Autophagic Receptor Research Articles

Dusquetide modulates innate immune response through binding to p62

SQSTM1/p62 is an autophagic receptor that plays a major role in mediating stress and innate immune responses. Preclinical studies identified p62 as a target of the prototype innate defense regulator (IDR); however, the molecular mechanism of this process remains unclear. Here, we describe the structural basis and biological consequences of the interaction of p62 with the next generation of IDRs, dusquetide. Both electrostatic and hydrophobic contacts drive the formation of the complex between dusquetide and the ZZ domain of p62. We show that dusquetide penetrates the cell membrane and associates with p62 invivo. Dusquetide binding modulates the p62-RIP1 complex, increases p38 phosphorylation, and enhances CEBP/B expression without activating autophagy. Our findings provide molecular details underlying the IDR action that may help in the development of new strategies to pharmacologically target p62.

Inhibition of autophagic flux by S-nitrosylation of SQSTM1/p62 promotes neuronal secretion and cell-to-cell transmission of SNCA/α-synuclein in Parkinson disease and Lewy body dementia.

Autophagy (in the form of macroautophagy) is the major intracellular protein quality control system for removal of damaged organelles and abnormally aggregated proteins. We and others have shown that dysregulated autophagic pathways contribute to accumulation and spread of misfolded proteins in many neurodegenerative disorders, including Parkinson disease (PD) and Lewy body dementia (LBD). Additionally, generation of excessive reactive oxygen and nitrogen species, such as nitric oxide (NO), accelerates neuronal and synaptic damage mediated, at least in part, via aberrant protein S-nitrosylation. Using cell-based models, including human induced pluripotent stem cell (hiPSC)-derived neurons, CRISPR-Cas9 technology, and transgenic PD/LBD mice, plus vetting in human postmortem brains, we found that S-nitrosylation of the autophagic receptor protein SQSTM1/p62 (forming SNO-SQSTM1/p62) inhibits autophagic flux, thus contributing to accumulation of misfolded SNCA/α-synuclein. Consequently, this impairment in autophagy increases extracellular vesicle-dependent secretion and spread of aggregated SNCA. Taken together, our evidence suggests that aberrant formation of SNO-SQSTM1/p62 represents a pathogenic event contributing not only to inhibition of autophagic flux and potentiation of neuronal damage, but also to propagation of α-synucleinopathy between cells in the diseased brain.

Abstract 522: Macrophage Galectin 3 Protects Against Atherosclerosis By Promoting Repair, Removal And Replacement Of Damaged Lysosomes

Understanding the derangements in cellular processes that underlie macrophage dysfunction is critical to the development of future therapeutics. The autophagy-lysosome system in macrophages plays critical role in the degradation and recycling of long-lived/damaged intracellular material. Atherosclerotic plaque macrophages develop an impairment in autophagy and lysosomes which is a contributing factor to plaque progression. Thus, one can surmise that harnessing programs of lysosomal repair and lysosomal biogenesis in macrophages can have atheroprotective effects. A class of important regulators of the autophagy-lysosomal system are cytosolic lectins. When the lysosome membrane becomes damaged, Galectin-3 (Gal3) recognizes the damage and recruits autophagic receptors and delivers the cargo to autophagosomes. Additionally, Gal3 may recruit the ESCRT complex and facilitate the repair and restoration of lysosomes. We hypothesized that Gal3 provides a protective role in atherogenic macrophages, by repairing or removing damaged lysosomes, thus preventing the relevant deleterious signaling pathways such as inflammasome activation and apoptosis. We utilized Gal3-KO macrophages and mice to study the role of Gal3 in atherosclerosis. First, we show that Gal3-KO macrophages are highly sensitive to lysosome membrane damaging agents including atherogenic oxLDL and cholesterol crystals, exhibiting decreased autophagy and protein aggregate clearance compared to the WT controls. Furthermore, the recruitment of lysosome repair mechanisms involving the ESCRT complex are blunted. Such blunting of critical repair mechanisms results in increased macrophage apoptosis and inflammasome/IL-1β activation. These findings are also recapitulated in vivo, where bone marrow transplants of atherogenic LDLR-null mice with Gal3-deficient bone marrow results in increased plaque size and altered plaque composition with significant macrophage accumulation, apoptosis, and necrotic core formation, characteristic features of advanced plaques. Overall, our data demonstrate important roles for Gal3 in macrophage lysosome repair mechanisms which are a critical response against atherosclerosis.

