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The ubiquitin-proteasome pathway degrades the majority of proteins in mammalian cells and plays an essential role in the generation of antigenic peptides presented by major histocompatibility class I molecules. Proteasome inhibitors are of great interest as research tools and drug candidates. Most work on proteasome inhibitors has focused on the inhibition of the chymotryptic-like (beta5) sites; little attention has been paid to the inhibition of two other types of active sites, the trypsin-like (beta2) and the caspase-like (beta1). We report here the development of the first cell-permeable and highly selective inhibitors (4 and 5) of the proteasome's caspase-like site. The selectivity of the compounds is directly and unambiguously established by Staudinger-Bertozzi labeling of proteasome subunits covalently modified with azide-functionalized inhibitor 5. This labeling reveals that the caspase-like site of the immunoproteasome (beta1i) is a preferred target of this compound. These compounds can be used as tools to study roles of beta1 and beta1i sites in generation of specific antigenic peptides and their potential role as co-targets of anti-cancer drugs.
\n \n\n \n \nHypoxia inducible factor (HIF) is regulated by dual pathways involving oxygen-dependent prolyl and asparaginyl hydroxylation of its \u03b1-subunits. Prolyl hydroxylation at two sites within a central degradation domain promotes association of HIF-\u03b1 with the von Hippel-Lindau ubiquitin E3 ligase and destruction by the ubiquitin-proteasome pathways. Asparaginyl hydroxylation blocks the recruitment of p300/CBP co-activators to a C-terminal activation domain in HIF-\u03b1. These hydroxylations are catalyzed by members of the Fe(II) and 2-oxoglutarate (2-OG) oxygenase family. Activity of the enzymes is suppressed by hypoxia, increasing both the abundance and activity of the HIF transcriptional complex. We have used hydroxy residue-specific antibodies to compare and contrast the regulation of each site of prolyl hydroxylation (Pro(402), Pro(564)) with that of asparaginyl hydroxylation (Asn(803)) in human HIF-1\u03b1. Our findings reveal striking differences in the sensitivity of these hydroxylations to hypoxia and to different inhibitor types of 2-OG oxygenases. Hydroxylation at the three sites in endogenous human HIF-1\u03b1 proteins was suppressed by hypoxia in the order Pro(402) > Pro(564) > Asn(803). In contrast to some predictions from in vitro studies, prolyl hydroxylation was substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions; studies of a range of different small molecule 2-OG analogues demonstrated the feasibility of selectively inhibiting either prolyl or asparaginyl hydroxylation within cells.
\n \n\n \n \nUNLABELLED: 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.
\n \n\n \n \nObjective: To identify a potential diagnostic endometriosis marker using matrix-enhanced laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based urinary proteomics. Design: Prospective randomized pilot study. Setting: University hospital, tertiary referral center for endometriosis. Patient(s): 53 women undergoing laparoscopic surgery for pain and/or infertility comprising 30 women without endometriosis and 23 with endometriosis. Intervention(s): Laparoscopy and urine specimens. Main Outcome Measure(s): Urinary peptide profiles. Result(s): We observed distinct patterns of peptide profiles in the urine samples of women presenting with typical clinical symptoms of endometriosis. Six statistically significant putative peptide markers were identified (four during the periovulatory phase and two during the luteal phase) by comparing controls with moderate/severe endometriosis patients. The periovulatory peptide mass of 1,767.1 Da and the luteal peptide mass of 1,824.3 Da both showed a sensitivity of 75% and a specificity of 85% and 71%, respectively. Also detected were seven peptide markers (two during the periovulatory phase and five during the luteal phase) by comparing the urinary peptide profiles of patients with minimal/mild to moderate/severe endometriosis. The periovulatory peptide mass of 3,280.9 Da and the luteal peptide mass of 1,933.8 Da showed a sensitivity of 82% and 75% and a specificity of 88% and 75%, respectively. Conclusion(s): Urinary proteomic analysis may provide a novel method of diagnosing and staging endometriosis. \u00a9 2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
\n \n\n \n \nOBJECTIVE: To identify a potential diagnostic endometriosis marker using matrix-enhanced laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based urinary proteomics. DESIGN: Prospective randomized pilot study. SETTING: University hospital, tertiary referral center for endometriosis. PATIENT(S): 53 women undergoing laparoscopic surgery for pain and/or infertility comprising 30 women without endometriosis and 23 with endometriosis. INTERVENTION(S): Laparoscopy and urine specimens. MAIN OUTCOME MEASURE(S): Urinary peptide profiles. RESULT(S): We observed distinct patterns of peptide profiles in the urine samples of women presenting with typical clinical symptoms of endometriosis. Six statistically significant putative peptide markers were identified (four during the periovulatory phase and two during the luteal phase) by comparing controls with moderate/severe endometriosis patients. The periovulatory peptide mass of 1,767.1 Da and the luteal peptide mass of 1,824.3 Da both showed a sensitivity of 75% and a specificity of 85% and 71%, respectively. Also detected were seven peptide markers (two during the periovulatory phase and five during the luteal phase) by comparing the urinary peptide profiles of patients with minimal/mild to moderate/severe endometriosis. The periovulatory peptide mass of 3,280.9 Da and the luteal peptide mass of 1,933.8 Da showed a sensitivity of 82% and 75% and a specificity of 88% and 75%, respectively. CONCLUSION(S): Urinary proteomic analysis may provide a novel method of diagnosing and staging endometriosis.
