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1.
Curr Protoc ; 1(9): e245, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34516047

RESUMO

Studies in various tissues have revealed a central role of metabolic pathways in regulating adult stem cell function in tissue regeneration and tumor initiation. The unique metabolic dependences or preferences of adult stem cells, therefore, are emerging as a new category of therapeutic target. Recently, advanced methods including high-resolution metabolomics, proteomics, and transcriptomics have been developed to address the growing interest in stem cell metabolism. A practical framework integrating the omics analyses is needed to systematically perform metabolic characterization in a cell-type-specific manner. Here, we leverage recent advances in transcriptomics and proteomics research to identify cell-type-specific metabolic features by reconstructing cell identity using genes and the encoded enzymes involved in major metabolic pathways. We provide protocols for cell isolation, transcriptome and proteome analyses, and metabolite profiling and measurement. The workflow for mapping cell-type-specific metabolic signatures presented here, although initially developed for intestinal crypt cells, can be easily implemented for cell populations in other tissues, and is highly compatible with most public datasets. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Intestinal crypt isolation and cell population purification Basic Protocol 2: Transcriptome analyses for cell-type-specific metabolic gene expression Basic Protocol 3: Proteome analyses for cell-type-specific metabolic enzyme levels Basic Protocol 4: Metabolite profiling and measurement.


Assuntos
Proteoma , Transcriptoma , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/genética , Proteoma/genética , Proteômica
2.
Adv Protein Chem Struct Biol ; 127: 93-126, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34340775

RESUMO

The biological complexity cannot be captured by genes or proteins alone. The protein posttranslational modifications (PTMs) impart functional diversity to the proteome and regulate protein structure, activity, localization and interactions. Their dynamics drive cellular signaling, growth and development while their dysregulation causes many diseases. Mass spectrometry based quantitative profiling of PTMs and bioinformatics analysis tools allow systems level insights into their network architecture. High-resolution profiling of PTM networks will advance disease understanding and precision medicine. It can accelerate the discovery of biomarkers and drug targets. This requires better tools for unbiased, high-throughput and accurate PTM identification, site localization and automated annotation on a systems level.


Assuntos
Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteômica , Biologia de Sistemas , Humanos , Espectrometria de Massas , Proteoma/genética
3.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361011

RESUMO

Many lepidopteran larvae produce silk feeding shelters and cocoons to protect themselves and the developing pupa. As caterpillars evolved, the quality of the silk, shape of the cocoon, and techniques in forming and leaving the cocoon underwent a number of changes. The silk of Pseudoips prasinana has previously been studied using X-ray analysis and classified in the same category as that of Bombyx mori, suggesting that silks of both species have similar properties despite their considerable phylogenetic distance. In the present study, we examined P. prasinana silk using 'omics' technology, including silk gland RNA sequencing (RNA-seq) and a mass spectrometry-based proteomic analysis of cocoon proteins. We found that although the central repetitive amino acid sequences encoding crystalline domains of fibroin heavy chain molecules are almost identical in both species, the resulting fibers exhibit quite different mechanical properties. Our results suggest that these differences are most probably due to the higher content of fibrohexamerin and fibrohexamerin-like molecules in P. prasinana silk. Furthermore, we show that whilst P. prasinana cocoons are predominantly made of silk similar to that of other Lepidoptera, they also contain a second, minor silk type, which is present only at the escape valve.


Assuntos
Bombyx/genética , Evolução Molecular , Fibroínas/genética , Animais , Bombyx/classificação , Bombyx/metabolismo , Glândulas Exócrinas/metabolismo , Fibroínas/química , Filogenia , Proteoma/genética , Proteoma/metabolismo , Transcriptoma
4.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361015

