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Lipophagy, the selective engulfment of lipid droplets (LDs) by autophagosomes for lysosomal degradation, is critical to lipid and energy homeostasis. Here we show that the lipid transfer protein ORP8 is located on LDs and mediates the encapsulation of LDs by autophagosomal membranes. This function of ORP8 is independent of its lipid transporter activity and is achieved through direct interaction with phagophore-anchored LC3/GABARAPs. Upon lipophagy induction, ORP8 has increased localization on LDs and is phosphorylated by AMPK, thereby enhancing its affinity for LC3/GABARAPs. Deletion of ORP8 or interruption of ORP8-LC3/GABARAP interaction results in accumulation of LDs and increased intracellular triglyceride. Overexpression of ORP8 alleviates LD and triglyceride deposition in the liver of ob/ob mice, and Osbpl8-/- mice exhibit liver lipid clearance defects. Our results suggest that ORP8 is a lipophagy receptor that plays a key role in cellular lipid metabolism., (©The Author(s) 2022. Published by Oxford University Press on behalf of Higher Education Press.)

As one of the major acetyltransferases in mammalian cells, p300 (also known as EP300) and its highly related protein CBP (also known as CREBBP), collectively termed p300/CBP, is characterized as a key regulator in gene transcription by modulating the acetylation of histones. In recent decades, proteomic analyses have revealed that p300 is also involved in the regulation of various cellular processes by acetylating many non-histone proteins. Among the identified substrates, some are key players involved in different autophagy steps, which together establish p300 as a master regulator of autophagy. Accumulating evidence has shown that p300 activity is controlled by many distinct cellular pathways to regulate autophagy in response to cellular or environmental stimuli. In addition, several small molecules have been shown to regulate autophagy by targeting p300, suggesting that manipulation of p300 activity is sufficient for controlling autophagy. Importantly, dysfunction of p300-regulated autophagy has been implicated in a number of human disorders, such as cancer, aging and neurodegeneration, highlighting p300 as a promising target for the drug development of autophagy-related human disorders. Here, we focus on the roles of p300-mediated protein acetylation in the regulation of autophagy and discuss implications for autophagy-related human disorders., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The cGAS-STING pathway plays an important role in host defense by sensing pathogen DNA, inducing type I IFNs, and initiating autophagy. However, the molecular mechanism of autophagosome formation in cGAS-STING pathway-induced autophagy is still unclear. Here, we report that STING directly interacts with WIPI2, which is the key protein for LC3 lipidation in autophagy. Binding to WIPI2 is necessary for STING-induced autophagosome formation but does not affect STING activation and intracellular trafficking. In addition, the specific interaction between STING and the PI3P-binding motif of WIPI2 leads to the competition of WIPI2 binding between STING and PI3P, and mutual inhibition between STING-induced autophagy and canonical PI3P-dependent autophagy. Furthermore, we show that the STING-WIPI2 interaction is required for the clearance of cytoplasmic DNA and the attenuation of cGAS-STING signaling. Thus, the direct interaction between STING and WIPI2 enables STING to bypass the canonical upstream machinery to induce LC3 lipidation and autophagosome formation., (© 2023 The Authors.)

Macroautophagy/autophagy, a highly conserved lysosome-dependent degradation pathway, has been intensively studied in regulating cell metabolism by degradation of intracellular components. In this study, we link autophagy to RNA metabolism by uncovering a regulatory role of autophagy in ribosomal RNA (rRNA) synthesis. Autophagy-deficient cells exhibit much higher 47S precursor rRNA level, which is caused by the accumulation of SQSTM1/p62 (sequestosome 1) but not other autophagy receptors. Mechanistically, SQSTM1 accumulation potentiates the activation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) signaling and promotes the assembly of RNA polymerase I pre-initiation complex at ribosomal DNA (rDNA) promoters, which leads to an increase of 47S rRNA transcribed from rDNA. Functionally, autophagy deficiency promotes protein synthesis, cell growth and cell proliferation, both of which are dependent on SQSTM1 accumulation. Taken together, our findings suggest that autophagy deficiency is involved in RNA metabolism by activating rDNA transcription and provide novel mechanisms for the reprogramming of cell metabolism in autophagy-related diseases including multiple types of cancers. Abbreviations: 5-FUrd: 5-fluorouridine; AMPK: AMP-activated protein kinase; ATG: autophagy related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; ChIP: chromatin immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NFKB/NF-κB: nuclear factor kappa B; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PIC: pre-initiation complex; POLR1: RNA polymerase I; POLR1A/RPA194: RNA polymerase I subunit A; POLR2A: RNA polymerase II subunit A; rDNA: ribosomal DNA; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; rRNA: ribosomal RNA; RUBCN/Rubicon: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; SUnSET: surface sensing of translation; TAX1BP1: Tax1 binding protein 1; UBTF/UBF1: upstream binding transcription factor; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild-type.

