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1.
Mol Cell Proteomics ; 23(9): 100831, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39168282

RESUMO

Characterizing the antibody reactome for circulating antibodies provide insight into pathogen exposure, allergies, and autoimmune diseases. This is important for biomarker discovery, clinical diagnosis, and prognosis of disease progression, as well as population-level insights into the immune system. The emerging technology phage display immunoprecipitation and sequencing (PhIP-seq) is a high-throughput method for identifying antigens/epitopes of the antibody reactome. In PhIP-seq, libraries with sequences of defined lengths and overlapping segments are bioinformatically designed using naturally occurring proteins and cloned into phage genomes to be displayed on the surface. These libraries are used in immunoprecipitation experiments of circulating antibodies. This can be done with parallel samples from multiple sources, and the DNA inserts from the bound phages are barcoded and subjected to next-generation sequencing for hit determination. PhIP-seq is a powerful technique for characterizing the antibody reactome that has undergone rapid advances in recent years. In this review, we comprehensively describe the history of PhIP-seq and discuss recent advances in library design and applications.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Imunoprecipitação , Humanos , Imunoprecipitação/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anticorpos , Biblioteca de Peptídeos , Animais , Bacteriófagos/genética
2.
Vet Res ; 55(1): 131, 2024 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-39375775

RESUMO

African swine fever (ASF) is a devastating disease caused by African swine fever virus (ASFV) and leads to significant economic losses in the pig farming industry. Given the absence of an effective vaccine or treatment, the mortality rate of ASF is alarmingly close to 100%. Consequently, the ability to rapidly and accurately detect ASFV on site and promptly identify infected pigs is critical for controlling the spread of this pandemic. The dynamics of the ASF virus load and antibody response necessitate the adoption of various detection strategies at different stages of infection, a topic that has received limited attention to date. This review offers detailed guidance for choosing appropriate ASF diagnostic techniques tailored to the clinical manifestations observed from the acute to chronic phases, including asymptomatic cases. We comprehensively summarize and evaluate the latest advancements in ASFV detection methods, such as CRISPR-based diagnostics, biosensors, and microfluidics. Additionally, we address the challenges of false negatives or positives due to ASF variants or the use of injected live attenuated vaccines. This review provides an exhaustive list of diagnostic tests suitable for detecting each stage of symptoms and potential target genes for developing new detection methods. In conclusion, we highlight the current challenges and future directions in ASFV detection, underscoring the need for continued research and innovation in this field.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Febre Suína Africana/diagnóstico , Febre Suína Africana/virologia , Animais , Suínos , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/fisiologia
3.
Cell ; 136(6): 1073-84, 2009 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-19303850

RESUMO

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) conduct many critical functions through nonhistone substrates in metazoans, but only chromatin-associated nonhistone substrates are known in Saccharomyces cerevisiae. Using yeast proteome microarrays, we identified and validated many nonchromatin substrates of the essential nucleosome acetyltransferase of H4 (NuA4) complex. Among these, acetylation sites (Lys19 and 514) of phosphoenolpyruvate carboxykinase (Pck1p) were determined by tandem mass spectrometry. Acetylation at Lys514 was crucial for enzymatic activity and the ability of yeast cells to grow on nonfermentable carbon sources. Furthermore, Sir2p deacetylated Pck1p both in vitro and in vivo. Loss of Pck1p activity blocked the extension of yeast chronological life span caused by water starvation. In human hepatocellular carcinoma (HepG2) cells, human Pck1 acetylation and glucose production were dependent on TIP60, the human homolog of ESA1. Our findings demonstrate a regulatory function for the NuA4 complex in glucose metabolism and life span by acetylating a critical metabolic enzyme.


Assuntos
Gluconeogênese , Histona Acetiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Histona Acetiltransferases/genética , Histona Desacetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisina Acetiltransferase 5 , Complexos Multiproteicos/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Análise Serial de Proteínas , Sirtuínas/metabolismo , Água/metabolismo
4.
Acta Biochim Biophys Sin (Shanghai) ; 56(8): 1145-1155, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39099413

RESUMO

Glycosylation, a crucial posttranslational modification, plays a significant role in numerous physiological and pathological processes. Lectin microarrays, which leverage the high specificity of lectins for sugar binding, are ideally suited for profiling the glycan spectra of diverse and complex biological samples. In this review, we explore the evolution of lectin detection technologies, as well as the applications and challenges of lectin microarrays in analyzing the glycome profiles of various clinical samples, including serum, saliva, tissues, sperm, and urine. This review not only emphasizes significant advancements in the high-throughput analysis of polysaccharides but also provides insight into the potential of lectin microarrays for diagnosing and managing diseases such as tumors, autoimmune diseases, and chronic inflammation. We aim to provide a clear, concise, and comprehensive overview of the use of lectin microarrays in clinical settings, thereby assisting researchers in conducting clinical studies in glycobiology.


