Unfolded Protein Response and Activated Degradative Pathways Regulation in GNE Myopathy

Although intracellular beta amyloid (Aβ) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.     

The B56γ3 Regulatory Subunit of Protein Phosphatase 2A (PP2A) Regulates S Phase-specific Nuclear Accumulation of PP2A and the G1 to S Transition

Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a scaffold subunit (A), a catalytic subunit (C), and a variable regulatory subunit (B). The regulatory B subunits determine the substrate specificity and subcellular localization of the PP2A holoenzyme. Here, we demonstrate that the subcellular localization of the B56γ3 regulatory subunit is regulated in a cell cycle-specific manner. Notably, B56γ3 becomes enriched in the nucleus at the G₁/S border and in S phase. The S phase-specific nuclear enrichment of B56γ3 is accompanied by increases of nuclear A and C subunits and nuclear PP2A activity. Overexpression of B56γ3 promotes nuclear localization of the A and C subunits, whereas silencing both B56γ2 and B56γ3 blocks the S phase-specific increase in the nuclear localization and activity of PP2A. In NIH3T3 cells, B56γ3 overexpression reduces p27 phosphorylation at Thr-187, concomitantly elevates p27 protein levels, delays the G₁ to S transition, and retards cell proliferation. Consistently, knockdown of endogenous B56γ3 expression reduces p27 protein levels and increases cell proliferation in HeLa cells. These findings demonstrate that the dynamic nuclear distribution of the B56γ3 regulatory subunit controls nuclear PP2A activity, which regulates cell cycle controllers, such as p27, to restrain cell cycle progression, and may be responsible for the tumor suppressor function of PP2A.     

Lipids Modulate the Increase of BK Channel Calcium Sensitivity by the β1 Subunit

Co-expression of the auxiliary β1 subunit with the pore forming α subunit of BK dramatically alters apparent calcium sensitivity. Investigation of the mechanism underlying the increase in calcium sensitivity of BK in smooth muscle has concentrated on the energetic effect of β1′s interaction with α. We take a novel approach, exploring whether β1 modification of calcium sensitivity reflects altered interaction between the channel protein and surrounding lipids. We reconstituted hSlo BK α and BK α+β1 channels into two sets of bilayers. One set contained POPE with POPS, POPG, POPA and POPC, where the length of acyl chains is constant, but surface charge differs. The second set is a series of neutral bilayers formed from DOPE with phosphatidylcholines (PCs) of varying acyl chain lengths: C (14∶1), C (18∶1), C (22∶1) and C (24∶1), and with brain sphingomyelin (SPM), in which surface charge is constant, but bilayer thickness varies. The increase in calcium sensitivity caused by the β1 subunit was preserved in negatively charged lipid bilayers but not in neutral bilayers, indicating that modification of apparent Ca²⁺ sensitivity by β1 is modulated by membrane lipids, requiring negatively charged lipids in the membrane. Moreover, the presence of β1 reduces BK activity in thin bilayers of PC 14∶1 and thick bilayers containing SPM, but has no significant effect on activity of BK in PC 18∶1, PC 22∶1 and PC 24∶1 bilayers. These data suggest that auxiliary β1 subunits fine-tune channel gating not only through direct subunit-subunit interactions but also by modulating lipid-protein interactions.     

Characterization of the Testis-specific Proteasome Subunit α4s in Mammals

The 26 S proteasome is responsible for regulated proteolysis in eukaryotic cells. It is composed of one 20 S core particle (CP) flanked by one or two 19 S regulatory particles. The CP is composed of seven different α-type subunits (α1-α7) and seven different β-type subunits, three of which are catalytic. Vertebrates encode four additional catalytic β subunits that are expressed predominantly in immune tissues and produce distinct subtypes of CPs particularly well suited for the acquired immune system. In contrast, the diversity of α subunits remains poorly understood. Recently, another α subunit, referred to as α4s, was reported. However, little is known about α4s. Here we provide a detailed characterization of α4s and the α4s-containing CP. α4s is exclusively expressed in germ cells that enter the meiotic prophase and is incorporated into the CP in place of α4. A comparison of structural models revealed that the differences in the primary sequences between α4 and α4s are located on the outer surface of the CP, suggesting that α4s interacts with specific molecules via these unique regions. α4s-containing CPs account for the majority of the CPs in mouse sperm. The catalytic β subunits in the α4s-containing CP are β1, β2, and β5, and immunosubunits are not included in the α4s-containing CP. α4s-containing CPs have a set of peptidase activities almost identical to those of α4-containing CPs. Our results provide a basis for understanding the role of α4s and male germ cell-specific proteasomes in mammals.     

