Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies

p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.     

Degradation of Some Polyubiquitinated Proteins Requires an Intrinsic Proteasomal Binding Element in the Substrates

Lysine 48-linked polyubiquitin chains usually target proteins for 26 S proteasomal degradation; however, this modification is not a warrant for destruction. Here, we found that efficient degradation of a physiological substrate UbcH10 requires not only an exogenous polyubiquitin chain modification but also its unstructured N-terminal region. Interestingly, the unstructured N-terminal region of UbcH10 directly binds the 19 S regulatory complex of the 26 S proteasome, and it mediates the initiation of substrate translocation. To promote ubiquitin- dependent degradation of the folded domains of UbcH10, its N-terminal region can be displaced by exogenous proteasomal binding elements. Moreover, the unstructured N-terminal region can initiate substrate translocation even when UbcH10 is artificially cyclized without a free terminus. Polyubiquitinated circular UbcH10 is completely degraded by the 26 S proteasome. Accordingly, we propose that degradation of some polyubiquitinated proteins requires two binding interactions: a polyubiquitin chain and an intrinsic proteasomal binding element in the substrates (likely an unstructured region); moreover, the intrinsic proteasomal binding element initiates substrate translocation regardless of its location in the substrates.     

Autophagy blockage promotes the pyroptosis of ox-LDL-treated macrophages by modulating the p62/Nrf2/ARE axis

Journal of Physiology and Biochemistry - Atherosclerosis, a chronic comprehensive cardiovascular disease, is characterized by the lipid infiltration, formation of foam cells derived from...     

Regulation of Autophagy by the p300 Acetyltransferase

Autophagy is a regulated process of intracellular catabolism required for normal cellular maintenance, as well as serving as an adaptive response under various stress conditions, including starvation. The molecular regulation of autophagy in mammalian cells remains incompletely understood. Here we demonstrate a role for protein acetylation in the execution and regulation of autophagy. In particular, we demonstrate that the p300 acetyltransferase can regulate the acetylation of various known components of the autophagy machinery. Knockdown of p300 reduces acetylation of Atg5, Atg7, Atg8, and Atg12, although overexpressed p300 increases the acetylation of these same proteins. Furthermore, p300 and Atg7 colocalize within cells, and the two proteins physically interact. The interaction between p300 and Atg7 is dependent on nutrient availability. Finally, we demonstrate that knockdown of p300 can stimulate autophagy, whereas overexpression of p300 inhibits starvation-induced autophagy. These results demonstrate a role for protein acetylation and particularly p300 in the regulation of autophagy under conditions of limited nutrient availability.Macro-autophagy, herein referred to as autophagy, is an evolutionary conserved process first characterized in lower organisms (1). In yeast, over 20 separate genes (designated ATG1, ATG2, etc.) have been demonstrated to be essential to carry out the autophagy program. This process is thought to provide a mechanism for the efficient removal of both long lived proteins and damaged cellular organelles. This regulated degradation provides several essential functions for the cell. First, it allows for the removal of damaged and potentially harmful cellular contents. In addition, in breaking down various intracellular components, the autophagy process provides essential building blocks for the cell to use in the re-synthesis of necessary macromolecules. To accomplish this recycling effort, the coordinated actions of various Atg gene products are required. In particular, the Atg gene products together orchestrate the formation of a double membrane structure known as the autophagosome that engulfs the intended cellular cargo targeted for degradation. The autophagosome eventually fuses with the vacuole in yeast or the lysosome in mammals.In both yeast and mammalian cells, autophagy can be stimulated by the withdrawal of nutrients. Under these conditions, autophagic degradation of nonessential components may be essential to meet ongoing energetic needs in the presence of limited extracellular nutrients. This point was underscored by the analysis of mice containing a targeted deletion of Atg5 (2). In the absence of Atg5, there is a lack of both basal and starvation-induced autophagy. Mice lacking Atg5 are born normally but succumb within the 1st day of life. This post-natal lethality is thought to be due in large part for the requirement of autophagy to supply the energetic needs of neonates. These needs are particularly critical during the small window of time where the animal no longer has a placental circulation and before the pup can begin to nurse and thus obtain external nutrients.Relatively little is known regarding how signals such as nutrient availability are able to be transduced to ultimately regulate the level of cellular autophagy. One important pathway that impinges on the process is signaling thorough the target of rapamycin (TOR)2 network (3). Evidence suggests that TOR signaling inhibits autophagy, and indeed agents such as rapamycin that can inhibit TOR are known to result in increased autophagy. We recently have observed that in addition to this mode of regulation, the NAD-dependent deacetylase Sirt1 is also a regulator of autophagy in mammalian cells and tissues (4). In particular, we demonstrated that in the absence of Sirt1 levels of acetylation for various components of the autophagy machinery are increased and that starvation-induced autophagy is impaired. Interestingly, like the Atg5 knock-out animals, Sirt1^-/- mice are also born normally but die within the few hours to days after birth. Consistent with a defect in autophagy, electron micrographs of hearts from Sirt1^-/- mice demonstrated an accumulation of abnormal appearing organelles, including mitochondria, a phenotype previously observed in Atg-deficient animals (5). Here we have further characterized the role of acetylation in the regulation of autophagy, and in particular, we demonstrate a role for the p300 acetyltransferase in this process.     