The fusion of autophagosomes and endosomes/lysosomes, also called autophagosome maturation, ensures the degradation of autophagic cargoes. It is an important regulatory step of the macroautophagy/autophagy process. STX17 is the key autophagosomal SNARE protein that mediates autophagosome maturation. Here, we report that the acetylation of STX17 regulates its SNARE activity and autophagic degradation. The histone acetyltransferase CREBBP/CBP and the deacetylase HDAC2 specifically regulate the acetylation of STX17. In response to cell starvation and MTORC1 inhibition, the inactivation of CREBBP leads to the deacetylation of STX17 at its SNARE domain. This deacetylation promotes the interaction between STX17 and SNAP29 and the formation of the STX17-SNAP29-VAMP8 SNARE complex with no effect on the recruitment of STX17 to autophagosomal membranes. Deacetylation of STX17 also enhances the interaction between STX17 and the tethering complex HOPS, thereby further promoting autophagosome-lysosome fusion. Our study suggests a mechanism by which acetylation regulates the late-stage of autophagy, and possibly other STX17-related intracellular membrane fusion events. Abbreviations: ACTB: actin beta; CREBBP/CBP: CREB binding protein; Ctrl: control; GFP: green fluorescent protein; GST: glutathione S-transferase; HDAC: histone deacetylase; HOPS: homotypic fusion and protein sorting complex; KO: knockout; LAMP1/2: lysosomal associated membrane protein 1/2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; MS: mass spectrometry; MTORC1: mechanistic target of rapamycin kinase complex 1; NAM: nicotinamide; PtdIns3K: phosphatidylinositol 3-kinase; RFP: red fluorescent protein; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylamide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TSA: trichostatin A; TSC1/2: TSC complex subunit 1/2; VAMP8: vesicle associated membrane protein 8; WT: wild type.

Cells integrate intracellular and extracellular cues to precisely control the balance of anabolic and catabolic processes, which is essential for cells to maintain homeostasis. The nuclear protein TP53INP2 (tumor protein p53 inducible nuclear protein 2) has emerged as one of the key players participating in both anabolic and catabolic processes. In the nucleus including the nucleolus, TP53INP2 binds to multiple transcription-related factors to modulate transcription, such as the transcription of thyroid hormone-related genes and ribosomal DNA. Interestingly, upon nutrient deprivation, TP53INP2 rapidly moves from the nucleus to the cytoplasm and participates in the regulation of macroautophagy/autophagy. By acting as a nutrient status sensor, TP53INP2 switches its role between transcription and autophagy by changing its subcellular localization and helps the cell to cope with environmental changes., Abbreviations: Atg: autophagy related; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; rDNA: ribosomal DNA; TP53INP2: tumor protein p53 inducible nuclear protein 2; UIM: ubiquitin-interacting motif.

Metformin, an ancient drug commonly used for treating type II diabetes, has been associated to anti-cancer capacity in a variety of developing cancers, though the mechanism remains elusive. Here, we aimed to examine the inhibitory effect of metformin in osteosarcoma. Herein, we demonstrated that metformin treatment blocked proliferation progression by causing accumulation of G2/M phase in U2OS and 143B cells. Furthermore, metformin treatment triggered programmed cell death process in osteosarcoma cell lines. Further research indicated the induction of apoptosis and autophagy triggered by metformin could remarkably attenuate after the treatment of ROS scavenger NAC and JNK inhibitor SP600125. Additionally, our results showed that NAC-suppressed JNK/c-Jun signaling pathway could have been activated through metformin treatment. Lastly, metformin could inhibit osteosarcoma growth under safe dose in vivo . Thus, we propose that metformin could induce cell cycle arrest as well as programmed cell death, including apoptosis and autophagy, through ROS-dependent JNK/c-Jun cascade in human osteosarcoma. This metformin-induced pathway provides further insights into its antitumor potential molecular mechanism and illuminates potential cancer targets for osteosarcoma., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

