RCHY1 and OPTN: an E3-ligase and an autophagy receptor required for melanophagy, respectively.

Dysregulation of melanin homeostasis is implicated in causing skin pigmentation disorders, such as melasma due to hyperpigmentation and vitiligo due to hypopigmentation. Although the synthesis of melanin has been well studied, the removal of the formed skin pigment requires more research. We determined that ß-mangostin, a plant-derived metabolite, induces the degradation of already-formed melanin in the mouse B16F10 cell line. The whitening effect of ß-mangostin is mediated by macroautophagy/autophagy, as it was abolished by the knockdown of ATG5 or RB1CC1/FIP200, and by treatment with 3-methyladenine, a phosphatidylinositol 3-kinase complex inhibitor. However, the exact autophagy mechanism of melanosome degradation remains unknown. Selective autophagy for a specific cellular organelle requires specific E3-ligases and autophagic receptors for the target organelle. In this study, an E3-ligase, RCHY1, and an autophagy receptor, OPTN (optineurin), were identified as being essential for melanophagy in the ß-mangostin-treated B16F10 cell line. As per our knowledge, this is the first report of a specific mechanism for the degradation of melanosomes, the target organelle of melanophagy. These findings are expected to broaden the scope of melanin homeostasis research and can be exploited for the development of therapeutics for skin pigmentation disorders.

Impaired neuronal macroautophagy in the prelimbic cortex contributes to comorbid anxiety-like behaviors in rats with chronic neuropathic pain.

A large proportion of patients with chronic pain experience co-morbid anxiety. The medial prefrontal cortex (mPFC) is proposed to underlie this comorbidity, but the molecular and neuronal mechanisms are not fully understood. Here, we reported that impaired neuronal macroautophagy in the prelimbic cortical (PrL) subregion of the mPFC paralleled the occurrence of anxiety-like behaviors in rats with chronic spared nerve injury (SNI). Intriguingly, such macroautophagy impairment was mainly observed in a FOS/c-Fos+ neuronal subpopulation in the PrL. Chemogenetic inactivation of this comorbid anxiety-related neuronal ensemble relieved pain-induced anxiety-like behaviors. Rescuing macroautophagy impairment in this neuronal ensemble relieved chronic pain-associated anxiety and mechanical allodynia and restored synaptic homeostasis at the molecular level. By contrast, artificial disruption of macroautophagy induced early-onset co-morbid anxiety in neuropathic rats, but not general anxiety in normal rats. Taken together, our work identifies causal linkage between PrL neuronal macroautophagy dysfunction and comorbid anxiety in neuropathic pain and provides novel insights into the role of PrL by differentiating its contribution in pain-induced comorbid anxiety from its modulation over general anxiety-like behaviors.Abbreviation: AAV: adeno-associated viruses; ACC: anterior cingulate cortex; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; CAMK2/CaMKII: calcium/calmodulin-dependent protein kinase II; CNO: clozapine-N-oxide; CQ: chloroquine; DIA: data independent acquisition; DIO: double floxed inverse orf; DLG4/PSD-95: discs large MAGUK scaffold protein 4; Dox: doxycycline; GABA: γ-aminobutyric acid; GFP: green fluorescent protein; GO: gene ontology; Gi: inhibitory guanine nucleotide-binding proteins; HsCHRM4/M4D: human cholinergic receptor muscarinic 4; HsSYN: human synapsin; KEGG: Kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; LC3-II: PE conjugated microtubule-associated protein 1 light chain3; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mPFC: medial prefrontal cortex; P2A: 2A self-cleaving peptide; PPI: protein-protein interaction networks; PrL: prelimbic cortex; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; rtTA: reverse tetracycline-transactivator; SDS-PAGE: sodium dodecylsulfate-polyacrylamide gel electrophoresis; SHANK3: SH3 and multiple ankyrin repeat domains 3; SLC1A1/EAAC1: solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, systemXag), member 1; SNAP23: synaptosomal-associated protein 23; SNI:spared nerve injury; SQSTM1/p62: sequestosome 1; SYT3: synaptotagmin 3; TRE: tetracycline-responsive element; TRE3G: third-generation tetracycline-responsive element.

Chaperon-mediated autophagy can regulate diquat-induced apoptosis by inhibiting α-synuclein accumulation cooperatively with macroautophagy.

