RESUMO
The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.
Assuntos
Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/química , Cristalografia , Metilação , Metiltransferases/química , Metiltransferases/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Análogos de Capuz de RNA/química , Análogos de Capuz de RNA/metabolismo , Capuzes de RNA/química , RNA Mensageiro/química , RNA Viral/química , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Síncrotrons , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
Serial synchrotron crystallography enables the study of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D-printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin Mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed using a high-performance computing pipeline. Here, the system was applied to two ß-lactamases: a class D serine ß-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-ß-lactamase from Stenotrophomonas maltophilia K279a.
Assuntos
Stenotrophomonas maltophilia , Biologia , Cristalografia , ProteínasRESUMO
The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Modelos Moleculares , RNA Viral/genética , Espalhamento a Baixo Ângulo , Proteínas não Estruturais Virais , Replicação Viral , Difração de Raios XRESUMO
It has been previously established that hypoxia leads to tumor development, treatment resistance, and a poor prognosis. Under oxygen deprivation, hypoxia-inducible factors (HIFs) are stimulated to activate the genes necessary for tumor development in a low-oxygen environment. These genes encode regulators of angiogenesis, epithelial-mesenchymal transition, and cellular metabolism. A disulfide isomerase, anterior gradient 2 (AGR2), has been shown to increase hypoxia-inducible factor 1, alpha subunit (HIF-1α) stability in breast cancer. Our goal was to determine if AGR2 affects the level of transcription factor hypoxia-inducible factor 2, alpha subunit (HIF-2α). As a model, we used the clear cell renal cell carcinoma (ccRCC) cell line Caki-1. The cells were transduced with lentiviral vector (Tet-On) encoding AGR2. After induction of AGR2 expression, cells were grown under either hypoxic (0.5% O2 ) or normoxic (21% O2 ) conditions. Our data showed that AGR2 upregulated both HIF-1α and HIF-2α expression in Caki-1 cells increasing the expression of HIF-activated genes (glucose transporter 1, phosphoglycerate kinase 1, vascular endothelial growth factor A, and transforming growth factor-alpha) under the hypoxic conditions. Under the normoxic conditions, AGR2 strongly activated CCAAT-enhancer binding protein beta (C/EBPß). Upregulation of C/EBPß correlated with increased expression and secretion of the interleukin-6 and interleukin-8, inducing angiogenesis and inflammation in Caki-1 cells. In summary, our studies revealed that AGR2 has essential functions in ccRCC progression through upregulation of C/EBPß and HIF-2α expressions, which affects cell signaling and metabolism.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Carcinoma de Células Renais/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mucoproteínas/genética , Proteínas Oncogênicas/genética , Carcinogênese/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Interleucina-6/genética , Interleucina-8/genética , Proteínas de Neoplasias/genética , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
YY1 (Yin Yang 1) is a zinc finger protein with an essential role in various biological functions via DNA- and protein-protein interactions with numerous partners. YY1 is involved in the regulation of a broad spectrum of cellular processes such as embryogenesis, proliferation, tumorigenesis, and snRNA transcription. The more than 100 reported targets of the YY1 protein suggest that it contains intrinsically disordered regions that are involved in such diverse interactions. Here, we present a study of the structural properties of human YY1 using several biochemical and biophysical techniques (fluorescence, circular dichroism, gel filtration chromatography, proteolytic susceptibility) together with various bioinformatics approaches. To facilitate our exploration of the YY1 structure, the full-length protein as well as an N-terminal fragment (residues 1-295) and the C-terminal DNA binding domain were used. We found the N-terminus to be a non-compact fragment of YY1 with little residual secondary structure and lacking a well-defined tertiary structure. The results of our study indicate that YY1 belongs to the family of intrinsically disordered proteins (IDPs), which exist natively in a partially unfolded conformation.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Desdobramento de Proteína , Fator de Transcrição YY1/química , Cromatografia em Gel , Dicroísmo Circular , Clonagem Molecular , Biologia Computacional/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Dobramento de Proteína , Proteólise , Tripsina/química , Fator de Transcrição YY1/genéticaRESUMO
