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1.
J Plant Physiol ; 264: 153487, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34358944

RESUMO

AtCYP38, a thylakoid lumen localized immunophilin, is found to be essential for photosystem II assembly and maintenance, but how AtCYP38 functions in chloroplast remains unknown. Based on previous functional studies and its crystal structure, we hypothesize that AtCYP38 should function via binding its targets or cofactors in the thylakoid lumen. To identify potential interacting proteins of AtCYP38, we first adopted ATTED-II and STRING web-tools, and found 12 proteins functionally related to AtCYP38. We then screened a yeast two-hybrid library including an Arabidopsis genome wide cDNA with different domain of AtCYP38, and five thylakoid lumen-localized targets were identified. In order to specifically search interacting proteins of AtCYP38 in the thylakoid lumen, we generated a yeast two-hybrid mini library including the thylakoid lumenal proteins and lumenal fractions of thylakoid membrane proteins, and we obtained six thylakoid membrane proteins and nine thylakoid lumenal proteins as interacting proteins of AtCYP38. The interactions between AtCYP38 and several potential targets were further confirmed via pull-down and co-immunoprecipitation assays. Together, a couple of new potential candidate interacting proteins of AtCYP38 were identified, and the results will lay a foundation for unveiling the regulatory mechanisms in photosynthesis by AtCYP38.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Ciclofilinas/metabolismo , Proteínas de Arabidopsis/fisiologia , Ciclofilinas/fisiologia , Imunoprecipitação , Complexo de Proteína do Fotossistema II/metabolismo , Domínios e Motivos de Interação entre Proteínas , Técnicas do Sistema de Duplo-Híbrido
2.
Vet Microbiol ; 258: 109099, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33984791

RESUMO

Poxviruses have evolved multiple strategies to modulate host-derived factors to create an optimal environment for viral efficient replication. Our previous study indicated that cyclophilin B (CypB) is a critical factor for ORFV replication in MDBK cells. However, the precise molecular mechanism by which CypB facilitates ORFV replication remains less understood. In the present study, the function of CypB in ORFV replication is further evaluated. The overexpression of CypB was observed to facilitate ORFV replication in OFTu cells and HeLa cells, however, RNA interference (RNAi)-mediated reduction of endogenous CypB decreased the levels of ORFV replication. Coimmunoprecipitation experiments revealed that the CypB interacted with ORFV ORF058 protein, a late protein involved in virus entry. The interaction of host factor CypB and ORF058 protein was further confirmed by confocal microscopy analysis and GST-pull down. In addition, the 52-55 aa was identified as the critical binding sites for CypB on ORF058 protein by GST-pull down with OFTu cells overexpressing CypB and purified GST-tagged truncated ORF058. In conclusion, we demonstrate that CypB is a critical host factor for ORFV replication in vitro by interacting with ORF058 protein, providing new insights into ORFV pathogenesis.


Assuntos
Ciclofilinas/metabolismo , Regulação da Expressão Gênica/imunologia , Vírus do Orf/efeitos dos fármacos , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Ovinos , Regulação para Cima
3.
Front Immunol ; 12: 609196, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33859635

RESUMO

Cyclophilins (Cyps) are a group of peptidyl-prolyl cis/trans isomerases that play crucial roles in regulatory mechanisms of cellular physiology and pathology in several inflammatory conditions. Their receptor, CD147, also participates in the development and progression of the inflammatory response. Nevertheless, the main function of Cyps and their receptor are yet to be deciphered. The release of CypA and the expression of the CD147 receptor in activated T lymphocytes were already described, however, no data are available about other Cyps in these cells. Therefore, in the present work intra and extracellular CypA, B and C levels were measured followed by induced inflammatory conditions. After activation of T lymphocytes by incubation with concanavalin A, both intra and extracellular Cyps levels and the CD147 membrane receptor expression were increased leading to cell migration towards circulating CypA and CypB as chemoattractants. When CypA was modulated by natural and synthetic compounds, the inflammatory cascade was avoided including T cell migration. Our results strengthen the relationship between CypA, B, and C, their receptor, and the inflammatory process in human T lymphocytes, associating CypC with these cells for the first time.