RESUMO

The sacred lotus (Nelumbo nucifera) can maintain a stable floral chamber temperature between 30 and 35 °C when blooming despite fluctuations in ambient temperatures between about 8 and 45 °C, but the regulatory mechanism of floral thermogenesis remains unclear. Here, we obtained comprehensive protein profiles from receptacle tissue at five developmental stages using data-independent acquisition (DIA)-based quantitative proteomics technology to reveal the molecular basis of floral thermogenesis of N. nucifera. A total of 6913 proteins were identified and quantified, of which 3513 differentially abundant proteins (DAPs) were screened. Among them, 640 highly abundant proteins during the thermogenic stages were mainly involved in carbon metabolism processes such as the tricarboxylic acid (TCA) cycle. Citrate synthase was identified as the most connected protein in the protein-protein interaction (PPI) network. Next, the content of alternative oxidase (AOX) and plant uncoupling protein (pUCP) in different tissues indicated that AOX was specifically abundant in the receptacles. Subsequently, a protein module highly related to the thermogenic phenotype was identified by the weighted gene co-expression network analysis (WGCNA). In summary, the regulation mechanism of floral thermogenesis in N. nucifera involves complex regulatory networks, including TCA cycle metabolism, starch and sucrose metabolism, fatty acid degradation, and ubiquinone synthesis, etc.


Assuntos
Adaptação Fisiológica , Flores/metabolismo , Redes Reguladoras de Genes , Nelumbo/genética , Mapas de Interação de Proteínas , Proteoma/metabolismo , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Ciclo do Ácido Cítrico , Flores/genética , Regulação da Expressão Gênica de Plantas , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nelumbo/crescimento & desenvolvimento , Nelumbo/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteoma/genética , Temperatura
5.
EBioMedicine ; 70: 103525, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34392148

RESUMO

BACKGROUND: While our battle with the COVID-19 pandemic continues, a multitude of Omics data have been generated from patient samples in various studies. Translation of these data into clinical interventions against COVID-19 remains to be accomplished. Exploring host response to COVID-19 in the upper respiratory tract can unveil prognostic markers and therapeutic targets. METHODS: We conducted a meta-analysis of published transcriptome and proteome profiles of respiratory samples of COVID-19 patients to shortlist high confidence upregulated host factors. Subsequently, mRNA overexpression of selected genes was validated in nasal swabs from a cohort of COVID-19 positive/negative, symptomatic/asymptomatic individuals. Guided by this analysis, we sought to check for potential drug targets. An FDA-approved drug, Auranofin, was tested against SARS-CoV-2 replication in cell culture and Syrian hamster challenge model. FINDINGS: The meta-analysis and validation in the COVID-19 cohort revealed S100 family genes (S100A6, S100A8, S100A9, and S100P) as prognostic markers of severe COVID-19. Furthermore, Thioredoxin (TXN) was found to be consistently upregulated. Auranofin, which targets Thioredoxin reductase, was found to mitigate SARS-CoV-2 replication in vitro. Furthermore, oral administration of Auranofin in Syrian hamsters in therapeutic as well as prophylactic regimen reduced viral replication, IL-6 production, and inflammation in the lungs. INTERPRETATION: Elevated mRNA level of S100s in the nasal swabs indicate severe COVID-19 disease, and FDA-approved drug Auranofin mitigated SARS-CoV-2 replication in preclinical hamster model. FUNDING: This study was supported by the DBT-IISc partnership program (DBT (IED/4/2020-MED/DBT)), the Infosys Young Investigator award (YI/2019/1106), DBT-BIRAC grant (BT/CS0007/CS/02/20) and the DBT-Wellcome Trust India Alliance Intermediate Fellowship (IA/I/18/1/503613) to ST lab.


Assuntos
COVID-19/genética , Nasofaringe/virologia , Proteoma/genética , Transcriptoma/genética , Adulto , Animais , Biomarcadores/metabolismo , COVID-19/patologia , COVID-19/virologia , Linhagem Celular , Chlorocebus aethiops , Estudos de Coortes , Feminino , Células HEK293 , Humanos , Inflamação/genética , Inflamação/virologia , Interleucina-6/genética , Masculino , Mesocricetus , Pessoa de Meia-Idade , Nasofaringe/patologia , Pandemias , Prognóstico , RNA Mensageiro/genética , SARS-CoV-2/patogenicidade , Regulação para Cima/genética , Células Vero , Replicação Viral/genética
6.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361002