BACKGROUND & AIMS: Hepatic fibrosis is characterized by enhanced deposition of extracellular matrix (ECM), which results from the wound healing response to chronic, repeated injury of any etiology. Upon injury, hepatic stellate cells (HSCs) activate and secrete ECM proteins, forming scar tissue, which leads to liver dysfunction. Monocyte-chemoattractant protein-induced protein 1 (MCPIP1) possesses anti-inflammatory activity, and its overexpression reduces liver injury in septic mice. In addition, mice with liver-specific deletion of Zc3h12a develop features of primary biliary cholangitis. In this study, we investigated the role of MCPIP1 in liver fibrosis and HSC activation. METHODS: We analyzed MCPIP1 levels in patients' fibrotic livers and hepatic cells isolated from fibrotic murine livers. In vitro experiments were conducted on primary HSCs, cholangiocytes, hepatocytes, and LX-2 cells with MCPIP1 overexpression or silencing. RESULTS: MCPIP1 levels are induced in patients' fibrotic livers compared with their nonfibrotic counterparts. Murine models of fibrosis revealed that its level is increased in HSCs and hepatocytes. Moreover, hepatocytes with Mcpip1 deletion trigger HSC activation via the release of connective tissue growth factor. Overexpression of MCPIP1 in LX-2 cells inhibits their activation through the regulation of TGFB1 expression, and this phenotype is reversed upon MCPIP1 silencing. CONCLUSIONS: We demonstrated that MCPIP1 is induced in human fibrotic livers and regulates the activation of HSCs in both autocrine and paracrine manners. Our results indicate that MCPIP1 could have a potential role in the development of liver fibrosis.
Assuntos
Comunicação Autócrina , Células Estreladas do Fígado , Cirrose Hepática , Comunicação Parácrina , Ribonucleases , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Animais , Humanos , Cirrose Hepática/patologia , Cirrose Hepática/metabolismo , Camundongos , Ribonucleases/metabolismo , Ribonucleases/genética , Masculino , Modelos Animais de Doenças , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fígado/patologia , Fígado/metabolismoRESUMO
Human protein Yin Yang 1 (YY1) controls the transcription of hundreds of genes both positively and negatively through interactions with a wide range of partner proteins. Results presented here from proteolytic sensitivity, calorimetry, circular dichroism, fluorescence, NMR, size-exclusion chromatography, SELEX, and EMSA show that purified YY1 forms dimers via its disordered N-terminal region with strong zinc-ion concentration dependence. The YY1 dimer is shown to bind tandem repeats of a canonical recognition DNA sequence with high affinity, and analysis of human YY1 regulatory sites shows that many contain repeats of its recognition elements. YY1 dimerization may compete with partner protein interactions, making control by zinc ion concentration a previously unrecognized factor affecting YY1 gene regulation. Indeed, YY1 is known to be important in many pathogenic processes, including neoplasia, in which zinc ion concentrations are altered. The present results incentivize studies in vivo or in vitro that explore the role of zinc ion concentration in YY1-mediated gene expression.
Assuntos
Fator de Transcrição YY1 , Zinco , Humanos , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo , Zinco/metabolismo , Dimerização , Regulação da Expressão Gênica , Sequência de BasesRESUMO
Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NP NTD ) from SARS-CoV-2 using small angle X-ray scattering (SAXS). Our solution-based results distinguished the mAbs' flexibility and how this flexibility impacts the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NP NTD , we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the Fabs is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich ELISA, we show the rigid mAb-pairings with linear polymerization led to increased NP NTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices (LFA), key for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.
