Proteasome Gene Expression Research Articles

Structure, Assembly and Homeostatic Regulation of the 26S Proteasome

The 26S proteasome is the major protease responsible for degradation of regulatory and abnormal proteins in the cell. Proteasomal degradation controls many cellular processes including, but not limited to, cell cycle control, transcription, DNA repair, apoptosis, quality control and antigen presentation. To elucidate how the proteasome is regulated is crucial to our understanding of the molecular details of proteasomal degradation and its functions in diverse biological pathways. In this article, I will highlight recent advances in understanding the proteasome structure and assembly and the regulation of proteasome gene expression. The implications of these new developments in cancer therapy will also be discussed.

Proteasomal Degradation Is Transcriptionally Controlled by TCF11 via an ERAD-Dependent Feedback Loop

Coordinated regulation of the ubiquitin-proteasome system (UPS) is crucial for the cell to adjust its protein degradation capacity to changing proteolytic requirements. We have shown previously that mammalian cells upregulate proteasome gene expression in response to proteasome inhibition. Here, we report the identification of the transcription factor TCF11 (long isoform of Nrf1) as a key regulator for 26S proteasome formation in human cells to compensate for reduced proteolytic activity. Under noninducing conditions, TCF11 resides in the endoplasmic reticulum (ER) membrane. There, TCF11 is targeted to ER-associated protein degradation requiring the E3 ubiquitin ligase HRD1 and the AAA ATPase p97. Proteasome inhibitors trigger the accumulation of oxidant-damaged proteins and promote the nuclear translocation of TCF11 from the ER, permitting activation of proteasome gene expression by binding to antioxidant response elements in their promoter regions. Thus, we uncovered the transcriptional control loop regulating human proteasome-dependent protein degradation to counteract proteotoxic stress caused by proteasome inhibition.

Feedback regulation of proteasome gene expression and its implications in cancer therapy

Proteasomal protein degradation is one of the major regulatory mechanisms in the cell. Aberrant proteasome activity is directly related to the pathogenesis of many human diseases including cancers. How proteasome homeostasis is controlled is a fundamental question toward our understanding of proteasome dysregulation in cancer cells. The recent discovery of the Rpn4-proteasome negative feedback circuit provides mechanistic insight into the regulation of proteasome gene expression. This finding also has important implications in cancer therapy that uses small molecule inhibitors to target the proteasome.

Allicin-induced global gene expression profile of Saccharomyces cerevisiae

To understand the response mechanisms of fungus cells upon exposure to the natural fungicide allicin, we performed commercial oligonucleotide microarrays to determine the overall transcriptional response of allicin-treated Saccharomyces cerevisiae strain L1190. Compared with the transcriptional profiles of untreated cultures, 147 genes were significantly upregulated, and 145 genes were significantly downregulated in the allicin-treated cells. We interpreted the microarray data with the hierarchical clustering tool, T-profiler. Major transcriptional responses were induced by allicin and included the following: first, Rpn4p-mediated responses involved in proteasome gene expression; second, the Rsc1p-mediated response involved in iron ion transporter activity; third, the Gcn4p-mediated response, also known as general amino acid control; finally, the Yap1p-, Msn2/4p-, Crz1p-, and Cin5p-mediated multiple stress response. Interestingly, allicin treatment, similar to mycotoxin patulin and artificial fungicide thiuram treatment, was found to induce genes involved in sulfur amino acid metabolism and the defense system for oxidative stress, especially DNA repair, which suggests a potential mutagenicity for allicin. Quantitative real-time reverse transcription-polymerase chain reaction was performed for selected genes to verify the microarray results. To our knowledge, this is the first report of the global transcriptional profiling of allicin-treated S. cerevisiae by microarray.

