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1.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069882

RESUMO

Geographically, East Asia had the highest liver cancer burden in 2017. Besides this, liver cancer-related deaths were high in Japan, accounting for 3.90% of total deaths. The development of liver cancer is influenced by several factors, and genetic alteration is one of the critical factors among them. Therefore, the detailed mechanism driving the oncogenic transformation of liver cells needs to be elucidated. Recently, many researchers have focused on investigating the liver cancer genome and identified somatic mutations (MTs) of several transcription factors. In this line, next-generation sequencing of the cancer genome identified that oxidative stress-related transcription factor NRF2 (NFE2L2) is mutated in different cancers, including hepatocellular carcinoma (HCC). Here, we demonstrated that NRF2 DLG motif mutations (NRF2 D29A and L30F), found in Japanese liver cancer patients, upregulate the transcriptional activity of NRF2 in HCC cell lines. Moreover, the transcriptional activity of NRF2 mutations is not suppressed by KEAP1, presumably because NRF2 MTs disturb proper NRF2-KEAP1 binding and block KEAP1-mediated degradation of NRF2. Additionally, we showed that both MTs upregulate the transcriptional activity of NRF2 on the MMP9 promoter in Hepa1-6 and Huh7 cells, suggesting that MT derived gain-of-function of NRF2 may be important for liver tumor progression. We also found that ectopic overexpression of oncogenic BRAF WT and V600E increases the transcriptional activity of NRF2 WT on both the 3xARE reporter and MMP9 promoter. Interestingly, NRF2 D29A and L30F MTs with oncogenic BRAF V600E MT synergistically upregulate the transcription activity of NRF2 on the 3xARE reporter and MMP9 promoter in Hepa1-6 and Huh7 cells. In summary, our findings suggest that MTs in NRF2 have pathogenic effects, and that NRF2 MTs together with oncogenic BRAF V600E MT synergistically cause more aberrant transcriptional activity. The high activity of NRF2 MTs in HCC with BRAF MT warrants further exploration of the potential diagnostic, prognostic, and therapeutic utility of this pathway in HCC.


Assuntos
Carcinoma Hepatocelular/genética , Fator 2 Relacionado a NF-E2/genética , Motivos de Aminoácidos/genética , Carcinogênese/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Japão , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metaloproteinase 9 da Matriz/metabolismo , Mutação , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/genética , Prognóstico , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética
2.
Nucleic Acids Res ; 49(8): 4768-4781, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33856462

RESUMO

Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.


Assuntos
Motivos de Aminoácidos , Candida albicans/química , Candida tropicalis/química , Domínio Catalítico , Telomerase/química , Motivos de Aminoácidos/genética , Candida albicans/enzimologia , Candida tropicalis/enzimologia , Catálise , Domínio Catalítico/genética , Cromatografia em Gel , Cristalografia por Raios X , Difusão Dinâmica da Luz , Escherichia coli/metabolismo , Técnicas In Vitro , Modelos Moleculares , Mutação , Proteínas Recombinantes , Telomerase/genética
3.
Biochem Biophys Res Commun ; 552: 91-97, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33744765

RESUMO

High mobility group A2 (HMGA2) is a chromatin-associated protein involved in the regulation of stem cell function, embryogenesis and cancer development. Although the protein does not contain a consensus SUMOylation site, it is shown to be SUMOylated. In this study, we demonstrate that the first lysine residue in the reported K66KAE SUMOylation motif in HMGA2 can be methylated in vitro and in vivo by the Set7/9 methyltransferase. By editing the lysine, the increased hydrophobicity of the resulting 6-N-methyl-lysine transforms the sequence into a consensus SUMO motif. This post-translational editing dramatically increases the subsequent SUMOylation of this site. Furthermore, similar putative methylation-dependent SUMO motifs are found in a number of other chromatin factors, and we confirm methylation-dependent SUMOylation of a site in one such protein, the Polyhomeotic complex 1 homolog (PHC1). Together, these results suggest that crosstalk between methylation and SUMOylation is a general mode for regulation of chromatin function.


