bims-auttor 2022-07-03 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2022‒07‒03
35 papers selected by
Viktor Korolchuk, Newcastle University

PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy.
A degradative to secretory autophagy switch mediates mitochondria clearance in the absence of the mATG8-conjugation machinery.
RhoA signaling increases mitophagy and protects cardiomyocytes against ischemia by stabilizing PINK1 protein and recruiting Parkin to mitochondria.
Zonated leucine sensing by Sestrin-mTORC1 in the liver controls the response to dietary leucine.
Monitoring Autophagy in Neural Stem and Progenitor Cells.
ER-phagy: selective autophagy of the endoplasmic reticulum.
The mTORC1-SLC4A7 axis stimulates bicarbonate import to enhance de novo nucleotide synthesis.
A "short-cut" response of autophagy to oxidative stress: oxygen-dependent activity of a lysine demethylase guides the activity of ULK1 during hypoxia.
Autophagy-Related Pathways in Vesicular Unconventional Protein Secretion.
LAMP2A mediates the loading of proteins into endosomes and selects exosomal cargo.
Nuciferine protects against high-fat diet-induced hepatic steatosis and insulin resistance via activating TFEB-mediated autophagy-lysosomal pathway.
Mitophagy and neuroinflammation: a compelling interplay.
Structural mechanism of protein recognition by the FW domain of autophagy receptor Nbr1.
Inositol triphosphate signaling triggers lysosome biogenesis via calcium release from endoplasmic reticulum stores.
Molecular mechanism of S-adenosylmethionine sensing by SAMTOR in mTORC1 signaling.
Stimulating the autophagic-lysosomal axis enhances host defense against fungal infection in a zebrafish model of invasive Aspergillosis.
PINK1/Parkin Pathway Activation for Mitochondrial Quality Control - Which Is the Best Molecular Target for Therapy?
NCOA4-mediated ferritinophagy is a pancreatic cancer dependency via maintenance of iron bioavailability for iron-sulfur cluster proteins.
Caveolin-1 scaffolding domain peptide abrogates autophagy dysregulation in pulmonary fibrosis.
TFEB-Mediated Lysosomal Restoration Alleviates High Glucose-Induced Cataracts Via Attenuating Oxidative Stress.
Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity.
Cvm1 is a component of multiple vacuolar contact sites required for sphingolipid homeostasis.
Hsp multichaperone complex buffers pathologically modified Tau.
Role for autophagy-related markers Beclin-1 and LC3 in endometriosis.
mTORC1 Inhibition Protects Human Regulatory T Cells From Granzyme-B-Induced Apoptosis.
CD137-CD137L Aggravates Calcification of Vascular Smooth Muscle Cell and Vasculature of ApoE-/- Mice Via Rab7-Mediated Autophagy.
A protocol to induce systemic autophagy and increase energy metabolism in mice using PEGylated arginine deiminase.
Mitochondrial protein import stress regulates the LC3 lipidation step of mitophagy through NLRX1 and RRBP1.
Defects in autophagy lead to selective in vivo changes in turnover of cytosolic and organelle proteins in Arabidopsis.
Sphk1-induced autophagy in microglia promotes neuronal injury following cerebral ischemia-reperfusion.
Combination of the NRF2 Inhibitor and Autophagy Inhibitor Significantly Inhibited Tumorigenicity of Castration-Resistant Prostate Cancer.
Translating across kingdoms: TOR promotes protein synthesis through conserved and divergent pathways in plants.
p62 Phase-Separation as the Foundation of Autophagy-Based Degraders.
Urolithin A improves mitochondrial health, reduces cartilage degeneration, and alleviates pain in osteoarthritis.
Plakophilin3 loss leads to an increase in autophagy and radio-resistance.

PLoS Genet. 2022 Jun;18(6): e1010264

PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy.

Yukako Oe, Keita Kakuda, Shin-Ichiro Yoshimura, Naohiro Hara, Junya Hasegawa, Seigo Terawaki, Yasuyoshi Kimura, Kensuke Ikenaka, Shiro Suetsugu, Hideki Mochizuki, Tamotsu Yoshimori, Shuhei Nakamura.

Autophagy is an indispensable process that degrades cytoplasmic materials to maintain cellular homeostasis. During autophagy, double-membrane autophagosomes surround cytoplasmic materials and either fuse with endosomes (called amphisomes) and then lysosomes, or directly fuse with lysosomes, in both cases generating autolysosomes that degrade their contents by lysosomal hydrolases. However, it remains unclear if there are specific mechanisms and/or conditions which distinguish these alternate routes. Here, we identified PACSIN1 as a novel autophagy regulator. PACSIN1 deletion markedly decreased autophagic activity under basal nutrient-rich conditions but not starvation conditions, and led to amphisome accumulation as demonstrated by electron microscopic and co-localization analysis, indicating inhibition of lysosome fusion. PACSIN1 interacted with SNAP29, an autophagic SNARE, and was required for proper assembly of the STX17 and YKT6 complexes. Moreover, PACSIN1 was required for lysophagy, aggrephagy but not mitophagy, suggesting cargo-specific fusion mechanisms. In C. elegans, deletion of sdpn-1, a homolog of PACSINs, inhibited basal autophagy and impaired clearance of aggregated protein, implying a conserved role of PACSIN1. Taken together, our results demonstrate the amphisome-lysosome fusion process is preferentially regulated in response to nutrient state and stress, and PACSIN1 is a key to specificity during autophagy.

DOI: https://doi.org/10.1371/journal.pgen.1010264
Nat Commun. 2022 Jun 28. 13(1): 3720

A degradative to secretory autophagy switch mediates mitochondria clearance in the absence of the mATG8-conjugation machinery.

Hayden Weng Siong Tan, Guang Lu, Han Dong, Yik-Lam Cho, Auginia Natalia, Liming Wang, Charlene Chan, Dennis Kappei, Reshma Taneja, Shuo-Chien Ling, Huilin Shao, Shih-Yin Tsai, Wen-Xing Ding, Han-Ming Shen.

PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses.

DOI: https://doi.org/10.1038/s41467-022-31213-7
Cell Death Differ. 2022 Jun 27.

RhoA signaling increases mitophagy and protects cardiomyocytes against ischemia by stabilizing PINK1 protein and recruiting Parkin to mitochondria.

Michelle Tu, Valerie P Tan, Justin D Yu, Raghav Tripathi, Zahna Bigham, Melissa Barlow, Jeffrey M Smith, Joan Heller Brown, Shigeki Miyamoto.

