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1.
J Agric Food Chem ; 67(33): 9411-9422, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31393126

RESUMO

Royal jelly (RJ) is secreted by young worker bees, and it plays key roles in the development and physiological function in honeybees and can improve human health. Although there have been analyses on the glycosylation modification of RJ proteins, none of these methods have been conducted on a site-specific analysis of glycosylation from these glycoproteins. Here, a combined glycomics and glycoproteomics strategy was developed for the site-specific analysis of N-linked glycosylation heterogeneity of RJ glycoproteins. First, global characterization of the N-glycome of RJ was performed using a direct infusion ion trap-sequential mass spectrometry (IT-MSn) method. Second, tryptic glycopeptides were enriched and separated by hydrophilic interaction liquid chromatography-ion trap-sequential mass spectrometry (HILIC-IT-MSn). A total of 50 N-glycopeptides and 30 N-glycans have been site-specific glycosylation profiled in major royal jelly protein 1 (MRJP1) and MRJP2 of RJ for the first time. Eighteen of the identified N-glycans have been structurally characterized by IT-MSn, including oligosaccharide composition, sequence, branching, and linkage. Two N-glycosylation sites (N177 and N394), 3 sites (N145, N178, and N92), and 1 site of N183 were identified in MRJP1, MRJP2, and MRJP3, respectively. There were 18, 17, and 2 N-glycans attached to MRJP1, MRJP2, and MRJP3, respectively. The diversity of N-glycans attached to each single glycosylation site of these glycoproteins confirmed that MRJP1 and MRJP2 heterogeneity was mostly associated with their glycoform populations. Understanding the properties of the site-specific glycosylation heterogeneity of the RJ glycoproteins can be potentially useful for producing a glycoprotein with desirable pharmacokinetic and biological activity.


Assuntos
Ácidos Graxos/química , Glicoproteínas/química , Proteínas de Insetos/química , Motivos de Aminoácidos , Animais , Abelhas , Cromatografia Líquida , Glicômica , Glicosilação , Espectrometria de Massas em Tandem
2.
BMC Plant Biol ; 19(1): 370, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438851

RESUMO

BACKGROUND: Accumulating evidences show that SPLs are crucial regulators of plant abiotic stress tolerance and the highly conserved module miR156/SPL appears to balance plant growth and stress responses. The halophyte Tamarix chinensis is highly resistant to salt tress. SPLs of T. chinensis (TcSPLs) and theirs roles in salt stress responses remain elusive. RESULTS: In this study, we conducted a systematic analysis of the TcSPLs gene family including 12 members belonging to 7 groups. The physicochemical properties and conserved motifs showed divergence among groups and similarity in each group. The microRNA response elements (MREs) are conserved in location and sequence, with the exception of first MRE within TcSPL5. The miR156-targeted SPLs are identified by dual-luciferase reporter assay of MRE-miR156 interaction. The digital expression gene profiles cluster suggested potential different functions of miR156-targeted SPLs vs non-targeted SPLs in response to salt stress. The expression patterns analysis of miR156-targeted SPLs with a reverse expression trend to TcmiR156 suggested 1 h (salt stress time) could be a critical time point of post-transcription regulation in salt stress responses. CONCLUSIONS: Our work demonstrated the post-transcription regulation of miR156-targeted TcSPLs and transcription regulation of non-targeted TcSPLs in salt stress responses, and would be helpful to expound the miR156/SPL-mediated molecular mechanisms underlying T. chinensis salt stress tolerance.


Assuntos
MicroRNAs/fisiologia , Proteínas de Plantas/fisiologia , RNA de Plantas/fisiologia , Estresse Salino/genética , Tamaricaceae/genética , Fatores de Transcrição/fisiologia , Motivos de Aminoácidos , Sequência Conservada , Genes de Plantas , Família Multigênica , Filogenia , Transcriptoma
3.
Nat Commun ; 10(1): 2898, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31263112

RESUMO

The HIV-1 envelope (Env) is the target for neutralizing antibodies and exists on the surface of virions in open or closed conformations. Difficult-to-neutralize viruses (tier 2) express Env in a closed conformation antigenic for broadly neutralizing antibodies (bnAbs) but not for third variable region (V3) antibodies. Here we show that select V3 macaque antibodies elicited by Env vaccination can neutralize 26% of otherwise tier 2 HIV-1 isolates in standardized virus panels. The V3 antibodies only bound to Env in its open conformation. Thus, Envs on tier 2 viruses sample a state where the V3 loop is not in its closed conformation position. Envelope second variable region length, glycosylation sites and V3 amino acids were signatures of neutralization sensitivity. This study determined that open conformations of Env with V3 exposed are present on a subset of otherwise neutralization-resistant virions, therefore neutralization of tier 2 HIV-1 does not always indicate bnAb induction.