p62 Plays a Protective Role in the Autophagic Degradation of Polyglutamine Protein Oligomers in Polyglutamine Disease Model Flies

Oligomer formation and accumulation of pathogenic proteins are key events in the pathomechanisms of many neurodegenerative diseases, such as Alzheimer disease, ALS, and the polyglutamine (polyQ) diseases. The autophagy-lysosome degradation system may have therapeutic potential against these diseases because it can degrade even large oligomers. Although p62/sequestosome 1 plays a physiological role in selective autophagy of ubiquitinated proteins, whether p62 recognizes and degrades pathogenic proteins in neurodegenerative diseases has remained unclear. In this study, to elucidate the role of p62 in such pathogenic conditions in vivo, we used Drosophila models of neurodegenerative diseases. We found that p62 predominantly co-localizes with cytoplasmic polyQ protein aggregates in the MJDtr-Q78 polyQ disease model flies. Loss of p62 function resulted in significant exacerbation of eye degeneration in these flies. Immunohistochemical analyses revealed enhanced accumulation of cytoplasmic aggregates by p62 knockdown in the MJDtr-Q78 flies, similarly to knockdown of autophagy-related genes (Atgs). Knockdown of both p62 and Atgs did not show any additive effects in the MJDtr-Q78 flies, implying that p62 function is mediated by autophagy. Biochemical analyses showed that loss of p62 function delays the degradation of the MJDtr-Q78 protein, especially its oligomeric species. We also found that loss of p62 function exacerbates eye degeneration in another polyQ disease fly model as well as in ALS model flies. We therefore conclude that p62 plays a protective role against polyQ-induced neurodegeneration, by the autophagic degradation of polyQ protein oligomers in vivo, indicating its therapeutic potential for the polyQ diseases and possibly for other neurodegenerative diseases.     

Effect of Sequestosome-1/p62 Expression on Autophagy of Human Periodontal Ligament Fibroblasts Induced by Porphyromonas gingivalis

Russian Journal of Bioorganic Chemistry - Porphyromonas gingivalis is the main pathogen of periodontitis. The purpose of this study was to analyze the effect of p62 protein on P. gingivalis-induced...     

Oligomers of the cytoplasmic domain of the p62/E2 membrane protein of Semliki Forest virus bind to the nucleocapsid in vitro.

We analyzed the interaction between the nucleocapsid and synthetic peptides corresponding to the complete or truncated cytoplasmic protein domain of the Semliki Forest virus p62/E2 glycoprotein. We found that the peptide corresponding to the full-length domain efficiently bound nucleocapsids when coupled to a solid matrix via specific antibodies, whereas the shorter one did not. In solution, a substantial fraction of the full-length peptide associated into oligomers. Binding studies showed that it was mostly these oligomers, rather than the monomeric form of the peptide, which were able to interact with the nucleocapsid. Thus, our findings demonstrate a direct interaction between the spike proteins and the viral nucleocapsid. Furthermore, they suggest that this interaction is directed through formation of complexes containing several p62 or E2 subunits.     

