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1.
Mol Microbiol ; 81(5): 1390-401, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21797942

RESUMO

Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane. It is widely recognized that LF, and the related fusion protein LFnDTA, must be completely denatured in order to transit through the PA63 formed pore and enter the eukaryotic cell cytosol. We demonstrate by protease protection assays that the molecular chaperone GRP78 mediates the unfolding of LFnDTA and LF at neutral pH and thereby converts these proteins from a trypsin resistant to sensitive conformation. We have used immunoelectron microscopy and gold-labelled antibodies to demonstrate that both GRP78 and GRP94 chaperones are present in the lumen of endosomal vesicles. Finally, we have used siRNA to demonstrate that knock-down of GRP78 results in the emergence of resistance to anthrax lethal toxin and oedema toxin action.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Bacillus anthracis/química , Bacillus anthracis/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Transporte Proteico/fisiologia , Animais , Antraz/metabolismo , Antraz/microbiologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Citosol/enzimologia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/genética , Glicoproteínas de Membrana/biossíntese , Camundongos , Desdobramento de Proteína , Interferência de RNA , RNA Interferente Pequeno , Tripsina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
2.
Mol Microbiol ; 76(4): 1010-9, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20398220

RESUMO

The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1 with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild-type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.


Assuntos
Complexo I de Proteína do Envoltório/metabolismo , Toxina Diftérica/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Bovinos , Linhagem Celular , Toxina Diftérica/química , Toxina Diftérica/genética , Humanos , Lisina/genética , Lisina/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Transporte Proteico
3.
J Histochem Cytochem ; 66(8): 549-561, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29587004

RESUMO

Phosphorylation is the most extensively studied posttranslational modification of proteins. There are approximately 500 kinases known in the human genome. The kinase-activated pathways regulate almost every aspect of cell function and a deregulated kinase cascade leads to impaired cellular function. Impaired regulation of several kinase cascades, including the epidermal growth factor receptor (EGFR) pathway, leading to tumor pathogenesis, is well documented. Thus, a phosphospecific test with prognostic or predictive value was expected in oncology. However, no phosphospecific IHC test is used in oncology clinics. Human topoisomerase I (topoI) inhibitors, camptothecin and its analogues (CPT), are used extensively to treat various solid tumors. Depending on tumor type, the response rate is only 13-32%. We have demonstrated that the deregulated kinase cascade is at the core of CPT resistance. DNA-PKcs, a kinase central to the DNA-double-strand break (DSB) response pathway, phosphorylates topoI at serine 10 (topoI-pS10), and cells with higher basal levels of topoI-pS10 degrade topoI rapidly and are resistant to this class of drug. The higher basal level of topoI phosphorylation is due to continual activation of DNA-PKcs, and one potential mechanism of this pathway activation is failure of upstream effector phosphatases such as phosphatase and tensin homolog (PTEN). Based on this understanding, we have developed an IHC-based test (P-topoIDx) that can stratify the responder and non-responder patient population.


Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/análise , Imuno-Histoquímica/métodos , Inibidores da Topoisomerase I/farmacologia , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Linhagem Celular Tumoral , DNA Topoisomerases Tipo I/imunologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação
4.
Oncotarget ; 8(27): 43733-43751, 2017 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-28415827

RESUMO

Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.


Assuntos
Camptotecina/farmacologia , DNA Topoisomerases Tipo I/metabolismo , Resistencia a Medicamentos Antineoplásicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores da Topoisomerase I/farmacologia , Ubiquitina/metabolismo , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Edição de Genes , Humanos , Autoantígeno Ku/metabolismo , Complexos Multiproteicos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Ligação Proteica , Proteína Quinase C/metabolismo , Proteólise , Interferência de RNA
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