Domain Of P62 Research Articles

P62 Promotes the Mitochondrial Localization of p53 through Its UBA Domain and Participates in Regulating the Sensitivity of Ovarian Cancer Cells to Cisplatin.

Chemotherapeutic drug-induced p53-dependent crosstalk among tumor cells affects the sensitivity of tumor cells to chemotherapeutic drugs, contributing to chemoresistance. Therefore, pharmacological targeting of p53 may contribute to overcoming drug resistance. The localization of p53 is closely related to its function. Thus, we assessed the effect of p62 on the coordination of p53 mitochondrial localization under chemotherapeutic drug treatment in ovarian cancer cells. We found that the combined use of the proteasome inhibitor epoxomicin and cisplatin led to the accumulation of p53 and sequestosome1(p62) in the mitochondria, downregulated mitochondrial DNA (mtDNA) transcription, inhibited mitochondrial functions, and ultimately promoted apoptosis by enhancing cisplatin sensitivity in ovarian cancer cells. Moreover, the ubiquitin-associated (UBA) domain of p62 was involved in regulating the mitochondrial localization of p53. Our findings suggest that the interaction between p62 and p53 may be a mechanism that determines the fate of tumor cells. In conclusion, p62 coordinated the mitochondrial localization of p53 through its UBA domain, inhibited mtDNA transcription, downregulated mitochondrial function, and promoted ovarian cancer cell death. Our study demonstrates the important role of p53 localization in tumor cell survival and apoptosis, and provides new insights into understanding the anti-tumor mechanism of targeting the ubiquitin–proteasome system in tumor cells.

An Experimentally Induced Mutation in the UBA Domain of p62 Changes the Sensitivity of Cisplatin by Up-Regulating HK2 Localisation on the Mitochondria and Increasing Mitophagy in A2780 Ovarian Cancer Cells.

The study of cisplatin sensitivity is the key to the development of ovarian cancer treatment strategies. Mitochondria are one of the main targets of cisplatin, its self-clearing ability plays an important role in determining the fate of ovarian cancer cells. First, we proved that the sensitivity of ovarian cancer cells to cisplatin depends on mitophagy, and p62 acts as a broad autophagy receptor to regulate this process. However, p62′s regulation of mitophagy does not depend on its location on the mitochondria. Our research shows that the mutation of the UBA domain of p62 increases the localisation of HK2 on the mitochondria, thereby increasing the phosphorylated ubiquitin form of parkin, then stabilising the process of mitophagy and ultimately cell survival. Collectively, our results showed that a mutation in the UBA domain of p62 regulates the level of apoptosis stimulated by cisplatin in ovarian cancer.

UXT chaperone prevents proteotoxicity by acting as an autophagy adaptor for p62-dependent aggrephagy

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.

R-catcher, a potent molecular tool to unveil the arginylome

Protein arginylation is a critical regulator of a variety of biological processes. The ability to uncover the global arginylation pattern and its associated signaling pathways would enable us to identify novel disease targets. Here, we report the development of a tool able to capture the N-terminal arginylome. This tool, termed R-catcher, is based on the ZZ domain of p62, which was previously shown to bind N-terminally arginylated proteins. Mutating the ZZ domain enhanced its binding specificity and affinity for Nt-Arg. R-catcher pulldown coupled to LC–MS/MS led to the identification of 59 known and putative arginylated proteins. Among these were a subgroup of novel ATE1-dependent arginylated ER proteins that are linked to diverse biological pathways, including cellular senescence and vesicle-mediated transport as well as diseases, such as Amyotrophic Lateral Sclerosis and Alzheimer’s disease. This study presents the first molecular tool that allows the unbiased identification of arginylated proteins, thereby unlocking the arginylome and provide a new path to disease biomarker discovery.

HDAC6, A Novel Cargo for Autophagic Clearance of Stress Granules, Mediates the Repression of the Type I Interferon Response During Coxsackievirus A16 Infection.