Assuntos
Proteína HMGA2/metabolismo , Lisina/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Linhagem Celular , Proteína HMGA2/química , Proteína HMGA2/genética , Humanos , Lisina/química , Lisina/genética , Metilação , Ligação Proteica , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Sumoilação , Fatores de Transcrição/química , Fatores de Transcrição/genética , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo
4.
Nat Commun ; 12(1): 1899, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33771996

RESUMO

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.


Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitose , Treonina/metabolismo , Motivos de Aminoácidos/genética , Animais , Aurora Quinase A/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Ciclina A2/genética , Ciclina A2/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Ativação Enzimática , Feminino , Humanos , Oócitos/metabolismo , Fosforilação , Prolina/genética , Prolina/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Serina/genética , Serina/metabolismo , Treonina/genética , Xenopus laevis
5.
Nat Commun ; 12(1): 394, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452244

RESUMO

Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) to kill host cells. Here, we show that the WXG100 proteins EsxE and EsxF are essential for TNT secretion. EsxE and EsxF form a water-soluble heterodimer (EsxEF) that assembles into oligomers and long filaments, binds to membranes, and forms stable membrane-spanning channels. Electron microscopy of EsxEF reveals mainly pentameric structures with a central pore. Mutations of both WXG motifs and of a GXW motif do not affect dimerization, but abolish pore formation, membrane deformation and TNT secretion. The WXG/GXW mutants are locked in conformations with altered thermostability and solvent exposure, indicating that the WXG/GXW motifs are molecular switches controlling membrane interaction and pore formation. EsxF is accessible on the bacterial cell surface, suggesting that EsxEF form an outer membrane channel for toxin export. Thus, our study reveals a protein secretion mechanism in bacteria that relies on pore formation by small WXG proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Mycobacterium tuberculosis/patogenicidade , Porinas/metabolismo , Sistemas de Secreção Tipo VII/metabolismo , Motivos de Aminoácidos/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/toxicidade , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Humanos , Bicamadas Lipídicas/metabolismo , Microscopia Eletrônica , Mutação , Mycobacterium tuberculosis/metabolismo , Porinas/genética , Multimerização Proteica , Células THP-1 , Tuberculose/microbiologia , Tuberculose/patologia , Sistemas de Secreção Tipo VII/genética
6.
Nat Chem Biol ; 17(4): 492-500, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33398169

RESUMO

De novo protein design has enabled the creation of new protein structures. However, the design of functional proteins has proved challenging, in part due to the difficulty of transplanting structurally complex functional sites to available protein structures. Here, we used a bottom-up approach to build de novo proteins tailored to accommodate structurally complex functional motifs. We applied the bottom-up strategy to successfully design five folds for four distinct binding motifs, including a bifunctionalized protein with two motifs. Crystal structures confirmed the atomic-level accuracy of the computational designs. These de novo proteins were functional as components of biosensors to monitor antibody responses and as orthogonal ligands to modulate synthetic signaling receptors in engineered mammalian cells. Our work demonstrates the potential of bottom-up approaches to accommodate complex structural motifs, which will be essential to endow de novo proteins with elaborate biochemical functions, such as molecular recognition or catalysis.


Assuntos
Engenharia de Proteínas/métodos , Motivos de Aminoácidos/genética , Sítios de Ligação/genética , Catálise , Ligantes , Modelos Moleculares , Ligação Proteica/genética , Dobramento de Proteína , Proteínas/química
7.
Nat Commun ; 12(1): 585, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33500419

RESUMO

The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1 and RMI2 to form the BTR complex, which dissolves double Holliday junctions to produce non-crossover homologous recombination (HR) products. BLM also promotes DNA-end resection, restart of stalled replication forks, and processing of ultra-fine DNA bridges in mitosis. How these activities of the BTR complex are regulated in cells is still unclear. Here, we identify multiple conserved motifs within the BTR complex that interact cooperatively with the single-stranded DNA (ssDNA)-binding protein RPA. Furthermore, we demonstrate that RPA-binding is required for stable BLM recruitment to sites of DNA replication stress and for fork restart, but not for its roles in HR or mitosis. Our findings suggest a model in which the BTR complex contains the intrinsic ability to sense levels of RPA-ssDNA at replication forks, which controls BLM recruitment and activation in response to replication stress.


