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1.
Nat Commun ; 12(1): 1703, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731717

RESUMO

The factors regulating cellular identity are critical for understanding the transition from health to disease and responses to therapies. Recent literature suggests that autophagy compromise may cause opposite effects in different contexts by either activating or inhibiting YAP/TAZ co-transcriptional regulators of the Hippo pathway via unrelated mechanisms. Here, we confirm that autophagy perturbation in different cell types can cause opposite responses in growth-promoting oncogenic YAP/TAZ transcriptional signalling. These apparently contradictory responses can be resolved by a feedback loop where autophagy negatively regulates the levels of α-catenins, LC3-interacting proteins that inhibit YAP/TAZ, which, in turn, positively regulate autophagy. High basal levels of α-catenins enable autophagy induction to positively regulate YAP/TAZ, while low α-catenins cause YAP/TAZ activation upon autophagy inhibition. These data reveal how feedback loops enable post-transcriptional determination of cell identity and how levels of a single intermediary protein can dictate the direction of response to external or internal perturbations.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , alfa Catenina/metabolismo , Animais , Células Cultivadas , Células Epiteliais , Retroalimentação Fisiológica , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Transdução de Sinais , alfa Catenina/química , alfa Catenina/genética
2.
Int J Mol Sci ; 22(4)2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567580

RESUMO

SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually in part due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor, several in silico methods and numerous experimental data reported recently to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both the UK and South African strains) should be favorable for the interaction with ACE2, while the K417N and E484K substitutions (South African strain) would seem neutral or even unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the K417N and E484K Spike replacements with regard to ACE2 binding. Our finding suggests that the UK strain should have higher affinity toward ACE2 and therefore likely increased transmissibility and possibly pathogenicity. If indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions located outside the direct Spike-ACE2 interface but not so much to the K417N and E484K replacements. Yet, it should be noted that amino acid changes at Spike position 484 can lead to viral escape from neutralizing antibodies. Further, these amino acid substitutions do not seem to induce major structural changes in this region of the Spike protein. This structure-function study allows us to rationalize some observations made for the UK strain but raises questions for the South African strain.


Assuntos
Substituição de Aminoácidos , /virologia , Simulação por Computador , Domínios e Motivos de Interação entre Proteínas/genética , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , /química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Sítios de Ligação , Humanos , Ligação Proteica , Receptores Virais/química , /metabolismo , África do Sul/epidemiologia , Glicoproteína da Espícula de Coronavírus/química , Reino Unido/epidemiologia
3.
PLoS One ; 16(2): e0246901, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33596252

RESUMO

The MERS-CoV, SARS-CoV, and SARS-CoV-2 are highly pathogenic viruses that can cause severe pneumonic diseases in humans. Unfortunately, there is a non-available effective treatment to combat these viruses. Domain-motif interactions (DMIs) are an essential means by which viruses mimic and hijack the biological processes of host cells. To disentangle how viruses achieve this process can help to develop new rational therapies. Data mining was performed to obtain DMIs stored as regular expressions (regexp) in 3DID and ELM databases. The mined regexp information was mapped on the coronaviruses' proteomes. Most motifs on viral protein that could interact with human proteins are shared across the coronavirus species, indicating that molecular mimicry is a common strategy for coronavirus infection. Enrichment ontology analysis for protein domains showed a shared biological process and molecular function terms related to carbon source utilization and potassium channel regulation. Some of the mapped motifs were nested on B, and T cell epitopes, suggesting that it could be as an alternative way for reverse vaccinology. The information obtained in this study could be used for further theoretic and experimental explorations on coronavirus infection mechanism and development of medicines for treatment.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Mimetismo Molecular/fisiologia , Domínios e Motivos de Interação entre Proteínas/imunologia , Betacoronavirus/genética , /virologia , Infecções por Coronavirus/genética , Bases de Dados Genéticas , Interações Hospedeiro-Patógeno , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas/genética , Proteoma , Vírus da SARS/genética , Vírus da SARS/metabolismo , /metabolismo , Proteínas Virais/metabolismo
4.
Biochem J ; 478(2): 357-375, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33394033

