Encoding Proteasome Subunits Research Articles

Proteasome gene expression is controlled by coordinated functions of multiple transcription factors.

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 days after denervation), it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g., type-2 diabetes, cancer).

Co-expression analysis of transcriptomic data from cancer and healthy specimens reveals rewiring of proteasome genes and an interaction with the XPO1 gene across several tumour types

BackgroundThe 26S proteasome is a large intracellular multiprotein complex, that plays a homeostatic role by degrading proteins that have been tagged by ubiquitin. It is composed of 64 subunits assembled according to a well-defined structure and stoichiometry. Several proteasome subunits have been found to be overexpressed in tumours. However, comprehensive data are lacking on the relative abundance of each subunit and the impact on proteasome composition or stoichiometry. In cancer treatment, proteasome inhibitors and inhibitors of XPO1 (Exportin-1) have unexpectedly a similar range of activity, but the interaction between the two pathways has not been studied.MethodsWe performed gene co-expression analysis of 38 genes encoding proteasome subunits and 38 genes encoding proteins involved in nucleocytoplasmic transport in specimens from the Cancer Genome Atlas (33 tumour types) and from the Gene Tissue Expression database (32 healthy tissue types). We obtained 65 matrices, each containing Pearson correlation factors for 2964 gene pairs. We applied cluster analysis to the correlation matrices and compared the distribution of Pearson correlation coefficients of thirteen tumour types with their healthy tissue counterpart.ResultsStrong positive correlation (R Pearson correlation > 0.8) was observed for pairs of proteasome genes in the majority of healthy tissues, whereas the correlation for co-expression was significantly lower (R ≤ 0.50) for most gene pairs in the majority of cancer types. Cluster analysis based on gene co-expression allowed to distinguish cancers from healthy tissues in a clear-cut manner, and to identify the genes that contributed most to the separation. The crossed analysis between proteasome and nucleocytoplasmic transport genes showed that the expression of XPO1 and a subset of proteasome genes, including in particular PSMD14, is correlated in several cancer types and not in their healthy counterpart.ConclusionsThis analysis reveals that in cancer the co-expression of proteasome genes is significantly altered, highlighting the genes that are more often deregulated. In addition, it finds that XPO1 expression is often correlated with the expression of proteasome genes. From a therapeutic perspective, these findings support the investigation of novel targets within the proteasome and strategies of co-targeting of the proteasome and nucleocytoplasmic transport.

The histone acetyl transferases CBP and p300 regulate stress response pathways in synovial fibroblasts at transcriptional and functional levels

The activation of stress response pathways in synovial fibroblasts (SF) is a hallmark of rheumatoid arthritis (RA). CBP and p300 are two highly homologous histone acetyl transferases and writers of activating histone 3 lysine 27 acetylation (H3K27ac) marks. Furthermore, they serve as co-factors for transcription factors and acetylate many non-histone proteins. Here we showed that p300 but not CBP protein expression was down regulated by TNF and 4-hydroxynonenal, two factors that mimic inflammation and oxidative stress in the synovial microenvironment. We used existing RNA-sequencing data sets as a basis for a further in-depth investigation of individual functions of CBP and p300 in regulating different stress response pathways in SF. Pathway enrichment analysis pointed to a profound role of CBP and/ or p300 in regulating stress response-related gene expression, with an enrichment of pathways associated with oxidative stress, hypoxia, autophagy and proteasome function. We silenced CBP or p300, and performed confirmatory experiments on transcriptome, protein and functional levels. We have identified some overlap of CBP and p300 target genes in the oxidative stress response pathway, however, with several genes being regulated in opposite directions. The majority of stress response genes was regulated by p300, with a specific function of p300 in regulating hypoxia response genes and genes encoding proteasome subunits. Silencing of p300 suppressed proteasome enzymatic activities. CBP and p300 regulated autophagy on transcriptome and functional levels. Whereas CBP was indispensable for autophagy synthesis, silencing of p300 affected late-stage autophagy. In line with impaired autophagy and proteasome function, poly-ubiquitinated proteins accumulated after silencing of p300.