α-Synuclein, which is associated with Parkinson's disease, is cleared by the ubiquitin-proteasome system and autophagy lysosome system. Chaperon-mediated autophagy (CMA) and macroautophagy are major subtypes of autophagy and play a critical role in pesticide-induced α-synucleinopathy. In this study, we explored the role of CMA in diquat (DQ)-induced α-synucleinopathy and characterized the relationship between CMA and macroautophagy in the clearance of pathologic α-synuclein for the prevention of DQ neurotoxicity. DQ was cytotoxic to SH-SY5Y cells in a concentration-dependent manner, as shown by decreased cell viability and increased cytotoxicity. DQ treatment was also found to induce autophagy such as CMA and macroautophagy by monitoring the expression of Lamp2A and microtubule-associated protein 1A/1B light chain 3B (LC3-II) respectively. Following DQ treatment, SH-SY5Y cells were found to have induced phosphorylated and detergent-insoluble α-synuclein deposits, and MG132, a proteasome inhibitor, effectively potentiated both CMA and macroautophagy for preventing α-synuclein aggregation. Interestingly, CMA impairment by Lamp2A-knock down decreased the LC3II expression compared to in DQ-treated cells transfected with control siRNA. In Lamp2-knock down cells, pathologic α-synuclein was increased 12 h after DQ treatment, but there was no change observed at 24 h. In DQ-treated cells, macroautophagy by 3-methyladenine and bafilomycin inhibition increased Lamp2A expression, indicating an increase in CMA activity. In addition, CMA modulation affected apoptosis, and inhibiting lysosome activity by NH4Cl increased apoptosis in DQ-treated cells. An increase in autophagy was confirmed to compensate for the decrease in lysosome activity. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, significantly enhanced the macroautophagy response of DQ-exposed cells without alterations in Lamp2A expression. Our results suggest that CMA can regulate DQ-induced α-synucleinopathy cooperatively with macroautophagy, and crosstalk between macroautophagy and CMA plays an important role in DQ-induced cytotoxicity. Taken together, autophagy modulation may be a useful treatment strategy in pesticide-induced neurodegenerative disorders through preventing α-synucleinopathy.

An autophagy enhancer ameliorates diabetes of human IAPP-transgenic mice through clearance of amyloidogenic oligomer.

We have reported that autophagy is crucial for clearance of amyloidogenic human IAPP (hIAPP) oligomer, suggesting that an autophagy enhancer could be a therapeutic modality against human diabetes with amyloid accumulation. Here, we show that a recently identified autophagy enhancer (MSL-7) reduces hIAPP oligomer accumulation in human induced pluripotent stem cell-derived ß-cells (hiPSC-ß-cells) and diminishes oligomer-mediated apoptosis of ß-cells. Protective effects of MSL-7 against hIAPP oligomer accumulation and hIAPP oligomer-mediated ß-cell death are significantly reduced in cells with knockout of MiTF/TFE family members such as Tfeb or Tfe3. MSL-7 improves glucose tolerance and ß-cell function of hIAPP+ mice on high-fat diet, accompanied by reduced hIAPP oligomer/amyloid accumulation and ß-cell apoptosis. Protective effects of MSL-7 against hIAPP oligomer-mediated ß-cell death and the development of diabetes are also significantly reduced by ß-cell-specific knockout of Tfeb. These results suggest that an autophagy enhancer could have therapeutic potential against human diabetes characterized by islet amyloid accumulation.

Selective autophagy of intracellular organelles: recent research advances.

Macroautophagy (hereafter called autophagy) is a highly conserved physiological process that degrades over-abundant or damaged organelles, large protein aggregates and invading pathogens via the lysosomal system (the vacuole in plants and yeast). Autophagy is generally induced by stress, such as oxygen-, energy- or amino acid-deprivation, irradiation, drugs, etc. In addition to non-selective bulk degradation, autophagy also occurs in a selective manner, recycling specific organelles, such as mitochondria, peroxisomes, ribosomes, endoplasmic reticulum (ER), lysosomes, nuclei, proteasomes and lipid droplets (LDs). This capability makes selective autophagy a major process in maintaining cellular homeostasis. The dysfunction of selective autophagy is implicated in neurodegenerative diseases (NDDs), tumorigenesis, metabolic disorders, heart failure, etc. Considering the importance of selective autophagy in cell biology, we systemically review the recent advances in our understanding of this process and its regulatory mechanisms. We emphasize the 'cargo-ligand-receptor' model in selective autophagy for specific organelles or cellular components in yeast and mammals, with a focus on mitophagy and ER-phagy, which are finely described as types of selective autophagy. Additionally, we highlight unanswered questions in the field, helping readers focus on the research blind spots that need to be broken.

