The Small Non-coding Vault RNA1-1 Acts as a Riboregulator of Autophagy.

Vault RNAs (vtRNA) are small non-coding RNAs transcribed by RNA polymerase III found in many eukaryotes. Although they have been linked to drug resistance, apoptosis, and viral replication, their molecular functions remain unclear. Here, we show that vault RNAs directly bind the autophagy receptor sequestosome-1/p62 in human and murine cells. Overexpression of human vtRNA1-1 inhibits, while its antisense LNA-mediated knockdown enhances p62-dependent autophagy. Starvation of cells reduces the steady-state and p62-bound levels of vault RNA1-1 and induces autophagy. Mechanistically, p62 mutants that fail to bind vtRNAs display increased p62 homo-oligomerization and augmented interaction with autophagic effectors. Thus, vtRNA1-1 directly regulates selective autophagy by binding p62 and interference with oligomerization, a critical step of p62 function. Our data uncover a striking example of the potential of RNA to control protein functions directly, as previously recognized for protein-protein interactions and post-translational modifications.

Autophagy preferentially degrades non-fibrillar polyQ aggregates.

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos.

Reactive sulfur species (RSS) have emerged as key regulators of protein quality control. However, the mechanisms by which RSS contribute to cellular processes are not fully understood. In this study, we identified a novel function of RSS in preventing parthanatos, a nonapoptotic form of cell death that is induced by poly (ADP-ribose) polymerase-1 and mediated by the aggresome-like induced structures (ALIS) composed of SQSTM1/p62. We found that sodium tetrasulfide (Na2S4), a donor of RSS, strongly suppressed oxidative stress-dependent ALIS formation and subsequent parthanatos. On the other hand, the inhibitors of the RSS-producing enzymes, such as 3-mercaptopyruvate sulfurtransferase and cystathionine γ-lyase, clearly enhanced ALIS formation and parthanatos. Interestingly, we found that Na2S4 activated heat shock factor 1 by promoting its dissociation from heat shock protein 90, leading to accelerated transcription of HSP70. Considering that the genetic deletion of HSP70 allowed the enhanced ALIS formation, these findings suggest that RSS prevent parthanatos by specifically suppressing ALIS formation through induction of HSP70. Taken together, our results demonstrate a novel mechanism by which RSS prevent cell death, as well as a novel physiological role of RSS in contributing to protein quality control through HSP70 induction, which may lead to better understanding of the bioactivity of RSS.

Rift Valley Fever Virus Nucleoprotein Triggers Autophagy to Dampen Antiviral Innate Immune Responses.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes severe and potentially fatal hemorrhagic fever in humans. Autophagy is a self-degradative process that can restrict viral replication at multiple infection steps. In this study, we evaluated the effects of RVFV-triggered autophagy on viral replication and immune responses. Our results showed that RVFV infection triggered autophagosome formation and induced complete autophagy. Impairing autophagy flux by depleting autophagy-related gene 5 (ATG5), ATG7, or sequestosome 1 (SQSTM1) or treatment with autophagy inhibitors markedly reduced viral RNA synthesis and progeny virus production. Mechanistically, our findings demonstrated that the RVFV nucleoprotein (NP) C-terminal domain interacts with the autophagy receptor SQSTM1 and promotes the SQSTM1-microtubule-associated protein 1 light chain 3 B (LC3B) interaction and autophagy. Deletion of the NP C-terminal domain impaired the interaction between NP and SQSTM1 and its ability to trigger autophagy. Notably, RVFV-triggered autophagy promoted viral infection in macrophages but not in other tested cell types, including Huh7 hepatocytes and human umbilical vein endothelial cells, suggesting cell type specificity of this mechanism. It was further revealed that RVFV NP-triggered autophagy dampens antiviral innate immune responses in infected macrophages to promote viral replication. These results provide novel insights into the mechanisms of RVFV-triggered autophagy and indicate the potential of targeting the autophagy pathway to develop antivirals against RVFV. IMPORTANCE We showed that RVFV infection induced the complete autophagy process. Depletion of the core autophagy genes ATG5, ATG7, or SQSTM1 or pharmacologic inhibition of autophagy in macrophages strongly suppressed RVFV replication. We further revealed that the RVFV NP C-terminal domain interacted with SQSTM1 and enhanced the SQSTM1/LC3B interaction to promote autophagy. RVFV NP-triggered autophagy strongly inhibited virus-induced expression of interferon-stimulated genes in infected macrophages but not in other tested cell types. Our study provides novel insights into the mechanisms of RVFV-triggered autophagy and highlights the potential of targeting autophagy flux to develop antivirals against this virus.

