RESUMO
BORIS (for brother of the regulator of imprinted sites), a paralogue of the transcription factor, CTCF, is a novel member of the cancer-testis antigen family. The aims of the present study were as follows: (1) to investigate BORIS expression in breast cells and tumours using immunohistochemical staining, western and real-time RT-PCR analyses and (2) assess potential correlation between BORIS levels in tumours with clinical/pathological parameters. BORIS was detected in all 18 inspected breast cell lines, but not in a primary normal breast cell culture. In 70.7% (41 of 58 cases) BORIS was observed in breast tumours. High levels of BORIS correlated with high levels of progesterone receptor (PR) and oestrogen receptor (ER). The link between BORIS and PR/ER was further confirmed by the ability of BORIS to activate the promoters of the PR and ER genes in the reporter assays. Detection of BORIS in a high proportion of breast cancer patients implies potential practical applications of BORIS as a molecular biomarker of breast cancer. This may be important for diagnosis of the condition and for the therapeutic use of BORIS. The ability of BORIS to activate promoters of the RP and ER genes points towards possible involvement of BORIS in the establishment, progression and maintenance of breast tumours.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/metabolismo , Biomarcadores Tumorais/análise , Mama/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/genética , Expressão Gênica , Humanos , Imuno-Histoquímica , Regiões Promotoras Genéticas , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismoRESUMO
Two permuted variants of S6 ribosomal protein were obtained in direct and fusion expression systems, respectively. The product of direct expression contained the extra N-terminal methionine residue. The structural properties and conformational stability of these permuteins were compared using 1-D (1)H-NMR, circular dichroism, intrinsic fluorescence, differential scanning calorimetry and resistance to urea-induced unfolding. A pronounced difference in all the parameters studied has been demonstrated. This means that the structure of recombinant protein can be sensitive to peculiarities of the expression and purification procedures, leading particularly to the presence or absence of the Met at the first position in the target protein sequence.
Assuntos
Proteínas Ribossômicas/genética , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Escherichia coli/genética , Vetores Genéticos , Espectroscopia de Ressonância Magnética , Metionina/química , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteína S6 Ribossômica , Proteínas Ribossômicas/química , Thermus thermophilus/genética , Triptofano/química , UreiaRESUMO
Structural properties and conformational stability of de novo proteins -- albebetin and albeferon (albebetin with a grafted interferon fragment) -- were studied by means of CD spectroscopy, gel filtration and urea-induced unfolding. The results allow us to conclude that albebetin possesses the properties of the molten globule state. Grafting of the octapeptide to the N-terminus of this de novo protein affects its structure. We show here that albeferon maintains a secondary structure content of albebetin; it becomes more compact and much more stable toward urea-induced unfolding as compared to albebetin and even possesses some weak tertiary structure (at least around Tyr7). This means that the structure of the artificial protein albebetin can be improved by a simple procedure of octapeptide grafting to its N-terminus.
Assuntos
Oligopeptídeos/química , Proteínas/química , Cromatografia em Gel , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/genética , Engenharia de Proteínas , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Termodinâmica , Ureia/químicaRESUMO
Ribosomal protein S6 from Thermus thermophilus was modified to form the unusual unique topology designed earlier for a de novo protein albebetin. The S6 gene was cloned, sequenced and circularly permutated by means of genetic engineering methods. The permutated gene was expressed in Escherichia coli and the permutein was isolated and investigated by means of circular dichroism, fluorescence spectroscopy and scanning microcalorimetry. The permutated protein revealed a pronounced secondary structure close to that of the wild type S6 protein and a rigid tertiary structure possessing cooperative temperature melting. It means that the unusual new topology of albebetin is compatible with a rigid tertiary structure, it may be realized in natural proteins and it is not responsible for the absence of rigid structure in albebetin.