The Epstein-Barr virus deubiquitinase BPLF1 targets SQSTM1/p62 to inhibit selective autophagy.

Macroautophagy/autophagy plays an important role in the control of viral infections and viruses have evolved multiple strategies to interfere with autophagy to avoid destruction and promote their own replication and spread. Here we report that the deubiquitinase encoded in the N-terminal domain of the Epstein-Barr virus (EBV) large tegument protein, BPLF1, regulates selective autophagy. Mass spectrometry analysis identified several vesicular traffic and autophagy related proteins as BPLF1 interactors and potential substrates, suggesting that the viral protein targets this cellular defense during productive infection. Direct binding of BPLF1 to the autophagy receptor SQSTM1/p62 (sequestosome 1) was confirmed by co-immunoprecipitation of transfected BPLF1 and by in vitro affinity isolation of bacterially expressed proteins. Expression of the catalytically active BPLF1 was associated with decreased SQSTM1/p62 ubiquitination and failure to recruit LC3 to SQSTM1/p62-positive aggregates. Selective autophagy was inhibited as illustrated by the accumulation of large protein aggregates in BPLF1-positive cells co-transfected with an aggregate-prone HTT (huntingtin)-Q109 construct, and by a slower autophagy-dependent clearance of protein aggregates upon transfection of BPLF1 in cells expressing a tetracycline-regulated HTT-Q103. The inhibition of aggregate clearance was restored by overexpression of a SQSTM1/p62[E409A,K420R] mutant that does not require ubiquitination of Lys420 for cargo loading. These findings highlight a previously unrecognized role of the viral deubiquitinase in the regulation of selective autophagy, which may promote infection and the production of infectious virus.Abbreviations: BPLF1, BamH1 fragment left open reading frame-1; EBV, Epstein-Barr virus; GFP, green fluorescent protein; HTT, huntingtin; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; PB1, Phox and Bem1 domain; PE, phosphatidylethanolamine; SQSTM1/p62, sequestosome 1; UBA, ubiquitin-associated domain.

Exocytosis of Progeny Infectious Varicella-Zoster Virus Particles via a Mannose-6-Phosphate Receptor Pathway without Xenophagy following Secondary Envelopment.

The literature on the egress of different herpesviruses after secondary envelopment is contradictory. In this report, we investigated varicella-zoster virus (VZV) egress in a cell line from a child with Pompe disease, a glycogen storage disease caused by a defect in the enzyme required for glycogen digestion. In Pompe cells, both the late autophagy pathway and the mannose-6-phosphate receptor (M6PR) pathway are interrupted. We have postulated that intact autophagic flux is required for higher recoveries of VZV infectivity. To test that hypothesis, we infected Pompe cells and then assessed the VZV infectious cycle. We discovered that the infectious cycle in Pompe cells was remarkably different from that of either fibroblasts or melanoma cells. No large late endosomes filled with VZV particles were observed in Pompe cells; only individual viral particles in small vacuoles were seen. The distribution of the M6PR pathway (trans-Golgi network to late endosomes) was constrained in infected Pompe cells. When cells were analyzed with two different anti-M6PR antibodies, extensive colocalization of the major VZV glycoprotein gE (known to contain M6P residues) and the M6P receptor (M6PR) was documented in the viral highways at the surfaces of non-Pompe cells after maximum-intensity projection of confocal z-stacks, but neither gE nor the M6PR was seen in abundance at the surfaces of infected Pompe cells. Taken together, our results suggested that (i) Pompe cells lack a VZV trafficking pathway within M6PR-positive large endosomes and (ii) most infectious VZV particles in conventional cell substrates are transported via large M6PR-positive vacuoles without degradative xenophagy to the plasma membrane.IMPORTANCE The long-term goal of this research has been to determine why VZV, when grown in cultured cells, invariably is more cell associated and has a lower titer than other alphaherpesviruses, such as herpes simplex virus 1 (HSV1) or pseudorabies virus (PRV). Data from both HSV1 and PRV laboratories have identified a Rab6 secretory pathway for the transport of single enveloped viral particles from the trans-Golgi network within small vacuoles to the plasma membrane. In contrast, after secondary envelopment in fibroblasts or melanoma cells, multiple infectious VZV particles accumulated within large M6PR-positive late endosomes that were not degraded en route to the plasma membrane. We propose that this M6PR pathway is most utilized in VZV infection and least utilized in HSV1 infection, with PRV's usage being closer to HSV1's usage. Supportive data from other VZV, PRV, and HSV1 laboratories about evidence for two egress pathways are included.