Changes in cytokine and sequestosome-1 levels during twin pregnancy progression: Association with outcome.

BACKGROUND: Twin pregnancies are associated with complications and adverse outcomes. The number of twin pregnancies has increased in the last decades, due to the use of assisted reproductive techniques and delayed childbearing. Analysis of changes that occur during twin pregnancy progression and their association with outcome will lead to improved clinical interventions. OBJECTIVE: We evaluated if the plasma concentration of select cytokines and the level of sequestosome-1 (p62) in peripheral blood mononuclear cells (PBMCs) during each trimester of twin gestations was predictive of pregnancy outcome. STUDY DESIGN: This prospective, observational study was conducted at Careggi University Hospital, Florence, Italy. Plasma from 82 women with twin pregnancies was collected in each trimester for measurement of interleukin (IL)-1ß, IL-6, IL-10, IL-12 and tumor necrosis factor (TNF)-α. The intracellular PBMC concentration of p62, a protein involved in autophagy, kinase activity and cell differentiation, was also determined. RESULTS: IL-1ß (p < 0.001), IL-6 (p < 0.001), TNF-α (p < 0.001) and p62 (p < 0.05) increased from the 1st to the 2nd to the 3rd trimester. The TNF-α level was correlated with the IL-1ß concentration in the 1st and 3rd trimesters p < 0.01) and with the IL-6 concentration in each of the three trimesters (p < 0.01). The intracellular p62 level in PBMCs was negatively correlated with the concentration of IL-1ß in the 2nd trimester (p < 0.05) and negatively correlated with the IL-6 level in the 3rd trimester (p < 0.05). The TNF-α level was significantly higher in the 2nd (p < 0.05) and 3rd (p < 0.001) trimester in women with a spontaneous preterm delivery. The TNF-α concentrations in the 2nd (p < 0.05) and 3rd (p < 0.01) trimester, respectively, and 3rd trimester IL-6 (p < 0.01), were negatively associated with gestational age at delivery. The concentration of IL-6 was highest in the 2nd (p < 0.05) and 3rd (p < 0.05) trimesters in women who utilized assisted reproductive technologies. An elevated IL-1ß level in the 3rd trimester was associated with gestational diabetes mellitus (p < 0.05). CONCLUSION: Variations in cytokine levels between individual women during the three trimesters of twin gestations are predictive of spontaneous preterm delivery and the onset of gestational diabetes.

Functional and structural consequences of TBK1 missense variants in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

Mutations in the Tank-binding kinase 1 (TBK1) gene were identified in 2015 in individuals with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). They account for ∼3-4% of cases. To date, over 100 distinct mutations, including missense, nonsense, deletion, insertion, duplication, and splice-site mutations have been reported. While nonsense mutations are predicted to cause disease via haploinsufficiency, the mechanisms underlying disease pathogenesis with missense mutations is not fully elucidated. TBK1 is a kinase involved in neuroinflammation, which is commonly observed in these diseases. TBK1 also phosphorylates key autophagy mediators, thereby regulating proteostasis, a pathway that is dysregulated in ALS-FTLD. Recently, several groups have characterised various missense mutations with respect to their effects on the phosphorylation of known TBK1 substrates, TBK1 homodimerization, interaction with optineurin, and the regulation of autophagy and neuroinflammatory pathways. Further, the effects of either global or conditional heterozygous knock-out of Tbk1, or the heterozygous or homozygous knock-in of ALS-FTLD associated mutations, alone or when crossed with the SOD1G93A classical ALS mouse model or a TDP-43 mouse model, have been reported. In this review we summarise the known functional effects of TBK1 missense mutations. We also present novel modelling data that predicts the structural effects of missense mutations and discuss how they correlate with the known functional effects of these mutations.

The Calcineurin-TFEB-p62 Pathway Mediates the Activation of Cardiac Macroautophagy by Proteasomal Malfunction.