Suppression of protein quality control systemby TRIM30a sensitises tumour cells to NK cell-mediated immune surveillance.

Tumorigenesis entails circumventing cell-intrinsic regulatory mechanisms while avoiding extrinsic immune surveillance and other host defence systems. Nevertheless, how tumour cells' ability to eliminate misfolded proteins affects immune surveillance remains poorly understood. In this study, we find that overexpression of murine tripartite motif-containing protein 30a (TRIM30a) sensitises tumour cells to natural killer (NK) cells-mediated cytolysis. TRIM30a has no effect on tumour cell proliferation or apoptosis in vitro. However, TRIM30a-overexpressing tumour cells grow substantially slower than control tumour cells in immune-competent mice but not in NK cell-depleted mice. [Correction added on 04 October 2023, after first online publication: 'NK-depleted' has been changed to 'NK cell-depleted' in the preceding sentence.] Mechanistically, TRIM30a overexpression impedes the clearance of misfolded proteins and increases the production of reactive oxygen species induced by proteotoxic stress, implying that TRIM30a impairs protein quality control (PQC) systems in tumour cells. Furthermore, TRIM30a reduces expression of genes encoding proteasome subunits and antioxidant proteins. Our study demonstrates that TRIM30a is a potential tumour suppressor and immune modulator that promotes tumour cytolysis by NK cells, and suggests that an enhanced PQC and antioxidant capacity is an integral part of the immune escape mechanism during tumorigenesis.

Analytical screening of polymorphic variants of 20S proteasome genes when planning a study of pathogenetic effects of modification of NFKB1 post-translational processing

Aim. Formation of polymorphic variants panel of the proteasome genes 20S, potentially significant for the study as balance modifier factors of p105/p50 NFKB1.Materials and methods. Determination of genes that encode proteins of the multisubunit proteasome complex prospective for research purposes, was carried out on the basis of information retrieved from eLIBRARY and PubMed. The source of information for the formation of polymorphic variants panel of genes (SNP, single nucleotide polymorphism) was the Ensembl genomic browser, http://www.ensembl.org. The structure of genes is described by the NCBI (databases Gene, http:// www.ncbi.nlm.nih.gov/gene). The panel was filled with the minor allelic frequency in the population (MAF), the localization of SNP in the gene structure and the availability of data on the relationship with multifactorial diseases and other effects in mind. To calculate the genetic distances between populations, we used the methord of comparing the populations by frequencies of polymorphic marker alleles proposed by Ney, the obtained matrices are illustrated by the method of multidimensional scaling in space using Statistica v.8.0.Results. Discussion of the algorithm and results of analytical screening of polymorphic variants of 14 genes (PSMA1-PSMA7, PSMB1–PSMB7) encoding proteasome subunits 20S. The characteristics of the SNP panel are given, compiled with the selection criteria taken into account. According to the data on the frequencies of polymorphic gene variants, the features of global and European population gene pools (283 SNP), as well as samples from Russian populations (20 SNP) are analyzed. Based on the results of the analysis of information on the associations of selected SNPs with various diseases, a panel (42 SNPs) of 20S proteasome genes was formed, potentially significant for the study as factors modifying the p105/p50 NFKB1 balance.Conclusion. Annotation of the formed panel of SNP genes of the 20S proteasome with MAF>0.1 indicates the potential role of polymorphism in the pathogenesis of diseases of various profiles. This may be of research interest to the formed panel in context of implementation of traditional approaches – the search for candidate genes based on the analysis of associations with diseases, as well as the analysis of the influence of SNP on the level of genetic expression, synthesis of gene products, NFKB1 processing and p105/p50 balance in silico and on model objects.