Subcellular Distribution and Dynamics of Active Proteasome Complexes Unraveled by a Workflow Combining in Vivo Complex Cross-Linking and Quantitative Proteomics

Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins—which modulate proteasome activity, stability, localization, or substrate uptake—rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells.The proteasome is the proteolytic machinery of the ubiquitin-proteasome system (UPS)¹, the main pathway responsible for degradation of intracellular proteins. As the major cellular protease, the proteasome is a key player in eukaryotic protein homeostasis and dysregulation of the UPS has been involved in neurodegenerative diseases and cancers. Because of this, proteasomes have been identified as therapeutic targets, especially for some cancers (1). Therefore, understanding the structure and function relationship controlling proteasome activity is of major interest in biology.Mammalian proteasomes are composed of a central α7β7β7α7 barrel-shaped catalytic core particle (CP), the 20S proteasome, the structure of which has been determined (2). In cells, the 20S proteasome has been found as an isolated complex, and associated with one or two regulatory particles (RPs) of identical or different protein composition (3). Four RPs have been identified: 19S, PA28αβ, PA28γ, and PA200. The 26S proteasome is a particular complex in which the CP is capped by two 19S RPs, forming a 2.5 MDa complex. Because of a high level of heterogeneity and to the dynamics of the complex, the structure of the mammalian 26S proteasome has yet to be fully determined, but major progress has been made, resulting in a suggested spatial arrangement for the yeast 26S proteasome (4, 5). In the 19S complex, some specific subunits have specialized functions: poly-ubiquitinated (polyUb) substrate recognition, ATP-unfolding, and ubiquitin recycling. These allow ubiquitin-dependent protein degradation. In addition to the RPs, other proteasome interacting proteins (PIPs) bind proteasome complexes and affect their efficiency. These include Ecm29, which plays a role in yeast 26S proteasome assembly and stability (6–8).The CP degrades proteins through three main proteolytic activities, defined as trypsin-like (T-like), chymotrypsin-like (ChT-like), and peptidyl-glutamyl peptide hydrolyzing (PGPH). These activities are exerted by the three beta catalytic subunits, β2, β5, and β1, respectively. An alternative form of the 20S proteasome has been characterized, the immuno-proteasome, where the three standard catalytic subunits are replaced by the so called immuno-subunit counterparts (β2i, β5i, β1i), which can modulate its activity. The proportion of 20S immuno-proteasome varies in different cell types and is increased in cells stimulated by interferon γ (IFNγ) (9, 10). In addition, other 20S proteasome subtypes made up of a mixed assortment of standard catalytic and immuno-subunits were recently described (11). These intermediate 20S proteasome complexes exist in high proportions in many human organs, but also in human tumor cells and dendritic cells. By generating specific antigenic peptides, intermediate 20S proteasome complexes can trigger an immune response (11). Although changes in the CP composition modulate the relative contribution of the cleavage specificity of each catalytic site, overall proteasome activity is drastically increased by association between the CP and RPs.Cell imaging technologies or subcellular fractionation combined with protein blotting techniques have located proteasome complexes in several cellular compartments, mainly the cytosol, nucleus, and associated with the cytoplasmic face of the ER (12). Unlike these antibody-based techniques, quantitative proteomic approaches provide a global view of the cellular distribution of proteins in all their physiological forms (spliced, post-translationally modified, etc.) (13) and have revealed intracellular proteasome relocalization following DNA damage (14). Given the broad function of proteasomes, in quality control, antigenic peptide generation, or short-lived protein-tuned regulation, the cell is likely to adapt proteasome plasticity and dynamics to meet specific subcellular needs or to respond to stress or other stimuli. However, the precise intracellular subunit composition and distribution of proteasome complexes remains largely undetermined. This could be explained by the highly dynamic state of proteasome complexes, their heterogeneity and instability, which make them inherently difficult to study. To deal with this, efficient strategies are needed to purify and quantify fully assembled, active proteasome complexes in homogeneous cellular fractions. These strategies will help us to understand how cells adapt proteasome activity to their needs.In vivo formaldehyde cross-linking can be an efficient tool to study protein–protein interactions and cellular networks (15). It has recently been used to stabilize labile proteasome complexes, allowing the study of the proteasome network in yeast (16) and human cells (17) by quantitative proteomic analyses.In this article, we describe an integrated strategy combining in vivo cross-linking, efficient cell fractionation, affinity purification, and robust label-free quantitative proteomics. We have used this strategy to determine the intracellular distribution of fully assembled active proteasome complexes in human leukemic cells for the first time. Following IFNγ stimulation, our strategy also revealed recruitment of specific PIPs (known to participate in the UPS) to microsomal proteasome complexes. This suggests an important role for these complexes in the endoplasmic reticulum associated degradation (ERAD) pathway.     

