The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The 'AC to Hly-linking segment' thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins.

• MeSH
• adenylátcyklasový toxin chemie   genetika   metabolismus   MeSH
• adenylátcyklasy chemie   genetika   MeSH
• AMP cyklický metabolismus   MeSH
• Bordetella pertussis chemie   patogenita   MeSH
• hemolyziny genetika   MeSH
• lidé MeSH
• lipidové dvojvrstvy chemie   metabolismus   MeSH
• perforin chemie   MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• pertuse genetika   mikrobiologie   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Adenylyl cyclase (AC) in brain cortex from young (12-day-old) rats exhibits markedly higher activity than in adult (90-day-old) animals. In order to find some possibly different regulatory features of AC in these two age groups, here we modulated AC activity by dithiothreitol (DTT), Fe(2+), ascorbic acid and suramin. We did not detect any substantial difference between the effects of all these tested agents on AC activity in cerebrocortical membranes from young and adult rats, and the enzyme activity was always about two-fold higher in the former preparations. Nevertheless, several interesting findings have come out of these investigations. Whereas forskolin- and Mn(2+)-stimulated AC activity was significantly enhanced by the addition of DTT, increased concentrations of Fe(2+) ions or ascorbic acid substantially suppressed the enzyme activity. Lipid peroxidation induced by suitable combinations of DTT/Fe(2+) or by ascorbic acid did not influence AC activity. We have also observed that PKC- or protein tyrosine kinase-mediated phosphorylation apparently does not play any significant role in different activity of AC determined in cerebrocortical preparations from young and adult rats. Our experiments analysing the presumed modulatory role of suramin revealed that this pharmacologically important drug may act as a direct inhibitor of AC. The enzyme activity was diminished to the same extent by suramin in membranes from both tested age groups. Our present data show that AC is regulated similarly in brain cortex from both young and adult rats, but its overall activity is much lower in adulthood.

• MeSH
• adenylátcyklasy chemie   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• časové faktory MeSH
• dithiothreitol farmakologie   MeSH
• financování organizované MeSH
• fluoridy farmakologie   MeSH
• fosforylace MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• inhibitory adenylylcyklasy MeSH
• kolforsin farmakologie   MeSH
• krysa rodu rattus MeSH
• kyselina askorbová farmakologie   chemie   MeSH
• mangan farmakologie   MeSH
• mozek enzymologie   metabolismus   MeSH
• mozková kůra enzymologie   metabolismus   MeSH
• peroxidace lipidů MeSH
• potkani Wistar MeSH
• proteinkinasa C metabolismus   MeSH
• sloučeniny hliníku farmakologie   MeSH
• stárnutí MeSH
• suramin farmakologie   chemie   metabolismus   MeSH
• tyrosinkinasy metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• železo chemie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH

• MeSH
• acylace MeSH
• adenylátcyklasy farmakologie   chemie   MeSH
• bakteriální toxiny farmakologie   chemie   MeSH
• finanční podpora výzkumu jako téma MeSH
• fluorescein-5-isothiokyanát analogy a deriváty   MeSH
• fosfolipidy chemie   MeSH
• liposomy chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH