OMA1 clears traffic jam in TOM tunnel in mammals.

Using an engineered mitochondrial clogger, Krakowczyk et al. (https://doi.org/10.1083/jcb.202306051) identified the OMA1 protease as a critical component that eliminates import failure at the TOM translocase in mammalian cells, providing a novel quality control mechanism that is distinct from those described in yeast.

A mitochondrial iron-responsive pathway regulated by DELE1.

The heme-regulated kinase HRI is activated under heme/iron deficient conditions; however, the underlying molecular mechanism is incompletely understood. Here, we show that iron-deficiency-induced HRI activation requires the mitochondrial protein DELE1. Notably, mitochondrial import of DELE1 and its subsequent protein stability are regulated by iron availability. Under steady-state conditions, DELE1 is degraded by the mitochondrial matrix-resident protease LONP1 soon after mitochondrial import. Upon iron chelation, DELE1 import is arrested, thereby stabilizing DELE1 on the mitochondrial surface to activate the HRI-mediated integrated stress response (ISR). Ablation of this DELE1-HRI-ISR pathway in an erythroid cell model enhances cell death under iron-limited conditions, suggesting a cell-protective role for this pathway in iron-demanding cell lineages. Our findings highlight mitochondrial import regulation of DELE1 as the core component of a previously unrecognized mitochondrial iron responsive pathway that elicits stress signaling following perturbation of iron homeostasis.

TIM23 facilitates PINK1 activation by safeguarding against OMA1-mediated degradation in damaged mitochondria.

PINK1 is activated by autophosphorylation and forms a high-molecular-weight complex, thereby initiating the selective removal of damaged mitochondria by autophagy. Other than translocase of the outer mitochondrial membrane complexes, members of PINK1-containing protein complexes remain obscure. By mass spectrometric analysis of PINK1 co-immunoprecipitates, we identify the inner membrane protein TIM23 as a component of the PINK1 complex. TIM23 downregulation decreases PINK1 levels and significantly delays autophosphorylation, indicating that TIM23 promotes PINK1 accumulation in response to depolarization. Moreover, inactivation of the mitochondrial protease OMA1 not only enhances PINK1 accumulation but also represses the reduction in PINK1 levels induced by TIM23 downregulation, suggesting that TIM23 facilitates PINK1 activation by safeguarding against degradation by OMA1. Indeed, deficiencies of pathogenic PINK1 mutants that fail to interact with TIM23 are partially restored by OMA1 inactivation. These findings indicate that TIM23 plays a distinct role in activating mitochondrial autophagy by protecting PINK1.

Discovery of bactericides as an acute mitochondrial membrane damage inducer.

Mitochondria evolved from endosymbiotic bacteria to become essential organelles of eukaryotic cells. The unique lipid composition and structure of mitochondrial membranes are critical for the proper functioning of mitochondria. However, stress responses that help maintain the mitochondrial membrane integrity are not well understood. One reason for this lack of insight is the absence of efficient tools to specifically damage mitochondrial membranes. Here, through a compound screen, we found that two bis-biguanide compounds, chlorhexidine and alexidine, modified the activity of the inner mitochondrial membrane (IMM)-resident protease OMA1 by altering the integrity of the IMM. These compounds are well-known bactericides whose mechanism of action has centered on their damage-inducing activity on bacterial membranes. We found alexidine binds to the IMM likely through the electrostatic interaction driven by the membrane potential as well as an affinity for anionic phospholipids. Electron microscopic analysis revealed that alexidine severely perturbated the cristae structure. Notably, alexidine evoked a specific transcriptional/proteostasis signature that was not induced by other typical mitochondrial stressors, highlighting the unique property of alexidine as a novel mitochondrial membrane stressor. Our findings provide a chemical-biological tool that should enable the delineation of mitochondrial stress-signaling pathways required to maintain the mitochondrial membrane homeostasis.

Nutritional intervention after an early assessment by a flexible endoscopic evaluation of swallowing is associated with a shorter hospital stay for patients with acute cerebral infarction: A retrospective study.

