RESUMO
Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.
Assuntos
Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Peptídeo Hidrolases/metabolismo , Proteoma/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/genética , Animais , Sítios de Ligação/fisiologia , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Conformação Molecular , Peptídeo Hidrolases/genética , Proteoma/genéticaRESUMO
ClpX binds substrates bearing specific classes of peptide signals, denatures these proteins, and translocates them through a central pore into ClpP for degradation. ClpX with the V154F po e mutation is severely defective in binding substrates bearing C-motif 1 degradation signals and is also impaired in a subsequent step of substrate engagement. In contrast, this mutant efficiently processes substrates with other classes of recognition signals both in vitro and in vivo. These results demonstrate that the ClpX pore functions in the recognition and catalytic engagement of specific substrates, and that ClpX recognizes different substrate classes in at least two distinct fashions.
Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Endopeptidase Clp , Proteínas de Escherichia coli , Cinética , Chaperonas Moleculares , Dados de Sequência Molecular , Mutagênese , Fragmentos de Peptídeos/química , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Especificidade por SubstratoRESUMO
ClpX and related AAA+ ATPases of the Clp/Hsp100 family are able to denature native proteins. Here, we explore the role of protein stability in ClpX denaturation and subsequent ClpP degradation of model substrates bearing ssrA degradation tags at different positions. ClpXP degraded T. thermophilus RNase-H* with a C-terminal ssrA tag very efficiently, despite the very high global stability of this thermophilic protein. In fact, global thermodynamic stability appears to play little role in susceptibility to degradation, as a far less stable RNase-H*-ssrA mutant was degraded more slowly than wild type by ClpXP and a completely unfolded mutant variant was degraded less than twice as fast as the wild-type parent. When ssrA peptide tags were covalently linked to surface cysteines at positions 114 or 140 of RNase-H*, the conjugates were proteolyzed very slowly. This resistance to degradation was not caused by inaccessibility of the ssrA tag or an inability of ClpXP to degrade proteins with side-chain linked ssrA tags. Our results support a model in which ClpX denatures proteins by initially unfolding structural elements attached to the degradation tag, suggest an important role for the position of the degradation tag and direction of force application, and correlate well with the mapping of local protein stability within RNase-H* by native-state hydrogen exchange.