Assuntos
Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Motivos de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Glicosilação , Infecções por HIV/virologia , HIV-1/química , HIV-1/genética , Humanos , Macaca mulatta , Testes de Neutralização , Conformação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
4.
J Agric Food Chem ; 67(32): 9079-9087, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31353905

RESUMO

Organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transporters expressed in the human liver. However, epigallocatechin gallate (EGCG), which is the most predominant catechin in green tea, has opposite effects on the function of OATP1B1 and OATP1B3. In the present study, the critical structural domains and amino acid residues for the activation of OATP1B3 by EGCG have been determined by characterizing the function of a series of OATP1B3-derived chimeric transporters, site-directed mutagenesis, and kinetic studies. Our results showed that G45 and F555 in transmembrane domains 1 and 10 are the most important amino acid residues for OATP1B3 activation. Kinetic studies showed that the activation of OATP1B3 by EGCG at a low substrate concentration was due to its increased substrate binding affinity. However, EGCG caused increased Km and decreased Vmax for 1B3-G45A and 1B3-F555H. The flexibility at position 45 and aromaticity at position 555 might be important for OATP1B3 activation. While 1B3-G45A and 1B3-F555H could not be activated by EGCG, their transport activity for EGCG was comparable to that of wild-type OATP1B3. In conclusion, the present study elucidated the molecular mechanism for OATP1B3 activation by EGCG.


Assuntos
Catequina/análogos & derivados , Extratos Vegetais/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/química , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Motivos de Aminoácidos , Camellia sinensis/química , Catequina/química , Catequina/metabolismo , Células HEK293 , Humanos , Cinética , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/química , Transportador 1 de Ânion Orgânico Específico do Fígado/genética , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Modelos Moleculares , Extratos Vegetais/química , Domínios Proteicos , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/genética
5.
Molecules ; 24(13)2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31288444

RESUMO

Recently, we have found that calcium binding proteins of the EF-hand superfamily (i.e., a large family of proteins containing helix-loop-helix calcium binding motif or EF-hand) contain two types of conserved clusters called cluster I ('black' cluster) and cluster II ('grey' cluster), which provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domains. Cluster I is more conservative and mostly incorporates aromatic amino acids, whereas cluster II includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. Recoverin is EF-hand Ca2+-binding protein containing two 'black' clusters comprised of F35, F83, Y86 (N-terminal domain) and F106, E169, F172 (C-terminal domain) as well as two 'gray' clusters comprised of F70, Q46, F49 (N-terminal domain) and W156, K119, V122 (C-terminal domain). To understand a role of these residues in structure and function of human recoverin, we sequentially substituted them for alanine and studied the resulting mutants by a set of biophysical methods. Under metal-free conditions, the 'black' clusters mutants (except for F35A and E169A) were characterized by an increase in the α-helical content, whereas the 'gray' cluster mutants (except for K119A) exhibited the opposite behavior. By contrast, in Ca2+-loaded mutants the α-helical content was always elevated. In the absence of calcium, the substitutions only slightly affected multimerization of recoverin regardless of their localization (except for K119A). Meanwhile, in the presence of calcium mutations in N-terminal domain of the protein significantly suppressed this process, indicating that surface properties of Ca2+-bound recoverin are highly affected by N-terminal cluster residues. The substitutions in C-terminal clusters generally reduced thermal stability of recoverin with F172A ('black' cluster) as well as W156A and K119A ('gray' cluster) being the most efficacious in this respect. In contrast, the mutations in the N-terminal clusters caused less pronounced differently directed changes in thermal stability of the protein. The substitutions of F172, W156, and K119 in C-terminal domain of recoverin together with substitution of Q46 in its N-terminal domain provoked significant but diverse changes in free energy associated with Ca2+ binding to the protein: the mutant K119A demonstrated significantly improved calcium binding, whereas F172A and W156A showed decrease in the calcium affinity and Q46A exhibited no ion coordination in one of the Ca2+-binding sites. The most of the N-terminal clusters mutations suppressed membrane binding of recoverin and its inhibitory activity towards rhodopsin kinase (GRK1). Surprisingly, the mutant W156A aberrantly activated rhodopsin phosphorylation regardless of the presence of calcium. Taken together, these data confirm the scaffolding function of several cluster-forming residues and point to their critical role in supporting physiological activity of recoverin.


