bims-apauto 2022-09-18 papers

bims-apauto

Biomed News

on Apoptosis and autophagy

Issue of 2022–09–18
ten papers selected by
Su Hyun Lee, Seoul National University

Conjugation of the ubiquitin family proteins to phospholipids.
A HaloTag-based reporter processing assay to monitor autophagic flux.
Sequential conversion of PtdIns3P to PtdIns(3,5)P2 via endosome maturation couples nutrient signaling to lysosome reformation and basal autophagy.
Slingshot homolog-1 mediates the secretion of small extracellular vesicles containing misfolded proteins by regulating autophagy cargo receptors and actin dynamics.
TRIM27 is an autophagy substrate facilitating mitochondria clustering and mitophagy via phosphorylated TBK1.
The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy.
Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα.
The emerging mechanisms and functions of microautophagy.
The emerging roles of circular RNA-mediated autophagy in tumorigenesis and cancer progression.
Inhibition of the IRE1/JNK pathway in renal tubular epithelial cells attenuates ferroptosis in acute kidney injury.

Autophagy. 2022 Sep 12.

Conjugation of the ubiquitin family proteins to phospholipids.

Jun-Ichi Sakamaki, Noboru Mizushima.

  Conjugation of Atg8-family proteins to phosphatidylethanolamine (PE) is important for autophagosome formation. PE conjugation has been thought to be specific to Atg8 among the ubiquitin-family proteins. However, this dogma has not been experimentally verified. Our recent study revealed that ubiquitin is also conjugated to PE on endosomes and the vacuole (or lysosomes). Other ubiquitin-like proteins, such as NEDD8 and ISG15, also covalently bind to phospholipids. We propose that conjugation to phospholipids could be a common feature of the ubiquitin family.

Keywords:  Atg8; Doa4; Tul1; endosome; lysosome; phosphatidylethanolamine; phospholipids; ubiquitin; ubiquitin-like proteins; vacuole

DOI:  https://doi.org/10.1080/15548627.2022.2123637
Autophagy. 2022 Sep 12.

A HaloTag-based reporter processing assay to monitor autophagic flux.

Willa Wen-You Yim, Hayashi Yamamoto, Noboru Mizushima.

  Monitoring mammalian macroautophagic/autophagic flux is necessary in most autophagy studies but has generally been difficult to do. Here, we discuss our recent report of a HaloTag-based processing method that offers a straightforward readout for autophagic flux. We found that the self-labeling protein HaloTag becomes resistant to proteolysis when labeled with its ligand. Fusing HaloTag to an autophagy protein such as LC3 results in a reporter that is completely degraded when delivered into lysosomes but, when pulse-labeled with HaloTag ligand, releases free HaloTagligand when processed by lysosomal enzymes. The quantifiable amount of free HaloTagligand, observed by immunoblotting or in-gel fluorescence detection, reflects autophagic flux. Besides being compatible with fluorescence microscopy and flow cytometry applications, this quantitative assay can be readily adapted to monitor most autophagy pathways or the autophagic degradation of a protein of interest.

Keywords:  HaloTag; autophagic activity; autophagic flux; autophagy; lysosomal degradation; lysosome; processing assay; protein degradation; protein turnover; pulse-labeling

DOI:  https://doi.org/10.1080/15548627.2022.2123638
Autophagy. 2022 Sep 14.

Sequential conversion of PtdIns3P to PtdIns(3,5)P2 via endosome maturation couples nutrient signaling to lysosome reformation and basal autophagy.

Samuel J Rodgers, Emily I Jones, Christina A Mitchell, Meagan J McGrath.