RATIONALE: The ubiquitin-proteasome system (UPS) and the autophagic-lysosomal pathway are pivotal to proteostasis. Targeting these pathways is emerging as an attractive strategy for treating cancer. However, a significant proportion of patients who receive a proteasome inhibitor-containing regime show cardiotoxicity. Moreover, UPS and autophagic-lysosomal pathway defects are implicated in cardiac pathogenesis. Hence, a better understanding of the cross-talk between the 2 catabolic pathways will help advance cardiac pathophysiology and medicine. OBJECTIVE: Systemic proteasome inhibition (PSMI) was shown to increase p62/SQSTM1 expression and induce myocardial macroautophagy. Here we investigate how proteasome malfunction activates cardiac autophagic-lysosomal pathway. METHODS AND RESULTS: Myocardial macroautophagy, TFEB (transcription factor EB) expression and activity, and p62 expression were markedly increased in mice with either cardiomyocyte-restricted ablation of Psmc1 (an essential proteasome subunit gene) or pharmacological PSMI. In cultured cardiomyocytes, PSMI-induced increases in TFEB activation and p62 expression were blunted by pharmacological and genetic calcineurin inhibition and by siRNA-mediated Molcn1 silencing. PSMI induced remarkable increases in myocardial autophagic flux in wild type mice but not p62 null (p62-KO) mice. Bortezomib-induced left ventricular wall thickening and diastolic malfunction was exacerbated by p62 deficiency. In cultured cardiomyocytes from wild type mice but not p62-KO mice, PSMI induced increases in LC3-II flux and the lysosomal removal of ubiquitinated proteins. Myocardial TFEB activation by PSMI as reflected by TFEB nuclear localization and target gene expression was strikingly less in p62-KO mice compared with wild type mice. CONCLUSIONS: (1) The activation of cardiac macroautophagy by proteasomal malfunction is mediated by the Mocln1-calcineurin-TFEB-p62 pathway; (2) p62 unexpectedly exerts a feed-forward effect on TFEB activation by proteasome malfunction; and (3) targeting the Mcoln1 (mucolipin1)-calcineurin-TFEB-p62 pathway may provide new means to intervene cardiac autophagic-lysosomal pathway activation during proteasome malfunction.

Sequestosome 1/p62 enhances chronic skin inflammation.

BACKGROUND: The molecular control of inflammation and epidermal thickening in skin lesions of patients with atopic dermatitis (AD) is not known. Sequestosome 1/p62 is a multifunctional adapter protein implicated in the control of key regulators of cellular homeostasis, such as proinflammatory and mechanistic target of rapamycin signaling. OBJECTIVE: We sought to determine whether p62 plays a role in the cutaneous and systemic manifestations of an AD-like mouse model. METHODS: AD-like skin lesions were induced by deletion of JunB/AP-1, specifically in epidermal keratinocytes (JunBΔep). The contribution of p62 to pathological changes was determined by inactivation of p62 in JunBΔepp62-/- double knockout mice. RESULTS: Expression of p62 was elevated in skin lesions of JunBΔep mice, resembling upregulation of p62 in AD and psoriasis. When p62 was inactivated, JunBΔep-associated defects in the differentiation of keratinocytes, epidermal thickening, skin infiltration by mast cells and neutrophils, and the development of macroscopic skin lesions were significantly reduced. p62 inactivation had little effect on circulating cytokines, but decreased serum IgE. Signaling through mechanistic target of rapamycin and natural factor kappa B was increased in JunBΔep but not in JunBΔepp62-/- double knockout skin, indicating an important role of p62 in enhancing these signaling pathways in the skin during AD-like inflammation. CONCLUSIONS: Our results provide the first in vivo evidence for a proinflammatory role of p62 in skin and suggest that p62-dependent signaling pathways may be promising therapeutic targets to ameliorate the skin manifestations of AD and possibly psoriasis.

P62-positive aggregates are homogenously distributed in the myocardium and associated with the type of mutation in genetic cardiomyopathy.