Secretory autophagy maintains proteostasis upon lysosome inhibition.

The endolysosome system plays central roles in both autophagic degradation and secretory pathways, including the release of extracellular vesicles and particles (EVPs). Although previous work reveals important interconnections between autophagy and EVP-mediated secretion, our understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition, which mediates EVP-associated release of autophagic cargo receptors, including p62/SQSTM1. This secretion is highly regulated and dependent on multiple ATGs required for autophagosome formation, as well as the small GTPase Rab27a. Furthermore, disrupting autophagosome maturation, either via genetic inhibition of autophagosome-to-autolysosome fusion or expression of SARS-CoV-2 ORF3a, is sufficient to induce EVP secretion of autophagy cargo receptors. Finally, ATG-dependent EVP secretion buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Thus, we propose secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis during endolysosomal dysfunction or impaired autophagosome maturation.

Chemical modulation of SQSTM1/p62-mediated xenophagy that targets a broad range of pathogenic bacteria

ABSTRACT The N-degron pathway is a proteolytic system in which the N-terminal degrons (N-degrons) of proteins, such as arginine (Nt-Arg), induce the degradation of proteins and subcellular organelles via the ubiquitin-proteasome system (UPS) or macroautophagy/autophagy-lysosome system (hereafter autophagy). Here, we developed the chemical mimics of the N-degron Nt-Arg as a pharmaceutical means to induce targeted degradation of intracellular bacteria via autophagy, such as Salmonella enterica serovar Typhimurium (S. Typhimurium), Escherichia coli, and Streptococcus pyogenes as well as Mycobacterium tuberculosis (Mtb). Upon binding the ZZ domain of the autophagic cargo receptor SQSTM1/p62 (sequestosome 1), these chemicals induced the biogenesis and recruitment of autophagic membranes to intracellular bacteria via SQSTM1, leading to lysosomal degradation. The antimicrobial efficacy was independent of rapamycin-modulated core autophagic pathways and synergistic with the reduced production of inflammatory cytokines. In mice, these drugs exhibited antimicrobial efficacy for S. Typhimurium, Bacillus Calmette–Guérin (BCG), and Mtb as well as multidrug-resistant Mtb and inhibited the production of inflammatory cytokines. This dual mode of action in xenophagy and inflammation significantly protected mice from inflammatory lesions in the lungs and other tissues caused by all the tested bacterial strains. Our results suggest that the N-degron pathway provides a therapeutic target in host-directed therapeutics for a broad range of drug-resistant intracellular pathogens. Abbreviations: ATG: autophagy-related gene; BCG: Bacillus Calmette–Guérin; BMDMs: bone marrow-derived macrophages; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CFUs: colony-forming units; CXCL: C-X-C motif chemokine ligand; EGFP: enhanced green fluorescent protein; IL1B/IL-1β: interleukin 1 beta; IL6: interleukin 6; LIR: MAP1LC3/LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PB1: Phox and Bem1; SQSTM1/p62: sequestosome 1; S. Typhimurium: Salmonella enterica serovar Typhimurium; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; UBA: ubiquitin-associated.

Vault RNA1-1 riboregulates the autophagic function of p62 by binding to lysine 7 and arginine 21, both of which are critical for p62 oligomerization.

