Abstract
In humans, low serum carnosinase (CN1) activity protects patients with type 2 diabetes from diabetic nephropathy. We now characterized the interaction of thiol-containing compounds with CN1 cysteine residue at position 102, which is important for CN1 activity. Reduced glutathione (GSH), N-acetylcysteine and cysteine (3.2 ± 0.4, 2.0 ± 0.3, 1.6 ± 0.2 µmol/mg/h/mM; p < .05) lowered dose-dependently recombinant CN1 (rCN1) efficiency (5.2 ± 0.2 µmol/mg/h/mM) and normalized increased CN1 activity renal tissue samples of diabetic mice. Inhibition was allosteric. Substitution of rCN1 cysteine residues at position 102 (Mut1C102S) and 229 (Mut2C229S) revealed that only cysteine-102 is influenced by cysteinylation. Molecular dynamic simulation confirmed a conformational rearrangement of negatively charged residues surrounding the zinc ions causing a partial shift of the carnosine ammonium head and resulting in a less effective pose of the substrate within the catalytic cavity and decreased activity. Cysteine-compounds influence the dynamic behaviour of CN1 and therefore present a promising option for the treatment of diabetes.
Highlights
Carnosinase (CN1, EC 3.4.13.20) plays an important role in the development of nephropathy in diabetic patients
Human serum carnosinase activity The activity of recombinant CN1, produced in CHO cells, was concentration-dependently reduced by reduced glutathione (GSH), cysteine, N-acetylcysteine (Table 1) and cysteine (Figure 1 shows the effect of cysteine)
In diabetic mice and rats, decreased carnosine content was found in retina, kidney and liver[24,26,43] whereas renal CN1 was increased[21]
Summary
Carnosinase (CN1, EC 3.4.13.20) plays an important role in the development of nephropathy in diabetic patients. Its function as antioxidant is debated[10,11,12,13,14] It restores erythrocyte deformability[15], inhibits cellular senescence[16,17] as well as the production of matrix proteins such as fibronectin and collagen type VI by podocytes[2]. No serum CN1 is present and carnosine supplementation of diabetic mice increases serum and tissue carnosine levels and mitigates DN, reduces renal vasculopathy[21], normalizes vascular permeability[21], improves wound healing[22] and decreases insulin growth factor binding protein-1 (IGFBP1) production through suppression of HIF-1a, and improves glucose homeostasis[23]. In streptozotocininduced diabetic rats, carnosine prevents apoptosis of glomerular cells, podocyte loss[24,25], and vascular damage[26]
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