Assuntos
Síndrome de Bloom/genética , Replicação do DNA , DNA de Cadeia Simples/metabolismo , RecQ Helicases/metabolismo , Proteína de Replicação A/metabolismo , Motivos de Aminoácidos/genética , Sistemas CRISPR-Cas/genética , Dano ao DNA , DNA Topoisomerases Tipo I/metabolismo , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Mitose/genética , Mutação , Ligação Proteica/genética , Domínios Proteicos/genética , RecQ Helicases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reparo de DNA por Recombinação/genética
8.
Arch Virol ; 166(3): 831-840, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33486631

RESUMO

Ovine pulmonary adenomatosis (OPA) is caused by jaagsiekte sheep retrovirus (JSRV) and is a chronic, progressive, and infectious neoplastic lung disease in sheep, which causes significant economic losses to the sheep industry. Neither a vaccine nor serological diagnostic methods to detect OPA are available. We performed a JSRV infection survey in sheep using blood samples (n = 1,372) collected in the three northeastern provinces of China (i.e., Inner Mongolia, Heilongjiang, and Jilin) to determine JSRV infection status in sheep herds using a real-time PCR assay targeting the gag gene of JSRV. The ovine endogenous retrovirus sequence was successfully amplified in all sheep samples tested (296 from the Inner Mongolia Autonomous Region, 255 from Jilin province, and 821 from Heilongjiang province). Subsequently, we attempted to distinguish exogenous JSRV (exJSRV) and endogenous JSRV (enJSRV) infections in these JSRV-positive samples using a combination assay that identifies a ScaI restriction site in an amplified 229-bp fragment of the gag gene of JSRV and a "LHMKYXXM" motif in the cytoplasmic tail region of the JSRV envelope protein. The ScaI restriction site is present in all known oncogenic JSRVs but absent in ovine endogenous retroviruses, while the "LHMKYXXM" motif is in all known exJSRVs but not in enJSRVs. Interestingly, one JSRV strain (HH13) from Heilongjiang province contained the "LHMKYXXM" motif but not the ScaI enzyme site. Phylogenetic analysis showed that strain HH13 was closely related to strain enJSRV-21 reported in the USA, indicating that HH13 could be an exogenous virus. Our results provide valuable information for further research on the genetic evolution and pathogenesis of JSRV.


Assuntos
Retrovirus Endógenos/genética , Produtos do Gene env/genética , Retrovirus Jaagsiekte de Ovinos/genética , Adenomatose Pulmonar Ovina/epidemiologia , Adenomatose Pulmonar Ovina/patologia , Motivos de Aminoácidos/genética , Animais , Sequência de Bases , China/epidemiologia , DNA Viral/análise , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Evolução Molecular , Genoma Viral/genética , Retrovirus Jaagsiekte de Ovinos/classificação , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Ovinos
9.
Cell Mol Life Sci ; 78(4): 1745-1763, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32780150

RESUMO

SAM68 is an mRNA-binding protein involved in mRNA processing in the nucleus that forms membraneless compartments called SAM68 Nuclear Bodies (SNBs). We found that intersectin 1 (ITSN1), a multidomain scaffold protein harboring five soluble SH3 domains, interacts with SAM68 proline-rich motifs (PRMs) surrounded by self-adhesive low complexity domains. While SAM68 is poorly soluble in vitro, the interaction of ITSN1 SH3 domains and mRNA with SAM68 enhances its solubility. In HeLa cells, the interaction between the first ITSN1 SH3 domain (SH3A) and P0, the N-terminal PRM of SAM68, induces the dissociation of SNBs. In addition, we reveal the ability of another SH3 domain (SH3D) of ITSN1 to bind to mRNAs. ITSN1 and mRNA may thus act in concert to promote SAM68 solubilization, consistent with the absence of mRNA in SNBs in cells. Together, these results support the notion of a specific chaperoning of PRM-rich SAM68 within nuclear ribonucleoprotein complexes by ITSN1 that may regulate the processing of a fraction of nuclear mRNAs, notably SAM68-controlled splicing events related to higher neuronal functions or cancer progression. This observation may also serve as a putative model of the interaction between other PRM-rich RBPs and signaling proteins harboring SH3 domains.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Motivos de Aminoácidos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a RNA/genética , Domínios de Homologia de src/genética , Sequência de Aminoácidos/genética , Proteínas de Transporte/genética , Núcleo Celular/genética , Endocitose/genética , Células HeLa , Humanos , Prolina/genética , Ligação Proteica/genética , Splicing de RNA/genética , Solubilidade
10.
Biochimie ; 180: 229-242, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33197551