Cellular processes can be regulated at multiple levels, including transcriptional, post-transcriptional, and post-translational mechanisms. We have recently shown that the small, noncoding vault RNA1–1 negatively riboregulates p62 oligomerization in selective autophagy through direct interaction with the autophagic receptor. This function is highly specific for this Pol III transcript, but the determinants of this specificity and a mechanistic explanation of how vault RNA1–1 inhibits p62 oligomerization are lacking. Here, we combine biochemical and functional experiments to answer these questions. We show that the PB1 domain and adjacent linker region of p62 (aa 1–122) are necessary and sufficient for specific vault RNA1–1 binding, and we identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1–1 further reveals a central flexible loop within vault RNA1–1 that is required for the specific interaction with p62. Overall, our data provide molecular insight into how a small RNA riboregulates protein–protein interactions critical to the activation of specific autophagy.

EIF3k inhibits NF-κB signaling by targeting MyD88 for ATG5-mediated autophagic degradation in teleost fish

Optimal activation of NF-κB signaling is crucial for the initiation of inflammatory responses and eliminating invading bacteria. Bacteria have likewise evolved the ability to evade immunity; however, mechanisms by which bacteria dysregulate host NF-κB signaling are unclear. In this study, we identify eukaryotic translation initiation factor eIF3k, a nonessential member of the eIF3 translation initiation complex, as a suppressor of the NF-κB pathway. Mechanistically, we show that eIF3k expression induced by Vibrio harveyi enhances E3 ligase Nrdp1-mediated K27-linked ubiquitination of MyD88, an upstream regulator of NF-κB pathway activation. Furthermore, we show that eIF3k acts as a bridge linking ubiquitin-tagged MyD88 and ATG5, an important mediator of autophagy. We demonstrate that the MyD88-eIF3k-ATG5 complex is transported to the autophagosome for degradation, and that innate immune signaling is subsequently terminated and does not attack invading V. harveyi. Therefore, our study identifies eIF3k as a specific inhibitor of the MyD88-dependent NF-κB pathway and suggests that eIF3k may act as a selective autophagic receptor that synergizes with ATG5 to promote the autophagic degradation of MyD88, which helps V. harveyi to evade innate immunity. We conclude that V. harveyi can manipulate a host's autophagy process to evade immunity in fish and also provide a new perspective on mammalian resistance to bacterial invasion.

Komagataella phaffii Cue5 Piggybacks on Lipid Droplets for Its Vacuolar Degradation during Stationary Phase Lipophagy.

Recently, we developed Komagataella phaffii (formerly Pichia pastoris) as a model for lipophagy, the selective autophagy of lipid droplets (LDs). We found that lipophagy pathways induced by acute nitrogen (N) starvation and in stationary (S) phase have different molecular mechanisms. Moreover, both types of lipophagy are independent of Atg11, the scaffold protein that interacts with most autophagic receptors and, therefore, is essential for most types of selective autophagy in yeast. Since yeast aggrephagy, the selective autophagy of ubiquitinated protein aggregates, is also independent of Atg11 and utilizes the ubiquitin-binding receptor, Cue5, we studied the relationship of K. phaffii Cue5 with differentially induced LDs and lipophagy. While there was no relationship of Cue5 with LDs and lipophagy under N-starvation conditions, Cue5 accumulated on LDs in S-phase and degraded together with LDs via S-phase lipophagy. The accumulation of Cue5 on LDs and its degradation by S-phase lipophagy strongly depended on the ubiquitin-binding CUE domain and Prl1, the positive regulator of lipophagy 1. However, unlike Prl1, which is required for S-phase lipophagy, Cue5 was dispensable for it suggesting that Cue5 is rather a new substrate of this pathway. We propose that a similar mechanism (Prl1-dependent accumulation on LDs) might be employed by Prl1 to recruit another ubiquitin-binding protein that is essential for S-phase lipophagy.