Assuntos
Glicômica , Lectinas , Polissacarídeos , Humanos , Lectinas/metabolismo , Lectinas/química , Polissacarídeos/metabolismo , Polissacarídeos/análise , Glicômica/métodos , Análise em Microsséries/métodos , Glicosilação , Neoplasias/metabolismo , Neoplasias/diagnóstico , Doenças Autoimunes/metabolismo , Doenças Autoimunes/diagnóstico
5.
Acta Biochim Biophys Sin (Shanghai) ; 56(8): 1118-1129, 2024 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-39066577

RESUMO

Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus's entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.


Assuntos
SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Glicosilação , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células HEK293 , SARS-CoV-2/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/genética , Polissacarídeos/metabolismo , Polissacarídeos/química , Polipeptídeo N-Acetilgalactosaminiltransferase , Simulação de Dinâmica Molecular , Glicosiltransferases/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Multimerização Proteica , COVID-19/virologia , COVID-19/metabolismo
6.
Proteomics ; 23(2): e2200306, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36205637

RESUMO

The majority of people in China have been immunized with the inactivated viral vaccine BBIBP-CorV. The emergence of the Omicron variant raised the concerns about protection efficacy of the inactivated viral vaccine in China. However, longitudinal neutralization data describing protection efficacy against Omicron variant is still lacking. Here we present one-year longitudinal neutralization data of BBIBP-CorV on authentic Omicron, Delta, and wild-type strains using 224 sera collected from 14 volunteers who have finished three doses BBIBP-CorV. The sera were also subjected for monitoring the SARS-CoV-2 specific IgG, IgA, and IgM responses on protein and peptide microarrays. The neutralization titers showed different protection efficacies against the three strains. By incorporating IgG and IgA signals of proteins and Spike protein derived peptide on microarray, panels as potential surrogate biomarkers for rapid estimation of neutralization titers were established. These data support the necessity of the 3rd dose of BBIBP-CorV vaccination. After further validation and assay development, the panels could be used for reliable, convenient and fast evaluation of the efficacy of vaccination.


Assuntos
COVID-19 , Humanos , COVID-19/prevenção & controle , SARS-CoV-2 , Vacinas contra COVID-19 , Imunoglobulina G , Vacinação , Imunoglobulina A , Anticorpos Antivirais
7.
EMBO J ; 38(18): e100948, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31418899

RESUMO

As a ubiquitous bacterial secondary messenger, c-di-GMP plays key regulatory roles in processes such as bacterial motility and transcription regulation. CobB is the Sir2 family protein deacetylase that controls energy metabolism, chemotaxis, and DNA supercoiling in many bacteria. Using an Escherichia coli proteome microarray, we found that c-di-GMP strongly binds to CobB. Further, protein deacetylation assays showed that c-di-GMP inhibits the activity of CobB and thereby modulates the biogenesis of acetyl-CoA. Interestingly, we also found that one of the key enzymes directly involved in c-di-GMP production, DgcZ, is a substrate of CobB. Deacetylation of DgcZ by CobB enhances its activity and thus the production of c-di-GMP. Our work establishes a novel negative feedback loop linking c-di-GMP biogenesis and CobB-mediated protein deacetylation.


Assuntos
GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Sirtuínas/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , GMP Cíclico/metabolismo , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Análise Serial de Proteínas/métodos , Proteômica/métodos , Sistemas do Segundo Mensageiro
8.
Mol Cell Proteomics ; 20: 100059, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33109704

RESUMO

Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next-generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and potentially conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the binding capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.