WIPI2 contributes to autophagy by recruiting the ATG12-ATG5-ATG16L1 complex to PtdIns3P-rich membranes, which enables the growth and elongation of phagophores. So far, PtdIns3P and ATG16L1 are the best known WIPI2 interaction partners. In the screening of novel binding proteins for WIPI2, we recently identified interactions between WIPI2 and MTORC1 and the E3 ubiquitin ligase HUWE1. With this clue, we uncovered that WIPI2 is a phosphorylation substrate of MTORC1 and a ubiquitination target of HUWE1. Further, by determining the phosphorylation site on WIPI2 targeted by MTORC1, we show that MTORC1-dependent phosphorylation directs WIPI2 to interact with HUWE1, leading to WIPI2 ubiquitination and subsequent degradation by proteasomes. Finally, we provide evidence that this quantity control mechanism of WIPI2 is utilized by cells for not only the regulation of basal autophagy but also nutrient deprivation-stimulated autophagy. Together, these results indicate that WIPI2 protein level regulated by MTORC1 and HUWE1 is a pivotal determinant of cellular autophagy intensity. The findings firm up the role of MTORC1 as a master regulator of autophagy, suggesting that the quantity control of WIPI2 is a potential intervention point in autophagy-related physiopathological processes. Abbreviations: HUWE1, HECT, UBA and WWE domain containing 1; MTORC1, mechanistic target of rapamycin complex 1; WIPI2, WD repeat domain phosphoinositide interacting protein 2.

mTORC1, the major homeostatic sensor and responder, regulates cell catabolism mainly by targeting autophagy. Here, we show that mTORC1 directly controls autophagosome formation via phosphorylation of WIPI2, a critical protein in isolation membrane growth and elongation. mTORC1 phosphorylates Ser395 of WIPI2, directing WIPI2 to interact specifically with the E3 ubiquitin ligase HUWE1 for ubiquitination and proteasomal degradation. Physiological or pharmacological inhibition of mTORC1 in cells promotes WIPI2 stabilization, autophagosome formation, and autophagic degradation. In mouse liver, fasting significantly increases the WIPI2 protein level, while silencing HUWE1 enhances autophagy, and introducing WIPI2 improves lipid clearance. Thus, regulation of the intracellular WIPI2 protein level by mTORC1 and HUWE1 is a key determinant of autophagy flux and may coordinate the initiation, progression, and completion of autophagy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Acetylation is increasingly recognized as one of the major post-translational mechanisms for the regulation of multiple cellular functions in mammalian cells. Acetyltransferase p300, which acetylates histone and non-histone proteins, has been intensively studied in its role in cell growth and metabolism. However, the mechanism underlying the activation of p300 in cells remains largely unknown. Here, we identify the homeostatic sensor mTORC1 as a direct activator of p300. Activated mTORC1 interacts with p300 and phosphorylates p300 at 4 serine residues in the C-terminal domain. Mechanistically, phosphorylation of p300 by mTORC1 prevents the catalytic HAT domain from binding to the RING domain, thereby eliminating intra-molecular inhibition. Functionally, mTORC1-dependent phosphorylation of p300 suppresses cell-starvation-induced autophagy and activates cell lipogenesis. These results uncover p300 as a direct target of mTORC1 and suggest that the mTORC1-p300 pathway plays a pivotal role in cell metabolism by coordinately controlling cell anabolism and catabolism., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The class III phosphoinositide 3-kinase VPS34 plays a key role in the regulation of vesicular trafficking and macroautophagy. So far, we know little about the molecular mechanism of VPS34 activation besides its interaction with regulatory proteins to form complexes. Here, we report that VPS34 is specifically acetylated by the acetyltransferase p300, and p300-mediated acetylation represses VPS34 activity. Acetylation at K771 directly diminishes the affinity of VPS34 for its substrate PI, while acetylation at K29 hinders the VPS34-Beclin 1 core complex formation. Inactivation of p300 induces VPS34 deacetylation, PI3P production, and autophagy, even in AMPK -/- , TSC2 -/- , or ULK1 -/- cells. In fasting mice, liver autophagy correlates well with p300 inactivation/VPS34 deacetylation, which facilitates the clearance of lipid droplets in hepatocytes. Thus, p300-dependent VPS34 acetylation/deacetylation is the physiological key to VPS34 activation, which controls the initiation of canonical autophagy and of non-canonical autophagy in which the upstream kinases of VPS34 can be bypassed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cells control their metabolism through modulating the anabolic and catabolic pathways. TP53INP2/DOR (tumor protein p53 inducible nuclear protein 2), participates in cell catabolism by serving as a promoter of autophagy. Here we uncover a novel function of TP53INP2 in protein synthesis, a major biosynthetic and energy-consuming anabolic process. TP53INP2 localizes to the nucleolus through its nucleolar localization signal (NoLS) located at the C-terminal domain. Chromatin immunoprecipitation (ChIP) assays detected an association of TP53INP2 with the ribosomal DNA (rDNA), when exclusion of TP53INP2 from the nucleolus repressed rDNA promoter activity and the production of ribosomal RNA (rRNA) and proteins. The removal of TP53INP2 also impaired the association of the POLR1/RNA polymerase I preinitiation complex (PIC) with rDNA. Further, TP53INP2 interacts directly with POLR1 PIC, and is required for the assembly of the complex. These data indicate that TP53INP2 promotes ribosome biogenesis through facilitating rRNA synthesis at the nucleolus, suggesting a dual role of TP53INP2 in cell metabolism, assisting anabolism on the nucleolus, and stimulating catabolism off the nucleolus.