Mitophagy, a mitochondria-specific form of autophagy, removes dysfunctional mitochondria and is hence an essential process contributing to mitochondrial quality control. PTEN-induced kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin are critical molecules involved in stress-induced mitophagy, but the intracellular signaling mechanisms by which this pathway is regulated are unclear. We tested the hypothesis that signaling through RhoA, a small GTPase, induces mitophagy via modulation of the PINK1/Parkin pathway as a protective mechanism against ischemic stress. We demonstrate that expression of constitutively active RhoA as well as sphingosine-1-phosphate induced activation of endogenous RhoA in cardiomyocytes result in an accumulation of PINK1 at mitochondria. This is accompanied by translocation of Parkin to mitochondria and ubiquitination of mitochondrial proteins leading to recognition of mitochondria by autophagosomes and their lysosomal degradation. Expression of RhoA in cardiomyocytes confers protection against ischemia, and this cardioprotection is attenuated by siRNA-mediated PINK1 knockdown. In vivo myocardial infarction elicits increases in mitochondrial PINK1, Parkin, and ubiquitinated mitochondrial proteins. AAV9-mediated RhoA expression potentiates these responses and a concurrent decrease in infarct size is observed. Interestingly, induction of mitochondrial PINK1 accumulation in response to RhoA signaling is neither mediated through its transcriptional upregulation nor dependent on depolarization of the mitochondrial membrane, the canonical mechanism for PINK1 accumulation. Instead, our results reveal that RhoA signaling inhibits PINK1 cleavage, thereby stabilizing PINK1 protein at mitochondria. We further show that active RhoA localizes at mitochondria and interacts with PINK1, and that the mitochondrial localization of RhoA is regulated by its downstream effector protein kinase D. These findings demonstrate that RhoA activation engages a unique mechanism to regulate PINK1 accumulation, induce mitophagy and protect against ischemic stress, and implicates regulation of RhoA signaling as a potential strategy to enhance mitophagy and confer protection under stress conditions.

DOI: https://doi.org/10.1038/s41418-022-01032-w
Science. 2022 Jul;377(6601): 47-56

Zonated leucine sensing by Sestrin-mTORC1 in the liver controls the response to dietary leucine.

Andrew L Cangelosi, Anna M Puszynska, Justin M Roberts, Andrea Armani, Thao P Nguyen, Jessica B Spinelli, Tenzin Kunchok, Brianna Wang, Sze Ham Chan, Caroline A Lewis, William C Comb, George W Bell, Aharon Helman, David M Sabatini.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase controls growth in response to nutrients, including the amino acid leucine. In cultured cells, mTORC1 senses leucine through the leucine-binding Sestrin proteins, but the physiological functions and distribution of Sestrin-mediated leucine sensing in mammals are unknown. We find that mice lacking Sestrin1 and Sestrin2 cannot inhibit mTORC1 upon dietary leucine deprivation and suffer a rapid loss of white adipose tissue (WAT) and muscle. The WAT loss is driven by aberrant mTORC1 activity and fibroblast growth factor 21 (FGF21) production in the liver. Sestrin expression in the liver lobule is zonated, accounting for zone-specific regulation of mTORC1 activity and FGF21 induction by leucine. These results establish the mammalian Sestrins as physiological leucine sensors and reveal a spatial organization to nutrient sensing by the mTORC1 pathway.

DOI: https://doi.org/10.1126/science.abi9547
Methods Mol Biol. 2022 ;2515 99-116

Monitoring Autophagy in Neural Stem and Progenitor Cells.

Romina L Filippelli, Samaneh Kamyabiazar, Natasha C Chang.

  Autophagy is a critical cellular program that is necessary for cellular survival and adaptation to nutrient and metabolic stress. In addition to homeostatic maintenance and adaptive response functions, autophagy also plays an active role during development and tissue regeneration. Within the neural system, autophagy is important for stem cell maintenance and the ability of neural stem cells to undergo self-renewal. Autophagy also contributes toward neurogenesis and provides neural progenitor cells with sufficient energy to mediate cytoskeleton remodeling during the differentiation process. In differentiated neural cells, autophagy maintains neuronal homeostasis and viability by preventing the accumulation of toxic and pathological intracellular aggregates. However, prolonged autophagy or dysregulated upregulation of autophagy can result in autophagic cell death. Moreover, mutations or defects in autophagy that result in neural stem cell instability and cell death underlie many neurodegenerative disorders, such as Parkinson's disease. Thus, autophagy plays a multi-faceted role during neurogenesis from the stem cell to the differentiated neural cell. In this chapter, we describe methods to monitor autophagy at the protein and transcript level to evaluate alterations within the autophagy program in neural stem and progenitor cells. We describe immunoblotting and immunocytochemistry approaches for evaluating autophagy-dependent protein modifications, as well as quantitative real-time PCR to assess transcript levels of autophagy genes. As autophagy is a dynamic process, we highlight the importance of using late-stage inhibitors to be able to assess autophagic flux and quantify the level of autophagy occurring within cells.

Keywords:  Autophagic flux; Autophagy; Bafilomycin A1; Chloroquine; MAP1LC3B; Neural stem cell; Nutrient deprivation; p62/SQSTM1

DOI:  https://doi.org/10.1007/978-1-0716-2409-8_7
EMBO Rep. 2022 Jun 27. e55192

ER-phagy: selective autophagy of the endoplasmic reticulum.

Keisuke Mochida, Hitoshi Nakatogawa.

  Eukaryotic cells adequately control the mass and functions of organelles in various situations. Autophagy, an intracellular degradation system, largely contributes to this organelle control by degrading the excess or defective portions of organelles. The endoplasmic reticulum (ER) is an organelle with distinct structural domains associated with specific functions. The ER dynamically changes its mass, components, and shape in response to metabolic, developmental, or proteotoxic cues to maintain or regulate its functions. Therefore, elaborate mechanisms are required for proper degradation of the ER. Here, we review our current knowledge on diverse mechanisms underlying selective autophagy of the ER, which enable efficient degradation of specific ER subdomains according to different demands of cells.

Keywords:  ER-phagy; autophagy; endoplasmic reticulum; intracellular degradation

DOI:  https://doi.org/10.15252/embr.202255192
Mol Cell. 2022 Jun 24. pii: S1097-2765(22)00544-5. [Epub ahead of print]

The mTORC1-SLC4A7 axis stimulates bicarbonate import to enhance de novo nucleotide synthesis.

Eunus S Ali, Anna Lipońska, Brendan P O'Hara, David R Amici, Michael D Torno, Peng Gao, John M Asara, Mee-Ngan F Yap, Marc L Mendillo, Issam Ben-Sahra.

  Bicarbonate (HCO3-) ions maintain pH homeostasis in eukaryotic cells and serve as a carbonyl donor to support cellular metabolism. However, whether the abundance of HCO3- is regulated or harnessed to promote cell growth is unknown. The mechanistic target of rapamycin complex 1 (mTORC1) adjusts cellular metabolism to support biomass production and cell growth. We find that mTORC1 stimulates the intracellular transport of HCO3- to promote nucleotide synthesis through the selective translational regulation of the sodium bicarbonate cotransporter SLC4A7. Downstream of mTORC1, SLC4A7 mRNA translation required the S6K-dependent phosphorylation of the translation factor eIF4B. In mTORC1-driven cells, loss of SLC4A7 resulted in reduced cell and tumor growth and decreased flux through de novo purine and pyrimidine synthesis in human cells and tumors without altering the intracellular pH. Thus, mTORC1 signaling, through the control of SLC4A7 expression, harnesses environmental bicarbonate to promote anabolic metabolism, cell biomass, and growth.