RESUMO
Monoclonal antibodies (mAbs) are the basis of treatments and diagnostics for pathogens and other biological phenomena. We conducted a structural characterization of mAbs against the N-terminal domain of nucleocapsid protein (NPNTD) from SARS-CoV-2 using small-angle X-ray scattering and transmission electron microscopy. Our solution-based results distinguished the mAbs' flexibility and how this flexibility affects the assembly of multiple mAbs on an antigen. By pairing two mAbs that bind different epitopes on the NPNTD, we show that flexible mAbs form a closed sandwich-like complex. With rigid mAbs, a juxtaposition of the antigen-binding fragments is prevented, enforcing a linear arrangement of the mAb pair, which facilitates further mAb polymerization. In a modified sandwich enzyme-linked immunosorbent assay, we show that rigid mAb-pairings with linear polymerization led to increased NPNTD detection sensitivity. These enhancements can expedite the development of more sensitive and selective antigen-detecting point-of-care lateral flow devices, which are critical for early diagnosis and epidemiological studies of SARS-CoV-2 and other pathogens.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Epitopos/imunologia , Proteínas do Nucleocapsídeo/imunologia , SARS-CoV-2/enzimologia , Animais , HumanosRESUMO
Periodontal disease (PD) is an inflammatory disease of the supporting tissues of the teeth that develops in response to formation of a dysbiotic biofilm on the subgingival tooth surface. Although exacerbated inflammation leads to alveolar bone destruction and may cause tooth loss, the molecular basis of PD initiation and progression remains elusive. Control over the inflammatory reaction and return to homeostasis can be efficiently restored by negative regulators of Toll-like receptor (TLR) signaling pathways such as monocyte chemoattractant protein-induced protein 1 (MCPIP-1), which is constitutively expressed in gingival keratinocytes and prevents hyperresponsiveness in the gingiva. Here, we found that inflammophilic periodontal species influence the stability of MCPIP-1, leading to an aggravated response of the epithelium to proinflammatory stimulation. Among enzymes secreted by periodontal species, gingipains-cysteine proteases from Porphyromonas gingivalis-are considered major contributors to the pathogenic potential of bacteria, strongly influencing the components of the innate and adaptive immune system. Gingipain proteolytic activity leads to a rapid degradation of MCPIP-1, exacerbating the inflammatory response induced by endotoxin. Collectively, these results establish a novel mechanism of corruption of inflammatory signaling by periodontal pathogens, indicating new possibilities for treatment of this chronic disease. IMPORTANCE Periodontitis is a highly prevalent disease caused by accumulation of a bacterial biofilm. Periodontal pathogens use a number of virulence strategies that are under intensive study to find optimal therapeutic approaches against bone loss. In our work, we present a novel mechanism utilized by the key periodontal pathogen Porphyromonas gingivalis, based on the selective degradation of the negative regulator of inflammation, MCPIP-1. We found that the diminished levels of MCPIP-1 in gingival keratinocytes-cells at the forefront of the fight against bacteria-cause sensitization to endotoxins produced by other oral species. This results in an enhanced inflammatory response, which promotes the growth of inflammophilic pathobionts and damage of tooth-supporting tissues. Our observation is relevant to understanding the molecular basis of periodontitis and the development of new methods for treatment.
Assuntos
Gengiva/citologia , Inflamação , Queratinócitos/imunologia , Lipopolissacarídeos/metabolismo , Porphyromonas gingivalis/imunologia , Porphyromonas gingivalis/metabolismo , Ribonucleases/metabolismo , Transdução de Sinais , Animais , Biofilmes/crescimento & desenvolvimento , Células Cultivadas , Feminino , Cisteína Endopeptidases Gingipaínas , Queratinócitos/metabolismo , Queratinócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Periodontite/microbiologia , Porphyromonas gingivalis/fisiologia , Ribonucleases/genética , Ribonucleases/imunologia , Organismos Livres de Patógenos EspecíficosRESUMO
SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.