PAC1 Gene Knockout Reveals an Essential Role of Chaperone-Mediated 20S Proteasome Biogenesis and Latent 20S Proteasomes in Cellular Homeostasis

The 26S proteasome, a central enzyme for ubiquitin-dependent proteolysis, is a highly complex structure comprising 33 distinct subunits. Recent studies have revealed multiple dedicated chaperones involved in proteasome assembly both in yeast and in mammals. However, none of these chaperones is essential for yeast viability. PAC1 is a mammalian proteasome assembly chaperone that plays a role in the initial assembly of the 20S proteasome, the catalytic core of the 26S proteasome, but does not cause a complete loss of the 20S proteasome when knocked down. Thus, both chaperone-dependent and -independent assembly pathways exist in cells, but the contribution of the chaperone-dependent pathway remains unclear. To elucidate its biological significance in mammals, we generated PAC1 conditional knockout mice. PAC1-null mice exhibited early embryonic lethality, demonstrating that PAC1 is essential for mammalian development, especially for explosive cell proliferation. In quiescent adult hepatocytes, PAC1 is responsible for producing the majority of the 20S proteasome. PAC1-deficient hepatocytes contained normal amounts of the 26S proteasome, but they completely lost the free latent 20S proteasome. They also accumulated ubiquitinated proteins and exhibited premature senescence. Our results demonstrate the importance of the PAC1-dependent assembly pathway and of the latent 20S proteasomes for maintaining cellular integrity.The 26S proteasome is a eukaryotic ATP-dependent protease responsible for the degradation of proteins tagged with polyubiquitin chains (21). The ubiquitin-dependent proteolysis by the proteasome plays a pivotal role in various cellular processes by catalyzing the selective degradation of short-lived regulatory proteins as well as damaged proteins. Thus, the proteasome is essential for the viability of all eukaryotic cells.The 26S proteasome is a large protein complex consisting of two portions; one is the catalytic 20S proteasome of approximately 700 kDa (also called the 20S core particle), and the other is the 19S regulatory particle (RP; also called PA700) of approximately 900 kDa, both of which are composed of a set of multiple distinct subunits (70). The 20S proteasome is a cylindrically shaped stack of four heptameric rings, where the outer and inner rings each are composed of seven homologous α subunits (α1 to α7) and seven homologous β subunits (β1 to β7), respectively (5). The proteolytic active sites reside within the central chamber enclosed by the two inner β-rings, while a small channel formed by the outer α-ring, which is primarily closed, restricts the access of native proteins to the catalytic chamber. Thus, the 20S proteasome is a latent enzyme. Appending 19S RP, which consists of 19 different subunits, to the α-ring enables the 20S proteasome to degrade native proteins; 19S RP accepts ubiquitin chains of substrate proteins, removes ubiquitin chains while unfolding the substrates, and feeds the substrates into the interior proteolytic chamber of the 20S proteasome through the α-ring that is opened when the C-terminal tails of the ATPase subunits of 19S RP are inserted into the intersubunit spaces of the α-ring (24, 62, 74). However, it also has been reported that some denatured or unstructured proteins can be degraded directly by the 20S proteasome even in the absence of 19S RP and ubiquitination (37, 39).Much attention has been focused on how such a highly elaborate structure is achieved. Recent studies have identified various proteasome-dedicated chaperones that assist in the assembly of the proteasome in eukaryotic cells (23, 40, 56, 57, 65, 66). In yeast, while most of the proteasome subunits are essential for viability, the deletion of any of these chaperones does not cause lethality. In fact, many, if not all, of the deletions exhibit subtle phenotypes. In mammalian cells, although the knockdown of the assembly chaperones reduced proteasome assembly and thus proteasome activity, leading to slow cell growth, the degree of reduction was much lower than that which occurred following the knockdown of the proteasome subunit itself (33, 35, 40). These results indicate that the assembly chaperones play an auxiliary role in proteasome biogenesis.Proteasome assembly chaperone 1 (PAC1) is one of the assembly chaperones originally identified in mammalian cells (34). PAC1 plays a role in α-ring formation that occurs during the initial assembly of the 20S proteasome; it also prevents the aberrant dimerization of the α-ring. As is the case for most assembly chaperones, the knockdown of PAC1 in mammalian cells decreases proteasome activity but to a lesser extent than that in, for example, β2 knockdown (34, 35). Therefore, both PAC1-dependent and -independent assembly pathways exist in cells, but the importance of the PAC1-dependent pathway remains elusive. To further elucidate the biological significance of PAC1 and PAC1-dependent proteasome biogenesis, we generated conditional mouse mutants carrying an inactivating mutation in Psmg1, the gene coding for PAC1 protein, in the whole body, the nervous system, and in the liver. Our results demonstrate that PAC1 is essential for the development of a mouse, and that it plays important roles in maintaining cellular integrity in quiescent tissue. Our study revealed for the first time the importance of chaperone-mediated proteasome biogenesis in a whole-body mammalian system and may provide valuable knowledge in medical drug development targeting proteasomes.     

Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets.Although first described over a century ago, new roles and functions for platelets continue to emerge. Derived by budding from megakaryocytes and devoid of a nucleus, platelets were formerly not thought to produce proteins and their one role was to initiate and perform blood clotting. However, this view has changed in recent years; platelets have mRNA, microRNAs to regulate their mRNA, the machinery to synthesize proteins and they use it (1, 2). Furthermore, in addition to their function in hemostasis, it has been recognized that platelets play a role in inflammatory processes (3, 4). Through their interactions with the endothelium and other blood cells, platelets are believed to play a critical role in defense, wound repair, and more (5). Understanding of many of the new aspects of platelet function is still limited, but these recent advances raise the question of what other features are awaiting discovery that might be hidden in these small cell fragments.There are limited methods available with which to study platelets; DNA-based methods cannot be applied, and although mRNA is present in platelets, its low level only allows for restricted analysis. Mass spectrometry (MS)-based proteomics is particularly well set up to study platelets, and previous studies have analyzed the platelet proteome (6–11), various subproteomes (12–16), and have shed light on aspects of platelet signaling and function (17–21). In this study, proteomic analysis of human platelets was conducted, generating an inventory of platelet proteins, which was then explored by comparison to proteomic data sets of nucleated cells with the aim of identifying new biology-related functions. This approach revealed consistently high expression of the proteasome, the protein complex that is the main protein degradation machinery in cells (Fig. 1). The presence of the proteasome in platelets has been described earlier (22). It is known to be active and its activity increases in response to agonist stimulation (23); however, a detailed analysis of the many subunits of this multimeric complex has not been performed and its role in platelets, which produce less protein than nucleated cells, is not fully understood. The proteasome''s core complex, the 20S proteasome, is composed of 28 nonidentical subunits, arranged in four rings, two comprising of seven α subunits and two of seven β subunits. Three of the β subunits (β1, β2, and β5) are catalytically active. The 20S proteasome forms the 26S proteasome together with the 19S regulator, which contains ATPase subunits and is responsible for the ATP¹ dependence of the 26S proteasome. The immunoproteasome, which is constitutively expressed in cells of the immune system or is synthesized following induction by interferon γ (IFNγ) in all other nucleated cells, is formed when the catalytically active β subunits are replaced by their immunoproteasome counterparts (β1i, β2i, and β5i). IFNγ also up-regulates the 11S regulator, which consists of PA28 α and β subunits, and both the immunoproteasome and the 11S proteasome are thought to be involved in improved peptide generation for major histocompatibility complex (MHC) I antigen presentation (24).Open in a separate windowFig. 1.Composition of the proteasome and immunoproteasome. The standard 20S core (middle) is composed of 28 nonidentical subunits that are arranged in four rings; two composed of seven α subunits and two composed of seven β subunits. Three of the β subunits (β1, β2, and β5) are catalytically active. The 19S regulator is composed of a base, containing six ATPase subunits and two non-ATPase subunits, and a lid, which contains up to ten non-ATPase subunits. The 20S proteasome and two 19S regulators form the 26S proteasome (left). The immunoproteasome, which is induced by IFNγ, contains three different catalytically active subunits (β1i, β2i, and β5i). The 11S regulator, which consists of heptameric complexes containing PA28α and β subunits, is also induced by IFNγ and can replace the 19S regulator (right).Here, discovery of the high expression of the proteasome in our platelet proteomic data set was followed up with traditional biochemical assays to explore in detail the composition of the proteasome in platelets. Not only were all components of the 26S proteasome detected in our global platelet data sets, but immunoproteasome subunits were also identified. We validated that all members of the 20S proteasome were present and assembled in human platelets. Furthermore, we show that the standard as well as the immunoproteasome catalytic subunits are active. The presence of not only active proteasome but active immunoproteasome subunits in platelets opens up the possibility of new roles for these anucleate players, and further illustrates the critical role proteomics plays in improving our understanding of platelet function.     

