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Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.
Assuntos
Biotinilação , Proteoma , Proteômica , Proteômica/métodos , Reprodutibilidade dos Testes , Humanos , Proteoma/metabolismo , Espectrometria de Massas/métodos , Células HEK293RESUMO
Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.
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Proximity labeling (PL) coupled with mass spectrometry has emerged as a powerful technique to map proximal protein interactions in living cells. Large-scale sample processing for proximity proteomics necessitates a high-throughput workflow to reduce hands-on time and increase quantitative reproducibility. To address this issue, we developed a scalable and automated PL pipeline, including generation and characterization of monoclonal cell lines, automated enrichment of biotinylated proteins in a 96-well format, and optimization of the quantitative mass spectrometry (MS) acquisition method. Combined with data-independent acquisition (DIA) MS, our pipeline outperforms manual enrichment and data-dependent acquisition (DDA) MS regarding reproducibility of protein identification and quantification. We apply the pipeline to map subcellular proteomes for endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, using serotonin receptor (5HT2A) as a model, we investigated agonist-induced dynamics in protein-protein interactions. Importantly, the approach presented here is universally applicable for PL proteomics using all biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.
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Amyloid precursor protein (APP) metabolism is central to Alzheimer's disease (AD) pathogenesis, but the key etiological driver remains elusive. Recent failures of clinical trials targeting amyloid-ß (Aß) peptides, the proteolytic fragments of amyloid precursor protein (APP) that are the main component of amyloid plaques, suggest that the proteostasis-disrupting, key pathogenic species remain to be identified. Previous studies suggest that APP C-terminal fragment (APP.C99) can cause disease in an Aß-independent manner. The mechanism of APP.C99 pathogenesis is incompletely understood. We used Drosophila models expressing APP.C99 with the native ER-targeting signal of human APP, expressing full-length human APP only, or co-expressing full-length human APP and ß-secretase (BACE), to investigate mechanisms of APP.C99 pathogenesis. Key findings are validated in mammalian cell culture models, mouse 5xFAD model, and postmortem AD patient brain materials. We find that ribosomes stall at the ER membrane during co-translational translocation of APP.C99, activating ribosome-associated quality control (RQC) to resolve ribosome collision and stalled translation. Stalled APP.C99 species with C-terminal extensions (CAT-tails) resulting from inadequate RQC are prone to aggregation, causing endolysosomal and autophagy defects and seeding the aggregation of amyloid ß peptides, the main component of amyloid plaques. Genetically removing stalled and CAT-tailed APP.C99 rescued proteostasis failure, endolysosomal/autophagy dysfunction, neuromuscular degeneration, and cognitive deficits in AD models. Our finding of RQC factor deposition at the core of amyloid plaques from AD brains further supports the central role of defective RQC of ribosome collision and stalled translation in AD pathogenesis. These findings demonstrate that amyloid plaque formation is the consequence and manifestation of a deeper level proteostasis failure caused by inadequate RQC of translational stalling and the resultant aberrantly modified APP.C99 species, previously unrecognized etiological drivers of AD and newly discovered therapeutic targets.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide/biossíntese , Placa Amiloide/patologia , Biossíntese de Proteínas/fisiologia , Proteostase/fisiologia , Ribossomos/metabolismo , Animais , Drosophila , Humanos , Camundongos , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
The molecular bases underlying cognitive impairments in Alzheimer's disease remain elusive. In this study, we sought to determine the molecular correlates of memory deficits in APP/PS1 mice, a widely used animal model of Alzheimer's disease. To this end, we tested 18-month-old APP/PS1 mice in the Morris water maze and ranked them by their spatial memory performance. We found that some APP/PS1 mice performed poorly, whereas others performed as well as nontransgenic mice. We took advantage of this intragroup variability to identify the best predictor of cognitive deficits. In this APP/PS1 cohort, soluble and insoluble amyloid-ß levels did not correlate significantly with cognitive performance. However, we found that cognitive performance within the APP/PS1 group had a strong inverse correlation with Aß plaque load and mammalian target of rapamycin activation and positively correlated with autophagy activation. Our data suggest that mammalian target of rapamycin signaling may account cognitive performance in APP/PS1 mice.