  Macroautophagy/autophagy occurs basally under nutrient-rich conditions in most mammalian cells, contributing to protein and organelle quality control, and protection against ageing and neurodegeneration. During autophagy, lysosomes are heavily utilized via their fusion with autophagosomes and must be repopulated to maintain autophagic degradative capacity. During starvation-induced autophagy, lysosomes are generated via de novo biogenesis under the control of TFEB (transcription factor EB), or by the recycling of autolysosome membranes via autophagic lysosome reformation (ALR). However, these lysosome repopulation processes do not operate under nutrient-rich conditions. In our recent study, we identify a sequential phosphoinositide conversion pathway that enables lysosome repopulation under nutrient-rich conditions to facilitate basal autophagy. Phosphatidylinositol-3,4-bisphosphate (PtdIns[3,4]P2) signals generated downstream of phosphoinositide 3-kinase alpha (PI3Kα) during growth factor stimulation are converted to phosphatidylinositol-3-phosphate (PtdIns3P) on endosomes by INPP4B (inositol polyphosphate-4-phosphatase type II B). We show that PtdIns3P is retained as endosomes mature into endolysosomes, and serves as a substrate for PIKFYVE (phosphoinositide kinase, FYVE-type zinc finger containing) to generate phosphatidylinositol-3,5-bisphosphate (PtdIns[3,5]P2) to promote SNX2-dependent lysosome reformation, basal autophagic flux and protein aggregate degradation. Therefore, endosome maturation couples nutrient signaling to lysosome repopulation during basal autophagy by delivering PI3Kα-derived PtdIns3P to endolysosomes for PtdIns(3,5)P2-dependent lysosome reformation.

Keywords:  INPP4B; PI3Kα; PIKFYVE; endosome; lysosome; phosphoinositides; proteostasis

DOI:  https://doi.org/10.1080/15548627.2022.2124499
Front Aging Neurosci. 2022 ;14 933979

Slingshot homolog-1 mediates the secretion of small extracellular vesicles containing misfolded proteins by regulating autophagy cargo receptors and actin dynamics.

Sara Cazzaro, Cenxiao Fang, Hirah Khan, Richard Witas, Teresa R Kee, Jung-A A Woo, David E Kang.

  Increasing evidence indicates that the accumulation misfolded proteins in Alzheimer's disease (AD) arises from clearance defects in the autophagy-lysosome pathway. Misfolded proteins such as Aβ and tau are secreted in small extracellular vesicles (i.e., exosomes) and are propagated from cell to cell in part through secreted small extracellular vesicles (sEVs). Recent studies suggest that autophagic activity and exosome secretion are coregulated events, and multiple autophagy-related proteins are found in sEVs, including the cargo receptors Sqstm1/p62 and optineurin. However, whether and how autophagy cargo receptors per se regulate the secretion of sEVs is unknown. Moreover, despite the prominent role of actin dynamics in secretory vesicle release, its role in EV secretion is unknown. In this study, we leveraged the dual axes of Slingshot Homolog-1 (SSH1), which inhibits Sqstm1/p62-mediated autophagy and activates cofilin-mediated actin dynamics, to study the regulation of sEV secretion. Here we show that cargo receptors Sqstm1/p62 and optineurin inhibit sEV secretion, an activity that requires their ability to bind ubiquitinated cargo. Conversely, SSH1 increases sEV secretion by dephosphorylating Sqstm1/p62 at pSer403, the phospho-residue that allows Sqstm1/p62 to bind ubiquitinated cargo. In addition, increasing actin dynamics through the SSH1-cofilin activation pathway also increases sEV secretion, which is mimicked by latrunculin B treatment. Finally, Aβ42 oligomers and mutant tau increase sEV secretion and are physically associated with secreted sEVs. These findings suggest that increasing cargo receptor engagement with autophagic cargo and reducing actin dynamics (i.e., SSH1 inhibition) represents an attractive strategy to promote misfolded protein degradation while reducing sEV-mediated cell to cell spread of pathology.

Keywords:  SSH1; actin dynamics; amyloid; autophagy; exosome; optineurin; p62; tau

DOI:  https://doi.org/10.3389/fnagi.2022.933979
FEBS J. 2022 Sep 16.

TRIM27 is an autophagy substrate facilitating mitochondria clustering and mitophagy via phosphorylated TBK1.

Juncal Garcia-Garcia, Anne Kristin McLaren Berge, Katrine Stange Overå, Kenneth Bowitz Larsen, Zambarlal Bhujabal, Andreas Brech, Yakubu Princely Abudu, Trond Lamark, Terje Johansen, Eva Sjøttem.