102P Co-expression analysis of genes encoding proteasome subunits and XPO1-related proteins in the Cancer Genome Atlas (TCGA) and the Gene Tissue Expression (GTEx) databases as a tool to devise new treatment strategies

102P Co-expression analysis of genes encoding proteasome subunits and XPO1-related proteins in the Cancer Genome Atlas (TCGA) and the Gene Tissue Expression (GTEx) databases as a tool to devise new treatment strategies

Circadian transcriptional pathway atlas highlights a proteasome switch in intermittent fasting

While intermittent fasting is a safe strategy to benefit health, it remains unclear whether a "timer" exists invivo to record fasting duration and trigger a transcriptional switch. Here, we map a circadian transcriptional pathway atlas from 600 samples across four metabolic tissues of mice under five feeding regimens. Results show that 95.6% of detected canonical pathways are rhythmic in a tissue-specific and feeding-regimen-specific manner, while only less than 25% of them induce changes in transcriptional function. Fasting for 16h initiates a circadian resonance of 43 pathways in the liver, and the resonance punctually switches following refeeding. The hepatic proteasome coordinates the resonance, and most genes encoding proteasome subunits display a 16-h fasting-dependent transcriptional switch. These findings indicate that the hepatic proteasome may serve as a fasting timer and a coordinator of pathway transcriptional resonance, which provide a target for revealing the underlying mechanism of intermittent fasting.

Proteasome activity modulates amyloid toxicity.

Alzheimer's disease (AD) is responsible for 60%-80% of identified cases of dementia. While the generation and accumulation of amyloid precursor protein (APP) fragments is accepted as a key step in AD pathogenesis, the precise role of these fragments remains poorly understood. To overcome this deficit, we induced the expression of the soluble C-terminal fragment of APP (C99), the rate-limiting peptide for the generation of amyloid fragments, in yeast that contain thermosensitive mutations in genes encoding proteasome subunits. Our previous work with this system demonstrated that these proteasome-deficient yeast cells, expressing C99 when proteasome activity was blunted, generated amyloid fragments similar to those observed in AD patients. We now report the phenotypic repercussions of inducing C99 expression in proteasome-deficient cells. We show increased levels of protein aggregates, cellular stress and chaperone expression, electron-dense accumulations in the nuclear envelope/ER, abnormal DNA condensation, and an induction of apoptosis. Taken together, these findings suggest that the generation of C99 and its associated fragments in yeast cells with compromised proteasomal activity results in phenotypes that may be relevant to the neuropathological processes observed in AD patients. These data also suggest that this yeast model should be useful for testing therapeutics that target AD-associated amyloid, since it allows for the assessment of the reversal of the perturbed cellular physiology observed when degradation pathways are dysfunctional.

Structures of the Mouse Central Nervous System Contain Different Quantities of Proteasome Gene Transcripts

Proteasomes are multisubunit complexes that degrade most intracellular proteins. Three of the 14 subunits of the 20S proteasome, specifically β1, β2, and β5, demonstrate catalytic activity and hydrolyze peptide bonds after acidic, basic, and hydrophobic amino acids, respectively. Within proteasome, the constitutive catalytic subunits β1, β2, and β5 can be substituted by the immune βli, β2i, and β5i subunits, respectively. However, proteasomes do not always contain all the immune subunits at once; some proteasomes contain both immune and constitutive catalytic subunits simultaneously. Incorporation of immune subunits modifies the pattern of peptides produced by proteasomes. This is essential for antigen presentation and cellular response to stress as well as for a number of intracellular signaling pathways. We have developed a quantitative PCR-based system for the determination of the absolute levels of murine constitutive and immune proteasome subunits gene expression. Using the obtained system, we have estimated the expression levels of genes encoding proteasome subunits in the mouse central nervous system (CNS) tissues. We have shown that the quantity of transcripts of proteasome catalytic subunits in different CNS structures differed significantly. These data allow us to assume that the studied brain regions can be divided into two groups, with relatively "high" (cerebral cortex and spinal cord) and "low" (hippocampus and cerebellum) levels of proteasome subunit genes expression. Moreover, it was possible to distinguish structures with similar and significantly different gene expression profiles of proteasome catalytic subunits. Thus, the gene expression profiles in the cortex, spinal cord, and cerebellum were similar, but different from the expression profile in the hippocampus. Based on the obtained data, we suggest that there are differences in the proteasome pool, as well as in the functional load on the ubiquitin-proteasome system in different parts of the CNS.

