Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial steps that govern Bak activation are poorly understood. To further clarify these pivotal events, we have investigated whether post-translational modifications of Bak impinge on its activation potential. In this study, we report that on apoptotic stimulation Bak undergoes dephosphorylation at tyrosine residue 108 (Y108), a critical event that is necessary but not sufficient for Bak activation, but is required both for early exposure of the occluded N-terminal domain and multimerisation. RNA interference (RNAi) screening identified non-receptor tyrosine phosphatases (PTPNs) required for Bak dephosphorylation and apoptotic induction through chemotherapeutic agents. Specifically, modulation of PTPN5 protein expression by siRNA and overexpression directly affected both Bak-Y108 phosphorylation and the initiation of Bak activation. We further show that MEK/ERK signalling directly affects Bak phosphorylation through inhibition of PTPN5 to promote cell survival. We propose a model of Bak activation in which the regulation of Bak dephosphorylation constitutes the initial step in the activation process, which reveals a previously unsuspected mechanism controlling the initiation of mitochondrial apoptosis.
JOURNAL OF ENDOUROLOGY, 24 pp. A17-A18.2010. COMPARATIVE QUANTITATIVE PROTEOMIC PROFILING OF URINE TO IDENTIFY CANDIDATE REGULATORS OF UROLITHIASIS
BACKGROUND: We have reported improved islet isolation outcomes using a new digestion protocol where the pancreas is perfused only with collagenase, and neutral protease (NP) is administered during the digestion phase. Since the inception of this protocol, we have had some cases where administration of NP was not required. Our new protocol was utilized in 94 islet isolations. The timing of adding NP was dependent on the progression of digestion but in 10 cases the progression was rapid and most islets in the assessment samples were free from the exocrine tissue. As a result NP was not added at all for these isolations (no-NP group). In the remaining 84 isolations, NP was added during digestion phase (control group). RESULTS: Pancreata in the each group were digested with a similar collagenase dose. Digestion time was shorter in the no-NP (15.0±1.8 vs 19.5±0.6 min, P=0.004). At post-digestion, the no-NP had fewer trapped islets (10.9±2.8 vs 28.1±2.4%, P=0.009). Post-purification islet yield was similar (355±45 x10 ( 3) vs 318±17 x10 ( 3) IE, P=0.29). Five preparations in the no-NP were used for clinical transplantation, leading to a 64.3±15.2% reduction in insulin usage. Interestingly, cold ischemia time was longer in the no-NP (10.3±0.9 vs 7.9±0.4 h, P=0.04). One particular collagenase lot having the highest NP activity contamination was used in 7 isolations in the no-NP. Our experience indicates that supplementation of collagenase with NP is not always necessary for effective isolation. Cold ischemia time and NP contamination should be evaluated for optimal NP dosage.
Matrix metalloproteinase-9 (MMP-9) expression is known to enhance the invasion and metastasis of tumor cells. In previous work based on a proteomic screen, we identified the serpin protease nexin-1 (PN-1) as a potential target of MMP-9. Here, we show that PN-1 is a substrate for MMP-9 and establish a link between PN-1 degradation by MMP-9 and regulation of invasion. PN-1 levels increased in prostate carcinoma cells after downregulation of MMP-9 and in tissues of MMP-9-deficient mice, consistent with PN-1 degradation by MMP-9. We identified three MMP-9 cleavage sites in PN-1 and showed that mutations in those sites made PN-1 more resistant to MMP-9. Urokinase plasminogen activator (uPA) is inhibited by PN-1. MMP-9 augmented uPA activity in the medium of PC3-ML cells by degrading PN-1. Prostate cancer cells, overexpressing PN-1 or treated with MMP-9 shRNA, had reduced cell invasion in Matrigel. PN-1 siRNA restored uPA activity and the invasive capacity. PN-1 mutated in the serpin inhibitory domain, the reactive center loop, failed to inhibit uPA and to reduce Matrigel invasion. This study shows a novel molecular pathway in which MMP-9 regulates uPA activity and tumor cell invasion through cleavage of PN-1.