Mechanistic insights into an atypical interaction between ATG8 and SH3P2 in Arabidopsis thaliana

ABSTRACT In selective macroautophagy/autophagy, cargo receptors are recruited to the forming autophagosome by interacting with Atg8 (autophagy-related 8)-family proteins and facilitate the selective sequestration of specific cargoes for autophagic degradation. In addition, Atg8 interacts with a number of adaptors essential for autophagosome biogenesis, including ATG and non-ATG proteins. The majority of these adaptors and receptors are characterized by an Atg8-family interacting motif (AIM) for binding to Atg8. However, the molecular basis for the interaction mode between ATG8 and regulators or cargo receptors in plants remains largely unclear. In this study, we unveiled an atypical interaction mode for Arabidopsis ATG8f with a plant unique adaptor protein, SH3P2 (SH3 domain-containing protein 2), but not with the other two SH3 proteins. By structure analysis of the unbound form of ATG8f, we identified the unique conformational changes in ATG8f upon binding to the AIM sequence of a plant known autophagic receptor, NBR1. To compare the binding affinity of SH3P2-ATG8f with that of ATG8f-NBR1, we performed a gel filtration assay to show that ubiquitin-associated domain of NBR1 outcompetes the SH3 domain of SH3P2 for ATG8f interaction. Biochemical and cellular analysis revealed that distinct interfaces were employed by ATG8f to interact with NBR1 and SH3P2. Further subcellular analysis showed that the AIM-like motif of SH3P2 is essential for its recruitment to the phagophore membrane but is dispensable for its trafficking in endocytosis. Taken together, our study provides an insightful structural basis for the ATG8 binding specificity toward a plant-specific autophagic adaptor and a conserved autophagic receptor. Abbreviations: ATG, autophagy-related; AIM, Atg8-family interacting motif; BAR, Bin-Amphiphysin-Rvs; BFA, brefeldin A; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; CCV, clathrin-coated-vesicle; CLC2, clathrin light chain 2; Conc A, concanamycin A; ER, endoplasmic reticulum; LDS, LIR docking site; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; LIR, LC3-interacting region; PE, phosphatidylethanolamine; SH3P2, SH3 domain containing protein 2; SH3, Src-Homology-3; UBA, ubiquitin-associated; UIM, ubiquitin-interacting motif.

Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy.

Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their interrelationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosome-associated proteome remodeling during lysophagy. Among the proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both HeLa cells and induced neurons (iNeurons). While the related receptor Optineurin (OPTN) can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.

Selective autophagic receptor NbNBR1 prevents NbRFP1-mediated UPS-dependent degradation of βC1 to promote geminivirus infection.

Autophagy is an evolutionarily conserved, lysosomal/vacuolar degradation mechanism that targets cell organelles and macromolecules. Autophagy and autophagy-related genes have been studied for their antiviral and pro-viral roles in virus-infected plants. Here, we demonstrate the pro-viral role of a selective autophagic receptor NbNBR1 in geminivirus-infected Nicotiana benthamiana plants. The βC1 protein encoded by tomato yellow leaf curl China betasatellite (TYLCCNB) that is associated with tomato yellow leaf curl China virus (TYLCCNV) enhanced the expression level of NbNBR1. Then NbNBR1 interacted with βC1 to form cytoplasmic granules. Interaction of NbNBR1 with βC1 could prevent degradation of βC1 by the NbRFP1, an E3 ligase. Overexpression of NbNBR1 in N. benthamiana plants increased βC1 accumulation and promoted virus infection. In contrast, silencing or knocking out NbNBR1 expression in N. benthamiana suppressed βC1 accumulation and inhibited virus infection. A single amino acid substitution in βC1 (βC1K4A) abolished its interaction with NbNBR1, leading to a reduced level of βC1K4A. The TYLCCNV/TYLCCNBK4A mutant virus caused milder disease symptoms and accumulated much less viral genomic DNAs in the infected plants. Collectively, the results presented here show how a viral satellite-encoded protein hijacks host autophagic receptor NbNBR1 to form cytoplasmic granules to protect itself from NbRFP1-mediated degradation and facilitate viral infection.