Shuttling of macromolecules between different cellular compartments helps regulate the timing and extent of different cellular activities. Here, we report that LC3, a key initiator of autophagy that cycles between the nucleus and cytoplasm, becomes selectively activated in the nucleus during starvation through deacetylation by the nuclear deacetylase Sirt1. Deacetylation of LC3 at K49 and K51 by Sirt1 allows LC3 to interact with the nuclear protein DOR and return to the cytoplasm with DOR, where it is able to bind Atg7 and other autophagy factors and undergo phosphatidylethanolamine conjugation to preautophagic membranes. The association of deacetylated LC3 with autophagic factors shifts LC3's distribution from the nucleus toward the cytoplasm. Thus, an acetylation-deacetylation cycle ensures that LC3 effectively redistributes in an activated form from nucleus to cytoplasm, where it plays a central role in autophagy to enable the cell to cope with the lack of external nutrients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Number 2 Feibi Recipe (N2FBR) is a traditional Chinese medicine formula for treating idiopathic pulmonary fibrosis. N2FBR inhibits H2O2-mediated oxidative stress damage in alveolar epithelial cells by increasing autophagy, as we previously demonstrated. However, it is unknown if similar mechanisms occur in vivo. We established a pulmonary fibrosis model by instilling bleomycin (BLM) from the airway to examine the effects of N2FBR on pulmonary fibrosis and investigate its probable mechanism in this work. We discovered that N2FBR treatment effectively alleviated interstitial fibrosis as well as collagen deposition, primarily in upregulating SOD, GSH-Px, T-AOC and downregulating MDA content. N2FBR also increased the expression of LC3B, Beclin-1, LAMP1, TFEB and downregulated the expression of p62, legumain. N2FBR treatment boosted the production of autophagosomes, according to the results of the TEM observation. Furthermore, we explored that N2FBR exerted its anti-oxidative stress and pro-autophagy effects via GSK-3β/mTOR signalling pathway. Therefore, these results provide further evidence for the protective effect of N2FBR in pulmonary fibrosis. Our findings could have ramifications for the development of antifibrosis therapies.

