Accumulation of altered aspartyl residues in erythrocyte proteins from patients with Down's syndrome

Abstract

Spontaneous protein deamidation of labile Asn residues, generating l-isoaspartates and d-aspartates, is associated with cell aging and is enhanced by an oxidative microenvironment; to minimize the damage, the isoaspartate residues can be 'repaired' by a specific l-isoaspartate (d-aspartate) protein O-methyltransferase (PIMT). As both premature aging and chronic oxidative stress are typical features of Down's syndrome (DS), we tested the hypothesis that deamidated proteins may build up in trisomic patients. Blood samples were obtained from children with karyotypically confirmed full trisomy 21 and from age-matched healthy controls. Using recombinant PIMT as a probe, we demonstrated a dramatic rise of l-isoaspartates in erythrocyte membrane proteins from DS patients. The content of d-aspartate was also significantly increased. The integrity of the repair system was checked by evaluating methionine transport, PIMT specific activity, and intracellular concentrations of adenosylmethionine and adenosylhomocysteine. The overall methylation pathway was directly monitored by incubating fresh red blood cells with methyl-labeled methionine; a three-fold increase of protein methyl esters was detected in trisomic children. Deamidated species include ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3; ankyrin and band 4.1 were significantly hypermethylated in DS. When DS red blood cells were subjected to oxidative treatment in vitro, the increase of protein deamidation paralleled lipid peroxidation and free radical generation. We observed a similar pattern in Epstein-Barr virus B-lymphocytes from trisomic patients. In conclusion, our findings support the hypothesis that protein instability at asparagine sites is a biochemical feature of DS, presumably depending upon the oxidative microenvironment. The possible pathophysiological implications are discussed.

Protein Isoaspartate Methyltransferase Prevents Apoptosis Induced by Oxidative Stress in Endothelial Cells: Role of Bcl-Xl Deamidation and Methylation

“…In this respect, it has been previously shown that a) human erythrocytes treated with either t -BHP or H 2 O 2 accumulate deamidated isomerized proteins in the cell membrane, which are methyl esterified ex vivo by PCMT [31]; b) human erythrocytes from G6PD-deficient subjects are particularly prone to undergo deamidation at membrane protein level, upon oxidative stress, compared to normal red cells [9]; c) melanoma cells also accumulate PCMT substrates upon UV irradiation, according to a mechanism which is mediated by an oxidation [12]. Finally, we detected increased isoaspartyl formation in the erythrocytes from patients with Down Syndrome, a condition which is characterized by increased oxidative stress [32].…”

DYRK1A, a Novel Determinant of the Methionine-Homocysteine Cycle in Different Mouse Models Overexpressing this Down-Syndrome-Associated Kinase

“…A previous study showed a lower concentration of plasma Hcy and SAH levels in DS children than in plasma of control subjects [11]. Galletti et al also demonstrated that the intracellular concentration of SAH is significantly reduced in DS erythrocytes, paralleling the low plasma Hcy levels, and the intracellular SAM concentration is the same as in the control cells [13]. Therefore the diminution of SAH levels could be due to other genes located on chromosome 21.…”

“…As increased expression of DYRK1A and low plasma Hcy levels have been associated with DS [6], [11]–[13], we also analyzed the contribution of DYRK1A on Hcy levels in a mouse trisomic complex, the Ts65Dn mice. We found an increased expression of DYRK1A in liver and a decreased level of plasma Hcy level in Ts65Dn.…”

“…On the one hand, we recently reported a reduction of Dyrk1a protein level in liver of CBS-deficient mice, a murine model of hyperhomocysteinemia [10], suggesting a link between DYRK1A related pathways and the Hcy cycle. On the other hand, an increased expression of DYRK1A and low plasma Hcy levels have been associated with DS [6], [11]–[13]. To analyze further the relation between DYRK1A and Hcy metabolism, we used four transgenic models to demonstrate the effect of the over-expression of Dyrk1a on Hcy metabolism: a model of hyperhomocysteinemia due to CBS deficiency [14] and three models with duplications of increasing complexity and over-expression of Dyrk1a [15]: a BAC transgenic with one copy of the murine Dyrk1a gene; a YAC transgenic for a human chromosome 21 fragment carrying five genes including DYRK1A; a partial trisomy 16 mouse carrying an extra copy of a region of MMU16 syntenic for a region of HSA21 between Mrpl39 and Znf295 containing 138 genes (also including Dyrk1a) and considered to be a valid mouse model of human Down syndrome [16].…”

Defective DNA repair and increased chromatin binding of DNA repair factors in Down syndrome fibroblasts

“…Down syndrome (DS) is a human genetic disease due to trisomy of chromosome 21, and it is characterized by an elevated oxidative stress status [1][2][3][4][5][6][7] as demonstrated by increase in lipid peroxidation [8], protein deamidation [9] and DNA oxidation [5,10,11]. Although the source of the oxidative stress found in DS individuals has not been fully elucidated, there is sufficient evidence indicating that it is due to an imbalance in the metabolism of reactive oxygen species (ROS).…”