BACKGROUND AND OBJECTIVES: It is important to evaluate the swallowing function of patients with acute cerebral infarction. The effects of nutritional intervention after an early assessment by a flexible endoscopic evaluation of swallowing (FEES) were evaluated. METHODS AND STUDY DESIGN: This retrospective study included 274 patients who were hospitalized for acute cerebral infarction and underwent a FEES between 2016 and 2018. The effects of early nutritional intervention after an assessment by a FEES within 48 h from admission were evaluated. The patients were divided into a shorter hospital stay group (<30 days) and a longer group (≥30 days). A multivariate analysis was performed to identify the predictive factors for a shorter hospital stay. RESULTS: The overall patient characteristics were as follows: 166 men; median age, 81 years old; and median body mass index (BMI), 21.1 kg/m2. No significant differences in the age, sex, or BMI were found between the shorter and longer hospital stay groups. A FEES within 48 h of admission (odds ratio [OR], 2.040; 95% confidence interval [CI], 1.120-3.700; p=0.019), FILS level ≥6 at admission (OR, 2.300; 95% CI, 1.190-4.440; p=0.013), and an administered energy dose of ≥18.5 kcal/kg on hospital day 3 (OR, 2.360; 95% CI, 1.180-4.690; p=0.015) were independently associated with a hospital stay <30 days. CONCLUSIONS: Patients with acute cerebral infarction are more likely to have a shorter hospital stay (<30 days) if they undergo a FEES early after admission and receive optimal nutritional intervention.

Thiol-Cleavable Biotin for Chemical and Enzymatic Biotinylation and Its Application to Mitochondrial TurboID Proteomics.

Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-bead digestion in numerous biological applications. However, the popular on-bead digestion method faces major challenges of streptavidin contamination, overwhelming signals from endogenous biotinylated proteins, the lost information on biotinylation sites, and limited sequence coverage of enriched proteins. Here, we explored thiol-cleavable biotin as an alternative approach to elute biotinylated proteins from streptavidin-coated beads for both chemical biotinylation and biotin ligase-based proximity labeling. All possible amino acid sites for biotinylation were thoroughly evaluated in addition to the primary lysine residue. We found that biotinylation at lysine residues notably reduces the trypsin digestion efficiency, which can be mitigated by the thiol-cleavable biotinylation method. We then evaluated the applicability of thiol-cleavable biotin as a substrate for proximity labeling in living cells, where TurboID biotin ligase was engineered onto the mitochondrial inner membrane facing the mitochondrial matrix. As a proof-of-principle study, thiol-cleavable biotin-assisted TurboID proteomics achieved remarkable intraorganelle spatial resolution with significantly enriched proteins localized in the mitochondrial inner membrane and mitochondrial matrix.

Cellular metabolic stress responses via organelles.

Metabolite fluctuations following nutrient metabolism or environmental stresses impact various intracellular signaling networks and stress responses to maintain cellular and organismal homeostasis. It has been shown that subcellular organelles, such as the endoplasmic reticulum, the Golgi apparatus, lysosomes and mitochondria serve as crucial hubs linking alterations in metabolite levels to cellular responses. This role is coordinated by molecular machineries that are associated with the lipid membranes of organelles, which sense the fluctuations in specific metabolites and activate the appropriate signaling and effector molecules. Moreover, recent studies have demonstrated that membraneless organelles, such as the nucleolus and stress granules, are involved in the metabolic stress response. Metabolite-induced post-translational modifications appear to play an important role in this process. Here, we review the molecular mechanisms of metabolite sensing and metabolite-mediated stress responses through membrane-bound and membraneless organelles in mammalian cells.

Acetylation-mediated remodeling of the nucleolus regulates cellular acetyl-CoA responses.

The metabolite acetyl-coenzyme A (acetyl-CoA) serves as an essential element for a wide range of cellular functions including adenosine triphosphate (ATP) production, lipid synthesis, and protein acetylation. Intracellular acetyl-CoA concentrations are associated with nutrient availability, but the mechanisms by which a cell responds to fluctuations in acetyl-CoA levels remain elusive. Here, we generate a cell system to selectively manipulate the nucleo-cytoplasmic levels of acetyl-CoA using clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing and acetate supplementation of the culture media. Using this system and quantitative omics analyses, we demonstrate that acetyl-CoA depletion alters the integrity of the nucleolus, impairing ribosomal RNA synthesis and evoking the ribosomal protein-dependent activation of p53. This nucleolar remodeling appears to be mediated through the class IIa histone deacetylases (HDACs). Our findings highlight acetylation-mediated control of the nucleolus as an important hub linking acetyl-CoA fluctuations to cellular stress responses.