PTEN status determines chemosensitivity to proteasome inhibition in cholangiocarcinoma

Patient-derived xenografts (PDXs) and PDX-derived cells (PDCs) are useful in preclinical research. We performed a drug screening assay using PDCs and identified proteasome inhibitors as promising drugs for cholangiocarcinoma (CCA) treatment. Furthermore, we determined that phosphate and tensin homology deleted on chromosome ten (PTEN) deficiency promotes protein synthesis and proteasome subunit expression and proteolytic activity, creating a dependency on the proteasome for cancer cell growth and survival. Thus, targeting the proteasome machinery with the inhibitor bortezomib inhibited the proliferation and survival of CCA cells lacking functional PTEN. Therapeutic evaluation of PDXs, autochthonous mouse models, and patients confirmed this dependency on the proteasome. Mechanistically, we found that PTEN promoted the nuclear translocation of FOXO1, resulting in the increased expression of BACH1 and MAFF BACH1 and MAFF are transcriptional regulators that recognize the antioxidant response element, which is present in genes encoding proteasome subunits. PTEN induced the accumulation and nuclear translocation of these proteins, which directly repressed the transcription of genes encoding proteasome subunits. We revealed that the PTEN-proteasome axis is a potential target for therapy in PTEN-deficient CCA and other PTEN-deficient cancers.

Molecular Insight Into the IRE1α-Mediated Type I Interferon Response Induced by Proteasome Impairment in Myeloid Cells of the Brain.

Proteostasis is critical for cells to maintain the balance between protein synthesis, quality control, and degradation. This is particularly important for myeloid cells of the central nervous system as their immunological function relies on proper intracellular protein turnover by the ubiquitin-proteasome system. Accordingly, disruption of proteasome activity due to, e.g., loss-of-function mutations within genes encoding proteasome subunits, results in systemic autoinflammation. On the molecular level, pharmacological inhibition of proteasome results in endoplasmic reticulum (ER) stress-activated unfolded protein response (UPR) as well as an induction of type I interferons (IFN). Nevertheless, our understanding as to whether and to which extent UPR signaling regulates type I IFN response is limited. To address this issue, we have tested the effects of proteasome dysfunction upon treatment with proteasome inhibitors in primary murine microglia and microglia-like cell line BV-2. Our data show that proteasome impairment by bortezomib is a stimulus that activates all three intracellular ER-stress transducers activation transcription factor 6, protein kinase R-like endoplasmic reticulum kinase and inositol-requiring protein 1 alpha (IRE1α), causing a full activation of the UPR. We further demonstrate that impaired proteasome activity in microglia cells triggers an induction of IFNβ1 in an IRE1-dependent manner. An inhibition of the IRE1 endoribonuclease activity significantly attenuates TANK-binding kinase 1-mediated activation of type I IFN. Moreover, interfering with TANK-binding kinase 1 activity also compromised the expression of C/EBP homologous protein 10, thereby emphasizing a multilayered interplay between UPR and type IFN response pathway. Interestingly, the induced protein kinase R-like endoplasmic reticulum kinase-activation transcription factor 4-C/EBP homologous protein 10 and IRE1-X-box-binding protein 1 axes caused a significant upregulation of proinflammatory cytokine interleukin 6 expression that exacerbates STAT1/STAT3 signaling in cells with dysfunctional proteasomes. Altogether, these findings indicate that proteasome impairment disrupts ER homeostasis and triggers a complex interchange between ER-stress sensors and type I IFN signaling, thus inducing in myeloid cells a state of chronic inflammation.