Assuntos
Recoverina/química , Recoverina/metabolismo , Alanina/química , Motivos de Aminoácidos , Substituição de Aminoácidos , Cálcio/metabolismo , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Recoverina/genética , Rodopsina/metabolismo
6.
J Agric Food Chem ; 67(31): 8626-8631, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31287307

RESUMO

An almond allergen with two known short peptide sequences was reported as the almond 2S albumin but was later suspected to be almond vicilin. However, this allergen was not designated by the World Health Organization/International Union of Immunological Societies. This study aimed to determine the true identity of this elusive almond allergen. cDNAs were synthesized from total RNA of the Nonpareil almond. The complete sequence of the previously reported almond allergen was determined from its coding sequence. The deduced protein was produced recombinantly and was confirmed to be a food allergen by testing with 18 almond-allergic sera. The allergen is a potential cysteine-rich antimicrobial protein with characteristic C[X]3C-[X]10-12-C[X]3C motifs of the hairpinin antimicrobial protein. This first member of a novel family of food allergens was named Pru du 8. The signature motif of the hairpinin antimicrobial protein can be found in the N-terminal region of some vicilin allergens (e.g., Ara h 1). It can also be found in the signal peptide of other vicilin allergens (e.g., Car i 2). In many species, however, vicilins do not contain such a motif, indicating that the presence of the signature motifs of the hairpinin antimicrobial protein in vicilins might be a result of translocation during evolution.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Prunus dulcis/imunologia , Alérgenos/química , Alérgenos/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/genética , DNA Complementar/genética , Hipersensibilidade Alimentar/imunologia , Humanos , Prunus dulcis/química , Prunus dulcis/genética , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA
7.
DNA Cell Biol ; 38(8): 824-839, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31295023

RESUMO

Tea plant is an important economic crop on a global scale. Its yield and quality are affected by abiotic stress. The calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) family genes play irreplaceable roles in plant development and stress resistance. More and more CBL-CIPK genes have been identified, but a few CBL-CIPK genes have been cloned and characterized in tea plants. In this study, 7 CsCBLs and 18 CsCIPKs were identified based on the tea plant genome. Physicochemical properties, phylogenetic, conserved motifs, gene structure, homologous gene network, and promoter upstream elements of these 25 genes were analyzed. Conserved motifs of these genes varied with phylogenetic tree node. From the genetic structure, members of the tea plant CIPK gene family can be divided into two types: intron rich and no intron. Many stress-related elements were found in the 2000 bp upstream of the promoter, and PlantCARE predicted that CsCBL4 contained 30 stress-related elements. PlantPAN2 shows that CsCIPK6 contains 48 ABRELATERD1; CsCIPK17 contains 37 GT1CONSENSUS; CsCIPK3 contains 64 MYBCOREATCYCB1; CsCBL3 contains 52 SORLIP1AT; CsCBL5 contains 65 SURECOREATSULTR11; and CsCIPK11 contains 83 WBOXATNPR1. In addition, eight genes were selected for quantitative real-time PCR (RT-qPCR) to detect their expression profiles under high-temperature, low-temperature, salt, and drought treatments. These genes were found to be responsive to one or more abiotic stress treatments. The expression levels of CsCBL4, CsCIPK2, and CsCIPK14 were similar, and they were homologous to AtSOS3 and AtSIP3 and AtSIP4 in Arabidopsis, which were involved in the SOS pathway. This study provides insight into the potential functions of the CsCBL and CsCIPK of tea plant.