Tripartite motif containing protein 27 (TRIM27/also called RFP) is a multifunctional ubiquitin E3 ligase involved in numerous cellular functions, such as proliferation, apoptosis, regulation of the NF-kB pathway, endosomal recycling, and the innate immune response. TRIM27 interacts directly with TANK-binding kinase 1 (TBK1) and regulates its stability. TBK1 in complex with autophagy receptors are recruited to ubiquitin chains assembled on the mitochondrial outer membrane promoting mitophagy. Here we identify TRIM27 as an autophagy substrate, depending on ATG7, ATG9 and autophagy receptors for its lysosomal degradation. We show that TRIM27 forms ubiquitylated cytoplasmic bodies that colocalize with autophagy receptors. Surprisingly, we observed that induced expression of EGFP-TRIM27 in HEK293 FlpIn TRIM27 Knock-Out cells mediates mitochondrial clustering. TRIM27 interacts with autophagy receptor SQSTM1/p62, and the TRIM27 mediated mitochondrial clustering is facilitated by SQSTM/p62. We show that phosphorylated TBK1 is recruited to the clustered mitochondria. Moreover, induced mitophagy activity is reduced in HEK293 FlpIn TRIM27 Knock Out cells, while re-introduction of EGFP-TRIM27 completely restores the mitophagy activity. Inhibition of TBK1 reduces mitophagy in HEK293 FlpIn cells and in the reconstituted EGFP-TRIM27 expressing cells, but not in HEK293 FlpIn TRIM27 Knock Out cells. Altogether, these data reveal novel roles for TRIM27 in mitophagy, facilitating mitochondrial clustering via SQSTM1/p62 and mitophagy via stabilization of phosphorylated TBK1 on mitochondria.

DOI: https://doi.org/10.1111/febs.16628
EMBO Rep. 2022 Sep 14. e54993

The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy.

Ting Huang, Gaoyue Jiang, Yabin Zhang, Yuqing Lei, Shiyan Liu, Huihui Li, Kefeng Lu.

  Macroautophagy/autophagy is a conserved process in eukaryotic cells that mediates the degradation and recycling of intracellular substrates. Proteins encoded by autophagy-related (ATG) genes are essentially involved in the autophagy process and must be tightly regulated in response to various circumstances, such as nutrient-rich and starvation conditions. However, crucial transcriptional activators of ATG genes have remained obscure. Here, we identify the RNA polymerase II subunit Rpb9 as an essential regulator of autophagy by a high-throughput screen of a Saccharomyces cerevisiae gene knockout library. Rpb9 plays a crucial and specific role in upregulating ATG1 transcription, and its deficiency decreases autophagic activities. Rpb9 promotes ATG1 transcription by binding to its promoter region, which is mediated by Gcn4. Furthermore, the function of Rpb9 in autophagy and its regulation of ATG1/ULK1 transcription are conserved in mammalian cells. Together, our results indicate that Rpb9 specifically activates ATG1 transcription and thus positively regulates the autophagy process.

Keywords:  ATG1; Rpb9; autophagy; starvation; transcription

DOI:  https://doi.org/10.15252/embr.202254993
Proc Natl Acad Sci U S A. 2022 Sep 20. 119(38): e2210769119

Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα.

Jonathan R Hart, Xiao Liu, Chen Pan, Anyi Liang, Lynn Ueno, Yingna Xu, Alexandra Quezada, Xinyu Zou, Su Yang, Qingtong Zhou, Steve Schoonooghe, Gholamreza Hassanzadeh-Ghassabeh, Tian Xia, Wenqing Shui, Dehua Yang, Peter K Vogt, Ming-Wei Wang.

  Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.

Keywords:  chemical cross-linking; conformational changes; mass spectrometry; nanobody; phosphoinositide 3-kinase (PI3K)

DOI:  https://doi.org/10.1073/pnas.2210769119
Nat Rev Mol Cell Biol. 2022 Sep 12.

The emerging mechanisms and functions of microautophagy.

Liming Wang, Daniel J Klionsky, Han-Ming Shen.

'Autophagy' refers to an evolutionarily conserved process through which cellular contents, such as damaged organelles and protein aggregates, are delivered to lysosomes for degradation. Different forms of autophagy have been described on the basis of the nature of the cargoes and the means used to deliver them to lysosomes. At present, the prevailing categories of autophagy in mammalian cells are macroautophagy, microautophagy and chaperone-mediated autophagy. The molecular mechanisms and biological functions of macroautophagy and chaperone-mediated autophagy have been extensively studied, but microautophagy has received much less attention. In recent years, there has been a growth in research on microautophagy, first in yeast and then in mammalian cells. Here we review this form of autophagy, focusing on selective forms of microautophagy. We also discuss the upstream regulatory mechanisms, the crosstalk between macroautophagy and microautophagy, and the functional implications of microautophagy in diseases such as cancer and neurodegenerative disorders in humans. Future research into microautophagy will provide opportunities to develop novel interventional strategies for autophagy- and lysosome-related diseases.