Positive symptoms of psychosis correlate with expression of ubiquitin proteasome genes in peripheral blood

Several brain- and blood-based gene expression studies in patients with psychotic disorders (e.g., schizophrenia) have identified genes in the ubiquitin proteasome system (UPS) pathway as putative biomarkers. However, to date an examination of the UPS pathway in the broader context of symptom severity in psychosis has not been conducted. The purpose of this study was to investigate the correlation between clinical scores on the Scales for the Assessment of Positive and Negative Symptoms (SAPS-SANS) and expression of 43 highly annotated genes within the UPS pathway in blood from patients with psychosis. A sample of 19 psychotic patients diagnosed with schizophrenia (n = 13) or bipolar disorder (n = 6) were recruited. Pearson's partial correlations, adjusting for gender, ethnicity, age, education, medication, smoking, and past 6-month substance use, were performed between each of the selected UPS genes and both scales. Significant Bonferroni-adjusted positive associations were observed between SAPS scores and two ubiquitin conjugation genes (i.e., UBE2K, SIAH2), while a negative association was observed with one deubiquitination gene (i.e., USP2). No gene expression levels were significantly associated with scores on the SANS after correction for multiple testing. Our findings suggest that dysregulation of the UPS, specifically ubiquitin conjugation and deubiquitination, may point to a possible underlying biological mechanism for severity of positive but not negative symptoms.

Abstract 1318: Proteasome and Fanconi Anemia/BRCA pathways as prognostic determinants of glioblastoma response to radiation and temozolomide: a proposed clinical trial

Abstract OBJECTIVE: Despite the standard regimen of maximal resection followed by concurrent ionizing radiation (IR) and temozolomide (TMZ), long-term survivors of patients afflicted with glioblastoma (GBM) remain rare. We sought to identify molecular determinants of resistance to these agents using a combined functional genomic and bioinformatic approach. METHODS: Parallel RNAi screens were performed to identify gene silencings that modulate IR and TMZ resistance. A viability-based screen was performed in multiple glioma lines with the Hannon-Elledge library of 74,705 short hairpin RNAs targeting nearly all human genes. A focused screen was also performed to identify modulators of the Fanconi Anemia (FA)/BRCA pathway, a pathway we previously identified as a determinant of IR and TMZ resistance. The most promising candidates were confirmed by independent RNAi and pharmacologic inhibition in vitro and in vivo xenografts. Correlations between relative expression levels of the candidate genes and patient survival were made using data from seven series of clinically annotated GBM gene expression profiles. RESULTS: PSMA1, a core subunit of the 20S proteasome, was a top hit in both screens. Proteasome inhibition by either RNAi or pharmacologic inhibition sensitized GBM cell lines to IR and TMZ in both in vitro and in vivo xenograft models. This inhibition also resulted in decreased FANCD2 monoubiquitination, a marker for FA pathway activation. Using our collated set of 531 patients, we observed that patients with low (< mean) PSMA1 showed higher OS vs. those with high expression, median OS = 14.8 vs. 12.2 months (log-rank P = 0.017). This correlation was recapitulated with several other proteasome genes. Since recent studies revealed distinct GBM subtypes, we tested whether the prognostic value of proteasome gene expression was subtype-specific. We found that the difference was only significant in the proneural subtype, median OS = 16.5 vs. 9.4 mo. (P = 0.012). Realizing that proteasome inhibition exerts phenotypes beyond FA pathway inhibition, we determined the extent to which FA/BRCA expression impacts prognosis. High FA/BRCA expression correlated with lower OS only for proneural tumors (P = 0.015); this observation suggested that proteasome function impacts tumor survival, in large part, by modulating the FA/BRCA pathway. CONCLUSION: Proteasome inhibition sensitizes GBM cells to IR and TMZ and results in impaired FA pathway activation. Our combined functional genomic and bioinformatic analysis suggests a clinical trial: patients with proneural gliomas with high FA/BRCA expression should be treated with proteasome inhibitors such as bortezomib to augment standard therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1318.

Identification of proteasome gene regulation in a rat model for HIV protease inhibitor-induced hyperlipidemia

Patients treated with highly active antiretroviral therapy may develop metabolic side effects such as hyperlipidemia, insulin resistance, lipoatrophy and lactic acidosis. The pathophysiology of these metabolic abnormalities is unknown, although some, e.g., lactic acidosis and lipoatrophy, are more associated with nucleoside use while protease inhibitors (PIs) have been shown to contribute to hyperlipidemia and insulin resistance. Identifying new PIs that are not associated with dyslipidemia has been hindered by the lack of mechanistic information and the unavailability of relevant animal models. In order to understand the molecular mechanism behind the hyperlipidemia associated with other protease inhibitors, and to develop a more effective, faster screen for compounds with this liability, we have analyzed expression profiles from PI-treated animals. Previously, we have shown that treatment of rats with ritonavir results in increases in the expression of proteasomal subunit genes in the liver. We show this increase is similar in rats treated with bortezomib, a proteasome inhibitor. In addition, we have treated rats with additional protease inhibitors, including atazanavir, which is associated with lower rates of lipid elevations in the clinic when administered in the absence of ritonavir. Our results indicate a strong correlation between proteasomal induction and lipid elevations, and have allowed us to develop a rapid screen for identifying novel PIs that do not induce the proteasome.