Assuntos
Camellia sinensis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinases/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Camellia sinensis/fisiologia , Sequência Conservada , Secas , Evolução Molecular , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Anotação de Sequência Molecular , Filogenia , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética
8.
Nat Commun ; 10(1): 2746, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227712

RESUMO

Nicotinic acetylcholine receptors (nAChRs) mediate and modulate synaptic transmission throughout the brain, and contribute to learning, memory, and behavior. Dysregulation of α7-type nAChRs in neuropsychiatric as well as immunological and oncological diseases makes them attractive targets for pharmaceutical development. Recently, we identified NACHO as an essential chaperone for α7 nAChRs. Leveraging the robust recombinant expression of α7 nAChRs with NACHO, we utilized genome-wide cDNA library screening and discovered that several anti-apoptotic Bcl-2 family proteins further upregulate receptor assembly and cell surface expression. These effects are mediated by an intracellular motif on α7 that resembles the BH3 binding domain of pro-apoptotic Bcl-2 proteins, and can be blocked by BH3 mimetic Bcl-2 inhibitors. Overexpression of Bcl-2 member Mcl-1 in neurons enhanced surface expression of endogenous α7 nAChRs, while a combination of chemotherapeutic Bcl2-inhibitors suppressed neuronal α7 receptor assembly. These results demonstrate that Bcl-2 proteins link α7 nAChR assembly to cell survival pathways.


Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neurônios/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Motivos de Aminoácidos/genética , Animais , Benzotiazóis/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células HEK293 , Humanos , Isoquinolinas/farmacologia , Chaperonas Moleculares/metabolismo , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Cultura Primária de Células , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Ratos , Transmissão Sináptica/efeitos dos fármacos , Tiofenos/farmacologia , Regulação para Cima , Receptor Nicotínico de Acetilcolina alfa7/genética
9.
Gene ; 710: 161-169, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31153884

RESUMO

The glycerol-3-phosphate dehydrogenase (GPD) gene family plays a major role in glycerol synthesis and adaptation to abiotic stresses. Few studies on GPD family genes from the halotolerant algae Dunaliella salina are available. In this study, seven DsaGPD genes were identified by mining D. salina sequencing data. Among them, DsaGPD5 contained the canonical NAD+-GPD protein domain, called si-GPD. In comparison, DsaGPD1-4 not only contained the canonical NAD+-GPD domain but also a unique domain, the haloacid dehalogenase (HAD)-like superfamily domain, in their N-terminal region, called bi-GPD. DsaGPD6, 7 contained the FAD+-GPD domain. In the transient expression system, DsaGPD1, 3, 4 were found in the cytosol of Arabidopsis thaliana protoplast, DsaGPD2, 5 in the chloroplast, and DsaGPD6, 7 in the mitochondria. MEME analysis showed that six conserved motifs were present in both si-GPDs and bi-GPDs, whereas seven highly conserved motifs were only present in bi-GPDs. The quantitative real-time PCR results showed significant induction of the DsaGPD genes under abiotic stresses, indicating their tolerance-related role in D. salina. DsaGPD2 and DsaGPD5 may be the osmoregulator form and glyceride form in the chloroplast, respectively. The evolutionary forces acting on si-GPDs and bi-GPDs were different in the same organism: bi-GPDs were under purifying selection, while si-GPDs were mainly under positive selection. Furthermore, evolution of the N_HAD domain and C_GPD domain in bi-GPDs is highly correlated. In summary, this study characterizes DsaGPD gene family members and provides useful information for elucidating the salt tolerance mechanism in D. salina.


Assuntos
Clorofíceas/enzimologia , Mineração de Dados/métodos , Glicerolfosfato Desidrogenase/química , Glicerolfosfato Desidrogenase/genética , Proteínas de Algas/química , Proteínas de Algas/genética , Motivos de Aminoácidos , Clorofíceas/genética , Cloroplastos/enzimologia , Evolução Molecular , Mitocôndrias/enzimologia , Família Multigênica , Filogenia , Domínios Proteicos , Análise de Sequência de DNA
10.
Nat Commun ; 10(1): 2493, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175300

RESUMO

Tauopathies are neurodegenerative diseases characterized by intracellular amyloid deposits of tau protein. Missense mutations in the tau gene (MAPT) correlate with aggregation propensity and cause dominantly inherited tauopathies, but their biophysical mechanism driving amyloid formation is poorly understood. Many disease-associated mutations localize within tau's repeat domain at inter-repeat interfaces proximal to amyloidogenic sequences, such as 306VQIVYK311. We use cross-linking mass spectrometry, recombinant protein and synthetic peptide systems, in silico modeling, and cell models to conclude that the aggregation-prone 306VQIVYK311 motif forms metastable compact structures with its upstream sequence that modulates aggregation propensity. We report that disease-associated mutations, isomerization of a critical proline, or alternative splicing are all sufficient to destabilize this local structure and trigger spontaneous aggregation. These findings provide a biophysical framework to explain the basis of early conformational changes that may underlie genetic and sporadic tau pathogenesis.


