NUDEL-OLIGOPEPTIDASE: ANÁLISE DA DISTRIBUIÇÃO DO RNAm NO SNC DE RATOS NEONATOS E ADULTOS E CARACTERIZAÇÃO DO RESÍDUO CRÍTICO PARA A ATIVIDADE CATALÍTICA. / NUDEL-oligopeptidase: analysis of mRNA distribution in the central nervous system (CNS) of newborn and adult rats, and characterization of the amino acid residue critical for the catalytic activity.

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

Thirty years ago the first endooligopeptidases were described, and named Endo A (EOPA) and Endo B. EOPA was later characterized as a thiol-activated peptidase showing specificity for substrates comprising 7 to 13 amino acid residues. In 2000, studying the molecular mechanisms of the brain disorder lissencephaly, three independent groups isolated a protein named NUDEL (nuclear distributing-like protein), in a screen for LIS1 interacting proteins, and showed that it is essential for neuronal migration and axonal outgrowth during brain development. Surprisingly, the cDNA and the deduced amino acid sequences of NUDEL revealed to be the same molecule as EOPA, and in 2005 the name NUDEL-oligopeptidase was suggested. Recently, NUDELoligopeptidase was also identified as the protein that binds to several proteins found in neuronal cells, such as Lis1, dineyn, DISC1, neurofilaments, suggesting its involvement in the Developmental processes and maintenance of the CNS. The first aim of this work was to map the distribution of the NUDEL-oligopeptidase mRNA in different areas of the CNS of rats at different ages, using the in situ hybridization, Northern Blotting and RTPCR methods. Different regions of the CNS showed some level of NUDELoligopeptidase expression, while the neocortex and the hippocampus clearly presented the highest levels, a fact that was observed for all analyzed age groups. In fact, the hippocampal region showed higher levels in the younger animals (5, 10 and 15 days), while the neocortex region showed higher levels in 90 days old animals. The second aim of the present work was to determine the critical cysteine residue for the catalytic activity of the NUDEL-oligopeptidase, using the site-directed mutagenesis method. The three cysteine residues found in the deduced amino acid sequence at positions 203, 273 and 293 of the NUDEL-oligopeptidase protein were mutagenized and expressed. The enzymatic analysis of recombinant wild-type and mutants proteins showed that the cysteine residue at position 273 is essential for the catalytic activity of the NUDELoligopeptidase.

ASSUNTO(S)

2. mutação sítio dirigida 4. hibridização in situ biologia molecular 3. desenvolvimento do snc 4. neuropatologias. 1. oligopeptidase

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