bims-mitpro 2025-02-02 papers

Issue of 2025–02–02
five papers selected by
Andreas Kohler, Umeå University

Proc Natl Acad Sci U S A. 2025 Feb 04. 122(5): e2422447122

Serine phosphorylation facilitates protein degradation by the human mitochondrial ClpXP protease.

ClpXP is a two-component mitochondrial matrix protease. The caseinolytic mitochondrial matrix peptidase chaperone subunit X (ClpX) recognizes and translocates protein substrates into the degradation chamber of the caseinolytic protease P (ClpP) for proteolysis. ClpXP degrades damaged respiratory chain proteins and is necessary for cancer cell survival. Despite the critical role of ClpXP in mitochondrial protein quality control, the specific degrons, or modifications that tag substrate proteins for degradation by human ClpXP, are still unknown. We demonstrated that phosphorylated serine (pSer) targets substrates to ClpX and facilitates their degradation by ClpXP in biochemical assays. In contrast, ClpP hyperactivated by the small-molecule drug ONC201 lost the preference for phosphorylated substrates. Hydrogen deuterium exchange mass spectrometry combined with biochemical assays showed that pSer binds the RKL loop of ClpX. ClpX variants with substitutions in the RKL loop failed to recognize phosphorylated substrates. In intact cells, ClpXP also preferentially degraded substrates with pSer. Moreover, ClpX substrates with the pSer were selectively found in aggregated mitochondrial proteins. Our work uncovers a mechanism for substrate recognition by ClpXP, with implications for targeting acute myeloid leukemia and other disorders involving ClpXP dysfunction.

Keywords: AAA+ proteases; degron; mitochondrial proteostasis; phosphorylation; protein degradation

DOI: https://doi.org/10.1073/pnas.2422447122

Mol Biol Cell. 2025 Jan 29. mbcE25010035

Mechanism of chaperone recruitment and retention on mitochondrial precursors.

Szymon Juszkiewicz, Sew-Yeu Peak-Chew, Ramanujan S Hegde.

Nearly all mitochondrial proteins are imported into mitochondria from the cytosol. How nascent mitochondrial precursors acquire and sustain import-competence in the cytosol under normal and stress conditions is incompletely understood. Here, we show that under normal conditions, the Hsc70 and Hsp90 systems interact with and redundantly minimize precursor degradation. During acute import stress, Hsp90 buffers precursor degradation, preserving proteins in an import-competent state until stress resolution. Unexpectedly, buffering by Hsp90 relies critically on a mitochondrial targeting signal (MTS), the absence of which greatly decreases precursor-Hsp90 interaction. Site-specific photo-crosslinking and biochemical reconstitution showed how the MTS directly engages co-chaperones of Hsc70 (St13 and Stip1) and Hsp90 (p23 and Cdc37) to facilitate chaperone retention on the mature domain. Thus, the MTS has a previously unappreciated role in regulating chaperone dynamics on mitochondrial precursors to buffer their degradation and maintain import competence, functions that may facilitate restoration of mitochondrial homeostasis after acute import stress.

DOI: https://doi.org/10.1091/mbc.E25-01-0035

Cell Death Dis. 2025 Jan 28. 16(1): 52

The E3-ligase Siah2 activates mitochondrial quality control in neurons to maintain energy metabolism during ischemic brain tolerance.

Maria Josè Sisalli, Elena D'Apolito, Ornella Cuomo, Giovanna Lombardi, Michele Tufano, Lucio Annunziato, Antonella Scorziello.

Mitochondrial quality control is crucial for the homeostasis of the mitochondrial network. The balance between mitophagy and biogenesis is needed to reduce cerebral ischemia-induced cell death. Ischemic preconditioning (IPC) represents an adaptation mechanism of CNS that increases tolerance to lethal cerebral ischemia. It has been demonstrated that hypoxia-induced Seven in absentia Homolog 2 (Siah2) E3-ligase activation influences mitochondrial dynamics promoting the degradation of mitochondrial proteins. Therefore, in the present study, we investigated the role of Siah2 in the IPC-induced neuroprotection in in vitro and in vivo models of IPC. To this aim, cortical neurons were exposed to 30-min oxygen and glucose deprivation (OGD, sublethal insult) followed by 3 h OGD plus reoxygenation (lethal insult). Our results revealed that the mitochondrial depolarization induced by hypoxia activates Siah2 at the mitochondrial level and increases LC3-II protein expression, a marker of mitophagy, an effect counteracted by the reoxygenation phase. By contrast, hypoxia reduced the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a marker of mitochondrial biogenesis, whereas its expression was increased after reoxygenation thus improving mitochondrial membrane potential, mitochondrial calcium content, and mitochondrial morphology, hence leading to neuroprotection in IPC. Furthermore, Siah2 silencing confirmed these results. Collectively, these findings indicate that the balance between mitophagy and mitochondrial biogenesis, due to the activation of the Siah2-E3-ligase, might play a role in IPC-induced neuroprotection.

