D-tyrosyl-trna Off The Acyl Enzyme (h-dtd), Functional Studies

It has long been assumed that L-forms of amino acids were predominantly existing inorganisms and incorporated into proteins. But in recent years, high concentration of freeD-amino acids is found present in merchanisms. D-amino acids are reported toxic toprokaryotic and eukaryotic systems, in E.coli, D-Tyr's toxicity was found comes fromcharging of tRNA^Tyr and starvation of L-Tyr-tRNA^Tyr(1). D-Tyr-tRNA^Tyr deacylase (DTD)motif/gene can be found in most of the organisms and able to deacylate mis-aminoacylated D-aminoacyl-tRNAs and showed anti D-amino acids toxicity activity(1-3, 5-8).D-amino acids can be found in the human cental nervers system (CNS) and endocrinerelated organs with high concentration. More and more work was focused on thephysiological and pathophysiological roles of D-amino acids in human CNS and peripheraltissues. The cognition of D-serine as a endogenous ligand of N-methyl-D-Aspartatereceptor updated our concept of "neurotransmitters", D-Asp was shown to influence thesecretion of several hormones. Editing-defective tRNA synthetase causesed proteinmisfolding and deposition is examined able to cause neurodegeneration, for the neurons donot divide and there is no dilution of the deposition(9). Deposition of D-Asp and D-Ser inβ-amyloid in neurons during ageing are thought to be related to Alzehheimer disease(10,11). Quality control of tRNAs are important in genetic information transmission.We found that h-DTD was enriched in CNS magcellullar neurons; it has very similardistribution with D-Asp and D-Ser. h-DTD was found near to the nuclear pore apparatus(NPC) with immune electron microscopy assay. Fragmentation locaization results indicatethat the C tail of h-DTD, which is reported responsible for DNA binding, does notinfluence h-DTD's perinuclear localization. Endogenous h-DTD of HeLa cells in differentcell cycle progress displayed different distribution, some can be observed maily inperinuclear region, and some can be found with in the nucleus. In cell of M phase theendogenous h-DTD displayed spindle like distribution as the NPC marker (Ab24609). 7.5mM D-Tyr treatment decreased the aminoacylation level of tRNA^Tyr, and resultedtRNA^Tyr nuclear accumulation, tRNA^Tyr was depleted by both D-Tyr treatment and h-DTDsilencing, h-DTD silencing exaggerated D-Tyr's inhibition of protein synthesis. Depressionof HeLa cells' viability by D-amino acids (D-Tyr, D-Asp & D-Ser) was detected with adose dependent manner. Over expression of h-DTD decreased the depression rate of D-Tyrtreatment, h-DTD silenced cells became more sensitive to D-Tyr treatment. Treatment of7.5 mM D-Tyr on non-silenced HeLa cells resulted an increase in the population of S &G2/M phase cells, h-DTD silencing can also increase in the population of S & G2/M phasecells, h-DTD silenced cells treated with 7.5 mM D-Tyr can result in the most increase inpopulation of S & G2/M phase cells. At nuclear pore position h-DTD can hydrolizeD-aminoacylated tRNAs and keep the tRNAs with D-amino acid preference from export. Itcontrols the tRNA quality and showed anti D-amino acid toxicity activity. Being anindividual editing molecule able to act on several D-amino acylated tRNAs, will h-DTD beinvolved in anti neurodegeneration mechanism is what we are concerned.