BMC Biochemistry
1471-2091
Cơ quản chủ quản: BioMed Central Ltd.
Lĩnh vực:
BiochemistryMolecular Biology
Các bài báo tiêu biểu
HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF, FGF-1, and SDF-1 Abstract
Background
Heparin/heparan sulfate (HS) proteoglycans are found in the extracellular matrix (ECM) and on the cell surface. A considerable body of evidence has established that heparin and heparan sulfate proteoglycans (HSPGs) interact with numerous protein ligands including fibroblast growth factors, vascular endothelial growth factor (VEGF), cytokines, and chemokines. These interactions are highly dependent upon the pattern of sulfation modifications within the glycosaminoglycan chains. We previously cloned a cDNA encoding a novel human endosulfatase, HSulf-2, which removes 6-O-sulfate groups on glucosamine from subregions of intact heparin. Here, we have employed both recombinant HSulf-2 and the native enzyme from conditioned medium of the MCF-7-breast carcinoma cell line. To determine whether HSulf-2 modulates the interactions between heparin-binding factors and heparin, we developed an ELISA, in which soluble factors were allowed to bind to immobilized heparin.
Results
Our results show that the binding of VEGF, FGF-1, and certain chemokines (SDF-1 and SLC) to immobilized heparin was abolished or greatly diminished by pre-treating the heparin with HSulf-2. Furthermore, HSulf-2 released these soluble proteins from their association with heparin. Native Sulf-2 from MCF-7 cells reproduced all of these activities.
Conclusion
Our results validate Sulf-2 as a new tool for deciphering the sulfation requirements in the interaction of protein ligands with heparin/HSPGs and expand the range of potential biological activities of this enzyme.
Tập 7 Số 1 - 2006
Characterization of the aggregates formed during recombinant protein expression in bacteria Abstract
Background
The first aim of the work was to analyze in detail the complexity of the aggregates formed upon overexpression of recombinant proteins in E. coli . A sucrose step gradient succeeded in separating aggregate subclasses of a GFP-GST fusion protein with specific biochemical and biophysical features, providing a novel approach for studying recombinant protein aggregates.
Results
The total lysate separated into 4 different fractions whereas only the one with the lowest density was detected when the supernatant recovered after ultracentrifugation was loaded onto the sucrose gradient. The three further aggregate sub-classes were otherwise indistinctly precipitated in the pellet. The distribution of the recombinant protein among the four subclasses was strongly dependent on the DnaK availability, with larger aggregates formed in Dnak- mutants. The aggregation state of the GFP-GST recovered from each of the four fractions was further characterized by examining three independent biochemical parameters. All of them showed an increased complexity of the recombinant protein aggregates starting from the top of the sucrose gradient (lower mass aggregates) to the bottom (larger mass aggregates). These results were also confirmed by electron microscopy analysis of the macro-structure formed by the different aggregates. Large fibrils were rapidly assembled when the recombinant protein was incubated in the presence of cellular extracts, but the GFP-GST fusion purified soon after lysis failed to undergo amyloidation, indicating that other cell components probably participate in the active formation of large aggregates. Finally, we showed that aggregates of lower complexity are more efficiently disaggregated by a combination of molecular chaperones.
Conclusion
An additional analytical tool is now available to investigate the aggregation process and separate subclasses by their mass. It was possible to demonstrate the complexity of the aggregation pattern of a recombinant protein expressed in bacteria and to characterize biochemically the different aggregate subclasses. Furthermore, we have obtained evidence that the cellular environment plays a role in the development of the aggregates and the problem of the artifact generation of aggregates has been discussed using in vitro models. Finally, the possibility of separating aggregate fractions with different complexities offers new options for biotechnological strategies aimed at improving the yield of folded and active recombinant proteins.
Tập 6 Số 1 - 2005
Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening Abstract
Background
The mitotic exit network (MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae . The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified.
Results
Using a combination of peptide library selection, phosphorylation of opitmal peptide variants, and screening of a phosphosite array, we found that Dbf2-Mob1 preferentially phosphorylated serine over threonine and required an arginine three residues upstream of the phosphorylated serine in its substrate. This requirement for arginine in peptide substrates could not be substituted with the similarly charged lysine. This specificity determined for peptide substrates was also evident in many of the proteins phosphorylated by Dbf2-Mob1 in a proteome chip analysis.
Conclusion
We have determined by peptide library selection and phosphosite array screening that the protein kinase Dbf2-Mob1 preferentially phosphorylated substrates that contain an RXXS motif. A subsequent proteome microarray screen revealed proteins that can be phosphorylated by Dbf2-Mob1 in vitro. These proteins are enriched for RXXS motifs, and may include substrates that mediate the function of Dbf2-Mob1 in mitotic exit and cytokinesis. The relatively low degree of sequence restriction at the site of phosphorylation suggests that Dbf2 achieves specificity by docking its substrates at a site that is distinct from the phosphorylation site
Tập 6 Số 1 - 2005
The protein kinase DYRK1A phosphorylates the splicing factor SF3b1/SAP155 at Thr434, a novel in vivo phosphorylation site Abstract
Background
The U2 small nuclear ribonucleoprotein particle (snRNP) component SF3b1/SAP155 is the only spliceosomal protein known to be phosphorylated concomitant with splicing catalysis. DYRK1A is a nuclear protein kinase that has been localized to the splicing factor compartment. Here we describe the identification of DYRK1A as a protein kinase that phosphorylates SF3b1 in vitro and in cultivated cells.