Refining current knowledge on the yeast FLR1 regulatory network by combined experimental and computational approaches

Multidrug resistance is often the result of the activation of drug efflux pumps able to catalyze the extrusion of the toxic compound to the outer medium, this activation being frequently controlled at the transcriptional level. Transcriptional regulation in the model eukaryote S. cerevisiae is the result of the interaction and cross-talk between networks of transcription factors. This is the case of the transcriptional activation of the FLR1 gene occurring in response to stress induced by the agricultural fungicide mancozeb in yeast. FLR1 up-regulation depends on the integrated action of Yap1, a key regulator of oxidative stress response, Pdr3 and Yrr1, two of the transcription factors controlling multidrug resistance, and Rpn4, a regulator of proteasome gene expression, which interplay to produce the observed transcriptional up-shift. Based on the expression profiles of FLR1, YAP1, PDR3, YRR1 and RPN4 registered during yeast adaptation to stress induced by mancozeb and using a qualitative modeling approach, a model of the FLR1 regulatory network was built, and the response of S. cerevisiae to mancozeb stress was simulated. The use of a qualitative approach is especially useful to overcome the lack of enough quantitative data on kinetic parameters and molecular concentrations, permitting the immediate focus on the qualitative behavior of the system. This Systems Biology approach allowed the identification of essential features of the early yeast response to fungicide stress. The resulting model allowed the formulation of new hypotheses, in a quick and cost effective manner, on the qualitative behavior of the system following mancozeb challenge, some of which were validated experimentally. In particular, Pdr3 and Yrr1 were shown to directly control FLR1 up-regulation in mancozeb-challenged cells, based on the analysis of the effect of the inactivation of their putative binding sites in the FLR1 promoter. Furthermore, the inter-dependent role of Yap1 and Yrr1 in the regulation of PDR3 and RPN4 was brought to light, this joint activity possibly being extensible to eight other genes involved in multidrug resistance. The FLR1 network structure was revised, based on the comparison between simulated and experimental gene expression data in the double deletion mutant strains Δyrr1Δpdr3 and Δyrr1Δrpn4, and an additional, still unidentified, transcription factor was found to be required to fully explain the behavior of the network.

Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation

BackgroundIntestinal barrier function is important for preserving health, as a compromised barrier allows antigen entry and can induce inflammatory diseases. Probiotic bacteria can play a role in enhancing intestinal barrier function; however, the mechanisms are not fully understood. Existing studies have focused on the ability of probiotics to prevent alterations to tight junctions in disease models, and have been restricted to a few tight junction bridging proteins. No studies have previously investigated the effect of probiotic bacteria on healthy intestinal epithelial cell genes involved in the whole tight junction signalling pathway, including those encoding for bridging, plaque and dual location tight junction proteins. Alteration of tight junction signalling in healthy humans is a potential mechanism that could lead to the strengthening of the intestinal barrier, resulting in limiting the ability of antigens to enter the body and potentially triggering undesirable immune responses.ResultsThe effect of Lactobacillus plantarum MB452 on tight junction integrity was determined by measuring trans-epithelial electrical resistance (TEER) across Caco-2 cell layers. L. plantarum MB452 caused a dose-dependent TEER increase across Caco-2 cell monolayers compared to control medium. Gene expression was compared in Caco-2 cells untreated or treated with L. plantarum MB452 for 10 hours. Caco-2 cell RNA was hybridised to human oligonucleotide arrays. Data was analysed using linear models and differently expressed genes were examined using pathway analysis tools. Nineteen tight junction-related genes had altered expression levels in response to L. plantarum MB452 (modified-P < 0.05, fold-change > 1.2), including those encoding occludin and its associated plaque proteins that anchor it to the cytoskeleton. L. plantarum MB452 also caused changes in tubulin and proteasome gene expression levels which may be linked to intestinal barrier function. Caco-2 tight junctions were visualised by fluorescent microscopy of immuno-stained occludin, zona occludens (ZO)-1, ZO-2 and cingulin. Caco-2 cells treated with L. plantarum MB452 had higher intensity fluorescence of each of the four tight junction proteins compared to untreated controls.ConclusionsThis research indicates that enhancing the expression of genes involved in tight junction signalling is a possible mechanism by which L. plantarum MB452 improves intestinal barrier function.

Expression regulation of the proteasomal genes in eukaryotes

The ubiquitin-proteasome system (UPS) is involved in the degradation of many intracellular proteins and is necessary for a proper functioning of the cell under normal conditions and its survival in stress. This review considers the general principles of the structure and functioning of the UPS. Main attention is paid to the expression regulation of the proteasomal genes in yeasts and structural and functional analyses of the Rpn4p transcription factor, which activates the proteasomal genes in Saccharomyces cerevisiae. Data on the regulation of proteasomal gene expression in higher eukaryotes are discussed.

Changes in the Expression of Proteasome Genes in Tumor Cells Following Short-Term Proteasome Inhibitor Therapy Predicts Survival in Multiple Myeloma Treated with Bortezomib-Containing Multi-Agent Chemotherapy

Introduction: The mechanism of action and resistance to chemotherapy is poorly understood and measures of efficacy typically rely on clinical outcome data. Recent advances suggest that prospective gene expression profiling (GEP) can be used to more accurately define not only short-term but lasting treatment benefits. We recently reported that baseline tumor cell gene signatures, encompassing 70 and as few as 17 genes, can discriminate risk groups of myeloma patients both in the untreated and previously treated settings. However, a subset of predicted low-risk cases followed an aggressive clinical course accompanied by a shift from 70-gene-defined low-to high-risk over time, either reflecting clonal evolution or outgrowth of aggressive clones present, but undetectable, at diagnosis. Accurately identifying this patient population is a first step in preempting such transformation. We hypothesized that changes in gene expression patterns of tumor cells following a short term in-vivo challenge with a single agent chemotherapeutic might expose these latently aggressive cells. Unlike in-vitro testing, clinical drug administration also allows for assessing tumor cell perturbation in the context of host interactions. Building on recent observations that clinical outcome in myeloma patients could be correlated with 48hr GEP changes induced in vivo following single agent administration of thalidomide, lenalidomide, and dexamethasone, we now examined whether such short-term tumor-cell GEP and proteomic alterations could fine-tune clinical outcome prediction beyond the well established 70-gene-based baseline prediction model.Methods: Affymetrix U133Plus2.0 microarray analysis and mass spectrometry were performed on CD138-enriched tumor cells prior to and 48hr following a single test-dose application of bortezomib at 1.0mg/m2 in 142 newly diagnosed patients with MM. A total of 1051 genes (P < .005) were differentially expressed at 48 hours. Both change in expression, adjusting for baseline expression, and post-drug expression levels of each gene were examined for correlations with event-free survival in a Cox proportional hazards model. Post-drug expression was chosen and 113 genes were retained (p <= .05). The difference of the mean log2 expression of genes with hazard ratios (HR) of < 1.0 (favorable) and genes with HR >=1 (unfavorable) was used to create a score which, in the context of running log rank statistics, was used to classify patients into high- and low-risk groups. The independent prognostic power of the score for event-free and overall survival was investigated, together with baseline prognostic variables, by multivariate analysis. This method was tested in a 10-fold cross-validation procedure using the same data set. The model is currently being validated in an independent set of 100 cases and results will be reported.Results: Changes in the expression of proteasome genes, and their related proteins, predominated a list of 113 outcome-related genes. A high-risk score, associated with upregulation of proteasome genes, seen in 24% of cases, was associated with median survival of less than 24 months, dramatically contrasting with a 3-year survival estimate of greater than 80% in the 76% in whom proteasome genes were not activated (p<0.0001). The cross-validated post-bortezomib score was an independent predictor of outcome in multivariate analysis of standard and genetic variables, including the well established and validated baseline 70-gene risk score. Importantly, 12% of patients in the 70-gene model-defined low-risk category were upgraded to high-risk by the 113-gene post-bortezomib model, with poor outcomes resembling those in the 70-gene-defined high-risk baseline model. Moreover, the 113-gene post-bortezomib score alone accounted for an unprecedented 57% of outcome variability by R2 statistics, with hazard ratio values of 5.45 for overall and 7.84 for event-free survival.Conclusions: The rapid activation of proteasome genes and their corresponding proteins in MM cells within 48hr of a single bortezomib test-dose exposure as an indicator of poor clinical outcome suggests a novel and perhaps central mechanism of resistance to this new class of cancer therapeutics and perhaps standard genotoxic agents, which were part of the overall treatment. We are now testing the hypothesis whether the high-risk associated with the post-bortezomib proteasome activation can be overcome by higher doses of bortezomib or the addition of agents targeting other critical pathways.

