Proteasome Inhibitor Bortezomib Ameliorates Intestinal Injury in Mice

Background: Bortezomib is a proteasome inhibitor that has shown impressive efficacy in the treatment of multiple myeloma. In mice, the addition of dextran sulfate sodium (DSS) to drinking water leads to acute colitis that can serve as an experimental animal model for human ulcerative colitis. Methodology/Principal Findings: Bortezomib treatment was shown to potently inhibit murine DSS-induced colitis. The attenuation of DSS-induced colitis was associated with decreased inflammatory cell infiltration in the colon. Specifically, bortezomib-treated mice showed significantly decreased numbers of CD4 + and CD8 + T cells in the colon and mesenteric lymph nodes. Bortezomib treatment significantly diminished interferon (IFN)-c expression in the colon and mesenteric lymph nodes. Furthermore, cytoplasmic IFN-c production by CD4 + and CD8 + T cells in mesenteric lymph nodes was substantially decreased by bortezomib treatment. Notably, bortezomib enhanced T cell apoptosis by inhibiting nuclear factor-kB activation during DSS-induced colitis. Conclusions/Significance: Bortezomib treatment is likely to induce T cell death, thereby suppressing DSS-induced colitis by reducing IFN-c production

Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases. Apomyoglobin emerges as a new model substrate to further explore the role of ATPases and protein structure in proteasomal degradatio

Enhanced killing of androgen-independent prostate cancer cells using inositol hexakisphosphate in combination with proteasome inhibitors

Effective treatments for androgen-independent prostate cancer (AIPCa) are lacking. To address this, emerging therapeutics such as proteasome inhibitors are currently undergoing clinical trials. Inositol hexakisphosphate (IP6) is an orally non-toxic phytochemical that exhibits antitumour activity against several types of cancer including PCa. We have previously shown that treatment of PC3 cells with IP6 induces the transcription of a subset of nuclear factor-κB (NF-κB)-responsive and pro-apoptotic BCL-2 family genes. In this study, we report that although NF-κB subunits p50/p65 translocate to the nucleus of PC3 cells in response to IP6, inhibition of NF-κB-mediated transcription using non-degradable inhibitor of κB (IκB)-α does not modulate IP6 sensitivity. Treatment with IP6 also leads to increased protein levels of PUMA, BIK/NBK and NOXA between 4 and 8 h of treatment and decreased levels of MCL-1 and BCL-2 after 24 h. Although blocking transcription using actinomycin D does not modulate PC3 cell sensitivity to IP6, inhibition of protein translation using cycloheximide has a significant protective effect. In contrast, blocking proteasome-mediated protein degradation using MG-132 significantly enhances the ability of IP6 to reduce cellular metabolic activity in both PC3 and DU145 AIPCa cell lines. This effect of combined treatment on mitochondrial depolarisation is particularly striking and is also reproduced by another proteasome inhibitor (ALLN). The enhanced effect of combined MG132/IP6 treatment is almost completely inhibited by cycloheximide and correlates with changes in BCL-2 family protein levels. Altogether these results suggest a role for BCL-2 family proteins in mediating the combined effect of IP6 and proteasome inhibitors and warrant further pre-clinical studies for the treatment of AIPCa

A Gammaherpesvirus Cooperates with Interferon-alpha/beta-Induced IRF2 to Halt Viral Replication, Control Reactivation, and Minimize Host Lethality

The gammaherpesviruses, including Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), establish latency in memory B lymphocytes and promote lymphoproliferative disease in immunocompromised individuals. The precise immune mechanisms that prevent gammaherpesvirus reactivation and tumorigenesis are poorly defined. Murine gammaherpesvirus 68 (MHV68) is closely related to EBV and KSHV, and type I (alpha/beta) interferons (IFNαβ) regulate MHV68 reactivation from both B cells and macrophages by unknown mechanisms. Here we demonstrate that IFNβ is highly upregulated during latent infection, in the absence of detectable MHV68 replication. We identify an interferon-stimulated response element (ISRE) in the MHV68 M2 gene promoter that is bound by the IFNαβ-induced transcriptional repressor IRF2 during latency in vivo. The M2 protein regulates B cell signaling to promote establishment of latency and reactivation. Virus lacking the M2 ISRE (ISREΔ) overexpresses M2 mRNA and displays uncontrolled acute replication in vivo, higher latent viral load, and aberrantly high reactivation from latency. These phenotypes of the ISREΔ mutant are B-cell-specific, require IRF2, and correlate with a significant increase in virulence in a model of acute viral pneumonia. We therefore identify a mechanism by which a gammaherpesvirus subverts host IFNαβ signaling in a surprisingly cooperative manner, to directly repress viral replication and reactivation and enforce latency, thereby minimizing acute host disease. Since we find ISREs 5′ to the major lymphocyte latency genes of multiple rodent, primate, and human gammaherpesviruses, we propose that cooperative subversion of IFNαβ-induced IRFs to promote latent infection is an ancient strategy that ensures a stable, minimally-pathogenic virus-host relationship