Vacuolar accumulation and colocalization is not a proper criterion for cytoplasmic soluble proteins undergoing selective autophagy

ABSTRACT Autophagy is an important cytoprotective process that mediates degradation of dysfunctional or unnecessary cellular components. In the process of autophagy, a double-membrane organelle termed the autophagosome is formed to sequestrate portions of cytoplasm and subsequently delivered into lysosome or vacuole for degradation. The accumulation of autophagic bodies in the vacuoles after treatment with concanamycin A (ConcA) is a widely used protocol for monitoring the occurrence of autophagy in plants. Here, it was found that the cytoplasmic soluble GFP was accumulated in vacuoles upon ConcA treatment. Importantly, the GFP signal showed good colocalization with the autophagic marker mCherry-ATG8f in vacuoles based on two commonly used methods, the Pearson-Spearman correlation colocalization analysis and the plot profile analysis. Further results showed that the free GFP did not interact with ATG8s. Thus, analysis of accumulation and colocalization only in vacuoles is not a trustworthy way to judge whether degradation of cytoplasmic protein is dependent on the selective autophagy pathway in plants. In this short perspective, we propose several primary steps to distinguish that the cytoplasmic proteins are degraded by selective or bulk autophagy, hoping they could contribute to identify and clarify the selective autophagic cargos and receptors in plants.

Galectin-9 Targets NLRP3 for Autophagic Degradation to Limit Inflammation.

NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome has been implicated in a variety of inflammatory disorders, and its activation should be tightly controlled to avoid detrimental effects. NLRP3 protein expression is considered as the rate-limiting step for NLRP3 inflammasome activation. In this study, we show that galectin-9 (encoded by lgals9) attenuated NLRP3 inflammasome activation by promoting the protein degradation of NLRP3 in primary peritoneal macrophages of C57BL/6J mice. Lgals9 deficiency enhances NLRP3 inflammasome activation and promotes NLRP3-dependent inflammation in C57BL/6J mice in vivo. Mechanistically, galectin-9 interacts with NLRP3, promotes the formation of NLRP3/p62 (an autophagic cargo receptor, also known as SQSTM1) complex, and thus facilitates p62-dependent autophagic degradation of NLRP3 in primary peritoneal macrophages of C57BL/6J mice and HEK293T cells. Therefore, we identify galectin-9 as an "eat-me" signal for selective autophagy of NLRP3 and uncover the potential roles of galectins in controlling host protein degradation. Furthermore, our work suggests galectin-9 as a priming therapeutic target for the diseases caused by improper NLRP3 inflammasome activation.

Exploring the role of the multiple sclerosis susceptibility gene CLEC16A in T cells.

C-type lectin-like domain family 16 member A (CLEC16A) is associated with autoimmune disorders, including multiple sclerosis (MS), but its functional relevance is not completely understood. CLEC16A is expressed in several immune cells, where it affects autophagic processes and receptor expression. Recently, we reported that the risk genotype of an MS-associated single nucleotide polymorphism in CLEC16A intron 19 is associated with higher expression of CLEC16A in CD4+ T cells. Here, we show that CLEC16A expression is induced in CD4+ T cells upon T cell activation. By the use of imaging flow cytometry and confocal microscopy, we demonstrate that CLEC16A is located in Rab4a-positive recycling endosomes in Jurkat TAg T cells. CLEC16A knock-down in Jurkat cells resulted in lower cell surface expression of the T cell receptor, however, this did not have a major impact on T cell activation response in vitro in Jurkat nor in human, primary CD4+ T cells.

A protective variant of the autophagy receptor CALCOCO2/NDP52 in Multiple Sclerosis (MS)

ABSTRACT Multiple sclerosis (MS) is an autoimmune disease of the central nervous system, which has been found associated with dysfunctional mitochondria. In order to advance our understanding of the complex molecular mechanisms underlying this disease, we analyzed mitophagy, a process fundamental for the elimination of damaged mitochondria through the autophagic process, in peripheral blood mononuclear cells (PBMCs) of MS patients. Through a genetic analysis carried out on 203 MS patients and 1000 healthy controls, we identified a natural variant of CALCOCO2/NDP52, a well-known autophagic receptor, associated with and protective in MS. Structural modeling of the CALCOCO2 variant and functional studies highlighted an amino acid substitution (G140E) located near the LC3-interacting region (LIR) motif of CALCOCO2, crucial in controlling mitophagy. In addition, we found that among PBMCs, CALCOCO2 is mainly expressed in B cells and, by mediating mitophagy, it reduces pro-inflammatory cytokine production following stimulation of these cells. Here we summarize these recent findings, discuss the putative protective roles of CALCOCO2 in B cells and its novel association with an autoimmune disease such as MS. Abbreviations: LIR: LC3-interacting region; MS: multiple sclerosis; PBMC: peripheral blood mononuclear cells; RR-MS: relapsing-remitting MS; TLR: toll like receptor.

Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy

Selective autophagy of damaged mitochondria, protein aggregates, and other cargoes is essential for health. Cargo initiates phagophore biogenesis, which entails the conjugation of LC3 to phosphatidylethanolamine. Current models suggest that clustered ubiquitin chains on a cargo trigger a cascade from autophagic cargo receptors through the core complexes ULK1 and class III phosphatidylinositol 3-kinase complex I, WIPI2, and the ATG7, ATG3, and ATG12ATG5-ATG16L1 machinery of LC3 lipidation. This was tested using giant unilamellar vesicles (GUVs), GST-Ub4 as a model cargo, the cargo receptors NDP52, TAX1BP1, and OPTN, and the autophagy core complexes. All three cargo receptors potently stimulated LC3 lipidation on GUVs. NDP52- and TAX1BP1-induced LC3 lipidation required all components, but not ULK1 kinase activity. However, OPTN bypassed the ULK1 requirement. Thus, cargo-dependent stimulation of LC3 lipidation is common to multiple autophagic cargo receptors, yet the details of core complex engagement vary between the different receptors.

Reduction of WDR81 impairs autophagic clearance of aggregated proteins and cell viability in neurodegenerative phenotypes.

Neurodegenerative diseases are characterized by neuron loss and accumulation of undegraded protein aggregates. These phenotypes are partially due to defective protein degradation in neuronal cells. Autophagic clearance of aggregated proteins is critical to protein quality control, but the underlying mechanisms are still poorly understood. Here we report the essential role of WDR81 in autophagic clearance of protein aggregates in models of Huntington’s disease (HD), Parkinson’s disease (PD) and Alzheimer’s disease (AD). In hippocampus and cortex of patients with HD, PD and AD, protein level of endogenous WDR81 is decreased but autophagic receptor p62 accumulates significantly. WDR81 facilitates the recruitment of autophagic proteins onto Htt polyQ aggregates and promotes autophagic clearance of Htt polyQ subsequently. The BEACH and MFS domains of WDR81 are sufficient for its recruitment onto Htt polyQ aggregates, and its WD40 repeats are essential for WDR81 interaction with covalent bound ATG5-ATG12. Reduction of WDR81 impairs the viability of mouse primary neurons, while overexpression of WDR81 restores the viability of fibroblasts from HD patients. Notably, in Caenorhabditis elegans, deletion of the WDR81 homolog (SORF-2) causes accumulation of p62 bodies and exacerbates neuron loss induced by overexpressed α-synuclein. As expected, overexpression of SORF-2 or human WDR81 restores neuron viability in worms. These results demonstrate that WDR81 has crucial evolutionarily conserved roles in autophagic clearance of protein aggregates and maintenance of cell viability under pathological conditions, and its reduction provides mechanistic insights into the pathogenesis of HD, PD, AD and brain disorders related to WDR81 mutations.

The CDT of Helicobacter hepaticus induces pro-survival autophagy and nucleoplasmic reticulum formation concentrating the RNA binding proteins UNR/CSDE1 and P62/SQSTM1.