Keywords:  SLC4A7/NBCn1; bicarbonate metabolism; mTOR signaling; purine metabolism; pyrimidine metabolism

DOI:  https://doi.org/10.1016/j.molcel.2022.06.008
Autophagy. 2022 Jun 26. 1-3

A "short-cut" response of autophagy to oxidative stress: oxygen-dependent activity of a lysine demethylase guides the activity of ULK1 during hypoxia.

Ge Yu, Daniel J Klionsky.

  Hypoxia is a type of stress caused by an insufficient supply of oxygen. Macroautophagy/autophagy, a well-conserved pathway, is induced during hypoxia; however, the exact mechanism by which autophagy is regulated in a hypoxic environment remains to be elucidated. A recent study by Li et al. shed light on how hypoxia can regulate early steps of autophagy induction. In this study, the authors discovered a novel symmetrical dimethylation of ULK1 at arginine 170 (R170me2s) that accumulates during hypoxia and increases ULK1 kinase activity by promoting autophosphorylation of ULK1 at T180. The authors identified PRMT5 and KDM5C as the primary methyltransferase and demethylase regulating ULK1 R170me2s and show that the lack of oxygen directly leads to reduced activity of KDM5C, which is likely the cause of accumulation of ULK1 R170me2s during hypoxia. Furthermore, the authors showed that ULK1 R170me2s promotes mitochondrial turnover and maintains cell viability in response to hypoxia stress. Together these data provide a new perspective on how the oxygen level regulates autophagy induction and show the physiological role of ULK1 R170me2s.

Keywords:  Autophagy; KDM5C; PRMT5; ULK1; hypoxia; mitophagy; oxidative stress; oxygen-sensitive methylation; tumor

DOI:  https://doi.org/10.1080/15548627.2022.2089957
Front Cell Dev Biol. 2022 ;10 892450

Autophagy-Related Pathways in Vesicular Unconventional Protein Secretion.

Shin Hye Noh, Ye Jin Kim, Min Goo Lee.

  Cellular proteins directed to the plasma membrane or released into the extracellular space can undergo a number of different pathways. Whereas the molecular mechanisms that underlie conventional ER-to-Golgi trafficking are well established, those associated with the unconventional protein secretion (UPS) pathways remain largely elusive. A pathway with an emerging role in UPS is autophagy. Although originally known as a degradative process for maintaining intracellular homeostasis, recent studies suggest that autophagy has diverse biological roles besides its disposal function and that it is mechanistically involved in the UPS of various secretory cargos including both leaderless soluble and Golgi-bypassing transmembrane proteins. Here, we summarize current knowledge of the autophagy-related UPS pathways, describing and comparing diverse features in the autophagy-related UPS cargos and autophagy machineries utilized in UPS. Additionally, we also suggest potential directions that further research in this field can take.

Keywords:  GRASP; autophagy; golgi bypass; multi-vesicular body; unconventional protein secretion (UPS)

DOI:  https://doi.org/10.3389/fcell.2022.892450
Autophagy. 2022 Jun 27. 1-3

LAMP2A mediates the loading of proteins into endosomes and selects exosomal cargo.

João Vasco Ferreira, Ana da Rosa Soares, Paulo Pereira.

  Exosomes are a subtype of extracellular vesicles (EVs), released by all cell types, that originate from the invagination of the endosomal limiting membrane. These EVs can transport biological information in the form of proteins and RNA and have been the focus of intensive research over the last decade. It is becoming apparent that EVs can have important roles in health and disease. EVs are also promising noninvasive biomarkers of disease (liquid biopsies) and valuable vectors for innovative therapies. However, little is known about the mechanisms that regulate the loading of cytosolic proteins into exosomes. We recently showed that soluble proteins containing amino acid sequences biochemically related to the KFERQ motif are loaded into nascent exosomes at the endosomal limiting membrane, in a process mediated by LAMP2A. Because of the subcellular localization and machinery involved, this mechanism has many similarities with chaperone-mediated autophagy (CMA) and endosomal microautophagy (e-Mi), but also some important differences. In this punctum we will focus on the mechanistic details of exosomal LAMP2A loading of cargo (e-LLoC) as well as on its implications for intercellular and interorgan communication.

Keywords:  LAMP2A; autophagy; chaperones; eLLoC; endosomes; exosomes; inter-organ communication; intercellular communication; lysosome

DOI:  https://doi.org/10.1080/15548627.2022.2092315
Acta Pharm Sin B. 2022 Jun;12(6): 2869-2886

Nuciferine protects against high-fat diet-induced hepatic steatosis and insulin resistance via activating TFEB-mediated autophagy-lysosomal pathway.

Xiliang Du, Chiara Di Malta, Zhiyuan Fang, Taiyu Shen, Xiaodi Niu, Meng Chen, Bo Jin, Hao Yu, Lin Lei, Wenwen Gao, Yuxiang Song, Zhe Wang, Chuang Xu, Zhijun Cao, Guowen Liu, Xinwei Li.

  Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis and insulin resistance and there are currently no approved drugs for its treatment. Hyperactivation of mTOR complex 1 (mTORC1) and subsequent impairment of the transcription factor EB (TFEB)-mediated autophagy-lysosomal pathway (ALP) are implicated in the development of NAFLD. Accordingly, agents that augment hepatic TFEB transcriptional activity may have therapeutic potential against NAFLD. The objective of this study was to investigate the effects of nuciferine, a major active component from lotus leaf, on NAFLD and its underlying mechanism of action. Here we show that nuciferine activated ALP and alleviated steatosis, insulin resistance in the livers of NAFLD mice and palmitic acid-challenged hepatocytes in a TFEB-dependent manner. Mechanistic investigation revealed that nuciferine interacts with the Ragulator subunit hepatitis B X-interacting protein and impairs the interaction of the Ragulator complex with Rag GTPases, thereby suppressing lysosomal localization and activity of mTORC1, which activates TFEB-mediated ALP and further ameliorates hepatic steatosis and insulin resistance. Our present results indicate that nuciferine may be a potential agent for treating NAFLD and that regulation of the mTORC1-TFEB-ALP axis could represent a novel pharmacological strategy to combat NAFLD.

Keywords:  Autophagy; Fatty liver; Lotus leaf; Lysosome; MiT/TFE; Ragulator; TFEB; mTORC1

DOI:  https://doi.org/10.1016/j.apsb.2021.12.012
Curr Neuropharmacol. 2022 Jun 28.

Mitophagy and neuroinflammation: a compelling interplay.

Nikolaos Charmpilas, Evandro Fei Fang, Konstantinos Palikaras.

