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.Key words: genomic stability, DNA repair, double-strand breaks, ATM, proteasome, PA28γ (PSME3)     

Characterization of Nuclear Localization Signals (NLSs) and Function of NLSs and Phosphorylation of Serine Residues in Subcellular and Subnuclear Localization of Transformer-2β (Tra2β)

The serine/arginine-rich (SR) proteins are one type of major actors in regulation of pre-mRNA splicing. Their functions are closely related to the intracellular spatial organization. The RS domain and phosphorylation status of SR proteins are two critical factors in determining the subcellular distribution. Mammalian Transformer-2β (Tra2β) protein, a member of SR proteins, is known to play multiple important roles in development and diseases. In the present study, we characterized the subcellular and subnuclear localization of Tra2β protein and its related mechanisms. The results demonstrated that in the brain the nuclear and cytoplasmic localization of Tra2β were correlated with its phosphorylation status. Using deletional mutation analysis, we showed that the nuclear localization of Tra2β was determined by multiple nuclear localization signals (NLSs) in the RS domains. The point-mutation analysis disclosed that phosphorylation of serine residues in the NLSs inhibited the function of NLS in directing Tra2β to the nucleus. In addition, we identified at least two nuclear speckle localization signals within the RS1 domain, but not in the RS2 domain. The nuclear speckle localization signals determined the localization of RS1 domain-contained proteins to the nuclear speckle. The function of the signals did not depend on the presence of serine residues. The results provide new insight into the mechanisms by which the subcellular and subnuclear localization of Tra2β proteins are regulated.     

PA28α overexpressing female mice maintain exploratory behavior and capacity to prevent protein aggregation in hippocampus as they age

With age, protein damage accumulates and increases the risk of age‐related diseases. The proteasome activator PA28αβ is involved in protein damage clearance during early embryogenesis and has demonstrated protective effects against proteinopathy. We have recently discovered that adult female mice overexpressing PA28α (PA28αOE) have enhanced learning and memory, and protein extracts from their hippocampi prevent aggregation more efficiently than wild type. In this study, we investigated the effect of overexpressing PA28α on aging using C57BL/6N×BALB/c F2 hybrid mice. We found that the hippocampal anti‐aggregation effect was maintained in young adult (7 months) to middle‐aged (15 months) and old (22 months) PA28αOE females. While the PA28αOE influence on learning and memory gradually decreased with aging, old PA28αOE females did not display the typical drop in explorative behavior—a behavioral hallmark of aging—but were as explorative as young mice. PA28αOE lowered PA28‐dependent proteasome capacity in both heart and hippocampus, and there was no indication of lower protein damage load in PA28αOE. The life span of PA28αOE was also similar to wild type. In both wild type and PA28αOE, PA28‐dependent proteasome capacity increased with aging in the heart, while 26S and 20S proteasome capacities were unchanged in the timepoints analyzed. Thus, PA28αOE females exhibit improved hippocampal ability to prevent aggregation throughout life and enhanced cognitive capabilities with different behavioral outcomes dependent on age; improved memory at early age and a youth‐like exploration at old age. The cognitive effects of PA28αβ combined with its anti‐aggregation molecular effect highlight the therapeutical potential of PA28αβ in combating proteinopathies.     

High yield bacterial expression and purification of active recombinant PA28αβ complex

The PA28 complexes (also termed REG or 11S complexes) are described as activators of the 20S proteasome, a major intracellular protease in eukaryotic cells. They bind to the ends of the barrel-shaped 20S proteasome, and activate its peptidase activities. The interferon γ inducible PA28αβ, made of the two related subunits PA28α and β, is under sustained investigation as it plays important roles in the production by the proteasome of class I antigen peptides. However, in vitro studies of this complex have been impaired by the difficulty of producing large amount of this protein, mainly due to the poor solubility of its β subunit when expressed in Escherichia coli. Here we describe the construction of a bicistronic vector, allowing simultaneous production of functional human PA28α and β subunits in E. coli. Co-expression of the two proteins allows efficient formation of active PA28αβ complexes, that remain soluble and can be easily purified by regular chromatographic procedures.     

Identification of Amino Acid Residues of ERH Required for Its Recruitment to Nuclear Speckles and Replication Foci in HeLa Cells

ERH is a small, highly evolutionarily conserved nuclear protein of unknown function. Its three-dimensional structure is absolutely unique and it can form a homodimer through a β sheet surface. ERH has been shown to interact, among others, with PDIP46/SKAR and Ciz1. When coexpressed with the latter protein, ERH accumulates in replication foci in the nucleus of HeLa cells. Here, we report that when ERH is coexpressed with PDIP46/SKAR in HeLa cells, it is recruited to nuclear speckles, and identify amino acid residues critical for targeting ERH to both these subnuclear structures. ERH H3A Q9A shows a diminished recruitment to nuclear speckles but it is recruited to replication foci. ERH E37A T51A is very poorly recruited to replication foci while still accumulating in nuclear speckles. Consequently, ERH H3A Q9A E37A T51A is recruited neither to nuclear speckles nor to replication foci. The lack of interactions of these three ERH forms with PDIP46/SKAR and/or Ciz1 was further confirmed in vitro by GST pull-down assay. The residues whose substitutions interfere with the accumulation in nuclear speckles are situated on the β sheet surface of ERH, indicating that only the monomer of ERH can interact with PDIP46/SKAR. Substitutions affecting the recruitment to replication foci map to the other side of ERH, near a long loop between the α1 and α2 helices, thus both the monomer and the dimer of ERH could interact with Ciz1. The construction of the ERH mutants not recruited to nuclear speckles or replication foci will facilitate further studies on ERH actions in these subnuclear structures.     

