RESUMO
Considering the enormous importance of protein turns as participants in various biological events, such as protein-protein interactions, great efforts have been made to develop their conformationally and proteolytically stable mimetics. Ferrocene-1,1'-diamine was previously shown to nucleate the stable turn structures in peptides prepared by conjugation with Ala (III) and Ala-Pro (VI). Here, we prepared the homochiral conjugates of ferrocene-1,1'-diamine with l-/d-Phe (32/35), l-/d-Val (33/36), and l-/d-Leu (34/37) to investigate (1) whether the organometallic template induces the turn structure upon conjugation with amino acids, and (2) whether the bulky or branched side chains of Phe, Val, and Leu affect hydrogen bonding. Detailed spectroscopic (IR, NMR, CD), X-ray, and DFT studies revealed the presence of two simultaneous 10-membered interstrand hydrogen bonds, i.e., two simultaneous ß-turns in goal compounds. A preliminary biological evaluation of d-Leu conjugate 37 showed its modest potential to induce cell cycle arrest in the G0/G1 phase in the HeLa cell line but these results need further investigation.
Assuntos
Diaminas , Dipeptídeos , Humanos , Metalocenos/química , Ligação de Hidrogênio , Células HeLa , Cristalografia por Raios X , Estereoisomerismo , Dipeptídeos/química , Peptídeos/química , Aminoácidos/química , Conformação ProteicaRESUMO
Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a 'gate' for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Triptofano/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Domínio Catalítico/genética , Dicroísmo Circular , DNA/química , DNA/genética , DNA Helicases/química , DNA Helicases/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Humanos , Mutação de Sentido Incorreto , Conformação Proteica , Homologia de Sequência de Aminoácidos , Temperatura , Triptofano/química , Triptofano/genéticaRESUMO
Background: Many genome- and epigenome-wide association studies (GWAS and EWAS) and studies of promoter methylation of candidate genes for inflammatory bowel disease (IBD) have demonstrated significant associations between genetic and epigenetic changes and IBD. Independent GWA studies have identified genetic variants in the BACH2, IL6ST, LAMB1, IKZF1, and MGAT3 loci to be associated with both IBD and immunoglobulin G (IgG) glycosylation. Methods: Using bisulfite pyrosequencing, we analyzed CpG methylation in promoter regions of these five genes from peripheral blood of several hundred IBD patients and healthy controls (HCs) from two independent cohorts, respectively. Results: We found significant differences in the methylation levels in the MGAT3 and BACH2 genes between both Crohn's disease and ulcerative colitis when compared to HC. The same pattern of methylation changes was identified for both genes in CD19+ B cells isolated from the whole blood of a subset of the IBD patients. A correlation analysis was performed between the MGAT3 and BACH2 promoter methylation and individual IgG glycans, measured in the same individuals of the two large cohorts. MGAT3 promoter methylation correlated significantly with galactosylation, sialylation, and bisecting GlcNAc on IgG of the same patients, suggesting that activity of the GnT-III enzyme, encoded by this gene, might be altered in IBD. The correlations between the BACH2 promoter methylation and IgG glycans were less obvious, since BACH2 is not a glycosyltransferase and therefore may affect IgG glycosylation only indirectly. Conclusions: Our results suggest that epigenetic deregulation of key glycosylation genes might lead to an increase in pro-inflammatory properties of IgG in IBD through a decrease in galactosylation and sialylation and an increase of bisecting GlcNAc on digalactosylated glycan structures. Finally, we showed that CpG methylation in the promoter of the MGAT3 gene is altered in CD3+ T cells isolated from inflamed mucosa of patients with ulcerative colitis from a third smaller cohort, for which biopsies were available, suggesting a functional role of this glyco-gene in IBD pathogenesis.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Metilação de DNA , Imunoglobulina G/metabolismo , Doenças Inflamatórias Intestinais/genética , N-Acetilglucosaminiltransferases/genética , Estudos de Casos e Controles , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Doença de Crohn/genética , Doença de Crohn/metabolismo , Epigênese Genética , Feminino , Estudo de Associação Genômica Ampla , Humanos , Doenças Inflamatórias Intestinais/imunologia , Masculino , Polissacarídeos/metabolismo , Regiões Promotoras Genéticas , Estudos Prospectivos , Análise de Sequência de DNARESUMO
BACKGROUND: The RAS association domain family protein 1a (RASSF1A) is a prominent tumor suppressor gene showing altered promoter methylation in testicular germ cell tumors (TGCT). RASSF1A promoter hypermethylation might represent an early event in TGCT tumorigenesis. We investigated whether the RASSF1A promoter methylation in peripheral blood of TGCT patients can be associated with testicular cancer risk. MATERIALS AND METHODS: Following a meta-analysis, we performed a cohort study including 32 testicular cancer patients and 32 healthy controls. Promoter methylation of the RASSF1A and O6-methylguanine-DNA-methyltransferase (MGMT) genes was analyzed using bisulfite pyrosequencing of DNA from peripheral blood. RESULTS: Meta-analysis showed an odds ratio (OR) of 7.69 for RASSF1A promoter methylation as a risk factor for TGCT. Cohort study found altered methylation of the RASSF1A promoter in blood of TGCT patients. Methylation was higher in TGCT patients before BEP chemotherapy. CONCLUSION: The meta-analysis indicates a role of the RASSF1A promoter hypermethylation from peripheral blood in TCGT. We confirmed that finding in our cohort study, which represents the first report of changed RASSF1A promoter methylation in peripheral blood TGCT.
Assuntos
Metilação de DNA/genética , Neoplasias Embrionárias de Células Germinativas/genética , Regiões Promotoras Genéticas , Neoplasias Testiculares/genética , Proteínas Supressoras de Tumor/genética , Sequência de Bases , Estudos de Coortes , Ilhas de CpG/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Predisposição Genética para Doença , Humanos , Masculino , Razão de Chances , Prognóstico , Viés de Publicação , Fatores de RiscoRESUMO
Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2'-deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that the MGAT3 gene might be responsible for the glycan changes consistently induced by both doses of 5-aza-2dC. Core-fucosylated tetra-antennary structures were decreased in quantity likely as a result of hypomethylated MGAT3 gene promoter followed by increased expression of this gene.