Serine Racemase is a Cysteine Racemase and Physiologic Down Regulator of Insulin Promoter Methylation

Abstract

Endogenous D-stereoisomers are being recognized as functionally important molecules in mammals. Here we report the first identification of endogenous D-cysteine in mammalian pancreas. Serine Racemase (SR) is the biosynthetic enzyme for D- cysteine. D-cysteine is present in substantial amounts in the eyes, brain and pancreas of mice. To characterize endogenous D-cysteine, we used SR deficient mice lacking racemizing ability and show 3.5 fold reduction in pancreatic D-cysteine. SR-/- mice produce 6-10 fold higher levels of insulin in the pancreas and plasma and are stored as amyloid aggregates in secretory vesicles and exosomes respectively. Lack of SR and endogenous D-cysteine globally decreased levels of nucleotides and cAMP, followed by reduced phosphorylation of CREB (S133) including lower expression of DNA methyltransferase (DNMT) 1, 3A and 3B, and reduced DNMT enzymatic and promoter activities in the pancreas. This results in decreased DNA methylation globally and specifically of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1 transporters in cells. Dietary supplementation with methyl donors rescues the high insulin levels and low DNMT activity in SR-/- mice. Our data show that SR racemizes cysteine in the pancreas and is a physiologic down regulator of insulin promoter methylation.

A Novel Stereospecific Bioluminescent Assay for Detection of Endogenous d-Cysteine

“…Our recent work in the brain showed that endogenous d -cysteine is enriched in the developing brain of mice at E9.5 where it is present at a concentration of 4.5 mM and decreases substantially to 50 μM at postnatal development, which suggests that it may play roles during development. d -Cysteine in the developing brain controls neural progenitor cell proliferation via transcription factors Foxo1- and 3-mediated AKT signaling. , Our work in the pancreas (unpublished) shows that d -cysteine controls islet size and insulin secretion, which echoes a similar theme in the brain of controlling cell proliferation during organ development . Endogenous d -cysteine is present not only in mice but also in humans.…”

“…8−10 D-Cysteine is present in substantial amounts in the eye, brain, and pancreas of mice. 11 Our work shows that endogenous D-cysteine is enriched in the developing mammalian brain at E9.5 (embryonic day 9.5), is a regulator of neural progenitor cell proliferation, and may play roles in neural development. 6 Here, we report a novel, stereospecific, bioluminescent luciferase assay that allows the quantitative detection of endogenous D-cysteine in mammalian tissues.…”

“…7,6 Our work in the pancreas (unpublished) shows that D-cysteine controls islet size and insulin secretion, which echoes a similar theme in the brain of controlling cell proliferation during organ development. 11 Endogenous Dcysteine is present not only in mice but also in humans. We estimated its levels in healthy adult postmortem brains.…”

“…In mammals, d -amino acids are being recognized as functionally important molecules. , Cysteine is the fastest spontaneously racemizing amino acid in vitro with a t 1/2 of 2 days compared with other amino acids, like serine ( t 1/2 = 3 days) and aspartate ( t 1/2 = 30 days). As a result, its d -stereoisomer has precluded detection in multiple biological screens and, hence, has never been reported. − We recently utilized a novel, stereospecific bioluminescent assay to identify endogenous d -cysteine in mammalian brain. , Endogenous d -cysteine is racemized from l -cysteine by a PLP-dependent enzyme, serine racemase (E.C 5.1.1.18), the enzyme that racemizes l -serine to d -serine and vice versa. − d -Cysteine is present in substantial amounts in the eye, brain, and pancreas of mice . Our work shows that endogenous d -cysteine is enriched in the developing mammalian brain at E9.5 (embryonic day 9.5), is a regulator of neural progenitor cell proliferation, and may play roles in neural development .…”