The cofilin oligomer did not contain actin. Notably, ADF that only depolymerizes F-actin was present mainly in monomeric form. Furthermore, we found that formation of the cofilin oligomer is regulated by Ser-3 cofilin phosphorylation. Cofilin but not phosphorylated cofilin was present in the endogenous cofilin oligomer. In vitro, formation of cofilin oligomers was drastically reduced after phosphorylation by LIMK2. In endothelial cells, LIMK-mediated cofilin phosphorylation after thrombin-stimulation of EGFP- or DsRed2-tagged cofilin transfected cells reduced cofilin aggregate formation, whereas inhibition of cofilin phosphorylation after Rho-kinase inhibitor (Y27632) treatment of endothelial cells promoted formation of cofilin aggregates. In platelets, cofilin dephosphorylation after thrombin-stimulation and Y27632 treatment led to an increased formation of the cofilin oligomer. Conclusion/Significance: Based on our results, we propose that an equilibrium exists between the monomeric and oligomeric forms of cofilin in intact cells that is regulated by cofilin phosphorylation. Cofilin phosphorylation at Ser-3 may induce conformational changes on the protein-protein interacting surface of the cofilin oligomer, thereby preventing and/or disrupting cofilin oligomer formation. Cofilin oligomerization might explain the dual action of cofilin on actin dynamics in vivo.
Protein sequence alignment of human non-muscle cofilin (CFL1-Human) and human ADF (ADF-Human). Cysteine residues are highlighted in yellow. The positions of cysteine in human cofilin are indicated. (RTF)
Live cell imaging of human endothelial cells transfected with DsRed2-cofilin. Endothelial cells transfected with cofilin-DsRed2 were stimulated with thrombin (1 U/ml) and then observed under the Nikon TE2000E PFS microscope with 63x magnification at 37°C. Pictures were taken every 30 seconds for 30 minutes using NIS elements software. Movie was edited with QuickTime Pro from Apple Inc. (MOV)
Immunoblotting of cofilin-EGFP and cofilin-S3D-EGFP transfected and cross-linked endothelial cells. Endothelial cells were transfected with EGFP, cofilin-EGFP or cofilin-S3D-EGFP plasmid. After 20 hours of transfection, cells (0.8–1×106 cells/20 µl) were treated with DMSO (1 µl) or BMOE at a final concentration of 1 mM. The cell lysates were subjected to SDS-PAGE on a gradient gel (4–15%) and were then immunoblotted with anti-cofilin or anti-EGFP antibody. A band of ∼100 kDa was apparent in both anti-EGFP and anti-cofilin immunoblots (arrow) of lysates of cofilin-EGFP and cofilin-S3D-EGFP transfected cells after BMOE cross-linking. (PDF)
ADF does not form a distinct 65 kDa oligomer after cross-linking of endothelial cells. Endothelial cells (0.8–1×106 cells/20 µl) were incubated with DMSO (1 µl) or BMOE (1 mM). For formaldehyde cross-linking, endothelial cells (1×106 cells/ml) were treated with formaldehyde at a final concentration (1%). The cell lysates were subjected to SDS-PAGE on gradient gel (4–15%) and were then immunoblotted with an anti-ADF antibody. A smear of ADF cross-linked proteins was observed for both BMOE and formaldehyde cross-linked endothelial cells. Proteins were detected by fluorescence imaging of secondary antibodies labeled with infrared dyes. (PDF)
Various cytokines derived from placental cells are essential for normal placenta development and successful pregnancy. Interleukin-6 (IL-6) is a multifunctional cytokine produced by extravillous and cytotrophoblasts regulating the functions of these cells e.g. migration, invasion, trophoblast differentiation, and proliferation. In macrophages, newly synthesized IL-6 accumulates in the Golgi complex and exits in tubulovesicular carriers fused with recycling endosomes and secreted as a soluble protein. Sphingosine-1-phosphate (S1P) induces various cytokine secretions including IL-6 in different cell types. The signaling mechanisms regulating the IL-6 secretion are unknown. In this study, we found that S1PR2 was the major S1P receptor being expressed in BeWo cells. S1P regulated IL-6 protein secretion in early phase (6 hours) and gene expression in later phase (24 hours). IL-6 secretion was completely inhibited via inhibitor of transcription (Actinomycin D) or protein synthesis (Cycloheximide) confirming that IL-6 releases constitutively from BeWo cells. By using specific S1PR2 inhibitor JTE-013 and S1PR2 gene silencing, we found that S1PR2 was the main receptor that regulates IL-6 secretion. Furthermore, S1P induced RhoGTPases dependent pathways that are required for IL-6 secretion. Pretreatment of cells with specific Rho-kinase inhibitor (Y27632) and Rac1 inhibitor (NSC23766) drastically inhibited S1P-induced IL-6 secretion. By using a specific Phosphoinositide 3-kinase (PI3 K) inhibitor (LY294002), we found that basal activity of PI3 K was required for secretion but was independent of S1P/S1PR2 axis activation. In summary, we report first time that binding of S1P to S1PR2 activates multiple RhoGTPases dependent pathways that coordinate with PI3 K pathway for secretion of IL-6 in BeWo cells.
OVO-like proteins (OVOL) are members of the zinc finger protein family and serve as transcription factors to regulate gene expression in various differentiation processes. Recent studies have shown that OVOL genes are involved in epithelial development and differentiation in a wide variety of organisms; yet there is a lack of comprehensive studies that describe OVOL proteins from an evolutionary perspective. Using comparative genomic analysis, we traced three different OVOL genes (OVOL1-3) in vertebrates. One gene, OVOL3, was duplicated during a whole-genome-duplication event in fish, but only the copy (OVOL3b) was retained. From early-branching metazoa to humans, we found that a core domain, comprising a tetrad of C2H2 zinc fingers, is conserved. By domain comparison of the OVOL proteins, we found that they evolved in different metazoan lineages by attaching intrinsically-disordered (ID) segments of N/C-terminal extensions of 100 to 1000 amino acids to this conserved core. These ID regions originated independently across different animal lineages giving rise to different types of OVOL genes over the course of metazoan evolution. We illustrated the molecular evolution of metazoan OVOL genes over a period of 700 million years (MY). This study both extends our current understanding of the structure/function relationship of metazoan OVOL genes, and assembles a good platform for further characterization of OVOL genes from diverged organisms.