The coupling of translational control and stress responses.

The translation of messenger RNA (mRNA) into protein is a multistep process by which genetic information transcribed into an mRNA is decoded to produce a specific polypeptide chain of amino acids. Ribosomes play a central role in translation by coordinately working with various translation regulatory factors and aminoacyl-transfer RNAs. Various stresses attenuate the ribosomal synthesis in the nucleolus as well as the translation rate in the cytosol. To efficiently reallocate cellular energy and resources, mammalian cells are endowed with mechanisms that directly link the suppression of translation-related processes to the activation of stress adaptation programmes. This review focuses on the integrated stress response (ISR) and the nucleolar stress response (NSR) both of which are activated by various stressors and selectively upregulate stress-responsive transcription factors. Emerging findings have delineated the detailed molecular mechanisms of the ISR and NSR and expanded their physiological and pathological significances.

Loss of CHCHD2 and CHCHD10 activates OMA1 peptidase to disrupt mitochondrial cristae phenocopying patient mutations.

Dominant mutations in the mitochondrial paralogs coiled-helix-coiled-helix (CHCHD) domain 2 (C2) and CHCHD10 (C10) were recently identified as causing Parkinson's disease and amyotrophic lateral sclerosis/frontotemporal dementia/myopathy, respectively. The mechanism by which they disrupt mitochondrial cristae, however, has been uncertain. Using the first C2/C10 double knockout (DKO) mice, we report that C10 pathogenesis and the normal function of C2/C10 are intimately linked. Similar to patients with C10 mutations, we found that C2/C10 DKO mice have disrupted mitochondrial cristae, because of cleavage of the mitochondrial-shaping protein long form of OPA1 (L-OPA1) by the stress-induced peptidase OMA1. OMA1 was found to be activated similarly in affected tissues of mutant C10 knock-in (KI) mice, demonstrating that L-OPA1 cleavage is a novel mechanism for cristae abnormalities because of both C10 mutation and C2/C10 loss. Using OMA1 activation as a functional assay, we found that C2 and C10 are partially functionally redundant, and some but not all disease-causing mutations have retained activity. Finally, C2/C10 DKO mice partially phenocopied mutant C10 KI mice with the development of cardiomyopathy and activation of the integrated mitochondrial integrated stress response in affected tissues, tying mutant C10 pathogenesis to C2/C10 function.

The mitochondrial protein PGAM5 suppresses energy consumption in brown adipocytes by repressing expression of uncoupling protein 1.

Accumulating evidence suggests that brown adipose tissue (BAT) is a potential therapeutic target for managing obesity and related diseases. PGAM family member 5, mitochondrial serine/threonine protein phosphatase (PGAM5), is a protein phosphatase that resides in the mitochondria and regulates many biological processes, including cell death, mitophagy, and immune responses. Because BAT is a mitochondria-rich tissue, we have hypothesized that PGAM5 has a physiological function in BAT. We previously reported that PGAM5-knockout (KO) mice are resistant to severe metabolic stress. Importantly, lipid accumulation is suppressed in PGAM5-KO BAT, even under unstressed conditions, raising the possibility that PGAM5 deficiency stimulates lipid consumption. However, the mechanism underlying this observation is undetermined. Here, using an array of biochemical approaches, including quantitative RT-PCR, immunoblotting, and oxygen consumption assays, we show that PGAM5 negatively regulates energy expenditure in brown adipocytes. We found that PGAM5-KO brown adipocytes have an enhanced oxygen consumption rate and increased expression of uncoupling protein 1 (UCP1), a protein that increases energy consumption in the mitochondria. Mechanistically, we found that PGAM5 phosphatase activity and intramembrane cleavage are required for suppression of UCP1 activity. Furthermore, utilizing a genome-wide siRNA screen in HeLa cells to search for regulators of PGAM5 cleavage, we identified a set of candidate genes, including phosphatidylserine decarboxylase (PISD), which catalyzes the formation of phosphatidylethanolamine at the mitochondrial membrane. Taken together, these results indicate that PGAM5 suppresses mitochondrial energy expenditure by down-regulating UCP1 expression in brown adipocytes and that its phosphatase activity and intramembrane cleavage are required for UCP1 suppression.