RESUMO

Platelets are involved in tumor angiogenesis and cancer progression. Previous studies indicated that cancer could affect platelet content. In the current study, we investigated whether cancer-associated proteins can be discerned in the platelets of cancer patients, and whether antitumor treatment may affect the platelet proteome. Platelets were isolated from nine patients with different cancer types and ten healthy volunteers. From three patients, platelets were isolated before and after the start of antitumor treatment. Mass spectrometry-based proteomics of gel-fractionated platelet proteins were used to compare patients versus controls and before and after treatment initiation. A total of 4059 proteins were detected, of which 50 were significantly more abundant in patients, and 36 more in healthy volunteers. Eight of these proteins overlapped with our previous cancer platelet proteomics study. From these data, we selected potential biomarkers of cancer including six upregulated proteins (RNF213, CTSG, PGLYRP1, RPL8, S100A8, S100A9) and two downregulated proteins (GPX1, TNS1). Antitumor treatment resulted in increased levels of 432 proteins and decreased levels of 189 proteins. In conclusion, the platelet proteome may be affected in cancer patients and platelets are a potential source of cancer biomarkers. In addition, we found in a small group of patients that anticancer treatment significantly changes the platelet proteome.


Assuntos
Plaquetas/metabolismo , Neoplasias do Sistema Digestório/sangue , Proteoma/metabolismo , Idoso , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias do Sistema Digestório/tratamento farmacológico , Neoplasias do Sistema Digestório/genética , Neoplasias do Sistema Digestório/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteoma/genética
7.
BMC Genomics ; 22(1): 520, 2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34238212

RESUMO

BACKGROUND: A comprehensive evaluation of the -omic profiles of venom is important for understanding the potential function and evolution of snake venom. Here, we conducted an integrated multi-omics-analysis to unveil the venom-transcriptomic and venomic profiles in a same group of spine-bellied sea snakes (Hydrophis curtus) from the South China Sea, where the snake is a widespread species and might generate regionally-specific venom potentially harmful to human activities. The capacity of two heterologous antivenoms to immunocapture the H. curtus venom was determined for an in-depth evaluation of their rationality in treatment of H. curtus envenomation. In addition, a phylogenetic analysis by maximum likelihood was used to detect the adaptive molecular evolution of full-length toxin-coding unigenes. RESULTS: A total of 90,909,384 pairs of clean reads were generated via Illumina sequencing from a pooled cDNA library of six specimens, and yielding 148,121 unigenes through de novo assembly. Sequence similarity searching harvested 63,845 valid annotations, including 63,789 non-toxin-coding and 56 toxin-coding unigenes belonging to 22 protein families. Three protein families, three-finger toxins (3-FTx), phospholipase A2 (PLA2), and cysteine-rich secretory protein, were detected in the venom proteome. 3-FTx (27.15% in the transcriptome/41.94% in the proteome) and PLA2 (59.71%/49.36%) were identified as the most abundant families in the venom-gland transcriptome and venom proteome. In addition, 24 unigenes from 11 protein families were shown to have experienced positive selection in their evolutionary history, whereas four were relatively conserved throughout evolution. Commercial Naja atra antivenom exhibited a stronger capacity than Bungarus multicinctus antivenom to immunocapture H. curtus venom components, especially short neurotoxins, with the capacity of both antivenoms to immunocapture short neurotoxins being weaker than that for PLA2s. CONCLUSIONS: Our study clarified the venom-gland transcriptomic and venomic profiles along with the within-group divergence of a H. curtus population from the South China Sea. Adaptive evolution of most venom components driven by natural selection appeared to occur rapidly during evolutionary history. Notably, the utility of commercial N. atra and B. multicinctus antivenoms against H. curtus toxins was not comprehensive; thus, the development of species-specific antivenom is urgently needed.


Assuntos
Hydrophiidae , Animais , China , Venenos Elapídicos , Humanos , Filogenia , Proteoma/genética , Transcriptoma
8.
Adv Exp Med Biol ; 1208: 373-386, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34260034

RESUMO

Autophagy is an evolutionarily conserved intracellular degradation process. Autophagy is closely involved in human health and diseases. In recent years, mass spectrometry-based proteomic methods have become important and powerful tools for autophagy studies. These types of techniques have been especially helpful to reveal the range of degradation substrates of autophagy through large-scale, unbiased analysis of cellular proteomes. At present, a variety of mass spectrometry-based proteomics methods have been successfully applied to autophagy research.