Alignment of OVOL proteins from different vertebrates, B. floridae and N. vectensis. We created this alignment using MUSCLE ,  and further edited for visualization using GENEDOC . Secondary structures of human OVOL1 were predicted using PSIPRED  and these secondary structures are marked above the alignment. Four C2H2 zinc finger motifs (I-IV) are marked by the orange bar. The rodent OVOL3 protein terminates at position 10 in C2H2 motif IV. Grey and back shades indicate 70% and over conserved residues (with similar residues) and 100% conserved residues, respectively. (PDF)
Similarities and differences among Drosophila OVOA-D and mouse OVOL1-OVOL3 using protein sequence alignment. Zinc finger motif is a highly conserved region (red shading). The presence of multiple stretches of the same amino acids are visible in this alignment in the N-terminal regions. (PDF)
Protein sequence alignment of selected regions of OVOL. We generated this alignment using MUSCLE ,  at default parameters. This alignment was used for the reconstruction of the Bayesian phylogenetic tree (Figure 7B). (TXT)
Background Mesothelial cells are critical in the pathogenesis of post-surgical intraabdominal adhesions as well as in the deterioration of the peritoneal membrane associated with long-term peritoneal dialysis. Mesothelial denudation is a pathophysiolocigally important finding in these processes. Matrix metalloproteinase (MMP) biology underlies aspects of mesothelial homeostasis as well as wound repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) moderate MMP activity. Methods and Finding By modifying human TIMP-1 through the addition of a glycosylphosphatidylinositol (GPI) anchor, a recombinant protein was generated that efficiently focuses TIMP-1 on the cell surface. Treatment of primary mesothelial cells with TIMP-1-GPI facilitates their mobilization and migration leading to a dramatic increase in the rate of wound experimental closure. Mesothelial cells treated with TIMP-1-GPI showed a dose dependent increase in cell proliferation, reduced secretion of MMP-2, MMP-9, TNF-α and urokinase-type plasminogen activator (uPA), but increased tissue plasminogen activator (t-PA). Treatment resulted in reduced expression and processing of latent TGF-β1. Conclusions TIMP-1-GPI stimulated rapid and efficient in vitro wound closure. The agent enhanced mesothelial cell proliferation and migration and was bioactive in the nanogram range. The application of TIMP-1-GPI may represent a new approach for limiting or repairing damaged mesothelium.
TIMP-1-GPI treatment moderates the steady state TNF-α, PAI-1 and t-PA gene expression. Primary human mesothelial cells were left untreated, or were treated with 7 ng/ml TIMP-1-GPI, 14 ng/ml TIMP-1-GPI, 14 ng/ml of heat treated TIMP-1-GPI or 14 ng/ml of rhTIMP-1. After 48 hrs RNA was isolated and subjected to analysis using TaqMan RT-PCR. The genes analyzed were (A) t-PA, (B) TNF-α and (C) PAI-1. (EPS)
Micro-synteny analysis of MC3 receptors. A. Ortholog of MC3R is conserved in tetrapods. B. MC3R like gene is found in only zebrafish whereas other ray-finned fishes have another thyrotropin-releasing hormone receptor 3 (TRHR3) gene at this locus instead of MC3R. Takifugu has two copies of TRHR3, which are named as TRHR3a-b. (TIFF)
Intron insertions in MC5 receptor during diversification of ray-finned fishes. There are three intron inserted at positions 41a, 77c and 140c (numbering human MC5R with suffix a–c for intron phasing; blue background) in MC5Rs of four fishes - Takifugu, Tetraodon, stickleback and medaka, but not in MC5R like genes from zebrafish, elephant shark and tetrapods. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 . Residues conserved above 70% are marked by white on black background. In mouse and rat MC5R, there is one intron inserted in N-terminal extension marked as 34c-MMU (numbering according to mouse MC5R; red background). (PDF)
Intron insertions in P2Y6 receptor during diversification of ray-finned fishes. One novel intron is inserted at positions 120c (human MC5R amino acid numbering with suffix a–c for intron phasing) in the P2Y6 receptor of four fishes - Takifugu, Tetraodon, stickleback and medaka (blue background), but not in P2Y2 genes from zebrafish, and tetrapods. Residues conserved above 70% are marked by white on black background. ### indicates location of highly conserved DRY motif. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 . (PDF)
List of selected GPCRs used in this study, other than MC receptors. Codon usage in the DRY motif is shown and codon usage of the R residue is marked by red color. Proto-splice site forming codons are indicated by bold letters. The presence and the absence of intron is marked by blue background and grey background for R residue respectively. (DOC)
Comprehensive protein alignment of MC receptors from evolutionary important organisms. There are three MC receptors such MC1R, MC3R and MC3R were detected from elephant shark (C. milii) genome. Lampreys L. fluviatilis and P. marinus have two copies of MC receptors named as MCAR and MCBR. There are total 69 protein sequences of MC receptors used in this alignment and all of these MCR have conserved DRY motif (marked as ###) at the end of transmembrane helix 3 (TM3). Seven transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 . Residues conserved above 70% are marked by white on black background. (PDF)
Intron insertions in P2Y2 receptor during diversification of ray-finned fishes. There are three introns inserted at positions 124a, 181c and 267a (human MC5R amino acid numbering with suffix a–c for intron phasing) in P2Y2 receptor of four fishes - Takifugu, Tetraodon, stickleback and medaka (blue background), but not in P2Y2 genes from zebrafish, elephant shark and tetrapods. Residues conserved above 70% are marked by white on black background. ### indicates location of highly conserved DRY motif. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 . (PDF)
List of GPCRs sharing a common DRY intron from different vertebrates. Vertebrates with compacted genomes are marked in yellow background. Codon usage of DRY motif is shown with | as point of intron insertion. Only mammalian PRLHR gene has no intron, shown in orange background. # Only partial sequence available that renders detection of DRY motif. *Upon BLAT search in Tetraodon Genome browser (http://www.genoscope.cns.fr/externe/tetranew/) using Takifugu NMBR, we obtained putative full length NMBR from Tetraodon localized as fragements on chrUn_random in current assembly of tetraodon genome. @Accession id from Ensembl. $Gene is partial in databases starting from the middle of DRY motif. (XLSX)
