RESUMO
The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent "disassembly enzyme" for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631-1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP- and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70-facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone.
Assuntos
Auxilinas/metabolismo , Cadeias Pesadas de Clatrina/química , Cadeias Pesadas de Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Cadeias Pesadas de Clatrina/isolamento & purificação , Vesículas Revestidas por Clatrina/ultraestrutura , Insetos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Ratos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The nitration of tyrosine has been suggested to play a role in the pathogenesis of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). METHODS: In the present study, we identified four targets of protein nitration, T-complex polypeptide 1 alpha subunit (TCP-1), neurofilament L (NFL), glial fibrillary acidic protein (GFAP) and clathrin heavy chain (CHC), in the normal rat cortex using a proteomics approach. CONCLUSIONS: There have been no reports on these proteins being identified by proteomics as nitrated forms in the brain. For further study, we have to investigate alterations in these nitrated proteins during aging and in neurodegenerative disorders.
