Inhibition of NUDEL (nuclear distribution element-like)-oligopeptidase activity by disrupted-in-schizophrenia 1

Abstract

Recently, nuclear distribution element-like (NUDEL) has been implicated to play a role in lissencephaly and schizophrenia through interactions with the lissencephaly gene 1 (Lis1) and disrupted-in-schizophrenia 1 (DISC1) products, respectively. Interestingly, NUDEL is the same protein as endooligopeptidase A (EOPA), a thiol-activated peptidase involved in conversion and inactivation of a number of bioactive peptides. In this study, we have cloned EOPA from the human brain and have confirmed that it is equivalent to NUDEL, leading us to suggest a single name, NUDEL-oligopeptidase. In the brain, the monomeric form of NUDEL-oligopeptidase is responsible for the peptidase activity whose catalytic mechanism is likely to involve a reactive cysteine, because mutation of Cys-273 fully abolished NUDEL-oligopeptidase activity without disrupting the protein's secondary structure. Cys-273 is very close to the DISC1-binding site on NUDEL-oligopeptidase. Intriguingly, DISC1 inhibits NUDEL-oligopeptidase activity in a competitive fashion. We suggest that the activity of NUDEL-oligopeptidase is under tight regulation through protein-protein interactions and that disruption of these interactions, as postulated in a Scottish DISC1 translocation schizophrenia cohort, may lead to aberrant regulation of NUDEL-oligopeptidase, perhaps providing a substrate for the pathology of schizophrenia.

Fig. 1.

Determination of the active NUDEL-oligopeptidase species. (A) Profile of the oligopeptidase activities in different molecular mass fractions after gel-filtration chromatography of rat brain cytosol. (B) Oligopeptidase activities as in A, with the cytosol pretreated with MO inhibitor (see Methods). (C) Analysis of fractions by Western blotting for DISC1 (Left) and NUDEL-oligopeptidase (Center). The presence of the NUDEL-oligopeptidase in fraction V was detected only after a 10-fold amount of this fraction was loaded onto the gel (Right). The arrows indicate the positions of the M_r marker in kDa.

Fig. 2.

DISC1 inhibits NUDEL-oligopeptidase activity. (A and B) Lineweaver–Burk plots (1/v versus 1/S) (A) and kinetic analyses (B) of rNUDEL-oligopeptidase inhibition by recombinant DISC1. The enzyme assays were performed by using qf-A⁶Bk_4-9 as substrate and different fixed concentrations of rDISC1. All enzymatic assays were performed in triplicate. DISC1 competitively inhibits enzyme activity. (C) Diagram illustrating the Lis1 and DISC1 interaction domains on NUDEL-oligopeptidase. The position of the cysteines (C203, C273, and C293; arrows) and leucine-glutamate (L266/E267) residues, critical for interaction with DISC1, are indicated.