Sequences of novel introns inserted into MC receptors of selected ray-finned fishes. Only introns mapping to core MCR domain (TM1–7) are considered. The numbering of intron positions refers to the full length amino acid numbering of human MC5R. (PDF)
Analysis of intron insertions in MC2R from selected ray-finned fishes. There is one intron inserted at position 140c (numbering human MC5R with suffix a–c for intron phasing; blue background) in MC2Rs of four fishes - Takifugu, Tetraodon, stickleback and medaka, but not in zebrafish and tetrapods. This intron is also conserved in MC5Rs of these selected ray-finned fishes. Takifugu and stickleback has one intron inserted in their MC2R at intron positions 230c (or 225c-TRU, according to Takifugu MC2R numbering) and 236a, respectively. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 . Residues conserved above 70% are marked by white on black background. (PDF)
Intron insertions in P2Y3-like (P2Y3L) receptor during diversification of ray-finned fishes. One novel intron is inserted at position 187c (human MC5R amino acid numbering with suffix a–c for intron phasing) in the P2Y3L receptor of four fishes - Takifugu, Tetraodon, stickleback and medaka (blue background), but not in P2Y3L genes from zebrafish, elephant shark and tetrapods. There is also species-specific introns in the P2Y3L gene such as S212a for stickleback, M234a in medaka and T252a for Takifugu (numbering is species specific numbering, due to gaps in the region and red background), localized in the loop between TM5 and TM6. Residues conserved above 70% are marked by white on black background. ### indicates location of highly conserved DRY motif. Gallus P2Y3 receptor was used as it a typical representative of tetrapod P2Y3. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 . (PDF)
Intron insertions in CHRM3 receptor during diversification of ray-finned fishes. There are two introns inserted at positions 67a, 166c (DRY intron or 140c by human MC5R numbering) in CHRM3 receptor of four fishes - Takifugu, Tetraodon, stickleback and medaka (blue background), but not in P2Y2 genes from zebrafish, elephant shark and tetrapods. Various species-specific introns in the large loop between TM5 and TM6 are marked by red background. Residues conserved above 70% are marked by white on black background. ### indicates location of highly conserved DRY motif. In this case, human CHRM3 amino acid numbering was followed as human MC5R was not suitable due to variable size of these receptor proteins with suffix a–c for intron phasing. Transmembrane regions are marked as TM1–TM7 (yellow bars) as predicted by TMHMM2.0 . (PDF)
Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution. We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes. The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality.
Protein sequence alignment of mammalian STK35L1. N-terminal region and kinase domain of STK35L1 are shaded in gray and yellow color respectively. The conserved bipartite NLS (boxed) is marked in red color. Stretches of arginine and lysine are colored in gold. (PDF)
Migration assay using the IBIDI insert. Endothelial cells transfected with STK35L1 siRNA (left side) or control siRNA (right side) were seeded into different reservoirs of an IBIDI insert. After 8 hours the insert was removed and the closure of the gap was observed on Nikon TE2000E-PFS fluorescence microscope equipped with incubation camber (37°C) and CO2 supply. The microscope function was controlled by NIS elements software. Pictures were taken every 7 minutes for 15 hours. Movie was edited with QuickTime Pro and iMovie software from Apple Inc. (MOV)
Prediction of protein-binding motifs within STK35L1 using the ELM web server. Predicted binding motifs within STK35L1 are shown. The consensus binding sequence for the given binding domains is labeled in red. LIG, binding for. (PDF)
Live cell imaging of human endothelial cells transfected with EGFP-PDM. EGFP-PDM containing the PDZ-binding motif (see text for details), distributes thought the cytoplasm and the nucleus. In migrating cells, it concentrates in membrane ruffles at the leading edge as indicated by white arrows. Pictures were taken every four minutes for 90 minutes. Movie was edited with QuickTime Pro and iMovie software from Apple Inc. (MOV)
Endothelial cell prolifiration using AlamarBlue®. HUVECs were seeded (25000 cells/well) in 24 well plates and were grown for 24 hours, 48 hours and 72 hours. Before four hours of every time points, cells were incubated with AlamarBlue reagent as described in Materials and methods. The absorbance of control siRNA treated cells was considered as 100% and the proliferation was calculated as % of control. (JPG)
Migration and proliferation of vascular endothelial cells are essential for repair of injured endothelium and angiogenesis. Cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors play an important role in vascular tissue injury and wound healing. Previous studies suggest a link between the cell cycle and cell migration: cells present in the G(1) phase have the highest potential to migrate. The molecular mechanism linking these two processes is not understood. In this study, we explored the function of STK35L1, a novel Ser/Thr kinase, localized in the nucleus and nucleolus of endothelial cells. Molecular biological analysis identified a bipartite nuclear localization signal, and nucleolar localization sequences in the N-terminal part of STK35L1. Nuclear actin was identified as a novel binding partner of STK35L1. A class III PDZ binding domains motif was identified in STK35L1 that mediated its interaction with actin. Depletion of STK35L1 by siRNA lead to an accelerated G(1) to S phase transition after serum-stimulation of endothelial cells indicating an inhibitory role of the kinase in G(1) to S phase progression. Cell cycle specific genes array analysis revealed that one gene was prominently downregulated (8.8 fold) in STK35L1 silenced cells: CDKN2A alpha transcript, which codes for p16(INK4a) leading to G(1) arrest by inhibition of CDK4/6. Moreover in endothelial cells seeded on Matrigel, STK35L1 expression was rapidly upregulated, and silencing of STK35L1 drastically inhibited endothelial sprouting that is required for angiogenesis. Furthermore, STK35L1 depletion profoundly impaired endothelial cell migration in two wound healing assays. The results indicate that by regulating CDKN2A and inhibiting G1- to S-phase transition STK35L1 may act as a central kinase linking the cell cycle and migration of endothelial cells. The interaction of STK35L1 with nuclear actin might be critical in the regulation of these fundamental endothelial functions.