Assuntos
Ciclofilinas/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Biomarcadores , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Ciclofilinas/farmacologia , Suscetibilidade a Doenças , Descoberta de Drogas , Expressão Gênica , Humanos , Inflamação/patologia , Ligantes , Ligação Proteica , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos
4.
Plant Physiol ; 185(2): 503-518, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33721893

RESUMO

Photosynthesis in leaves generates fixed-carbon resources and essential metabolites that support sink tissues, such as roots. Two of these metabolites, sucrose and auxin, promote growth in root systems, but the explicit connection between photosynthetic activity and control of root architecture has not been explored. Through a mutant screen to identify pathways regulating root system architecture, we identified a mutation in the Arabidopsis thaliana CYCLOPHILIN 38 (CYP38) gene, which causes accumulation of pre-emergent stage lateral roots. CYP38 was previously reported to stabilize photosystem II (PSII) in chloroplasts. CYP38 expression is enriched in shoots, and grafting experiments show that the gene acts non-cell-autonomously to promote lateral root emergence. Growth of wild-type plants under low-light conditions phenocopies the cyp38 lateral root emergence defect, as does the inhibition of PSII-dependent electron transport or Nicotinamide adenine dinucleotide phosphate (NADPH) production. Importantly, these perturbations to photosynthetic activity rapidly suppress lateral root emergence, which is separate from their effects on shoot size. Supplementary exogenous sucrose largely rescued primary root (PR) growth in cyp38, but not lateral root growth. Auxin (indole-3-acetic acid (IAA)) biosynthesis from tryptophan is dependent on reductant generated during photosynthesis. Consistently, we found that wild-type seedlings grown under low light and cyp38 mutants have highly diminished levels of IAA in root tissues. IAA treatment rescued the cyp38 lateral root defect, revealing that photosynthesis promotes lateral root emergence partly through IAA biosynthesis. These data directly confirm the importance of CYP38-dependent photosynthetic activity in supporting root growth, and define the specific contributions of two metabolites in refining root architecture under light-limited conditions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Ciclofilinas/metabolismo , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Ciclofilinas/genética , Transporte de Elétrons , Mutação , Fenótipo , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Transdução de Sinais
5.
Biochem Biophys Res Commun ; 552: 44-51, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33743348

RESUMO

Hepatocellular carcinoma (HCC) is the fifth common types of cancer with poor prognosis in the world. Honokiol (HNK), a natural biphenyl compound derived from the magnolia plant, has been reported to exert anticancer effects, but its mechanism has not been elucidated exactly. In the present study, HNK treatment significantly suppressed the migration ability of HepG2 and Hep3B human hepatocellular carcinoma. The treatment reduced the expression levels of the genes associated with cell migration, such as S100A4, MMP-2, MMP-9 and Vimentin. Interestingly, treatment with HNK significantly reduced the expression level of Cyclophilin B (CypB) which stimulates cancer cell migration. However, overexpressed CypB abolished HNK-mediated suppression of cell migration, and reversed the apoptotic effects of HNK. Altogether, we concluded that the suppression of migration activities by HNK was through down-regulated CypB in HCC. These finding suggest that HNK may be a promising candidate for HCC treatment via regulation of CypB.


Assuntos
Compostos de Bifenilo/farmacologia , Carcinoma Hepatocelular/genética , Movimento Celular/efeitos dos fármacos , Ciclofilinas/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lignanas/farmacologia , Neoplasias Hepáticas/genética , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ciclofilinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
6.
PLoS One ; 16(2): e0237956, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606679

RESUMO

Cyp33 is an essential human cyclophilin prolyl isomerase that plays myriad roles in splicing and chromatin remodeling. In addition to a canonical cyclophilin (Cyp) domain, Cyp33 contains an RNA-recognition motif (RRM) domain, and RNA-binding triggers proline isomerase activity. One prominent role for Cyp33 is through a direct interaction with the mixed lineage leukemia protein 1 (MLL1, also known as KMT2A) complex, which is a histone methyltransferase that serves as a global regulator of human transcription. MLL activity is regulated by Cyp33, which isomerizes a key proline in the linker between the PHD3 and Bromo domains of MLL1, acting as a switch between gene activation and repression. The direct interaction between MLL1 and Cyp33 is critical, as deletion of the MLL1-PHD3 domain responsible for this interaction results in oncogenesis. The Cyp33 RRM is central to these activities, as it binds both the PHD3 domain and RNA. To better understand how RNA binding drives the action of Cyp33, we performed RNA-SELEX against full-length Cyp33 accompanied by deep sequencing. We have identified an enriched Cyp33 binding motif (AAUAAUAA) broadly represented in the cellular RNA pool as well as tightly binding RNA aptamers with affinities comparable and competitive with the Cyp33 MLL1-PHD3 interaction. RNA binding extends beyond the canonical RRM domain, but not to the Cyp domain, suggesting an indirect mechanism of interaction. NMR chemical shift mapping confirms an overlapping, but not identical, interface on Cyp33 for RNA and PHD3 binding. This finding suggests RNA can disrupt the gene repressive Cyp33-MLL1 complex providing another layer of regulation for chromatin remodeling by MLL1.