Assuntos
COVID-19/imunologia , Mapeamento de Epitopos/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos/metabolismo , Proteínas de Escherichia coli/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Soros Imunes/sangue , Soros Imunes/imunologia , Biblioteca de Peptídeos
9.
Acta Biochim Biophys Sin (Shanghai) ; 55(10): 1539-1550, 2023 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-37528660

RESUMO

CRISPR-based detection technologies have been widely explored for molecular diagnostics. However, the challenge lies in converting the signal of different biomolecules, such as nucleic acids, proteins, small molecules, exosomes, and ions, into a CRISPR-based nucleic acid detection signal. Understanding the detection of different biomolecules using CRISPR technology can aid in the development of practical and promising detection approaches. Unfortunately, existing reviews rarely provide an overview of CRISPR-based molecular diagnostics from the perspective of different biomolecules. Herein, we first introduce the principles and characteristics of various CRISPR nucleases for molecular diagnostics. Then, we focus on summarizing and evaluating the latest advancements in CRISPR-based detection of different biomolecules. Through a comparison of different methods of amplification and signal readout, we discuss how general detection methods can be integrated with CRISPR. Finally, we conclude by identifying opportunities for the improvement of CRISPR in quantitative, amplification-free, multiplex, all-in-one, and point-of-care testing (POCT) purposes.


Assuntos
Exossomos , Ácidos Nucleicos , Exossomos/genética , Ácidos Nucleicos/genética , Endonucleases , Sistemas CRISPR-Cas/genética
10.
Proc Natl Acad Sci U S A ; 117(25): 14395-14404, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513696

RESUMO

Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all-trans retinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5'-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I-binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 (TRIM25) messenger RNA (mRNA). TRIM25 encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I-mediated antiviral signaling. We show that RIG-I could bind TRIM25 mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability of TRIM25 transcripts. RIG-I could increase the transcriptional expression of TRIM25 by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I-induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation.


Assuntos
Diferenciação Celular , Citocinas/metabolismo , Proteína DEAD-box 58/metabolismo , Granulócitos/fisiologia , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Linhagem Celular Tumoral , Citocinas/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Receptores Imunológicos , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/genética , Regulação para Cima
11.
Acta Biochim Biophys Sin (Shanghai) ; 54(10): 1453-1463, 2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36239351

RESUMO

Type 2 diabetes mellitus (T2DM) is recognized as a serious public health concern with increasing incidence. The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin has been used for the treatment of T2DM worldwide. Although sitagliptin has excellent therapeutic outcome, adverse effects are observed. In addition, previous studies have suggested that sitagliptin may have pleiotropic effects other than treating T2DM. These pieces of evidence point to the importance of further investigation of the molecular mechanisms of sitagliptin, starting from the identification of sitagliptin-binding proteins. In this study, by combining affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), we discover seven high-confidence targets that can interact with sitagliptin. Surface plasmon resonance (SPR) assay confirms the binding of sitagliptin to three proteins, i. e., LYPLAL1, TCP1, and CCAR2, with binding affinities (K D) ranging from 50.1 µM to 1490 µM. Molecular docking followed by molecular dynamic (MD) simulation reveals hydrogen binding between sitagliptin and the catalytic triad of LYPLAL1, and also between sitagliptin and the P-loop of ATP-binding pocket of TCP1. Molecular mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis indicates that sitagliptin can stably bind to LYPLAL1 and TCP1 in active sites, which may have an impact on the functions of these proteins. SPR analysis validates the binding affinity of sitagliptin to TCP1 mutant D88A is ~10 times lower than that to the wild-type TCP1. Our findings provide insights into the sitagliptin-targets interplay and demonstrate the potential of sitagliptin in regulating gluconeogenesis and in anti-tumor drug development.


Assuntos
Diabetes Mellitus Tipo 2 , Inibidores da Dipeptidil Peptidase IV , Fosfato de Sitagliptina , Humanos , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Diabetes Mellitus Tipo 2/induzido quimicamente , Inibidores da Dipeptidil Peptidase IV/farmacologia , Hipoglicemiantes/farmacologia , Simulação de Acoplamento Molecular , Fosfato de Sitagliptina/farmacologia
12.
J Proteome Res ; 20(12): 5241-5263, 2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34672606

