The enzymes prolyl 4-hydroxylase [PPH; EC 1.14.11.2], aspartyl 3-hydroxylase (AAH; EC 1.14.11.16) and deoxyhypusyl hydroxylase [DOHH; EC 1.14.99.29] are metalloenzymes that form, respectively, peptidyl hydroxyproline in the collagens and collectins, hydroxyaspartate/hydroxyasparagine in EGF modules of extracellular proteins, and hypusine only in the translation initiation factor eIF-5A. In each case, hydroxylation markedly affects, or even is the sole determinant of, the particular protein's biological function. The enzymes appear to follow a common catalytic pathway of O2 utilization, show similar active site architecture, and provide three cisoid coordination sites at their active site metal ion. We noted that deferiprone (DMHP), used therapeutically to alleviate tissue iron overload in chronically transfused patients, should fit the active site pocket of each enzyme and block the catalytically essential metal ion. We examined this hypothesis with regard to:
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Inhibition of PPH. Hydroxyproline occurs in all collagenous proteins and determines their conformation and biosynthesis. DMHP inhibited the purified human recombinant enzyme with an app. Ki of 140 μM. In cultured human lung and skin fibroblasts, 3H-hydroxyproline formation was inhibited without affecting 3H-proline incorporation, and secretion of procollagens type I and III, but not of fibronectin, was suppressed (IC50s≤ 200μM). Electron microscopy revealed shunting of the underhydroxylated material into the lysosomal compartment, without toxic morphological effects.
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Inhibition of DOHH. Hypusine formation is associated with commitment to DNA replication, i.e. GI exit. DMHP inhibited 3H-hypusine formation in human lymphoblastoid cells (IC50: 100 μM) and caused accumulation of precursor peptidyl 3H-deoxyhypusine. This reversible effect coincided with reversible arrest at the GI-S boundary. Flow cytometry revealed no further cytokinetic consequences.
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Inhibition of AAH. Hydroxyaspartate is critical for high-affinity calcium binding in extracellular proteins like LTBP, the chaperone for latent TGF-β and involved in its proper matrix deposition and consequently bioactivation. At DMHP concentrations that inhibit collagen secretion, the total amount of TGF-β in medium was increased while the endogenously active TGF-β was markedly decreased, suggesting that a collagen deficient matrix or underhydroxylated LTBP suppress endogenous activation of TGF-β.
Inhibition of PPH. Hydroxyproline occurs in all collagenous proteins and determines their conformation and biosynthesis. DMHP inhibited the purified human recombinant enzyme with an app. Ki of 140 μM. In cultured human lung and skin fibroblasts, 3H-hydroxyproline formation was inhibited without affecting 3H-proline incorporation, and secretion of procollagens type I and III, but not of fibronectin, was suppressed (IC50s≤ 200μM). Electron microscopy revealed shunting of the underhydroxylated material into the lysosomal compartment, without toxic morphological effects.
