טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDumin Elena
SubjectProtein-Interactors of Serine Racemase and Their
Role in Regulating Brain D-serine Levels
DepartmentDepartment of Medicine
Supervisor Professor Herman Wolosker
Full Thesis textFull thesis text - English Version


Abstract

Mammalian serine racemase is a brain enriched enzyme that converts L-into D-serine in the nervous system. D-Serine is an endogenous co-agonist at the"glycine site" of N-methyl D-aspartate receptors, which is required for the receptor/channel opening. Factors regulating the synthesis of D-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine racemase levels. We found that serine racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins and in vivo by co-immunoprecipitation studies from co-transfected HEK293 cells and brain homogenates. Golga3 and serine racemase co-localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine racemase levels, such as the ubiquitin-proteasome system, which affects protein stability.  We found that serine racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Serine racemase was ubiquitylated both in vitro and in vivo, indicating that the enzyme is a substrate for the ubiquitin system. Moreover, serine racemase protein levels were greatly increased by the proteasome inhibitors MG132 or lactacystine. Golga3 significantly increased serine racemase steady-state protein levels when transfected into HEK293 cells or when overexpressed in primary astrocyte cultures by recombinant lentivirus-mediated gene transfer. This was associated to a decreased in the ubiquitylation levels of serine racemase both in vitro and in vivo by Golga3.  We show that serine racemase has a relatively short half-life (about 4 hours) and that Golga3 co-transfection significantly increased the serine racemase protein half-life in pulse-chase experiments with [S35]-methionine. On the other hand, Golga3 did not affect the levels of an unrelated protein, GFPu, which is rapidly degraded, indicating that Golga3 per se does not inhibit the function of the ubiquitin-proteasome system. Serine racemase also significantly increased Golga3 steady-state protein levels and half-life in co-transfection experiments in HEK293 cells. Thus, serine racemase and Golga3 complex is more stable towards degradation by the ubiquitin-proteosome system. Our results suggest that the ubiquitin system is a main regulator of serine racemase and D-serine levels. Modulation of serine racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain D-serine levels and the NMDA receptor activity.