Assuntos
Autofagia , Proteômica , Humanos , Espectrometria de Massas , Proteoma/genética
9.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299022

RESUMO

Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse formation. However, little is known about NCAM2 functional mechanisms and its interactive partners during brain development. Here we used mass spectrometry to study the molecular interactome of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organization, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene.


Assuntos
Córtex Cerebral/metabolismo , Citoesqueleto/metabolismo , Espectrometria de Massas , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurogênese , Neurônios/metabolismo , Actinas/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Biologia Computacional , Citoplasma/genética , Citoplasma/metabolismo , Bases de Dados de Compostos Químicos , Ontologia Genética , Técnicas In Vitro , Filamentos Intermediários/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurogênese/genética , Proteínas Nogo , Fosforilação , Domínios Proteicos , Mapas de Interação de Proteínas , Proteoma/genética , Proteoma/metabolismo , Transcriptoma/genética
10.
Mol Cell ; 81(16): 3275-3293.e12, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34245671

RESUMO

Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.


Assuntos
Proteínas da Matriz do Complexo de Golgi/genética , Proteoma/genética , Proteômica , Estresse Fisiológico/genética , Matriz Extracelular/genética , Complexo de Golgi/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , Transdução de Sinais/genética
11.
Mol Cell ; 81(16): 3294-3309.e12, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34293321

RESUMO

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.


Assuntos
Adaptação Fisiológica/genética , Proteoma/genética , Estresse Fisiológico/genética , Transcriptoma/genética , Aclimatação/genética , Animais , Exposição Ambiental/efeitos adversos , Regulação Fúngica da Expressão Gênica/genética , Temperatura Alta/efeitos adversos , Saccharomycetales/genética
12.
Genes (Basel) ; 12(5)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067543

RESUMO

Cortical cytoskeletal proteins are significant in controlling various cellular mechanisms such as migration, cell adhesion, intercellular attachment, cellular signaling, exo- and endocytosis and plasma membrane integrity, stability and flexibility. Our earlier studies involving in vitro and ex vivo approaches led us to identify certain undiscovered characteristics of α-fodrin, a prominent cortical protein. The conventional functions attributed to this protein mainly support the plasma membrane. In the present study, we utilized a global protein expression analysis approach to detect underexplored functions of this protein. We report that downregulation of α-fodrin in glioblastoma cells, U-251 MG, results in upregulation of genes affecting the regulation of the cytoskeleton, cell cycle and apoptosis. Interestingly, certain key microtubule kinesins such as KIF23, KIF2B and KIF3C are downregulated upon α-fodrin depletion, as validated by real-time PCR studies.


Assuntos
Proteínas de Transporte/metabolismo , Cinesina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Proteoma/metabolismo , Apoptose , Proteínas de Transporte/genética , Ciclo Celular , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Cinesina/genética , Proteínas dos Microfilamentos/genética , Proteoma/genética
13.
Methods Mol Biol ; 2344: 227-238, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34115363

RESUMO

The completion and annotation of the human proteome require the availability of information related to protein function. Currently, more than 1800 human genes constitute the "dark proteome," which include missing proteins, uncharacterized human genes validated at protein level, smORFs, proteins from lncRNAs, or any uncharacterized transcripts. During the last years, different experimental workflows based on multi-omics analyses, bioinformatics, and in vitro and in vivo studies have been promoted by the Human Proteome Project Consortium to enhance the annotation of dark proteins. In this chapter, we describe a method that utilizes recombinant proteins and antibody arrays to establish a straightforward methodology in order to rapidly characterize potential functional features of dark proteins associated to intracellular signaling dynamics and extracellular immune response in human cell cultures. Further validating the method, this workflow was applied to probe changes in the activation patterns of kinases and transcription factors as well as in cytokine production modulated by the dark C1orf128 (PITHD1) protein in human olfactory neuroepithelial cells.