RESUMO

The study of proteins circulating in blood offers tremendous opportunities to diagnose, stratify, or possibly prevent diseases. With recent technological advances and the urgent need to understand the effects of COVID-19, the proteomic analysis of blood-derived serum and plasma has become even more important for studying human biology and pathophysiology. Here we provide views and perspectives about technological developments and possible clinical applications that use mass-spectrometry(MS)- or affinity-based methods. We discuss examples where plasma proteomics contributed valuable insights into SARS-CoV-2 infections, aging, and hemostasis and the opportunities offered by combining proteomics with genetic data. As a contribution to the Human Proteome Organization (HUPO) Human Plasma Proteome Project (HPPP), we present the Human Plasma PeptideAtlas build 2021-07 that comprises 4395 canonical and 1482 additional nonredundant human proteins detected in 240 MS-based experiments. In addition, we report the new Human Extracellular Vesicle PeptideAtlas 2021-06, which comprises five studies and 2757 canonical proteins detected in extracellular vesicles circulating in blood, of which 74% (2047) are in common with the plasma PeptideAtlas. Our overview summarizes the recent advances, impactful applications, and ongoing challenges for translating plasma proteomics into utility for precision medicine.


Assuntos
Proteoma , Proteômica/tendências , Envelhecimento/genética , COVID-19/genética , Bases de Dados de Proteínas , Hemostasia/genética , Humanos , Espectrometria de Massas , Proteoma/genética
13.
Allergy ; 76(2): 551-561, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33040337

RESUMO

BACKGROUND: The missing asymptomatic COVID-19 infections have been overlooked because of the imperfect sensitivity of the nucleic acid testing (NAT). Globally understanding the humoral immunity in asymptomatic carriers will provide scientific knowledge for developing serological tests, improving early identification, and implementing more rational control strategies against the pandemic. MEASURE: Utilizing both NAT and commercial kits for serum IgM and IgG antibodies, we extensively screened 11 766 epidemiologically suspected individuals on enrollment and 63 asymptomatic individuals were detected and recruited. Sixty-three healthy individuals and 51 mild patients without any preexisting conditions were set as controls. Serum IgM and IgG profiles were further probed using a SARS-CoV-2 proteome microarray, and neutralizing antibody was detected by a pseudotyped virus neutralization assay system. The dynamics of antibodies were analyzed with exposure time or symptoms onset. RESULTS: A combination test of NAT and serological testing for IgM antibody discovered 55.5% of the total of 63 asymptomatic infections, which significantly raises the detection sensitivity when compared with the NAT alone (19%). Serum proteome microarray analysis demonstrated that asymptomatics mainly produced IgM and IgG antibodies against S1 and N proteins out of 20 proteins of SARS-CoV-2. Different from strong and persistent N-specific antibodies, S1-specific IgM responses, which evolved in asymptomatic individuals as early as the seventh day after exposure, peaked on days from 17 days to 25 days, and then disappeared in two months, might be used as an early diagnostic biomarker. 11.8% (6/51) mild patients and 38.1% (24/63) asymptomatic individuals did not produce neutralizing antibody. In particular, neutralizing antibody in asymptomatics gradually vanished in two months. CONCLUSION: Our findings might have important implications for the definition of asymptomatic COVID-19 infections, diagnosis, serological survey, public health, and immunization strategies.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Portador Sadio/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/sangue , COVID-19/diagnóstico , Teste para COVID-19/métodos , Portador Sadio/sangue , Portador Sadio/diagnóstico , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade
14.
Mol Cell Proteomics ; 18(9): 1851-1863, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31308251

RESUMO

Systemic lupus erythematosus (SLE) is one of the most serious autoimmune diseases, characterized by highly diverse clinical manifestations. A biomarker is still needed for accurate diagnostics. SLE serum autoantibodies were discovered and validated using serum samples from independent sample cohorts encompassing 306 participants divided into three groups, i.e. healthy, SLE patients, and other autoimmune-related diseases. To discover biomarkers for SLE, a phage displayed random peptide library (Ph.D. 12) and deep sequencing were applied to screen specific autoantibodies in a total of 100 serum samples from 50 SLE patients and 50 healthy controls. A statistical analysis protocol was set up for the identification of peptides as potential biomarkers. For validation, 10 peptides were analyzed using enzyme-linked immunosorbent assays (ELISA). As a result, four peptides (SLE2018Val001, SLE2018Val002, SLE2018Val006, and SLE2018Val008) were discovered with high diagnostic power to differentiate SLE patients from healthy controls. Among them, two peptides, i.e. SLE2018Val001 and SLE2018Val002, were confirmed between SLE with other autoimmune patients. The procedure we established could be easily adopted for the identification of autoantibodies as biomarkers for many other diseases.