  Mitochondria are the main sites of energy production and a major source of metabolic stress. Not surprisingly, impairment of mitochondrial homeostasis is tightly associated with the development and progression of a broad spectrum of human pathologies, including neurodegenerative disorders. Mitophagy mediates the selective degradation of damaged organelles, thus promoting cellular viability and tissue integrity. Defective mitophagy triggers cellular senescence and prolonged neuroinflammation, leading eventually to cell death and brain homeostasis collapse. Here, we survey the intricate interplay between mitophagy and neuroinflammation, highlighting that mitophagy can be a focal point for therapeutic interventions to tackle neurodegeneration.

Keywords:  Ageing; energy homeostasis; immunity; inflammation; metabolism; mitochondria; mitophagy; neurodegeneration

DOI:  https://doi.org/10.2174/1570159X20666220628153632
Nat Commun. 2022 Jun 25. 13(1): 3650

Structural mechanism of protein recognition by the FW domain of autophagy receptor Nbr1.

Jianxiu Zhang, Ying-Ying Wang, Zhao-Qian Pan, Yulu Li, Jianhua Sui, Li-Lin Du, Keqiong Ye.

Neighbor of BRCA1 (Nbr1) is a conserved autophagy receptor that provides cargo selectivity to autophagy. The four-tryptophan (FW) domain is a signature domain of Nbr1, but its exact function remains unclear. Here, we show that Nbr1 from the filamentous fungus Chaetomium thermophilum uses its FW domain to bind the α-mannosidase Ams1, a cargo of selective autophagy in both budding yeast and fission yeast, and delivers Ams1 to the vacuole by conventional autophagy in heterologous fission yeast. The structure of the Ams1-FW complex was determined at 2.2 Å resolution by cryo-electron microscopy. The FW domain adopts an immunoglobulin-like β-sandwich structure and recognizes the quaternary structure of the Ams1 tetramer. Notably, the N-terminal di-glycine of Ams1 is specifically recognized by a conserved pocket of the FW domain. The FW domain becomes degenerated in fission yeast Nbr1, which binds Ams1 with a ZZ domain instead. Our findings illustrate the protein binding mode of the FW domain and reveal the versatility of Nbr1-mediated cargo recognition.

DOI: https://doi.org/10.1038/s41467-022-31439-5
Contact (Thousand Oaks). 2022 Jan;5 251525642210970

Inositol triphosphate signaling triggers lysosome biogenesis via calcium release from endoplasmic reticulum stores.

Mouhannad Malek, Volker Haucke.

  Lysosomes serve as cellular degradation and signaling centers that coordinate the turnover of macromolecules with cell metabolism. The adaptation of cellular lysosome content and activity via the induction of lysosome biogenesis is therefore key to cell physiology and to counteract disease. Previous work has established a pathway for the induction of lysosome biogenesis in signaling-inactive starved cells that is based on the repression of mTORC1-mediated nutrient signaling. How lysosomal biogenesis is facilitated in signaling-active fed cells is poorly understood. A recent study by Malek et al (Malek et al, 2022) partially fills this gap by unraveling a nutrient signaling-independent pathway for lysosome biogenesis that operates in signaling-active cells. This pathway involves the receptor-mediated activation of phospholipase C, inositol (1,4,5)-triphosphate (IP3)-triggered release of calcium ions from endoplasmic reticulum stores, and the calcineurin-induced activation of transcription factor EB (TFEB) and its relative TFE3 to induce lysosomal gene expression independent of calcium in the lysosome lumen. These findings contribute to our understanding of how lysosome biogenesis and function are controlled in response to environmental changes and cell signaling and may conceivably be of relevance for our understanding and the treatment of lysosome-related diseases as well as for aging and neurodegeneration.

Keywords:  calcium; inositol (1,4,5)-triphosphate; lysosome; nutrient signaling; phospholipase C

DOI:  https://doi.org/10.1177/25152564221097052
Sci Adv. 2022 Jul;8(26): eabn3868

Molecular mechanism of S-adenosylmethionine sensing by SAMTOR in mTORC1 signaling.

Xin Tang, Yifan Zhang, Guanchao Wang, Chunxiao Zhang, Fang Wang, Jiawen Shi, Tianlong Zhang, Jianping Ding.

The mechanistic target of rapamycin-mLST8-raptor complex (mTORC1) functions as a central regulator of cell growth and metabolism in response to changes in nutrient signals such as amino acids. SAMTOR is an S-adenosylmethionine (SAM) sensor, which regulates the mTORC1 activity through its interaction with the GTPase-activating protein activity toward Rags-1 (GATOR1)-KPTN, ITFG2, C12orf66 and SZT2-containing regulator (KICSTOR) complex. In this work, we report the crystal structures of Drosophila melanogaster SAMTOR in apo form and in complex with SAM. SAMTOR comprises an N-terminal helical domain and a C-terminal SAM-dependent methyltransferase (MTase) domain. The MTase domain contains the SAM-binding site and the potential GATOR1-KICSTOR-binding site. The helical domain functions as a molecular switch, which undergoes conformational change upon SAM binding and thereby modulates the interaction of SAMTOR with GATOR1-KICSTOR. The functional roles of the key residues and the helical domain are validated by functional assays. Our structural and functional data together reveal the molecular mechanism of the SAM sensing of SAMTOR and its functional role in mTORC1 signaling.

DOI: https://doi.org/10.1126/sciadv.abn3868
Autophagy. 2022 Jul 01. 1-14

Stimulating the autophagic-lysosomal axis enhances host defense against fungal infection in a zebrafish model of invasive Aspergillosis.

G Forn-Cuní, L Welvaarts, F M Stel, C J van den Hondel, M Arentshorst, Afj Ram, A H Meijer.

  The increasing prevalence of antifungal-resistant human pathogenic fungi, particularly azole-resistant Aspergillus fumigatus, is a life-threatening challenge to the immunocompromised population. Autophagy-related processes such as LC3-associated phagocytosis have been shown to be activated in the host response against fungal infection, but their overall effect on host resistance remains uncertain. To analyze the relevance of these processes in vivo, we used a zebrafish animal model of invasive Aspergillosis. To confirm the validity of this model to test potential treatments for this disease, we confirmed that immunosuppressive treatments or neutropenia rendered zebrafish embryos more susceptible to A. fumigatus. We used GFP-Lc3 transgenic zebrafish to visualize the autophagy-related processes in innate immune phagocytes shortly after phagocytosis of A. fumigatus conidia, and found that both wild-type and melanin-deficient conidia elicited Lc3 recruitment. In macrophages, we observed GFP-Lc3 accumulation in puncta after phagocytosis, as well as short, rapid events of GFP-Lc3 decoration of single and multiple conidia-containing vesicles, while neutrophils covered single conidia-containing vesicles with bright and long-lasting GFP-Lc3 signal. Next, using genetic and pharmacological stimulation of three independent autophagy-inducing pathways, we showed that the antifungal autophagy response improves the host survival against A. fumigatus infection, but only in the presence of phagocytes. Therefore, we provide proof-of-concept that stimulating the (auto)phagolysosomal pathways is a promising approach to develop host-directed therapies against invasive Aspergillosis, and should be explored further either as adjunctive or stand-alone therapy for drug-resistant Aspergillus infections.