RESUMO

Multiple starvation-induced, high-affinity nutrient transporters in yeast function as receptors for activation of the protein kinase A (PKA) pathway upon re-addition of their substrate. We now show that these transceptors may play more extended roles in nutrient regulation. The Gap1 amino acid, Mep2 ammonium, Pho84 phosphate and Sul1 sulfate transceptors physically interact in vitro and in vivo with the PKA-related Sch9 protein kinase, the yeast homolog of mammalian S6 protein kinase and protein kinase B. Sch9 is a phosphorylation target of TOR and well known to affect nutrient-controlled cellular processes, such as growth rate. Mapping with peptide microarrays suggests specific interaction domains in Gap1 for Sch9 binding. Mutagenesis of the major domain affects the upstart of growth upon the addition of L-citrulline to nitrogen-starved cells to different extents but apparently does not affect in vitro binding. It also does not correlate with the drop in L-citrulline uptake capacity or transceptor activation of the PKA target trehalase by the Gap1 mutant forms. Our results reveal a nutrient transceptor-Sch9-TOR axis in which Sch9 accessibility for phosphorylation by TOR may be affected by nutrient transceptor-Sch9 interaction under conditions of nutrient starvation or other environmental challenges.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sítios de Ligação , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Citrulina/metabolismo , Mutação , Domínios e Motivos de Interação entre Proteínas/genética , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/genética , Simportadores de Próton-Fosfato/genética , Simportadores de Próton-Fosfato/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética
5.
Sci Signal ; 14(665)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436497

RESUMO

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the µ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin ß3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.


Assuntos
/virologia , Interações entre Hospedeiro e Microrganismos/fisiologia , /patogenicidade , Internalização do Vírus , Sequência de Aminoácidos , /genética , Animais , Sequência Conservada , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Integrinas/química , Integrinas/genética , Integrinas/fisiologia , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/fisiologia , Modelos Biológicos , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/fisiologia
6.
PLoS Biol ; 18(9): e3000821, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886672

RESUMO

As a novel alternative to established surface display or combinatorial chemistry approaches for the discovery of therapeutic peptides, we present a method for the isolation of small, cysteine-rich domains from bovine antibody ultralong complementarity-determining regions (CDRs). We show for the first time that isolated bovine antibody knob domains can function as autonomous entities by binding antigen outside the confines of the antibody scaffold. This yields antibody fragments so small as to be considered peptides, each stabilised by an intricate, bespoke arrangement of disulphide bonds. For drug discovery, cow immunisations harness the immune system to generate knob domains with affinities in the picomolar to low nanomolar range, orders of magnitude higher than unoptimized peptides from naïve library screening. Using this approach, knob domain peptides that tightly bound Complement component C5 were obtained, at scale, using conventional antibody discovery and peptide purification techniques.


Assuntos
Anticorpos/química , Dissulfetos/isolamento & purificação , Domínios de Imunoglobulina , Fragmentos de Peptídeos/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Linfócitos B/fisiologia , Bovinos , Complemento C5/química , Complemento C5/genética , Complemento C5/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Dissulfetos/química , Dissulfetos/imunologia , Mapeamento de Epitopos/métodos , Humanos , Imunização , Domínios de Imunoglobulina/genética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas/genética
7.
J Theor Biol ; 505: 110425, 2020 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-32735992

