Macroautophagy/autophagy plays a crucial role in inhibiting viral replication and regulating the host\'s immune response. The autophagy receptor SQSTM1/p62 (sequestosome 1) restricts viral replication by directing specific viral proteins to phagophores for degradation. In this study, we investigate the reciprocal relationship between Zika virus (ZIKV) and selective autophagy mediated by SQSTM1/p62. We show that NS2B3 protease encoded by ZIKV cleaves human SQSTM1/p62 at arginine 265 (R265). This cleavage also occurs with endogenous SQSTM1 in ZIKV-infected cells. Furthermore, overexpression of SQSTM1 inhibits ZIKV replication in A549 cells, while its absence increases viral titer. We have also shown that SQSTM1 impedes ZIKV replication by interacting with NS3 and NS5 and directing them to autophagic degradation, and that NS2B3-mediated cleavage could potentially alter this antiviral function of SQSTM1. Taken together, our study highlights the role of SQSTM1-mediated selective autophagy in the host\'s antiviral defense against ZIKV and uncovers potential viral evasion strategies that exploit the host\'s autophagic machinery to ensure successful infection.Abbreviation: Cas9: CRISPR-associated protein 9; Co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DENV: dengue virus; GFP: green fluorescent protein; IFA: indirect immunofluorescence assay; KIR: KEAP1-interacting region; KO: knockout; LIR: MAP1LC3/LC3-interacting region; mAb: monoclonal antibody; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; pAb: polyclonal antibody; PB1: Phox/BEM1 domain; R265A, a SQSTM1 construct with the arginine (R) residue at position 265 replaced with glutamic acid (A); SQSTM1: sequestosome 1; SQSTM1-C, C-terminal fragment of SQSTM1; SQSTM1-N, N-terminal fragment of SQSTM1; SVV: Seneca Valley virus; TAX1BP1: Tax1 binding protein 1; TBD: TRAF6-binding domain; TCID50: 50% tissue culture infective dose; UBA: ubiquitin-associated domain; Ub: ubiquitin; WT: wild type; ZIKV: Zika virus; ZZ: ZZ-type zinc finger domain.

The production of type I interferon is tightly regulated to prevent excessive immune activation. However, the role of selective autophagy receptor SQSTM1 in this regulation in teleost remains unknown. In this study, we cloned the triploid fish SQSTM1 (3nSQSTM1), which comprises 1371 nucleotides, encoding 457 amino acids. qRT-PCR data revealed that the transcript levels of SQSTM1 in triploid fish were increased both in vivo and in vitro following spring viraemia of carp virus (SVCV) infection. Immunofluorescence analysis confirmed that 3nSQSTM1 was mainly distributed in the cytoplasm. Luciferase reporter assay results showed that 3nSQSTM1 significantly blocked the activation of interferon promoters induced by 3nMDA5, 3nMAVS, 3nTBK1, and 3nIRF7. Co-immunoprecipitation assays further confirmed that 3nSQSTM1 could interact with both 3nTBK1 and 3nIRF7. Moreover, upon co-transfection, 3nSQSTM1 significantly inhibited the antiviral activity mediated by TBK1 and IRF7. Mechanistically, 3nSQSTM1 decreased the TBK1 phosphorylation and its interaction with 3nIRF7, thereby suppressing the subsequent antiviral response. Notably, we discovered that 3nSQSTM1 also interacted with SVCV N and P proteins, and these viral proteins may exploit 3nSQSTM1 to further limit the host\'s antiviral innate immune responses. In conclusion, our study demonstrates that 3nSQSTM1 plays a pivotal role in negatively regulating the interferon signaling pathway by targeting 3nTBK1 and 3nIRF7.

Sequestosome1 (SQSTM1) is an autophagy receptor that mediates degradation of intracellular cargo, including protein aggregates, through multiple protein interactions. These interactions form the SQSTM1 protein network, and these interactions are mediated by SQSTM1 functional interaction domains, which include LIR, PB1, UBA and KIR. Technological advances in cell biology continue to expand our knowledge of the SQSTM1 protein network and of the relationship of the actions of the SQSTM1 protein network in cellular physiology and disease states. Here we apply proximity profile labeling to investigate the SQSTM1 protein interaction network by fusing TurboID with the human protein SQSTM1 (TurboID::SQSTM1). This chimeric protein displayed well-established SQSTM1 features including production of SQSTM1 intracellular bodies, binding to known SQSTM1 interacting partners, and capture of novel SQSTM1 protein interactors. Strikingly, aggregated tau protein altered the protein interaction network of SQSTM1 to include many stress-associated proteins. We demonstrate the importance of the PB1 and/or UBA domains for binding network members, including the K18 domain of tau. Overall, our work reveals the dynamic landscape of the SQSTM1 protein network and offers a resource to study SQSTM1 function in cellular physiology and disease state.

Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Emerging evidence has reported that acute lung injury (ALI), characterized by inflammation and oxidative stress in airway epithelium, is regulated by programmed cell death. Ferroptosis, a regulated form of cell death spurred by uncontrolled lipid peroxidation, has been proven to implicate various diseases. Inhibiting ferroptosis represents a feasible strategy for ALI through the suppression of lipid peroxidation, while the mechanism remains to be further elucidated. Here, we identified Sequestosome 1 (SQSTM1) as a negative regulator of airway epithelium ferroptosis during ALI. SQSTM1 knockdown cells manifested higher sensitivity to ferroptosis. Mechanistically, SQSTM1 was found to directly interact with vitamin D receptor (VDR) through its nuclear receptor (NR) box motif, facilitating its nuclear translocation and initiating autophagy at the transcriptional level. To further validate these findings, an in vivo preventive model utilizing spermidine, a proven inducer of SQSTM1 was established. The results consistently demonstrated that spermidine supplementation significantly induced SQSTM1 and ameliorated ALI by mitigating airway epithelial ferroptosis. Notably, these effects were abrogated in the absence of SQSTM1. Taken together, this study identified SQSTM1 as a negative regulator of airway epithelium ferroptosis in a VDR-mediated autophagy manner, making it a potential therapeutic target for the treatment of ALI.

UNASSIGNED: Intervertebral disc degeneration (IDD) is the leading cause of low back pain (LBP). The mechanism of IDD development and progression is not fully understood. Peripheral biomarkers are increasingly vital non-radioactive methods in early detection and diagnosis for IDD. Nevertheless, less attention has been paid to the role of mitophagy genes in the progress of IDD. This study aimed to identify the mitophagy disease-causing genes in the process of IDD and mitophagy diagnostic biomarkers for IDD.
UNASSIGNED: Mitophagy-related differentially expressed genes (MRDEGs) related to IDD were investigated by analyzing the microarray datasets of IDD cases from GEO, PathCards and Molecular Signatures Databases. We used R software, WGCNA, PPI, mRNA-miRNA, mRNA-TF, GO, KEGG, GSEA, GSVA and Cytoscape to analyze and visualize the data. We further used ssGSEA for immunoinfiltration analysis to obtain different immune cell infiltration. LASSO model was developed to screen for genes that met the diagnostic gene model requirements. Finally, qRT-PCR, Western blotting and HE were used to verify hub genes and their expression from clinical IDD samples.
UNASSIGNED: We identified 14 MRDEGs and 12 hub genes. GO, KEGG, GSEA and GSVA analyses demonstrated that hub genes were critical for the development of IDD. LASSO diagnostic model consisted of six hub genes, among which SQSTM1, ATG7 and OPTN were significantly different between the two IDD disease subtypes. At the same time, SQSTM1 also had a high correlation with immune characteristic subtypes. The results of qRT-PCR and Western blotting also indicated that these genes were significantly differentially expressed in nucleus pulposus cells (NPCs) of the IDD group.
UNASSIGNED: We explored an association between MRDEGs-associated signature in IDD and validated that hub genes like SQSTM1 might serve as biomarkers for diagnostic and therapeutic targets for IDD. Meanwhile, this study can provide new insights into the functional characteristics and mechanism of mitophagy in the development of IDD.

BACKGROUND: Autophagy plays multifaceted roles in regulating hepatocellular carcinoma (HCC) and the mechanisms involved are under-explored. Regulatory microRNAs (miRNAs) have been reported to target autophagy proteins but their roles in HCC is not well studied. Using HCC patient tissues, this study aims to investigate the association of autophagy with several clinicopathological parameters as well as identifying the autophagy-related miRNAs and the possible pathways.
RESULTS: Autophagy level in the HCC patient-derived cancer and non-cancer tissues was determined by immunohistochemistry (IHC) targeting SQSTM1, LC3A and LC3B proteins. Significance tests of clinicopathological variables were tested using the Fisher\'s exact or Chi-square tests. Gene and miRNA expression assays were carried out and analyzed using Nanostring platform and software followed by validation of other online bioinformatics tools, namely String and miRabel. Autophagy expression was significantly higher in cancerous tissues compared to adjacent non-cancer tissues. High LC3B expression was associated with advanced tumor histology grade and tumor location. Nanostring gene expression analysis revealed that SQSTM1, PARP1 and ATG9A genes were upregulated in HCC tissues compared to non-cancer tissues while SIRT1 gene was downregulated. These genes are closely related to an autophagy pathway in HCC. Further, using miRabel tool, three downregulated miRNAs (hsa-miR-16b-5p, hsa-miR-34a-5p, and hsa-miR-660-5p) and one upregulated miRNA (hsa-miR-539-5p) were found to closely interact with the abovementioned autophagy-related genes. We then mapped out the possible pathway involving the genes and miRNAs in HCC tissues.
CONCLUSIONS: We conclude that autophagy events are more active in HCC tissues compared to the adjacent non-cancer tissues. We also reported the possible role of several miRNAs in regulating autophagy-related genes in the autophagy pathway in HCC. This may contribute to the development of potential therapeutic targets for improving HCC therapy. Future investigations are warranted to validate the target genes reported in this study using a larger sample size and more targeted molecular technique.