Yeast adaptation to mancozeb involves the up-regulation of FLR1 under the coordinate control of Yap1, Rpn4, Pdr3, and Yrr1

FLR1 gene, encoding a multidrug resistance (MDR) transporter of the major facilitator superfamily (MFS) was found to confer resistance to the fungicide mancozeb in Saccharomyces cerevisiae. This agrochemical has been linked to the development of Parkinson disease and cancer. Yeast response to mancozeb was proved to involve the strong activation of FLR1 transcription (20-fold) during the fungicide-induced growth latency. This activation of FLR1 transcription is fully dependent on Yap1p and is reduced (by 50%) in the absence of Rpn4p, Yrr1p or Pdr3p. A model for the coordinate action over FLR1 transcription activation, in response to mancozeb, of these transcription factors that mediate oxidative stress response (Yap1p), proteasome gene expression (Rpn4p), and pleiotropic drug resistance (Pdr3p and Yrr1p), is proposed.

Modifications in endopeptidase and 20S proteasome expression and activities in cadmium treated tomato (Solanum lycopersicum L.) plants

The effects of cadmium (Cd) on cellular proteolytic responses were investigated in the roots and leaves of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 3 and 10 days in the presence of 0.3-300 microM Cd and compared to control plants grown in the absence of Cd. Roots of Cd treated plants accumulated four to fivefold Cd as much as mature leaves. Although 10 days of culture at high Cd concentrations inhibited plant growth, tomato plants recovered and were still able to grow again after Cd removal. Tomato roots and leaves are not modified in their proteolytic response with low Cd concentrations (< or =3 microM) in the incubation medium. At higher Cd concentration, protein oxidation state and protease activities are modified in roots and leaves although in different ways. The soluble protein content of leaves decreased and protein carbonylation level increased indicative of an oxidative stress. Conversely, protein content of roots increased from 30 to 50%, but the amount of oxidized proteins decreased by two to threefold. Proteolysis responded earlier in leaves than in root to Cd stress. Additionally, whereas cysteine- and metallo-endopeptidase activities, as well as proteasome chymotrypsin activity and subunit expression level, increased in roots and leaves, serine-endopeptidase activities increased only in leaves. This contrasted response between roots and leaves may reflect differences in Cd compartmentation and/or complexation, antioxidant responses and metabolic sensitivity to Cd between plant tissues. The up-regulation of the 20S proteasome gene expression and proteolytic activity argues in favor of the involvement of the 20S proteasome in the degradation of oxidized proteins in plants.

Genomic Evolution of the Proteasome System Among Hemiascomycetous Yeasts

Components of the proteasome-ubiquitin pathway are highly conserved throughout eukaryotic organisms. In S. cerevisiae, the expression of proteasomal genes is subject to concerted control by a transcriptional regulator, Rpn4p, interacting with a highly conserved cis-regulatory element, PACE, located in the upstream regions of these genes. Taking advantage of sequence data accumulated from 15 Hemiascomycetes, we performed an in silico study to address the problem of how this system might have evolved among these species. We found that in all these species the Rpn4p homologues are well conserved in terms of sequence and characteristic domain features. The "PACE patterns" turned out to be nearly identical among the Saccharomyces "sensu stricto" species, whereas in the evolutionary more distant species the putatively functional cis-regulatory motifs revealed deviations from the "canonical" PACE nonamere sequence in one or two nucleotides. Our findings suggest that during evolution of the Hemiascomycetes such slightly divergent ancestral motifs have converged into a unique PACE element for the majority of the proteasomal genes within the most recent species of this class. Likewise, the Rpn4 factors within the most recent species of this class show a higher degree of similarity in sequence than their ancestral counterparts. By contrast, we did not detect PACE-like motifs among the proteasomal genes in other eukaryotes, such as S. pombe, several filamentous fungi, A. thaliana, or humans, leaving the interesting question which type of concerted regulation of the proteasome system has developed in species other than the Hemiascomycetes.