Genetic cardiomyopathy is caused by mutations in various genes. The accumulation of potentially proteotoxic mutant protein aggregates due to insufficient autophagy is a possible mechanism of disease development. The objective of this study was to investigate the distribution in the myocardium of such aggregates in relation to specific pathogenic genetic mutations in cardiomyopathy hearts. Hearts from 32 genetic cardiomyopathy patients, 4 non-genetic cardiomyopathy patients and 5 controls were studied. Microscopic slices from an entire midventricular heart slice were stained for p62 (sequestosome-1, marker for aggregated proteins destined for autophagy). The percentage of cardiomyocytes with p62 accumulation was higher in cardiomyopathy hearts (median 3.3%) than in healthy controls (0.3%; P < .0001). p62 accumulation was highest in the desmin (15.6%) and phospholamban (7.2%) groups. P62 accumulation was homogeneously distributed in the myocardium. Fibrosis was not associated with p62 accumulation in subgroup analysis of phospholamban hearts. In conclusion, accumulation of p62-positive protein aggregates is homogeneously distributed in the myocardium independently of fibrosis distribution and associated with desmin and phospholamban cardiomyopathy. Proteotoxic protein accumulation is a diffuse process in the myocardium while a more localized second hit, such as local strain during exercise, might determine whether this leads to regional myocyte decay.

Use of the LC3B-fusion technique for biochemical and structural studies of proteins involved in the N-degron pathway.

The N-degron pathway, formerly the N-end rule pathway, is a protein degradation process that determines the half-life of proteins based on their N-terminal residues. In contrast to the well-established in vivo studies over decades, in vitro studies of this pathway, including biochemical characterization and high-resolution structures, are relatively limited. In this study, we have developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B, a key marker protein of autophagy, to tag the N terminus of the proteins involved in the N-degron pathway, which enables high yield of homogeneous target proteins with variable N-terminal residues for diverse biochemical studies including enzymatic and binding assays and substrate identification. Intriguingly, crystallization showed a markedly enhanced probability, even for the N-degron complexes. To validate our results, we determined the structures of select proteins in the N-degron pathway and compared them with the Protein Data Bank-deposited proteins. Furthermore, several biochemical applications of this technique were introduced. Therefore, this technique can be used as a general tool for the in vitro study of the N-degron pathway.

N-terminal arginylation generates a bimodal degron that modulates autophagic proteolysis.

The conjugation of amino acids to the protein N termini is universally observed in eukaryotes and prokaryotes, yet its functions remain poorly understood. In eukaryotes, the amino acid l-arginine (l-Arg) is conjugated to N-terminal Asp (Nt-Asp), Glu, Gln, Asn, and Cys, directly or associated with posttranslational modifications. Following Nt-arginylation, the Nt-Arg is recognized by UBR boxes of N-recognins such as UBR1, UBR2, UBR4/p600, and UBR5/EDD, leading to substrate ubiquitination and proteasomal degradation via the N-end rule pathway. It has been a mystery, however, why studies for the past five decades identified only a handful of Nt-arginylated substrates in mammals, although five of 20 principal amino acids are eligible for arginylation. Here, we show that the Nt-Arg functions as a bimodal degron that directs substrates to either the ubiquitin (Ub)-proteasome system (UPS) or macroautophagy depending on physiological states. In normal conditions, the arginylated forms of proteolytic cleavage products, D101-CDC6 and D1156-BRCA1, are targeted to UBR box-containing N-recognins and degraded by the proteasome. However, when proteostasis by the UPS is perturbed, their Nt-Arg redirects these otherwise cellular wastes to macroautophagy through its binding to the ZZ domain of the autophagic adaptor p62/STQSM/Sequestosome-1. Upon binding to the Nt-Arg, p62 acts as an autophagic N-recognin that undergoes self-polymerization, facilitating cargo collection and lysosomal degradation of p62-cargo complexes. A chemical mimic of Nt-Arg redirects Ub-conjugated substrates from the UPS to macroautophagy and promotes their lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux under stresses.

