RESUMO
BACKGROUND: Mycobacterium tuberculosis is a virulent bacillus causing tuberculosis, a disease responsible for million deaths each year worldwide. In order to understand its mechanism of pathogenesis in humans and to help control tuberculosis, functions of numerous Mycobacterium tuberculosis genes are being characterized. In this study we report the dual functionality of tlyA gene product of Mycobacterium tuberculosis annotated as Rv1694, a 268 amino acid long basic protein. RESULTS: The recombinant purified Rv1694 protein was found to exhibit hemolytic activity in vitro. It showed concentration and time-dependent hemolysis of rabbit and human erythrocytes. Multiple oligomeric forms (dimers to heptamers) of this protein were seen on the membranes of the lysed erythrocytes. Like the oligomers of conventional, well-known, pore-forming toxins, the oligomers of Rv1694 were found to be resistant to heat and SDS, but were susceptible to reducing agents like ß-mercaptoethanol as it had abolished the hemolytic activity of Rv1694 indicating the role of disulfide bond(s). The Rv1694 generated de novo by in vitro transcription and translation also exhibited unambiguous hemolysis confirming the self assembly and oligomerization properties of this protein. Limited proteolytic digestion of this protein has revealed that the amino terminus is susceptible while in solution but is protected in presence of membrane. Striking feature of Rv1694 is its presence on the cell wall of E. coli as visualized by confocal microscopy. The surface expression is consistent with the contact dependent haemolytic ability of E. coli expressing this protein. Also, immune serum specific to this protein inhibits the contact dependent hemolysis. Moreover, Rv1694 protein binds to and forms stable oligomers on the macrophage phagosomal membranes. In addition to all these properties, E. coli expressing Rv1694 was found to be susceptible to the antibiotic capreomycin as its growth was significantly slower than mock vector transformed E. coli. The S30 extract of E. coli expressing the Rv1694 had poor translational activity in presence of capreomycin, further confirming its methylation activity. Finally, incorporation of methyl group of [3H]-S-adenosylmethionine in isolated ribosomes also confirmed its methylation activity. CONCLUSIONS: The Rv1694 has an unusual dual activity. It appears to contain two diverse functions such as haemolytic activity and ribosomal RNA methylation activity. It is possible that the haemolytic activity might be relevant to intra-cellular compartments such as phagosomes rather than cell lysis of erythrocytes and the self-assembly trait may have a potential role after successful entry into macrophages by Mycobacterium tuberculosis.
Assuntos
Proteínas de Bactérias/química , Proteínas Hemolisinas/química , Metiltransferases/química , Mycobacterium tuberculosis/metabolismo , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capreomicina/farmacologia , Dicroísmo Circular , Metilação de DNA , Eritrócitos/efeitos dos fármacos , Eritrócitos/imunologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Hemólise , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Ligação Proteica , Estrutura Terciária de Proteína , RNA Ribossômico/metabolismo , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Plant lectins have been reported to affect the proliferation of different human cancer cell line probably by binding to the specific carbohydrate moieties. In the present study, Badan labeled single cysteine mutant (present in the caveolin-1 binding motif) of jacalin (rJacalin) was found to penetrate the target membrane, indicating a protein-protein or protein-membrane interaction apart from its primary mode of binding i.e. protein-carbohydrate interaction. Further, Jacalin treatment has resulted in the movement of the GFP-Caveolin-1 predominantly at the cell-cell contact region with much restricted dynamics. Jacalin treatment has resulted in the perinuclear accumulation of PP2A and dissociation of the PHAP1/PP2A complex. PP2A was found to act as a negative regulator of ERK signaling and a significant decrease in the phosphorylation level of MEK and AKT (T308) in A431. In addition, we have also identified several ER resident proteins including molecular chaperones like ORP150, Hsp70, Grp78, BiP of A431 cells, which were bound to the Jacalin-sepharose column. Among various ER chaperones that were identified, ORP150 was found to present on the cell surface of A431 cells.
Assuntos
Cavéolas/enzimologia , Retículo Endoplasmático/enzimologia , Chaperonas Moleculares/metabolismo , Lectinas de Plantas/farmacologia , Proteína Fosfatase 2/metabolismo , Sequência de Aminoácidos , Proliferação de Células , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico HSP70 , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas/metabolismo , Células Tumorais CultivadasRESUMO
Keratins, the epithelial-predominant members of the intermediate filament superfamily, are expressed in a pairwise, tissuespecific and differentiation-dependent manner. There are 28 type I and 26 type II keratins, which share a common structure comprising a central coiled coil α-helical rod domain flanked by two nonhelical head and tail domains. These domains harbor sites for major posttranslational modifications like phosphorylation and glycosylation, which govern keratin function and dynamics. Apart from providing structural support, keratins regulate various signaling machinery involved in cell growth, motility, apoptosis etc. However, tissue-specific functions of keratins in relation to cell proliferation and differentiation are still emerging. Altered keratin expression pattern during and after malignant transformation is reported to modulate different signaling pathways involved in tumor progression in a context-dependent fashion. The current review focuses on the literature related to the role of keratins in the regulation of cell proliferation, differentiation and transformation in different types of epithelia.