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.

Xenophagy in cancer.

Macroautophagy (herein autophagy) is an intracellular pathway in which cytoplasmic components are captured by double-membrane vesicles (autophagosomes) that eventually fuse with lysosomes to degrade the cargo. Basal levels of autophagy in all eukaryotic cells maintain cellular homeostasis and under conditions of stress, organelles and proteins not essential for survival are degraded. Apart from these functions, cargoes like aggregated proteins, damaged organelles and intracellular pathogens, which are otherwise harmful to cells, are also selectively captured by autophagy and are destined for degradation. In terms of infectious diseases, pathogens are cleared by a specific form of autophagy known as xenophagy. This lysosomal mediated degradation of pathogens also increases the antigen presentation of cells thereby inducing a further immune response. The process of xenophagy provides a broad spectrum of defense mechanism to capture bacterial, viral and protozoan pathogens. However, pathogens have developed ingenious mechanisms to modulate xenophagy to enhance their intracellular survival. Meanwhile, certain pathogens also induce deleterious effects such as chronic inflammation and overexpression of oncogenes in the host system. This over time can increase the susceptibility of the host for tumorigenesis. Hence targeting tumor through anti-microbial mechanisms like xenophagy could be a novel strategy for combinatorial anti-cancer therapy. The recent developments in understanding the role of xenophagy in combating cancer causing pathogens will be discussed in this review.

LC3C mediates selective autophagy of the MET RTK, inhibiting cancer cell invasion.

Macroautophagy/autophagy is an evolutionarily conserved degradative process with a central role in maintaining cellular homeostasis under conditions of stress, and recent evidence suggests this may occur in part through direct modification of cell signaling. The MET/HGF receptor tyrosine kinase (RTK) signaling axis is an important mediator of cell motility and invasion in normal cell functions and in cancer. We discovered a role for autophagy in regulating ligand-activated MET signaling and cellular responses. When autophagy is induced by starvation, the HGF-activated and internalized MET RTK is selectively recruited for autophagic degradation through complex formation with the MAP1LC3C autophagy protein. Decreased LC3C expression in cancer results in loss of autophagic degradation of MET and enhanced HGF-stimulated cell invasion implicated in metastatic progression. This emerging role for autophagy in selectively regulating signaling proteins has implications for understanding cellular adaptations to stress and the functions of autophagy at different stages of cancer progression.

Regulation of lysosome integrity and lysophagy by the ubiquitin-conjugating enzyme UBE2QL1.

Lysosomal membrane permeabilization or full rupture of lysosomes is a common and severe stress condition that is relevant for degenerative disease, infection and cancer. Cells respond with extensive ubiquitination of damaged lysosomes, which triggers selective macroautophagy/autophagy of the whole organelle, termed lysophagy. We screened an siRNA library targeting human E2-conjugating enzymes and identified UBE2QL1 as critical for efficient lysosome ubiquitination after chemically-induced lysosomal damage. UBE2QL1 translocates to lysosomes upon damage and associates with autophagy regulators. Loss of UBE2QL1-mediated ubiquitination reduces association of the autophagy receptor SQSTM1/p62 and the LC3-decorated phagophore, and prevents recruitment of the ubiquitin-targeted AAA-ATPase VCP/p97 that facilitates lysophagy. Even in unchallenged cells, UBE2QL1 depletion leads to MTOR dissociation and TFEB activation, and mutation of the homolog UBC-25 destabilizes lysosomes in C. elegans, indicating that UBE2QL1 is critical for maintaining lysosome integrity in addition to lysophagy.

DIPK2A promotes STX17- and VAMP7-mediated autophagosome-lysosome fusion by binding to VAMP7B.