Effect of EGFR on SQSTM1 Expression in Malignancy and Tumor Progression of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common types of malignant tumor. Sequestosome 1 (SQSTM1) serves as an adaptor of autophagy for degrading protein aggregates. The regulation of autophagy by EGFR and its clinical impacts are indicated in various types of cancer. However, the association of EGFR and SQSTM1 in OSCC is still unknown. Our results show that the expression levels of SQSTM1 and EGFR proteins are higher in tumor tissues than in the corresponding tumor-adjacent (CTAN) tissues of OSCC patients. The expression levels of SQSTM1 were positively associated with the EGFR expression level. High co-expression of SQSTM1 and EGFR is associated with poor prognosis in OSCC patients. Moreover, SQSTM1 expression is decreased in EGFR-knockdown cells. Cell growth and invasion/migration are also decreased in cells with single/combined knockdowns of EGFR and SQSTM1 or in SQSTM1-knockdown cells without EGFR kinase inhibitor Lapatinib treatment compared to that in scrambled cells. However, cell growth and invasion/metastasis were not significantly different between the scrambled cells and SQSTM1-knockdown cells in the presence of Lapatinib. This study is the first to indicate the biological roles and clinical significance of SQSTM1 regulation by EGFR in OSCC.

Autophagy activation promotes clearance of α-synuclein inclusions in fibril-seeded human neural cells.

There is much interest in delineating the mechanisms by which the α-synuclein protein accumulates in brains of individuals with Parkinson's disease (PD). Preclinical studies with rodent and primate models have indicated that fibrillar forms of α-synuclein can initiate the propagation of endogenous α-synuclein pathology. However, the underlying mechanisms by which α-synuclein fibrils seed pathology remain unclear. To investigate this further, we have used exogenous fibrillar α-synuclein to seed endogenous α-synuclein pathology in human neuronal cell lines, including primary human neurons differentiated from induced pluripotent stem cells. Fluorescence microscopy and immunoblot analyses were used to monitor levels of α-synuclein and key autophagy/lysosomal proteins over time in the exogenous α-synuclein fibril-treated neurons. We observed that temporal changes in the accumulation of cytoplasmic α-synuclein inclusions were associated with changes in the key autophagy/lysosomal markers. Of note, chloroquine-mediated blockade of autophagy increased accumulation of α-synuclein inclusions, and rapamycin-induced activation of autophagy, or use of 5'-AMP-activated protein kinase (AMPK) agonists, promoted the clearance of fibril-mediated α-synuclein pathology. These results suggest a key role for autophagy in clearing fibrillar α-synuclein pathologies in human neuronal cells. We propose that our findings may help inform the development of human neural cell models for screening of potential therapeutic compounds for PD or for providing insight into the mechanisms of α-synuclein propagation. Our results further add to existing evidence that AMPK activation may be a therapeutic option for managing PD.

Sequestosome 1 (p62) accumulation in breast cancer cells suppresses progesterone receptor expression via argonaute 2.

Sequestosome 1 (p62) is a multifunctional adapter protein involved in various physiological functions, such as selective autophagy and oxidative stress response. Hence, aberrant expression and defective regulation of p62 are thought to lead to the onset of various diseases, including cancer. The expression of p62 has been shown to be increased in breast cancer tissues, and is correlated with a poor prognosis. However, the role of p62 in the breast cancer pathophysiology is still unclear. Here, we aimed to analyze the effect of changes in p62 expression on breast cancer cell lines. DNA microarray analysis revealed that the expression of progesterone receptor (PR), which is one of the indices for the classification of breast cancer subtypes, was markedly suppressed by forced expression of p62. The protein expression of PR was also decreased by forced expression of p62, but increased by knockdown of p62. Moreover, we found that p62 knockdown induced the protein expression of argonaute 2 (AGO2). Luciferase reporter assay results showed that the gene expression of PR was promoted by AGO2. Furthermore, results revealed that overexpression of AGO2 partially rescued the decrease in PR expression induced by forced expression of p62. Collectively, our findings indicated that p62 accumulation suppressed the expression of AGO2, which in turn decreased the expression of PR, suggesting that p62 may serve as a marker of aggressive breast cancer and poor prognosis. Moreover, the p62-AGO2-PR axis was identified as a crucial signaling cascade in breast cancer progression.

p62/sequestosome 1 deficiency accelerates osteoclastogenesis in vitro and leads to Paget's disease-like bone phenotypes in mice.