RESUMO

Antimicrobial peptides (AMPs) play an essential role in plant defense against invading pathogens. Due to their biological properties, these molecules have been considered useful for drug development, as novel agents in disease therapeutics, applicable to both agriculture and medicine. New technologies of massive sequencing open opportunities to discover novel AMP encoding genes in wild plant species. This work aimed to identify cysteine-rich AMPs from Peltophorum dubium, a legume tree from South America. We performed whole-transcriptome sequencing of P. dubium seedlings followed by de novo transcriptome assembly, uncovering 78 AMP transcripts classified into five families: hevein-like, lipid-transfer proteins (LTPs), alpha hairpinins, defensins, and snakin/GASA (Giberellic Acid Stimulated in Arabidopsis) peptides. No transcripts with similarity to cyclotide or thionin genes were identified. Genomic DNA analysis by PCR confirmed the presence of 18 genes encoding six putative defensins and 12 snakin/GASA peptides and allowed the characterization of their exon-intron structure. The present work demonstrates that AMP prediction from a wild species is possible using RNA sequencing and de novo transcriptome assembly, regarding a starting point for studies focused on AMP gene evolution and expression. Moreover, this study allowed the detection of strong AMP candidates for drug development and novel biotechnological products.


Assuntos
Fabaceae/química , Genes de Plantas/genética , Genoma de Planta/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Plântula/genética , Plântula/metabolismo , Motivos de Aminoácidos/genética , Modelos Moleculares , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/classificação , Alinhamento de Sequência , Transcriptoma
11.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118887, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33075383

RESUMO

The G protein-coupled receptor (GPCR) dimer interface plays an important role in the formation and stabilization of the dimer. Therefore, identifying the potential receptor-receptor interface is an important part of studying GPCRs. Various strategies have been employed to study the GPCR dimer interface and explore its functional significance, but experimental methods lack robustness and calculations are laborious. Herein, we report a combined optimized experimental and calculation approach for identifying and structurally characterizing GPCR dimer interfaces, and constructing atomic resolution models. Using a transmembrane domain (TM) peptide containing a human immunodeficiency virus trans-acting transcriptional activator (HIV-TAT) protein transduction motif, matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDITOF-MS), and bioluminescence resonance energy transfer (BRET), we successfully identified Apelin receptor (APJ)/Nociceptin receptor 1 (ORL1) and APJ/Vasopressin receptor 2 (V2R) heterodimer interfaces, and two key sites mediating dimerization. This method can identify dimer interfaces of GPCR homodimers and heterodimers.


Assuntos
Dimerização , Conformação Proteica , Receptores Acoplados a Proteínas G/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Survivina/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Motivos de Aminoácidos/genética , Membrana Celular/química , Proteínas de Ligação ao GTP/química , Células HEK293 , Humanos , Modelos Moleculares , Domínios Proteicos/genética , Multimerização Proteica/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Survivina/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química
12.
PLoS One ; 15(11): e0242030, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33156866

RESUMO

Sequence logos have been widely used as graphical representations of conserved nucleic acid and protein motifs. Due to the complexity of the amino acid (AA) alphabet, rich post-translational modification, and diverse subcellular localization of proteins, few versatile tools are available for effective identification and visualization of protein motifs. In addition, various reduced AA alphabets based on physicochemical, structural, or functional properties have been valuable in the study of protein alignment, folding, structure prediction, and evolution. However, there is lack of tools for applying reduced AA alphabets to the identification and visualization of statistically significant motifs. To fill this gap, we developed an R/Bioconductor package dagLogo, which has several advantages over existing tools. First, dagLogo allows various formats for input sets and provides comprehensive options to build optimal background models. It implements different reduced AA alphabets to group AAs of similar properties. Furthermore, dagLogo provides statistical and visual solutions for differential AA (or AA group) usage analysis of both large and small data sets. Case studies showed that dagLogo can better identify and visualize conserved protein sequence patterns from different types of inputs and can potentially reveal the biological patterns that could be missed by other logo generators.