SQSTM1/p62 droplets play crucial roles in droplets-based macroautophagy/autophagy including selective autophagy and bulk autophagy. We observed that under several stress milieus, SQSTM1 droplets entirely colocalize with P-body markers, and these stress-induced SQSTM1 droplets contain mRNAs. We thus determined that under certain stress conditions, autophagic SQSTM1 droplets are converted to a type of enlarged P-bodies, designated SQSTM1/p62-dependent P-bodies (pd-PBs). Stress-enhanced SQSTM1 droplet formation drives the nucleation of pd-PBs through the interaction between SQSTM1 and the RNA-binding protein DDX6. Furthermore, pd-PBs sequester PYCARD, facilitating the assembly of NLRP3 inflammasomes, and in turn induce inflammation-related cytotoxicity. Our study suggests that under stress settings, autophagic SQSTM1 droplets are transformed to pd-PBs, underlining a critical role of SQSTM1 in P-body condensation.

Corticobasal syndrome is generally considered to be a sporadic condition. There are familial and isolated genetic cases, associated with GRN, MAPT, c9orf72 or PNRP variants. Some reports implicate other genes: LRRK2, CHMP2B, GBA, CYP27A1, PSEN1, APP, TARDBP and TBK1. Here, we report a case of a patient carrying a SQSTM1 Pro392Leu variant. We report a 57-year-old right-handed-woman with a history of progressive speech impairment, marked right side rigidity and bradykinesia, with rest tremor and stimulus sensitive myoclonus. She had predominantly right-sided apraxia. She had right side agraphestesia and astereognosis. MRI showed asymmetrical left frontotemporoparietal atrophy. DaTSCAN showed predominantly left involvement, PiB-PET was negative. CSF NfL was of 9356.5pg/mL. She carried a heterozygous variant P392L in SQSTM1. This case report expands the spectrum of phenotypes associated with SQSTM1 pathogenic variants. It also expands the list of genes associated with corticobasal syndrome, supporting the involvement of the ubiquitin-proteasome system in this condition.

Avian metapneumovirus subgroup C (aMPV/C), an important pathogen causing acute respiratory infection in chickens and turkeys, contributes to substantial economic losses in the poultry industry worldwide. aMPV/C has been reported to induce autophagy, which is beneficial to virus replication. Sequestosome 1 (SQSTM1/P62), a selective autophagic receptor, plays a crucial role in viral replication by clearing ubiquitinated proteins. However, the relationship between SQSTM1-mediated selective autophagy and aMPV/C replication is unclear. In this study, we found that the expression of SQSTM1 negatively regulates aMPV/C replication by reducing viral protein expression and viral titers. Further studies revealed that the interaction between SQSTM1 and aMPV/C M2-2 protein is mediated via the Phox and Bem1 (PB1) domain of the former, which recognizes a ubiquitinated lysine at position 67 of the M2-2 protein, and finally degrades M2-2 via SQSTM1-mediated selective autophagy. Collectively, our results reveal that SQSTM1 degrades M2-2 via a process of selective autophagy to suppress aMPV/C replication, thereby providing novel insights for the prevention and control of aMPV/C infection.IMPORTANCEThe selective autophagy plays an important role in virus replication. As an emerging pathogen of avian respiratory virus, clarification of the effect of SQSTM1, a selective autophagic receptor, on aMPV/C replication in host cells enables us to better understand the viral pathogenesis. Previous study showed that aMPV/C infection reduced the SQSTM1 expression accompanied by virus proliferation, but the specific regulatory mechanism between them was still unclear. In this study, we demonstrated for the first time that SQSTM1 recognizes the 67th amino acid of M2-2 protein by the interaction between them, followed by M2-2 degradation via the SQSTM1-mediated selective autophagy, and finally inhibits aMPV/C replication. This information supplies the mechanism by which SQSTM1 negatively regulates viral replication, and provides new insights for preventing and controlling aMPV/C infection.