Proteolysis of the Human DNA Polymerase Delta Smallest Subunit p12 by μ-Calpain in Calcium-Triggered Apoptotic HeLa Cells

Degradation of p12 subunit of human DNA polymerase delta (Pol δ) that results in an interconversion between Pol δ4 and Pol δ3 forms plays a significant role in response to replication stress or genotoxic agents triggered DNA damage. Also, the p12 is readily degraded by human calpain in vitro. However, little has been done for the investigation of its degree of participation in any of the more common apoptosis. Here, we first report that the p12 subunit is a substrate of μ-calpain. In calcium-triggered apoptotic HeLa cells, the p12 is degraded at 12 hours post-induction (hpi), restored thereafter by 24 hpi, and then depleted again after 36 hpi in a time-dependent manner while the other three subunits are not affected. It suggests a dual function of Pol δ by its interconversion between Pol δ4 and Pol δ3 that is involved in a novel unknown apoptosis mechanism. The proteolysis of p12 could be efficiently blocked by both calpain inhibitor ALLN and proteasome inhibitor MG132. In vitro pull down and co-immunoprecipitation assays show that the μ-calpain binds to p12 through the interaction of μ-calpain with Pol δ other three subunits, not p12 itself, and PCNA, implying that the proteolysis of p12 by μ-calpain might be through a Pol δ4/PCNA complex. The p12 cleavage sites by μ-calpain are further determined as the location within a 16-amino acids peptide 28-43 by in vitro cleavage assays. Thus, the p12/Pol δ is a target as a nuclear substrate of μ-calpain in a calcium-triggered apoptosis and appears to be a potential marker in the study of the chemotherapy of cancer therapies.     

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.     

Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.     

Phosphatidic acid regulation of PIPKI is critical for actin cytoskeletal reorganization

Type I phosphatidylinositol-4-phosphate 5-kinase (PIPKI) is the main enzyme generating the lipid second messenger phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which has critical functions in many cellular processes, such as cytoskeletal reorganization, membrane trafficking, and signal transduction. All three members of the PIPKI family are activated by phosphatidic acid (PA). However, how PA regulates the activity and functions of PIPKI have not been fully elucidated. In this study, we identify a PA-binding site on PIPKIγ. Mutation of this site inhibited the PA-stimulated activity and membrane localization of PIPKIγ as well as the formation of actin comets and foci induced by PIPKIγ. We also demonstrate that phospholipase D (PLD) generates a pool of PA involved in PIPKIγ regulation by showing that PLD inhibitors blocked the membrane localization of PIPKIγ and its ability to induce actin cytoskeletal reorganization. Targeting the PIPKIγ PA-binding-deficient mutant to membranes by a membrane localization sequence failed to restore the actin reorganization activity of PIPKIγ, suggesting that PA binding is not only involved in recruiting PIPKIγ to membranes but also may induce a conformational change. Taken together, these results reveal a new molecular mechanism through which PA regulates PIPKI and provides direct evidence that PA is important for the localization and functions of PIPKI in intact cells.     

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.     

Effect of Proline Analogues on Activity of Human Prolyl Hydroxylase and the Regulation of HIF Signal Transduction Pathway

Hypoxia inducible factor 1 (HIF-1) plays a pivotal role in cellular responses to hypoxia. Prolyl hydroxylase 3 (PHD3) degrades HIF-1α under normoxic conditions through the hydroxylation of HIF-1α for proteolysis. Inhibiting PHD3 activity is crucial for up-regulating HIF-1α, thereby acting as a potential target for treating hypoxia-related diseases. In this study, two proline analogues (PA1 and PA2) were screened as PHD3 inhibitors with apparent EC₅₀ values of 1.53 and 3.17 µM respectively, indicating good inhibition potency. Nine proteins, significantly regulated by PA1, were identified using 2-DE coupled with MALDI-TOF/TOF MS. Pyruvate kinase isozymes M1/M2 (PKM) and alpha-enolase 1 (ENO1), which are key modulators of glycolysis, are directly regulated by HIF-1α. Moreover, VEGF, a signal protein stimulating angiogenesis, was strongly promoted by PA1. Our findings suggest that PA1 stabilized HIF-1α as well as up-regulated glycolysis and angiogenesis proteins. Herein, for the first time, we systematically studied proline analogue PA1 as a PHD3 inhibitor, which provides innovative evidence for the treatment of HIF-related diseases.