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1.
Gene ; 722: 144105, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31521702

RESUMO

BACKGROUND: Caulophyllum robustum Maxim (CRM) is a medicinal compound of the Northeast and is commonly used in China for the treatment of rheumatic pain and rheumatoid arthritis (RA). A preliminary study found that CRM has good anti-inflammatory, analgesic and immunosuppressive effects. However, the specific links and targets for its function remain unclear. Our study aimed to provide a mechanism for the action of Caulophyllum robustum Maxim extraction (CRME) against RA and to establish a method for studying disease treatment using Chinese medicine. METHODS: The 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) method was used to detect the toxicity of CRME in L929 cells, and the concentration ranges of the blank, model, and CRME drug groups were determined. Differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) were identified between the three groups. Gene Ontology (GO) and pathway enrichment analyses were performed to analyze the biological functions and pathways of the differentially expressed genes. Expression of Hist1h2bj, Hist1h2ba, Zfp36, Ccl3, Cxcl2 and Egr1 in the blank, model and drug groups was detected by quantitative real-time PCR (qRT-PCR), and the role of CRME on the above factors was determined to ensure consistency with the chip data. RESULTS: A total of 329 significantly upregulated genes and 141 downregulated genes were identified between the blank and model groups. A total of 218 significantly upregulated genes and 191 downregulated genes were identified between the CRME drug group and model group. CRME has a significant role in multiple pathways involved in the occurrence and development of RA. Additionally, Hist1h2bj, Hist1h2ba, Zfp36, Ccl3, Cxcl2, and Egr1 were observed in modules of the lncRNA-mRNA weighted co-expression network, consistent with the chip data. CONCLUSIONS: CRME has regulatory effects on inflammatory factors, the histone family, chemokines and their ligands that are related to RA-related cytokines, the RA pathway, the TNF signaling pathway, the Toll receptor-like signaling pathway, the chemokine signaling pathways and other pathways are related to the course of RA.


Assuntos
Artrite Reumatoide/genética , Caulophyllum , Medicamentos de Ervas Chinesas/farmacologia , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Animais , Artrite Reumatoide/metabolismo , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Mapeamento de Interação de Proteínas
2.
Tumour Biol ; 41(11): 1010428319883721, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31718480

RESUMO

The involvement of microRNA in cancers plays a significant role in their pathogenesis. Specific expressions of these non-coding RNAs also serve as biomarkers for early colorectal cancer diagnosis, but their laboratory/molecular identification is challenging and expensive. The aim of this study was to identify potential microRNAs for colorectal cancer diagnosis using in silico approach. Sequence similarity search was employed to obtain the candidate microRNA from the datasets, and three target prediction software were employed to determine their target genes. To determine the involvement of these microRNAs in colorectal cancer, the microRNA gene list obtained was used alongside with colorectal cancer expressed genes from gbCRC and CoReCG databases for gene intersection analysis. The involvement of these genes in the cancer subtype was further strengthened with the DAVID database. KEGG and Gene Ontology were used for the pathway and functional analysis, while STRING was employed for the interactions of protein network and further visualized by Cytoscape. The cBioPortal database was used to prioritize the target genes; prognostic and expression analysis were finally performed on the candidate microRNAs and the prioritized targets. This study, therefore, identified five candidate microRNAs, two hub genes (CTNNB1 and epidermal growth factor receptor), and seven significant target genes associated with colorectal cancer. The molecular validation studies are ongoing to ascertain the biological fitness of these findings.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , MicroRNAs/genética , Transcriptoma/genética , Neoplasias Colorretais/patologia , Biologia Computacional , Simulação por Computador , Bases de Dados Genéticas , Detecção Precoce de Câncer , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/genética , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Mapeamento de Interação de Proteínas , beta Catenina/genética
3.
Biomed Khim ; 65(5): 374-379, 2019 Aug.
Artigo em Russo | MEDLINE | ID: mdl-31666408