\n \n\n \n \nProtein homoeostasis is a fundamental requirement for all living cells in order to survive in a dynamic surrounding. Proper levels of AIRAPL (arsenite-inducible RNA-associated protein-like protein) (ZFAND2B) are required in order to maintain cellular folding capacity in metazoans, and functional impairment of AIRAPL results in acceleration of aging and protein aggregation. However, the cellular roles of AIRAPL in this process are not known. In the present paper, we report that AIRAPL binds and forms a complex with p97 [VCP (valosin-containing protein)/Cdc48], Ubxd8 (ubiquitin regulatory X domain 8), Npl4-Ufd1, Derlin-1 and Bag6 on the ER (endoplasmic reticulum) membrane. In spite of the fact that AIRAPL complex partners are involved in the ERAD (ER-associated degradation) process, AIRAPL knockdown does not show any impairment in ERAD substrate degradation. However, translocation into the ER of a subset of ERAD- and non-ERAD-secreted proteins are regulated by AIRAPL. The ability to regulate translocation by the p97-AIRAPL complex is entirely dependent on the proteins' signal peptide. Our results demonstrate a p97 complex regulating translocation into the ER in a signal-peptide-dependent manner.
\n \n\n \n \nThe ubiquitin-proteasome system has emerged as essential sets of reactions involved in many biological processes in addition to the disposal of misfolded and damaged proteins. Studies in different research areas reveal its role in regulating cell growth, differentiation, apoptosis, signaling, and protein targeting. Small molecule inhibitors against the proteasome have been useful in determining the specific role of this enzyme in these processes. Here we review recent progress made in the development and application of molecules that target proteasomal proteolysis. In addition, an increasing number of other enzymes in this pathway, in particular deubiquitinating enzymes (DUBs) and N-glycanases, appear to be attractive alternative targets for developing inhibitors that can be used to interfere with biological processes linked to the ubiquitin-proteasome pathway. \u00a9 2008 Wiley-VCH Verlag GmbH & Co. KGaA.
\n \n\n \n \nNearly 90% of human melanomas contain inactivated wild-type p53, the underlying mechanisms for which are not fully understood. Here, we identify that cyclin B1/CDK1-phosphorylates iASPP, which leads to the inhibition of iASPP dimerization, promotion of iASPP monomer nuclear entry, and exposure of its p53 binding sites, leading to increased p53 inhibition. Nuclear iASPP is enriched in melanoma metastasis and associates with poor patient survival. Most wild-type p53-expressing melanoma cell lines coexpress high levels of phosphorylated nuclear iASPP, MDM2, and cyclin B1. Inhibition of MDM2 and iASPP phosphorylation with small molecules induced p53-dependent apoptosis and growth suppression. Concurrent p53 reactivation and BRAFV600E inhibition achieved additive suppression in vivo, presenting an alternative for melanoma therapy.
\n \n\n \n \nBoth humans and mice with a mutation in the autoimmune regulator (aire) gene develop multiorgan autoimmune disease. Aire was shown to exert its critical function in medullary epithelial cells of the thymus by promoting ectopic expression of peripheral tissue antigens. It was hypothesized that the widespread autoimmunity of Aire-deficient individuals reflects a lack of tolerance induction to the repertoire of peripheral tissue antigens expressed in the thymus of normal individuals. Here, we substantiate this hypothesis by identifying Mucin 6 as a stomach-specific antigen targeted by autoantibodies in gastritis-prone mice lacking thymic expression of aire and demonstrate that transcription of the Mucin 6 gene in thymic medullary epithelial cells is indeed Aire-dependent.
\n \n\n \n \nThe 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.