Assuntos
Doença de Alzheimer/fisiopatologia , Autofagia/fisiologia , Disfunção Cognitiva/metabolismo , Placa Amiloide/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Disfunção Cognitiva/fisiopatologia , Modelos Animais de Doenças , Hipocampo/metabolismo , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos Transgênicos , Memória Espacial/fisiologiaRESUMO
Translation of mRNAs is tightly regulated and constantly surveyed for errors. Aberrant translation can trigger co-translational protein and RNA quality control processes, impairments of which cause neurodegeneration by still poorly understood mechanism(s). Here we show that quality control of translation of mitochondrial outer membrane (MOM)-localized mRNA intersects with the turnover of damaged mitochondria, both orchestrated by the mitochondrial kinase PINK1. Mitochondrial damage causes stalled translation of complex-I 30 kDa subunit (C-I30) mRNA on MOM, triggering the recruitment of co-translational quality control factors Pelo, ABCE1, and NOT4 to the ribosome/mRNA-ribonucleoprotein complex. Damage-induced ubiquitination of ABCE1 by NOT4 generates poly-ubiquitin signals that attract autophagy receptors to MOM to initiate mitophagy. In the Drosophila PINK1 model, these factors act synergistically to restore mitophagy and neuromuscular tissue integrity. Thus ribosome-associated co-translational quality control generates an early signal to trigger mitophagy. Our results have broad therapeutic implications for the understanding and treatment of neurodegenerative diseases.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Mitocôndrias/metabolismo , Mitofagia/genética , Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitinação , Animais , Drosophila , Proteínas de Drosophila/genética , Endonucleases/metabolismo , Células HeLa , Humanos , Proteínas Mitocondriais/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Ribossomos/metabolismoRESUMO
Respiratory Complex I deficiency is implicated in numerous degenerative and metabolic diseases. In particular, mutations in several mitochondrial DNA (mtDNA)-encoded Complex I subunits including ND4, ND5 and ND6 have been identified in several neurological diseases. We previously demonstrated that these subunits played essential roles in Complex I assembly which in turn affected mitochondrial function. Here, we carried out a comprehensive study of the Complex I assembly pathway. We identified a new Complex I intermediate containing both membrane and matrix arms at an early assembly stage. We find that lack of the ND6 subunit does not hinder membrane arm formation; instead it recruits ND1 and ND5 enters the intermediate. While ND4 is important for the formation of the newly identified intermediate, the addition of ND5 stabilizes the complex and is required for the critical transition from Complex I to supercomplex assembly. As a result, the Complex I assembly pathway has been redefined in this study.
Assuntos
DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/metabolismo , Multimerização Proteica , Animais , Linhagem Celular , Complexo I de Transporte de Elétrons/química , Camundongos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
Defects in Complex I assembly is one of the emerging underlying causes of severe mitochondrial disorders. The assembly of Complex I has been difficult to understand due to its large size, dual genetic control and the number of proteins involved. Mutations in Complex I subunits as well as assembly factors have been reported to hinder its assembly and give rise to a range of mitochondria disorders. In this review, we summarize the recent progress made in understanding the Complex I assembly pathway. In particularly, we focus on the known as well as novel assembly factors and their role in assembly of Complex I and human disease.