Assuntos
Ciclofilinas/metabolismo , Regulação da Expressão Gênica/genética , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , Ciclofilinas/genética , Proteínas de Ligação a DNA/genética , Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Motivos de Nucleotídeos/genética , Ligação Proteica/genética , RNA/metabolismo , Motivo de Reconhecimento de RNA/genética , Técnica de Seleção de Aptâmeros/métodos , Fatores de Transcrição/metabolismo
7.
Exp Cell Res ; 400(2): 112514, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33582093

RESUMO

Cardiovascular diseases and atherosclerosis are currently some of the most widespread diseases of our time. Within cardiovascular disease, coronary artery disease and underlying atherosclerosis were recently linked with systemic and local inflammation. Cyclophilins participate in the initiation and progression of these inflammatory-related diseases. Cyclophilins are released into the extracellular space upon inflammatory stimuli and participate in the pathology of cardiovascular diseases. The cell surface receptor for extracellular cyclophilins, the CD147 receptor, also contributes to coronary artery disease pathogenesis. Nevertheless, the physiological relevance of cyclophilin's family and their receptor in cardiovascular diseases remains unclear. The present study aimed to better understand the role of cyclophilins in cardiovascular artery disease and their relationship with inflammation. Hence, cyclophilins and pro-inflammatory interleukins were measured in the serum of 30 subjects (divided into three groups according to coronary artery disease status: 10 patients with acute coronary syndrome, 10 patients with chronic coronary artery disease, and 10 control volunteers). In addition, cyclophilin levels and CD147 receptor expression were measured in T lymphocytes purified from these subjects. Cyclophilin A, B, and C, pro-inflammatory interleukins, and CD147 membrane expression were significantly elevated in patients with coronary artery disease.


Assuntos
Basigina/metabolismo , Comunicação Celular , Doença da Artéria Coronariana/patologia , Ciclofilinas/metabolismo , Interleucinas/metabolismo , Linfócitos T/imunologia , Estudos de Casos e Controles , Doença da Artéria Coronariana/imunologia , Doença da Artéria Coronariana/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T/metabolismo
8.
Int J Mol Sci ; 22(4)2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567582

RESUMO

Cyclophilin (Cyp) and Ca2+/calcineurin proteins are cellular components related to fungal morphogenesis and virulence; however, their roles in mediating the pathogenesis of Botrytis cinerea, the causative agent of gray mold on over 1000 plant species, remain largely unexplored. Here, we show that disruption of cyclophilin gene BcCYP2 did not impair the pathogen mycelial growth, osmotic and oxidative stress adaptation as well as cell wall integrity, but delayed conidial germination and germling development, altered conidial and sclerotial morphology, reduced infection cushion (IC) formation, sclerotial production and virulence. Exogenous cyclic adenosine monophosphate (cAMP) rescued the deficiency of IC formation of the ∆Bccyp2 mutants, and exogenous cyclosporine A (CsA), an inhibitor targeting cyclophilins, altered hyphal morphology and prevented host-cell penetration in the BcCYP2 harboring strains. Moreover, calcineurin-dependent (CND) genes are differentially expressed in strains losing BcCYP2 in the presence of CsA, suggesting that BcCyp2 functions in the upstream of cAMP- and Ca2+/calcineurin-dependent signaling pathways. Interestingly, during IC formation, expression of BcCYP2 is downregulated in a mutant losing BcJAR1, a gene encoding histone 3 lysine 4 (H3K4) demethylase that regulates fungal development and pathogenesis, in B. cinerea, implying that BcCyp2 functions under the control of BcJar1. Collectively, our findings provide new insights into cyclophilins mediating the pathogenesis of B. cinerea and potential targets for drug intervention for fungal diseases.