RESUMO

The interaction between the angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a pivotal role in virus entry into the host cells. Since recombinant ACE2 protein has been suggested as an anti-SARS-CoV-2 therapeutic agent, this study was conducted to design an ACE2 protein with more desirable properties. In this regard, the amino acids with central roles in enzymatic activity of the ACE2 were substituted. Moreover, saturation mutagenesis at the interaction interface between the ACE2 and RBD was performed to increase their interaction affinity. The best mutations to increase the structural and thermal stability of the ACE2 were also selected based on B factors and mutation effects. The obtained resulted revealed that the Arg273Gln and Thr445Gly mutation have drastically reduced the binding affinity of the angiotensin-II into the active site of ACE2. The Thr27Arg mutation was determined to be the most potent mutation to increase the binding affinity. The Asp427Arg mutation was done to decrease the flexibility of the region with high B factor. The Pro451Met mutation along with the Gly448Trp mutation was predicted to increase the thermodynamic stability and thermostability of the ACE2. The designed therapeutic ACE2 would have no enzymatic activity while it could bear stronger interaction with Spike glycoprotein of the SARS-CoV-2. Moreover, decreased in vivo enzymatic degradation would be anticipated due to increased thermostability. This engineered ACE2 could be exploited as a novel therapeutic agent against COVID-19 after necessary evaluations.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Desenho de Fármacos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Engenharia de Proteínas/métodos , Substituição de Aminoácidos , Betacoronavirus/genética , Sítios de Ligação , Evolução Molecular Direcionada , Humanos , Pandemias , Peptidil Dipeptidase A/uso terapêutico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo
8.
J Transl Med ; 18(1): 321, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32831104

RESUMO

BACKGROUND: The outbreak of coronavirus disease (COVID-19) was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through its surface spike glycoprotein (S-protein) recognition on the receptor Angiotensin-converting enzyme 2 (ACE2) in humans. However, it remains unclear how genetic variations in ACE2 may affect its function and structure, and consequently alter the recognition by SARS-CoV-2. METHODS: We have systemically characterized missense variants in the gene ACE2 using data from the Genome Aggregation Database (gnomAD; N = 141,456). To investigate the putative deleterious role of missense variants, six existing functional prediction tools were applied to evaluate their impact. We further analyzed the structural flexibility of ACE2 and its protein-protein interface with the S-protein of SARS-CoV-2 using our developed Legion Interfaces Analysis (LiAn) program. RESULTS: Here, we characterized a total of 12 ACE2 putative deleterious missense variants. Of those 12 variants, we further showed that p.His378Arg could directly weaken the binding of catalytic metal atom to decrease ACE2 activity and p.Ser19Pro could distort the most important helix to the S-protein. Another seven missense variants may affect secondary structures (i.e. p.Gly211Arg; p.Asp206Gly; p.Arg219Cys; p.Arg219His, p.Lys341Arg, p.Ile468Val, and p.Ser547Cys), whereas p.Ile468Val with AF = 0.01 is only present in Asian. CONCLUSIONS: We provide strong evidence of putative deleterious missense variants in ACE2 that are present in specific populations, which could disrupt the function and structure of ACE2. These findings provide novel insight into the genetic variation in ACE2 which may affect the SARS-CoV-2 recognition and infection, and COVID-19 susceptibility and treatment.


Assuntos
Betacoronavirus/fisiologia , Mutação de Sentido Incorreto , Peptidil Dipeptidase A/genética , Domínios e Motivos de Interação entre Proteínas/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Betacoronavirus/metabolismo , Sítios de Ligação/genética , Infecções por Coronavirus/etnologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Análise Mutacional de DNA/métodos , Bases de Dados Genéticas , Predisposição Genética para Doença/etnologia , Variação Genética , Geografia , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/etnologia , Pneumonia Viral/genética , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Estrutura Secundária de Proteína/genética , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus
9.
Proc Natl Acad Sci U S A ; 117(32): 19435-19445, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719131

RESUMO

The Ras/RAF/MEK/ERK pathway is an essential signaling cascade for various refractory cancers, such as those with mutant KRAS (mKRAS) and BRAF (mBRAF). However, there are unsolved ambiguities underlying mechanisms for this growth signaling thereby creating therapeutic complications. This study shows that a vital component of the pathway CRAF is directly impacted by an end product of the cascade, glutathione transferases (GST) P1 (GSTP1), driving a previously unrecognized autocrine cycle that sustains proliferation of mKRAS and mBRAF cancer cells, independent of oncogenic stimuli. The CRAF interaction with GSTP1 occurs at its N-terminal regulatory domain, CR1 motif, resulting in its stabilization, enhanced dimerization, and augmented catalytic activity. Consistent with the autocrine cycle scheme, silencing GSTP1 brought about significant suppression of proliferation of mKRAS and mBRAF cells in vitro and suppressed tumorigenesis of the xenografted mKRAS tumor in vivo. GSTP1 knockout mice showed significantly impaired carcinogenesis of mKRAS colon cancer. Consequently, hindering the autocrine loop by targeting CRAF/GSTP1 interactions should provide innovative therapeutic modalities for these cancers.


