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Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased alpha-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and alpha-synclein aggregation.

Ubiquitin-positive intraneuronal inclusions are a consistent feature of the major human neurodegenerative diseases, suggesting that dysfunction of the ubiquitin proteasome system is central to disease etiology. Research using inhibitors of the 20S proteasome to model Parkinson's disease is controversial. We report for the first time that specifically 26S proteasomal dysfunction is sufficient to trigger neurodegenerative disease. Here, we describe novel conditional genetic mouse models using the Cre/loxP system to spatially restrict inactivation of Psmc1 (Rpt2/S4) to neurons of either the substantia nigra or forebrain (e.g., cortex, hippocampus, and striatum). PSMC1 is an essential subunit of the 26S proteasome and Psmc1 conditional knock-out mice display 26S proteasome depletion in targeted neurons, in which the 20S proteasome is not affected. Impairment of specifically ubiquitin-mediated protein degradation caused intraneuronal Lewy-like inclusions and extensive neurodegeneration in the nigrostriatal pathway and forebrain regions. Ubiquitin and alpha-synuclein neuropathology was evident, similar to human Lewy bodies, but interestingly, inclusion bodies contained mitochondria. We support this observation by demonstrating mitochondria in an early form of Lewy body (pale body) from Parkinson's disease patients. The results directly confirm that 26S dysfunction in neurons is involved in the pathology of neurodegenerative disease. The model demonstrates that 26S proteasomes are necessary for normal neuronal homeostasis and that 20S proteasome activity is insufficient for neuronal survival. Finally, we are providing the first reproducible genetic platform for identifying new therapeutic targets to slow or prevent neurodegeneration.

Downregulation of p53 by MDM2-mediated proteasomal degradation makes cells resistant to apoptosis. The MDM2-p53 interaction is well characterized, but the mechanisms that regulate the interaction are not well understood. Here, we show that PA28gamma, a proteasome activator that inhibits apoptosis and promotes cell cycle progression through unknown mechanisms, exerts an effect as a cofactor in the MDM2-p53 interaction. The polymer form of PA28gamma interacts with both MDM2 and p53 proteins and facilitates their physical interaction. This promotes ubiquitination- and MDM2-dependent proteasomal degradation of p53, limiting its accumulation and resulting in inhibited apoptosis after DNA damage. Elimination of endogenous PA28gamma in human cancer cells abrogates MDM2-mediated p53 degradation, increases the activity of p53, and enhances apoptosis. These findings reveal the mechanism by which PA28gamma affects apoptosis and proliferation. Manipulation of the level of PA28gamma, an approach that would regulate the cellular content of p53, may improve the efficacy of current cancer therapies.

The cell-cycle regulator p21(Cip1) is degraded by proteasomes independently of ubiquitination. We now show that degradation of p21 in vivo does not require the 19S proteasome lid, which contains the ubiquitin-binding subunit. Instead, the major proteasomal pathway for p21 degradation involves an alternative proteasome lid, the REGgamma complex. REGgamma binds to p21 in vivo, and deletion of p21's REGgamma-binding site greatly extends its half-life. Knockdown of REGgamma by RNA interference stabilizes p21, p21 has a significantly extended half-life in REGgamma(-/-) murine embryonic fibroblasts, and the p21 abundance is elevated in REGgamma(-/-) mice. The role of REGgamma in cell-cycle regulation may extend beyond p21 regulation, because p16(INK4A) and p19(Arf) also bind to REGgamma and are stabilized in REGgamma-deficient cells.

The hepatitis C virus (HCV) core protein is a component of nucleocapsids and a pathogenic factor for hepatitis C. Several epidemiological and experimental studies have suggested that HCV infection is associated with insulin resistance, leading to type 2 diabetes. We have previously reported that HCV core gene-transgenic (PA28gamma(+/+)CoreTg) mice develop marked insulin resistance and that the HCV core protein is degraded in the nucleus through a PA28gamma-dependent pathway. In this study, we examined whether PA28gamma is required for HCV core-induced insulin resistance in vivo. HCV core gene-transgenic mice lacking the PA28gamma gene (PA28gamma(-/-)CoreTg) were prepared by mating of PA28gamma(+/+)CoreTg with PA28gamma-knockout mice. Although there was no significant difference in the glucose tolerance test results among the mice, the insulin sensitivity in PA28gamma(-/-)CoreTg mice was recovered to a normal level in the insulin tolerance test. Tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), production of IRS2, and phosphorylation of Akt were suppressed in the livers of PA28gamma(+/+)CoreTg mice in response to insulin stimulation, whereas they were restored in the livers of PA28gamma(-/-)CoreTg mice. Furthermore, activation of the tumor necrosis factor alpha promoter in human liver cell lines or mice by the HCV core protein was suppressed by the knockdown or knockout of the PA28gamma gene. These results suggest that the HCV core protein suppresses insulin signaling through a PA28gamma-dependent pathway.