Keywords:  Aspergillus; autophagic defense; fungal infection; host-pathogen interaction; immunomodulation; innate immunity; phagocytes; zebrafish

DOI:  https://doi.org/10.1080/15548627.2022.2090727
Front Aging Neurosci. 2022 ;14 890823

PINK1/Parkin Pathway Activation for Mitochondrial Quality Control - Which Is the Best Molecular Target for Therapy?

Laura F Silvian.

  There has been long-term interest in drugging the PINK1-Parkin pathway with therapeutics as a treatment for Parkinson's disease (PD). Despite significant structural data on Parkin as well as the PINK1 kinase and the multiple conformational changes it undergoes, activation of these targets is non-trivial. This review highlights small molecule screening results that suggests that activation of Parkin biochemically does not necessarily translate to activation of Parkin within cells. There are also issues with activation of PINK1 with kinetin analogs, which do not appear to rescue rodent models of PD. The counter-measure of activating the mitophagy pathway with deubiquitinase (DUB) inhibitors such as USP30 inhibitors is progressing in the clinic for kidney disease and the proof of biology for this target will be tested in these trials. An alternative mechanism of activating Parkin in response to oxidative stress via Parkin phosphorylation by the AMPK-ULK1 pathway may be a simpler way to lower the energy barrier Parkin activation.

Keywords:  AMPK; PINK1-Parkin pathway; Parkinson’s disease; ULK1 (unc-51 like autophagy activating kinase 1); cryoEM (cryo-electron microscopy); crystallography; molecular mechanisms of activation; small molecule

DOI:  https://doi.org/10.3389/fnagi.2022.890823
Cancer Discov. 2022 Jun 30. pii: candisc.0043.2022-1-10 21:36:20.420. [Epub ahead of print]

NCOA4-mediated ferritinophagy is a pancreatic cancer dependency via maintenance of iron bioavailability for iron-sulfur cluster proteins.

Naiara Santana-Codina, Maria Quiles Del Rey, Kevin S Kapner, Huan Zhang, Ajami Gikandi, Callum Malcolm, Clara Poupault, Miljan Kuljanin, Kristen M John, Douglas E Biancur, Brandon Chen, Nupur K Das, Kristen E Lowder, Connor J Hennessey, Wesley Huang, Annan Yang, Yatrik M Shah, Jonathan A Nowak, Andrew J Aguirre, Joseph D Mancias.

Pancreatic ductal adenocarcinomas (PDAC) depend on autophagy for survival; however, the metabolic substrates that autophagy provides to drive PDAC progression are unclear. Ferritin, the cellular iron storage complex, is targeted for lysosomal degradation (ferritinophagy) by the selective autophagy adaptor NCOA4, resulting in release of iron for cellular utilization. Using patient-derived and murine models of PDAC we now demonstrate that ferritinophagy is upregulated in PDAC to sustain iron availability thereby promoting tumor progression. Quantitative proteomics reveals that ferritinophagy fuels iron-sulfur cluster protein synthesis to support mitochondrial homeostasis. Targeting NCOA4 leads to tumor growth delay and prolonged survival but with development of compensatory iron acquisition pathways. Finally, enhanced ferritinophagy accelerates PDAC tumorigenesis, and an elevated ferritinophagy expression signature predicts for poor prognosis in PDAC patients. Together, our data reveal that maintenance of iron homeostasis is a critical function of PDAC autophagy, and we define NCOA4-mediated ferritinophagy as a therapeutic target in PDAC.

DOI: https://doi.org/10.1158/2159-8290.CD-22-0043
Sci Rep. 2022 Jun 30. 12(1): 11086

Caveolin-1 scaffolding domain peptide abrogates autophagy dysregulation in pulmonary fibrosis.

Shalini Venkatesan, Liang Fan, Hua Tang, Nagarjun V Konduru, Sreerama Shetty.

Idiopathic pulmonary fibrosis (IPF) is the most common and fatal form of interstitial lung disease. IPF is characterized by irreversible scarring of the lungs leading to lung function decline. Although the etiology remains poorly understood, dysregulated autophagy in alveolar-epithelial cells (AECs) together with interplay between apoptotic-AECs and proliferative-myofibroblasts have been strongly implicated in IPF pathogenesis. Recent studies have revealed that a caveolin-1-derived 7-mer peptide, CSP7, mitigates established PF at least in part by improving AEC viability. In the present study, we aimed to determine whether and how CSP7 regulates autophagy in fibrotic-lung AECs. We found that p53 and autophagic proteins were markedly upregulated in AECs from mice with single/multi-doses of bleomycin-or silica-induced PF. This was abolished following treatment of PF-mice with CSP7. Further, CSP7 abrogated silica- or bleomycin-induced p53 and autophagy proteins in AECs. Immunoprecipitation further revealed that CSP7 abolishes the interaction of caveolin-1 with LC3BII and p62 in AECs. AEC-specific p53-knockout mice resisted silica- or bleomycin-induced changes in autophagy proteins, or CSP7 treatment. Our findings provide a novel mechanism by which CSP7 inhibits dysregulated autophagy in injured AECs and mitigates existing PF. These results affirm the potential of CSP7 for treating established PF, including IPF and silicosis.

DOI: https://doi.org/10.1038/s41598-022-14832-4
Invest Ophthalmol Vis Sci. 2022 Jun 01. 63(6): 26

TFEB-Mediated Lysosomal Restoration Alleviates High Glucose-Induced Cataracts Via Attenuating Oxidative Stress.

Yan Sun, Xiaoran Wang, Baoxin Chen, Mi Huang, Pengjuan Ma, Lang Xiong, Jingqi Huang, Jieping Chen, Shan Huang, Yizhi Liu.

Purpose: Diabetic cataract (DC) is a visual disorder arising from diabetes mellitus (DM). Autophagy, a prosurvival intracellular process through lysosomal fusion and degradation, has been implicated in multiple diabetic complications. Herein, we performed in vivo and in vitro assays to explore the specific roles of the autophagy-lysosome pathway in DC.Methods: Streptozotocin-induced DM and incubation in high glucose (HG) led to rat lens opacification. Protein Simple Wes, Western blot, and immunoassay were utilized to investigate autophagic changes in lens epithelial cells (LECs) and lens fiber cells (LFCs). RNA-sequencing (RNA-seq) was performed to explore genetic changes in the lenses of diabetic rats. Moreover, autophagy-lysosomal functions were examined using lysotracker, Western blot, and immunofluorescence analyses in HG-cultured primary rabbit LECs.
Results: First, DM and HG culture led to fibrotic LECs, swelling LFCs, and eventually cataracts. Further analysis showed aberrant autophagic degradation in LECs and LFCs during cataract formation. RNA-seq data revealed that the differentially expressed genes (DEGs) were enriched in the lysosome pathway. In primary LECs, HG treatment resulted in decreased transcription factor EB (TFEB) and cathepsin B (CTSB) activity, and increased lysosomal size and pH values. Moreover, TFEB-mediated dysfunctional lysosomes resulted from excessive oxidative stress in LECs under HG conditions. Furthermore, TFEB activation by curcumin analog C1 alleviated HG-induced cataracts through enhancing lysosome biogenesis and activating protective autophagy, thereby attenuating HG-mediated oxidative damage.
Conclusions: In summary, we first identified that ROS-TFEB-dependent lysosomal dysfunction contributed to autophagy blockage in HG-induced cataracts. Additionally, TFEB-mediated lysosomal restoration might be a promising therapeutic method for preventing and treating DC through mitigating oxidative stress.