Assuntos
Botrytis/patogenicidade , Ciclofilinas/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , Esporos Fúngicos/crescimento & desenvolvimento , Adaptação Fisiológica , Ciclofilinas/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Folhas de Planta/microbiologia , Virulência
9.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530556

RESUMO

Although the mitochondrial permeability transition pore (PTP) is presumably formed by either ATP synthase or the ATP/ADP carrier (AAC), little is known about their differential roles in PTP activation. We explored the role of AAC and ATP synthase in PTP formation in Saccharomyces cerevisiae using bisindolylpyrrole (BP), an activator of the mammalian PTP. The yeast mitochondrial membrane potential, as indicated by tetramethylrhodamine methyl ester signals, dissipated over 2-4 h after treatment of cells with 5 µM BP, which was sensitive to cyclosporin A (CsA) and Cpr3 deficiency and blocked by porin1/2 deficiency. The BP-induced depolarization was inhibited by a specific AAC inhibitor, bongkrekate, and consistently blocked in a yeast strain lacking all three AACs, while it was not affected in the strain with defective ATP synthase dimerization, suggesting the involvement of an AAC-associated pore. Upon BP treatment, isolated yeast mitochondria underwent CsA- and bongkrekate-sensitive depolarization without affecting the mitochondrial calcein signals, indicating the induction of a low conductance channel. These data suggest that, upon BP treatment, yeast can form a porin1/2- and Cpr3-regulated PTP, which is mediated by AACs but not by ATP synthase dimers. This implies that yeast may be an excellent tool for the screening of PTP modulators.


Assuntos
Ciclofilinas/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Porinas/metabolismo , Pirróis/farmacologia , Leveduras/efeitos dos fármacos , Leveduras/fisiologia , Ciclofilinas/genética , Relação Dose-Resposta a Droga , Potencial da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Permeabilidade , Porinas/genética , Multimerização Proteica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/fisiologia
10.
Biochemistry ; 60(8): 597-606, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33591178

RESUMO

The multifunctional protein p53 is the central molecular sensor of cellular stresses. The canonical function of p53 is to transcriptionally activate target genes in response to, for example, DNA damage that may trigger apoptosis. Recently, p53 was also found to play a role in the regulation of necrosis, another type of cell death featured by the mitochondrial permeability transition (mPT). In this process, p53 directly interacts with the mPT regulator cyclophilin D, the detailed mechanism of which however remains poorly understood. Here, we report a comprehensive computational investigation of the p53-cyclophilin D interaction using molecular dynamics simulations and associated analyses. We have identified the specific cyclophilin D binding site on p53 that is located at proline 151 in the DNA binding domain. As a peptidyl-prolyl isomerase, cyclophilin D binds p53 and catalyzes the cis-trans isomerization of the peptide bond preceding proline 151. We have also characterized the effect of such an isomerization and found that the p53 domain in the cis state is overall more rigid than the trans state except for the local region around proline 151. Dynamical changes upon isomerization occur in both local and distal regions, indicating an allosteric effect elicited by the isomerization. We present potential allosteric communication pathways between proline 151 and distal sites, including the DNA binding surface. Our work provides, for the first time, a model for how cyclophilin D binds p53 and regulates its activity by switching the configuration of a specific site.


Assuntos
Ciclofilinas/metabolismo , DNA/metabolismo , Simulação de Dinâmica Molecular , Prolina/química , Proteína Supressora de Tumor p53/metabolismo , Sítios de Ligação , Catálise , Ciclofilinas/química , Ciclofilinas/genética , DNA/química , Humanos , Prolina/metabolismo , Domínios Proteicos , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genética
11.
Sci Rep ; 11(1): 91, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420123

RESUMO

Beauveria bassiana is a species complex whose isolates show considerable natural genetic variability. However, little is known about how this genetic diversity affects the fungus performance. Herein, we characterized the diversity of genes involved in various mechanisms of the infective cycle of 42 isolates that have different growth rates, thermotolerance and virulence. The analysed genes showed general genetic diversity measured as non-synonymous changes (NSC) and copy number variation (CNV), with most of them being subjected to positive episodic diversifying selection. Correlation analyses between NSC or CNV and the isolate virulence, thermotolerance and growth rate revealed that various genes shaped the biological features of the fungus. Lectin-like, mucin signalling, Biotrophy associated and chitinase genes NSCs correlated with the three biological features of B. bassiana. In addition, other genes (i.e. DNA photolyase and cyclophilin B) that had relatively conserved sequences, had variable CNs across the isolates which were correlated with the variability of either virulence or thermotolerance of B. bassiana isolates. The data obtained is important for a better understanding of population structure, ecological and potential impact when isolates are used as mycoinsecticides and can justify industrialization of new isolates.