Assuntos
Aminoácidos/genética , Algoritmos , Motivos de Aminoácidos/genética , Sequência Conservada/genética , Bases de Dados de Proteínas , Humanos , Matrizes de Pontuação de Posição Específica , Proteínas/genética , Proteômica/métodos , Alinhamento de Sequência/métodos , Software
13.
J Mol Biol ; 432(24): 166712, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33197462

RESUMO

La is an abundant phosphoprotein that protects polymerase III transcripts from 3'-5' exonucleolytic degradation and facilitates their folding. Consisting of the evolutionary conserved La motif (LAM) and two consecutive RNA Recognition Motifs (RRMs), La was also found to bind additional RNA transcripts or RNA domains like internal ribosome entry site (IRES), through sequence-independent binding modes which are poorly understood. Although it has been reported overexpressed in certain cancer types and depletion of its expression sensitizes cancer cells to certain chemotherapeutic agents, its role in cancer remains essentially uncharacterized. Herein, we study the effects of La overexpression in A549 lung adenocarcinoma cells, which leads to increased cell proliferation and motility. Expression profiling of several transcription and translation factors indicated that La overexpression leads to downregulation of global translation through hypophosphorylation of 4E-BPs and upregulation of IRES-mediated translation. Moreover, analysis of La localization after nutrition deprivation of the transfected cells showed a normal distribution in the nucleus and nucleoli. Although the RNA binding capacity of La has been primarily linked to the synergy between the conserved LAM and RRM1 domains which act as a module, we show that recombinant stand-alone LAM can specifically bind a pre-tRNA ligand, based on binding experiments combined with NMR analysis. We propose that LAM RNA binding properties could support the expanding and diverse RNA ligand repertoire of La, thus promoting its modulatory role, both under normal and pathogenic conditions like cancer.


Assuntos
Neoplasias Pulmonares/genética , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética , Relação Estrutura-Atividade , Células A549 , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Sítios Internos de Entrada Ribossomal/genética , Neoplasias Pulmonares/patologia , Fosfoproteínas/química , Ligação Proteica/genética , Biossíntese de Proteínas/genética , Motivo de Reconhecimento de RNA/genética
14.
Genes Dev ; 34(23-24): 1680-1696, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33184220

RESUMO

Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1-HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.


Assuntos
Motivos de Aminoácidos/genética , Proteínas de Drosophila/genética , Evolução Molecular , Proteínas de Homeodomínio/genética , Animais , Drosophila melanogaster/classificação , Drosophila melanogaster/genética , Estudo de Associação Genômica Ampla , Camundongos , Modelos Moleculares , Ligação Proteica/genética , Domínios Proteicos , Relação Estrutura-Atividade
15.
Biochem Biophys Res Commun ; 533(4): 1135-1141, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33041003

RESUMO

RNA G-quadruplex (rG4) structure and its association with rG4-binding proteins/peptides are important for its function. However, there is very limited study that investigates what factors are involved in rG4 that drive the rG4-protein/peptide interaction. Here we study and uncover the effect of RNA sequence context and stereochemistry on G-quadruplex-peptide interaction. Using rG4-binding RHAU53 peptide as an example, we report that the number of G-quartet, thermostability, overhanging nucleotides, and RNA base chirality have an impact on rG4-RHAU53 binding. Notably, our data also demonstrate that RHAU53 preferentially binds to 5' G-quartet over 3' G-quartet, and showcase that RHAU53 interacts with unnatural L-rG4 for the first time. Our findings reported here offer unique insights to the potential development of targeting tools that recognize rG4 structure and rG4-binding peptide/protein.