Assuntos
Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Queratinas/genética , Neoplasias/genética , Processamento de Proteína Pós-Traducional , Acetilação , Animais , Apoptose/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Glicosilação , Humanos , Queratinas/química , Queratinas/classificação , Queratinas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Estrutura Secundária de Proteína , Transdução de SinaisRESUMO
Taxol treatment of HeLa cells resulted in a transient recruitment of Caveolin-1 to the cell surface followed by internalization. Interestingly, 20min after 10-deacetylbaccatinIII (10-DAB) treatment, the caveolae displayed faster 'kiss and run' dynamics while BaccatinIII (BacIII) did not induce any change. Sustained phosphorylation of Caveolin-1 is observed upon treatment and between Taxol and 10-DAB, the former shows phosphorylated Raf-1, ERK1/2 and hyperphosphorylated Bcl-2 while the later showed much less magnitude of the same. BacIII treatment did not induce phosphorylation of Raf-1 or Bcl-2. It is possible that Taxol might act on multiple targets and the side chain may be crucial.
Assuntos
Antineoplásicos/farmacologia , Cavéolas/efeitos dos fármacos , Caveolina 1/metabolismo , Paclitaxel/farmacologia , Taxoides/farmacologia , Cavéolas/metabolismo , Caveolina 1/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-raf/metabolismoRESUMO
Keratins 5/14 (K5/14) are intermediate filament proteins expressed in the basal layer of stratified epithelial cells and are known targets of p63. Previous research in our laboratory showed that upon K5/14 downregulation in oral squamous cell carcinoma (OSCC)derived cells, there was an increase in intracellular Notch1 levels and differentiation markers such as involucrin, keratin 1 and a decrease in tumorigenic potential in vivo. However, the molecules involved in the K14 regulated cell differentiation and transformation are not known to date. In order to understand the possible role of TAp63, we downregulated TAp63 in a K14knockdown background. We observed that there was a decrease in the expression of Notch1. Expression levels of differentiation markers such as involucrin, K1, loricrin and filaggrin were also decreased. Furthermore, TAp63 downregulation led to an increase in invasion, migration and in vivo tumorigenic potential of these cells. We observed a decrease in ßcatenin signaling in K14downregulated cells. Notably, when TAp63 was downregulated in K14knockdown cells, there was increase in nonphospho ßcatenin levels. Hence, this study indicates that TAp63 plays an important role in K14downregulated cells possibly by regulating the Notch1 expression. K14 regulates the expression of TAp63 which in turn regulates expression of Notch1. The present study is a step forward in our quest to understand the functional significance of molecules that regulate the process of differentiation and tumorigenesis in stratified epithelial cells.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Queratina-14/metabolismo , Queratina-5/metabolismo , Neoplasias Bucais/metabolismo , Receptor Notch1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Regulação para Baixo , Proteínas Filagrinas , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Transplante de NeoplasiasAssuntos
Caspase 1/metabolismo , Cavéolas/metabolismo , Toxinas Bacterianas/farmacologia , Membrana Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Células HeLa , Humanos , Potássio/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
BACKGROUND: Wild type Staphylococcal alpha-hemolysin (alpha-HL) assembly on target mammalian cells usually results in necrotic form of cell death; however, caspase activation also occurs. The pathways of caspase activation due to binding/partial assembly by alpha-HL are unknown till date. RESULTS: Cells treated with H35N (a mutant of alpha-HL that remains as membrane bound monomer), have been shown to accumulate hypodiploid nuclei, activate caspases and induce intrinsic mitochondrial apoptotic pathway. We have earlier shown that the binding and assembly of alpha-HL requires functional form of Caveolin-1 which is an integral part of caveolae. In this report, we show that the caveolae of mammalian cells, which undergo a continuous cycle of 'kiss and run' dynamics with the plasma membrane, have become immobile upon the binding of the monomer. The cells treated with H35N were unable to recover despite activation of membrane repair mechanism involving caspase-1 dependent activation of sterol regulatory element binding protein-1. CONCLUSIONS: This is for the first time we show the range of cellular changes and responses that take place immediately after the binding of the monomeric form of staphylococcal alpha-hemolysin.