The human kinome containing 478 eukaryotic protein kinases has over 100 uncharacterized kinases with unknown substrates and biological functions. The Ser/Thr kinase 35 (STK35, Clik1) is a member of the NKF 4 (New Kinase Family 4) in the kinome with unknown substrates and biological functions. Various high throughput studies indicate that STK35 could be involved in various human diseases such as colorectal cancer and malaria. In this study, we found that the previously published coding sequence of the STK35 gene is incomplete. The newly identified sequence of the STK35 gene codes for a protein of 534 amino acids with a N-terminal elongation of 133 amino acids. It has been designated as STK35L (STK35 long). Since it is the first of further homologous kinases we termed it as STK35L1. The STK35L1 protein (58 kDa on SDS-PAGE), but not STK35 (44 kDa), was found to be expressed in all human cells studied (endothelial cells, HeLa, and HEK cells) and was down-regulated after silencing with specific siRNA. EGFP-STK35L1 was localized in the nucleus and the nucleolus. By combining syntenic and gene structure pattern data and homology searches, two further STK35L1 homologs, STK35L2 (previously known as PDIK1L) and STK35L3, were found. All these protein kinase homologs were conserved throughout the vertebrates. The STK35L3 gene was specifically lost during placental mammalian evolution. Using comparative genomics, we have identified orthologous sets of these three protein kinases genes and their possible ancestor gene in two sea squirt genomes. We found the full-length coding sequence of the STK35 gene and termed it as STK35L1. We identified a new third STK35-like gene, STK35L3, in vertebrates and a possible ancestor gene in sea squirt genome. This study will provide a comprehensive platform to explore the role of STK35L kinases in cell functions and human diseases.
Expression analysis of STK35. Expression analysis of STK35. To verify the expression of STK35 mRNA, RNA and cDNA were prepared from endothelial cells. 455 bp and 755 bp PCR products (Lane 2 and 3) were amplified with STK35 specific primers from cDNA pools. No PCR product was amplified in negative control (reverse transcriptase polymerase was excluded during RT-PCR). (0.11 MB TIF)
In platelets stimulated by thrombin to secrete and aggregate, cofilin is rapidly dephosphorylated leading to its activation. Cofilin by severing existing actin filaments and stimulating F-actin polymerization on newly created barbed ends dynamizes the actin cytoskeleton. We previously found that cofilin dephosphorylation is Ca(2+)-dependent and occurs upstream of degranulation in stimulated platelets. We report now in thrombin-stimulated platelets that Rac1 and class II PAKs (PAK4/5/6) were rapidly (within 5 seconds) activated, whereas PAK1/2 (class I PAKs) phosphorylation was slower. The Rac1-specific inhibitor NSC23766 blocked phosphorylation of class II PAKs, but not PAK1/2. Moreover, inhibition of the Ca(2+)/calmodulin-dependent phosphatase calcineurin inhibited Rac1 activation and class II PAKs phosphorylation. Prevention of Rac1 activation by calcineurin inhibition or NSC23766 also blocked cofilin dephosphorylation and platelet granule secretion indicating that a calcineurin/Rac1/class II PAKs pathway regulates cofilin dephosphorylation leading to secretion. We further found that PI3-kinases were activated downstream of Rac1, but were not involved in regulating cofilin dephosphorylation and secretion in thrombin-stimulated platelets. Our study unravels a Ca(2+)-dependent pathway of secretion in stimulated platelets as a signaling pathway linking Rac1 activation to actin dynamics: calcineurin-->Rac1-->class II PAKs-->cofilin activation. We further demonstrate that this pathway is separate and independent of the protein kinase C (PKC) pathway mediating secretion.
- Apr 2009
Lysophosphatidic acid (LPA) is a ligand for LPA(1-3) of the endothelial differentiation gene family G-protein-coupled receptors, and LPA(4-8) is related to the purinergic family G-protein-coupled receptor. Because the structure-activity relationship (SAR) of GPR92/LPA(5) is limited and whether LPA is its preferred endogenous ligand has been questioned in the literature, in this study we applied a combination of computational and experimental site-directed mutagenesis of LPA(5) residues predicted to interact with the headgroup of LPA. Four residues involved in ligand recognition in LPA(5) were identified as follows: R2.60N mutant abolished receptor activation, whereas H4.64E, R6.62A, and R7.32A greatly reduced receptor activation. We also investigated the SAR of LPA(5) using LPA analogs and other non-lysophospholipid ligands. SAR revealed that the rank order of agonists is alkyl glycerol phosphate > LPA > farnesyl phosphates > N-arachidonoylglycine. These results confirm LPA(5) to be a bona fide lysophospholipid receptor. We also evaluated several compounds with previously established selectivity for the endothelial differentiation gene receptors and found several that are LPA(5) agonists. A pharmacophore model of LPA(5) binding requirements was developed for in silico screening, which identified two non-lipid LPA(5) antagonists. Because LPA(5) transcripts are abundant in human platelets, we tested its antagonists on platelet activation and found that these non-lipid LPA(5) antagonists inhibit platelet activation. The present results suggest that selective inhibition of LPA(5) may provide a basis for future anti-thrombotic therapies.