Assuntos
Quadruplex G , Peptídeos/química , Peptídeos/genética , RNA/química , RNA/genética , Motivos de Aminoácidos/genética , Dicroísmo Circular , Modelos Moleculares , Espectrofotometria Ultravioleta , Termodinâmica
16.
J Mol Biol ; 432(23): 6005-6027, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33058872

RESUMO

In filamentous fungi, amyloid signaling sequences allow Nod-like receptors (NLRs) to activate downstream cell-death inducing proteins with HeLo and HeLo-like (HELL) domains and amyloid RHIM and RHIM-related motifs control immune defense pathways in mammals and flies. Herein, we show bioinformatically that analogous amyloid signaling motifs exist in bacteria. These short motifs are found at the N terminus of NLRs and at the C terminus of proteins with a domain we term BELL. The corresponding NLR and BELL proteins are encoded by adjacent genes. We identify 10 families of such bacterial amyloid signaling sequences (BASS), one of which (BASS3) is homologous to RHIM and a fungal amyloid motif termed PP. BASS motifs occur nearly exclusively in bacteria forming multicellular structures (mainly in Actinobacteria and Cyanobacteria). We analyze experimentally a subset of seven of these motifs (from the most common BASS1 family and the RHIM-related BASS3 family) and find that these sequences form fibrils in vitro. Using a fungal in vivo model, we show that all tested BASS-motifs form prions and that the NLR-side motifs seed prion-formation of the corresponding BELL-side motif. We find that BASS3 motifs show partial prion cross-seeding with mammalian RHIM and fungal PP-motifs and that proline mutations on key positions of the BASS3 core motif, conserved in RHIM and PP-motifs, abolish prion formation. This work expands the paradigm of prion amyloid signaling to multicellular prokaryotes and suggests a long-term evolutionary conservation of these motifs from bacteria, to fungi and animals.


Assuntos
Amiloide/genética , Evolução Molecular , Imunidade Inata/genética , Proteínas NLR/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Proteínas Amiloidogênicas/genética , Animais , Cianobactérias/genética , Drosophila/genética , Fungos/genética , Genoma Bacteriano/genética , Príons/genética , Transdução de Sinais/genética
17.
Cells ; 9(10)2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-33003441

RESUMO

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that Gq-coupled receptors and constitutively active Gαq/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical Gq/phospholipase C (PLC) signaling. Here, we further characterized Gq/11 regulation of SphK1. SphK1 translocation by the M3 receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCß3, but accelerated by wild-type PLCß3 and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M3 receptor-stimulated inositol phosphate production, suggesting competition at Gαq. Embryonic fibroblasts from Gαq/11 double-deficient mice were used to show that amino acids W263 and T257 of Gαq, which interact directly with PLCß3 and p63RhoGEF, were important for bradykinin B2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gαq binding motif, ALXXPI, in PLCß and p63RhoGEF. After M3 receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCß/PLCδ(PH) expression are important for regulation of SphK1 by Gq/11.


Assuntos
Membrana Celular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingosina/análogos & derivados , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Membrana Celular/genética , Cromatografia Líquida de Alta Pressão , Fibroblastos , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Fosfatos de Inositol/metabolismo , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ligação Proteica , Receptor B2 da Bradicinina/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/genética , Esfingosina/metabolismo , Espectrometria de Massas em Tandem , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
18.
Sci Rep ; 10(1): 16944, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037310

RESUMO

The Spike protein of the novel coronavirus SARS-CoV2 contains an insertion 680SPRRAR↓SV687 forming a cleavage motif RxxR for furin-like enzymes at the boundary of S1/S2 subunits. Cleavage at S1/S2 is important for efficient viral entry into target cells. The insertion is absent in other CoV-s of the same clade, including SARS-CoV1 that caused the 2003 outbreak. However, an analogous cleavage motif was present at S1/S2 of the Spike protein of the more distant Middle East Respiratory Syndrome coronavirus MERS-CoV. We show that a crucial third arginine at the left middle position, comprising a motif RRxR is required for furin recognition in vitro, while the general motif RxxR in common with MERS-CoV is not sufficient for cleavage. Further, we describe a surprising finding that the two serines at the edges of the insert SPRRAR↓SV can be efficiently phosphorylated by proline-directed and basophilic protein kinases. Both phosphorylations switch off furin's ability to cleave the site. Although phospho-regulation of secreted proteins is still poorly understood, further studies, supported by a recent report of ten in vivo phosphorylated sites in the Spike protein of SARS-CoV2, could potentially uncover important novel regulatory mechanisms for SARS-CoV2.