Pervasive and opposing effects of Unpredictable Chronic Mild Stress (UCMS) on hippocampal gene expression in BALB/cJ and C57BL/6J mouse strains

Background: BALB/cJ is a strain susceptible to stress and extremely susceptible to a defective hedonic impact in response to chronic stressors. The strain offers much promise as an animal model for the study of stress related disorders. We present a comparative hippocampal gene expression study on the effects of unpredictable chronic mild stress on BALB/cJ and C57BL/6J mice. Affymetrix MOE 430 was used to measure hippocampal gene expression from 16 animals of two different strains (BALB/cJ and C57BL/6J) of both sexes and subjected to either unpredictable chronic mild stress (UCMS) or no stress. Differences were statistically evaluated through supervised and unsupervised linear modelling and using Weighted Gene Coexpression Network Analysis (WGCNA). In order to gain further understanding into mechanisms related to stress response, we cross-validated our results with a parallel study from the GENDEP project using WGCNA in a meta-analysis design. Results: The effects of UCMS are visible through Principal Component Analysis which highlights the stress sensitivity of the BALB/cJ strain. A number of genes and gene networks related to stress response were uncovered including the Creb1 gene. WGCNA and pathway analysis revealed a gene network centered on Nfkb1. Results from the meta-analysis revealed a highly significant gene pathway centred on the Ubiquitin C (Ubc) gene. All pathways uncovered are associated with inflammation and immune response. Conclusions: The study investigated the molecular mechanisms underlying the response to adverse environment in an animal model using a GxE design. Stress-related differences were visible at the genomic level through PCA analysis highlighting the high sensitivity of BALB/cJ animals to environmental stressors. Several candidate genes and gene networks reported are associated with inflammation and neurogenesis and could serve to inform candidate gene selection in human studies and provide additional insight into the pathology of Major Depressive Disorder

Enhanced Inflammatory Potential of CD4(+) T-Cells That Lack Proteasome Immunosubunit Expression, in a T-Cell Transfer-Based Colitis Model

Proteasomes play a fundamental role in intracellular protein degradation and therewith regulate a variety of cellular processes. Exposure of cells to (pro)inflammatory cytokines upregulates the expression of three inducible catalytic proteasome subunits, the immunosubunits, which incorporate into newly assembled proteasome complexes and alter the catalytic activity of the cellular proteasome population. Single gene-deficient mice lacking one of the three immunosubunits are resistant to dextran sulfate sodium (DSS)-induced colitis development and, likewise, inhibition of one single immunosubunit protects mice against the development of DSS-induced colitis. The observed diminished disease susceptibility has been attributed to altered cytokine production and CD4+ T-cell differentiation in the absence of immunosubunits. To further test whether the catalytic activity conferred by immunosubunits plays an essential role in CD4+ T-cell function and to distinguish between the role of immunosubunits in effector T-cells versus inflamed tissue, we used a T-cell transfer-induced colitis model. Naïve wt or immunosubunit-deficient CD4+ T-cells were adoptively transferred into RAG1-/- and immunosubunit-deficient RAG1-/- mice and colitis development was determined six weeks later. While immunosubunit expression in recipient mice had no effect on colitis development, transferred immunosubunit-deficient T- cells were more potent in inducing colitis and produced more proinflammatory IL17 than wt T-cells. Taken together, our data show that modifications in proteasome-mediated proteolysis in T-cells, conferred by lack of immunosubunit incorporation, do not attenuate but enhance CD4+ T-cell-induced inflammation