Gene expression profiling of rat liver reveals a mechanistic basis for ritonavir-induced hyperlipidemia

The molecular mechanisms of action of a HIV protease inhibitor, ritonavir, on hepatic function were explored on a genomic scale using microarrays comprising genes expressed in the liver of Sprague–Dawley rats ( Rattus norvegicus). Analyses of hepatic transcriptional fingerprints led to the identification of several key cellular pathways affected by ritonavir treatment. These effects were compared to a compendium of gene expression responses for 52 unrelated compounds and to other protease inhibitors, including atazanavir and two experimental compounds. We identified genes involved in cholesterol and fatty acid biosynthesis, as well as genes involved in fatty acid and cholesterol breakdown, whose expressions were regulated in opposite manners by ritonavir and bezafibrate, a hypolipidemic agonist of the peroxisome proliferator-activated receptor α. Ritonavir also upregulated multiple proteasomal subunit transcripts as well as genes involved in ubiquitination, consistent with its known inhibitory effect on proteasomal activity. We also tested three other protease inhibitors in addition to ritonavir. Atazanavir did not impact ubiquitin or proteasomal gene expression, although the two other experimental protease inhibitors impacted both proteasomal gene expression and sterol regulatory element-binding protein-activated genes, similar to ritonavir. Identification of key metabolic pathways that are affected by ritonavir and other protease inhibitors will enable us to understand better the downstream effects of protease inhibitors, thus leading to better drug design and an effective method to mitigate the side effects of this important class of HIV therapeutics.

Prostaglandin J2 Alters Pro-survival and Pro-death Gene Expression Patterns and 26 S Proteasome Assembly in Human Neuroblastoma Cells

Many neurodegenerative disorders are characterized by two pathological hallmarks: progressive loss of neurons and occurrence of inclusion bodies containing ubiquitinated proteins. Inflammation may be critical to neurodegeneration associated with ubiquitin-protein aggregates. We previously showed that prostaglandin J2 (PGJ2), one of the endogenous products of inflammation, induces neuronal death and the accumulation of ubiquitinated proteins into distinct aggregates. We now report that temporal microarray analysis of human neuroblastoma SK-N-SH revealed that PGJ2 triggered a "repair" response including increased expression of heat shock, protein folding, stress response, detoxification and cysteine metabolism genes. PGJ2 also decreased expression of cell growth/maintenance genes and increased expression of apoptotic genes. Over time pro-death responses prevailed over pro-survival responses, leading to cellular demise. Furthermore, PGJ2 increased the expression of proteasome and other ubiquitin-proteasome pathway genes. This increase failed to overcome PGJ2 inhibition of 26 S proteasome activity. Ubiquitinated proteins are degraded by the 26 S proteasome, shown here to be the most active proteasomal form in SK-N-SH cells. We demonstrate that PGJ2 impairs 26 S proteasome assembly, which is an ATP-dependent process. PGJ2 perturbs mitochondrial function, which could be critical to the observed 26 S proteasome disassembly, suggesting a cross-talk between mitochondrial and proteasomal impairment. In conclusion neurotoxic products of inflammation, such as PGJ2, may play a role in neurodegenerative disorders associated with the aggregation of ubiquitinated proteins by impairing 26 S proteasome activity and inducing a chain of events that culminates in neuronal cell death. Temporal characterization of these events is relevant to understanding the underlying mechanisms and to identifying potential early biomarkers.

In vivo changes in gene expression profiles (GEP) after bortezomib (V) for multiple myeloma (MM): Differential effects on plasma cells (PC) and micro-environment (ME)

