bims-tofagi 2023-12-17 papers

on Mitophagy

Issue of 2023‒12‒17
six papers selected by
Michele Frison, University of Cambridge and Aitor Martínez Zarate, Euskal Herriko Unibertsitatea

GRAF1 integrates PINK1-Parkin signaling and actin dynamics to mediate cardiac mitochondrial homeostasis.
Mammalian phagophores with finger-like shapes emerge from recycling endosomes.
Caveolin-1 promotes mitochondrial health and limits mitochondrial ROS through ROCK/AMPK regulation of basal mitophagic flux.
Parkin-mediated ubiquitination inhibits BAK apoptotic activity by blocking its canonical hydrophobic groove.
Senecavirus a induces mitophagy to promote self-replication through direct interaction of 2C protein with K27-linked ubiquitinated TUFM catalyzed by RNF185.
CMT2A-linked MFN2 mutation, T206I promotes mitochondrial hyperfusion and predisposes cells towards mitophagy.

Nat Commun. 2023 Dec 11. 14(1): 8187

GRAF1 integrates PINK1-Parkin signaling and actin dynamics to mediate cardiac mitochondrial homeostasis.

Qiang Zhu, Matthew E Combs, Juan Liu, Xue Bai, Wenbo B Wang, Laura E Herring, Jiandong Liu, Jason W Locasale, Dawn E Bowles, Ryan T Gross, Michelle Mendiola Pla, Christopher P Mack, Joan M Taylor.

The serine/threonine kinase, PINK1, and the E3 ubiquitin ligase, Parkin, are known to facilitate LC3-dependent autophagosomal encasement and lysosomal clearance of dysfunctional mitochondria, and defects in this process contribute to a variety of cardiometabolic and neurological diseases. Although recent evidence indicates that dynamic actin remodeling plays an important role in PINK1/Parkin-mediated mitochondrial autophagy (mitophagy), the underlying signaling mechanisms remain unknown. Here, we identify the RhoGAP GRAF1 (Arhgap26) as a PINK1 substrate that regulates mitophagy. GRAF1 promotes the release of damaged mitochondria from F-actin anchors, regulates mitochondrial-associated Arp2/3-mediated actin remodeling and facilitates Parkin-LC3 interactions to enhance mitochondria capture by autophagosomes. Graf1 phosphorylation on PINK1-dependent sites is dysregulated in human heart failure, and cardiomyocyte-restricted Graf1 depletion in mice blunts mitochondrial clearance and attenuates compensatory metabolic adaptations to stress. Overall, we identify GRAF1 as an enzyme that coordinates cytoskeletal and metabolic remodeling to promote cardioprotection.

DOI: https://doi.org/10.1038/s41467-023-43889-6
Autophagy. 2023 Dec 14.

Mammalian phagophores with finger-like shapes emerge from recycling endosomes.

Claudia Puri, David C Rubinsztein.

  Autophagosomes are double-membraned vesicles that engulf cytoplasmic contents, which are ultimately degraded after autophagosome-lysosome fusion. The prevailing view, largely inferred from EM-based studies, was that mammalian autophagosomes evolved from disc-shaped precursors that invaginated and then were closed at the single opening. Many site(s) of origin of these precursors have been proposed. Using superresolution structured illumination microscopy and electron microscopy, we find that mammalian autophagosomes derive from finger-like outgrowths from the recycling endosome. These "fingers" survey a large cell volume and then close into a "fist" and the openings are sealed in an ESCRT-dependent fashion, while the precursors are still attached to the recycling endosome. We call this transient recycling endosome-attached, closed, autophagic structure an "autophago-dome". DNM2-dependent scission of the autophago-dome from the recycling endosomes liberates free autophagosomes from this compartment. These data reveal unexpected morphologies of autophagosome precursors and raise new questions about the control of this process.