Recruitment of Histone Deacetylase 3 to the Interferon-A Gene Promoters Attenuates Interferon Expression

Induction of Type I Interferon (IFN) genes constitutes an essential step leading to innate immune responses during virus infection. Sendai virus (SeV) infection of B lymphoid Namalwa cells transiently induces the transcriptional expression of multiple IFN-A genes. Although transcriptional activation of IFN-A genes has been extensively studied, the mechanism responsible for the attenuation of their expression remains to be determined.In this study, we demonstrate that virus infection of Namalwa cells induces transient recruitment of HDAC3 (histone deacetylase 3) to IFN-A promoters. Analysis of chromatin-protein association by Chip-QPCR demonstrated that recruitment of interferon regulatory factor (IRF)3 and IRF7, as well as TBP correlated with enhanced histone H3K9 and H3K14 acetylation, whereas recruitment of HDAC3 correlated with inhibition of histone H3K9/K14 acetylation, removal of IRF7 and TATA-binding protein (TBP) from IFN-A promoters and inhibition of virus-induced IFN-A gene transcription. Additionally, HDAC3 overexpression reduced, and HDAC3 depletion by siRNA enhanced IFN-A gene expression. Furthermore, activation of IRF7 enhanced histone H3K9/K14 acetylation and IFN-A gene expression, whereas activation of both IRF7 and IRF3 led to recruitment of HDAC3 to the IFN-A gene promoters, resulting in impaired histone H3K9 acetylation and attenuation of IFN-A gene transcription.Altogether these data indicate that reversal of histone H3K9/K14 acetylation by HDAC3 is required for attenuation of IFN-A gene transcription during viral infection

Negative Regulation of Interferon-β Gene Expression during Acute and Persistent Virus Infections

The production of type I interferons (IFNs) in response to viral infections is critical for antiviral immunity. However, IFN production is transient, and continued expression can lead to inflammatory or autoimmune diseases. Thus, understanding the mechanisms underlying the negative regulation of IFN expression could lead to the development of novel therapeutic approaches to the treatment of these diseases. We report that the transcription factor IRF3 plays a central role in the negative regulation of interferon-β (IFNβ) expression during both acute and persistent (chronic) virus infections. We show that the degradation of IRF3 during acute infections, rather than the activation of transcriptional repressors, leads to the down regulation of IFNβ expression. We also show that the block to IFNβ expression in mouse embryonic fibroblasts that are persistently infected with Sendai virus (SeV) correlates with the absence of transcriptionally active IRF3. Remarkably, ongoing protein synthesis and viral replication are required to maintain repression of the IFNβ gene in persistently infected cells, as the gene can be activated by the protein synthesis inhibitor cycloheximide, or by the antiviral drug ribavirin. Finally, we show that the SeV V protein inhibits IRF3 activity in persistently infected cells. Thus, in conjunction with the known interference with STAT1 by the SeV C protein, both IFN activation and its signaling pathways are blocked in persistently infected cells. We conclude that the transcription factor IRF3 is targeted for turnover and inactivation through distinct mechanisms from both the host cells and virus, leading to the inhibition of IFNβ gene expression during acute and persistent viral infections. These observations show that IRF3 plays a critical role, not only in the activation of the IFNβ gene, but also in the controlling the duration of its expression. (284 words

Muscle wasting in chronic kidney disease: the role of the ubiquitin proteasome system and its clinical impact

Muscle wasting in chronic kidney disease (CKD) and other catabolic diseases (e.g. sepsis, diabetes, cancer) can occur despite adequate nutritional intake. It is now known that complications of these various disorders, including acidosis, insulin resistance, inflammation, and increased glucocorticoid and angiotensin II production, all activate the ubiquitin–proteasome system (UPS) to degrade muscle proteins. The initial step in this process is activation of caspase-3 to cleave the myofibril into its components (actin, myosin, troponin, and tropomyosin). Caspase-3 is required because the UPS minimally degrades the myofibril but rapidly degrades its component proteins. Caspase-3 activity is easily detected because it leaves a characteristic 14kD actin fragment in muscle samples. Preliminary evidence from several experimental models of catabolic diseases, as well as from studies in patients, indicates that this fragment could be a useful biomarker because it correlates well with the degree of muscle degradation in dialysis patients and in other catabolic conditions