Assuntos
Lúpus Eritematoso Sistêmico/sangue , Biblioteca de Peptídeos , Peptídeos/sangue , Adulto , Área Sob a Curva , Doenças Autoimunes/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/genética , Reprodutibilidade dos Testes
15.
Acta Biochim Biophys Sin (Shanghai) ; 53(4): 389-399, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33537750

RESUMO

Display technology, especially phage display technology, has been widely applied in many fields. The theoretical core of display technology is the physical linkage between the protein/peptide on the surface of a phage and the coding DNA sequence inside the same phage. Starting from phage-displayed peptide/protein/antibody libraries and taking advantage of the ever-growing power of next-generation sequencing (NGS) for DNA sequencing/decoding, rich protein-related information can easily be obtained in a high-throughput way. Based on this information, many scientific and clinical questions can be readily addressed. In the past few years, aided by the development of NGS, droplet technology, and massive oligonucleotide synthesis, we have witnessed and continue to witness large advances of phage display technology, in both technology development and application. The aim of this review is to summarize and discuss these recent advances.


Assuntos
Ácidos Nucleicos/química , Biblioteca de Peptídeos
16.
Acta Biochim Biophys Sin (Shanghai) ; 53(9): 1134-1141, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34159380

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health threat since December 2019, and there is still no highly effective drug to control the pandemic. To facilitate drug target identification for drug development, studies on molecular mechanisms, such as SARS-CoV-2 protein interactions, are urgently needed. In this study, we focused on Nsp2, a non-structural protein with largely unknown function and mechanism. The interactome of Nsp2 was revealed through the combination of affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), and 84 proteins of high-confidence were identified. Gene ontology analysis demonstrated that Nsp2-interacting proteins are involved in several biological processes such as endosome transport and translation. Network analysis generated two clusters, including ribosome assembly and vesicular transport. Bio-layer interferometry (BLI) assay confirmed the bindings between Nsp2- and 4-interacting proteins, i.e. STAU2 (Staufen2), HNRNPLL, ATP6V1B2, and RAP1GDS1 (SmgGDS), which were randomly selected from the list of 84 proteins. Our findings provide insights into the Nsp2-host interplay and indicate that Nsp2 may play important roles in SARS-CoV-2 infection and serve as a potential drug target for anti-SARS-CoV-2 drug development.


Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2/química , Proteínas não Estruturais Virais/química , Sistemas de Liberação de Medicamentos , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas não Estruturais Virais/metabolismo
17.
Acta Biochim Biophys Sin (Shanghai) ; 53(5): 628-635, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33637989

RESUMO

PD-1 plays an important role as an immune checkpoint. Sintilimab is a newly approved PD-1 antibody for cancer immunotherapy with an unknown binding epitope on PD-1. In this study, to elucidate the molecular mechanism by which sintilimab blocks PD-1 activation, we applied Antibody binding epitope Mapping (AbMap) to identify the binding epitope of sintilimab. An epitope was successfully identified, i.e. SLAPKA, aa 127-132. By constructing a series of point mutations, the dominant residues S127, L128, A129, P130, and A132 of PD-1 were further validated by western blot analysis, biolayer interferometry, and flow cytometry. Structural analysis showed that the epitope is partially within the binding interface of PD-1 and PD-L1, and this epitope also partially overlaps with that of nivolumab and pembrolizumab. These results demonstrate that sintilimab can attenuate PD-1 activation by directly competing with the interaction between PD-1 and PD-L1 through binding with the key residues of the FG loop on PD-1. This study also demonstrates the high efficiency and accuracy of AbMap for determining the binding epitope of therapeutic antibodies.