Protein kinases form a superfamily of highly diverged proteins that mediate wide array of the signal transduction in eukaryotic cells; by modification of substrate activity, protein kinases also control many other cellular processes, including metabolism, transcription, cell cycle progression, cytoskeletal rearrangement and cell movement, apoptosis, and differentiation. Characterizing these kinases is major challenge in a biomedical lab, since they play major roles as important physiological and disease conditions such a cancer. We have characterized two STK35 homologs with aid of gene structure corrections; previously reported gene structures were partial and new validated by experimental support. These genes form a distinct sub-group of protein kinase and possess novel domain in N-terminal end. Orthologs are found across vertebrates and are not found in any invertebrate species during our analysis.
Lysophosphatidic acid (LPA), a component of mildly-oxidized LDL and the lipid rich core of atherosclerotic plaques, elicits platelet activation. LPA is the ligand of G protein-coupled receptors (GPCR) of the EDG family (LPA 1–3) and the newly identified LPA 4–7 subcluster. LPA 4 , LPA 5 and LPA 7 increase cellular cAMP levels that would induce platelet inhibition rather than activation. In the present study we quantified the mRNA levels of the LPA 1–7 GPCR in human platelets and found a rank order LPA 4 =LPA 5 >LPA 7 >LPA 6 =LPA 2 >>LPA 1 >LPA 3. We examined platelet shape change using a panel of LPA receptor subtype-selective agonists and antagonists and compared them with their pharmacological profiles obtained in heterologous LPA 1–5 receptor expression systems. Responses to different natural acyl and alkyl species of LPA, and octyl phosphatidic acid analogs, alpha-substituted phosphonate analogs, N-palmitoyl-tyrosine phosphoric acid, N-palmitoyl-serine phosphoric acid were tested. All of these compounds elicited platelet activation and also inhibited LPA-induced platelet shape change after pre-incubation, suggesting that receptor desensitization is likely responsible for the inhibition of this response. Fatty acid free albumin (10 µM) lacking platelet activity completely inhibited platelet shape change induced by LPA with an IC 50 of 1.1 µM but had no effect on the activation of LPA 1,2,3,&5 expressed in endogenously non-LPA-responsive RH7777 cells. However, albumin reduced LPA 4 activation and shifted the dose-response curve to the right. LPA 5 transiently expressed in RH7777 cells showed preference to alkyl-LPA over acyl-LPA that is similar to that in platelets. LPA did not increase cAMP levels in platelets. In conclusion, our results with the pharmacological compounds and albumin demonstrate that LPA does not induce platelet shape change simply through activation of LPA 1–5, and the receptor(s) mediating LPA-induced platelet activation remains elusive.
- Dec 2007
In order to gain a better understanding of the structure-function relation in hSGLT1, single Trp residues were introduced into a functional hSGLT1 mutant devoid of Trps at positions that previously had been postulated to be involved in sugar recognition/translocation and/or phlorizin binding. The mutant proteins were expressed in Pichia pastoris, purified, and reconstituted into liposomes. In transport experiments the putative sugar binding site mutants W457hSGLT1 and W460hSGLT1 showed a drastic decrease in affinity toward alpha-methyl-d-glucopyranoside with Km values of 13.3 and 5.26 mM compared to 0.4 mM of the Trp-less hSGLT1. In addition, a strong decrease in the inhibitory effect of phlorizin was observed. In Trp fluorescence studies the position of the emission maxima of the mutants, their sensitivity to N-bromosuccinimide oxidation, and their interaction with water soluble quenchers demonstrate that Trp457 and Trp460 are in contact with the hydrophilic extravesicular environment. In both mutants Trp fluorescence was quenched significantly, but differently, by various glucose analogues. They also show significant protection by d-glucose and phlorizin against acrylamide, KI, or TCE quenching. W602hSGLT1 and W609hSGLT1, the putative aglucone binding site mutants, exhibit normal sugar and phlorizin affinity, and show fluorescence properties which indicate that these residues are located in a very hydrophilic environment. Phlorizin and phloretin, but not d-glucose, protect both mutants against collisional quenchers. Depth-calculations using the parallax method suggest a location of Trp457 and Trp460 at an average distance of 10.8 A and 7.4 A from the center of the bilayer, while Trp602 and Trp609 are located outside the membrane. These results suggest that in the native carrier residues Gln at position 457 and Thr at position 460 reside in a hydrophilic access pathway extending 5-7 A into the membrane to which sugars as well as the sugar moiety of inhibitory glucosides bind. Residues Phe602 and Phe609 contribute by their hydrophobic aromatic residues toward binding of the aglucone part of phlorizin. Thereby in the phlorizin-carrier complex a close vicinity between these two subdomains of the transporter is established creating a phlorizin binding pocket with the previously estimated dimensions of 10 x 17 x 7 A.
- May 2007
Cofilin is an actin dynamizing protein and inactivated after Ser3 phosphorylation by LIM-kinases (LIMKs). We studied whether in platelets stimulated by lysophosphatidic acid (LPA), Rho-kinase or p21-activated kinase (PAK) mediates LIMK-1 activation leading to subsequent phosphorylation and inactivation of cofilin and the increase of F-actin. During LPA (0.1 microM)-induced shape change, a rapid Rho-kinase activation and a slower activation of PAK were observed. Rho-kinase activation led to rapid LIMK-1 (Thr508) phosphorylation. Despite of LIMK-1 activation, cofilin net phosphorylation was not increased. Cofilin rapidly associated with F-actin and preceded the F-actin increase. Pretreatment with the Rho-kinase inhibitor Y-27632 inhibited LIMK-1 phosphorylation, unmasked cofilin dephosphorylation and inhibited the reversible F-actin increase during shape change. In the presence of fibrinogen, LPA (10 microM) induced ATP-secretion from dense granules and aggregation, and cofilin was rapidly dephosphorylated and then rephosphorylated in a Rho-kinase/LIMK-1-dependent manner. In the absence of fibrinogen, cofilin de- and rephosphorylation after LPA (10 microM) was unchanged, but secretion and aggregation were absent. Cofilin dephosphorylation was completely blocked by BAPTA-AM indicating that it was mediated by an increase of cytosolic Ca(2+). We conclude that in LPA-stimulated platelets, Rho-kinase-dependent LIMK-1 activation mediates the F-actin increase during shape change without enhancing cofilin net phosphorylation. However, a rapid dephosphorylation of cofilin occurs during secretion and aggregation, which is Ca(2+)-dependent, upstream of secretion and aggregation and might regulate these platelet responses.
