Growth retardation in mice lacking the proteasome activator PA28gamma

The proteasome activator PA28 binds to both ends of the central catalytic machine, known as the 20 S proteasome, in opposite orientations to form the enzymatically active proteasome. The PA28 family is composed of three members designated alpha, beta, and gamma; PA28alpha and PA28beta form the heteropolymer mainly located in the cytoplasm, whereas PA28gamma forms a homopolymer that predominantly occurs in the nucleus. Available evidence indicates that the heteropolymer of PA28alpha and PA28beta is involved in the processing of intracellular antigens, but the function of PA28gamma remains elusive. To investigate the role of PA28gamma in vivo, we generated mice deficient in the PA28gamma gene. The PA28gamma-deficient mice were born without appreciable abnormalities in all tissues examined, but their growth after birth was retarded compared with that of PA28gamma(+/-) or PA28gamma(+/+) mice. We also investigated the effects of the PA28gamma deficiency using cultured embryonic fibroblasts; cells lacking PA28gamma were larger and displayed a lower saturation density than their wild-type counterparts. Neither the expression of PA28alpha/beta nor the subcellular localization of PA28alpha was affected in PA28gamma(-/-) cells. These results indicate that PA28gamma functions as a regulator of cell proliferation and body growth in mice and suggest that neither PA28alpha nor PA28beta compensates for the PA28gamma deficiency.     

Properties of the nuclear proteasome activator PA28gamma (REGgamma)

PA28 or 11S REG is a proteasome activator composed of homologous alpha- and beta-subunits and predominantly found in the cytosol. A homologous protein originally known as the Ki antigen but now called PA28gamma or REGgamma is predominantly localized in the nucleus. To further characterize the biochemical properties of PA28gamma, we expressed and purified homogenous recombinant human protein with and without an N-terminal 6-His extension. PA28gamma is a heptamer based on the molecular masses of the native and monomeric proteins. The heptameric 6-His fusion protein can dimerize. Recombinant PA28y stimulates the proteasome-mediated hydrolysis of synthetic substrates containing hydrophobic, basic, and acidic amino acids in the P1 position. Stimulation is dependent on substrate size. PA28y only minimally stimulates degradation of the oxidized B chain of insulin. PA28gamma may facilitate the later stages of protein metabolism in the nucleus and/or have a more specialized role in controlling the levels of biologically active peptides in the nucleus.     

The role of the proteasome system and the proteasome activator PA28 complex in the cellular immune response

The generation of antigenic peptides bound and presented to the immune system by MHC class I molecules predominantly depends on the function of the proteasome system. Stimulation of cells with interferon gamma induces the incorporation of three active site bearing beta-subunits into the 20S proteasome and the formation of the PA28 proteasome modulator complex. PA28 alters the cleavage properties of the proteasome and enhances MHC class I antigen presentation. Thus, by cytokine induced change of the proteasome system cells may alter the proteolytic properties of the 20S proteasome and may render an organism more flexible in its peptide generation capacity.     

The proteasome activator PA28 functions in collaboration with Hsp90 in vivo

We have previously shown that the proteasome activator PA28 is essential to Hsp90-dependent protein refolding in vitro, where PA28 mediates transfer of the Hsp90-bound substrate protein to the Hsc70/Hsp40 chaperone machine for its correct refolding. This observation suggests that PA28 may also collaborate with Hsp90 in cells. To examine this possibility, here we have used double-stranded RNA interference (RNAi) against PA28 in Caenorhabditis elegans mutants of daf-21, which encodes Hsp90. We show that C. elegans PA28 facilitates Hsp90-initiated protein refolding, albeit with an activity lower than that of mouse PA28 proteins. RNAi-mediated knockdown of PA28 significantly suppresses the Daf-c (dauer formation constitutive) phenotype of the daf-21 mutant, but it has no affect on the distinct defects of this mutant in sensing odorants. Taking these results together, we conclude that PA28 is likely to function in collaboration with Hsp90 in vivo.     

Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28.

The activation kinetics of constitutive and IFNgamma-stimulated 20S proteasomes obtained with homomeric (recPA28alpha, recPA28beta) and heteromeric (recPA28alphabeta) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling. The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28alpha/RecP28beta/RecP28alpha + RecP28beta) exhibit biphasic characteristics which can be attributed to a low-level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28alphabeta complex is about two orders of magnitude higher than towards the homomeric PA28alpha and PA28beta complexes. Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides.     