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Complexo I de Transporte de Elétrons/metabolismo , Doenças Mitocondriais/enzimologia , Proteínas Mitocondriais/metabolismo , Mutação , Animais , Complexo I de Transporte de Elétrons/genética , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genéticaRESUMO
Mitochondrial dysfunction has been long proposed to play a major role in tumorigenesis. Mitochondrial DNA (mtDNA) mutations, especially the mtDNA 4,977 bp deletion has been found in patients of various types of cancer. In order to comprehend the mtDNA 4,977 bp deletion status in various cancer types, we performed a meta-analysis composed of 33 publications, in which a total of 1613 cancer cases, 1516 adjacent normals and 638 healthy controls were included. When all studies were pooled, we found that cancerous tissue carried a lower mtDNA 4,977 bp deletion frequency than adjacent non-cancerous tissue (ORâ=â0.43, 95% CIâ=â0.20-0.92, Pâ=â0.03 for heterogeneity test, I(2)â=â91.5%) among various types of cancer. In the stratified analysis by cancer type the deletion frequency was even lower in tumor tissue than in adjacent normal tissue of breast cancer (ORâ=â0.19, 95% CIâ=â0.06-0.61, Pâ=â0.005 for heterogeneity test, I(2)=â82.7%). Interestingly, this observation became more significant in the stratified studies with larger sample sizes (ORâ=â0.70, 95% CIâ=â0.58-0.86, Pâ=â0.0005 for heterogeneity test, I(2)â=â95.1%). Furthermore, when compared with the normal tissue from the matched healthy controls, increased deletion frequencies were observed in both adjacent non-cancerous tissue (ORâ=â3.02, 95% CIâ=â2.13-4.28, P<0.00001 for heterogeneity test, I(2)=â53.7%), and cancerous tissue (ORâ=â1.36, 95% CIâ=â1.04-1.77, Pâ=â0.02 for heterogeneity test, I(2)=â83.5%). This meta-analysis suggests that the mtDNA 4,977 bp deletion is often found in cancerous tissue and thus has the potential to be a biomarker for cancer occurrence in the tissue, but at the same time being selected against in various types of carcinoma tissues. Larger and better-designed studies are still warranted to confirm these findings.
Assuntos
Biomarcadores Tumorais/genética , DNA Mitocondrial/genética , Mitocôndrias/genética , Neoplasias/genética , Deleção de Sequência , Idoso , Estudos de Casos e Controles , Bases de Dados Bibliográficas , Feminino , Humanos , Masculino , Mitocôndrias/patologia , Neoplasias/patologia , Razão de Chances , Tamanho da AmostraRESUMO
The mitochondrial respiratory chain consists of 5 enzyme complexes that are responsible for ATP generation. The paradigm of the electron transport chain as discrete enzymes diffused in the inner mitochondrial membrane has been replaced by the solid state supercomplex model wherein the respiratory complexes associate with each other to form supramolecular complexes. Defects in these supercomplexes, which have been shown to be functionally active and required for forming stable respiratory complexes, have been associated with many genetic and neurodegenerative disorders demonstrating their biomedical significance. In this review, we will summarize the functional and structural significance of supercomplexes and provide a comprehensive review of their assembly and the assembly factors currently known to play a role in this process.
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Mitocôndrias/enzimologia , Complexos Multienzimáticos/metabolismo , Trifosfato de Adenosina/metabolismo , Arilamina N-Acetiltransferase/metabolismo , Cardiolipinas/metabolismo , Transporte de Elétrons , Humanos , Mitocôndrias/metabolismo , Complexos Multienzimáticos/químicaRESUMO
Previously, we have shown that a heteroplasmic mutation in mitochondrial DNA-encoded complex I ND5 subunit gene resulted in an enhanced tumorigenesis through increased resistance to apoptosis. Here we report that the tumorigenic phenotype associated with complex I dysfunction could be reversed by introducing a yeast NADH quinone oxidoreductase (NDI1) gene. The NDI1 mediated electron transfer from NADH to Co-Q, bypassed the defective complex I and restored oxidative phosphorylation in the host cells. Alternatively, suppression of complex I activity by a specific inhibitor, rotenone or induction of oxidative stress by paraquat led to an increase in the phosphorylation of v-AKT murine thymoma viral oncogene (AKT) and enhanced the tumorigenesis. On the other hand, antioxidant treatment can ameliorate the reactive oxygen species-mediated AKT activation and reverse the tumorigenicity of complex I-deficient cells. Our results suggest that complex I defects could promote tumorigenesis through induction of oxidative stress and activation of AKT pathway.