RESUMO

Identification of new protein-protein interactions (PPI) and characterization of quantitative parameters of complex formation represent one of central tasks of protein interactomics. This work is a logical continuation of the cycle of our previous works devoted to the study of PPIs among the components of cytochrome P450-dependent monooxygenase system. Using an optical biosensor of Surface Plasmon Resonance (SPR biosensor), a comparative analysis on the determination of kinetic and equilibrium parameters of complex formation between the membrane-bound hemoprotein cytochrome b5 with cytochrome P450s was performed using two different protocols for protein immobilization: 1) covalent non-oriented one on to the carboxymethyl dextran chip type CM and 2) non-covalent oriented immobilization in the lipid environment on the chip type L1 with internal control of liposomes surface distribution. In the second protocol it was shown that the complex formation was characterized by 2.5 times higher affinity due to an decrease in rate dissociation constants. The appropriateness of using both experimental models is discussed.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/metabolismo , Lipossomos/metabolismo , Mapeamento de Interação de Proteínas , Humanos , Cinética , Lipídeos , Ressonância de Plasmônio de Superfície
4.
Curr Top Med Chem ; 19(21): 1872-1876, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31696799

RESUMO

PPIs are involved in diverse biochemical events and perform their functions through the formation of protein-protein complexes or PPI networks. The large and flat interacting surfaces of PPIs make discovery of small-molecule modulators a challenging task. New strategies and more effective chemical technologies are needed to facilitate the development of PPIs small-molecule inhibitors. Covalent modification of a nucleophilic residue located proximally to the immediate vicinity of PPIs can overcome the disadvantages of large interacting surfaces and provides high-affinity inhibitors with increased duration of action and prolonged target modulation. On the other hand, covalent inhibitors that target non-conserved protein residues demonstrate improved selectivity over related protein family members. Herein, we highlight the latest progress of small-molecule covalent PPIs inhibitors and hope to shed light on future PPIs inhibitor design and development. The relevant challenges and opportunities are also discussed.


Assuntos
Proteínas/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Mapeamento de Interação de Proteínas , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química
6.
DNA Cell Biol ; 38(10): 1056-1068, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31403329

RESUMO

The AP2/ERF (APETALA2/ETHYLENE RESPONSE FACTOR) transcription factor represents one of the largest plant-specific transcriptional regulators in plants. ERF plays important roles in the regulation of various developmental processes and acts as a mediator in plant external stress responses. However, the research of the ERF gene family is still limited in alfalfa (Medicago sativa L.), one of the most important forage legume species in the world. In the present study, a total of 159 ERF genes were identified, and the phylogenetic reconstruction, classification, conserved motifs, signal peptide prediction, and expression patterns under salt, drought, and low-temperature stresses of these ERF genes were comprehensively analyzed. The ERF genes family in alfalfa could be classified into 10 groups and predicted to be strongly homologous. Based on the structure and functions relationships, the III and IV subfamilies were more likely to play functions in abiotic stresses and 18 MsERF genes were selected for further quantitative real-time PCR validation in different stresses treatment. The results showed that all these MsERF genes were upregulated under three stresses except MsERF008. This study identified the possibility of abiotic tolerance candidate genes playing various roles in stress resistance at the whole-genome level, which would provide primary understanding for exploring ERF-mediated tolerance in alfalfa.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Medicago sativa/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Temperatura Baixa , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Secas , Perfilação da Expressão Gênica , Medicago sativa/classificação , Medicago sativa/metabolismo , Filogenia , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Mapeamento de Interação de Proteínas , Isoformas de Proteínas , Salinidade , Estresse Fisiológico
7.
Adv Exp Med Biol ; 1158: 83-100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452137

