Intracellular localization of proteasomal degradation of a viral antigen. (1/198)

To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.  (+info)

Pleiotropic defects caused by loss of the proteasome-interacting factors Rad23 and Rpn10 of Saccharomyces cerevisiae. (2/198)

Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant. A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered.  (+info)

Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis. (3/198)

Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-x(L)-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be approximately 30% lower in bcl-x(L) cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome beta-subunit, as determined by Western blot analyses, were similar in control and bcl-x(L) cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-x(L) cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-7-amino-4-methylcou marin (Ac-DEVD-AMC), was twice that observed in bcl-x(L) cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-x(L) cells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-x(L) cells by 12 h, although the maximal increases found in bcl-x(L) cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-x(L) provide some protection against proteasome inhibitor-induced apoptosis.  (+info)

How an inhibitor of the HIV-I protease modulates proteasome activity. (4/198)

The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.  (+info)

Protein farnesylation is critical for maintaining normal cell morphology and canavanine resistance in Schizosaccharomyces pombe. (5/198)

Protein farnesyltransferase (FTase) plays important roles in the growth and differentiation of eukaryotic cells. In this paper, we report the identification of the Schizosaccharomyces pombe gene cpp1(+) encoding the beta-subunit of FTase. The predicted amino acid sequence of the cpp1(+) gene product shares significant similarity with FTase beta-subunits from a variety of organisms. S. pombe FTase purified from E. coli exhibits high enzymatic activity toward the CAAX farnesylation motif substrates (where C represents cysteine, A represents aliphatic amino acid, and X is preferentially methionine, cysteine, serine, alanine, or glutamine) while showing little preference for CAAL geranylgeranylation motif substrates (where L represents leucine or phenylalanine). cpp1(+) is not essential for growth as shown by gene disruption; however, mutant cells exhibit rounded or irregular cell morphology. Expression of a geranylgeranylated mutant form, Ras1-CVIL, which can bypass farnesylation, rescues these morphological defects. We also identify a novel phenotype of cpp1(-) mutants, hypersensitivity to canavanine. This appears to be due to a 3-4-fold increase in the rate of arginine uptake as compared with wild-type cells. Expression of the geranylgeranylated mutant form of a novel farnesylated small GTPase, SpRheb, is able to suppress the elevated arginine uptake rate. These results demonstrate that protein farnesylation is critical for maintaining normal cell morphology through Ras1 and canavanine resistance through SpRheb.  (+info)

Competition between a sterol biosynthetic enzyme and tRNA modification in addition to changes in the protein synthesis machinery causes altered nonsense suppression. (6/198)

The Saccharomyces cerevisiae Mod5 protein catalyzes isopentenylation of A to i(6)A on tRNAs in the nucleus, cytosol, and mitochondria. The substrate for Mod5p, dimethylallyl pyrophosphate, is also a substrate for Erg20p that catalyzes an essential step in sterol biosynthesis. Changing the distribution of Mod5p so that less Mod5p is present in the cytosol decreases i(6)A on cytosolic tRNAs and alters tRNA-mediated nonsense suppression. We devised a colony color/growth assay to assess tRNA-mediated nonsense suppression and used it to search for genes, which, when overexpressed, affect nonsense suppression. We identified SAL6, TEF4, and YDL219w, all of which likely affect nonsense suppression via alteration of the protein synthesis machinery. We also identified ARC1, whose product interacts with aminoacyl synthetases. Interestingly, we identified ERG20. Midwestern analysis showed that yeast cells overproducing Erg20p have reduced levels of i(6)A on tRNAs. Thus, Erg20p appears to affect nonsense suppression by competing with Mod5p for substrate. Identification of ERG20 reveals that yeast have a limited pool of dimethylallyl pyrophosphate. It also demonstrates that disrupting the balance between enzymes that use dimethylallyl pyrophosphate as substrate affects translation.  (+info)

The Saccharomyces cerevisiae Rheb G-protein is involved in regulating canavanine resistance and arginine uptake. (7/198)

The new member of the Ras superfamily of G-proteins, Rheb, has been identified in rat and human, but its function has not been defined. We report here the identification of Rheb homologues in the budding yeast Saccharomyces cerevisiae (ScRheb) as well as in Schizosaccharomyces pombe, Drosophila melanogaster, zebrafish, and Ciona intestinalis. These proteins define a new class of G-proteins based on 1) their overall sequence similarity, 2) high conservation of their effector domain sequence, 3) presence of a unique arginine in their G1 box, and 4) presence of a conserved CAAX farnesylation motif. Characterization of an S. cerevisiae strain deficient in ScRheb showed that it is hypersensitive to growth inhibitory effects of canavanine and thialysine, which are analogues of arginine and lysine, respectively. Accordingly, the uptake of arginine and lysine was increased in the ScRheb-deficient strain. This increased arginine uptake requires the arginine-specific permease Can1p. The function of ScRheb is dependent on having an intact effector domain since mutations in the effector domain of ScRheb are incapable of complementing canavanine hypersensitivity of scrheb disruptant cells. Furthermore, the conserved arginine in the G1 box plays a role in the activity of ScRheb, as a mutation of this arginine to glycine significantly reduced the ability of ScRheb to complement canavanine hypersensitivity of ScRheb-deficient yeast. Finally, a mutation in the C-terminal CAAX farnesylation motif resulted in a loss of ScRheb function. This result, in combination with our finding that ScRheb is farnesylated, suggests that farnesylation plays a key role in ScRheb function. Our findings assign the regulation of arginine and lysine uptake as the first physiological function for this new farnesylated Ras superfamily G-protein.  (+info)

Proteasome mutants, pre4-2 and ump1-2, suppress the essential function but not the mitochondrial RNase P function of the Saccharomyces cerevisiae gene RPM2. (8/198)

The Saccharomyces cerevisiae nuclear gene RPM2 encodes a component of the mitochondrial tRNA-processing enzyme RNase P. Cells grown on fermentable carbon sources do not require mitochondrial tRNA processing activity, but still require RPM2, indicating an additional function for the Rpm2 protein. RPM2-null cells arrest after 25 generations on fermentable media. Spontaneous mutations that suppress arrest occur with a frequency of approximately 9 x 10(-6). The resultant mutants do not grow on nonfermentable carbon sources. We identified two loci responsible for this suppression, which encode proteins that influence proteasome function or assembly. PRE4 is an essential gene encoding the beta-7 subunit of the 20S proteasome core. A Val-to-Phe substitution within a highly conserved region of Pre4p that disrupts proteasome function suppresses the growth arrest of RPM2-null cells on fermentable media. The other locus, UMP1, encodes a chaperone involved in 20S proteasome assembly. A nonsense mutation in UMP1 also disrupts proteasome function and suppresses Deltarpm2 growth arrest. In an RPM2 wild-type background, pre4-2 and ump1-2 strains fail to grow at restrictive temperatures on nonfermentable carbon sources. These data link proteasome activity with Rpm2p and mitochondrial function.  (+info)