Contribution of the Unfolded Protein Response (UPR) to the Pathogenesis of Proteasome-Associated Autoinflammatory Syndromes (PRAAS)

Type I interferonopathies cover a phenotypically heterogeneous group of rare genetic diseases including the recently described proteasome-associated autoinflammatory syndromes (PRAAS). By definition, PRAAS are caused by inherited and/or de novo loss-of-function mutations in genes encoding proteasome subunits such as PSMB8, PSMB9, PSMB7, PSMA3, or proteasome assembly factors including POMP and PSMG2, respectively. Disruption of any of these subunits results in perturbed intracellular protein homeostasis including accumulation of ubiquitinated proteins which is accompanied by a type I interferon (IFN) signature. The observation that, similarly to pathogens, proteasome dysfunctions are potent type I IFN inducers is quite unexpected and, up to now, the underlying molecular mechanisms of this process remain largely unknown. One promising candidate for triggering type I IFN under sterile conditions is the unfolded protein response (UPR) which is typically initiated in response to an accumulation of unfolded and/or misfolded proteins in the endoplasmic reticulum (ER) (also referred to as ER stress). The recent observation that the UPR is engaged in subjects carrying POMP mutations strongly suggests its possible implication in the cause-and-effect relationship between proteasome impairment and interferonopathy onset. The purpose of this present review is therefore to discuss the possible role of the UPR in the pathogenesis of PRAAS. We will particularly focus on pathways initiated by the four ER-membrane proteins ATF6, PERK, IRE1-α, and TCF11/Nrf1 which undergo activation under proteasome inhibition. An overview of the current understanding of the mechanisms and potential cross-talk between the UPR and inflammatory signaling casacades is provided to convey a more integrated picture of the pathophysiology of PRAAS and shed light on potential biomarkers and therapeutic targets.

Starvation Induces Proteasome Autophagy with Different Pathways for Core and Regulatory Particles

The proteasome is responsible for the degradation of many cellular proteins. If and how this abundant and normally stable complex is degraded by cells is largely unknown. Here we show that in yeast, upon nitrogen starvation, proteasomes are targeted for vacuolar degradation through autophagy. Using GFP-tagged proteasome subunits, we observed that autophagy of a core particle (CP) subunit depends on the deubiquitinating enzyme Ubp3, although a regulatory particle (RP) subunit does not. Furthermore, upon blocking of autophagy, RP remained largely nuclear, although CP largely localized to the cytosol as well as granular structures within the cytosol. In all, our data reveal a regulated process for the removal of proteasomes upon nitrogen starvation. This process involves CP and RP dissociation, nuclear export, and independent vacuolar targeting of CP and RP. Thus, in addition to the well characterized transcriptional up-regulation of genes encoding proteasome subunits, cells are also capable of down-regulating cellular levels of proteasomes through proteaphagy.

Graded Proteasome Dysfunction in Caenorhabditis elegans Activates an Adaptive Response Involving the Conserved SKN-1 and ELT-2 Transcription Factors and the Autophagy-Lysosome Pathway.

The maintenance of cellular proteins in a biologically active and structurally stable state is a vital endeavor involving multiple cellular pathways. One such pathway is the ubiquitin-proteasome system that represents a major route for protein degradation, and reductions in this pathway usually have adverse effects on the health of cells and tissues. Here, we demonstrate that loss-of-function mutants of the Caenorhabditis elegans proteasome subunit, RPN-10, exhibit moderate proteasome dysfunction and unexpectedly develop both increased longevity and enhanced resistance to multiple threats to the proteome, including heat, oxidative stress, and the presence of aggregation prone proteins. The rpn-10 mutant animals survive through the activation of compensatory mechanisms regulated by the conserved SKN-1/Nrf2 and ELT-2/GATA transcription factors that mediate the increased expression of genes encoding proteasome subunits as well as those mediating oxidative- and heat-stress responses. Additionally, we find that the rpn-10 mutant also shows enhanced activity of the autophagy-lysosome pathway as evidenced by increased expression of the multiple autophagy genes including atg-16.2, lgg-1, and bec-1, and also by an increase in GFP::LGG-1 puncta. Consistent with a critical role for this pathway, the enhanced resistance of the rpn-10 mutant to aggregation prone proteins depends on autophagy genes atg-13, atg-16.2, and prmt-1. Furthermore, the rpn-10 mutant is particularly sensitive to the inhibition of lysosome activity via either RNAi or chemical means. We also find that the rpn-10 mutant shows a reduction in the numbers of intestinal lysosomes, and that the elt-2 gene also plays a novel and vital role in controlling the production of functional lysosomes by the intestine. Overall, these experiments suggest that moderate proteasome dysfunction could be leveraged to improve protein homeostasis and organismal health and longevity, and that the rpn-10 mutant provides a unique platform to explore these possibilities.