RESUMO

Mitochondria (mt) are double-membraned, dynamic organelles that play an essential role in a large number of cellular processes, and impairments in mt function have emerged as a causative factor for a growing number of human disorders. Given that most biological functions are driven by physical associations between proteins, the first step towards understanding mt dysfunction is to map its protein-protein interaction (PPI) network in a comprehensive and systematic fashion. While mass-spectrometry (MS) based approaches possess the high sensitivity ideal for such an endeavor, it also requires stringent biochemical purification of bait proteins to avoid detecting spurious, non-specific PPIs. Here, we outline a tagging-based affinity purification coupled with mass spectrometry (AP-MS) workflow for discovering new mt protein associations and providing novel insights into their role in mt biology and human physiology/pathology. Because AP-MS relies on the creation of proteins fused with affinity tags, we employ a versatile-affinity (VA) tag, consisting of 3× FLAG, 6 × His, and Strep III epitopes. For efficient delivery of affinity-tagged open reading frames (ORF) into mammalian cells, the VA-tag is cloned onto a specific ORF using Gateway recombinant cloning, and the resulting expression vector is stably introduced in target cells using lentiviral transduction. In this chapter, we show a functional workflow for mapping the mt interactome that includes tagging, stable transduction, selection and expansion of mammalian cell lines, mt extraction, identification of interacting protein partners by AP-MS, and lastly, computational assessment of protein complexes/PPI networks.


Assuntos
Cromatografia de Afinidade , Espectrometria de Massas , Proteínas Mitocondriais , Mapeamento de Interação de Proteínas/métodos , Fluxo de Trabalho , Animais , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/isolamento & purificação
8.
Neuron ; 103(3): 380-394, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394063

RESUMO

The Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) was touted as a memory molecule, even before its involvement in long-term potentiation (LTP) was shown. The enzyme has not disappointed, with subsequent demonstrations of remarkable structural and regulatory properties. Its neuronal functions now extend to long-term depression (LTD), and last year saw the first direct evidence for memory storage by CaMKII. Although CaMKII may have taken the spotlight, it is a member of a large family of diverse and interesting CaM kinases. Our aim is to place CaMKII in context of the other CaM kinases and then review certain aspects of this kinase that are of current interest.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Cognição/fisiologia , Humanos , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Modelos Moleculares , Família Multigênica , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/fisiologia , Fosforilação , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/classificação , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica
9.
DNA Cell Biol ; 38(10): 1112-1124, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31464520

RESUMO

In this study, we mined out hepatocellular carcinoma (HCC) driver genes from MEDLINE literatures by bioinformatics methods of pathway crosstalk and protein interaction network. Furthermore, the relationship between driver genes and their clinicopathological characteristics, as well as classification effectiveness was verified in the public databases. We identified 560 human genes reported to be associated with HCC in 1074 published articles. Functional analysis revealed that biological processes and biochemical pathways relating to tumor pathogenesis, cancer disease, tumor cell molecule, and hepatic disease were enriched in these genes. Pathway crosstalk analysis indicated that significant pathways could be divided into three modules: cancer disease, virus infection, and tumor signaling pathway. The HCC-related protein-protein interaction network comprised 10,212 nodes, and 56,400 edges were mined out to identify 18 modules corresponding to 14 driver genes. We verified that these 14 driver genes have high classification effectiveness to distinguish cancer samples from normal samples and the classification effectiveness was better than that of randomly selected genes. Present study provided pathway crosstalk and protein interaction network for understanding potential tumorigenesis genes underlying HCC. The 14 driver genes identified from this study are of great translational value in HCC diagnosis and treatment, as well as in clinical study on the pathogenesis of HCC.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias Hepáticas/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Idoso , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Biologia Computacional/métodos , Progressão da Doença , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Mapeamento de Interação de Proteínas , Curva ROC , Transdução de Sinais
10.
BMC Bioinformatics ; 20(1): 398, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315557