The sequestosome 1 gene encodes the p62 protein and is the major genetic risk factor associated with Paget's disease of bone. In 2004, p62 was reported to up-regulate osteoclast differentiation by activating the transcription factors Nfatc1 and NF-κB. Here, we characterized the osteoclastogenic potential of murine p62-/--derived cells compared with WT cells. Our data confirmed previous findings indicating that p62 is induced during murine osteoclast differentiation. Surprisingly, an indispensable role for p62 in in vitro osteoclast differentiation was not reproducible because p62-deficient osteoclasts exhibited robust activation of Nfatc1, NF-κB, and osteoclast marker enzymes. Thus, we concluded that in vitro osteoclast differentiation is not negatively influenced by knocking out p62. On the contrary, our results revealed that p62 deficiency accelerates osteoclastogenesis. Differentiation potential, multinucleation status, and soluble receptor activator of NF-κB ligand (sRANKL) sensitivity were significantly elevated in p62-deficient, murine bone marrow-derived stem cells. Moreover, femur ultrastructures visualized by micro-computed tomography revealed pronounced accumulation of adipocytes and trabecular bone material in distal femora of obese p62-/- mice. Increased tartrate-resistant acid phosphatase activity, along with increased trabecular bone and accumulation of adipocytes, was confirmed in both paraffin-embedded decalcified and methyl methacrylate-embedded nondecalcified bones from p62-/- mice. Of note, Paget's disease-like osteolytic lesions and increased levels of the bone turnover markers CTX-I and PINP were also observed in the p62-/- mice. Our results indicate that p62 predominantly suppresses murine in vitro osteoclast differentiation and highlight previously undetected Paget's disease-like phenotypes in p62-/- mice in vivo.

Galectin-8-mediated selective autophagy protects against seeded tau aggregation.

Assembled tau can transfer between cells and seed the aggregation of soluble tau. This process is thought to underlie the amplification and propagation of tau inclusions throughout the brain in neurodegenerative diseases, including Alzheimer's disease. An understanding of the mechanisms involved may provide strategies for limiting assembled tau propagation. Here, we sought to determine how assembled tau seeds gain access to the cytosol and whether this access triggers cellular defenses. We show that tau assemblies enter cells through clathrin-independent endocytosis and escape from damaged endomembranes into the cytosol, where they seed the aggregation of soluble tau. We also found that the danger receptor galectin-8 detects damaged endomembranes and activates autophagy through recruitment of the cargo receptor nuclear dot protein 52 (NDP52). Inhibition of galectin-8- and NDP52-dependent autophagy increased seeded tau aggregation, indicating that autophagy triggered by damaged endomembranes during the entry of assembled tau seeds protects against tau aggregation, in a manner similar to cellular defenses against cytosol-dwelling microorganisms. A second autophagy cargo receptor, p62, then targeted seeded tau aggregates. Our results reveal that by monitoring endomembrane integrity, cells reduce entry of tau seeds into the cytosol and thereby prevent seeded aggregation. The mechanisms described here may help inform the development of therapies aimed at inhibiting the propagation of protein assemblies in neurodegenerative diseases.

Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells.

In the adult brain, programmed death of neural stem cells is considered to be critical for tissue homeostasis and cognitive function and is dysregulated in neurodegeneration. Previously, we have reported that adult rat hippocampal neural (HCN) stem cells undergo autophagic cell death (ACD) following insulin withdrawal. Because the apoptotic capability of the HCN cells was intact, our findings suggested activation of unique molecular mechanisms linking insulin withdrawal to ACD rather than apoptosis. Here, we report that phosphorylation of autophagy-associated protein p62 by AMP-activated protein kinase (AMPK) drives ACD and mitophagy in HCN cells. Pharmacological inhibition of AMPK or genetic ablation of the AMPK α2 subunit by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing suppressed ACD, whereas AMPK activation promoted ACD in insulin-deprived HCN cells. We found that following insulin withdrawal AMPK phosphorylated p62 at a novel site, Ser-293/Ser-294 (in rat and human p62, respectively). Phosphorylated p62 translocated to mitochondria and induced mitophagy and ACD. Interestingly, p62 phosphorylation at Ser-293 was not required for staurosporine-induced apoptosis in HCN cells. To the best of our knowledge, this is the first report on the direct phosphorylation of p62 by AMPK. Our data suggest that AMPK-mediated p62 phosphorylation is an ACD-specific signaling event and provide novel mechanistic insight into the molecular mechanisms in ACD.

