Cleavage motifs of the yeast 20S proteasome β subunits deduced from digests of enolase 1

The 436-amino acid protein enolase 1 from yeast was degraded in vitro by purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active β subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.

The subunits MECL-1 and LMP2 are mutually required for incorporation into the 20S proteasome

Processing of antigens for presentation by major histocompatibility complex (MHC) class I molecules requires the activity of the proteasome. The 20S proteasome complex is composed of 14 different subunits, 2 of which can be substituted by the interferon γ (IFN-γ)-inducible and MHC-encoded subunits LMP2 and LMP7 (low molecular mass poylpeptides 2 and 7). A third subunit, MECL-1, is inducible by IFN-γ but is encoded outside the MHC. Here we show by cotransfection experiments that the incorporation of MECL-1 into the 20S proteasome is directly dependent on the expression of LMP2 but independent of LMP7. Conversely, the uptake of LMP2 is strongly enhanced by MECL-1 expression. The expression of MECL-1 caused a replacement of the homologous subunit Z in the 20S proteasome complex. LMP2 is required for MECL-1 incorporation at the level of proteasome precursor formation that guarantees the concerted incorporation of two IFN-γ-inducible proteasome subunits encoded inside and outside the MHC. The obligatory coincorporation of MECL-1 and LMP2 is an important parameter for the interpretation of results obtained with LMP2-deficient cell lines and mice as well as for the design of experiments addressing the function of MECL-1 in antigen presentation.

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.

Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation

The proteasome is responsible for degradation of substrates of the ubiquitin pathway. 20S proteasomes are cylindrical particles with subunits arranged in a stack of four heptameric rings. The outer rings are composed of α subunits, and the inner rings are composed of β subunits. A well-characterized archaeal proteasome has a single type of each subunit, and the N-terminal threonine of the β subunit is the active-site nucleophile. Yeast proteasomes have seven different β subunits and exhibit several distinct peptidase activities, which were proposed to derive from disparate active sites. We show that mutating the N-terminal threonine in the yeast Pup1 β subunit eliminates cleavage after basic residues in peptide substrates, and mutating the corresponding threonine of Pre3 prevents cleavage after acidic residues. Surprisingly, neither mutation has a strong effect on cell growth, and they have at most minor effects on ubiquitin-dependent proteolysis. We show that Pup1 interacts with Pup3 in each β subunit ring. Our data reveal that different proteasome active sites contribute very differently to protein breakdown in vivo, that contacts between particular subunits in each β subunit ring are critical for active-site formation, and that active sites in archaea and different eukaryotes are highly similar.

Proteasome regulation of activation-induced T cell death

Lactacystin, a microbial metabolite that inhibits protease activity only in the proteasome, was used to study the role of the proteasome in the activation-induced cell death (AICD) of T cells. Lactacystin induces DNA fragmentation and apoptosis in a T cell hybridoma (DO.11.10) in a dose-dependent manner. Between 1 and 10 μM, the mildly cytotoxic lactacystin inhibited the AICD of DO.11.10 cells cultured in anti-CD3-coated wells. Degradation of IκBβ and the translocation of the NF-κB (p50/RelA) into the nucleus, which occurred at 1.5 hr after anti-CD3 activation, were inhibited by lactacystin. Lactacystin did not inhibit the expression of nuclear transcription factor Oct-1. The activation-induced expression of the immediate–early gene, Nur77, and the T cell death genes, CD95 (Fas) and CD95 ligand (FasL), were inhibited. Functional expression of FasL cytotoxicity and the increase of cell surface Fas were also inhibited. Lactacystin must be added within 2 hr of activation to efficiently block AICD. In addition, lactacystin failed to inhibit the killing of DO.11.10 by FasL-expressing allo-specific cytotoxic effector cells. These observations strongly suggest a direct link between the proteasome-dependent degradation of IκBβ and the AICD that occurs through activation of the FasL gene and up-regulation of the Fas gene.

Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor α (RARα) and oncogenic RARα fusion proteins

Analyzing the pathways by which retinoic acid (RA) induces promyelocytic leukemia/retinoic acid receptor α (PML/RARα) catabolism in acute promyelocytic leukemia (APL), we found that, in addition to caspase-mediated PML/RARα cleavage, RA triggers degradation of both PML/RARα and RARα. Similarly, in non-APL cells, RA directly targeted RARα and RARα fusions to the proteasome degradation pathway. Activation of either RARα or RXRα by specific agonists induced degradation of both proteins. Conversely, a mutation in RARα that abolishes heterodimer formation and DNA binding, blocked both RARα and RXRα degradation. Mutations in the RARα DNA-binding domain or AF-2 transcriptional activation region also impaired RARα catabolism. Hence, our results link transcriptional activation to receptor catabolism and suggest that transcriptional up-regulation of nuclear receptors by their ligands may be a feedback mechanism allowing sustained target-gene activation.

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1

The Epstein–Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

Interaction of the Doa4 Deubiquitinating Enzyme with the Yeast 26S Proteasome

The Saccharomyces cerevisiae Doa4 deubiquitinating enzyme is required for the rapid degradation of protein substrates of the ubiquitin–proteasome pathway. Previous work suggested that Doa4 functions late in the pathway, possibly by deubiquitinating (poly)-ubiquitin-substrate intermediates associated with the 26S proteasome. We now provide evidence for physical and functional interaction between Doa4 and the proteasome. Genetic interaction is indicated by the mutual enhancement of defects associated with a deletion of DOA4 or a proteasome mutation when the two mutations are combined. Physical association of Doa4 and the proteasome was investigated with a new yeast 26S proteasome purification procedure, by which we find that a sizeable fraction of Doa4 copurifies with the protease. Another yeast deubiquitinating enzyme, Ubp5, which is related in sequence to Doa4 but cannot substitute for it even when overproduced, does not associate with the proteasome. DOA4-UBP5 chimeras were made by a novel PCR/yeast recombination method and used to identify an N-terminal 310-residue domain of Doa4 that, when appended to the catalytic domain of Ubp5, conferred Doa4 function, consistent with Ubp enzymes having a modular architecture. Unlike Ubp5, a functional Doa4-Ubp5 chimera associates with the proteasome, suggesting that proteasome binding is important for Doa4 function. Together, these data support a model in which Doa4 promotes proteolysis through removal of ubiquitin from proteolytic intermediates on the proteasome before or after initiation of substrate breakdown.

Yeast Cycloheximide-resistant crl Mutants Are Proteasome Mutants Defective in Protein Degradation

In 1988 McCusker and Haber generated a series of mutants which are resistant to the minimum inhibitory concentration of the protein synthesis inhibitor cycloheximide. These cycloheximide-resistant, temperature-sensitive (crl) mutants, in addition, exhibited other pleiotropic phenotypes, e.g., incorrect response to starvation, hypersensitivity against amino acid analogues, and other protein synthesis inhibitors. Temperature sensitivity of one of these mutants, crl3–2, had been found to be suppressed by a mutation, SCL1–1, which resided in an α-type subunit of the 20S proteasome. We cloned the CRL3 gene by complementation and found CRL3 to be identical to the SUG1/CIM3 gene coding for a subunit of the 19S cap complex of the 26S proteasome. Another mutation, crl21, revealed to be allelic with the 20S proteasomal gene PRE3. crl3–2 and crl21 mutant cells show significant defects in proteasome-dependent proteolysis, whereas the SCL1–1 suppressor mutation causes partial restoration of crl3–2-induced proteolytic defects. Notably, cycloheximide resistance was also detected for other proteolytically deficient proteasome mutants (pre1–1, pre2–1, pre3–1, pre4–1). Moreover, proteasomal genes were found within genomic sequences of 9 of 13 chromosomal loci to which crl mutations had been mapped. We therefore assume that most if not all crl mutations reside in the proteasome and that phenotypes found are a result of defective protein degradation.