Assuntos
Glutationa S-Transferase pi/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa S-Transferase pi/deficiência , Glutationa S-Transferase pi/genética , Humanos , Camundongos , Camundongos Knockout , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica , Estabilidade Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais
10.
Medicine (Baltimore) ; 99(27): e20759, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32629654

RESUMO

Sepsis is one of the leading causes of mortality in intensive care units (ICU). The growing incidence rate of sepsis and its high mortality rate result are very important sociosanitary problems. Sepsis is a result of infection which can cause systemic inflammatory and organ failure. But the pathogenesis and the molecular mechanisms of sepsis is still not well understood. The aim of the present study was to identify the candidate key genes in the progression of sepsis.Microarray datasets GSE28750, GSE64457, and GSE95233 were downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified, and function enrichment analyses were performed. The protein-protein interaction network (PPI) was constructed and the module analysis was performed using STRING and Cytoscape. Furthermore, to verify the results of the bioinformatics analyses, the expression levels of selected DEGs were quantified by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) in libobolysaccharide (LPS)-induced Human Umbilical Vein Endothelial Cells (HUVECs) to support the result of bioinformatics analysis.Thirteen hub genes were identified and biological process analysis revealed that these genes were mainly enriched in apoptotic process, inflammatory response, innate immune response. Hub genes with high degrees, including MAPK14, SLC2A3, STOM, and MMP8, were demonstrated to have an association with sepsis. Furthermore, RT-PCR results showed that SLC2A3 and MAPK14 were significantly upregulated in the HUVECs induced by LPS compared with controls.In conclusion, DEGs and hub genes identified in the present study help us understand the molecular mechanisms of sepsis, and provide candidate targets for diagnosis and treatment of sepsis.


Assuntos
Predisposição Genética para Doença/genética , Sepse/genética , Biologia Computacional , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Domínios e Motivos de Interação entre Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/etiologia , Transcriptoma
11.
Nucleic Acids Res ; 48(15): 8393-8407, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32619221

RESUMO

The glucocorticoid receptor is an important immunosuppressive drug target and metabolic regulator that acts as a ligand-gated transcription factor. Generally, GR's anti-inflammatory effects are attributed to the silencing of inflammatory genes, while its adverse effects are ascribed to the upregulation of metabolic targets. GR binding directly to DNA is proposed to activate, whereas GR tethering to pro-inflammatory transcription factors is thought to repress transcription. Using mice with a point mutation in GR's zinc finger, that still tether via protein-protein interactions while being unable to recognize DNA, we demonstrate that DNA binding is essential for both transcriptional activation and repression. Performing ChIP-Seq, RNA-Seq and proteomics under inflammatory conditions, we show that DNA recognition is required for the assembly of a functional co-regulator complex to mediate glucocorticoid responses. Our findings may contribute to the development of safer immunomodulators with fewer side effects.


Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Inflamação/genética , Receptores de Glucocorticoides/genética , Animais , DNA/metabolismo , Regulação da Expressão Gênica/genética , Glucocorticoides/genética , Glucocorticoides/metabolismo , Humanos , Inflamação/patologia , Camundongos , Domínios e Motivos de Interação entre Proteínas/genética , RNA-Seq , Ativação Transcricional/genética
12.
Nat Biotechnol ; 38(9): 1073-1078, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32704169

RESUMO

A robust serological test to detect neutralizing antibodies to SARS-CoV-2 is urgently needed to determine not only the infection rate, herd immunity and predicted humoral protection, but also vaccine efficacy during clinical trials and after large-scale vaccination. The current gold standard is the conventional virus neutralization test requiring live pathogen and a biosafety level 3 laboratory. Here, we report a SARS-CoV-2 surrogate virus neutralization test that detects total immunodominant neutralizing antibodies targeting the viral spike (S) protein receptor-binding domain in an isotype- and species-independent manner. Our simple and rapid test is based on antibody-mediated blockage of the interaction between the angiotensin-converting enzyme 2 (ACE2) receptor protein and the receptor-binding domain. The test, which has been validated with two cohorts of patients with COVID-19 in two different countries, achieves 99.93% specificity and 95-100% sensitivity, and differentiates antibody responses to several human coronaviruses. The surrogate virus neutralization test does not require biosafety level 3 containment, making it broadly accessible to the wider community for both research and clinical applications.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/genética , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos/imunologia , Anticorpos/farmacologia , Betacoronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Humanos , Testes de Neutralização , Pandemias , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Domínios e Motivos de Interação entre Proteínas/genética , Glicoproteína da Espícula de Coronavírus/química
13.
Leukemia ; 34(10): 2722-2735, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32576963

RESUMO

Mutations in genes encoding subunits of the SWI/SNF chromatin remodeling complex are frequently found in different human cancers. While the tumor suppressor function of this complex is widely established in solid tumors, its role in hematologic malignancies is largely unknown. Recurrent point mutations in BCL7A gene, encoding a subunit of the SWI/SNF complex, have been reported in diffuse large B-cell lymphoma (DLBCL), but their functional impact remains to be elucidated. Here we show that BCL7A often undergoes biallelic inactivation, including a previously unnoticed mutational hotspot in the splice donor site of intron one. The splice site mutations render a truncated BCL7A protein, lacking a portion of the amino-terminal domain. Moreover, restoration of wild-type BCL7A expression elicits a tumor suppressor-like phenotype in vitro and in vivo. In contrast, splice site mutations block the tumor suppressor function of BCL7A by preventing its binding to the SWI/SNF complex. We also show that BCL7A restoration induces transcriptomic changes in genes involved in B-cell activation. In addition, we report that SWI/SNF complex subunits harbor mutations in more than half of patients with germinal center B-cell (GCB)-DLBCL. Overall, this work demonstrates the tumor suppressor function of BCL7A in DLBCL, and highlights that the SWI/SNF complex plays a relevant role in DLBCL pathogenesis.


Assuntos
Genes Supressores de Tumor , Linfoma Difuso de Grandes Células B/genética , Proteínas dos Microfilamentos/genética , Mutação , Proteínas Oncogênicas/genética , Domínios e Motivos de Interação entre Proteínas/genética , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Cromatografia Líquida , Proteínas Cromossômicas não Histona/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Ativação Linfocitária/imunologia , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/terapia , Camundongos , Proteínas dos Microfilamentos/química , Imagem Molecular , Complexos Multiproteicos , Proteínas Oncogênicas/química , Ligação Proteica , Espectrometria de Massas em Tandem , Ensaios Antitumorais Modelo de Xenoenxerto
14.
J Immunol ; 205(4): 915-922, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32591393

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for millions of infections and hundreds of thousands of deaths globally. There are no widely available licensed therapeutics against SARS-CoV-2, highlighting an urgent need for effective interventions. The virus enters host cells through binding of a receptor-binding domain within its trimeric spike glycoprotein to human angiotensin-converting enzyme 2. In this article, we describe the generation and characterization of a panel of murine mAbs directed against the receptor-binding domain. One mAb, 2B04, neutralized wild-type SARS-CoV-2 in vitro with remarkable potency (half-maximal inhibitory concentration of <2 ng/ml). In a murine model of SARS-CoV-2 infection, 2B04 protected challenged animals from weight loss, reduced lung viral load, and blocked systemic dissemination. Thus, 2B04 is a promising candidate for an effective antiviral that can be used to prevent SARS-CoV-2 infection.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Mapeamento de Epitopos , Feminino , Células HEK293 , Humanos , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção , Células Vero
15.
Invest Ophthalmol Vis Sci ; 61(5): 41, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32446246