Assuntos
Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , COVID-19/virologia , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pirrolidinas/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , Timina/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Células A549 , Antivirais/química , Antivirais/farmacocinética , Domínio Catalítico , Cristalografia por Raios X , Endorribonucleases/química , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Pirrolidinas/química , Pirrolidinas/farmacocinética , Timina/química , Timina/farmacocinética , Uridina/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
Monocyte chemoattractant protein-induced protein-1 (MCPIP-1) is a potent inhibitor of inflammatory response to pathogens. Acting as endonuclease against transcripts of inflammatory cytokines or transcription factors MCPIP-1 can significantly reduce the cytokine storm, thus limiting the tissue damage. As the adequate resolution of inflammation depends also on the efficient clearance of accumulated neutrophils, we focused on the role of MCPIP-1 in apoptosis and retention of neutrophils. We used peritoneal neutrophils from cell-specific MCPIP-1 knockout mice and showed prolonged survival of these cells. Moreover, we confirmed that MCPIP-1-dependent degradation of transcripts of antiapoptotic genes, including BCL3, BCL2A1, BCL2L1, and for the first time MCL-1, serves as an early event in spontaneous apoptosis of primary neutrophils. Additionally, we identified previously unknown miRNAs as potential binding partners to the MCPIP-1 transcript and their regulation suggest a role in MCPIP-1 half-life and translation. These phenomena may play a role as a molecular switch that balances the MCPIP-1-dependent apoptosis. Besides that, we determined these particular miRNAs as integral components of the GM-CSF-MCPIP-1 axis. Taken together, we identified the novel anti-inflammatory role of MCPIP-1 as a regulator of accumulation and survival of neutrophils that simultaneously promotes an adequate resolution of inflammation.
Assuntos
Anti-Inflamatórios/metabolismo , Apoptose/fisiologia , Inflamação/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Feminino , Humanos , Camundongos , MicroRNAs , Neutrófilos/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Emergence of Enterobacteriaceae harboring metallo-ß-lactamases (MBL) has raised global threats due to their broad antibiotic resistance profiles and the lack of effective inhibitors against them. We have been studied one of the emerging environmental MBL, the L1 from Stenotrophomonas maltophilia K279a. We determined several crystal structures of L1 complexes with three different classes of ß-lactam antibiotics (penicillin G, moxalactam, meropenem, and imipenem), with the inhibitor captopril and different metal ions (Zn+2 , Cd+2 , and Cu+2 ). All hydrolyzed antibiotics and the inhibitor were found binding to two Zn+2 ions mainly through the opened lactam ring and some hydrophobic interactions with the binding pocket atoms. Without a metal ion, the active site is very similarly maintained as that of the native form with two Zn+2 ions, however, the protein does not bind the substrate moxalactam. When two Zn+2 ions were replaced with other metal ions, the same di-metal scaffold was maintained and the added moxalactam was found hydrolyzed in the active site. Differential scanning fluorimetry and isothermal titration calorimetry were used to study thermodynamic properties of L1 MBL compared with New Deli Metallo-ß-lactamase-1 (NDM-1). Both enzymes are significantly stabilized by Zn+2 and other divalent metals but showed different dependency. These studies also suggest that moxalactam and its hydrolyzed form may bind and dissociate with different kinetic modes with or without Zn+2 for each of L1 and NDM-1. Our analysis implicates metal ions, in forming a distinct di-metal scaffold, which is central to the enzyme stability, promiscuous substrate binding and versatile catalytic activity. STATEMENT: The L1 metallo-ß-lactamase from an environmental multidrug-resistant opportunistic pathogen Stenotrophomonas maltophilia K279a has been studied by determining 3D structures of L1 enzyme in the complexes with several ß-lactam antibiotics and different divalent metals and characterizing its biochemical and ligand binding properties. We found that the two-metal center in the active site is critical in the enzymatic process including antibiotics recognition and binding, which explains the enzyme's activity toward diverse antibiotic substrates. This study provides the critical information for understanding the ligand recognition and for advanced drug development.