Proteasome-dependent degradation of the human estrogen receptor

In eukaryotic cells, the ubiquitin–proteasome pathway is the major mechanism for the targeted degradation of proteins with short half-lives. The covalent attachment of ubiquitin to lysine residues of targeted proteins is a signal for the recognition and rapid degradation by the proteasome, a large multi-subunit protease. In this report, we demonstrate that the human estrogen receptor (ER) protein is rapidly degraded in mammalian cells in an estradiol-dependent manner. The treatment of mammalian cells with the proteasome inhibitor MG132 inhibits activity of the proteasome and blocks ER degradation, suggesting that ER protein is turned over through the ubiquitin–proteasome pathway. In addition, we show that in vitro ER degradation depends on ubiquitin-activating E1 enzyme (UBA) and ubiquitin-conjugating E2 enzymes (UBCs), and the proteasome inhibitors MG132 and lactacystin block ER protein degradation in vitro. Furthermore, the UBA/UBCs and proteasome inhibitors promote the accumulation of higher molecular weight forms of ER. The UBA and UBCs, which promote ER degradation in vitro, have no significant effect on human progesterone receptor and human thyroid hormone receptor β proteins.

Poly-ADP ribose polymerase activates nuclear proteasome to degrade oxidatively damaged histones

The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the cytoplasm. Nuclear proteins are also subject to oxidation, and the nucleus of mammalian cells contains proteasome. In human beings, tumor cells frequently are subjected to oxidation as a consequence of antitumor chemotherapy, and K562 human myelogenous leukemia cells have a higher nuclear proteasome activity than do nonmalignant cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs and the mechanisms of inducible tumor resistance to oxidation are of obvious importance. After hydrogen peroxide treatment of K562 cells, degradation of the model proteasome peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increases significantly within minutes. Both increased proteolytic susceptibility of the histone substrates (caused by modification by oxidation) and activation of the proteasome enzyme complex occur independently during oxidative stress. This rapid up-regulation of 20S proteasome activity is accompanied by, and depends on, poly-ADP ribosylation of the proteasome, as shown by inhibitor experiments, 14C-ADP ribose incorporation assays, immunoblotting, in vitro reconstitution experiments, and immunoprecipitation of (activated) proteasome with anti-poly-ADP ribose polymerase antibodies. The poly-ADP ribosylation-mediated activated nuclear 20S proteasome is able to remove oxidatively damaged histones more efficiently and therefore is proposed as an oxidant-stimulatable defense or repair system of the nucleus in K562 leukemia cells.

Identification and characterization of a mammalian protein interacting with 20S proteasome precursors

The assembly of individual mammalian proteasome subunits into catalytically active 20S proteasome is not well understood. Herein, we report the identification and characterization of human and mouse homologues of the yeast proteasome maturating factor Ump1p. We delineate the region of hUMP1 implicated in the specific interaction with proteasome precursors and show that hUMP1 protein is absent from the mature form of the 20S proteasome. We also show that the transcript level of mammalian UMP1 is increased after IFN-γ treatment and that mammalian UMP1 is functionally related to but not interchangeable with its yeast homologue.

Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6gag, a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6gag. Consistent with this, viruses with mutations in PR or p6gag were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

Inhibition of the ubiquitin-proteasome system in Alzheimer's disease

Alzheimer's disease is the most common cause of dementia in the elderly. Although several genetic defects have been identified in patients with a family history of this disease, the majority of cases involve individuals with no known genetic predisposition. A mutant form of ubiquitin, termed Ub+1, has been selectively observed in the brains of Alzheimer's patients, including those with nonfamilial Alzheimer's disease, but it has been unclear why Ub+1 expression should be deleterious. Here we show that Ub+1 is an efficient substrate for polyubiquitination in vitro and in transfected human cells. The resulting polyubiquitin chains are refractory to disassembly by deubiquitinating enzymes and potently inhibit the degradation of a polyubiquitinated substrate by purified 26S proteasomes. Thus, expression of Ub+1 in aging brain could result in dominant inhibition of the Ub-proteasome system, leading to neuropathologic consequences.

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.