Results
Overexpression of DYRK1A caused a markedly increased phosphorylation of SF3b1 in COS-7 cells as assessed by Western blotting with an antibody specific for phosphorylated Thr-Pro dipeptide motifs. Phosphopeptide mapping of metabolically labelled SF3b1 showed that the majority of the in vivo -phosphopeptides corresponded to sites also phosphorylated by DYRK1A in vitro . Phosphorylation with cyclin E/CDK2, a kinase previously reported to phosphorylate SF3b1, generated a completely different pattern of phosphopeptides. By mass spectrometry and mutational analysis of SF3b1, Thr434 was identified as the major phosphorylation site for DYRK1A. Overexpression of DYRK1A or the related kinase, DYRK1B, resulted in an enhanced phosphorylation of Thr434 in endogenous SF3b1 in COS-7 cells. Downregulation of DYRK1A in HEK293 cells or in HepG2 cells by RNA interference reduced the phosphorylation of Thr434 in SF3b1.
Conclusion
The present data show that the splicing factor SF3b1 is a substrate of the protein kinase DYRK1A and suggest that DYRK1A may be involved in the regulation of pre mRNA-splicing.
Tập 7 Số 1 - 2006
Deubiquitylating enzymes and disease Abstract
Abstract
Deubiquitylating enzymes (DUBs) can hydrolyze a peptide, amide, ester or thiolester bond at the C-terminus of UBIQ (ubiquitin), including the post-translationally formed branched peptide bonds in mono- or multi-ubiquitylated conjugates. DUBs thus have the potential to regulate any UBIQ-mediated cellular process, the two best characterized being proteolysis and protein trafficking. Mammals contain some 80–90 DUBs in five different subfamilies, only a handful of which have been characterized with respect to the proteins that they interact with and deubiquitylate. Several other DUBs have been implicated in various disease processes in which they are changed by mutation, have altered expression levels, and/or form part of regulatory complexes. Specific examples of DUB involvement in various diseases are presented. While no specific drugs targeting DUBs have yet been described, sufficient functional and structural information has accumulated in some cases to allow their rapid development.
Publication history
Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com ).
- 2008
The ORF2 glycoprotein of hepatitis E virus inhibits cellular NF-κB activity by blocking ubiquitination mediated proteasomal degradation of IκBα in human hepatoma cells Abstract Background Nuclear factor kappa B (NF-κB) is a key transcription factor that plays a crucial role in host survival during infection by pathogens. Therefore, it has been a priority of many pathogens to manipulate the cellular NF-κB activity in order to create a favorable environment for their survival inside the host. Results We observed that heterologous expression of the open reading frame 2 (ORF2) protein in human hepatoma cells led to stabilization of the cellular I kappa B alpha (IκBα) pool, with a concomitant reduction in the nuclear localization of the p65 subunit of NF-κB and inhibition of NF-κB activity. Although basal or TPA induced phosphorylation of IκBα was not altered, its ubiquitination was markedly reduced in ORF2 expressing cells. Further analysis revealed that ORF2 protein could directly associate with the F-box protein, beta transducin repeat containing protein (βTRCP) and ORF2 over expression resulted in reduced association of IκBα with the SKP1 and CUL1 components of the SCFβTRCP complex. Chromatin immunoprecipitation (ChIP) assay of the proximal promoter regions of MHC-I heavy chain and IL-8 genes using p65 antibody and LPS stimulated ORF2 expressing cell extract revealed decreased association of p65 with the above regions, indicating that ORF2 inhibited p65 binding at endogenous promoters. Conclusions In this report we suggest a mechanism by which ORF2 protein of HEV may inhibit host cell NF-κB activity during the course of a viral infection.
- 2012
Tyrosine phosphorylation of Munc18c on residue 521 abrogates binding to Syntaxin 4 Abstract
Background
Insulin stimulates exocytosis of GLUT4 from an intracellular store to the cell surface of fat and muscle cells. Fusion of GLUT4-containing vesicles with the plasma membrane requires the SNARE proteins Syntaxin 4, VAMP2 and the regulatory Sec1/Munc18 protein, Munc18c. Syntaxin 4 and Munc18c form a complex that is disrupted upon insulin treatment of adipocytes. Munc18c is tyrosine phosphorylated in response to insulin in these cells. Here, we directly test the hypothesis that tyrosine phosphorylation of Munc18c is responsible for the observed insulin-dependent abrogation of binding between Munc18c and Syntaxin 4.
Results
We show that Munc18c is directly phosphorylated by recombinant insulin receptor tyrosine kinase in vitro . Using pull-down assays, we show that phosphorylation abrogates binding of Munc18c to both Syntaxin 4 and the v-SNARE VAMP2, as does the introduction of a phosphomimetic mutation into Munc18c (Y521E).
Conclusion
Our data indicate that insulin-stimulated tyrosine phosphorylation of Munc18c impairs the ability of Munc18c to bind its cognate SNARE proteins, and may therefore represent a regulatory step in GLUT4 traffic.
- 2011