Although there is no evidence of significant Na-independent glucose flux in tissues naturally expressing SGLT1, previous kinetic and biophysical studies suggest that sodium/d-glucose cotransporter 1 (hSGLT1) can facilitate sodium-independent d-glucose transport and may contain more than one sugar binding site. In this work, we analyze the kinetic properties and conformational states of isolated hSGLT1 reconstituted in liposomes by transport and fluorescence studies in the absence of sodium. In the transport studies with hSGLT1, significant sodium-independent phlorizin inhibitable alpha-methyl d-glucopyranoside (alpha-MDG) uptake was observed which amounted to approximately 20% of the uptake observed in the presence of a sodium gradient. The apparent affinity constant for alpha-MDG was thereby 3.4 +/- 0.5 mM, a value approximately 10-fold higher than that in the presence of sodium. In the absence of sodium, various sugars significantly decreased the intrinsic Trp fluorescence of hSGLT1 in proteoliposomes exhibiting the following sequence of affinities: alpha-MDG > d-glucose approximately d-galactose > 6-deoxy-d-glucose > 2-deoxy-d-glucose > d-allose. Furthermore, significant protection effects of d-glucose or phlorizin against potassium iodide, acrylamide, or trichloroethanol quenching were observed. To locate the Trps involved in this reaction, we generated mutants in which all Trps were sequentially substituted with Phe. None of the replacements significantly affected sodium-dependent uptake. Uptake in the absence of sodium and typical fluorescence changes depended, however, on the presence of Trp at position 561. This Trp residue is conserved in all known SGLT1 forms (except Vibrio parahaemolyticus SGLT) and all SGLT isoforms in humans (except hSGLT3). If all these data are taken into consideration, it seems that Trp-561 in hSGLT1 forms part of a low-affinity sodium-independent binding and/or translocation site for d-glucose. The rate of sodium-independent translocation via hSGLT1 seems, however, to be tightly regulated in the intact cell by yet unknown factors.
Recruitment of human CD34+ progenitor cells toward vascular lesions and differentiation into vascular cells has been regarded as a critical initial step in atherosclerosis. Previously we found that adherent platelets represent potential mediators of progenitor cell homing besides their role in thrombus formation. On the other hand, foam cell formation represents a key process in atherosclerotic plaque formation. To investigate whether platelets are involved in progenitor cell recruitment and differentiation into endothelial cells and foam cells, we examined the interactions of platelets and CD34+ progenitor cells. Cocultivation experiments showed that human platelets recruit CD34+ progenitor cells via the specific adhesion receptors P-selectin/PSGL-1 and beta1- and beta2-integrins. Furthermore, platelets were found to induce differentiation of CD34+ progenitor cells into mature foam cells and endothelial cells. Platelet-induced foam cell generation could be prevented partially by HMG coenzyme A reductase inhibitors via reduction of matrix metalloproteinase-9 (MMP-9) secretion. Finally, agonists of peroxisome proliferator-activated receptor-alpha and -gamma attenuated platelet-induced foam cell generation and production of MMP-9. The present study describes a potentially important mechanism of platelet-induced foam cell formation and generation of endothelium in atherogenesis and atheroprogression. The understanding and modulation of these mechanisms may offer new treatment strategies for patients at high risk for atherosclerotic diseases.
LIM kinases (LIMKs) regulate actin dynamics through cofilin phosphorylation and also have a function in the nucleus. Recently we have shown that LIMK2 shuttles between cytoplasm and nucleus in endothelial cells and that nuclear import is inhibited by protein kinase C-mediated phosphorylation of Ser-283. Here we aimed to identify the structural features of LIMK2 responsible for nuclear import. We found that the kinase domain of LIMK2 is localized exclusively in the nucleus and, in contrast to the kinase domain of LIMK1, it accumulated in the nucleolus. Through site-directed mutagenesis, we identified the basic amino acid-rich motif KKRTLRKNDRKKR (amino acids 491-503) as the functional nuclear and nucleolar localization signal of LIMK2. After fusing this motif to enhanced green fluorescent protein, the fusion protein localized exclusively in the nucleus and nucleolus. Mutagenesis studies showed that phosphorylation of Thr-494, a putative protein kinase C phosphorylation site identified within the nuclear localization signal, inhibits nuclear import of the enhanced green fluorescent protein-PDZ kinase domain of LIMK2. After inhibiting nuclear export with leptomycin B, phosphorylation of either Ser-283 or Thr-494 reduced the nuclear import of LIMK2. Phosphorylation of both Ser-283 and Thr-494 sites inhibited nuclear import completely. Our findings identify a unique basic amino acid-rich motif (amino acids 491-503) in LIMK2 which is not present in LIMK1 that serves to target the protein not only to the nucleus but also to the nucleolus. Phosphorylation of Thr-494 within this motif negatively regulates nuclear import of LIMK2.
The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow–derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin− bone marrow–derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin− bone marrow–derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1α, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury.