Post-translational modifications, such as phosphorylation, ubiquitination and acetylation, play crucial roles in the regulation of autophagy. Acetylation has emerged as an important regulatory mechanism for autophagy. Acetylation regulates autophagy initiation and autophagosome formation by targeting core components of the ULK1 complex, the BECN1-PIK3C3 complex, and the LC3 lipidation system. Recent studies have shown that acetylation occurs on the key proteins participating in autophagic cargo assembly and autophagosome-lysosome fusion, such as SQSTM1/p62 and STX17. In addition, acetylation controls autophagy at the transcriptional level by targeting histones and the transcription factor TFEB. Here, we review the current knowledge on acetylation of autophagy proteins and their regulations and functions in the autophagy pathway with focus on recent findings. Abbreviations : ACAT1: acetyl-CoA acetyltransferase 1; ACSS2: acyl-CoA synthetase short chain family member 2; AMPK: AMP-activated protein kinase; ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCAR2/DBC1: cell cycle and apoptosis regulator 2; BECN1: beclin 1; CMA: chaperone-mediated autophagy; CREBBP/CBP: CREB binding protein; EP300/p300: E1A binding protein p300; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3: glycogen synthase kinase 3; HDAC6: histone deacetylase 6; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; KAT2A/GCN5: lysine acetyltransferase 2A; KAT2B/PCAF: lysine acetyltransferase 2B; KAT5/TIP60: lysine acetyltransferase 5; KAT8/MOF: lysine acetyltransferase 8; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PD: Parkinson disease; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKM2: pyruvate kinase M1/2; PtdIns3P: phosphatidylinositol-3-phosphate; PTM: post-translational modification; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RUBCN/Rubicon: rubicon autophagy regulator; RUBCNL/Pacer: rubicon like autophagy enhancer; SIRT1: sirtuin 1; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylamide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TFEB: transcription factor EB; TP53/p53: tumor protein p53; TP53INP2/DOR: tumor protein p53 inducible nuclear protein 2; UBA: ubiquitin-associated; ULK1: unc-51 like autophagy activating kinase 1; VAMP8: vesicle associated membrane protein 8; WIPI2: WD repeat domain, phosphoinositide interacting 2. [ABSTRACT FROM AUTHOR]

Autophagy has emerged as a key regulator of cell metabolism. Recently, we have demonstrated that autophagy is involved in RNA metabolism by regulating ribosomal RNA (rRNA) synthesis. We found that autophagy-deficient cells display much higher 47S precursor rRNA level, which is caused by the accumulation of SQSTM1/p62 (sequestosome 1) but not other autophagy receptors. Mechanistically, SQSTM1 accumulation potentiates the activation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) signaling, which facilitates the assembly of RNA polymerase I pre-initiation complex at ribosomal DNA (rDNA) promoter regions and leads to the activation of rDNA transcription. Finally, we showed that SQSTM1 accumulation is responsible for the increase in protein synthesis, cell growth and cell proliferation in autophagy-deficient cells. Taken together, our findings reveal a regulatory role of autophagy and autophagy receptor SQSTM1 in rRNA synthesis and may provide novel mechanisms for the hyperactivated rDNA transcription in autophagy-related human diseases. Abbreviations: 5-FUrd: 5-fluorouridine; LAP: MAP1LC3/LC3-associated phagocytosis; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PIC: pre-initiation complex; POLR1: RNA polymerase I; POLR1A: RNA polymerase I subunit A; rDNA: ribosomal DNA; RRN3: RRN3 homolog, RNA polymerase I transcription factor; rRNA: ribosomal RNA; SQSTM1/p62: sequestosome 1; TP53INP2: tumor protein p53 inducible nuclear protein 2; UBTF: upstream binding transcription factor. [ABSTRACT FROM AUTHOR]

Sirtuins are protein factors that can delay aging and alleviate age-related diseases through multiple molecular pathways, mainly by promoting DNA damage repair, delaying telomere shortening, and mediating the longevity effect of caloric restriction. In the last decade, sirtuins have also been suggested to exert mitochondrial quality control by mediating mitophagy, which targets damaged mitochondria and delivers them to lysosomes for degradation. This is especially significant for age-related diseases because dysfunctional mitochondria accumulate in aging organisms. Accordingly, it has been suggested that sirtuins and mitophagy have many common and interactive aspects in the aging process. This article reviews the mechanisms and pathways of sirtuin family-mediated mitophagy and further discusses its role in aging and age-related diseases. [ABSTRACT FROM AUTHOR]

