SUMMARY In Saccharomyces cerevisiae, chemical or genetic inhibition of proteasome activity induces new proteasome synthesis promoted by the transcription factor RPN4. This ensures that proteasome activity is matched to demand. This transcriptional feedback loop is conserved in mammals, but its molecular basis is not understood. Here we report that Nuclear factor erythroid derived 2-related factor 1 (Nrf1), a transcription factor of the cap ‘n’ collar basic leucine zipper family, but not the related Nrf2, is necessary for induced proteasome gene transcription in mouse embryonic fibroblasts (MEFs). Promoter-reporter assays revealed the importance of antioxidant response elements in Nrf1-mediated upregulation of proteasome subunit genes. Nrf1-/- MEFs were impaired in the recovery of proteasome activity after transient treatment with the covalent proteasome inhibitor YU101 and knockdown of Nrf1 in human cancer cells enhanced cell killing by YU101. Taken together, our results suggest that Nrf1-mediated proteasome homeostasis could be an attractive target for therapeutic intervention in cancer.

Synaptic vesicle (SV) pools must maintain a functional repertoire of proteins to efficiently release neurotransmitter. The accumulation of old or damaged proteins on SV membranes is linked to synaptic dysfunction and neurodegeneration. However, despite the importance of SV protein turnover for neuronal health, the molecular mechanisms underlying this process are largely unknown. Here, we have used dissociated rat hippocampal neurons to investigate the pathway for SV protein degradation. We find that neuronal activity drives the degradation of a subset of SV proteins and that the endosomal sorting complex required for transport (ESCRT) machinery and SVassociated GTPase Rab35 are key elements of this use-dependent degradative pathway. Specifically, neuronal activity induces Rab35 activation and binding to the ESCRT-0 protein Hrs, which we have identified as a novel Rab35 effector. These actions recruit the downstream ESCRT machinery to SV pools, thereby initiating SV protein degradation via the ESCRT pathway. Our findings show that the Rab35/ESCRT pathway facilitates the activity-dependent removal of specific proteins from SV pools, thereby maintaining presynaptic protein homeostasis.

No abstract

BackgroundInnate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-κB transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood.ResultsWe show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-κB/Relish-driven antimicrobial peptide gene expression in flies. In nub1 mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism.ConclusionsThis study demonstrates that a large number of genes that are activated by NF-κB/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.