Keywords:  ESCRT; RAB11; autophagy; phagophore; recycling endosome

DOI:  https://doi.org/10.1080/15548627.2023.2293439
FASEB J. 2024 Jan;38(1): e23343

Caveolin-1 promotes mitochondrial health and limits mitochondrial ROS through ROCK/AMPK regulation of basal mitophagic flux.

Logan R Timmins, Milene Ortiz-Silva, Bharat Joshi, Y Lydia Li, Fiona H Dickson, Timothy H Wong, Kurt R Vandevoorde, Ivan R Nabi.

  Caveolin-1 (CAV1), the main structural component of caveolae, is phosphorylated at tyrosine-14 (pCAV1), regulates signal transduction, mechanotransduction, and mitochondrial function, and plays contrasting roles in cancer progression. We report that CRISPR/Cas9 knockout (KO) of CAV1 increases mitochondrial oxidative phosphorylation, increases mitochondrial potential, and reduces ROS in MDA-MB-231 triple-negative breast cancer cells. Supporting a role for pCAV1, these effects are reversed upon expression of CAV1 phosphomimetic CAV1 Y14D but not non-phosphorylatable CAV1 Y14F. pCAV1 is a known effector of Rho-associated kinase (ROCK) signaling and ROCK1/2 signaling mediates CAV1 promotion of increased mitochondrial potential and decreased ROS production in MDA-MB-231 cells. CAV1/ROCK control of mitochondrial potential and ROS is caveolae-independent as similar results were observed in PC3 prostate cancer cells lacking caveolae. Increased mitochondrial health and reduced ROS in CAV1 KO MDA-MB-231 cells were reversed by knockdown of the autophagy protein ATG5, mitophagy regulator PINK1 or the mitochondrial fission protein Drp1 and therefore due to mitophagy. Use of the mitoKeima mitophagy probe confirmed that CAV1 signaling through ROCK inhibited basal mitophagic flux. Activation of AMPK, a major mitochondrial homeostasis protein inhibited by ROCK, is inhibited by CAV1-ROCK signaling and mediates the increased mitochondrial potential, decreased ROS, and decreased basal mitophagy flux observed in wild-type MDA-MB-231 cells. CAV1 regulation of mitochondrial health and ROS in cancer cells therefore occurs via ROCK-dependent inhibition of AMPK. This study therefore links pCAV1 signaling activity at the plasma membrane with its regulation of mitochondrial activity and cancer cell metabolism through control of mitophagy.

Keywords:  AMPK; caveolin-1; mitochondria; mitophagy; reactive oxygen species; rho kinase

DOI:  https://doi.org/10.1096/fj.202201872RR
Commun Biol. 2023 Dec 12. 6(1): 1260

Parkin-mediated ubiquitination inhibits BAK apoptotic activity by blocking its canonical hydrophobic groove.

Peng Cheng, Yuzhu Hou, Mingxing Bian, Xueru Fang, Yan Liu, Yuanfang Rao, Shuo Cao, Yanjun Liu, Shuai Zhang, Yanke Chen, Xu Dong, Zhu Liu.

BAK permeabilizes the mitochondrial outer membrane, causing apoptosis. This apoptotic activity of BAK is stimulated by binding prodeath activators within its canonical hydrophobic groove. Parkin, an E3 ubiquitin (Ub) ligase, can ubiquitinate BAK, which inhibits BAK apoptotic activity. However, the molecular mechanism underlying the inhibition of ubiquitination remains structurally uncharacterized. Here, we utilize truncated and soluble BAK to construct a mimetic of K113-ubiquitinated BAK (disulfide-linked UbG76C ~ BAKK113C) and further present its NMR-derived structure model. The classical L8-I44-H68-V70 hydrophobic patch of the conjugated Ub subunit binds within the canonical hydrophobic groove of BAK. This Ub occludes the binding of prodeath BID activators in the groove and impairs BID-triggered BAK activation and membrane permeabilization. Reduced interaction between Ub and BAK subunits allows BID to activate K113-ubiquitinated BAK. These mechanistic insights suggest a nonsignaling function of Ub in that it directly antagonizes stimuli targeting Ub-modified proteins rather than by recruiting downstream partners for cellular messaging.