DOI: https://doi.org/10.1167/iovs.63.6.26
J Biol Chem. 2022 Jun 24. pii: S0021-9258(22)00629-9. [Epub ahead of print] 102187

Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity.

Golam T Saffi, Cheng An Wang, Emily M Mangialardi, Jean Vacher, Roberto J Botelho, Leonardo Salmena.

  Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome "fusion-fission" cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P2 to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of 3H-myo-inositol labelled phosphoinositides and phosphoinositide immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.

Keywords:  Apilimod; Inpp4b; Lysosomes; PIKfyve; PtdIns(3)P; Vps34

DOI:  https://doi.org/10.1016/j.jbc.2022.102187
J Cell Biol. 2022 Aug 01. pii: e202103048. [Epub ahead of print]221(8):

Cvm1 is a component of multiple vacuolar contact sites required for sphingolipid homeostasis.

Daniel D Bisinski, Inês Gomes Castro, Muriel Mari, Stefan Walter, Florian Fröhlich, Maya Schuldiner, Ayelén González Montoro.

Membrane contact sites are specialized platforms formed between most organelles that enable them to exchange metabolites and influence the dynamics of each other. The yeast vacuole is a degradative organelle equivalent to the lysosome in higher eukaryotes with important roles in ion homeostasis and metabolism. Using a high-content microscopy screen, we identified Ymr160w (Cvm1, for contact of the vacuole membrane 1) as a novel component of three different contact sites of the vacuole: with the nuclear endoplasmic reticulum, the mitochondria, and the peroxisomes. At the vacuole-mitochondria contact site, Cvm1 acts as a tether independently of previously known tethers. We show that changes in Cvm1 levels affect sphingolipid homeostasis, altering the levels of multiple sphingolipid classes and the response of sphingolipid-sensing signaling pathways. Furthermore, the contact sites formed by Cvm1 are induced upon a decrease in sphingolipid levels. Altogether, our work identifies a novel protein that forms multiple contact sites and supports a role of lysosomal contacts in sphingolipid homeostasis.

DOI: https://doi.org/10.1083/jcb.202103048
Nat Commun. 2022 Jun 27. 13(1): 3668

Hsp multichaperone complex buffers pathologically modified Tau.

Antonia Moll, Lisa Marie Ramirez, Momchil Ninov, Juliane Schwarz, Henning Urlaub, Markus Zweckstetter.

Alzheimer's disease is a neurodegenerative disorder in which misfolding and aggregation of pathologically modified Tau is critical for neuronal dysfunction and degeneration. The two central chaperones Hsp70 and Hsp90 coordinate protein homeostasis, but the nature of the interaction of Tau with the Hsp70/Hsp90 machinery has remained enigmatic. Here we show that Tau is a high-affinity substrate of the human Hsp70/Hsp90 machinery. Complex formation involves extensive intermolecular contacts, blocks Tau aggregation and depends on Tau's aggregation-prone repeat region. The Hsp90 co-chaperone p23 directly binds Tau and stabilizes the multichaperone/substrate complex, whereas the E3 ubiquitin-protein ligase CHIP efficiently disassembles the machinery targeting Tau to proteasomal degradation. Because phosphorylated Tau binds the Hsp70/Hsp90 machinery but is not recognized by Hsp90 alone, the data establish the Hsp70/Hsp90 multichaperone complex as a critical regulator of Tau in neurodegenerative diseases.

DOI: https://doi.org/10.1038/s41467-022-31396-z
BMC Womens Health. 2022 Jun 29. 22(1): 264

Role for autophagy-related markers Beclin-1 and LC3 in endometriosis.

Zhiwei Kong, Tingting Yao.

  BACKGROUND: Endometriosis is a common and challenging disease in women of childbearing age with high personal and social costs. Many molecular differences between ectopic and eutopic endometrium present difficulties in the development of new drug therapies and therapies. Autophagy is a response to stress and has recently been studied in human cancers. Two important autophagy genes, Beclin-1 and LC3, have been reported in several human cancers. However, the reports of Beclin-1 and LC3 in endometriosis are limited and controversial.METHODS: In this study, we investigated the expression of Beclin-1 and LC3 by Immunohistochemistry.
RESULTS: We found their downregulation in endometriosis. We also found these two autophagy gene expression are negatively correlated with the stage of endometriosis.
CONCLUSIONS: Decreased expression of Beclin-1 and LC3 may be related to the occurrence and development of endometriosis.

Keywords:  Autophagy; Beclin-1; Endometriosis; Immunohistochemistry; LC3

DOI:  https://doi.org/10.1186/s12905-022-01850-7
Front Immunol. 2022 ;13 899975

mTORC1 Inhibition Protects Human Regulatory T Cells From Granzyme-B-Induced Apoptosis.

Siawosh K Eskandari, Hazim Allos, Basmah S Al Dulaijan, Gandolina Melhem, Ina Sulkaj, Juliano B Alhaddad, Anis J Saad, Christa Deban, Philip Chu, John Y Choi, Branislav Kollar, Bohdan Pomahac, Leonardo V Riella, Stefan P Berger, Jan S F Sanders, Judy Lieberman, Li Li, Jamil R Azzi.

  Regulatory T cells (Tregs) have shown great promise as a means of cellular therapy in a multitude of allo- and auto-immune diseases-due in part to their immunosuppressive potency. Nevertheless, the clinical efficacy of human Tregs in patients has been limited by their poor in vivo homeostasis. To avert apoptosis, Tregs require stable antigenic (CD3ζ/T-cell-receptor-mediated), co-stimulatory (CD28-driven), and cytokine (IL-2-dependent) signaling. Notably, this sequence of signals supports an activated Treg phenotype that includes a high expression of granzymes, particularly granzyme B (GrB). Previously, we have shown that aside from the functional effects of GrB in lysing target cells to modulate allo-immunity, GrB can leak out of the intracellular lysosomal granules of host Tregs, initiating pro-apoptotic pathways. Here, we assessed the role of inhibiting mechanistic target of rapamycin complex 1 (mTORC1), a recently favored drug target in the transplant field, in regulating human Treg apoptosis via GrB. Using ex vivo models of human Treg culture and a humanized mouse model of human skin allotransplantation, we found that by inhibiting mTORC1 using rapamycin, intracytoplasmic expression and functionality of GrB diminished in host Tregs; lowering human Treg apoptosis by in part decreasing the phosphorylation of S6K and c-Jun. These findings support the already clinically validated effects of mTORC1 inhibition in patients, most notably their stabilization of Treg bioactivity and in vivo homeostasis.