RESUMO

Purpose: To identify the pathogenic gene of infantile nystagmus syndrome (INS) in three Chinese families and explore the potential pathogenic mechanism of FERM domain-containing 7 (FRMD7) mutations. Methods: Genetic testing was performed via Sanger sequencing. Western blotting was used to analyze protein expression of FRMD7. Glutathione S-transferase pull-down and immunoprecipitation were conducted to investigate the proteins interacting with FRMD7. Rescue assays were performed in Caenorhabditis elegans to explore the potential role of FRMD7 in vivo. Results: We recruited three Chinese families with X-linked INS and identified a duplication and two missense mutations in FRMD7: c.998dupA/p.His333Glnfs*2, c.580G>A/p.Ala194Thr, and c.973A>G/p.Arg325Gly (one in each family). Expression levels of three mutants were similar to that of wild-type FRMD7 in vitro. Interestingly, the mutant p.His333Glnfs*2 exhibited a predominantly nuclear location, whereas wild-type FRMD7 localized to the cytoplasm. In addition, we found FRMD7 to directly interact with the loop between transmembrane domains 3 and 4 of GABRA2, a type A gamma-aminobutyric acid (GABA) receptor (GABAARs) subunit critical for receptor transport and localization, whereas the mutants p.Ala194Thr and p.Arg325Gly exhibited decreased binding to GABRA2. In frm-3 (a nematode homologue of FRMD7) null C. elegans, we found that FRMD7 mutants exhibited a poor rescue effect on the defects of locomotion and fluorescence recovery after photobleaching of GABAARs. Conclusions: Our findings identified three FRMD7 mutants in three Chinese families with X-linked INS and confirmed GABRA2 as a novel binding partner of FRMD7. These findings suggest that FRMD7 plays an important role by targeting GABAARs.


Assuntos
Proteínas do Citoesqueleto/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Proteínas de Membrana/genética , Mutação , Nistagmo Congênito/genética , Domínios e Motivos de Interação entre Proteínas/genética , Receptores de GABA-A/metabolismo , Animais , Grupo com Ancestrais do Continente Asiático/genética , Western Blotting , Células COS , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , China/epidemiologia , Chlorocebus aethiops , Proteínas do Citoesqueleto/metabolismo , Análise Mutacional de DNA , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Testes Genéticos , Humanos , Imunoprecipitação , Masculino , Proteínas de Membrana/metabolismo , Nistagmo Congênito/metabolismo , Linhagem , Plasmídeos/genética
16.
Genes Dev ; 34(13-14): 973-988, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32467224

RESUMO

Chromatin modifiers play critical roles in epidermal development, but the functions of histone deacetylases in this context are poorly understood. The class I HDAC, HDAC3, is of particular interest because it plays divergent roles in different tissues by partnering with tissue-specific transcription factors. We found that HDAC3 is expressed broadly in embryonic epidermis and is required for its orderly stepwise stratification. HDAC3 protein stability in vivo relies on NCoR and SMRT, which function redundantly in epidermal development. However, point mutations in the NCoR and SMRT deacetylase-activating domains, which are required for HDAC3's enzymatic function, permit normal stratification, indicating that HDAC3's roles in this context are largely independent of its histone deacetylase activity. HDAC3-bound sites are significantly enriched for predicted binding motifs for critical epidermal transcription factors including AP1, GRHL, and KLF family members. Our results suggest that among these, HDAC3 operates in conjunction with KLF4 to repress inappropriate expression of Tgm1, Krt16, and Aqp3 In parallel, HDAC3 suppresses expression of inflammatory cytokines through a Rela-dependent mechanism. These data identify HDAC3 as a hub coordinating multiple aspects of epidermal barrier acquisition.