Assuntos
Agregação Patológica de Proteínas/genética , Tauopatias/genética , Proteínas tau/genética , Motivos de Aminoácidos/genética , Simulação por Computador , Células HEK293 , Humanos , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Mutação de Sentido Incorreto , Agregação Patológica de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas tau/metabolismo , Proteínas tau/ultraestrutura
11.
Vet Microbiol ; 233: 140-146, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176400

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), and is characterized by respiratory diseases in piglet and reproductive disorders in sow. Identification of sustainable and effective measures to mitigate PRRSV transmission is a pressing problem. The nucleocapsid (N) protein of PRRSV plays a crucial role in inhibiting host innate immunity during PRRSV infection. In the current study, a new host-restricted factor, tripartite motif protein 25 (TRIM25), was identified as an inhibitor of PRRSV replication. Co-immunoprecipitation assay indicated that the PRRSV N protein interferes with TRIM25-RIG-I interactions by competitively interacting with TRIM25. Furthermore, N protein inhibits the expression of TRIM25 and TRIM25-mediated RIG-I ubiquitination to suppress interferon ß production. Furthermore, with increasing TRIM25 expression, the inhibitory effect of N protein on the ubiquitination of RIG-I diminished. These results indicate for the first time that TRIM25 inhibits PRRSV replication and that the N protein antagonizes the antiviral activity by interfering with TRIM25-mediated RIG-I ubiquitination. This not only provides a theoretical basis for the development of drugs to control PRRSV replication, but also better explains the mechanism through which the PRRSV N protein inhibits innate immune responses of the host.


Assuntos
Proteína DEAD-box 58/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Proteínas com Motivo Tripartido/antagonistas & inibidores , Proteínas com Motivo Tripartido/genética , Ubiquitinação , Motivos de Aminoácidos , Animais , Linhagem Celular , Cercopithecus aethiops , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Proteínas do Nucleocapsídeo/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Ligação Proteica , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Suínos , Transfecção , Replicação Viral
12.
Nat Commun ; 10(1): 2673, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209208

RESUMO

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , RNA/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Consenso/genética , Éxons/genética , Biblioteca Gênica , Genes Reporter/genética , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida/métodos , Domínios Proteicos/genética , Isoformas de Proteínas/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
13.
Chem Commun (Camb) ; 55(54): 7752-7755, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31204733

RESUMO

Metal-binding peptides are versatile building blocks in supramolecular chemistry. We recently reported a class of crystalline materials formed through a combination of coiled-coil peptide self-association and metal coordination. Here, we probe the serendipitously discovered metal binding motif that drives the assembly and apply these insights to exert rational control over structure and morphology in the materials.


Assuntos
Metaloproteínas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Cobre/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Metaloproteínas/síntese química , Engenharia de Proteínas/métodos , Multimerização Proteica , Piridinas/química
14.
Genes Dev ; 33(13-14): 814-827, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171703

RESUMO

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors.


Assuntos
Núcleo Celular/metabolismo , DNA/biossíntese , Leucemia Promielocítica Aguda/fisiopatologia , Mitose , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , Homeostase do Telômero/genética , Motivos de Aminoácidos , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Leucemia Promielocítica Aguda/genética , Fenótipo , Transporte Proteico , Proteína SUMO-1/metabolismo , Telômero/genética , Telômero/metabolismo
15.
J Sci Food Agric ; 99(13): 5881-5889, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31206698