Assuntos
Betacoronavirus/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Vírus da SARS/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Furina/metabolismo , Fosforilação , Proteólise , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Internalização do Vírus
19.
Nat Commun ; 11(1): 5043, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028863

RESUMO

Human T-cell lymphotropic virus type 1 (HTLV-1) is a deltaretrovirus and the most oncogenic pathogen. Many of the ~20 million HTLV-1 infected people will develop severe leukaemia or an ALS-like motor disease, unless a therapy becomes available. A key step in the establishment of infection is the integration of viral genetic material into the host genome, catalysed by the retroviral integrase (IN) enzyme. Here, we use X-ray crystallography and single-particle cryo-electron microscopy to determine the structure of the functional deltaretroviral IN assembled on viral DNA ends and bound to the B56γ subunit of its human host factor, protein phosphatase 2 A. The structure reveals a tetrameric IN assembly bound to two molecules of the phosphatase via a conserved short linear motif. Insight into the deltaretroviral intasome and its interaction with the host will be crucial for understanding the pattern of integration events in infected individuals and therefore bears important clinical implications.


Assuntos
Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Integrases/ultraestrutura , Proteína Fosfatase 2/ultraestrutura , Vírus Linfotrópico T Tipo 1 de Símios/enzimologia , Proteínas Virais/ultraestrutura , Integração Viral , Motivos de Aminoácidos/genética , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Viral/metabolismo , DNA Viral/ultraestrutura , Vírus Linfotrópico T Tipo 1 Humano/enzimologia , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Integrases/genética , Integrases/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Paraparesia Espástica Tropical/patologia , Paraparesia Espástica Tropical/virologia , Multimerização Proteica , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Vírus Linfotrópico T Tipo 1 de Símios/genética , Imagem Individual de Molécula , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
Diabetes ; 69(11): 2523-2535, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32868339

RESUMO

HLA-DQA1 and -DQB1 genes have significant and potentially causal associations with autoimmune type 1 diabetes (T1D). To follow up on the earlier analysis on high-risk HLA-DQ2.5 and DQ8.1, the current analysis uncovers seven residues (αa1, α157, α196, ß9, ß30, ß57, and ß70) that are resistant to T1D among subjects with DQ4-, 5-, 6-, and 7-resistant DQ haplotypes. These 7 residues form 13 common motifs: 6 motifs are significantly resistant, 6 motifs have modest or no associations (P values >0.05), and 1 motif has 7 copies observed among control subjects only. The motifs "DAAFYDG," "DAAYHDG," and "DAAYYDR" have significant resistance to T1D (odds ratios [ORs] 0.03, 0.25, and 0.18; P = 6.11 × 10-24, 3.54 × 10-15, and 1.03 × 10-21, respectively). Remarkably, a change of a single residue from the motif "DAAYHDG" to "DAAYHSG" (D to S at ß57) alters the resistance potential, from resistant motif (OR 0.15; P = 3.54 × 10-15) to a neutral motif (P = 0.183), the change of which was significant (Fisher P value = 0.0065). The extended set of linked residues associated with T1D resistance and unique to each cluster of HLA-DQ haplotypes represents facets of all known features and functions of these molecules: antigenic peptide binding, peptide-MHC class II complex stability, ß167-169 RGD loop, T-cell receptor binding, formation of homodimer of α-ß heterodimers, and cholesterol binding in the cell membrane rafts. Identification of these residues is a novel understanding of resistant DQ associations with T1D. Our analyses endow potential molecular approaches to identify immunological mechanisms that control disease susceptibility or resistance to provide novel targets for immunotherapeutic strategies.


Assuntos
Motivos de Aminoácidos/genética , Diabetes Mellitus Tipo 1/genética , Antígenos HLA-DQ/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequência de Aminoácidos , Regulação da Expressão Gênica , Predisposição Genética para Doença , Antígenos HLA-DQ/genética , Haplótipos , Humanos , Modelos Moleculares , Conformação Proteica
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