Assuntos
Simulação de Dinâmica Molecular , Papillomaviridae , Sítios de Ligação , DNA , Ligação ProteicaRESUMO
Kruppel-like Factors (KLF) are responsible for regulating many genes involved in physiological and pathological processes. They are characterized by three conserved zinc-fingers in the DNA-binding domain, wherein mutations could affect the binding efficiency and transcription regulation. This study aimed to perform bioinformatics analysis to determine the most deleterious non-synonymous variants in KLFs involved in cardiac development and diseases, and their effects over the protein structure and stability. Eight hundred and fifty non-synonymous variants were found in seven KLFs related to cardiac diseases. Seventeen algorithms were used to predict the effect of selected variants over the structure and function of seven KLFs. The Top3 variants were selected in each category of conserved and non-conserved residues in the zinc-finger (ZF) domain. KLF5 p.Cys410Phe was the only variant predicted as deleterious in all algorithms, occurring in a conserved residue of zinc ion interaction. KLF15 p.Arg364Pro was the only variant predicted to affect the DNA-binding, and also occurs in a conserved ZF-domain. Our bioinformatics analysis determined potential variants that may lead to development of cardiac diseases, as well as reinforced the importance of KLF analysis in vitro and in vivo.
Assuntos
Cardiopatias/genética , Fatores de Transcrição Kruppel-Like/genética , Mutação de Sentido Incorreto , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Biologia Computacional , Sequência Conservada , Humanos , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/metabolismo , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Dedos de Zinco/genéticaRESUMO
Cervical cancer is primarily caused by Human papillomavirus (HPV) infection, but other factors such as smoking habits, co-infections and genetic background, can also contribute to its development. Although this cancer is avoidable, it is the fourth most frequent type of cancer in females worldwide and can only be treated with chemotherapy and radical surgery. There is a need for biomarkers that will enable early diagnosis and targeted therapy for this type of cancer. Therefore, a systems biology pipeline was applied in order to identify potential biomarkers for cervical cancer, which show significant reports in three molecular aspects: DNA sequence variants, DNA methylation pattern and alterations in mRNA/protein expression levels. CDH1, CDKN2A, RB1 and TP53 genes were selected as putative biomarkers, being involved in metastasis, cell cycle regulation and tumour suppression. Other ten genes (CDH13, FHIT, PTEN, MLH1, TP73, CDKN1A, CACNA2D2, TERT, WIF1, APC) seemed to play a role in cervical cancer, but the lack of studies prevented their inclusion as possible biomarkers. Our results highlight the importance of these genes. However, further studies should be performed to elucidate the impact of DNA sequence variants and/or epigenetic deregulation and altered expression of these genes in cervical carcinogenesis and their potential as biomarkers for cervical cancer diagnosis and prognosis.
Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Polimorfismo de Nucleotídeo Único , Transcriptoma , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Antígenos CD , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p18/genética , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Análise de Sequência de DNA , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Spermadhesins are the major proteins of boar seminal plasma and form a group of polypeptides probably involved in reproduction. In previous work, a member of the spermadhesin family from buck seminal plasma, called BSFP, was characterized by mass spectrometry and N-terminal sequencing. The present study aimed to clone and characterize the BSFP gene and investigate its expression along the genital tract using real-time polymerase chain reaction (PCR). The cDNAs of the seminal vesicle, testis, epididymis, bulbourethral gland, and ductus deferens were prepared from a buck. Following 3'- and 5'-end amplifications using seminal vesicle cDNA, we cloned and sequenced four highly similar (97-98%) nucleotide sequences encoding spermadhesins, which were named Bodhesin-1(Bdh-1), Bdh-2, Bdh-3, and Bdh-4. All deduced amino acid sequences contained the CUB domain signature and were 49-52% similar to boar AWN. Among the four Bdh amino acid sequences, Bdh-2 was the most similar to the BSFP N-terminal fragment. By using real-time PCR, it was verified specific amplifications for all Bdh in the seminal vesicle, testis, epididymis, and bulbourethral gland, with the exception of Bdh-2 in epididymis. The amplicons had a melting temperature and size of approximately 78 degrees C and 130 bp, respectively. Bdh expression was higher in the seminal vesicle when compared to the other tissues. The present work confirms that goat is the fifth mammalian species, after pig, cattle, horse, and sheep, in which spermadhesin molecules are found. To the best of our knowledge, this is the first report on buck spermadhesin genes using molecular cloning and expression profile.