RESUMO

BACKGROUND: The suppression of α-glucosidase activity to retard glucose absorption is an important therapy for type-2 diabetes. Corosolic acid (CRA) is a potential antidiabetic component in many plant-based foods and herbs. In this study, the interplay mechanism between α-glucosidase and corosolic acid was investigated by several methods, including three-dimensional fluorescence spectra, circular dichroism spectra, and molecular simulation. RESULTS: Corosolic acid significantly inhibited α-glucosidase reversibly in an uncompetitive manner and its IC50 value was 1.35 × 10-5 mol L-1 . A combination of CRA with myricetin exerted a weak synergy against α-glucosidase. The intrinsic fluorescence of α-glucosidase was quenched via a static quenching course and the binding constant was 3.47 × 103 L mol-1 at 298 K. The binding of CRA to α-glucosidase was mainly driven by hydrophobic forces and resulted in a partial extension of the protein polypeptide chain with a loss of α-helix content. The molecular simulation illustrated that CRA bound to the entrance part of the active center of α-glucosidase and interacted with the amino acid residues Ser157, Arg442, Phe303, Arg315, Tyr158, and Gln353, which could hinder the release of substrate and catalytic reaction product, eventually suppressing the catalytic activity of α-glucosidase. CONCLUSIONS: These results may suggest new insights into corosolic acid from food sources as a potential α-glucosidase inhibitor that could better control diabetes. © 2019 Society of Chemical Industry.


Assuntos
Inibidores Enzimáticos/química , Triterpenos/química , alfa-Glucosidases/química , Motivos de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Humanos , Hipoglicemiantes/química , Simulação de Acoplamento Molecular
16.
Genome Biol ; 20(1): 115, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159833

RESUMO

BACKGROUND: Hox transcription factors specify segmental diversity along the anterior-posterior body axis in metazoans. While the different Hox family members show clear functional specificity in vivo, they all show similar binding specificity in vitro and a satisfactory understanding of in vivo Hox target selectivity is still lacking. RESULTS: Using transient transfection in Kc167 cells, we systematically analyze the binding of all eight Drosophila Hox proteins. We find that Hox proteins show considerable binding selectivity in vivo even in the absence of canonical Hox cofactors Extradenticle and Homothorax. Hox binding selectivity is strongly associated with chromatin accessibility, being highest in less accessible chromatin. Individual Hox proteins exhibit different propensities to bind less accessible chromatin, and high binding selectivity is associated with high-affinity binding regions, leading to a model where Hox proteins derive binding selectivity through affinity-based competition with nucleosomes. Extradenticle/Homothorax cofactors generally facilitate Hox binding, promoting binding to regions in less accessible chromatin but with little effect on the overall selectivity of Hox targeting. These cofactors collaborate with Hox proteins in opening chromatin, in contrast to the pioneer factor, Glial cells missing, which facilitates Hox binding by independently generating accessible chromatin regions. CONCLUSIONS: These studies indicate that chromatin accessibility plays a key role in Hox selectivity. We propose that relative chromatin accessibility provides a basis for subtle differences in binding specificity and affinity to generate significantly different sets of in vivo genomic targets for different Hox proteins.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Drosophila , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/metabolismo
17.
Plant Physiol Biochem ; 141: 388-397, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31226508

RESUMO

Members of the Jasmonate ZIM domain (JAZ) proteins act as transcriptional repressors in the jasmonate (JA) hormonal response. To characterize the potential roles of JAZ gene family in plant development and abiotic stress response, fifteen JAZs were identified based on the genome of Nicotiana tabacum. Structural analysis confirmed the presence of single Jas and TIFY motif. Tissue expression pattern analysis indicated that NtJAZ-2, -3, -5, and -10 were highly expressed in roots and NtJAZ-11 was expressed only in the cotyledons. The transcript level of NtJAZ-3, -5, -9, and -10 in the stem epidermis was higher than that in the stem without epidermis. Dynamic expression of NtJAZs exposed to abiotic stress and phytohormone indicated that the expression of most NtJAZs was activated by salicylic acid, methyl jasmonate, gibberellic acid, cold, salt, and heat stresses. With abscisic acid treatment, NtJAZ-1, -2, and -3 were not activated; NtJAZ-4, -5, and -6 were up-regulated; and the remaining NtJAZ genes were inhibited. With drought stress, the expression of NtJAZ-1, -2, -3, -4, -5, -6, -7, and -8 was up-regulated, whereas the transcript of the remaining genes was inhibited. Moreover, high concentration MeJA (more than 1 mM MeJA) had an effect on secreting trichome induction, but inhabited the plant growth. Nine NtJAZs may play important role in secreting trichome induction. These results indicate that the JAZ proteins are convergence points for various phytohormone signal networks, which are involved in abiotic stress responses.