Autophagosome and lysosome fusion is an important macroautophagy/autophagy process for cargo degradation, and SNARE proteins, including STX17, SNAP29, VAMP7 and VAMP8, are key players in this process. However, the manner in which this process is precisely regulated is poorly understood. Here, we show that VAMP7B, a SNARE domain-disrupted isoform of R-SNARE protein VAMP7, competes with SNARE domain functional isoform VAMP7A to bind to STX17 and inhibits autophagosome-lysosome fusion. Moreover, we show that DIPK2A, a late endosome- and lysosome-localized protein, binds to VAMP7B, which inhibits the interaction of VAMP7B with STX17 and enhances the binding of STX17 to VAMP7A, thus enhancing autophagosome-lysosome fusion. Furthermore, DIPK2A participates in autophagic degradation of mitochondria proteins and alleviates apoptosis. Thus, we reveal a new aspect of autophagosome-lysosome fusion in which different isoforms of VAMP7 compete with STX17 and their regulation by DIPK2A.Abbreviations: DIPK2A: divergent protein kinase domain 2A; EEA1: early endosome antigen 1; GOLGA2: golgin A2; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; MT-CO2: mitochondrially encoded cytochrome c oxidase II; PARP1: poly(ADP-ribose) polymerase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB5A: RAB5A, member RAS oncogene family; RAB7A: RAB7A, member RAS oncogene family; REEP: receptor accessory protein; RTN4: reticulon 4; SNARE: SNAP receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TOMM20: translocase of outer mitochondrial membrane 20; VAMP7: vesicle associated membrane protein 7; VAMP8: vesicle associated membrane protein 8.

Pejvakin-mediated pexophagy protects auditory hair cells against noise-induced damage.

Noise overexposure causes oxidative stress, leading to auditory hair cell damage. Adaptive peroxisome proliferation involving pejvakin, a peroxisome-associated protein from the gasdermin family, has been shown to protect against this harmful oxidative stress. However, the role of pejvakin in peroxisome dynamics and homeostasis remains unclear. Here we show that sound overstimulation induces an early and rapid selective autophagic degradation of peroxisomes (pexophagy) in auditory hair cells from wild-type, but not pejvakin-deficient (Pjvk-/-), mice. Noise overexposure triggers recruitment of the autophagosome-associated protein MAP1LC3B (LC3B; microtubule-associated protein 1 light chain 3ß) to peroxisomes in wild-type, but not Pjvk-/-, mice. We also show that pejvakin-LC3B binding involves an LC3-interacting region within the predicted chaperone domain of pejvakin. In transfected cells and in vivo transduced auditory hair cells, cysteine mutagenesis experiments demonstrated the requirement for both C328 and C343, the two cysteine residues closest to the C terminus of pejvakin, for reactive oxygen species-induced pejvakin-LC3B interaction and pexophagy. The viral transduction of auditory hair cells from Pjvk-/- mice in vivo with both Pjvk and Lc3b cDNAs completely restored sound-induced pexophagy, fully prevented the development of oxidative stress, and resulted in normal levels of peroxisome proliferation, whereas Pjvk cDNA alone yielded only a partial correction of the defects. Overall, our results demonstrate that pexophagy plays a key role in noise-induced peroxisome proliferation and identify defective pexophagy as a cause of noise-induced hearing loss. They suggest that pejvakin acts as a redox-activated pexophagy receptor/adaptor, thereby identifying a previously unknown function of gasdermin family proteins.

LAMTOR2/LAMTOR1 complex is required for TAX1BP1-mediated xenophagy.

Xenophagy, also known as antibacterial autophagy, plays a role in host defence against invading pathogens such as Group A Streptococcus (GAS) and Salmonella. In xenophagy, autophagy receptors are used in the recognition of invading pathogens and in autophagosome maturation and autolysosome formation. However, the mechanism by which autophagy receptors are regulated during bacterial infection remains poorly elucidated. In this study, we identified LAMTOR2 and LAMTOR1, also named p14 and p18, respectively, as previously unrecognised xenophagy regulators that modulate the autophagy receptor TAX1BP1 in response to GAS and Salmonella invasion. LAMTOR1 was localized to bacterium-containing endosomes, and LAMTOR2 was recruited to bacterium-containing damaged endosomes in a LAMTOR1-dependent manner. LAMTOR2 was dispensable for the formation of autophagosomes targeting damaged membrane debris surrounding cytosolic bacteria, but it was critical for autolysosome formation, and LAMTOR2 interacted with the autophagy receptors NBR1, TAX1BP1, and p62 and was necessary for TAX1BP1 recruitment to pathogen-containing autophagosomes. Notably, knockout of TAX1BP1 caused a reduction in autolysosome formation and subsequent bacterial degradation. Collectively, our findings demonstrated that the LAMTOR1/2 complex is required for recruiting TAX1BP1 to autophagosomes and thereby facilitating autolysosome formation during bacterial infection.