Post-translational modifications on histones are an important mechanism for the regulation of gene expression and are involved in all aspects of cell growth and differentiation, as well as pathological processes including neurodegeneration, autoimmunity, and cancer. A major challenge within the chromatin field is to develop methods for the quantitative analysis of histone modifications. Here we report a mass spectrometry (MS) approach based on ultraperformance liquid chromatography high/low collision switching (UPLC-MS(E)) to monitor histone modifications in cells. This approach is exemplified by the analysis of trimethylated lysine-9 levels in histone H3, following a simple chemical derivatization procedure with d(6)-acetic anhydride. This method was used to study the inhibition of histone demethylases with pyridine-2,4-dicarboxylic acid (PDCA) derivatives in cells. Our results show that the PDCA-dimethyl ester inhibits JMJD2A catalyzed demethylation of lysine-9 on histone H3 in human HEK 293T cells. Demethylase inhibition, as observed by MS analyses, was supported by immunoblotting with modification-specific antibodies. The results demonstrate that PDCA derived small molecules are cell permeable demethylase inhibitors and reveal that quantitative MS is a useful tool for measuring post-translational histone modifications in cells.
CLINICAL AND EXPERIMENTAL RHEUMATOLOGY, 28 (4), pp. 607-607.2010. INVESTIGATING THE ROLE OF ENDOPLASMIC RETICULUM AMINOPEPTIDASE-1 (ERAP1) IN ANKYLOSING SPONDYLITIS
CLINICAL AND EXPERIMENTAL RHEUMATOLOGY, 28 (4), pp. 631-631.2010. INVESTIGATING THE ROLE OF ENDOPLASMIC RETICULUM AMINOPEPTIDASE-1 (ERAP1) IN ANKYLOSING SPONDYLITIS
Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.
Cyclin-dependent kinase-associated protein 1 (Cks1) is involved in the control of the transcription of a subset of genes in addition to its role in controlling the cell cycle in the budding yeast Saccharomyces cerevisiae. By directly ligating Cks1 onto a GAL1 promoter-driven reporter, we demonstrated that Cks1 acts as a transcription activator. Using this method, we dissected the downstream events from Cks1 recruitment at the promoter. We showed that subsequent to promoter binding, Cdc28 binding is required to modulate the level of gene expression. The ubiquitin-binding domain of Cks1 is essential for implementing downstream transcription events, which appears to recruit the proteasome via ubiquitylated proteasome subunits. We propose that the selective ability of Cks1 to bind ubiquitin allows this small molecule the flexibility to bind large protein complexes with specificity and that this may represent a novel mechanism of regulating transcriptional activation.
Microbial pathogens exploit the ubiquitin system to facilitate infection and manipulate the immune responses of the host. In this study, susceptibility to Yersinia enterocolitica and Yersinia pseudotuberculosis invasion was found to be increased upon overexpression of the deubiquitinating enzyme otubain 1 (OTUB1), a member of the ovarian tumour domain-containing protein family. Conversely, OTUB1 knockdown interfered with Yersinia invasion in HEK293T cells as well as in primary monocytes. This effect was attributed to a modulation of bacterial uptake. We demonstrate that the Yersinia-encoded virulence factor YpkA (YopO) kinase interacts with a post-translationally modified form of OTUB1 that contains multiple phosphorylation sites. OTUB1, YpkA and the small GTPase ras homologue gene family member A (RhoA) were found to be part of the same protein complex, suggesting that RhoA levels are modulated by OTUB1. Our results show that OTUB1 is able to stabilize active RhoA prior to invasion, which is concomitant with an increase in bacterial uptake. This effect is modulated by post-translational modifications of OTUB1, suggesting a new entry point for manipulating Yersinia interactions with the host.