Keywords:  granzyme B; grapoptosis; human tregs; mTORC1; rapamycin; treg homeostasis

DOI:  https://doi.org/10.3389/fimmu.2022.899975
J Cardiovasc Transl Res. 2022 Jun 28.

CD137-CD137L Aggravates Calcification of Vascular Smooth Muscle Cell and Vasculature of ApoE-/- Mice Via Rab7-Mediated Autophagy.

Ping Yang, Guangyao Zang, Yang Yan, Wei Zhong, Bo Li, Yao Xu, Chen Shao, Zhongqun Wang, Jun Pu, Wei Yuan.

  Vascular calcification is an independent risk factor for acute cardiovascular events and a predictor of adverse prognosis; the abnormal fusion and degradation of autophagosomes and lysosomes are closely related to the calcification of VSMC and aortic AS plaque in ApoE-/- mice. Rab7 is a member of the Ras protein family and acts as a molecular switch in the fusion between autophagosomes and lysosomes. In this study, we found that the activation of the CD137-CD137L signal promoted calcification by inhibiting the expression and activity of Rab7, which regulates the degradation of autophagic cargo in vascular smooth muscle cells (VSMCs) and aortic atherosclerosis (AS) plaques in ApoE-/- mice. Knockdown of Rab7 impaired its tethering with the downstream molecule FYVE and coiled-coil containing 1 (FYCO1), which transports autophagosomes to lysosomes through microtubule motor kinesins and fuses with lysosomes to degrade the autophagic content. Overexpression of Rab7-alleviated calcification caused by the activation of the CD137 signaling pathway. In addition, FYCO1 knockdown promoted calcification even though the expression and activity of Rab7 were normal. Our results suggest that Rab7 is the target of CD137 signaling; Rab7 cannot interact with its downstream molecule FYCO1 when its activity and expression were inhibited by the activation of CD137 signaling pathway, thus inhibiting the autophagic degradation and promoting calcification.

Keywords:  Autophagy; CD137; Calcification; Rab7; VSMC; Vascular calcification

DOI:  https://doi.org/10.1007/s12265-022-10272-7
STAR Protoc. 2022 Jun 23. pii: S2666-1667(22)00369-0. [Epub ahead of print]3(3): 101489

A protocol to induce systemic autophagy and increase energy metabolism in mice using PEGylated arginine deiminase.

Yiming Zhang, Brian J DeBosch.

  Obesity is a prevalent metabolic disorder worldwide. Here, we describe a comprehensive protocol using pegylated arginine deiminase (ADI-EPG 20) to apply the concept that arginine depletion induces systemic autophagy to drive whole-body energy metabolism and weight loss in mice. We detail the steps for cohort setup, mouse husbandry, and treatment and provide expected results under these conditions. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022a, 2022b).

Keywords:  Metabolism; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2022.101489
Mol Cell. 2022 Jun 16. pii: S1097-2765(22)00540-8. [Epub ahead of print]

Mitochondrial protein import stress regulates the LC3 lipidation step of mitophagy through NLRX1 and RRBP1.

Samuel A Killackey, Yuntian Bi, Fraser Soares, Ikram Hammi, Nathaniel J Winsor, Ali A Abdul-Sater, Dana J Philpott, Damien Arnoult, Stephen E Girardin.

  Protein import into mitochondria is a highly regulated process, yet how cells clear mitochondria undergoing dysfunctional protein import remains poorly characterized. Here we showed that mitochondrial protein import stress (MPIS) triggers localized LC3 lipidation. This arm of the mitophagy pathway occurs through the Nod-like receptor (NLR) protein NLRX1 while, surprisingly, without the engagement of the canonical mitophagy protein PINK1. Mitochondrial depolarization, which itself induces MPIS, also required NLRX1 for LC3 lipidation. While normally targeted to the mitochondrial matrix, cytosol-retained NLRX1 recruited RRBP1, a ribosome-binding transmembrane protein of the endoplasmic reticulum, which relocated to the mitochondrial vicinity during MPIS, and the NLRX1/RRBP1 complex in turn controlled the recruitment and lipidation of LC3. Furthermore, NLRX1 controlled skeletal muscle mitophagy in vivo and regulated endurance capacity during exercise. Thus, localization and lipidation of LC3 at the site of mitophagosome formation is a regulated step of mitophagy controlled by NLRX1/RRBP1 in response to MPIS.

Keywords:  NLRX1; Nod-like receptors; mitochondria; mitochondrial protein import; mitophagy

DOI:  https://doi.org/10.1016/j.molcel.2022.06.004
Plant Cell. 2022 Jun 29. pii: koac185. [Epub ahead of print]

Defects in autophagy lead to selective in vivo changes in turnover of cytosolic and organelle proteins in Arabidopsis.

Lei Li, Chun Pong Lee, Xinxin Ding, Yu Qin, Akila Wijerathna-Yapa, Martyna Broda, Marisa S Otegui, A Harvey Millar.

  Identification of autophagic protein cargo in plants in autophagy related genes (ATG) mutants is complicated by changes in protein synthesis and protein degradation. To detect autophagic cargo, we measured protein degradation rate in shoots and roots of Arabidopsis (Arabidopsis thaliana) atg5 and atg11 mutants. These data show that less than a quarter of proteins changing in abundance are probable cargo and revealed roles of ATG11 and ATG5 in degradation of specific glycolytic enzymes and of other cytosol, chloroplast and ER-resident proteins, and a specialized role for ATG11 in degradation of proteins from mitochondria and chloroplasts. Protein localization in transformed protoplasts and degradation assays in the presence of inhibitors confirm a role for autophagy in degrading glycolytic enzymes. Autophagy induction by Pi limitation changed metabolic profiles and the protein synthesis and degradation rates of atg5 and atg11 plants. A general decrease in the abundance of amino acids and increase in secondary metabolites in autophagy mutants was consistent with altered catabolism and changes in energy conversion caused by reduced degradation rate of specific proteins. Combining measures of changes in protein abundance and degradation rates, we also identify ATG11 and ATG5 associated protein cargo of low Pi induced autophagy in chloroplasts and ER-resident proteins involved in secondary metabolism.

Keywords:  autophagy; chloroplast; mitochondrion; phosphate starvation; protein degradation

DOI:  https://doi.org/10.1093/plcell/koac185
Eur J Neurosci. 2022 Jun 29.

Sphk1-induced autophagy in microglia promotes neuronal injury following cerebral ischemia-reperfusion.

Yuanyuan Zeng, Wei Zhang, Tengteng Xue, Dayong Zhang, Manhua Lv, Yongjia Jiang.

