RESUMO
Protein biotinylation has been widely used in biotechnology with various labeling and enrichment strategies. However, different enrichment strategies have not been systematically evaluated due to the lack of a benchmarking model for fair comparison. Most biotinylation proteomics workflows suffer from lengthy experimental steps, non-specific bindings, limited throughput, and experimental variability. To address these challenges, we designed a two-proteome model, where biotinylated yeast proteins were spiked in unlabeled human proteins, allowing us to distinguish true enrichment from non-specific bindings. Using this benchmarking model, we compared common biotinylation proteomics methods and provided practical selection guidelines. We significantly optimized and shortened sample preparation from 3 days to 9 hours, enabling fully-automated 96-well plate sample processing. Next, we applied this optimized and automated workflow for proximity labeling to investigate the intricate interplay between mitochondria and lysosomes in living cells under both healthy state and mitochondrial damage. Our results suggested a time-dependent proteome remodeling and dynamic translocation within mitochondria and between mitochondria and lysosomes upon mitochondrial damage. This newly established benchmarking model and the fully-automated 9-hour workflow can be readily applied to the broad fields of protein biotinylation to study protein interaction and organelle dynamics.
RESUMO
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.
Assuntos
Mamíferos , Metaloproteases , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais , Animais , Mitocôndrias , Peptídeo Hidrolases , Saccharomyces cerevisiae/genética , Metaloproteases/metabolismo , Proteínas Mitocondriais/metabolismoRESUMO
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.
Assuntos
Ferro , eIF-2 Quinase , Ferro/metabolismo , eIF-2 Quinase/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Células Eritroides/metabolismo , Heme/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
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.
Assuntos
Mitocôndrias , Membranas Mitocondriais , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Quinases/metabolismoRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Biguanidas/farmacologia , Clorexidina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células HeLa , Homeostase , Humanos , Membranas/metabolismo , Metaloendopeptidases/efeitos dos fármacos , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosfolipídeos/metabolismoRESUMO
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.
Assuntos
Deglutição , Hospitais , Idoso de 80 Anos ou mais , Infarto Cerebral/diagnóstico , Infarto Cerebral/terapia , Humanos , Tempo de Internação , Masculino , Estudos RetrospectivosRESUMO
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.
Assuntos
Biotina/química , Proteínas Mitocondriais , Proteômica/métodos , Compostos de Sulfidrila/química , Biotina/metabolismo , Biotinilação , Células HEK293 , Células HeLa , Humanos , Mitocôndrias/química , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
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.
Assuntos
Núcleo Celular/patologia , Retículo Endoplasmático/patologia , Complexo de Golgi/patologia , Homeostase , Lisossomos/patologia , Mitocôndrias/patologia , Estresse Fisiológico , Animais , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Lisossomos/metabolismo , Mitocôndrias/metabolismoRESUMO
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.
Assuntos
Acetilcoenzima A/biossíntese , Nucléolo Celular/metabolismo , ATP Citrato (pro-S)-Liase/deficiência , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Acetatos/metabolismo , Acetilação , Linhagem Celular , Nucléolo Celular/ultraestrutura , Expressão Gênica , Técnicas de Inativação de Genes , Células HCT116 , Histona Desacetilases/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Ribossômicas/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Animais , Humanos , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Demência Frontotemporal/genética , Metaloproteases/genética , Proteínas Mitocondriais/genética , Doença de Parkinson/genética , Fatores de Transcrição/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Cardiomiopatias/diagnóstico por imagem , Cardiomiopatias/genética , Cardiomiopatias/patologia , Modelos Animais de Doenças , Demência Frontotemporal/patologia , Predisposição Genética para Doença , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Mutação/genética , Doença de Parkinson/patologiaRESUMO
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.
Assuntos
Adipócitos Marrons/metabolismo , Regulação para Baixo , Metabolismo Energético , Proteínas Mitocondriais/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteína Desacopladora 1/genética , Animais , Células Cultivadas , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/genética , Consumo de Oxigênio , Fosfoproteínas Fosfatases/genética , Proteína Desacopladora 1/metabolismoRESUMO
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.
Assuntos
Mitocôndrias/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Mitofagia/genética , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Domínios Proteicos/genética , Transporte Proteico , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
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.
Assuntos
Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/enzimologia , Proteínas Quinases/metabolismo , Motivos de Aminoácidos , Antiparkinsonianos/farmacologia , Transporte Biológico , Desenho de Fármacos , Ativação Enzimática , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/genética , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.