Assuntos
Beauveria/genética , Beauveria/patogenicidade , Insetos/microbiologia , Animais , Beauveria/classificação , Beauveria/crescimento & desenvolvimento , Quitinases/genética , Quitinases/metabolismo , Ciclofilinas/genética , Ciclofilinas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Variação Genética , Filogenia , Virulência
12.
Science ; 371(6535)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33509932

RESUMO

The minor spliceosome mediates splicing of the rare but essential U12-type precursor messenger RNA. Here, we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-angstrom resolution. The 5' splice site and branch point sequence of the U12-type intron are recognized by the U6atac and U12 small nuclear RNAs (snRNAs), respectively. Five newly identified proteins stabilize the conformation of the catalytic center: The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome, the RBM48-ARMC7 complex binds the γ-monomethyl phosphate cap at the 5' end of U6atac snRNA, the U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 small nuclear ribonucleoprotein (snRNP), and CRIPT stabilizes U12 snRNP. Our study provides a framework for the mechanistic understanding of the function of the human minor spliceosome.


Assuntos
Spliceossomos/química , Spliceossomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/metabolismo , Microscopia Crioeletrônica , Ciclofilinas/química , Ciclofilinas/metabolismo , Humanos , Íntrons , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Domínios Proteicos , Precursores de RNA/química , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , RNA Nuclear Pequeno/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo
13.
Planta ; 252(4): 50, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32939624

RESUMO

MAIN CONCLUSION: The phloem-mobile protein SlCyp1 traffics to distant parts of the shoot to regulate its gravitropic response. In addition, SlCyp1 targets specific cells in the root to promote lateral root development. The tomato (Solanum lycopersicum) Cyclophilin 1 (SlCyp1) gene encodes a peptidyl-prolyl isomerase required for auxin response, lateral root development and gravitropic growth. The SlCyp1 protein is a phloem-mobile signal that moves from shoot to root to regulate lateral root development (Spiegelman et al., Plant J 83:853-863, 2015; J Exp Bot 68:953-964, 2017a). Here, we explored the mechanism of SlCyp1 movement by fusing it to the fluorescent protein mCherry. We found that, once trafficked to the root, SlCyp1 is unloaded from the phloem to the surrounding tissues, including the pericycle and lateral root primordia. Interestingly, SlCyp1 not only moves to the root system, but also to distant parts of the shoot. Grafting of the SlCyp1 mutant diageotropica (dgt) scions on VFN8 control rootstocks resulted in recovery of dgt shoot gravitropism, which was associated with the restoration of auxin-response capacity. Application of the cyclophilin inhibitor cyclosporine A suppressed gravitropic recovery, indicating that SlCyp1 must be active in the target tissue to affect the gravitropic response. These results provide new insights on the mechanism of SlCyp1 transport and functioning as a long-distance signal regulating shoot gravitropism.


Assuntos
Ciclofilinas , Gravitropismo , Lycopersicon esculentum , Brotos de Planta , Ciclofilinas/genética , Ciclofilinas/metabolismo , Ácidos Indolacéticos/metabolismo , Lycopersicon esculentum/genética , Floema , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento
14.
Acc Chem Res ; 53(9): 1782-1790, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32658452