Conservation of the Nrf2-Mediated Gene Regulation of Proteasome Subunits and Glucose Metabolism in Zebrafish.

The Keap1-Nrf2 system is an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress. Besides the exogenous stress response, Nrf2 has been found to regulate numerous cellular functions, including protein turnover and glucose metabolism; however, the evolutionary origins of these functions remain unknown. In the present study, we searched for novel target genes associated with the zebrafish Nrf2 to answer this question. A microarray analysis of zebrafish embryos that overexpressed Nrf2 revealed that 115 candidate genes were targets of Nrf2, including genes encoding proteasome subunits and enzymes involved in glucose metabolism. A real-time quantitative PCR suggested that the expression of 3 proteasome subunits (psma3, psma5, and psmb7) and 2 enzymes involved in glucose metabolism (pgd and fbp1a) were regulated by zebrafish Nrf2. We thus next examined the upregulation of these genes by an Nrf2 activator, diethyl maleate, using Nrf2 mutant zebrafish larvae. The results of real-time quantitative PCR and whole-mount in situ hybridization showed that all of these 5 genes were upregulated by diethyl maleate treatment in an Nrf2-dependent manner, especially in the liver. These findings implied that the Nrf2-mediated regulation of the proteasome subunits and glucose metabolism is evolutionarily conserved among vertebrates.

Genetic variations in the PSMA3, PSMA6 and PSMC6 genes are associated with type 1 diabetes in Latvians and with expression level of number of UPS-related and T1DM-susceptible genes in HapMap individuals.

The ubiquitin-proteasome system (UPS), a key player of proteostasis network in the body, was implicated in type 1 diabetes mellitus (T1DM) pathogenesis. Polymorphisms in genes encoding proteasome subunits may potentially affect system efficiency. However, data in this field are still limited. To fulfil this gap, single nucleotide polymorphisms in the PSMB5 (rs11543947), PSMA6 (rs2277460, rs1048990), PSMC6 (rs2295826, rs2295827) and PSMA3 (rs2348071) genes were genotyped on susceptibility to T1DM in Latvians. The rs11543947 was found to be neutral and other loci manifested disease susceptibility, with rs1048990 and rs2348071 being the most significantly associated (P < 0.001; OR 2.042 [1.376-3.032] and OR 2.096 [1.415-3.107], respectively). Risk effect was associated with female phenotype for rs2277460 and family history for rs2277460, rs2295826 and rs2295827. Five-locus genotypes being at risk simultaneously at any two or more loci showed strong (P < 0.0001) T1DM association. The T1DM protective effects (P < 0.001) were shown for five-locus genotype and haplotype homozygous on common alleles and composed of common alleles, respectively. Using SNPexp data set, correlations have been revealed between the rs1048990, rs2295826, rs2295827 and rs2348071 T1DM risk genotypes and expression levels of 14 genes related to the UPS and 42 T1DM-susceptible genes encoding proteins involved in innate and adaptive immunity, antiviral response, insulin signalling, glucose-energy metabolism and other pathways implicated in T1DM pathogenesis. Genotype-phenotype and genotype-genotype clusterings support genotyping results. Our results provide evidence on new T1DM-susceptible loci in the PSMA3, PSMA6 and PSMC6 proteasome genes and give a new insight into the T1DM pathogenesis.