Autophagic cargoes ensure selective autophagy for the recognition and removal of various cytosolic aggregated proteins, damaged organelles, or pathogens. Stress granules (SGs), as antiviral immune complexes, serve a positive role in the type I interferon (IFN) response and can be targeted by autophagy (termed granulophagy). However, the cargo of granulophagy remains elusive, and it is still unknown whether granulophagy plays a role in viral infection. Here, we found that histone deacetylase 6 (HDAC6), a component of viral RNA-induced SGs, is a novel granulophagic cargo that is recognized by p62/Sequestosome 1 (SQSTM1) and mediates the degradation of SGs in coxsackievirus A16 (CA16)-infected cells. CA16 viral RNA activated the protein kinase RNA-activated (PKR)/eukaryotic translation initiation factor 2-alpha (eIF2α) pathway to promote SG assembly. The SGs were degraded by CA16-triggered autophagy via the interaction between the ubiquitin-associated (UBA) domain of p62 and the ubiquitin-binding domain (UBD) of HDAC6, which was bridged by a poly-ubiquitin chain. We also found that granulophagy repressed the type I interferon response and facilitated viral replication. These results suggest that HDAC6 might be the first identified granulophagic cargo and granulophagy could be a strategy that viruses apply to repress the antiviral immune response.

Targeting the p62-ZZ/N-End Rule Pathway in Multiple Myeloma Overcomes Proteasome Inhibitor-Resistance Via Induction of Necroptosis and Enhances the Bone Anabolic Effects of Proteasome Inhibitors

Multiple myeloma (MM) is incurable and 80% of MM patients develop MM bone disease (MMBD). MMBD lesions do not heal due to the persistent suppression of bone formation, which markedly increases mortality and contributes to MM drug resistance. Current treatments for MMBD, such as bisphosphonates and denosumab, target bone destruction but do not result in new bone formation. Although proteasome inhibitors (PIs) have greatly improved survival of MM patients, they only have transient bone anabolic effects. Further, development of drug resistance to PIs remains a major clinical problem. We previously showed that the ZZ domain of p62 (sequestosome-1),plays an important role in both MM growth and suppression of osteoblast (OB) differentiation in the MM microenvironment, by regulating multiple signaling pathways and acting as a cargo-receptor for autophagy. Recently, our collaborators showed that the p62-ZZ is a high-affinity N-recognin of the N-end rule pathway (NERP). p62-ZZ also serves as the molecular switch for necroptotic versus apoptotic cell death pathways. We previously reported that MM cells or TNFα prevent OB differentiation by inducing persistent epigenetic repression of the Runx2-P1 promoter in MM patient bone marrow stromal cells (BMSCs), via the transcriptional repressor Gfi1. We found that blocking p62-ZZ by saturating it with a novel synthetic p62-ZZ/NERP competing ligand, XRK3F2, prevented and reversed MM-induced Gfi1 occupancy at the Runx2-P1 promoter, allowing BMSCs to increase OB marker gene expression and to mineralize. These results suggest that targeting the p62-ZZ/N-end rule pathway would enhance the bone anabolic effects of PIs. To test this hypothesis, we first exposed normal OBs to different doses of bortezomib (Btz) or XRK3F2 or their combination. The combination significantly increased OB differentiation markers Runx2 (60%), Osterix (20%) and ATF4 (60%), and induced mineral deposition compared with either drug alone. The combination also blocked TNFα up-regulation of Gfi1 and suppression of OB differentiation. Interestingly, none of the concentrations tested decreased OB viability. Studies with MM patient-BMSCs showed that XRK3F2 reversed suppression of OB differentiation induced by MM cells, allowing them to mineralize. Importantly, our preliminary in vivo data showed that administration of XRK3F2 to mice with established MM induced dramatic cortical bone formation in MM-containing bones but had no effect on tumor burden. We and others previously showed that MM cells subjected to sustained proteasomal inhibition, rely on p62-mediated autophagic degradation to reduce the proteotoxic load caused by excessive immunoglobulin synthesis. We recently found that targeting the p62-ZZ domain in human MM cells, increases Btz-induced MM cell death, independently of their p53 status. The combination also significantly reduced cell viability in Btz resistant cells although no caspase 3 activation was observed, suggesting a caspase-3 independent cell death. To determine the mechanism(s) responsible for MM cell death induced by the combination, we pretreated MM cell lines and primary CD138+ MM cells with Z-DEVD (20μM), bafilomycin (Baf, 40nM), or necrostatin1 (NEC1, 50μM). The anti-MM effects of XRK3F2 or Btz+XRK3F2 were fully blocked by NEC1, an inhibitor of necroptosis, but not by inhibitors of caspase-dependent apoptosis (Z-DEVD) or autophagy (Baf), supporting that p62-ZZ regulates necroptosis in MM cells. RNAseq analysis of the additive effect of Btz+XRK3F2 on gene expression showed a total of 583 differentially regulated genes, including 374 significantly down-regulated and 209 significantly upregulated. GO term analysis of up-regulated DEGs identified an enrichment in the endoplasmic reticulum (ER) stress and ER unfolded protein response, and regulation of transcription in response to stress and autophagy. In summary, our results demonstrate that targeting the p62-ZZ/N-end rule pathway in combination with PIs in MM significantly reduces MM cell viability by activating multiple death pathway and overcomes PI-resistance of MM cells. In addition, targeting the p62-ZZ in OBs potentiates the bone anabolic action of PIs and reverses the persistent OB suppression induced by MM cells to allow bone formation. Thus, p62-ZZ plays a critical role in MM and bone cells and identifies p62-ZZ as an important molecular target for the treatment of MMBD. Disclosures Xie: Oxis Biotech: Consultancy; ID4Pharma: Other: Founder. Roodman:Amgen: Membership on an entity's Board of Directors or advisory committees.