RESUMO

RNA-based technologies to control gene expression, such as RNA interference (RNAi) and CRISPR-Cas9, have become powerful tools in molecular biology and genomics. The exciting potential that RNAi and CRISPR-Cas9 may also become new therapeutic approaches has reinvigorated interest in chemically modifying RNA to improve its properties for in vivo applications. Chemical modifications can improve enzymatic stability, in vivo delivery, cellular uptake, and sequence specificity as well as minimize off-target activity of short interfering RNAs (siRNAs) and CRISPR associated RNAs. While numerous good solutions for improving stability toward enzymatic degradation have emerged, optimization of the latter functional properties remains challenging. In this Account, we discuss synthesis, structure, and biological activity of novel nonionic analogues of RNA that have the phosphodiester backbone replaced by amide linkages (AM1). Our long-term goal is to use the amide backbone to improve the stability and specificity of siRNAs and other functional RNAs. Our work in this area was motivated by early discoveries that nonionic backbone modifications, including AM1, did not disturb the overall structure or thermal stability of RNA duplexes. We hypothesized that the reduced negative charge and hydrophobic nature of the AM1 backbone modification might be useful in optimizing functional applications through enhanced cellular uptake, and might suppress unwanted off-target effects of siRNAs. NMR and X-ray crystallography studies showed that AM1 was an excellent mimic of phosphodiester linkages in RNA. The local conformational changes caused by the amide linkages were easily accommodated by small adjustments in RNA's conformation. Further, the amide carbonyl group assumed an orientation that is similar to one of the nonbridging P-O bonds, which may enable amide/phosphate mimicry by conserving hydrogen bonding interactions. The crystal structure of a short amide-modified DNA-RNA hybrid in complex with RNase H indicated that the amide N-H could also act as an H-bond donor to stabilize RNA-protein interactions, which is an interaction mode not available to phosphate groups. Functional assays established that amides were well tolerated at internal positions in both strands of siRNAs. Surprisingly, amide modifications in the middle of the guide strand and at the 5'-end of the passenger strand increased RNAi activity compared to unmodified siRNA. Most importantly, an amide linkage between the first and second nucleosides of the passenger strand completely abolished its undesired off-target activity while enhancing the desired RNAi activity. These results suggest that RNAi may tolerate more substantial modifications of siRNAs than the chemistries tried so far. The findings are also important and timely because they demonstrate that amide modifications may reduce off-target activity of siRNAs, which remains an important roadblock for clinical use of RNAi. Taken together, our work suggests that amide linkages have underappreciated potential to optimize the biological and pharmacological properties of RNA. Expanded use of amide linkages in RNA to enhance CRISPR and other technologies requiring chemically stable, functional mimics of noncoding RNAs is expected.


Assuntos
Amidas/química , Interferência de RNA , RNA Interferente Pequeno/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Ciclofilinas/antagonistas & inibidores , Ciclofilinas/genética , Ciclofilinas/metabolismo , Dimerização , Edição de Genes , Células HeLa , Humanos , Nucleosídeos/química , Estabilidade de RNA , RNA Interferente Pequeno/metabolismo
15.
Bioorg Med Chem Lett ; 30(14): 127251, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32527551

RESUMO

HCV utilizes cellular protein cyclophilins in the virus replication cycle and cyclophilin inhibitors have become a new class of anti-HCV agents. In our screening of natural products, we identified a unique cyclosporin analogue, FR901459, as a cyclophilin inhibitor with potent anti-HCV activity. In this work, we developed an efficient synthetic methodology to prepare FR901459 derivatives via an N, O-acyl migration reaction. This method allows us to efficiently manipulate the amino acid residues at the 3 position while avoiding lengthy total synthesis for each compound. By using this methodology, we discovered 4, which has superior anti-HCV activity and decreased immunosuppressive activity compared to FR901459.


Assuntos
Antivirais/farmacologia , Ciclosporina/farmacologia , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Imunossupressores/farmacologia , Antivirais/síntese química , Antivirais/química , Ciclofilinas/antagonistas & inibidores , Ciclofilinas/metabolismo , Ciclosporina/síntese química , Ciclosporina/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Imunossupressores/síntese química , Imunossupressores/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade
16.
Nucleic Acids Res ; 48(10): 5670-5683, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329775

RESUMO

Human CWC27 is an uncharacterized splicing factor and mutations in its gene are linked to retinal degeneration and other developmental defects. We identify the splicing factor CWC22 as the major CWC27 partner. Both CWC27 and CWC22 are present in published Bact spliceosome structures, but no interacting domains are visible. Here, the structure of a CWC27/CWC22 heterodimer bound to the exon junction complex (EJC) core component eIF4A3 is solved at 3Å-resolution. According to spliceosomal structures, the EJC is recruited in the C complex, once CWC27 has left. Our 3D structure of the eIF4A3/CWC22/CWC27 complex is compatible with the Bact spliceosome structure but not with that of the C complex, where a CWC27 loop would clash with the EJC core subunit Y14. A CWC27/CWC22 building block might thus form an intermediate landing platform for eIF4A3 onto the Bact complex prior to its conversion into C complex. Knock-down of either CWC27 or CWC22 in immortalized retinal pigment epithelial cells affects numerous common genes, indicating that these proteins cooperate, targeting the same pathways. As the most up-regulated genes encode factors involved in inflammation, our findings suggest a possible link to the retinal degeneration associated with CWC27 deficiencies.