Discovery of an NRF1-specific inducer from a large-scale chemical library using a direct NRF1-protein monitoring system.

NRF1 (NF-E2-p45-related factor 1) plays an important role in the regulation of genes encoding proteasome subunits, a cystine transporter, and lipid-metabolizing enzymes. Global and tissue-specific disruptions of the Nrf1 gene in mice result in embryonic lethality and spontaneous development of severe tissue defects, respectively, suggesting NRF1 plays a critical role in vivo. Mechanistically, the continuous degradation of the NRF1 protein by the proteasome is regarded as a major regulatory nexus of NRF1 activity. To develop NRF1-specific inducers that act to overcome the phenotypes related to the lack of NRF1 activity, we constructed a novel NRF1ΔC-Luc fusion protein reporter and developed cell lines that stably express the reporter in Hepa1c1c7 cells for use in high-throughput screening. In screening of a chemical library with this reporter system, we identified two hit compounds that significantly induced luciferase activity. Through an examination of a series of derivatives of one of the hit compounds, we identified T1-20, which induced a 70-fold increase in luciferase activity. T1-20 significantly increased the level of NRF1 protein in the mouse liver, indicating that the compound is also functional in vivo. Thus, these results show the successful identification of the first small chemical compounds which specifically and significantly induce NRF1.

Coordinated regulation of protein synthesis and degradation by mTORC1.

Eukaryotic cells coordinately control anabolic and catabolic processes to maintain cell and tissue homeostasis. Mechanistic target of rapamycin complex 1 (mTORC1) promotes nutrient-consuming anabolic processes, such as protein synthesis. Here we show that as well as increasing protein synthesis, mTORC1 activation in mouse and human cells also promotes an increased capacity for protein degradation. Cells with activated mTORC1 exhibited elevated levels of intact and active proteasomes through a global increase in the expression of genes encoding proteasome subunits. The increase in proteasome gene expression, cellular proteasome content, and rates of protein turnover downstream of mTORC1 were all dependent on induction of the transcription factor nuclear factor erythroid-derived 2-related factor 1 (NRF1; also known as NFE2L1). Genetic activation of mTORC1 through loss of the tuberous sclerosis complex tumour suppressors, TSC1 or TSC2, or physiological activation of mTORC1 in response to growth factors or feeding resulted in increased NRF1 expression in cells and tissues. We find that this NRF1-dependent elevation in proteasome levels serves to increase the intracellular pool of amino acids, which thereby influences rates of new protein synthesis. Therefore, mTORC1 signalling increases the efficiency of proteasome-mediated protein degradation for both quality control and as a mechanism to supply substrate for sustained protein synthesis.

Translational Homeostasis via the mRNA Cap-Binding Protein, eIF4E

Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.

Genetic identification of factors that modulate ribosomal DNA transcription in Saccharomyces cerevisiae.

Ribosomal RNA (rRNA) is transcribed from the ribosomal DNA (rDNA) genes by RNA polymerase I (Pol I). Despite being responsible for the majority of transcription in growing cells, Pol I regulation is poorly understood compared to Pol II. To gain new insights into rDNA transcriptional regulation, we developed a genetic assay in Saccharomyces cerevisiae that detects alterations in transcription from the centromere-proximal rDNA gene of the tandem array. Changes in Pol I transcription at this gene alter the expression of an adjacent, modified URA3 reporter cassette (mURA3) such that reductions in Pol I transcription induce growth on synthetic media lacking uracil. Increases in Pol I transcription induce growth on media containing 5-FOA. A transposon mutagenesis screen was performed with the reporter strain to identify genes that play a role in modulating rDNA transcription. Mutations in 68 different genes were identified, several of which were already known to function in chromatin modification and the regulation of Pol II transcription. Among the other classes of genes were those encoding proteasome subunits and multiple kinases and phosphatases that function in nutrient and stress signaling pathways. Fourteen genes were previously uncharacterized and have been named as regulators of rDNA transcription (RRT).