Assuntos
Anticorpos/imunologia , Células Neuroepiteliais/imunologia , Bulbo Olfatório/imunologia , Análise Serial de Proteínas , Proteínas/imunologia , Proteoma/imunologia , Anticorpos/genética , Humanos , Células Neuroepiteliais/patologia , Bulbo Olfatório/patologia , Proteínas/genética , Proteoma/genética
14.
J Biol Chem ; 297(1): 100877, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34139237

RESUMO

The human ZC3H14 gene, which encodes a ubiquitously expressed polyadenosine zinc finger RNA-binding protein, is mutated in an inherited form of autosomal recessive, nonsyndromic intellectual disability. To gain insight into neurological functions of ZC3H14, we previously developed a Drosophila melanogaster model of ZC3H14 loss by deleting the fly ortholog, Nab2. Studies in this invertebrate model revealed that Nab2 controls final patterns of neuron projection within fully developed adult brains, but the role of Nab2 during development of the Drosophila brain is not known. Here, we identify roles for Nab2 in controlling the dynamic growth of axons in the developing brain mushroom bodies, which support olfactory learning and memory, and regulating abundance of a small fraction of the total brain proteome. The group of Nab2-regulated brain proteins, identified by quantitative proteomic analysis, includes the microtubule-binding protein Futsch, the neuronal Ig-family transmembrane protein turtle, the glial:neuron adhesion protein contactin, the Rac GTPase-activating protein tumbleweed, and the planar cell polarity factor Van Gogh, which collectively link Nab2 to the processes of brain morphogenesis, neuroblast proliferation, circadian sleep/wake cycles, and synaptic development. Overall, these data indicate that Nab2 controls the abundance of a subset of brain proteins during the active process of wiring the pupal brain mushroom body and thus provide a window into potentially conserved functions of the Nab2/ZC3H14 RNA-binding proteins in neurodevelopment.


Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Neurogênese , Proteoma/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Contactinas/genética , Contactinas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Memória , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoma/metabolismo , Proteínas de Ligação a RNA/genética
15.
J Biol Chem ; 297(1): 100880, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34144036

RESUMO

More than half a century ago, reversible protein phosphorylation was linked to mitochondrial metabolism through the regulation of pyruvate dehydrogenase. Since this discovery, the number of identified mitochondrial protein phosphorylation sites has increased by orders of magnitude, driven largely by technological advances in mass spectrometry-based phosphoproteomics. However, the majority of these modifications remain uncharacterized, rendering their function and relevance unclear. Nonetheless, recent studies have shown that disruption of resident mitochondrial protein phosphatases causes substantial metabolic dysfunction across organisms, suggesting that proper management of mitochondrial phosphorylation is vital for organellar and organismal homeostasis. While these data suggest that phosphorylation within mitochondria is of critical importance, significant gaps remain in our knowledge of how these modifications influence organellar function. Here, we curate publicly available datasets to map the extent of protein phosphorylation within mammalian mitochondria and to highlight the known functions of mitochondrial-resident phosphatases. We further propose models by which phosphorylation may affect mitochondrial enzyme activities, protein import and processing, and overall organellar homeostasis.


Assuntos
Proteínas Mitocondriais/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Animais , Humanos , Proteínas Mitocondriais/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/genética , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteoma/genética
16.
Genes (Basel) ; 12(5)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070083

RESUMO

Type I toxin-antitoxin (TA) systems are widespread genetic modules in bacterial genomes. They express toxic peptides whose overexpression leads to growth arrest or cell death, whereas antitoxins regulate the expression of toxins, acting as labile antisense RNAs. The Staphylococcus aureus (S. aureus) genome contains and expresses several functional type I TA systems, but their biological functions remain unclear. Here, we addressed and challenged experimentally, by proteomics, if the type I TA system, the SprG1/SprF1 pair, influences the overall gene expression in S. aureus. Deleted and complemented S. aureus strains were analyzed for their proteomes, both intracellular and extracellular, during growth. Comparison of intracellular proteomes among the strains points to the SprF1 antitoxin as moderately downregulating protein expression. In the strain naturally expressing the SprG1 toxin, cytoplasmic proteins are excreted into the medium, but this is not due to unspecific cell leakages. Such a toxin-driven release of the cytoplasmic proteins may modulate the host inflammatory response that, in turn, could amplify the S. aureus infection spread.