Poh1 deubiquitinase activity is required for proteolytic processing of polyubiquitinated substrates by the 26S proteasome, linking deubiquitination to complete substrate degradation. Poh1 RNA interference (RNAi) in HeLa cells resulted in a reduction in cell viability and an increase in polyubiquitinated protein levels, supporting the link between Poh1 and the ubiquitin proteasome pathway. To more specifically test for any requirement of the zinc metalloproteinase motif of Poh1 to support cell viability and proteasome function, we developed a RNAi complementation strategy. Effects on cell viability and proteasome activity were assessed in cells with RNAi of endogenous Poh1 and induced expression of wild-type Poh1 or a mutant form of Poh1, in which two conserved histidines of the proposed catalytic site were replaced with alanines. We show that an intact zinc metalloproteinase motif is essential for cell viability and 26S proteasome function. As a required enzymatic component of the proteasome, Poh1 is an intriguing therapeutic drug target for cancer.

Primary CD8+ T cell (T(CD8+)) responses to viruses are directed toward multiple Ags and shaped by both the level of Ag presentation and the underlying Ag-specific T(CD8+) repertoire. The relative importance of these factors in deciding the hierarchy of T(CD8+) responses and how they are influenced by the immunoproteasome are not well understood. Using an influenza infection model in mice deficient in various immunoproteasome subunits, we observe that Ag presentation and T(CD8+) repertoire are altered in an epitope-specific and immunoproteasome subunit-dependent manner. More importantly, we find that the level of Ag presentation and the extent of the underlying repertoire can work either alone or in concert to determine definitively the magnitude of the individual T(CD8+) responses and hence the overall T(CD8+) hierarchy. Together, these results provide a clearer understanding of how immunodominance hierarchies are established.

Proteasomes are self-compartmentalizing proteases first discovered in eukaryotes but also occurring in archaea and in bacteria belonging to the order Actinomycetales. In bacteria, proteasomes have so far no known function. In order to evaluate the influence of the 20S proteasome on the production of heterologous proteins by Streptomyces lividans TK24, the production of a number of heterologous proteins, including soluble human tumour necrosis factor receptor II (shuTNFRII) and salmon calcitonin (sCT), was compared with the wild-type TK24, a proteasome-deficient mutant designated PRO41 and a strain complemented for the disrupted proteasome genes (strain PRO41R). S. lividans cells lacking intact proteasome genes are phenotypically indistinguishable from the wild-type or the complemented strain containing functional proteasomes. Using the expression and secretion signals of the subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (Vsi) for shuTNFRII and those of tyrosinase of Streptomyces antibioticus (MelC1) for the production of sCT, both proteins were secreted in significantly higher amounts in the strain PRO41 than in the wild-type S. lividans TK24 or the complemented strain PRO41R. However, the secretion of other heterologous proteins such as shuTNFRI was not enhanced in the proteasome-deficient strain. This suggests that S. lividans TK24 can degrade some heterologous proteins in a proteasome-dependent fashion. The proteasome-deficient strain may therefore be useful for the efficient production of these heterologous proteins.

Substrates destined for degradation by the 26 S proteasome are labelled with polyubiquitin chains. Rpn11/Mpr1, situated in the lid subcomplex, partakes in the processing of these chains or in their removal from substrates bound to the proteasome. Rpn11 also plays a role in maintaining mitochondrial integrity, tubular structure and proper function. The recent finding that Rpn11 participates in proteasome-associated deubiquitination focuses interest on the MPN+ (Mpr1, Pad1, N-terminal)/JAMM (JAB1/MPN/Mov34) metalloprotease site in its N-terminal domain. However, Rpn11 damaged at its C-terminus (the mpr1-1 mutant) causes pleiotropic effects, including proteasome instability and mitochondrial morphology defects, resulting in both proteolysis and respiratory malfunctions. We find that overexpression of WT (wild-type) RPN8, encoding a paralogous subunit that does not contain the catalytic MPN+ motif, corrects proteasome conformations and rescues cell cycle phenotypes, but is unable to correct defects in the mitochondrial tubular system or respiratory malfunctions associated with the mpr1-1 mutation. Transforming mpr1-1 with various RPN8-RPN11 chimaeras or with other rpn11 mutants reveals that a WT C-terminal region of Rpn11 is necessary, and more surprisingly sufficient, to rescue the mpr1-1 mitochondrial phenotype. Interestingly, single-site mutants in the catalytic MPN+ motif at the N-terminus of Rpn11 lead to reduced proteasome-dependent deubiquitination connected with proteolysis defects. Nevertheless, these rpn11 mutants suppress the mitochondrial phenotypes associated with mpr1-1 by intragene complementation. Together, these results point to a unique role for the C-terminal region of Rpn11 in mitochondrial maintenance that may be independent of its role in proteasome-associated deubiquitination.