  Microglial hyperactivation mediated by sphingosine kinase 1/sphingosine-1-phosphate (SphK1/S1P) signaling and the consequent inflammatory mediator production serve as the key drivers of cerebral ischemia-reperfusion injury (CIRI). Although SphK1 reportedly controls autophagy and microglial activation, it remains uncertain as to whether SphK1 is similarly capable of regulating damage mediated by CIRI-activated microglia. In the current study, we adopted both in vitro oxygen-glucose deprivation reperfusion (OGDR) models and in vivo rat models of focal CIRI to ascertain this possibility. It was found that CIRI up-regulated SphK1 and induced autophagy in microglia, while inhibiting these changes significantly impaired to prevented neuronal apoptosis. Results of mechanistic investigation revealed that SphK1 promoted autophagy via the tumor necrosis factor receptor associated factor 2 (TRAF2) pathway. Altogether, our findings unfolded to revealed a novel mechanism, whereby SphK1-induced autophagy in microglia contributed to the pathogenesis of CIRI, potentially highlighting novel avenues for future therapeutic intervention in ischemic stroke patients.

Keywords:  Autophagy; Cerebral ischemia-reperfusion; Microglia; Neuroinflammation; Sphingosine Kinase 1; Sphingosine-1-phosphate

DOI:  https://doi.org/10.1111/ejn.15749
Comput Math Methods Med. 2022 ;2022 4182401

Combination of the NRF2 Inhibitor and Autophagy Inhibitor Significantly Inhibited Tumorigenicity of Castration-Resistant Prostate Cancer.

Yong Zhang, Zhixiang Xin, Baijun Dong, Wei Xue.

Prostate cancer (PCa) is the most frequent cancer in men. Developing new treatment methods for CRPC will be a significant challenge in the clinical treatment of PCa. In conclusion, the results of this study show that NRF2 is downregulated in untreated PCa samples compared to normal PCa samples; however, it was upregulated in mCRPC samples compared to HSPC samples. These results demonstrated that NRF2 may serve as a tumor suppressor in tumorigenesis but promote PCa androgen-independent transferring after ADT treatment. Bioinformatics analysis showed that NRF2 was related to multiple signaling, such as the AGE-RAGE pathway, MAPK pathway, NF-kappa B signaling, PI3K-Akt signaling pathway, and VEGF signaling pathway. Moreover, we revealed that the NRF2 inhibitor significantly inhibited tumorigenicity of CRPC cells in vitro. Of note, combination of the NRF2 inhibitor and autophagy inhibitor had a more significantly suppressive role than either ML385 or CQ, indicating that combination of CQ (autophagy inhibitor) and ML385 (NRF2 inhibitor) is a potential treatment of CRPC. Finally, we conformed that high levels of autophagy regulators LC3B, ULK1, and beclin1 significantly correlated to longer PSA recurrence-free survival time. We think that this study could provide more evidence to confirm that NRF2 is a crucial regulator and targeting NRF2 and autophagy is a potential therapy option for CRPC.

DOI: https://doi.org/10.1155/2022/4182401
J Exp Bot. 2022 Jun 30. pii: erac267. [Epub ahead of print]

Translating across kingdoms: TOR promotes protein synthesis through conserved and divergent pathways in plants.

M Regina Scarpin, Carl H Simmons, Jacob O Brunkard.

  mRNA translation is the growth rate-limiting step in genome expression. TARGET OF RAPAMYCIN (TOR) evolved a central regulatory role in eukaryotes as a signaling hub that monitors nutrient availability to maintain homeostasis and promote growth, largely by increasing the rate of translation initiation and protein synthesis. The dynamic pathways engaged by TOR to regulate translation remain debated even in well-studied yeast and mammalian models, however, despite decades of intense investigation. Recent studies have firmly established that TOR also regulates mRNA translation in plants through conserved mechanisms, such as the TOR-LARP1-5'TOP signaling axis, and through pathways specific to plants. Here, we will review recent advances in our understanding of the regulation of mRNA translation in plants by TOR.

Keywords:   Arabidopsis thaliana ; 5′TOP motifs; Evolution; Kinase signaling; LARP1; Protein synthesis; Ribosomes; TARGET OF RAPAMYCIN; TOR; Translation

DOI:  https://doi.org/10.1093/jxb/erac267
Biochemistry. 2022 Jun 29.

p62 Phase-Separation as the Foundation of Autophagy-Based Degraders.

Daiki Takahashi, Hirokazu Arimoto.

DOI: https://doi.org/10.1021/acs.biochem.2c00252
Aging Cell. 2022 Jul 01. e13662

Urolithin A improves mitochondrial health, reduces cartilage degeneration, and alleviates pain in osteoarthritis.

Davide D'Amico, Merissa Olmer, Andréane M Fouassier, Pamela Valdés, Pénélope A Andreux, Chris Rinsch, Martin Lotz.

  Osteoarthritis (OA) is the most common age-related joint disorder with no effective therapy. According to the World Health Organization, OA affects over 500 million people and is characterized by degradation of cartilage and other joint tissues, severe pain, and impaired mobility. Mitochondrial dysfunction contributes to OA pathology. However, interventions to rescue mitochondrial defects in human OA are not available. Urolithin A (Mitopure) is a natural postbiotic compound that promotes mitophagy and mitochondrial function and beneficially impacts muscle health in preclinical models of aging and in elderly and middle-aged humans. Here, we showed that Urolithin A improved mitophagy and mitochondrial respiration in primary chondrocytes from joints of both healthy donors and OA patients. Furthermore, Urolithin A reduced disease progression in a mouse model of OA, decreasing cartilage degeneration, synovial inflammation, and pain. These improvements were associated with increased mitophagy and mitochondrial content, in joints of OA mice. These findings indicate that UA promotes joint mitochondrial health, alleviates OA pathology, and supports Urolithin A's potential to improve mobility with beneficial effects on structural damage in joints.

Keywords:  Mitopure; chondrocytes; mitochondria; mitophagy; osteoarthritis; urolithin

DOI:  https://doi.org/10.1111/acel.13662
Biochem Biophys Res Commun. 2022 Jun 22. pii: S0006-291X(22)00914-7. [Epub ahead of print]620 1-7

Plakophilin3 loss leads to an increase in autophagy and radio-resistance.

Nazia Chaudhary, Neha Joshi, Rinki Doloi, Anusha Shivashankar, Rahul Thorat, Sorab N Dalal.

  Loss of the desmosomal plaque protein plakophilin3 (PKP3) leads to increased tumor progression and metastasis. As metastatic tumors are often resistant to therapy, we wished to determine whether PKP3 loss led to increased radioresistance. PKP3 knockdown cells showed increased resistance to radiation in vitro and in vivo. The increase in resistance was accompanied by an increased ability to clear reactive oxygen species (ROS) and increased autophagy. The increase in autophagy was required for radioresistance and ROS clearance as inhibiting autophagy using either chloroquine or knocking down ATG3 re-sensitized the PKP3 knockdown clones to radiotherapy. These experiments suggest that autophagy inhibitors could target therapy-resistant PKP3 deficient tumors.

Keywords:  Autophagy; Colorectal cancer; Plakophilin3; Radio-resistance

DOI:  https://doi.org/10.1016/j.bbrc.2022.06.060