Discoveries of Protein S- and N-Homocysteinylation
In early studies, Hcy was identified in plasma and urine from patients with CBS or MTHFR deficiency [290, 291], but was undetectable in normal individuals. What was surprising in those studies was the inability to detect Hcy in tissues from CBS- or MTHFR-
- PDF / 71,173 Bytes
- 3 Pages / 439.37 x 666.142 pts Page_size
- 42 Downloads / 200 Views
Discoveries of Protein S- and N-Homocysteinylation
In early studies, Hcy was identified in plasma and urine from patients with CBS or MTHFR deficiency [290, 291], but was undetectable in normal individuals. What was surprising in those studies was the inability to detect Hcy in tissues from CBSor MTHFR-deficient patients [292, 293]. This suggested that a significant quantity of Hcy must have escaped detection by the conventional methods of amino acid analysis [294], possibly because Hcy was bound to protein via disulfide bonds and removed during the deproteinization step. Indeed, a simple treatment with the disulfide-reducing agent 2-mercaptoethanol led to the discovery of Hcy in normal individuals and revealed that most plasma and tissue Hcy is linked to protein via disulfide bonds [91]. It was also demonstrated that exogenous Hcy added to plasma quickly becomes protein bound and can be quantitatively released by the 2-mercaptoethanol treatment. These findings provided the first evidence for a redox reaction of Hcy with plasma proteins that is now called protein S-homocysteinylation [41, 295]. Subsequent studies have identified albumin [103] and IgG [79] as the major carriers of S-linked Hcy in plasma. Albumin also carries most of plasma Cys [111], which forms a disulfide bond with the conserved Cys34 thiol [108, 296]. Protein N-homocysteinylation was first discovered in studies of Hcy-thiolactone metabolism in cultured human cells using [35S]Met and [35S]Hcy as tracers. Those studies demonstrated that fibroblasts from CBS-deficient patients and the oncogenically transformed cells from breast cancer patients produce more Hcythiolactone and N-Hcy-protein than normal cells from unaffected individuals [73]. The treatment of cells with the antifolate drug aminopterin increases N-Hcyprotein levels, in addition to increasing Hcy-thiolactone and Hcy. In human endothelial cells, the extent of protein N-homocysteinylation increases with
H. Jakubowski, Homocysteine in Protein Structure/Function and Human Disease, DOI 10.1007/978-3-7091-1410-0_4, © Springer-Verlag Wien 2013
55
56
4 Discoveries of Protein S- and N-Homocysteinylation
Fig. 4.1 Reactions of Hcy-thiolactone and Hcy in human serum. (a) Kinetics of protein N-homocysteinylation (filled circles) and protein S-homocysteinylation (filled squares) in the presence of 12 μM [35S]Hcy-thiolactone. (b) Kinetics of protein S-homocysteinylation (filled squares) with 12 μM [35S]Hcy. At the point indicated by an arrow, 10 mm DTT was added (Reproduced from [81])
increasing Hcy concentration (Fig. 3.7) and decreases with increasing levels of folic acid (which lowers Hcy levels) and HDL (which hydrolyzes Hcy-thiolactone) (Table 3.9) [74]. Subsequent studies have revealed that the incubation of human serum with [35S]Hcy-thiolactone results in a progressive incorporation of the [35S] radiolabel into protein (Fig. 4.1a). At 3 h, most of the [35S] becomes protein bound and is precipitable by trichloroacetic acid. Treatment with dithiothreitol (DTT) of the [35S]Hcy-thiolactone-m
Data Loading...