DOI: https://doi.org/10.1038/s42003-023-05650-z
Autophagy. 2023 Dec 12.

Senecavirus a induces mitophagy to promote self-replication through direct interaction of 2C protein with K27-linked ubiquitinated TUFM catalyzed by RNF185.

Meirong Chen, Xin Zhang, Fanshu Kong, Peng Gao, Xinna Ge, Lei Zhou, Jun Han, Xin Guo, Yongning Zhang, Hanchun Yang.

  Senecavirus A (SVA) is a newly emerging picornavirus associated with swine vesicular lesions and neonatal mortality, threatening the global pig industry. Despite sustained efforts, the molecular mechanisms of SVA pathogenesis have not yet been fully elucidated. Here, we demonstrate for the first time that SVA infection can induce complete mitophagy in host cells, which depends on SVA replication. Mitophagy has been subsequently proven to promote SVA replication in host cells. Genome-wide screening of SVA proteins involved in inducing mitophagy showed that although VP2, VP3, 2C, and 3A proteins can independently induce mitophagy, only the 2C protein mediates mitophagy through direct interaction with TUFM (Tu translation elongation factor, mitochondrial). The glutamic acids at positions 196 and 211 of TUFM were shown to be two key sites for its interaction with 2C protein. Moreover, TUFM was discovered to interact directly with BECN1 and indirectly with the ATG12-ATG5 conjugate. Further experiments revealed that TUFM needs to undergo ubiquitination modification before being recognized by the macroautophagy/autophagy receptor protein SQSTM1/p62, and E3 ubiquitin ligase RNF185 catalyzes K27-linked polyubiquitination of TUFM through the interaction between RNF185's transmembrane domain 1 and TUFM to initiate SVA-induced mitophagy. The ubiquitinated TUFM is recognized and bound by SQSTM1, which in turn interacts with MAP1LC3/LC3, thereby linking the 2C-anchored mitochondria to the phagophore for sequestration into mitophagosomes, which ultimately fuse with lysosomes to achieve complete mitophagy. Overall, our results elucidated the molecular mechanism by which SVA induces mitophagy to promote self-replication and provide new insights into SVA pathogenesis.

Keywords:  2C protein; RNF185; SVA; TUFM; mitophagy; replication

DOI:  https://doi.org/10.1080/15548627.2023.2293442
Mitochondrion. 2023 Dec 11. pii: S1567-7249(23)00105-8. [Epub ahead of print] 101825

CMT2A-linked MFN2 mutation, T206I promotes mitochondrial hyperfusion and predisposes cells towards mitophagy.

Rajdeep Das, Sebabrata Maity, Palamou Das, Izaz Monir Kamal, Saikat Chakrabarti, Oishee Chakrabarti.

Mutations in Mitofusin2 (MFN2) associated with the pathology of the debilitating neuropathy Charcot-Marie-Tooth type 2A (CMT2A) are known to alter mitochondrial morphology. Previously, such mutations have been shown to elicit two diametrically opposite phenotypes - while some mutations have been causally linked to enhanced mitochondrial fragmentation, others have been shown to induce hyperfusion. Our study identifies one such MFN2 mutant, T206I that causes mitochondrial hyperfusion. Cells expressing this MFN2 mutant have elongated and interconnected mitochondria. T206I-MFN2 mutation in the GTPase domain increases MFN2 stability and renders cells susceptible to stress. We show that cells expressing T206I-MFN2 have a higher predisposition towards mitophagy under conditions of serum starvation. We also detect increased DRP1 recruitment onto the outer mitochondrial membrane, though the total DRP1 protein level remains unchanged. Here we have characterized a lesser studied CMT2A-linked MFN2 mutant to show that its presence affects mitochondrial morphology and homeostasis.

DOI: https://doi.org/10.1016/j.mito.2023.101825