Cofilin is a regulator of actin filament dynamics. We studied whether during platelet activation Rho kinase stimulates LIM kinase (LIMK) leading to subsequent phosphorylation and inactivation of cofilin. Platelet shape change and aggregation/secretion were induced by low and high concentrations of thrombin, respectively. We found that during these platelet responses Rho kinase activation was responsible for mediating rapid Thr508 phosphorylation and activation of LIMK-1 and for the F-actin increase during shape change and, in part, during secretion. Surprisingly, during shape change cofilin phosphorylation was unaltered, and during aggregation/secretion cofilin was first rapidly dephosphorylated by an okadaic acid-insensitive phosphatase and then slowly rephosphorylated by LIMK-1. LIMK-1 phosphorylation and cofilin dephosphorylation and rephosphorylation during aggregation were independent of integrin alpha(IIb)beta(3) engagement. Cofilin phosphorylation did not regulate cofilin association with F-actin and was unrelated to the F-actin increase in thrombin-activated platelets. Our study identifies LIMK-1 as being activated by Rho kinase in thrombin-stimulated platelets. Two counteracting pathways, a cofilin phosphatase and LIMK-1, are activated during platelet aggregation/secretion regulating cofilin phosphorylation sequentially and independently of integrin alpha(IIb)beta(3) engagement. Rho kinase-mediated F-actin increase during platelet shape change and secretion involves a mechanism other than LIMK-1-mediated cofilin phosphorylation, raising the possibility of another LIMK substrate regulating platelet actin assembly.
- Dec 2005
Studies on the structure-function relationship of transporters require the availability of sufficient amounts of the protein in a functional state. In this paper, we report the functional expression, purification, and reconstitution of the human sodium/d-glucose cotransporter1 (hSGLT1) in Pichia pastoris and ligand-induced conformational changes of hSGLT1 in solution as studied by intrinsic tryptophan fluorescence. hSGLT1 gene containing FLAG tag at position 574 was cloned into pPICZB plasmid, and the resulting expression vector pPICZB-hSGLT1 was introduced into P. pastoris strain GS115 by electroporation. Purification of recombinant hSGLT1 by nickel-affinity chromatography yields about 3 mg of purified recombinant hSGLT1 per 1-liter of cultured Pichia cells. Purified hSGLT1 migrates on SDS-PAGE with an apparent mass of 55 kDa. Kinetic analysis of hSGLT1 in proteoliposomes revealed sodium-dependent, secondary active, phlorizin-sensitive, and stereospecific alpha-methyl-d-glucopyranoside transport, demonstrating its full catalytic activity. The position of the maximum intrinsic tryptophan fluorescence and titration with hydrophilic collisional quenchers KI, acrylamide, and trichloroethanol suggested that most of Trps in hSGLT1 in solution are in a hydrophobic environment. In the presence of sodium, sugars that have been identified earlier as substrate for the transporter increase intrinsic fluorescence in a saturable manner by a maximum of 15%. alpha-Methyl-d-glucopyranoside had the highest affinity (K(d) = 0.71 mM), followed by d-glucose, d-galactose, d-mannose, and d-allose which showed a much lower affinity. l-Glucose was without effect. d-Glucose also increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary phlorizin, the well-established inhibitor of SGLT1, decreased intrinsic fluorescence by a maximum of 50%, and induced a blue shift of maximum (5 nm). Again, the effects were sodium-dependent and saturable and a high affinity K(d) of 5 muM was observed. In addition the surface of hSGLT1 was labeled with 1-anilinonaphthalene-8-sulfonic acid, a reporter molecule for the surface hydrophobicity. In the presence of sodium, addition of d-glucose decreased ANS fluorescence whereas phlorizin increased ANS fluorescence. Thus three conformational states of SGLT1 could be defined which differ in their packing density and hydrophobicity of their surface. They reflect properties of the empty carrier, the d-glucose loaded carrier facing the outside of membrane and the complex of the outside-orientated carrier with phlorizin.
- Aug 2005
LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-delta isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-delta-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G1-to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.
Lipid-rich atherosclerotic plaques are vulnerable, and their rupture can cause the formation of a platelet- and fibrin-rich thrombus leading to myocardial infarction and ischemic stroke. Although the role of plaque-based tissue factor as stimulator of blood coagulation has been recognized, it is not known whether plaques can cause thrombus formation through direct activation of platelets. We isolated lipid-rich atheromatous plaques from 60 patients with carotid stenosis and identified morphologically diverse collagen type I- and type III-positive structures in the plaques that directly stimulated adhesion, dense granule secretion, and aggregation of platelets in buffer, plasma, and blood. This material also elicited platelet-monocyte aggregation and platelet-dependent blood coagulation. Plaques exposed to flowing blood at arterial wall shear rate induced platelets to adhere to and spread on the collagenous structures, triggering subsequent thrombus formation. Plaque-induced platelet thrombus formation was observed in fully anticoagulated blood (i.e., in the absence of tissue factor-mediated coagulation). Mice platelets lacking glycoprotein VI (GPVI) were unable to adhere to atheromatous plaque or form thrombi. Human platelet thrombus formation onto plaques in flowing blood was completely blocked by GPVI inhibition with the antibody 10B12 but not affected by integrin alpha2beta1 inhibition with 6F1 mAb. Moreover, the initial platelet response, shape change, induced by plaque was blocked by GPVI inhibition but not with alpha2beta1 antagonists (6F1 mAb or GFOGER-GPP peptide). Pretreatment of plaques with collagenase or anti-collagen type I and anti-collagen type III antibodies abolished plaque-induced platelet activation. Our results indicate that morphologically diverse collagen type I- and collagen type III-containing structures in lipid-rich atherosclerotic plaques stimulate thrombus formation by activating platelet GPVI. This platelet collagen receptor, essential for plaque-induced thrombus formation, presents a promising new anti-thrombotic target for the prevention of ischemic cardiovascular diseases.