Transforming Growth Factor β Integrates Smad 3 to Mechanistic Target of Rapamycin Complexes to Arrest Deptor Abundance for Glomerular Mesangial Cell Hypertrophy

In many renal diseases, transforming growth factor β (TGFβ)-stimulated canonical Smad 3 and noncanonical mechanistic target of rapamycin (mTOR) promote increased protein synthesis and mesangial cell hypertrophy. The cellular underpinnings involving these signaling molecules to regulate mesangial cell hypertrophy are not fully understood. Deptor has recently been identified as an mTOR interacting protein and functions as an endogenous inhibitor of the kinase activity for both TORC1 and TORC2. Prolonged incubation of mesangial cells with TGFβ reduced the levels of deptor concomitant with an increase in TORC1 and TORC2 activity. Sustained TGFβ activation was required to inhibit association of deptor with mTOR, whereas rapid activation had no effect. Using the mTOR inhibitor PP242, we found that TGFβ-induced both early and sustained activation of TORC1 and TORC2 was necessary for deptor suppression. PP242-induced reversal of deptor suppression by TGFβ was associated with a significant inhibition of TGFβ-stimulated protein synthesis and hypertrophy. Interestingly, expression of siRNA against Smad 3 or Smad 7, which blocks TGFβ receptor-specific Smad 3 signaling, prevented TGFβ-induced suppression of deptor abundance and TORC1/2 activities. Furthermore, overexpression of Smad 3 decreased deptor expression similar to TGFβ stimulation concomitant with increased TORC1 and TORC2 activities. Finally, knockdown of deptor reversed Smad 7-mediated inhibition of protein synthesis and mesangial cell hypertrophy induced by TGFβ. These data reveal the requirement of both early and late activation of mTOR for TGFβ-induced protein synthesis. Our results support that TGFβ-stimulated Smad 3 acts as a key node to instill a feedback loop between deptor down-regulation and TORC1/2 activation in driving mesangial cell hypertrophy.     

