טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentDikopoltsev Elena
SubjectRegulation of D-serine Signaling: Focus on Serine Racemase
Enzyme
DepartmentDepartment of Medicine
Supervisor Professor Herman Wolosker
Full Thesis textFull thesis text - English Version


Abstract

The NMDAR has a central role in neuronal cell activation and cell to cell communications in the brain and as such takes part in multiple brain processes and is critical in neurotransmission [1]. For activation of the NMDAR the binding of a co-agonist is necessary, being either glycine or the unusual amino-acid D-serine [2-10], that is produced by the enzyme Serine Racemase. D-serine plays a crucial role in NMDAR- dependent processes, ranging from neurotransmission to neurodegeneration. SR represents an important regulatory point on D-serine dynamics. Initially, SR was thought to be a glia enriched- enzyme [11], and various SR-interacting proteins were described in astrocytes [12-17], modulating its activity through various mechanisms. Recent data indicate that SR is mainly localized to neurons [18-23], together with its product, D-serine [18, 20, 21]. So far, only one interacting protein, Golga3, modulating SR activity in neurons has been described [24]. Here we identified a new interactor of SR, the FBXO22 protein, which binds to SR both in-vitro and in-vivo. FBXO22a isoform is the main SR interactor, and is also part of the Skp1-Cul1-F-box (SCF) ubiquitin-ligase complex. Nevertheless, FBXO22a has no effect on SR ubiquitination or turnover, and siRNA-mediated knock-down of endogenous FBXO22 had no effect on SR ubiquitination. Using a cell line that expresses endogenous SR, A172 glioblastoma cells, we show that knock-down of endogenous FBXO22 has no effect on endogenous SR turnover. We next demonstrated that SR binds free FBXO22 species and does not interact with the SCF complex. We found that SR knock-out has no effect on FBXO22a levels and on its substrate, KDM4A. siRNA to FBXO22a significantly decreased D-serine production, by about 40%, suggesting FBXO22a is required for optimal SR activity. This was also observed using shRNA-mediated knock-down of endogenous FBXO22 in rat primary astrocyte cultures. On the other hand, we did not observe any effect of FBXO22 isoforms on in-vitro activity of SR, suggesting FBXO22a indirectly affects SR. siRNA-mediated knock-down of FBXO22a increased the levels of membrane-associated SR in glioblastoma cells. Similarly, shRNA-mediated knock-down of FBXO22a in primary neuronal cultures increased levels of membrane-bound SR. Immunocytochemistry experiments confirmed the biochemical findings in A172 glioblastoma cells. Our data indicate that FBXO22 appears to be required for keeping SR in a soluble form at the cytosol.

The last part of the research focuses on the identification of a novel phosphorylation site at Threonine71 of mouse-SR. This phosphorylation is found in both cytosolic and membranous fractions of SR, and is the main phosphorylation site, with a 90% decrease in phosphorylation following mutation to Alanine. The phosphorylation modulates SR activity, since T71A mutant displays a 50% lower activity. Our data indicate that phosphorylation of SR appears to be an important regulatory step for mouse-SR.