The mechanism by which Wnt receptors transduce signals to activate downstream beta-catenin-mediated target gene transcription remains incompletely understood but involves Frizzled (Fz) receptor-mediated plasma membrane recruitment and activation of the cytoplasmic effector Dishevelled (Dvl). Here, we identify the deubiquitinating enzyme CYLD, the familial cylindromatosis tumor suppressor gene, as a negative regulator of proximal events in Wnt/beta-catenin signaling. Depletion of CYLD from cultured cells markedly enhances Wnt-induced accumulation of beta-catenin and target gene activation. Moreover, we demonstrate hyperactive Wnt signaling in human cylindroma skin tumors that arise from mutations in CYLD. At the molecular level, CYLD interacts with and regulates K63-linked ubiquitination of Dvl. Enhanced ubiquitination of the polymerization-prone DIX domain in CYLD-deficient cells positively links to the signaling activity of Dvl. Together, our results argue that loss of CYLD instigates tumor growth in human cylindromatosis through a mechanism in which hyperubiquitination of polymerized Dvl drives enhancement of Wnt responses.
UNLABELLED: The central proteomics facilities pipeline (CPFP) provides identification, validation, and quantitation of peptides and proteins from LC-MS/MS datasets through an easy to use web interface. It is the first analysis pipeline targeted specifically at the needs of proteomics core facilities, reducing the data analysis load on staff, and allowing facility clients to easily access and work with their data. Identification of peptides is performed using multiple search engines, their output combined and validated using state-of-the-art techniques for improved results. Cluster execution of jobs allows analysis capacity to be increased easily as demand grows. AVAILABILITY: Released under the Common Development and Distribution License at http://cpfp.sourceforge.net/. Demonstration available at https://cpfp-master.molbiol.ox.ac.uk/cpfp_demo.
Ankylosing Spondylitis (AS) is a common, inflammatory rheumatic disease, which primarily affects the axial skeleton and is associated with sacroiliitis, uveitis and enthesitis. Unlike other autoimmune rheumatic diseases, such as rheumatoid arthritis or systemic lupus erythematosus, autoantibodies have not yet been reported to be a feature of AS. We therefore wished to determine if plasma from patients with AS contained autoantibodies and if so, characterize and quantify this response in comparison to patients with Rheumatoid Arthritis (RA) and healthy controls. Two high-density nucleic acid programmable protein arrays expressing a total of 3498 proteins were screened with plasma from 25 patients with AS, 17 with RA and 25 healthy controls. Autoantigens identified were subjected to Ingenuity Pathway Analysis in order to determine patterns of signalling cascades or tissue origin. 44% of patients with Ankylosing Spondylitis demonstrated a broad autoantibody response, as compared to 33% of patients with RA and only 8% of healthy controls. Individuals with AS demonstrated autoantibody responses to shared autoantigens, and 60% of autoantigens identified in the AS cohort were restricted to that group. The AS patients' autoantibody responses were targeted towards connective, skeletal and muscular tissue, unlike those of RA patients or healthy controls. Thus, patients with AS show evidence of systemic humoral autoimmunity and multispecific autoantibody production. Nucleic Acid Programmable Protein Arrays constitute a powerful tool to study autoimmune diseases.
Inheritance of biological information to future generations depends on the replication of DNA and the Mendelian principle of distribution of genes. In addition, external and environmental factors can influence traits that can be propagated to offspring, but the molecular details of this are only beginning to be understood. The discoveries of DNA methylation and post-translational modifications on chromatin and histones provided entry points for regulating gene expression, an area now defined as epigenetics and epigenomics. Mass spectrometry turned out to be instrumental in uncovering molecular details involved in these processes. The central role of histone post-translational modifications in epigenetics related biological processes has revitalized mass spectrometry based investigations. In this special report, current approaches and future challenges that lay ahead due to the enormous complexity are discussed.