NF-κB Signaling Activation Induced by Chloroquine Requires Autophagosome, p62 Protein, and c-Jun N-terminal Kinase (JNK) Signaling and Promotes Tumor Cell Resistance.

Macroautophagy (hereafter autophagy) is a catabolic cellular self-eating process by which unwanted organelles or proteins are delivered to lysosomes for degradation through autophagosomes. Although the role of autophagy in cancer has been shown to be context-dependent, the role of autophagy in tumor cell survival has attracted great interest in targeting autophagy for cancer therapy. One family of potential autophagy blockers is the quinoline-derived antimalarial family, including chloroquine (CQ). However, the molecular basis for tumor cell response to CQ remains poorly understood. We show here that in both squamous cell carcinoma cells and melanoma tumor cells, CQ induced NF-κB activation and the expression of its target genes HIF-1α, IL-8, BCL-2, and BCL-XL through the accumulation of autophagosomes, p62, and JNK signaling. The activation of NF-κB further increased p62 gene expression. Either genetic knockdown of p62 or inhibition of NF-κB sensitized tumor cells to CQ, resulting in increased apoptotic cell death following treatment. Our findings provide new molecular insights into the CQ response in tumor cells and CQ resistance in cancer therapy. These findings may facilitate development of improved therapeutic strategies by targeting the p62/NF-κB pathway.

Adaptor protein p62 promotes skin tumor growth and metastasis and is induced by UVA radiation.

Skin cancer is the most common cancer, and exposure to ultraviolet (UV) radiation, namely UVA and UVB, is the major risk factor for skin cancer development. UVA is significantly less effective in causing direct DNA damage than UVB, but UVA has been shown to increase skin cancer risk. The mechanism by which UVA contributes to skin cancer remains unclear. Here, using RNA-Seq, we show that UVA induces autophagy and lysosomal gene expression, including the autophagy receptor and substrate p62. We found that UVA activates transcription factor EB (TFEB), a known regulator of autophagy and lysosomal gene expression, which, in turn, induces p62 transcription. Next, we identified a novel relationship between p62 and cyclooxygenase-2 (COX-2), a prostaglandin synthase critical for skin cancer development. COX-2 expression was up-regulated by UVA-induced p62, suggesting that p62 plays a role in UVA-induced skin cancer. Moreover, we found that p62 stabilizes COX-2 protein through the p62 ubiquitin-associated domain and that p62 regulates prostaglandin E2 production in vitro In a syngeneic squamous cell carcinoma mouse model, p62 knockdown inhibited tumor growth and metastasis. Furthermore, p62-deficient tumors exhibited reduced immune cell infiltration and increased cell differentiation. Because prostaglandin E2 is known to promote pro-tumorigenic immune cell infiltration, increase proliferation, and inhibit keratinocyte differentiation in vivo, this work suggests that UVA-induced p62 acts through COX-2 to promote skin tumor growth and progression. These findings expand our understanding of UVA-induced skin tumorigenesis and tumor progression and suggest that targeting p62 can help prevent or treat UVA-associated skin cancer.

Normal pancreatic ß-cell function in mice with RIP-Cre-mediated inactivation of p62/SQSTM1.

Recent studies have suggested that decreased pancreatic ß-cell function and mass are common features of patients with type 2 diabetes mellitus. Pancreatic ß-cell homeostasis is regulated by various types of signaling molecules and stress responses. Sequestosome 1/p62 (SQSTM1, hereafter referred to as p62) is a ubiquitin-binding adaptor protein involved in cell signaling, oxidative stress, and autophagy. Because p62 appears to play an important role in maintaining mitochondrial quality control, it is possible that the loss of p62 in pancreatic ß cells contributes to mitochondrial dysfunction, and thus leading to impaired glucose tolerance. In this study we investigated the physiological roles of p62 by inactivating p62 in a ß-cell specific manner. We found that firstly, rat insulin-2 promoter-Cre (RIP-Cre)-mediated p62 inactivation did not cause body weight gain, although ubiquitous inactivation of p62 was previously shown to result in severe obesity. Secondly, we found no gross structural disorganization of the islets of p62-deficient mice. Consistent with normal islet morphology, no impairment in glucose tolerance was observed in mice with RIP-Cre-mediated p62 deletion. These results suggest that p62 is dispensable for normal islet organization and ß-cell function.