Endogenous Inhibitor of Ligand Binding to the Muscarinic Acetylcholine Receptor

An endogenous inhibitor of L-[3H]quinuclinidinyl benzilate binding to the brain muscarinic acetylcholine receptor was identified. [3H]Quinuclinidinyl benzilate binding to rat brain synaptosomes was measured using a filtration assay. The inhibitor was prepared from several calf tissues and was found in highest specific activity in thymus. The loss of binding activity was slow, requiring a 30-40 min preincubation of the synaptosomes with the inhibitor, and reversed by removing the inhibitor by washing the membranes. Scatchard analysis of the binding data showed that the inhibition was noncompetitive resulting from both a decrease in affinity and a decrease in the number of binding sites. Zn2+ was required in low concentrations for this effect. Muscarinic acetylcholine receptor in synaptic membranes and in membranes free of most peripheral membrane proteins was still sensitive to inhibition. Preliminary characterization of the inhibitory molecule showed that it is of low molecular weight, moderately heat-stable, and acidic. The inhibitor was inactivated by reagents that are nonspecific for nucleophiles, but not by reagents specific for primary amine or thiol groups.     

Nix Is Critical to Two Distinct Phases of Mitophagy,Reactive Oxygen Species-mediated Autophagy Induction and Parkin-Ubiquitin-p62-mediated Mitochondrial Priming

Damaged mitochondria can be eliminated by autophagy, i.e. mitophagy, which is important for cellular homeostasis and cell survival. Despite the fact that a number of factors have been found to be important for mitophagy in mammalian cells, their individual roles in the process had not been clearly defined. Parkin is a ubiquitin-protein isopeptide ligase able to translocate to the mitochondria that are to be removed. We showed here in a chemical hypoxia model of mitophagy induced by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP) that Parkin translocation resulted in mitochondrial ubiquitination and p62 recruitment to the mitochondria. Small inhibitory RNA-mediated knockdown of p62 significantly diminished mitochondrial recognition by the autophagy machinery and the subsequent elimination. Thus Parkin, ubiquitin, and p62 function in preparing mitochondria for mitophagy, here referred to as mitochondrial priming. However, these molecules were not required for the induction of autophagy machinery. Neither Parkin nor p62 seemed to affect autophagy induction by CCCP. Instead, we found that Nix was required for the autophagy induction. Nix promoted CCCP-induced mitochondrial depolarization and reactive oxygen species generation, which inhibited mTOR signaling and activated autophagy. Nix also contributed to mitochondrial priming by controlling the mitochondrial translocation of Parkin, although reactive oxygen species generation was not involved in this step. Deletion of the C-terminal membrane targeting sequence but not mutations in the BH3 domain disabled Nix for these functions. Our work thus distinguished the molecular events responsible for the different phases of mitophagy and placed Nix upstream of the events.     

Induction of Yersinia enterocolitica Stress Proteins by Phagocytosis with Macrophage

Yersinia enterocolitica is a facultative intracellular pathogen which invades to epithelial cells and survives in phagocytes. Since the internal environment of phagocytes should be stressful conditions for the phagocytosed Yersinia, the bacteria should respond to protect themselves from otherwise lethal results. We analyzed the stress-induced proteins which possibly contribute to survival of Yersinia within the phagocytes. Y. enterocolitica was radiolabeled during the growth in macrophage-like J774-1 cells, and the bacterial proteins were analyzed by two-dimensional gel electrophoresis. At least 16 proteins were selectively induced in response to phagocytosis, and several out of 16 proteins were also induced by heat shock at 42 C or oxidative stresses in vitro. Of those, two major stress proteins were identified to be homologues of DnaK and CRPA by immunoblotting analysis. These results have indicated that Y. enterocolitica exhibits a global stress response to the hostile environment in the phagocytosed macrophage.     

Inhibitor of Apoptosis Proteins Physically Interact with and Block Apoptosis Induced by Drosophila Proteins HID and GRIM

Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183–10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins.     

Ubiquitin Is Covalently Attached to the p6Gag Proteins of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus and to the p12Gag Protein of Moloney Murine Leukemia Virus

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6^Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12^Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6^Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.     

Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.