PP1α, PP1β and Wip-1 regulate H4S47 phosphorylation and deposition of histone H3 variant H3.3

Phosphorylation of histone H4 serine 47 (H4S47ph) is catalyzed by Pak2, a member of the p21-activated serine/threonine protein kinase (Pak) family and regulates the deposition of histone variant H3.3. However, the phosphatase(s) involved in the regulation of H4S47ph levels was unknown. Here, we show that three phosphatases (PP1α, PP1β and Wip1) regulate H4S47ph levels and H3.3 deposition. Depletion of each of the three phosphatases results in increased H4S47ph levels. Moreover, PP1α, PP1β and Wip1 bind H3-H4 in vitro and in vivo, whereas only PP1α and PP1β, but not Wip1, interact with Pak2 in vivo. These results suggest that PP1α, PP1β and Wip1 regulate the levels of H4S47ph through directly acting on H4S47ph, with PP1α and PP1β also likely regulating the activity of Pak2. Finally, depletion of PP1α, PP1β and Wip1 leads to increased H3.3 occupancy at candidate genes tested, elevated H3.3 deposition and enhanced association of H3.3 with its chaperones HIRA and Daxx. These results reveal a novel role of three phosphatases in chromatin dynamics in mammalian cells.     

Biochemical Characterization of the 20S Proteasome from the Methanoarchaeon Methanosarcina thermophila

The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native α ring structures and β prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the β prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the α₇ ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr¹ residue with an Ala in the β subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of β subunit active-site Lys³³ with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys³³ in polarization of the Thr¹ N, which serves to strip a proton from the active-site Thr¹ Oγ nucleophile. Replacement of Asp⁵¹ with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.     

N-Terminal α7 Deletion of the Proteasome 20S Core Particle Substitutes for Yeast PI31 Function

The proteasome core particle (CP) is a conserved protease complex that is formed by the stacking of two outer α-rings and two inner β-rings. The α-ring is a heteroheptameric ring of subunits α1 to α7 and acts as a gate that restricts entry of substrate proteins into the catalytic cavity formed by the two abutting β-rings. The 31-kDa proteasome inhibitor (PI31) was originally identified as a protein that binds to the CP and inhibits CP activity in vitro, but accumulating evidence indicates that PI31 is required for physiological proteasome activity. To clarify the in vivo role of PI31, we examined the Saccharomyces cerevisiae PI31 ortholog Fub1. Fub1 was essential in a situation where the CP assembly chaperone Pba4 was deleted. The lethality of Δfub1 Δpba4 was suppressed by deletion of the N terminus of α7 (α7ΔN), which led to the partial activation of the CP. However, deletion of the N terminus of α3, which activates the CP more efficiently than α7ΔN by gate opening, did not suppress Δfub1 Δpba4 lethality. These results suggest that the α7 N terminus has a role in CP activation different from that of the α3 N terminus and that the role of Fub1 antagonizes a specific function of the α7 N terminus.     

Regulation of PSMB5 Protein and β Subunits of Mammalian Proteasome by Constitutively Activated Signal Transducer and Activator of Transcription 3 (STAT3): POTENTIAL ROLE IN BORTEZOMIB-MEDIATED ANTICANCER THERAPY*

The ubiquitin-proteasome system facilitates the degradation of ubiquitin-tagged proteins and performs a regulatory role in cells. Elevated proteasome activity and subunit expression are found in several cancers. However, the inherent molecular mechanisms responsible for increased proteasome function in cancers remain unclear despite the well investigated and defined role of the mammalian proteasome. This study was initiated to elucidate the mechanisms involved in the regulation of β subunits of the mammalian proteasome. Suppression of STAT3 tyrosine phosphorylation coordinately decreased the mRNA and protein levels of the β subunits of the 20 S core complex in DU145 cells. Notably, PSMB5, a molecular target of bortezomib, was shown to be a target of STAT3. Knockdown of STAT3 decreased PSMB5 protein. Inhibition of phospho-STAT3 substantially reduced PSMB5 protein levels in cells expressing constitutively active-STAT3. Accumulation of activated STAT3 resulted in the induction of PSMB5 promoter and protein levels. In addition, a direct correlation was observed between the endogenous levels of PSMB5 and constitutively active STAT3. PSMB5 and STAT3 protein levels remained unaltered following the inhibition of proteasome activity. The EGF-induced concerted increase of β subunits was blocked by inhibition of the EGF receptor or STAT3 but not by the PI3K/AKT or MEK/ERK pathways. Decreased proteasome activities were due to reduced protein levels of catalytic subunits of the proteasome in STAT3-inhibited cells. Combined treatments with bortezomib and inhibitor of STAT3 abrogated proteasome activity and enhanced cellular apoptosis. Overall, we demonstrate that aberrant activation of STAT3 regulates the expression of β subunits, in particular PSMB5, and the catalytic activity of the proteasome.     