RESUMO

BACKGROUND: Utilization of quantitative proteomics data on the network level is still a challenge in proteomics data analysis. Currently existing models use sophisticated, sometimes hard to implement analysis techniques. Our aim was to generate a relatively simple strategy for quantitative proteomics data analysis in order to utilize as much of the data generated in a proteomics experiment as possible. RESULTS: In this study, we applied label-free proteomics, and generated a network model utilizing both qualitative, and quantitative data, in order to examine the early host response to Human Immunodeficiency Virus type 1 (HIV-1). A weighted network model was generated based on the amount of proteins measured by mass spectrometry, and analysis of weighted networks and functional sub-networks revealed upregulation of proteins involved in translation, transcription, and DNA condensation in the early phase of the viral life-cycle. CONCLUSION: A relatively simple strategy for network analysis was created and applied to examine the effect of HIV-1 on host cellular proteome. We believe that our model may prove beneficial in creating algorithms, allowing for both quantitative and qualitative studies of proteome change in various biological and pathological processes by quantitative mass spectrometry.


Assuntos
HIV-1/fisiologia , Proteômica/métodos , HIV-1/genética , Humanos , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Transdução Genética
11.
Adv Exp Med Biol ; 1162: 39-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31332733

RESUMO

Cannabinoids have been widely used for recreational and medicinal purposes. The increasing legalization of cannabinoid use and the growing success in Medicinal Chemistry of cannabinoids have fueled recent interest in cannabinoid-sensing sites in receptor proteins. Here, we review structural data from high-resolution cryo-EM and crystallography studies that depict phytocannabinoid, endocannabinoid, and synthetic cannabinoid molecules bound to various proteins. The latter include antigen-binding fragment (Fab), cellular retinol binding protein 2 (CRBP2), fatty acid-binding protein 5 (FABP5), peroxisome proliferator-activated receptor γ (PPAR γ), and cannabinoid receptor types 1 and 2 (CB1 and CB2). Cannabinoid-protein complexes reveal the complex design of cannabinoid binding sites that are usually presented by conventional ligand-binding pockets on respective proteins. However, subtle differences in cannabinoid interaction with amino acids within the binding pocket often result in diverse consequences for protein function. The rapid increase in available structural data on cannabinoid-protein interactions will ultimately direct drug design efforts toward rendering highly potent cannabinoid-related pharmacotherapies that are devoid of side effects.


Assuntos
Canabinoides/química , Endocanabinoides/química , Sítios de Ligação , Proteínas de Ligação a Ácido Graxo/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , PPAR gama/química , Mapeamento de Interação de Proteínas , Receptores de Canabinoides/química , Proteínas Celulares de Ligação ao Retinol/química
12.
Adv Exp Med Biol ; 1140: 1-26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347039

RESUMO

Within the past years, we have witnessed a great improvement is mass spectrometry (MS) and proteomics approaches in terms of instrumentation, protein fractionation, and bioinformatics. With the current technology, protein identification alone is no longer sufficient. Both scientists and clinicians want not only to identify the proteins, but also to identify the protein's post-translational modifications (PTMs), protein isoforms, protein truncation, protein-protein interactions (PPI), and protein quantitation. Here, we describe the principle of MS and proteomics, and strategies to identify proteins, protein's PTMs, protein isoforms, protein truncation, PPIs, and protein quantitation. We also discuss the strengths and weaknesses within this field. Finally, in our concluding remarks we assess the role of mass spectrometry and proteomics in the scientific and clinical settings, in the near future. This chapter provides an introduction and overview for subsequent chapters that will discuss specific MS proteomic methodologies and their application to specific medical conditions. Other chapters will also touch upon areas that expand beyond proteomics, such as lipidomics and metabolomics.