DOI: https://doi.org/10.1038/s41580-022-00529-z
Cell Death Discov. 2022 Sep 14. 8(1): 385

The emerging roles of circular RNA-mediated autophagy in tumorigenesis and cancer progression.

Yuan Yuan, Xiaojing Zhang, Xinmin Fan, Yin Peng, Zhe Jin.

Circular RNA (circRNA) is characterized by a specific covalently closed ring structure. The back-splicing of precursor mRNA is the main way of circRNA generation, and various cis/trans-acting elements are involved in regulating the process. circRNAs exhibit multiple biological functions, including serving as sponges of microRNAs, interacting with proteins to regulate their stabilities and abilities, and acting as templates for protein translation. Autophagy participates in many physiological and pathological processes, especially it plays a vital role in tumorigenesis and carcinoma progression. Increasing numbers of evidences have revealed that circRNAs are implicated in regulating autophagy during tumor development. Until now, the roles of autophagy-associated circRNAs in carcinoma progression and their molecular mechanisms remain unclear. Here, the emerging regulatory roles and mechanisms of circRNAs in autophagy were summarized. Furtherly, the effects of autophagy-associated circRNAs on cancer development were described. We also prospected the potential of autophagy-associated circRNAs as novel therapeutic targets of tumors and as biomarkers for cancer diagnosis and prognosis.

DOI: https://doi.org/10.1038/s41420-022-01172-5
Front Pharmacol. 2022 ;13 927641

Inhibition of the IRE1/JNK pathway in renal tubular epithelial cells attenuates ferroptosis in acute kidney injury.

Yan Liang, Zhenjie Liu, Lingyun Qu, Yingzi Wang, Yali Zhou, Lulu Liang, Yanhong Guo, Lin Tang.

  Backgroud: Ferroptosis is a form of regulated cell death in ischemia-reperfusion (I/R) injury models. Acute kidney injury (AKI) induced by I/R injury can result in cell death, and subcellular structural changes, including expansion of the endoplasmic reticulum (ER), mitochondrial shrinkage, and other morphological changes. Inositol requiring enzyme 1 (IRE1) a proximal ER stress sensor, activates c-Jun NH2-terminal kinases (JNK) in response to ER stress, which is inextricably linked to ER. Method: To determine the resulting damage and relationship between ferroptosis and the IRE1/JNK pathway in AKI, we modeled AKI in I/R renal injury mice and hypoxia/reoxygenation (H/R) HK-2 cells, as in vivo and in vitro experiments, respectively. Results: In I/R renal injury mice, we found that abnormal renal function; damage of renal tubular epithelial cells; activation of the IRE1/JNK pathway and ferroptosis. Our in vitro study showed a large number of reactive oxygen species and more ferroptotic mitochondria in H/R HK-2 cells. By inhibiting IRE1/JNK in I/R renal injury mice, we observed decreased blood urea nitrogen, creatinine, and tissue injury, compared with the I/R group, we also found the markers of ferroptosis changed, including decreased 4-hydroxynonenal and increased glutathione peroxidase 4, as well as in H/R induced IRE1/JNK knock-down HK-2 cell lines (stable depletion). Furthermore, inhibition of ferroptosis could also attenuate the IRE1/JNK pathway in mice following I/R and HK-2 cells following H/R. Conclusion: We observed cross-talk between the IRE1/JNK pathway and ferroptosis in I/R or H/R induced AKI. Our findings suggest that ferroptosis plays an important role in I/R induced AKI, and that inhibition of the IRE1/JNK pathway can protect against I/R induced renal injury by inhibiting ferroptosis. The inhibition of the IRE1/JNK pathway could therefore be a feasible therapeutic target for treatment of AKI.

Keywords:  AKI (acute kidney injury); ER stress; IRE1 (inositol-requiring enzyme 1); JNK (c-Jun N-terminal kinase); ferroptosis; tubular epithelial cells

DOI:  https://doi.org/10.3389/fphar.2022.927641