Ribosomal DNA (rDNA) transcription drives cell growth and cell proliferation via the product ribosomal RNA (rRNA), the essential component of ribosome. Given the fundamental role of rRNA in ribosome biogenesis, rDNA transcription has emerged as one of the effective targets for a number of human diseases including various types of cancers. In this study, we identify curcumin, an ancient drug, as a novel natural inhibitor of rDNA transcription. Curcumin treatment impairs the assembly of the RNA polymerase I preinitiation complex at rDNA promoters and represses rDNA promoter activity, which leads to the decrease of rRNA synthesis. In addition, curcumin treatment stimulates autophagosome formation and promotes autophagic degradation in cells. Mechanistically, curcumin inactivates the mechanistic target of rapamycin complex 1 (mTORC1), the upstream regulator of rDNA transcription and autophagy induction, by inhibiting mTOR lysosomal localization. Functionally, curcumin treatment inhibits protein synthesis, cell growth and cell proliferation. Taken together, these findings identify curcumin as an effective inhibitor of rDNA transcription and provide novel mechanisms for the anticancer properties of curcumin. Abbreviations: Atg: autophagy-related; GFP: green fluorescent protein; LAMP2: lysosomal associated membrane protein 2; LC3: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; mTORC1: mechanistic target of rapamycin complex 1; rDNA: ribosomal DNA; rRNA: ribosomal RNA; TP53INP2: tumor protein p53 inducible nuclear protein 2. [ABSTRACT FROM AUTHOR]

Autophagy receptor p62/SQSTM1 promotes the assembly and removal of ubiquitylated proteins by forming p62 bodies and mediating their encapsulation in autophagosomes. Here we show that under nutrient-deficient conditions, cellular p62 specifically undergoes acetylation, which is required for the formation and subsequent autophagic clearance of p62 bodies. We identify K420 and K435 in the UBA domain as the main acetylation sites, and TIP60 and HDAC6 as the acetyltransferase and deacetylase. Mechanically, acetylation at both K420 and K435 sites enhances p62 binding to ubiquitin by disrupting UBA dimerization, while K435 acetylation also directly increases the UBA-ubiquitin affinity. Furthermore, we show that acetylation of p62 facilitates polyubiquitin chain-induced p62 phase separation. Our results suggest an essential role of p62 acetylation in the selective degradation of ubiquitylated proteins in cells under nutrient stress, by specifically regulating the assembly of p62 bodies. The autophagy receptor p62 mediates the assembly and removal of ubiquitylated protein aggregates by forming p62 bodies. Here, the authors identify an acetylation-dependent mechanism that regulates formation and autophagic clearance of p62 bodies under nutrient-deficient conditions. [ABSTRACT FROM AUTHOR]

The tumor protein p53‐inducible nuclear protein 2 (TP53INP2) has been reported to participate in autophagy by interacting with autophagosome‐localized autophagy‐related protein 8 (Atg8) family proteins, including LC3. Here, we uncover a novel function for TP53INP2 in the autophagic degradation of proteins. We identify the ubiquitin‐interacting motif (UIM) of TP53INP2 that mediates its binding to ubiquitin and ubiquitinated proteins. TP53INP2 lacking the UIM is able to displace autophagic adaptor p62 from LC3, which leads to accumulation of ubiquitinated proteins in cells. Furthermore, overexpression of TP53INP2 lacking the UIM sensitizes cells to chloroquine treatment. Our findings indicate that TP53INP2 may act as a novel autophagic adaptor through recruiting ubquitinated substrates to autophagosomes for degradation. [ABSTRACT FROM AUTHOR]