Assuntos
Espectrometria de Massas , Proteômica , Biologia Computacional , Humanos , Mapeamento de Interação de Proteínas , Isoformas de Proteínas , Processamento de Proteína Pós-Traducional
13.
Adv Exp Med Biol ; 1140: 169-198, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347048

RESUMO

Mass Spectrometry (MS) has revolutionized the way we study biomolecules, especially proteins, their interactions and posttranslational modifications (PTM). As such MS has established itself as the leading tool for the analysis of PTMs mainly because this approach is highly sensitive, amenable to high throughput and is capable of assigning PTMs to specific sites in the amino acid sequence of proteins and peptides. Along with the advances in MS methodology there have been improvements in biochemical, genetic and cell biological approaches to mapping the interactome which are discussed with consideration for both the practical and technical considerations of these techniques. The interactome of a species is generally understood to represent the sum of all potential protein-protein interactions. There are still a number of barriers to the elucidation of the human interactome or any other species as physical contact between protein pairs that occur by selective molecular docking in a particular spatiotemporal biological context are not easily captured and measured.PTMs massively increase the complexity of organismal proteomes and play a role in almost all aspects of cell biology, allowing for fine-tuning of protein structure, function and localization. There are an estimated 300 PTMS with a predicted 5% of the eukaryotic genome coding for enzymes involved in protein modification, however we have not yet been able to reliably map PTM proteomes due to limitations in sample preparation, analytical techniques, data analysis, and the substoichiometric and transient nature of some PTMs. Improvements in proteomic and mass spectrometry methods, as well as sample preparation, have been exploited in a large number of proteome-wide surveys of PTMs in many different organisms. Here we focus on previously published global PTM proteome studies in the Apicomplexan parasites T. gondii and P. falciparum which offer numerous insights into the abundance and function of each of the studied PTM in the Apicomplexa. Integration of these datasets provide a more complete picture of the relative importance of PTM and crosstalk between them and how together PTM globally change the cellular biology of the Apicomplexan protozoa. A multitude of techniques used to investigate PTMs, mostly techniques in MS-based proteomics, are discussed for their ability to uncover relevant biological function.


Assuntos
Espectrometria de Massas , Mapeamento de Interação de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Humanos , Simulação de Acoplamento Molecular , Proteoma
14.
Nat Methods ; 16(8): 737-742, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31308550

RESUMO

Protein complexes are key macromolecular machines of the cell, but their description remains incomplete. We and others previously reported an experimental strategy for global characterization of native protein assemblies based on chromatographic fractionation of biological extracts coupled to precision mass spectrometry analysis (chromatographic fractionation-mass spectrometry, CF-MS), but the resulting data are challenging to process and interpret. Here, we describe EPIC (elution profile-based inference of complexes), a software toolkit for automated scoring of large-scale CF-MS data to define high-confidence multi-component macromolecules from diverse biological specimens. As a case study, we used EPIC to map the global interactome of Caenorhabditis elegans, defining 612 putative worm protein complexes linked to diverse biological processes. These included novel subunits and assemblies unique to nematodes that we validated using orthogonal methods. The open source EPIC software is freely available as a Jupyter notebook packaged in a Docker container (https://hub.docker.com/r/baderlab/bio-epic/).


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/análise , Software , Animais , Proteínas de Caenorhabditis elegans/isolamento & purificação
15.
Nucleic Acids Res ; 47(14): 7235-7246, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31265076

RESUMO

Despite large experimental and computational efforts aiming to dissect the mechanisms underlying disease risk, mapping cis-regulatory elements to target genes remains a challenge. Here, we introduce a matrix factorization framework to integrate physical and functional interaction data of genomic segments. The framework was used to predict a regulatory network of chromatin interaction edges linking more than 20 000 promoters and 1.8 million enhancers across 127 human reference epigenomes, including edges that are present in any of the input datasets. Our network integrates functional evidence of correlated activity patterns from epigenomic data and physical evidence of chromatin interactions. An important contribution of this work is the representation of heterogeneous data with different qualities as networks. We show that the unbiased integration of independent data sources suggestive of regulatory interactions produces meaningful associations supported by existing functional and physical evidence, correlating with expected independent biological features.