Enhanced Binding Affinity via Destabilization of the Unbound State: A Millisecond Hydrogen-Deuterium Exchange Study of the Interaction between p53 and a Pleckstrin Homology Domain.

The incorporation of intrinsically disordered domains enables proteins to engage a wide variety of targets, with phosphorylation often modulating target specificity and affinity. Although phosphorylation can clearly act as a chemical driver of complexation in structured proteins, e.g., by abrogating or permitting new charge-charge interactions, the basis for enhancement of the hydrophobically driven interactions that are typical of disordered protein-target complexation is less clear. To determine how phosphorylation can positively impact target recruitment in disordered domains, we have examined the interaction between the disordered N-terminal transactivation domain (TAD) of p53 and the pleckstrin homology (PH) domain of p62. Using time-resolved electrospray ionization with hydrogen-deuterium exchange, we demonstrate that phosphorylation has little effect on the conformation of the p53 TAD when it is bound to the PH domain but instead increases the degree of conformational disorder in the unbound state. We propose that this increase in the degree of disorder creates a wider free energy gap between the free and bound states, providing a target-independent mechanism for enhanced binding when the phosphorylated and unphosphorylated p53-target complexes have similar free energies.

The Interaction Mode of the Acidic Region of the Cell Cycle Transcription Factor DP1 with TFIIH

The heterodimeric transcription factor E2F1-DP1 plays crucial roles in coordinating gene expression during G1/S cell cycle progression. For transcriptional activation, the transactivation domain (TAD) of E2F1 is known to interact with the TATA-binding protein of TFIID and the p62 subunit of TFIIH. It is generally believed that DP1 facilitates E2F1 binding to target DNA and does not possess a TAD. Here, we show that an acidic region of DP1, whose function has remained elusive, binds to the plekstrin homology (PH) domain of p62 with higher affinity than that of E2F1 and contributes to transcriptional activation. The structure of the complex revealed that DP1 forms a twisted U-shaped, string-like conformation and binds to the surface of the PH domain by anchoring Phe403 into a pocket in the PH domain. The transcriptional activity of E2F1-DP1 was reduced when Phe403 of DP1 was mutated. These findings indicate that the acidic region of DP1 acts as a TAD by contacting TFIIH.

Renal cell carcinoma escapes death by p53 depletion through transglutaminase 2-chaperoned autophagy.