Timely elimination of damaged mitochondria is essential to protect cells from the potential harm of disordered mitochondrial metabolism and release of proapoptotic proteins. In mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. Mitochondrial sequestration by autophagosomes, followed by delivery to the lysosomal compartment for degradation (mitophagy), is a major mechanism of mitochondrial turnover. Here we show that mice lacking the essential autophagy gene Atg7 in the hematopoietic system develop severe anemia. Atg7(-/-) erythrocytes accumulate damaged mitochondria with altered membrane potential leading to cell death. We find that mitochondrial loss is initiated in the bone marrow at the Ter119(+)/CD71(High) stage. Proteomic analysis of erythrocyte ghosts suggests that in the absence of autophagy other cellular degradation mechanisms are induced. Importantly, neither the removal of endoplasmic reticulum nor ribosomes is affected by the lack of Atg7. Atg7 deficiency also led to severe lymphopenia as a result of mitochondrial damage followed by apoptosis in mature T lymphocytes. Ex vivo short-lived hematopoietic cells such as monocytes and dendritic cells were not affected by the loss of Atg7. In summary, we show that the selective removal of mitochondria by autophagy, but not other organelles, during erythropoeisis is essential and that this is a necessary developmental step in erythroid cells.
The aim of this study was to identify potential protein targets for insulin sensitization in human adipose tissue using unbiased proteomic approaches. Ten moderately obese, but otherwise healthy, subjects were treated with rosiglitazone 4 mg b.i.d. for 14 days and global protein and gene expression changes were monitored. Proteomic analysis revealed distinct up- or downregulation (greater than twofold) in 187 protein spots on the two-dimensional (2-D) gel images between day 0 and day 1 adipose tissue samples. When comparing the protein spots on the gels from day 0 with that of 14-day-treated samples, 122 spots showed differential expression. There was a striking increase in the expression of proteins involved in glucose transporter-4 (GLUT4) granule transport and fusion (actin, myosin-9, tubulin, vimentin, annexins, moesin, LIM, and SH3 domain protein-1), signaling (calmodulin, guanine nucleotide-binding proteins), redox regulation (superoxide dismutase, catalase, ferritin, transferrin, heat shock proteins), and adipogenesis (collagens, galectin-1, nidogen-1, laminin, lamin A/C). However, there was an intriguing absence of correlated changes in mRNA expression, suggesting adaptation at a post-transcriptional level in response to rosiglitazone. Thus, the major changes observed were among proteins involved in cytoskeletal rearrangement, insulin and calcium signaling, and inflammatory and redox signals that decisively upregulate GLUT4 granule trafficking in human adipose tissue. Such orchestrated changes in expression of multiple proteins provide insights into the mechanism underlying the increased efficiency in glucose uptake and improvement of insulin sensitivity in response to rosiglitazone treatment.
The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, beta-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.
The aim of this study was to investigate the quality and reproducibility of mass spectra derived from a matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) platform in a patient population undergoing carotid endarterectomy. Plasma samples were either digested with trypsin or left undigested, fractionated with either C18 or weak cation exchange (WCX) columns and analysed by MALDI-TOF MS. Quality of mass spectra for each method was assessed by baseline correction (lower area under the curve ratio indicating higher quality) and signal-to-noise ratio. Mean coefficient of variation (CV%) assessed reproducibility between repeated experiments and methods. Identified mass peak intensity differences were assessed for consistency across repeated experiments. Plasma from six patients was analysed. The quality of mass spectra was significantly better when derived from digested plasma fractionated by either WCX or C18 methods compared to undigested plasma fractionated by WCX (analysis of variance, p < 0.05). Mean CV% for repeated experiments was 18% and 28% for WCX and C18 fractionated digested plasma, respectively. A small number of differences in mass peak intensities were consistently observed in repeated experiments. Repeated experiments are required to confidently identify non-random mass peak intensity differences as putative plasma biomarkers that merit further investigation.
The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, b-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies. © 2010 Kramer et al.