Assuntos
Diferenciação Celular/genética , Células Epidérmicas/citologia , Epiderme/embriologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Animais , Embrião de Mamíferos , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais/genética , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
17.
Med Sci Sports Exerc ; 52(7): 1485-1494, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32168105

RESUMO

PURPOSE: To screen for candidate hub genes associated with the effects of exercise on melanoma tumor tissues and to review the potential signaling pathways involved in this process using bioinformatics analysis. METHODS: The GSE62628 expression profile was downloaded from Gene Expression Omnibus database. This data set contains 10 melanoma tumor tissues from two groups of exercise and nonexercise mice. The R software was utilized to identify differentially expressed genes between samples, and functional annotation and pathway analysis were performed. Results were visualized using Cytoscape software. RESULTS: In total, 315 differentially expressed genes were obtained, including 294 upregulated and 21 downregulated genes. The functional analysis showed that these genes were mainly enriched in immune response, inflammatory response, and positive regulation of the ERK1/2 cascade in biological process functional groups. The top 10 candidate hub genes were C3, Kng1, C3ar1, Ptafr, Fgg, Alb, Pf4, Orm1, Aldh3b1, and Apob. The pathway analysis of the most significant module identified from the protein-protein interaction network revealed that the complement and coagulation cascades, Staphylococcus aureus infection, cytokine-cytokine receptor interaction, chemokine signaling pathway and phagosome were mainly involved. C3, C3ar1, Kng1, Ptafr, and Fgg may be the critical genes in the complement and coagulation cascades pathway, and S. aureus in the infection pathway. CONCLUSIONS: Exercise may ameliorate the immune response and inflammatory response in melanoma tissue, and further studies exploring their relationships are warranted.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma Experimental/genética , Condicionamento Físico Animal/fisiologia , Transdução de Sinais , Animais , Coagulação Sanguínea/fisiologia , Quimiocinas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Biologia Computacional , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Camundongos , Análise em Microsséries , Domínios e Motivos de Interação entre Proteínas/genética , Mapas de Interação de Proteínas , Receptores de Citocinas/metabolismo , Infecções Estafilocócicas/metabolismo , Regulação para Cima
18.
Sci Rep ; 10(1): 5618, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221380

RESUMO

Many molecular system biology approaches recognize various interactions and functional associations of proteins that occur in cellular processing. Further understanding of the characterization technique reveals noteworthy information. These types of known and predicted interactions, gained through multiple resources, are thought to be important for experimental data to satisfy comprehensive and quality needs. The current work proposes the "WeiBI (WeiBiologicalInteractions)" database that clarifies direct and indirect partnerships associated with biological interactions. This database contains information concerning protein's functional partnerships and interactions along with their integration into a statistical model that can be computationally predicted for humans. This novel approach in WeiBI version 1.0 collects information using an improved algorithm by transferring interactions between more than 115570 entries, allowing statistical analysis with the automated background for the given inputs for functional enrichment. This approach also allows the input of an entity's list from a database along with the visualization of subsets as an interaction network and successful performance of the enrichment analysis for a gene set. This wisely improved algorithm is user-friendly, and its accessibility and higher accuracy make it the best database for exploring interactions among genomes' network and reflects the importance of this study. The proposed server "WeiBI" is accessible at http://weislab.com/WeiDOCK/?page=PKPD.


Assuntos
Genoma/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas/genética , Algoritmos , Bases de Dados de Proteínas , Humanos , Internet , Modelos Estatísticos , Mapeamento de Interação de Proteínas/métodos , Software , Interface Usuário-Computador
19.
PLoS One ; 15(2): e0228487, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32027716