TP53INP2/DOR (tumor protein p53-inducible nuclear protein 2) contributes to mammalian macroautophagy/autophagy by carrying nuclear deacetylated MAP1LC3/LC3 to the cytoplasm. Here, we report that in the cytoplasm, TP53INP2 further functions in autophagosome biogenesis by promoting LC3B-ATG7 interaction. Cytoplasmic expression of the N-terminal region of TP53INP2, which includes the LC3-interacting region, effectively triggered LC3B–PE production and autophagosome formation. In the cytoplasm, TP53INP2 colocalized to early autophagic membrane structures containing ATG14, ZFYVE1/DFCP1 or WIPI2. While knockdown of TP53INP2 did not affect the formation of these autophagic structures, deletion of BECN1 or Atg5, or mutations preventing TP53INP2 from LC3 interaction, disrupted the membrane binding of TP53INP2. TP53INP2 interacted directly with ATG7 to form a LC3B-TP53INP2-ATG7 complex in the cytoplasm. Loss of TP53INP2-LC3 or TP53INP2-ATG7 interaction significantly reduced LC3B-ATG7 binding. Together, these results suggest that after shifting from the nucleus, cytoplasmic TP53INP2 is targeted to early autophagic membranes accompanied by LC3, where it contributes to autophagosome biogenesis by mediating LC3-ATG7 interaction. Abbreviations: 3-MA, 3-methyladenine; 3NES, 3 repeated nuclear export signal; 3NLS, 3 repeated nuclear localization signal; ACTB, actin beta; ATG, autophagy related; BECN1, Beclin 1; mCherry, monomeric red fluorescent protein mCherry; GFP, green fluorescent protein; GST, glutathione S-transferase; KO, knockout; LC3B/MAP1LC3B, microtubule-associated protein 1 light chain 3 beta; LC3B[G120], LC3B mutant lacking amino acids after glycine 120; LDH, lactate dehydrogenase; LMNB1, lamin B1; LIR, LC3-interacting region; MTORC1, mechanistic target of rapamycin complex 1; PE, phosphatidylethanolamine; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol 3-phosphate; rDNA, ribosomal DNA; RFP, red fluorescent protein; RNAi, RNA interference; SQSTM1, sequestosome 1; TP53INP2, tumor protein p53-inducible nuclear protein 2; TP53INP2[1-28], TP53INP2 mutant containing amino acids 1 to 28; TP53INP2[28-45], TP53INP2 mutant containing amino acids 28 to 45; TP53INP2[LIRΔ], TP53INP2 mutant lacking amino acids 1 to 144; TP53INP2[NLSΔ], TP53INP2 mutant lacking amino acids 145 to 221; TP53INP2W35,I38A, TP53INP2 mutant in which tryptophan 35 and isoleucine 38 are replaced with alanine; TP53INP2W35,I38A[NLSΔ], TP53INP2 mutant lacking amino acids 145 to 221, and tryptophan 35 and isoleucine 38 are replaced with alanine; TP53INP2W35,I38A[Δ1-28],[NLSΔ], TP53INP2 mutant lacking amino acids 1 to 28 and amino acids 145 to 221, and tryptophan 35 and isoleucine 38 are replaced with alanine; TP53INP2[Δ1-28],[NLSΔ], TP53INP2 mutant lacking amino acids 1 to 28 and amino acids 145 to 221; TP53INP2[Δ67-111],[NLSΔ], TP53INP2 mutant lacking amino acids 67 to 111 and amino acids 145 to 221; TP53INP2[Δ112-144],[NLSΔ], TP53INP2 mutant lacking amino acids 112 to 144 and amino acids 145 to 221; TUBB, tubulin beta class I; ULK1, unc-51 like autophagy activating kinase 1; VMP1, vacuole membrane protein 1; WIPI2, WD repeat domain phosphoinositide-interacting 2; WT, wild-type; ZFYVE1/DFCP1, zinc finger FYVE-type containing 1. [ABSTRACT FROM AUTHOR]

Lysosomes are acidic intracellular organelles with autophagic functions that are critical for protein degradation and mitochondrial homeostasis, while abnormalities in lysosomal physiological functions are closely associated with neurological disorders. Transmembrane protein 175 (TMEM175), an ion channel in the lysosomal membrane that is essential for maintaining lysosomal acidity, has been proven to coordinate with V-ATPase to modulate the luminal pH of the lysosome to assist the digestion of abnormal proteins and organelles. However, there is considerable controversy about the characteristics of TMEM175. In this review, we introduce the research progress on the structural, modulatory, and functional properties of TMEM175, followed by evidence of its relevance for neurological disorders. Finally, we discuss the potential value of TMEM175 as a therapeutic target in the hope of providing new directions for the treatment of neurodegenerative diseases. • TMEM175 is a highly permeable ion channel for both protons and potassium ions in the lysosomal membrane. • TMEM175 affects lysosomal autophagy by regulating intra-lysosomal acidity and alkalinity. • TMEM175 mutation is a high-risk factor for neurodegenerative disease. [ABSTRACT FROM AUTHOR]