Chronic hyperglycemia leads to deterioration of insulin release from pancreatic β-cells as well as insulin action on peripheral tissues. However, the mechanism underlying β-cell dysfunction resulting from glucose toxicity has not been fully elucidated. The aim of the present study was to define a set of alterations in mitochondrial protein profiles of pancreatic β-cell line in response to glucotoxic condition using 2-DE and tandem mass spectrometry. INS1E cells were incubated in the presence of 5.5 and 20 mM glucose for 72 hrs and mitochondria were isolated. Approximately 75 protein spots displayed significant changes (p < 0.05) in relative abundance in the presence of 20 mM glucose compared to controls. Mitochondrial proteins down regulated under glucotoxic conditions includes ATP synthase α chain and δ chain, malate dehydrogenase, aconitase, trifunctional enzyme β subunit, NADH cytochrome b5 reductase and voltage-dependent anion-selective channel protein (VDAC) 2. VDAC1, 75 kDa glucose-regulated protein, heat shock protein (HSP) 60 and HSP10 were found to be upregulated. The orchestrated changes in expression of VDACs and multiple other proteins involved in nutrient metabolism, ATP synthesis, cellular defense, glycoprotein folding and mitochondrial DNA stability may explain cellular dysfunction in glucotoxicity resulting in altered insulin secretion.
Tri-functional activity-based protein probes that encompass an electrophilic trap, a photo-reactive group and a bio-orthogonal ligation handle are described. With these, and in a three-step chemical proteomics approach, proteasomal catalytic sites are covalently and irreversibly modified, followed by photocrosslinking of these to flanking subunits and Staudinger-Bertozzi ligation for visualization and identification of the resulting conjugates.
Activation of the cell-death mediator Bak commits a cell to mitochondrial apoptosis. The initial steps that govern Bak activation are poorly understood. To further clarify these pivotal events, we have investigated whether post-translational modifications of Bak impinge on its activation potential. In this study, we report that on apoptotic stimulation Bak undergoes dephosphorylation at tyrosine residue 108 (Y108), a critical event that is necessary but not sufficient for Bak activation, but is required both for early exposure of the occluded N-terminal domain and multimerisation. RNA interference (RNAi) screening identified non-receptor tyrosine phosphatases (PTPNs) required for Bak dephosphorylation and apoptotic induction through chemotherapeutic agents. Specifically, modulation of PTPN5 protein expression by siRNA and overexpression directly affected both Bak-Y108 phosphorylation and the initiation of Bak activation. We further show that MEK/ERK signalling directly affects Bak phosphorylation through inhibition of PTPN5 to promote cell survival. We propose a model of Bak activation in which the regulation of Bak dephosphorylation constitutes the initial step in the activation process, which reveals a previously unsuspected mechanism controlling the initiation of mitochondrial apoptosis. © 2010 European Molecular Biology Organization.
Among the hallmarks of aged organisms are an accumulation of misfolded proteins and a reduction in skeletal muscle mass ("sarcopenia"). We have examined the effects of aging and dietary restriction (which retards many age-related changes) on components of the ubiquitin proteasome system (UPS) in muscle. The hindlimb muscles of aged (30 months old) rats showed a marked loss of muscle mass and contained 2-3-fold higher levels of 26S proteasomes than those of adult (4 months old) controls. 26S proteasomes purified from muscles of aged and adult rats showed a similar capacity to degrade peptides, proteins, and an ubiquitylated substrate, but differed in levels of proteasome-associated proteins (e.g. the ubiquitin ligase E6AP and deubiquitylating enzyme USP14). Also, the activities of many other deubiquitylating enzymes were greatly enhanced in the aged muscles. Nevertheless, their content of polyubiquitylated proteins was higher than in adult animals. The aged muscles contained higher levels of the ubiquitin ligase CHIP, involved in eliminating misfolded proteins, and MuRF1, which ubiquitylates myofibrillar proteins. These muscles differed from ones rapidly atrophying due to disease, fasting, or disuse in that Atrogin-1/MAFbx expression was low and not inducible by glucocorticoids. Thus, the muscles of aged rats showed many adaptations indicating enhanced proteolysis by the UPS, which may enhance their capacity to eliminate misfolded proteins and seems to contribute to the sarcopenia. Accordingly, dietary restriction decreased or prevented the aging-associated increases in proteasomes and other UPS components and reduced muscle wasting.
Total publications on this page: 23
Total citations for publications on this page: 1143