Nitric oxide decreases activity and levels of the 11S proteasome activator PA28 in the vasculature

The 11S proteasome activator (PA28) binds to the 20S proteasome and increases its ability to degrade small peptides. Expression of PA28 subunits (α, β, γ) is induced by interferon-γ stimulation. Inflammation plays a role in the development of neointimal hyperplasia, and we have previously shown that nitric oxide (NO) reduces neointimal hyperplasia in animal models and 26S proteasome activity in rat aortic smooth muscle cells (RASMC). Here, we show that PA28 increased 26S proteasome activity in RASMC, as measured by a fluorogenic assay, and the NO donor S-nitroso N-acetylpenicillamine significantly inhibits this activation. This effect was abrogated by the reducing agents dithiothreitol and HgCl(2), suggesting that NO affects the activity of PA28 through S-nitrosylation. NO did not appear to affect PA28 levels or intracellular localization in RASMC in vitro. Three days following rat carotid artery balloon injury, levels of PA28α, β and γ subunits were decreased compared to uninjured control arteries (n=3/group) in vivo. The NO donor proline NONOate further decreased PA28α, β and γ levels by 1.9-, 2.3- and 3.4-fold, respectively, compared to uninjured control arteries. Fourteen days following arterial injury, levels of PA28α, β and γ subunits were increased throughout the arterial wall compared to uninjured control arteries, but were greatest for the α and β subunits. NO continued to decrease the levels of all three PA28 subunits throughout the arterial wall at this time point. Since the PA28 subunits are involved in the breakdown of peptides during inflammation, PA28 inhibition may be one mechanism by which NO inhibits neointimal hyperplasia.     

A critical role for the proteasome activator PA28 in the Hsp90-dependent protein refolding

The 90-kDa heat shock protein, Hsp90, was previously shown to capture firefly luciferase during thermal inactivation and prevent it from undergoing an irreversible off-pathway aggregation, thereby maintaining it in a folding-competent state. While Hsp90 by itself was not sufficient to refold the denatured luciferase, addition of rabbit reticulocyte lysate remarkably restored the luciferase activity. Here we demonstrate that Hsc70, Hsp40, and the 20 S proteasome activator PA28 are the effective components in reticulocyte lysate. Purified Hsc70, Hsp40, and PA28 were necessary and sufficient to fully reconstitute Hsp90-initiated refolding. Kinetics of substrate binding support the idea that PA28 acts as the molecular link between the Hsp90-dependent capture of unfolded proteins and the Hsc70- and ATP-dependent refolding process.     

Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks

The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs)—a highly cytotoxic DNA lesion—activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28γ (REGγ; PSME3) as a novel DDR player. PA28γ depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28γ deficiency abrogates the balance between the two major DSB repair pathways—nonhomologous end-joining and homologous recombination repair. Furthermore, PA28γ is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28γ-proteasome axis of the DDR that is required for timely coordination of DSB repair.     

The proteasome activator PA28γ is a novel component of the DNA double-strand break response network

Comment on: Levy-Barda A, et al. Cell Cycle 2011; 10:4300-10.     

Purification procedures determine the proteasome activation properties of REG gamma (PA28 gamma)

The proteasome activation properties of recombinant REG gamma molecules depend on purification procedures. Prior to ammonium sulfate precipitation recombinant REG gamma activates the trypsin-like catalytic subunit of the proteasome; afterwards it activates all three catalytic subunits. The expanded activation specificity is accompanied by reduced stability of the REG gamma heptamer providing support for the idea that a "tight" REG gamma heptamer suppresses the proteasome's chymotrypsin-like and postglutamyl-preferring active sites. In an attempt to determine whether REG gamma synthesized in mammalian cells also exhibits restricted activation properties, extracts were prepared from several mammalian organs and cell lines. Surprisingly, endogenous REG gamma was found to be largely monomeric. In an alternate approach, COS7 cells were cotransfected with plasmids expressing FLAG-REG gamma and REG gamma. The expressed FLAG-REG gamma molecules were shown to form oligomers with untagged REG gamma subunits, and the mixed oligomers preferentially activated the proteasome's trypsin-like subunit. Thus, REG gamma molecules synthesized in mammalian cells also exhibit restricted activation properties.     

Alteration of 20S proteasome-subtypes and proteasome activator PA28 in skeletal muscle of rat after induction of diabetes mellitus

Insulin-dependent diabetes mellitus is known to go along with enhanced muscle protein breakdown. Since evidence has been presented that the ubiquitin-proteasome system is significantly involved in muscle wasting under this condition, we have investigated, whether this biological role goes along with alterations of the proteasome system in skeletal muscle of streptozotocin-diabetic rats. Previously, we have found a drop of overall proteasome activity in muscle extracts of rats after induction of diabetes but no change in total amount of 20S proteasome was detected. In the present investigation under the same diabetic conditions we have measured a significant decrease in the amount of proteasome activator PA28, a finding that explains the loss of total proteasome activity. Since increased mRNA levels of proteasome subunits have been measured in muscle tissue of rats after induction of diabetes, we have isolated and purified 20S proteasomes from muscle tissue of control and 6 days diabetic rats. The specific chymotrypsin-like, trypsin-like, and peptidylglutamylpeptide-hydrolysing activities of proteasomes from diabetic and control rats were found to be not significantly different. Therefore, we have fractionated 20S proteasomes into their subtypes and detected that induction of diabetes mellitus effects a redistribution of subtypes of all three proteasome populations but only the increase in subtype V (immuno-subtype) was statistically significant. This altered subtype pattern obviously meets the requirements to the system under wasting conditions. Since this process goes along with de novo biogenesis of 20S proteasomes, it most likely explains the phenomenon of elevated mRNA concentrations of proteasome subunits after induction of diabetes mellitus.     