Assuntos
Proteínas de Plantas/metabolismo , Proteínas Repressoras/metabolismo , Estresse Fisiológico , Tabaco/genética , Tabaco/metabolismo , Tricomas/metabolismo , Acetatos/metabolismo , Motivos de Aminoácidos , Arabidopsis/metabolismo , Cromossomos/metabolismo , Ciclopentanos/metabolismo , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Cadeias de Markov , Oxilipinas/metabolismo , Filogenia , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição/genética
18.
Arch Virol ; 164(9): 2309-2314, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31172288

RESUMO

The surface (SU) and transmembrane (TM) glycoproteins of many retroviruses are linked by disulphide bonds, and the interaction of SU with a cellular receptor results in disulphide bond isomerisation triggered by the CXXC motif in SU. This reaction leads to the fusion of viral and host cell membranes. In this work, we show that the cysteine at amino acid position 212 in the CAIC motif of the SU glycoprotein of bovine leukaemia virus has a free thiol group. A C-to-A mutation at position 212, either individually or in combination with a C-to-A mutation at position 215, was found to inhibit the maturation process, suggesting its involvement in the formation of the covalent bond with TM.


Assuntos
Cisteína/metabolismo , Leucose Enzoótica Bovina/virologia , Vírus da Leucemia Bovina/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Internalização do Vírus , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Sequência Conservada , Cisteína/genética , Vírus da Leucemia Bovina/química , Vírus da Leucemia Bovina/isolamento & purificação , Vírus da Leucemia Bovina/fisiologia , Glicoproteínas de Membrana/genética , Mutação
19.
Nat Commun ; 10(1): 2834, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249292

RESUMO

Environmental information perceived by chloroplasts can be translated into retrograde signals that alter the expression of nuclear genes. Singlet oxygen (1O2) generated by photosystem II (PSII) can cause photo-oxidative damage of PSII but has also been implicated in retrograde signaling. We previously reported that a nuclear-encoded chloroplast FtsH2 metalloprotease coordinates 1O2-triggered retrograde signaling by promoting the degradation of the EXECUTER1 (EX1) protein, a putative 1O2 sensor. Here, we show that a 1O2-mediated oxidative post-translational modification of EX1 is essential for initiating 1O2-derived signaling. Specifically, the Trp643 residue in DUF3506 domain of EX1 is prone to oxidation by 1O2. Both the substitution of Trp643 with 1O2-insensitive amino acids and the deletion of the DUF3506 domain abolish the EX1-mediated 1O2 signaling. We thus provide mechanistic insight into how EX1 senses 1O2 via Trp643 located in the DUF3506 domain.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastídeos/metabolismo , Oxigênio Singlete/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Oxirredução , Plastídeos/química , Plastídeos/genética , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Transdução de Sinais
20.
BMC Plant Biol ; 19(1): 191, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31072335

RESUMO

BACKGROUND: BRASSINAZOLE-RESISTANT (BZR) family genes encode plant-specific transcription factors (TFs) that participate in brassinosteroid signal transduction. BZR TFs have vital roles in plant growth, including cell elongation. However, little is known about BZR genes in sugar beet (Beta vulgaris L.). RESULTS: Therefore, we performed a genome-wide investigation of BvBZR genes in sugar beet. Through an analysis of the BES1_N conserved domain, six BvBZR gene family members were identified in the sugar beet genome, which clustered into three subgroups according to a phylogenetic analysis. Each clade was well defined by the conserved motifs, implying that close genetic relationships could be identified among the members of each subfamily. According to chromosomal distribution mapping, 2, 1, 1, 1, and 1 genes were located on chromosomes 1, 4, 5, 6, and 8, respectively. The cis-acting elements related to taproot growth were randomly distributed in the promoter sequences of the BvBZR genes. Tissue-specific expression analyses indicated that all BvBZR genes were expressed in all three major tissue types (roots, stems, and leaves), with significantly higher expression in leaves. Subcellular localization analysis revealed that Bv1_fxre and Bv6_nyuw are localized in the nuclei, consistent with the prediction of Wolf PSORT. CONCLUSION: These findings offer a basis to predict the functions of BZR genes in sugar beet, and lay a foundation for further research of the biological functions of BZR genes in sugar beet.


Assuntos
Beta vulgaris/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Fatores de Transcrição/genética , Motivos de Aminoácidos , Beta vulgaris/efeitos dos fármacos , Cromossomos de Plantas/genética , Sequência Conservada/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Proteínas de Fluorescência Verde/metabolismo , Motivos de Nucleotídeos/genética , Filogenia , Reguladores de Crescimento de Planta/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
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