Humans are frequently exposed to bacterial genotoxins of the gut microbiota, such as colibactin and cytolethal distending toxin (CDT). In the present study, whole genome microarray-based identification of differentially expressed genes was performed in vitro on HT29 intestinal cells while following the ectopic expression of the active CdtB subunit of Helicobacter hepaticus CDT. Microarray data showed a CdtB-dependent upregulation of transcripts involved in positive regulation of autophagy concomitant with the downregulation of transcripts involved in negative regulation of autophagy. CdtB promotes the activation of autophagy in intestinal and hepatic cell lines. Experiments with cells lacking autophagy related genes, ATG5 and ATG7 infected with CDT- and colibactin-producing bacteria revealed that autophagy protects cells against the genotoxin-induced apoptotic cell death. Autophagy induction could also be associated with nucleoplasmic reticulum (NR) formation following DNA damage induced by these bacterial genotoxins. In addition, both genotoxins promote the accumulation of the autophagic receptor P62/SQSTM1 aggregates, which colocalized with foci concentrating the RNA binding protein UNR/CSDE1. Some of these aggregates were deeply invaginated in NR in distended nuclei together or in the vicinity of UNR-rich foci. Interestingly, micronuclei-like structures and some vesicles containing chromatin and γH2AX foci were found surrounded with P62/SQSTM1 and/or the autophagosome marker LC3. This study suggests that autophagy and P62/SQSTM1 regulate the abundance of micronuclei-like structures and are involved in cell survival following the DNA damage induced by CDT and colibactin. Similar effects were observed in response to DNA damaging chemotherapeutic agents, offering new insights into the context of resistance of cancer cells to therapies inducing DNA damage.

The PB1 protein of influenza A virus inhibits the innate immune response by targeting MAVS for NBR1-mediated selective autophagic degradation.

Influenza A virus (IAV) has evolved various strategies to counteract the innate immune response using different viral proteins. However, the mechanism is not fully elucidated. In this study, we identified the PB1 protein of H7N9 virus as a new negative regulator of virus- or poly(I:C)-stimulated IFN induction and specifically interacted with and destabilized MAVS. A subsequent study revealed that PB1 promoted E3 ligase RNF5 to catalyze K27-linked polyubiquitination of MAVS at Lys362 and Lys461. Moreover, we found that PB1 preferentially associated with a selective autophagic receptor neighbor of BRCA1 (NBR1) that recognizes ubiquitinated MAVS and delivers it to autophagosomes for degradation. The degradation cascade mediated by PB1 facilitates H7N9 virus infection by blocking the RIG-I-MAVS-mediated innate signaling pathway. Taken together, these data uncover a negative regulatory mechanism involving the PB1-RNF5-MAVS-NBR1 axis and provide insights into an evasion strategy employed by influenza virus that involves selective autophagy and innate signaling pathways.

A Role for the Autophagic Receptor, SQSTM1/p62, in Trafficking NF-κB/RelA to Nucleolar Aggresomes.

Elevated NF-κB activity is a contributory factor in many hematologic and solid malignancies. Nucleolar sequestration of NF-κB/RelA represses this elevated activity and mediates apoptosis of cancer cells. Here, we set out to understand the mechanisms that control the nuclear/nucleolar distribution of RelA and other regulatory proteins, so that agents can be developed that specifically target these proteins to the organelle. We demonstrate that RelA accumulates in intranucleolar aggresomes in response to specific stresses. We also demonstrate that the autophagy receptor, SQSTM1/p62, accumulates alongside RelA in these nucleolar aggresomes. This accumulation is not a consequence of inhibited autophagy. Indeed, our data suggest nucleolar and autophagosomal accumulation of p62 are in active competition. We identify a conserved motif at the N-terminus of p62 that is essential for nucleoplasmic-to-nucleolar transport of the protein. Furthermore, using a dominant-negative mutant deleted for this nucleolar localization signal (NoLS), we demonstrate a role for p62 in trafficking RelA and other aggresome-related proteins to nucleoli, to induce apoptosis. Together, these data identify a novel role for p62 in trafficking nuclear proteins to nucleolar aggresomes under conditions of cell stress, thus maintaining cellular homeostasis. They also provide invaluable information on the mechanisms that regulate the nuclear/nucleolar distribution of RelA that could be exploited for therapeutic purpose. IMPLICATIONS: The data open up avenues for the development of a unique class of therapeutic agents that act by targeting RelA and other aberrantly active proteins to nucleoli, thus killing cancer cells.