Assuntos
Antitoxinas/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Expressão Gênica/genética , Staphylococcus aureus/genética , Sistemas Toxina-Antitoxina/genética , Citoplasma/genética , Genoma Bacteriano/genética , Proteoma/genética , RNA Antissenso/genética
17.
J Proteome Res ; 20(7): 3689-3700, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34085531

RESUMO

Novel therapies and biomarkers are needed for the treatment of acute ischemic stroke (AIS). This study aimed to provide comprehensive insights into the dynamic proteome changes and underlying molecular mechanisms post-ischemic stroke. TMT-coupled proteomic analysis was conducted on mouse brain cortex tissue from five time points up to 4 weeks poststroke in the distal hypoxic-middle cerebral artery occlusion (DH-MCAO) model. We found that nearly half of the detected proteome was altered following stroke, but only ∼8.6% of the changes were at relatively large scales. Clustering on the changed proteome defined four distinct expression patterns characterized by temporal and quantitative changes in innate and adaptive immune response pathways and cytoskeletal and neuronal remodeling. Further analysis on a subset of 309 "top hits", which temporally responded to stroke with relatively large and sustained changes, revealed that they were mostly secreted proteins, highly correlated to different cortical cytokines, and thereby potential pharmacodynamic biomarker candidates for inflammation-targeting therapies. Closer examination of the top enriched neurophysiologic pathways identified 57 proteins potentially associated with poststroke recovery. Altogether, our study generated a rich dataset with candidate proteins worthy of further validation as biomarkers and/or therapeutic targets for stroke. The proteomics data are available in the PRIDE Archive with identifier PXD025077.


Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Animais , Camundongos , Proteoma/genética , Proteômica
18.
J Proteome Res ; 20(8): 4075-4088, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34185526

RESUMO

Understanding the molecular basis of sexual dimorphism in the cardiovascular system may contribute to the improvement of the outcome in biological, pharmacological, and toxicological studies as well as on the development of sex-based drugs and therapeutic approaches. Label-free protein quantification using high-resolution mass spectrometry was applied to detect sex-based proteome differences in the heart of zebrafish Danio rerio. Out of almost 3000 unique identified proteins in the heart, 79 showed significant abundance differences between male and female fish. The functional differences were mapped using enrichment analyses. Our results suggest that a large amount of materials needed for reproduction (e.g., sugars, lipids, proteins, etc.) may impose extra pressure on blood, vessels, and heart on their way toward the ovaries. In the present study, the female's heart shows a clear sexual dimorphism by changing abundance levels of numerous proteins, which could be a way to safely overcome material-induced elevated pressures. These proteins belong to the immune system, oxidative stress response, drug metabolization, detoxification, energy, metabolism, and so on. In conclusion, we showed that sex can induce dimorphism at the molecular level in nonsexual organs such as heart and must be considered as an important factor in cardiovascular research. Data are available via ProteomeXchange with identifier PXD023506.


Assuntos
Coração , Caracteres Sexuais , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Feminino , Masculino , Proteoma/genética , Proteômica , Peixe-Zebra/genética
19.
Mol Cell ; 81(13): 2851-2867.e7, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34118193

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs, and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Among them are several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.


Assuntos
COVID-19/metabolismo , Proteoma/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Células A549 , COVID-19/genética , Humanos , Proteoma/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas Virais/genética
20.
Mol Cell ; 81(12): 2501-2503, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34143967

RESUMO

In this issue of Molecular Cell, Lin et al. (2021) develop a tri-functional amino acid probe for the discovery and characterization of protein domains that sense or "read" protein post-translational modifications, a chemical tool that can facilitate our understanding of how signaling networks act at the molecular level.


Assuntos
Proteoma , Leitura , Linguística , Processamento de Proteína Pós-Traducional , Proteoma/genética , Transdução de Sinais
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