Interferon (IFN)-induced immunoproteasomes (i-proteasomes) have been associated with improved processing of major histocompatibility complex (MHC) class I antigens. Here, we show that i-proteasomes function to protect cell viability under conditions of IFN-induced oxidative stress. IFNs trigger the production of reactive oxygen species, which induce protein oxidation and the formation of nascent, oxidant-damaged proteins. We find that the ubiquitylation machinery is concomitantly upregulated in response to IFNs, functioning to target defective ribosomal products (DRiPs) for degradation by i-proteasomes. i-proteasome-deficiency in cells and in murine inflammation models results in the formation of aggresome-like induced structures and increased sensitivity to apoptosis. Efficient clearance of these aggregates by the enhanced proteolytic activity of the i-proteasome is important for the preservation of cell viability upon IFN-induced oxidative stress. Our findings suggest that rather than having a specific role in the production of class I antigens, i-proteasomes increase the peptide supply for antigen presentation as part of a more general role in the maintenance of protein homeostasis.

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.

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Peptide generation by the proteasome is rate-limiting in MHC class I-restricted antigen presentation in response to IFN-␥. IFN-␥-induced de novo formation of immunoproteasomes, therefore, essentially supports the rapid adjustment of the mammalian immune system. Here, we report that the molecular interplay between the proteasome maturation protein (POMP) and the proteasomal ␤5i subunit low molecular weight protein 7 (LMP7) has a key position in this immune adaptive program. IFN-␥-induced coincident biosynthesis of POMP and LMP7 and their direct interaction essentially accelerate immunoproteasome biogenesis compared with constitutive 20S proteasome assembly. The dynamics of this process is determined by rapid LMP7 activation and the immediate LMP7-dependent degradation of POMP. Silencing of POMP expression impairs recruitment of both ␤5 subunits into the proteasome complex, resulting in decreased proteasome activity, reduced MHC class I surface expression, and induction of apoptosis. Furthermore, our data reveal that immunoproteasomes exhibit a considerably shortened half-life, compared with constitutive proteasomes. In consequence, our studies demonstrate that the cytokine-induced rapid immune adaptation of the proteasome system is a tightly regulated and transient response allowing cells to return rapidly to a normal situation once immunoproteasome function is no longer required.antigen presentation ͉ immunoproteasome ͉ MHC class I

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The 26 S proteasome is a high molecular mass proteinase complex that is built by at least 32 different protein subunits. Such protease complexes in bacteria and yeast are systems that undergo a highly sophisticated network of gene expression regulation. However, regulation of mammalian proteasome gene expression has been neglected so far as a possible control mechanism for the amount of proteasomes in the cell. Here, we show that treatment of cells with proteasome inhibitors and the concomitant impairment of proteasomal enzyme activity induce a transient and concerted up-regulation of all mammalian 26 S proteasome subunit mRNAs. Proteasome inhibition in combination with inhibition of transcription revealed that the observed up-regulation is mediated by coordinated transcriptional activation of the proteasome genes and not by post-transcriptional events. Our experiments also demonstrate that inhibitor-induced proteasome gene activation results in enhanced de novo protein synthesis of all subunits and in increased de novo formation of proteasomes. This phenomenon is accompanied by enhanced expression of the proteasome maturation factor POMP. Thus, our experiments present the first evidence that the amount of proteasomes in mammalia is regulated at the transcriptional level and that there exists an autoregulatory feedback mechanism that allows the compensation of reduced proteasome activity.

A member of the clpC subfamily of stress response-related Clp ATPases was cloned from Bacillus subtilis. The B. subtilis cIpC gene was induced in response to various stresses, including heat shock. Its product was identified as a general stress protein (Gspl2) described previously. A dramatic increase in the amount of clpC mRNA immediately after exposure to multiple stresses suggested regulation on a transcriptional level.Induction by heat shock was independent of the alternative sigma factor SigB, indicating a new mechanism of heat shock induction in B. subtilis. A clpC insertional mutant had an impaired tolerance for heat shock and salt stress. Furthermore, the mutation triggered the formation of elongated cells, a phenomenon particularly pronounced during stress.The Clp proteins constitute a family of proteins which are highly conserved and universal (for reviews, see references 14 and 39). Members of this family have been shown to exist in Escherichia coli (40) 131, and 62 to 69 amino acids, respectively (39). All these Clp proteins possess an ATPase activity, and they have been postulated to function as molecular chaperones, to be involved in the regulation of ATP-dependent proteolysis, or both (13,23,39). ClpB of E. coli is identical to the previously identified Sig32-dependent heat shock protein F84.1 and is required for survival at 50°C (40). The Clphomologous protein of S. cerevisiae HsplO4 is also a heat shock protein and is involved in the protection of S. cerevisiae against various stresses (28).Bacillus subtilis is able to induce a set of conserved heat shock proteins in response to heat stress (1,30,41,43). The heat shock response of B. subtilis has not been studied extensively yet. However, the mechanism of induction seems to be different from that in E. coli. Two alternative sigma factors, Sig32 and Sig24, are required for the induction of heat shock genes in E. coli (9, 10). Presently, two groups of heat shock genes can be differentiated in B. subtilis according to their regulation (17, 46). The groESL operon and the dnaK locus, which contain SigA-dependent promoters, belong to the first group. A highly conserved inverted repeat immediately downstream of the start point of transcription seems to be involved in the heat induction of these genes (20,36,49,50 MATERUILS AND METHODSBacterial strains, plasmids, and culture conditions. The bacterial strains and plasmids used are listed in Table 1. E. coli was routinely grown in Luria broth medium. B. subtilis was cultivated under vigorous agitation at 37°C in a synthetic medium described previously (42). The different stress conditions were provoked as described earlier (46). Briefly, the culture was divided during exponential growth and one half of the culture was grown at 37°C (control), whereas the other half of the culture was exposed to a temperature of 48, 50, or 52°C or a final concentration of 4% (wt/vol) sodium chloride or 5% (vol/vol) ethanol. Limiting the different nutrients was accomplished either by cultivating the bacteria in synthetic ...

The proteasome system is a central component of a cascade of proteolytic processing steps required to generate antigenic peptides presented at the cell surface to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules. The nascent protein pool or DRiPs (defective ribosomal products) appear to represent an important source for MHC class I epitopes. Owing to the destructive activities of aminopeptidases in the cytosol, at most 1% of the peptides generated by the ubiquitin-proteasome system seems to be made available to the immune system. Interferon-gamma (IFN-gamma) helps to override these limitations by the formation of immunoproteasomes, the activator complex PA28, and the induction of several aminopeptidases. Both immunoproteasomes and PA28 use cleavage sites already used by constitutive proteasomes but with altered and in some cases dramatically enhanced frequency. Therefore, two proteolytic cascades appear to have evolved to provide MHC class I epitopes. The 'constitutive proteolytic cascade' is designed to efficiently degrade proteins to single amino acid residues, allowing only a small percentage of peptides to be presented at the cell surface. In contrast, the IFN-gamma-controlled proteolytic cascade generates larger amounts of appropriate antigenic peptides, assuring more peptides to overcome the proteolytic restrictions of the constitutive system, thereby enhancing MHC class I antigen presentation.