Assuntos
Anticorpos Monoclonais Humanizados/química , Antineoplásicos Imunológicos/química , Mapeamento de Epitopos , Epitopos/química , Receptor de Morte Celular Programada 1/química , Anticorpos Monoclonais Humanizados/imunologia , Antineoplásicos Imunológicos/imunologia , Epitopos/imunologia , Humanos , Receptor de Morte Celular Programada 1/imunologia
18.
Mol Cell Proteomics ; 17(9): 1720-1736, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29871872

RESUMO

Proteins, as the major executer for cell progresses and functions, its abundance and the level of post-translational modifications, are tightly monitored by regulators. Genetic perturbation could help us to understand the relationships between genes and protein functions. Herein, to explore the impact of the genome-wide interruption on certain protein, we developed a cell lysate microarray on kilo-conditions (CLICK) with 4837 knockout (YKO) and 322 temperature-sensitive (ts) mutant strains of yeast (Saccharomyces cerevisiae). Taking histone marks as examples, a general workflow was established for the global identification of upstream regulators. Through a single CLICK array test, we obtained a series of regulators for H3K4me3, which covers most of the known regulators in S. cerevisiae We also noted that several group of proteins are involved in negatively regulation of H3K4me3. Further, we discovered that Cab4p and Cab5p, two key enzymes of CoA biosynthesis, play central roles in histone acylation. Because of its general applicability, CLICK array could be easily adopted to rapid and global identification of upstream protein/enzyme(s) that regulate/modify the level of a protein or the posttranslational modification of a non-histone protein.


Assuntos
Redes Reguladoras de Genes , Código das Histonas/genética , Saccharomyces cerevisiae/genética , Acil Coenzima A/metabolismo , Acilação , Química Click , Histonas/metabolismo , Lisina/metabolismo , Metilação , Modelos Biológicos , Mutação/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico
19.
Expert Rev Proteomics ; 16(10): 815-827, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31469014

RESUMO

Introduction: Protein microarray is a powerful tool for both biological study and clinical research. The most useful features of protein microarrays are their miniaturized size (low reagent and sample consumption), high sensitivity and their capability for parallel/high-throughput analysis. The major focus of this review is functional proteome microarray. Areas covered: For proteome microarray, this review will discuss some recently constructed proteome microarrays and new concepts that have been used for constructing proteome microarrays and data interpretation in past few years, such as PAGES, M-NAPPA strategy, VirD technology, and the first protein microarray database. this review will summarize recent proteomic scale applications and address the limitations and future directions of proteome microarray technology. Expert opinion: Proteome microarray is a powerful tool for basic biological and clinical research. It is expected to see improvements in the currently used proteome microarrays and the construction of more proteome microarrays for other species by using traditional strategies or novel concepts. It is anticipated that the maximum number of features on a single microarray and the number of possible applications will be increased, and the information that can be obtained from proteome microarray experiments will more in-depth in the future.


Assuntos
Ensaios de Triagem em Larga Escala/tendências , Análise Serial de Proteínas/tendências , Proteoma/genética , Proteômica , Bases de Dados de Proteínas , Humanos
20.
Mol Cell Proteomics ; 16(2): 288-299, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27965274

RESUMO

The high genetic variability of RNA viruses is a significant factor limiting the discovery of effective biomarkers, the development of vaccines, and characterizations of the immune response during infection. Protein microarrays have been shown to be a powerful method in biomarker discovery and the identification of novel protein-protein interaction networks, suggesting that this technique could also be very useful in studies of infectious RNA viruses. However, to date, the amount of genetic material required to produce protein arrays, as well as the time- and labor-intensive procedures typically needed, have limited their more widespread application. Here, we introduce a method, protein microarray fabrication through gene synthesis (PAGES), for the rapid and efficient construction of protein microarrays particularly for RNA viruses. Using dengue virus as an example, we first identify consensus sequences from 3,604 different strains and then fabricate complete proteomic microarrays that are unique for each consensus sequence. To demonstrate their applicability, we show that these microarrays can differentiate sera from patients infected by dengue virus, related pathogens, or from uninfected patients. We anticipate that the microarray and expression library constructed in this study will find immediate use in further studies of dengue virus and that, more generally, PAGES will become a widely applied method in the clinical characterization of RNA viruses.


Assuntos
Vírus da Dengue/metabolismo , Dengue/virologia , Análise Serial de Proteínas/métodos , Proteínas Virais/análise , Proteínas Virais/sangue , Adulto , Idoso , Dengue/sangue , Vírus da Dengue/genética , Feminino , Biblioteca Gênica , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Mapas de Interação de Proteínas , Proteômica/métodos , Proteínas Virais/genética , Adulto Jovem
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