In renal cell carcinoma, transglutaminase 2 (TGase 2) crosslinks p53 in autophagosomes, resulting in p53 depletion and the tumor's evasion of apoptosis. Inhibition of TGase 2 stabilizes p53 and induces tumor cells to enter apoptosis. This study explored the mechanism of TGase 2-dependent p53 degradation. We found that TGase 2 competes with human double minute 2 homolog (HDM2) for binding to p53; promotes autophagy-dependent p53 degradation in renal cell carcinoma (RCC) cell lines under starvation; and binds to p53 and p62 simultaneously without ubiquitin-dependent recognition of p62. The bound complex does not have crosslinking activity. A binding assay using a series of deletion mutants of p62, p53 and TGase 2 revealed that the PB1 (Phox and Bem1p-1) domain of p62 (residues 85–110) directly interacts with the β-barrel domains of TGase 2 (residues 592–687), whereas the HDM2-binding domain (transactivation domain, residues 15–26) of p53 interacts with the N terminus of TGase 2 (residues 1–139). In addition to the increase in p53 stability due to TGase 2 inhibition, the administration of a DNA-damaging anti-cancer drug such as doxorubicin-induced apoptosis in RCC cell lines and synergistically reduced tumor volume in a xenograft model. Combination therapy with a TGase 2 inhibitor and a DNA-damaging agent may represent an effective therapeutic approach for treating RCC.

Selective autophagic receptor p62 regulates the abundance of transcriptional coregulator ARIP4 during nutrient starvation.

Transcriptional coregulators contribute to several processes involving nuclear receptor transcriptional regulation. The transcriptional coregulator androgen receptor-interacting protein 4 (ARIP4) interacts with nuclear receptors and regulates their transcriptional activity. In this study, we identified p62 as a major interacting protein partner for ARIP4 in the nucleus. Nuclear magnetic resonance analysis demonstrated that ARIP4 interacts directly with the ubiquitin-associated (UBA) domain of p62. ARIP4 and ubiquitin both bind to similar amino acid residues within UBA domains; therefore, these proteins may possess a similar surface structure at their UBA-binding interfaces. We also found that p62 is required for the regulation of ARIP4 protein levels under nutrient starvation conditions. We propose that p62 is a novel binding partner for ARIP4, and that its binding regulates the cellular protein level of ARIP4 under conditions of metabolic stress.

Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62

Atypical protein kinase C (aPKC) enzymes signal on protein scaffolds, yet how they are maintained in an active conformation on scaffolds is unclear. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of the atypical PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity; however, we have previously shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu-Zhang, A. X., Schramm, C. L., Nabavi, S., Malinow, R., and Newton, A. C. (2012) J. Biol. Chem. 287, 12879-12885). Here we sought to identify a bona fide target of ZIP and, in so doing, unveiled a novel mechanism by which aPKCs are maintained in an active conformation on a protein scaffold. Specifically, we used protein-protein interaction network analysis, structural modeling, and protein-protein docking to predict that ZIP binds an acidic surface on the Phox and Bem1 (PB1) domain of p62, an interaction validated by peptide array analysis. Using a genetically encoded reporter for PKC activity fused to the p62 scaffold, we show that ZIP inhibits the activity of wild-type aPKC, but not a construct lacking the pseudosubstrate. These data support a model in which the pseudosubstrate of aPKCs is tethered to the acidic surface on p62, locking aPKC in an open, signaling-competent conformation. ZIP competes for binding to the acidic surface, resulting in displacement of the pseudosubstrate of aPKC and re-engagement in the substrate-binding cavity. This study not only identifies a cellular target for ZIP, but also unveils a novel mechanism by which scaffolded aPKC is maintained in an active conformation.

Crystal Structure of the Ubiquitin-associated (UBA) Domain of p62 and Its Interaction with Ubiquitin

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.

1P-043 溶液中における単量体・二量体平衡にあるp62のユビキチン結合ドメインの解析(蛋白質・構造機能相関(1),第46回日本生物物理学会年会)

1P-043 溶液中における単量体・二量体平衡にあるp62のユビキチン結合ドメインの解析(蛋白質・構造機能相関(1),第46回日本生物物理学会年会)

NMR structure of the ubiquitin associated (UBA) domain of p62 (SQSTM1) in complex with ubiquitin. RDC refined

NMR structure of the ubiquitin associated (UBA) domain of p62 (SQSTM1) in complex with ubiquitin. RDC refined

NMR structure of the ubiquitin associated (UBA) domain of p62 (SQSTM1). RDC refined

NMR structure of the ubiquitin associated (UBA) domain of p62 (SQSTM1). RDC refined