PINK1 import regulation at a crossroad of mitochondrial fate: the molecular mechanisms of PINK1 import.

PTEN-induced kinase 1 (PINK1) is a mitochondrial kinase whose activity is tightly regulated by the mitochondrial health status. In response to mitochondrial damage, activated PINK1 can promote mitophagy, an autophagic elimination of damaged mitochondria, by cooperating with Parkin ubiquitin ligase. Loss-of-function of PINK1/Parkin-mediated mitophagy results in the accumulation of dysfunctional mitochondria, which could be one aetiology of Parkinson's disease (PD). Within step-by-step signalling cascades of PINK1/Parkin-mediated mitophagy, mitochondrial damage-dependent PINK1 kinase activation is a critical step to trigger the mitophagy signal. Recent investigation of this process reveals that this stress-dependent PINK1 kinase activation is achieved by its regulated import into different mitochondrial compartments. Thus, PINK1 import regulation stands at an important crossroad to determine the mitochondrial fate-'keep' or 'remove'? In this review, we will summarize how the PINK1 import is regulated in a mitochondrial health status-dependent manner and how this process could be pharmacologically modulated to activate the PINK1/Parkin pathway.

Reciprocal Roles of Tom7 and OMA1 during Mitochondrial Import and Activation of PINK1.

Mutations in PTEN-induced kinase 1 (PINK1) can cause recessive early-onset Parkinson's disease (PD). Import arrest results in PINK1 kinase activation specifically on damaged mitochondria, triggering Parkin-mediated mitophagy. Here, we show that PINK1 import is less dependent on Tim23 than on mitochondrial membrane potential (ΔΨm). We identified a negatively charged amino acid cluster motif that is evolutionarily conserved just C-terminal to the PINK1 transmembrane. PINK1 that fails to accumulate at the outer mitochondrial membrane, either by mutagenesis of this negatively charged motif or by deletion of Tom7, is imported into depolarized mitochondria and cleaved by the OMA1 protease. Some PD patient mutations also are defective in import arrest and are rescued by the suppression of OMA1, providing a new potential druggable target for PD. These results suggest that ΔΨm loss-dependent PINK1 import arrest does not result solely from Tim23 inactivation but also through an actively regulated "tug of war" between Tom7 and OMA1.

PINK1 import regulation; a fine system to convey mitochondrial stress to the cytosol.

Insights from inherited forms of parkinsonism suggest that insufficient mitophagy may be one etiology of the disease. PINK1/Parkin-dependent mitophagy, which helps maintain a healthy mitochondrial network, is initiated by activation of the PINK1 kinase specifically on damaged mitochondria. Recent investigation of this process reveals that import of PINK1 into mitochondria is regulated and yields a stress-sensing mechanism. In this review, we focus on the mechanisms of mitochondrial stress-dependent PINK1 activation that is exerted by regulated import of PINK1 into different mitochondrial compartments and how this offers strategies to pharmacologically activate the PINK1/Parkin pathway.

Mitochondrial fission facilitates the selective mitophagy of protein aggregates.

Within the mitochondrial matrix, protein aggregation activates the mitochondrial unfolded protein response and PINK1-Parkin-mediated mitophagy to mitigate proteotoxicity. We explore how autophagy eliminates protein aggregates from within mitochondria and the role of mitochondrial fission in mitophagy. We show that PINK1 recruits Parkin onto mitochondrial subdomains after actinonin-induced mitochondrial proteotoxicity and that PINK1 recruits Parkin proximal to focal misfolded aggregates of the mitochondrial-localized mutant ornithine transcarbamylase (ΔOTC). Parkin colocalizes on polarized mitochondria harboring misfolded proteins in foci with ubiquitin, optineurin, and LC3. Although inhibiting Drp1-mediated mitochondrial fission suppresses the segregation of mitochondrial subdomains containing ΔOTC, it does not decrease the rate of ΔOTC clearance. Instead, loss of Drp1 enhances the recruitment of Parkin to fused mitochondrial networks and the rate of mitophagy as well as decreases the selectivity for ΔOTC during mitophagy. These results are consistent with a new model that, instead of promoting mitophagy, fission protects healthy mitochondrial domains from elimination by unchecked PINK1-Parkin activity.