DOI: https://doi.org/10.1038/s41419-025-07339-z

Life Metab. 2025 Feb;4(1): loae040

Glucose-6-phosphate dehydrogenase regulates mitophagy by maintaining PINK1 stability.

Yik-Lam Cho, Hayden Weng Siong Tan, Jicheng Yang, Basil Zheng Mian Kuah, Nicole Si Ying Lim, Naiyang Fu, Boon-Huat Bay, Shuo-Chien Ling, Han-Ming Shen.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway (PPP) in glycolysis. Glucose metabolism is closely implicated in the regulation of mitophagy, a selective form of autophagy for the degradation of damaged mitochondria. The PPP and its key enzymes such as G6PD possess important metabolic functions, including biosynthesis and maintenance of intracellular redox balance, while their implication in mitophagy is largely unknown. Here, via a whole-genome CRISPR-Cas9 screening, we identified that G6PD regulates PINK1 (phosphatase and tensin homolog [PTEN]-induced kinase 1)-Parkin-mediated mitophagy. The function of G6PD in mitophagy was verified via multiple approaches. G6PD deletion significantly inhibited mitophagy, which can be rescued by G6PD reconstitution. Intriguingly, while the catalytic activity of G6PD is required, the known PPP functions per se are not involved in mitophagy regulation. Importantly, we found a portion of G6PD localized at mitochondria where it interacts with PINK1. G6PD deletion resulted in an impairment in PINK1 stabilization and subsequent inhibition of ubiquitin phosphorylation, a key starting point of mitophagy. Finally, we found that G6PD deletion resulted in lower cell viability upon mitochondrial depolarization, indicating the physiological function of G6PD-mediated mitophagy in response to mitochondrial stress. In summary, our study reveals a novel role of G6PD as a key positive regulator in mitophagy, which bridges several important cellular processes, namely glucose metabolism, redox homeostasis, and mitochondrial quality control.

Keywords: G6PD; NADPH; PINK1; PPP; ROS; mitophagy

DOI: https://doi.org/10.1093/lifemeta/loae040

Sci Rep. 2025 Jan 29. 15(1): 3686

Altered mitochondrial unfolded protein response and FGF21 secretion in MASLD progression and the effect of exercise intervention.

Xinmeng Yuan, Wen Sun, Ye Xu, Mengqi Xiang, Yaran Gao, Wanyu Feng, Hongjian Xiao, Liumei Zhang, Qiang Tang, Jiao Lu, Yuan Zhang.

A high-calorie diet and lack of exercise are the most important risk factors contributing to metabolic dysfunction-associated steatotic liver disease (MASLD) initiation and progression. The precise molecular mechanisms of mitochondrial function alteration during MASLD development remain to be fully elucidated. In this study, a total of 60 male C57BL/6J mice were maintained on a normal or amylin liver NASH (AMLN) diet for 6 or 10 weeks. Some of the mice were then subjected to voluntary wheel running, while the other mice were fed a normal or AMLN diet until 14 and 18 weeks. The results showed that hepatic lipid deposition and the PERK-eIF2α-ATF4 pathway were significantly increased with prolonged duration of AMLN diet. However, expression of mitochondrial unfolded protein response (UPRmt) genes and mitokine FGF21 secretion were significantly enhanced in the 14-week AMLN diet mice, but were markedly reduced with the excessive lipid deposition induced by longer AMLN diet. Additionally, the exercise intervention acts as a regulator to optimize UPRmt signal transduction and to enhance mitochondrial homeostasis by improving mitochondrial function, reversing the UPRmt activation pattern, and increasing FGF21 secretion, which plays a pivotal role in delaying the occurrence and development of MASLD.

Keywords: AMLN diet; Exercise intervention; Hepatic lipid accumulation; MASLD progression; Mitochondrial unfolded protein response

DOI: https://doi.org/10.1038/s41598-025-87190-6