Assuntos
Algoritmos , Biologia Computacional/métodos , Epigenômica/métodos , Redes Reguladoras de Genes , Regiões Promotoras Genéticas/genética , Elementos Reguladores de Transcrição/genética , Cromatina/genética , Cromatina/metabolismo , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Humanos , Células K562 , Polimorfismo de Nucleotídeo Único , Mapeamento de Interação de Proteínas/métodos , Reprodutibilidade dos Testes
16.
Medicine (Baltimore) ; 98(27): e16277, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31277155

RESUMO

Kaposi sarcoma (KS) is an endothelial tumor etiologically related to Kaposi sarcoma herpesvirus (KSHV) infection. The aim of our study was to screen out candidate genes of KSHV infected endothelial cells and to elucidate the underlying molecular mechanisms by bioinformatics methods. Microarray datasets GSE16354 and GSE22522 were downloaded from Gene Expression Omnibus (GEO) database. the differentially expressed genes (DEGs) between endothelial cells and KSHV infected endothelial cells were identified. And then, functional enrichment analyses of gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were performed. After that, Search Tool for the Retrieval of Interacting Genes (STRING) was used to investigate the potential protein-protein interaction (PPI) network between DEGs, Cytoscape software was used to visualize the interaction network of DEGs and to screen out the hub genes. A total of 113 DEGs and 11 hub genes were identified from the 2 datasets. GO enrichment analysis revealed that most of the DEGs were enrichen in regulation of cell proliferation, extracellular region part and sequence-specific DNA binding; KEGG pathway enrichments analysis displayed that DEGs were mostly enrichen in cell cycle, Jak-STAT signaling pathway, pathways in cancer, and Insulin signaling pathway. In conclusion, the present study identified a host of DEGs and hub genes in KSHV infected endothelial cells which may serve as potential key biomarkers and therapeutic targets, helping us to have a better understanding of the molecular mechanism of KS.


Assuntos
Biomarcadores Tumorais/genética , Biologia Computacional/métodos , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 8 , Mapas de Interação de Proteínas/genética , Sarcoma de Kaposi/genética , Biomarcadores Tumorais/biossíntese , DNA de Neoplasias/genética , Células Endoteliais/patologia , Células Endoteliais/virologia , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Humanos , Mapeamento de Interação de Proteínas/métodos , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia
17.
Nucleic Acids Res ; 47(15): 7734-7752, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31350900

RESUMO

DAXX displays complex biological functions. Remarkably, DAXX overexpression is a common feature in diverse cancers, which correlates with tumorigenesis, disease progression and treatment resistance. Structurally, DAXX is modular with an N-terminal helical bundle, a docking site for many DAXX interactors (e.g. p53 and ATRX). DAXX's central region folds with the H3.3/H4 dimer, providing a H3.3-specific chaperoning function. DAXX has two functionally critical SUMO-interacting motifs. These modules are connected by disordered regions. DAXX's structural features provide a framework for deciphering how DAXX mechanistically imparts its functions and how its activity is regulated. DAXX modulates transcription through binding to transcription factors, epigenetic modifiers, and chromatin remodelers. DAXX's localization in the PML nuclear bodies also plays roles in transcriptional regulation. DAXX-regulated genes are likely important effectors of its biological functions. Deposition of H3.3 and its interactions with epigenetic modifiers are likely key events for DAXX to regulate transcription, DNA repair, and viral infection. Interactions between DAXX and its partners directly impact apoptosis and cell signaling. DAXX's activity is regulated by posttranslational modifications and ubiquitin-dependent degradation. Notably, the tumor suppressor SPOP promotes DAXX degradation in phase-separated droplets. We summarize here our current understanding of DAXX's complex functions with a focus on how it promotes oncogenesis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Transdução de Sinais , Sumoilação , Transcrição Genética , Ubiquitinação
18.
Nat Protoc ; 14(8): 2318-2343, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31270507