- Feb 2005
The human endometrium prepares for implantation of the blastocyst by reorganization of its whole cellular network. Endometrial stroma cells change their phenotype starting around the 23rd day of the menstrual cycle. These predecidual stroma cells first appear next to spiral arteries, and after implantation these cells further differentiate into decidual stroma cells. The phenotypical changes in these cells during decidualization are characterized by distinct changes in the actin filaments and filament-related proteins such as alpha-actinin. The carboxy-terminal LIM domain protein with a molecular weight of 36 kDa (CLP36) is a cytoskeletal component that has been shown to associate with contractile actin filaments and to bind to alpha-actinin supporting a role for CLP36 in cytoskeletal reorganization and signal transduction by binding to signaling proteins. The expression patterns of CLP36, alpha-actinin and actin were studied in endometrial stroma cells from different stages of the menstrual cycle and in decidual stroma cells from the 6th week of gestation until the end of pregnancy. During the menstrual cycle, CLP36 is only expressed in the luminal and glandular epithelium but not in endometrial stroma cells. During decidualization and throughout pregnancy, a parallel upregulation of CLP36 and smooth muscle actin, an early marker of decidualization in the baboon, was observed in endometrial decidual cells. Since both proteins maintain a high expression level throughout pregnancy, a role of both proteins is suggested in the stabilization of the cytoskeleton of these cells that come into close contact with invading trophoblast cells.
- Sep 2003
Lysophosphatidic acid (LPA) is a platelet-activating component of mildly oxidized LDL (mox-LDL) and lipids isolated from human atherosclerotic plaques. Specific antagonists of platelet LPA receptors could be useful inhibitors of thrombus formation in patients with cardiovascular disease. Short-chain analogs of phosphatidic acid (PA) were examined for their effect on two initial platelet responses, platelet shape change and Ca2+ mobilization. Dioctylglycerol pyrophosphate [DGPP(8:0)] and dioctylphosphatidic acid [PA(8:0)], recently described selective antagonists of the LPA1 and LPA3 receptors, inhibited platelet activation evoked by LPA but not by other platelet stimuli. DGPP(8:0) was more potent than PA(8:0). DGPP(8:0) also inhibited platelet shape change induced by mox-LDL and lipid extracts from human atherosclerotic plaques. Notably, we demonstrate for the first time that the lipid-rich core isolated from soft plaques was able to directly induce shape change. This effect was completely abrogated by prior incubation of platelets with DGPP(8:0). Moreover, coapplication of the lipid-rich core or LPA together with subthreshold concentrations of ADP or epinephrine synergistically induced platelet aggregation; this effect was inhibited by DGPP(8:0). Analysis by liquid chromatography-mass spectrometry revealed the presence of LPA alkyl- and acyl-molecular species with high platelet-activating potency (16:0-alkyl-LPA, 20:4-acyl-LPA). LPA molecules present in the core region of atherosclerotic plaques trigger rapid platelet activation through the stimulation of LPA1 and LPA3 receptors. Antagonists of platelet LPA receptors might provide a new strategy to prevent thrombus formation in patients with cardiovascular diseases.
Various stimuli like thrombin induce endothelial cell shape change and stress fiber formation via Rho/Rho-kinase-mediated reorganization of the actin cytoskeleton. LIM-kinases regulate actin cytoskeletal reorganization through phosphorylation of cofilin at Ser3. The LIMK family kinases possess characteristic structural features, consisting of two LIM domains, a PDZ domain and a C-terminal kinase domain. In cell transfection studies it has been shown that LIMK2 is phosphorylated at Thr505 by Rho-kinase thereby activating the enzyme. Recently it has been reported that nuclear LIMKs suppress cyclin D1 expression in a manner independent of cofilin phosphorylation and actin polymerization. In this study, we found that endothelial cells express both LIMK1 and LIMK2. By using live cell imaging, we confirm previous findings that thrombin induces stress fiber formation, ruffle formation and cell contraction. Furthermore, the cell-cell contacts were disrupted and F-actin fibers connecting two cells were broken. Thrombin induced a rapid and sustained Rho-kinase activation and subsequent phosphorylation of LIM-kinase and cofilin. Pretreatment of endothelial cells with the specific Rho-kinase inhibitor Y27632 inhibited MYPT1 phosphorylation, LIM-kinase and cofilin phosphorylation and blocked stress fiber formation in thrombin-stimulated cells. Notably, thrombin induced actin stress fiber formation was abolished in cells transfected with dominant negative LIMK2. LIMK2 was mainly localized in the cytoplasm. By using Leptomycin B (a specific inhibitor of CRM-1 dependent nuclear export) and FRAP and FLIP analysis, we demonstrate that LIMK2 in resting endothelial cells shuttles between the nucleus and cytoplasm. The LIM domains of LIMK2, but not of LIMK1 inhibited its nuclear import thereby keeping LIMK2 mainly in the cytoplasm. Mutational analysis of the unique basic amino acid-rich motif (amino acids 480-503) indicated that this motif regulates the nuclear and nucleolar localization of LIMK2. Activation of PKC in PMA-stimulated endothelial cells stimulated the phosphorylation of LIMK2 at Ser283 and the translocation of LIMK2 and the PDZ-kinase construct of LIMK2 from the nucleus to the cytoplasm. Of the various PKC isoforms, PKC- and PKC- were found to be mainly responsible for Ser283 phosphorylation and the regulation of translocation of LIMK2. Mutational analysis indicated that LIMK2 phosphorylation at Ser283 and Thr494 play a role in the regulation of nucleocytoplasmic shuttling of LIMK2 by PKC. These results show that LIM-kinase activation is mediated by Rho-kinase in stimulated endothelial cells, and that LIM-kinase-mediated cofilin phosphorylation plays an essential role in thrombin-induced stress fiber formation. LIMK2 shuttles between nucleus and cytoplasm in resting endothelial cells. Phosphorylation of LIMK2 at Ser283 and Thr494 by PKC regulates nucleocytoplasmic shuttling and suggests that LIMK2 might also have a function in the nucleus such as the suppression of cyclin D1 expression.