Assuntos
Biocatálise , Metais Pesados/metabolismo , Stenotrophomonas maltophilia/enzimologia , beta-Lactamases/química , beta-Lactamases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Biocatálise/efeitos dos fármacos , Lactamas/química , Lactamas/farmacologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Stenotrophomonas maltophilia/efeitos dos fármacos , Inibidores de beta-Lactamases/química , Inibidores de beta-Lactamases/farmacologiaRESUMO
Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is rapidly spreading around the world. There is no existing vaccine or proven drug to prevent infections and stop virus proliferation. Although this virus is similar to human and animal SARS-CoVs and Middle East Respiratory Syndrome coronavirus (MERS-CoVs), the detailed information about SARS-CoV-2 proteins structures and functions is urgently needed to rapidly develop effective vaccines, antibodies, and antivirals. We applied high-throughput protein production and structure determination pipeline at the Center for Structural Genomics of Infectious Diseases to produce SARS-CoV-2 proteins and structures. Here we report two high-resolution crystal structures of endoribonuclease Nsp15/NendoU. We compare these structures with previously reported homologs from SARS and MERS coronaviruses.
Assuntos
Betacoronavirus/química , Endorribonucleases/química , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Oligonucleotídeos/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Proteínas não Estruturais Virais/química , Sequência de Aminoácidos , Betacoronavirus/genética , Betacoronavirus/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Endorribonucleases/genética , Endorribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Modelos Moleculares , Oligonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , SARS-CoV-2 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
NF-κB signaling, acting through NFKB1 dependent canonical and NFKB2 dependent non-canonical pathways plays a critical role in inflammatory and immune responses. Recent studies have associated mutations in these two genes with a common variable immunodeficiency (CVID). While evaluating a female patient seeking a diagnosis explaining her recurrent infections, we found a novel heterozygous c.1831C > T (p.Arg611∗) nonsense mutation in the NFKB2 gene which introduces a Stop codon in the ankyrin repeat domain of p100. Whole exome sequencing (WES) analysis, followed by Sanger sequencing, identified this previously unknown mutation in two other family members. Penetrance of the c.1831C > T variant was assessed by flow-cytometry and protein expression in peripheral blood mononuclear cells (PBMC); whereas, activation of the NF-κB2 signaling pathway was examined through immunoblotting and real-time PCR. Heterozygous c.1831C > T variant led to the expansion of lymphocyte B subpopulations with concomitant reduction of plasmablasts, low IgG levels, and accumulation of p52 in PBMC. On the other hand, tested subjects had normal levels of IgM, IgA, IgE and no impairment in lymphocytes proliferation. Although evaluated patients did not fulfill all clinical features of CVID, their health should be monitored in the future for possible late manifestation of the disease. In conclusion, we showed that NFKB2 haplodeficiency caused by c.1831C > T nonsense mutation is asymptomatic, possibly due to the compensatory mechanisms and allele redundancy.
RESUMO
MCPIP1, also known as Regnase-1, is a ribonuclease crucial for regulation of stability of transcripts related to inflammatory processes. Here, we report that MCPIP1 acts as an endonuclease by degrading several stem-loop RNA structures and single-stranded RNAs. Our studies revealed cleavage sites present in the stem-loops derived from the 3' untranslated region of the interleukin-6 transcript. Furthermore, MCPIP1 induced endonuclease cleavage at the loop motif of stem-loop structures. Additionally, we observed that MCPIP1 could cleave single-stranded RNA fragments. However, RNA substrates shorter than 6 nucleotides were not further affected by MCPIP1 nucleolytic activity. In this study, we also determined the dissociation constants of full-length MCPIP1D141N and its ribonuclease domain PIN D141N with twelve oligonucleotides substrates. The equilibrium binding constants (Kd) for MCPIP1D141N and the RNA targets were approximately 10 nM. Interestingly, we observed that the presence of a zinc finger in the PIN domain increases the affinity of this protein fragment to 25-nucleotide-long stem-loop RNA but not to shorter ones. Furthermore, size exclusion chromatography of the MCPIP1 and PIN proteins suggested that MCPIP1 undergoes homooligomerization during interaction with RNA substrates. Our results provide insight into the mechanism of MCPIP1 substrate recognition and its affinity towards various oligonucleotides.