Assuntos
Ciclofilinas/química , Fator de Iniciação 4A em Eucariotos/química , Proteínas de Ligação a RNA/química , Spliceossomos/química , Linhagem Celular , Ciclofilinas/genética , Ciclofilinas/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Éxons , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Inflamação/genética , Modelos Moleculares , Domínios Proteicos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Spliceossomos/metabolismo
17.
Biochem Pharmacol ; 177: 113995, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32339494

RESUMO

Few discoveries have influenced drug discovery programs more than the finding that mitochondrial membranes undergo swings in permeability in response to cellular perturbations. The conductor of these permeability changes is the aptly named mitochondrial permeability transition pore which, although not yet precisely defined, is comprised of several integral proteins that differentially act to regulate the flux of ions, proteins and metabolic byproducts during the course of cellular physiological functions but also pathophysiological insults. Pursuit of the pore's exact identity remains a topic of keen interest, but decades of research have unearthed provocative functions for the integral proteins leading to their evaluation to develop novel therapeutics for a wide range of clinical indications. Chief amongst these targeted, integral proteins have been the Voltage Dependent Anion Channel (VDAC) and the F1FO ATP synthase. Research associated with the roles and ligands of VDAC has been extensive and we will expand upon 3 examples of ligand:VDAC interactions for consideration of drug discovery projects: Tubulin:VDAC1, Hexokinase I/II:VDAC1 and olesoxime:VDAC1. The discoveries that cyclosporine blocks mitochondrial permeability transition via binding to cyclophilin D, and that cyclophilin D is an important component of F1FO ATP synthase, has heightened interest in the F1FO ATP synthase as a focal point for drug discovery, and we will discuss 2 plausible campaigns associated with disease indications. To date no drug has emerged from prospective targeting these integral proteins; however, continued exploration such as the approaches suggested in this Commentary will increase the likelihood of providing important therapeutics for severely unmet medical needs.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Colestenonas/uso terapêutico , Ciclosporina/uso terapêutico , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/genética , Canal de Ânion 1 Dependente de Voltagem/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Ciclofilinas/genética , Ciclofilinas/metabolismo , Regulação da Expressão Gênica , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Permeabilidade/efeitos dos fármacos , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Canal de Ânion 1 Dependente de Voltagem/antagonistas & inibidores , Canal de Ânion 1 Dependente de Voltagem/metabolismo
18.
Protein Pept Lett ; 27(8): 744-750, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32096737

RESUMO

BACKGROUND: Peptidyl-prolyl cis-trans isomerase (PPIases) enzyme plays a vital role in protein folding. It catalyses the cis-trans isomerisation of peptide bonds, an essential step for newly synthesized protein to acquire its correct functional conformation in both prokaryotes and eukaryotes. OBJECTIVE: The present study showed the biochemical and molecular characterisation of cyclophilins (PpiB), a type of peptidyl-prolyl isomerases proteins from the pathogenic bacteria Salmonella Typhimurium. METHODS: Salmonella Typhimurium is one of the leading serovars responsible for human and animal salmonellosis globally, with the majority of human cases originating through the food chain. Here successful expression and purification of PpiB protein have been demonstrated and LC-MS based analyses showed high protein score and similarity with other PPi protein. Further the enzymatic activity of the purified recombinant PpiB was determined using Succinyl-Ala-Phe-Pro- Phe-p nitroanilide as substrate and enzyme-catalysed reaction. RESULT: Km and Vmax were calculated and found to be Vm = 1.023 ± .06400 min/µg, Km = 0.6219 ± 0.1701 µM, respectively. We have reported for the first time the presence of Salmonella PPIase-B (PpiB) protein isoforms in salmonella genome having PPi activity. CONCLUSION: Taken together, our data clearly showed that Salmonella Cyclophilin B (PpiB) protein is active and involved in diverse biological processes and highly similar to the different domain of Cyclophilin proteins.