\n \n\n \n \nApoptosis is a key tumor suppression mechanism that can be initiated by activation of the proapoptotic factor Bax. The Ku70 DNA end-joining protein has recently been shown to suppress apoptosis by sequestering Bax from mitochondria. The mechanism by which Bax is regulated remains unknown. Here, we identify eight lysines in Ku70 that are targets for acetylation in vivo. Five of these, K539, K542, K544, K533, and K556, lie in the C-terminal linker domain of Ku70 adjacent to the Bax interaction domain. We show that CBP and PCAF efficiently acetylate K542 in vitro and associate with Ku70 in vivo. Mimicking acetylation of K539 or K542 or treating cells with deacetylase inhibitors abolishes the ability of Ku70 to suppress Bax-mediated apoptosis. We demonstrate that increased acetylation of Ku70 disrupts the Ku70-Bax interaction and coincides with cytoplasmic accumulation of CBP. These results shed light on the role of acetyltransferases as tumor suppressors.
\n \n\n \n \nTimely 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.
\n \n\n \n \nBortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.
\n \n\n \n \nCathepsins B and L are widely expressed cysteine proteases implicated in both intracellular proteolysis and extracellular matrix remodeling. However, specific roles remain to be validated in vivo. Here we show that combined deficiency of cathepsins B and L in mice is lethal during the second to fourth week of life. Cathepsin B(-/-)/L(-/-) mice reveal a degree of brain atrophy not previously seen in mice. This is because of massive apoptosis of select neurons in the cerebral cortex and the cerebellar Purkinje and granule cell layers. Neurodegeneration is accompanied by pronounced reactive astrocytosis and is preceded by an accumulation of ultrastructurally and biochemically unique lysosomal bodies in large cortical neurons and by axonal enlargements. Our data demonstrate a pivotal role for cathepsins B and L in maintenance of the central nervous system.
\n \n\n \n \nThe mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase 1 (PRMT1) and symmetric dimethylating PRMT5 and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favors proliferation by antagonizing methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell-cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN downregulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity.
\n \n\n \n \nEliciting neutralizing antibodies capable of inactivating a broad spectrum of HIV-1 strains is a major goal of HIV-1 vaccine design. The challenge is that envelopes (Envs) of circulating viruses are almost certainly different from any Env used in a vaccine. A novel immunogen composed of a highly diverse set of gp140 Envs including subtypes A, B, C, D and F was developed to stimulate a more cross-neutralizing antibody response. Env heterotrimers composed of up to 54 different gp140s were produced with the aim of focusing the response to the conserved regions of Env while reducing the dominance of any individual hypervariable region. Heterotrimeric gp140 Envs of inter- and intra-subtype combinations were shown to bind CD4 and a panel of neutralizing monoclonal antibodies with similar affinity to monovalent UG37 gp140. Macaques immunized with six groups of heterotrimer mixtures showed slightly more potent neutralizing antibody responses in TZM-BL tier 1 and A3R5 tier 2 pseudovirus assays than macaques immunized with monovalent Env gp140, and exhibited a marginally greater focus on the CD4-binding site. Carbopol enhanced neutralization when used as an adjuvant instead of RIBI in combination with UG37 gp140. These data indicate that cross-subtype heterotrimeric gp140 Envs may elicit some improvement of the neutralizing antibody response in macaques compared to monovalent gp140 Env.
\n \n\n \n \nPost-translational protein modifications such as acetylation have significant regulatory roles in metabolic processes, but their relationship to both variation in gene expression and DNA sequence is unclear. We address this question in the Goto-Kakizaki (GK) rat inbred strain, a model of polygenic type 2 diabetes. Expression of the NAD-dependent deacetylase Sirtuin-3 is down-regulated in GK rats compared to normoglycemic Brown Norway (BN) rats. We show first that a promoter SNP causes down-regulation of Sirtuin-3 expression in GK rats. We then use mass-spectrometry to identify proteome-wide differential lysine acetylation of putative Sirtuin-3 protein targets in livers of GK and BN rats. These include many proteins in pathways connected to diabetes and metabolic syndrome. We finally sequence GK and BN liver transcriptomes and find that mRNA expression of these targets does not differ significantly between GK and BN rats, in contrast to other components of the same pathways. We conclude that physiological differences between GK and BN rats are mediated by a combination of differential protein acetylation and gene transcription and that genetic variation can modulate acetylation independently of expression.
\n \n\n \n \nBACKGROUND: Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC. METHODS: Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis. RESULTS: Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages. CONCLUSIONS: The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose definitive cut-offs and combinations for evaluation of performance.
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