7603 Introduction: V, a proteasome inhibitor with potent anti-myeloma activity, is thought to act through effects on malignant PC and the ME. However, the in vivo effects of this drug have never been examined at the molecular level. Methods: Serial GEP analyses were performed of paired purified PC and bone marrow biopsies from 46 patients, obtained prior to and 48hr following administration of a single V dose at 1mg/m2. RNA was isolated from whole biopsies and purified PCs from each sample, converted to cRNA, and hybridized to Affymetrix U133 Plus2 microarrays. SAM analysis with 5% false discovery rate was employed to identify drug-altered genes. Results: Following V, 36 ME-associated genes (MAGs) were up-regulated, among them the osteoinductive factor osteoglycin (OGN), consistent with a V effect on osteoblastogenesis; CYR61, an angiogenesis inducer, significantly over-expressed in myeloma relative to normal marrow, was one of only 3 significantly down-regulated genes. V is known to induce expression of proteasome genes and PSMA6, PSMA1, and PSMA14 were among 15 genes up-regulated in PC. In contrast, early growth response (EGR1, EGR2, and EGR3), Krupple-like factor (KLR4, KLR5, KLR6, and KLR7) and nuclear receptor (NR4A1, NR4A2, and NR4A3) family members were down-regulated in PC by V. These differential PC and ME expression changes were only noted in low-risk MM, lacking over-expression of CKS1B. Conclusion: We report here, for the first time, on the differential molecular consequences of a single in-vivo dose of V on both tumor cells and cells of the microenvironment. The clinical implications of these findings are being further investigated and will be presented. [Table: see text]

Identification of gene expression changes in transgenic C. elegans overexpressing human α-synuclein

Alpha-synuclein containing cellular inclusions are a hallmark of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. A genome wide expression screen was performed in C. elegans overexpressing both wild-type and A53T human alpha-synuclein. 433 genes were up- and 67 genes down-regulated by statistical and fold change (> or <2) criteria. Gene ontology (GO) categories within the regulated gene lists indicated over-representation of development and reproduction, mitochondria, catalytic activity, and histone groups. Seven genes (pdr-1, ubc-7, pas-5, pas-7, pbs-4, RPT2, PSMD9) with function in the ubiquitin-proteasome system and 35 mitochondrial function genes were up-regulated. Nine genes that form histones H1, H2B, and H4 were down-regulated. These results demonstrate the effects of alpha-synuclein on proteasome and mitochondrial complex gene expression and provide further support for the role of these complexes in mediating neurotoxicity. The results also indicate an effect on nuclear protein genes that suggests a potential new avenue for investigation.

Gankyrin: a new oncoprotein and regulator of pRb and p53

Gankyrin is a new oncoprotein with potent cell cycle and apoptotic properties that is overexpressed early in hepatocarcinogenesis and in hepatocellular carcinomas. Gankyrin regulates the phosphorylation of the retinoblastoma protein (pRb) by CDK4 and enhances the ubiquitylation of p53 by the RING ubiquitin ligase MDM2. Purified preparations of the 26S proteasome contain gankyrin, which specifically interacts with the S6b (Rpt3) ATPase of the 19S regulator. In conclusion, gankyrin is a small versatile cell cycle regulator that illustrates the essential interplay between the ubiquitin proteasome system and gene expression in the cell. Here, we discuss the activities of gankyrin and present a model for its function in the regulation of pRb and p53.

Regulation of the Neuronal Proteasome by Zif268 (Egr1)

Most forms of neuronal plasticity are associated with induction of the transcription factor Zif268 (Egr1/Krox24/NGF-IA). In a genome-wide scan, we obtained evidence for potential modulation of proteasome subunit and regulatory genes by Zif268 in neurons, a finding of significance considering emerging evidence that the proteasome modulates synaptic function. Bioinformatic analysis indicated that the candidate proteasome Zif268 target genes had a rich concentration of putative Zif268 binding sites immediately upstream of the transcriptional start sites. Regulation of the mRNAs encoding the psmb9 (Lmp2) and psme2 (PA28beta) proteasome subunits, along with the proteasome-regulatory kinase serum/glucocorticoid-regulated kinase (SGK) and the proteasome-associated antigen peptide transporter subunit 1 (Tap1), was confirmed after transfection of a neuronal cell line with Zif268. Conversely, these mRNAs were upregulated in cerebral cortex tissue from Zif268 knock-out mice relative to controls, confirming that Zif268 suppresses their expression in the CNS. Transfected Zif268 reduced the activity of psmb9, SGK, and Tap1 promoter-reporter constructs. Altered psmb9, SGK, and Tap1 mRNA levels were also observed in an in vivo model of neuronal plasticity involving Zif268 induction: the effect of haloperidol administration on striatal gene expression. Consistent with these effects on proteasome gene expression, increased Zif268 expression suppressed proteasome activity, whereas Zif268 knock-out mice exhibited elevated cortical proteasome activity. Our findings reveal that Zif268 regulates the expression of proteasome and related genes in neuronal cells and provide new evidence that altered expression of proteasome activity after Zif268 induction may be a key component of long-lasting CNS plasticity.