Assuntos
Proteínas de Bactérias/química , Ciclofilinas/química , Peptidilprolil Isomerase/química , Dobramento de Proteína , Salmonella typhimurium/enzimologia , Animais , Proteínas de Bactérias/metabolismo , Ciclofilinas/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Peptidilprolil Isomerase/metabolismo
19.
Aging (Albany NY) ; 12(3): 3010-3024, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041924

RESUMO

Oxygen and glucose deprivation (OGD)-re-oxygenation (OGDR) stimulation to the human endometrial cells mimics ischemia-reperfusion injury. Cyclophilin D (CypD)-dependent programmed necrosis pathway mediates OGDR-induced cytotoxicity to human endometrial cells. We here identified a novel CypD-targeting miRNA, microRNA-1203 (miR-1203). In T-HESC and primary human endometrial cells, ectopic overexpression of miR-1203, using a lentiviral construct, potently downregulated the CypD 3'-untranslated region (3'-UTR) activity and its expression. Both were however upregulated in endometrial cells with forced miR-1203 inhibition by its anti-sense sequence. Functional studies demonstrated that ectopic miR-1203 overexpression in endometrial cells alleviated OGDR-induced programmed necrosis, inhibiting mitochondrial CypD-p53-adenine nucleotide translocator 1 association, mitochondrial depolarization, reactive oxygen species production, and medium lactate dehydrogenase release. Contrarily OGDR-induced programmed necrosis and cytotoxicity were intensified with forced miR-1203 inhibition in endometrial cells. Significantly, ectopic miR-1203 overexpression or inhibition failed to change OGDR-induced cytotoxicity in CypD-knockout T-HESC cells. Furthermore, ectopic miR-1203 overexpression was unable to protect T-HESC endometrial cells from OGDR when CypD was restored by an UTR-depleted CypD construct. Collectively, these results show that miR-1203 targets and silences CypD to protect human endometrial cells from OGDR.


Assuntos
Ciclofilinas/metabolismo , Endométrio/citologia , Glucose/metabolismo , MicroRNAs/metabolismo , Oxigênio/metabolismo , Linhagem Celular , Sobrevivência Celular , Ciclofilinas/genética , Feminino , Deleção de Genes , Regulação da Expressão Gênica/fisiologia , Humanos , MicroRNAs/genética
20.
Sci Rep ; 10(1): 1275, 2020 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988345

RESUMO

In Trichomonas vaginalis, the TvCyP1-catalyzed conformational switches of two glycinyl-prolyl imide bonds in Myb3 were previously shown to regulate the trafficking of Myb3 from cytoplasmic membrane compartments towards the nucleus. In this study, TvCyP2 was identified as a second cyclophilin that binds to Myb3 at the same dipeptide motifs. The enzymatic proficiency of TvCyP2, but not its binding to Myb3, was aborted by a mutation of Arg75 in the catalytic domain. TvCyP2 was localized to the endoplasmic reticulum with a weak signal that extensively extends into the cytoplasm as well as to the plasma membrane according to an immunofluorescence assay. Moreover, TvCyP2 was co-enriched with TvCyP1 and Myb3 in various membrane fractions purified by differential and gradient centrifugation. TvCyP2 was found to proficiently enzymatically regulate the distribution of TvCyP1 and Myb3 among purified membrane fractions, and to localize TvCyP1 in hydrogenosomes and on plasma membranes. Protein complexes immunoprecipitated from lysates of cells overexpressing TvCyP1 and TvCyP2 were found to share some common components, like TvCyP1, TvCyP2, TvBip, Myb3, TvHSP72, and the hydrogenosomal heat shock protein 70 (HSP70). Direct interaction between TvCyP1 and TvCyP2 was confirmed by a GST pull-down assay. Fusion of vesicles with hydrogenosomes was observed by transmission electron microscopy, whereas TvCyP1, TvCyP2, and Myb3 were each detected at the fusion junction by immunoelectron microscopy. These observations suggest that T. vaginalis may have evolved a novel protein trafficking pathway to deliver proteins among the endomembrane compartments, hydrogenosomes and plasma membranes.


Assuntos
Família 2 do Citocromo P450/metabolismo , Transporte Proteico/fisiologia , Trichomonas vaginalis/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Ciclofilinas/metabolismo , Ciclofilinas/fisiologia , Família 2 do Citocromo P450/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Proteínas de Membrana/metabolismo , Mapeamento de Interação de Proteínas , Proteínas de Protozoários/metabolismo , Fatores de Transcrição/metabolismo
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