RESUMO

Understanding how enzymes achieve their tremendous catalytic power is a major question in biochemistry. Greater understanding is also needed for enzyme engineering applications. In many cases, enzyme efficiency and specificity depend on residues not in direct contact with the substrate, termed remote residues. This work focuses on Escherichia coli ornithine transcarbamoylase (OTC), which plays a central role in amino acid metabolism. OTC has been reported to undergo an induced-fit conformational change upon binding its first substrate, carbamoyl phosphate (CP), and several residues important for activity have been identified. Using computational methods based on the computed chemical properties from theoretical titration curves, sequence-based scores derived from evolutionary history, and protein surface topology, residues important for catalytic activity were predicted. The roles of these residues in OTC activity were tested by constructing mutations at predicted positions, followed by steady-state kinetics assays and substrate binding studies with the variants. First-layer mutations R57A and D231A, second-layer mutation H272L, and third-layer mutation E299Q, result in 57- to 450-fold reductions in kcat/KM with respect to CP and 44- to 580-fold reductions with respect to ornithine. Second-layer mutations D140N and Y160S also reduce activity with respect to ornithine. Most variants had decreased stability relative to wild-type OTC, with variants H272L, H272N, and E299Q having the greatest decreases. Variants H272L, E299Q, and R57A also show compromised CP binding. In addition to direct effects on catalytic activity, effects on overall protein stability and substrate binding were observed that reveal the intricacies of how these residues contribute to catalysis.


Assuntos
Escherichia coli/enzimologia , Ornitina Carbamoiltransferase/química , Ornitina Carbamoiltransferase/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sequência de Bases , Sítios de Ligação , Carbamoil-Fosfato/química , Carbamoil-Fosfato/metabolismo , Catálise , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Ornitina/metabolismo , Ornitina Carbamoiltransferase/genética , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Especificidade por Substrato/genética
20.
BMC Cancer ; 20(1): 110, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041553

RESUMO

BACKGROUND: TP53 mutations occur in only about 3% of primary and 10-20% of relapse B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). However, alternative mechanisms may contribute to functionally impairing the p53 pathway in the absence of a mutation. Candidate mechanisms include overexpression of p53 mRNA variants encoding either dominant-negative p53 protein isoforms such as Delta40p53 and Delta133p53, or modulatory isoforms such as p53beta, which counteract the effects of Delta133p53 on replicative senescence in T-lymphocytes. METHODS: We used semi-quantitative reverse-transcriptase PCR (RT-PCR) and Western blot to investigate the expression of full length p53 (TAp53), Delta40p53, Delta133p53 or p53beta in diagnostic marrow from a clinical cohort of 50 BCP-ALL patients without TP53 mutation (29 males and 21 females, age range 2-14 years) and in the bone marrow cells of 4 healthy donors (used as controls). RESULTS: Irrespective of isoforms, levels of p53 mRNA were low in controls but were increased by 2 to 20-fold in primary or relapse BCP-ALL. TAp53 was increased in primary BCP-ALL, Delta40p53 was elevated in relapse BCP-ALL, whereas Delta133p53 and p53beta were increased in both. Next, mRNA levels were used as a basis to infer the ratio between protein isoform levels. This inference suggested that, in primary BCP-ALL, p53 was predominantly in active oligomeric conformations dominated by TAp53. In contrast, p53 mostly existed in inactive quaternary conformations containing ≥2 Delta40 or Delta133p53 in relapse BCP-ALL. Western blot analysis of blasts from BCP-ALL showed a complex pattern of N-terminally truncated p53 isoforms, whereas TAp53beta was detected as a major isoform. The hypothesis that p53 is in an active form in primary B-ALL was consistent with elevated level of p53 target genes CDKN1A and MDM2 in primary cases, whereas in relapse BCP-ALL, only CDKN1A was increased as compared to controls. CONCLUSION: Expression of p53 isoforms is deregulated in BCP-ALL in the absence of TP53 mutation, with increased expression of alternative isoforms in relapse BCP-ALL. Variations in isoform expression may contribute to functional deregulation of the p53 pathway in BCP-ALL, specifically contributing to its down-regulation in relapse forms.


Assuntos
Biomarcadores Tumorais , Regulação Neoplásica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Domínios e Motivos de Interação entre Proteínas/genética , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Mutação , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Isoformas de Proteínas/genética , Multimerização Proteica/genética , RNA Mensageiro , Recidiva , Proteína Supressora de Tumor p53/química
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