Assuntos
Estabilidade de RNA , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Humanos , Cinética , Especificidade por Substrato , Dedos de ZincoRESUMO
We used RNA sequencing (RNA-Seq) technology to investigate changes in the transcriptome profile in the Caki-1 clear cell renal cell carcinoma (ccRCC) cells, which overexpress monocyte chemoattractant protein-induced protein 1 (MCPIP1). RNA-Seq data showed changes in 11.6% and 41.8% of the global transcriptome of Caki-1 cells overexpressing wild-type MCPIP1 or its D141N mutant, respectively. Gene ontology and KEGG pathway functional analyses showed that these transcripts encoded proteins involved in cell cycle progression, protein folding in the endoplasmic reticulum, hypoxia response and cell signalling. We identified 219 downregulated transcripts in MCPIP1-expressing cells that were either unchanged or upregulated in D141N-expressing cells. We validated downregulation of 15 transcripts belonging to different functional pathways by qRT-PCR. The growth and viability of MCPIP1-expressing cells was reduced because of elevated p21Cip1 levels. MCPIP1-expressing cells also showed reduced levels of DDB1 transcript that encodes component of the E3 ubiquitin ligase that degrades p21Cip1. These results demonstrate that MCPIP1 influences the growth and viability of ccRCC cells by increasing or decreasing the transcript levels for proteins involved in cell cycle progression, protein folding, hypoxia response, and cell signaling.
RESUMO
CCAAT/enhancer-binding protein beta (C/EBPß) is a transcription factor controlling a broad range of genes essential for homeostasis, including genes related to immune functions, inflammation, metabolism and growth. Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) also called as Regnase-1 is an RNase and has been shown to decrease the stability of short-lived transcripts coding for inflammation-related proteins, including IL-1ß, IL-6, IL-2, IL-8, IL-12b, IER-3, c-Rel. We found previously that the half-life of the C/EBPß transcript is regulated by MCPIP. To understand the mechanism driving down-regulation of C/EBPß by MCPIP1, we applied an in vitro cleavage assay, followed by a luciferase-reporter assay and RNA immunoprecipitation (RIP). We demonstrated that MCPIP1 recognizes regions of the 3'UTR of C/EBPß mRNA and promotes its decay by introducing direct endonucleolytic cleavage.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , RNA Mensageiro/metabolismo , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/genética , Células Hep G2 , Humanos , Imunoprecipitação/métodos , Interleucina-12/metabolismo , Interleucina-1beta/metabolismo , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismoRESUMO
Pattern recognition receptors are critical for the detection of invading microorganisms. They activate multiple pathways that lead to the induction of proinflammatory responses and pathogen clearance. The intensity and duration of this immune reaction must be tightly controlled spatially and temporally in every tissue by different negative regulators. We hypothesized that monocyte chemoattractant protein-1-induced protein-1 (MCPIP-1) might play a role in maintaining immune homeostasis in the epithelium both under physiological conditions and upon bacterial infection. To this end, we examined the distribution of the MCPIP-1 transcript and protein in various tissues. The MCPIP-1 protein level was higher in epithelial cells than in myeloid cells. MCPIP-1 exerted RNase activity towards the interleukin (IL)-8 transcript and the lifespan of IL-8 was determined by the presence of the stem-loops/hairpin structures at the 3'UTR region of IL-8 mRNA. Moreover, using fully active, purified recombinant MCPIP-1 protein, we elucidated the mechanism by which MCPIP-1 controls the IL-8 mRNA level. In conclusion, we uncovered a novel IL-8-dependent mechanism via which MCPIP-1 maintains epithelial homeostasis. This study reveals for the first time that MCPIP-1 plays a crucial anti-inflammatory role not only in myeloid cells but also in epithelial cells.