The proteasome activator 11 S REG or PA28: chimeras implicate carboxyl-terminal sequences in oligomerization and proteasome binding but not in the activation of specific proteasome catalytic subunits

The REG homologs, alpha, beta and gamma, activate mammalian proteasomes in distinct ways. REGalpha and REGbeta activate the trypsin-like, chymotrypsin-like and peptidylglutamyl-preferring active sites, whereas REGgamma only activates the proteasome's trypsin-like subunit. The three REG homologs differ in carboxyl-terminal sequences that are located next to activation loops on their proteasome binding surface. To assess the importance of these carboxyl-terminal sequences in the activation of specific proteasome beta catalytic subunits, we characterized chimeras in which 8 or 12 residues were exchanged among the three proteins. Like the wild-type molecule, REGalpha chimeras activated all three proteasome catalytic subunits regardless of the carboxyl-terminal sequence. However, REGalpha-beta chimeras activated the proteasome at lower concentrations than wild-type REGalpha and higher levels of REGalpha-gamma chimeras were needed for maximal activation because exchanged carboxyl-terminal sequences can stabilize (REGalpha-beta) or destabilize (REGalpha-gamma) the REGalpha heptamer. REGgamma chimeras were equivalent to REGgamma in their activation properties, but they bound the proteasome less tightly than the wild-type molecule. REGbeta chimeras also bound the proteasome more weakly than wild-type REGbeta and were virtually unable to activate it. Our findings demonstrate that the carboxyl-terminal sequences of REG subunits can affect heptamer stability and proteasome affinity, but they do not determine which proteasome beta subunits become activated.     

Immune defects in 28-kDa proteasome activator gamma-deficient mice

Protein complexes of the 28-kDa proteasome activator (PA28) family activate the proteasome and may alter proteasome cleavage specificity. Initial investigations have demonstrated a role for the IFN-gamma-inducible PA28alpha/beta complex in Ag processing. Although the noninducible and predominantly nuclear PA28gamma complex has been implicated in affecting proteasome-dependent signaling pathways, such as control of the mitotic cell cycle, there is no previous evidence demonstrating a role for this structure in Ag processing. We therefore generated PA28gamma-deficient mice and investigated their immune function. PA28gamma(-/-) mice display a slight reduction in CD8+ T cell numbers and do not effectively clear a pulmonary fungal infection. However, T cell responses in two viral infection models appear normal in both magnitude and the hierarchy of antigenic epitopes recognized. We conclude that PA28gamma(-/-) mice, like PA28alpha(-/-)/beta(-/-) mice, are deficient in the processing of only specific Ags.     

Hepatitis B virus HBx peptide 116-138 and proteasome activator PA28 compete for binding to the proteasome alpha4/MC6 subunit

PA28 is a modulator of the 20S proteasome. The PA28 binding sites on the 20S proteasome are still not well defined. Using yeast two-hybrid interaction assays and proteasome inactivation kinetics we provide evidence that the proteasome alpha4 subunit is one of the PA28 binding sites. This finding is supported by the observation that a hepatitis B virus X protein-derived polypeptide habouring the alpha4 proteasome subunit binding motif impairs the activation of 20S proteasomes by PA28.     

20S proteasome, hsp90, p97 fusion protein, PA28 activator copurifying oligomers and ATPase activities

Whether hsp90 acts in an ATP-dependent or independent way is of crucial importance for understanding the molecular mechanism of this chaperone and, to day, the involvement of ATP hydrolysis in hsp90 function is still a controversial subject. ATPase activities may be detected in partially purified hsp90's preparations from rabbit muscle. We demonstrate that the major contaminant associated with hsp90 is the p97 fusion protein and that these oligomeric structures are copurifying together with the 20S proteasome and its PA28 activator. Improving the purification procedure permits to separate hsp90 and p97 to homogeneity. Then, our attempts failed to detect any significant ATPase activity in the hsp90 fraction. Thus, p97 would be principally responsible for the ATPase activity detected in partially purified hsp90 preparations from rabbit muscle.     

PA200, a nuclear proteasome activator involved in DNA repair

We have identified a novel 200 kDa nuclear protein that activates the proteasome. The protein, which we call PA200, has been purified to homogeneity from bovine testis and has been shown to activate proteasomal hydrolysis of peptides, but not proteins. Following gamma-irradiation of HeLa cells the uniform nuclear distribution of PA200 changes to a strikingly punctate pattern, a behavior characteristic of many DNA repair proteins. Homologs of PA200 are present in worms, plants and yeast. Others have shown that mutation of yeast PA200 results in hypersensitivity to bleomycin, and exposure of yeast to DNA damaging agents induces the PA200 message. Taken together, these findings implicate PA200 in DNA repair, possibly by recruiting proteasomes to double strand breaks.