RESUMO

This protocol describes a workflow for utilizing large-scale cross-linking with mass spectrometry (XL-MS) to make systems-level structural biology measurements in complex biological samples, including cells, isolated organelles, and tissue samples. XL-MS is a structural biology technique that provides information on the molecular structure of proteins and protein complexes using chemical probes that report the proximity of probe-reactive amino acids within proteins, typically lysine residues. Information gained through XL-MS studies is often complementary to more traditional methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. The use of MS-cleavable cross-linkers, including protein interaction reporter (PIR) technologies, enables XL-MS studies on protein structures and interactions in extremely complex biological samples, including intact living cells. PIR cross-linkers are designed to contain chemical bonds at specific locations within the cross-linker molecule that can be selectively cleaved by collision-induced dissociation or UV light. When broken, these bonds release the intact peptides that were cross-linked, as well as a reporter ion. Conservation of mass dictates that the sum of the two released peptide masses and the reporter mass equals the measured precursor mass. This relationship is used to identify cross-linked peptide pairs. Release of the individual peptides permits accurate measurement of their masses and independent amino acid sequence determination by tandem MS, allowing the use of standard proteomics search engines such as Comet for peptide sequence assignment, greatly simplifying data analysis of cross-linked peptide pairs. Search results are processed with XLinkProphet for validation and can be uploaded into XlinkDB for interaction network and structural analysis.


Assuntos
Espectrometria de Massas/métodos , Biologia Molecular/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Animais , Células Cultivadas , Escherichia coli , Humanos , Lisina/análise , Lisina/química , Camundongos , Peptídeos/análise , Peptídeos/química , Proteínas/análise , Proteômica , Biologia de Sistemas
19.
Anal Bioanal Chem ; 411(23): 6155-6163, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300857

RESUMO

Electrophoresis has demonstrated utility as tool for screening of small molecule modulators of protein-protein interactions and enzyme targets. Screening of large chemical libraries requires high-throughput separations. Such fast separation can be accessed by microchip electrophoresis. Here, microchip gel electrophoresis separations of proteins are achieved in 2.6 s with 1200 V/cm and 3-mm separation lengths. However, such fast separations can still suffer from limited overall throughput from sample introduction constraints. Automated introduction of microfluidic droplets has been demonstrated to overcome this limitation. Most devices for coupling microfluidic droplets to microchip electrophoresis are only compatible with free-solution separations. Here, we present a device that is compatible with coupling droplets to gel and free-solution electrophoresis. In this device, automated sample introduction is based on a novel mechanism of carrier phase separation using the difference in density of the carrier phase and the running buffer. This device is demonstrated for microchip gel electrophoresis and free-solution electrophoresis separations of protein-protein interaction and enzyme samples, respectively. Throughputs of about 10 s per sample are achieved and over 1000 separations are demonstrated without reconditioning of the device. Graphical abstract.


Assuntos
Eletroforese em Microchip/instrumentação , Mapeamento de Interação de Proteínas/instrumentação , Biocatálise , Desenho de Equipamento , Géis/química , Mapas de Interação de Proteínas , Proteínas/metabolismo
20.
Nat Commun ; 10(1): 3015, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289271

RESUMO

The protein-protein interaction (PPI) network of an organism serves as a skeleton for its signaling circuitry, which mediates cellular response to environmental and genetic cues. Understanding this circuitry could improve the prediction of gene function and cellular behavior in response to diverse signals. To realize this potential, one has to comprehensively map PPIs and their directions of signal flow. While the quality and the volume of identified human PPIs improved dramatically over the last decade, the directions of these interactions are still mostly unknown, thus precluding subsequent prediction and modeling efforts. Here we present a systematic approach to orient the human PPI network using drug response and cancer genomic data. We provide a diffusion-based method for the orientation task that significantly outperforms existing methods. The oriented network leads to improved prioritization of cancer driver genes and drug targets compared to the state-of-the-art unoriented network.


Assuntos
Biologia Computacional/métodos , Neoplasias/genética , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Análise de Dados , Bases de Dados Genéticas/estatística & dados numéricos , Bases de Dados de Produtos Farmacêuticos/estatística & dados numéricos , Conjuntos de Dados como Assunto , Humanos , Mapas de Interação de Proteínas/genética , Software
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