Protein Phosphatase 2A Is Regulated by Protein Kinase Cα (PKCα)-dependent Phosphorylation of Its Targeting Subunit B56α at Ser41

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser⁴¹ of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca²⁺ release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser⁴¹. This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca²⁺ homeostasis.     

Interferon-γ Induces Immunoproteasomes and the Presentation of MHC I-Associated Peptides on Human Salivary Gland Cells

A prominent histopathological feature of Sjögren''s syndrome, an autoimmune disease, is the presence of lymphocytic infiltrates in the salivary and lachrymal glands. Such infiltrates are comprised of activated lymphocytes and macrophages, and known to produce multiple cytokines including interferon-gamma (IFN-γ). In this study, we have demonstrated that IFN-γ strongly induces the expression of immunoproteasome beta subunits (β1i, β2i and β5i) and immunoproteasome activity but conversely inhibits the expression of proteasome beta subunits (β1, β2 and β5) in human salivary gland (HSG) cells. Mass spectrometric analysis has revealed potential MHC I-associated peptides on the HSG cells, including a tryptic peptide derived from salivary amylase, due to IFN-γ stimulation. These results suggest that IFN-γ induces immunoproteasomes in HSG cells, leading to enhanced presentation of MHC I-associated peptides on cell surface. These peptide-presenting salivary gland cells may be recognized and targeted by auto-reactive T lymphocytes. We have also found that lactacystin, a proteasome inhibitor, inhibits the expression of β1 subunit in HSG cells and blocks the IFN-γ-induced expression of β1i and immunoproteasome activity. However, the expression of β2i and β5i in HSG cells is not affected by lactacystin. These results may add new insight into the mechanism regarding how lactacystin blocks the action of proteasomes or immunoproteasomes.     

Inhibition of the Host Proteasome Facilitates Papaya Ringspot Virus Accumulation and Proteosomal Catalytic Activity Is Modulated by Viral Factor HcPro

The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant–pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro – PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation.     

Site-1 protease-activated formation of lysosomal targeting motifs is independent of the lipogenic transcription control

Site-1 protease (S1P) cleaves membrane-bound lipogenic sterol regulatory element-binding proteins (SREBPs) and the α/β-subunit precursor protein of the N-acetylglucosamine-1-phosphotransferase forming mannose 6-phosphate (M6P) targeting markers on lysosomal enzymes. The translocation of SREBPs from the endoplasmic reticulum (ER) to the Golgi-resident S1P depends on the intracellular sterol content, but it is unknown whether the ER exit of the α/β-subunit precursor is regulated. Here, we investigated the effect of cholesterol depletion (atorvastatin treatment) and elevation (LDL overload) on ER-Golgi transport, S1P-mediated cleavage of the α/β-subunit precursor, and the subsequent targeting of lysosomal enzymes along the biosynthetic and endocytic pathway to lysosomes. The data showed that the proteolytic cleavage of the α/β-subunit precursor into mature and enzymatically active subunits does not depend on the cholesterol content. In either treatment, lysosomal enzymes are normally decorated with M6P residues, allowing the proper sorting to lysosomes. In addition, we found that, in fibroblasts of mucolipidosis type II mice and Niemann-Pick type C patients characterized by aberrant cholesterol accumulation, the proteolytic cleavage of the α/β-subunit precursor was not impaired. We conclude that S1P substrate-dependent regulatory mechanisms for lipid synthesis and biogenesis of lysosomes are different.