Abstract
Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as “black” AKU chondrocytes, while those coming from the white portion were referred to as “white” AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both “white” and “black” AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in “black” AKU chondrocytes. J. Cell. Physiol. 227: 3333–3343, 2012. © 2011 Wiley Periodicals, Inc.
Highlights
Alkaptonuria (AKU; MIM no. 203500) is an autosomal recessive inherited rare disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase (HGO; EC 1.13.11.) that normally splits the aromatic ring of homogentisic acid (HGA; 2,5-dihydroxyphenylacetic acid), an intermediary product of the catabolism of tyrosine and phenylalanine (La Du et al, 1958; Fernandez-Canon et al, 1996; Phornphutkul et al, 2002)
Our findings indicated that AKU cells experience inflammatory stimuli and are characterized by relevant alterations in the expression of proteins involved in cell defence, protein folding and cell organization, sharing similarities with osteoarthritis (OA)
Since exposure to HGA may induce cytotoxicity (Kirkpatrick et al, 1984; Angeles et al, 1989), we investigated cell apoptosis and found that ‘‘white’’ and ‘‘black’’ chondrocytic cultures were both characterized by a higher% of apoptotic cells when compared to the control (þ1.5- and þ2.0-fold-change, respectively) (Fig. 2B)
Summary
Alkaptonuria (AKU; MIM no. 203500) is an autosomal recessive inherited rare disease resulting from a deficiency of the enzyme homogentisate 1,2-dioxygenase (HGO; EC 1.13.11.) that normally splits the aromatic ring of homogentisic acid (HGA; 2,5-dihydroxyphenylacetic acid), an intermediary product of the catabolism of tyrosine and phenylalanine (La Du et al, 1958; Fernandez-Canon et al, 1996; Phornphutkul et al, 2002). The same oxidation occurs within the body, where HGA is accumulated, allowing the formation of oxidized/polymerized melanin-like products imparting to connective tissues (especially skin, cardiovascular system, and joints) a characteristic pathologic pigmentation known as ‘‘ochronosis’’ (Zannoni et al, 1962; O’Brien et al, 1963; Phornphutkul et al, 2002; Helliwell et al, 2008). We recently introduced novel human ochronotic cell and serum models in which we demonstrated how HGA induces protein oxidation and aggregation (Braconi et al, 2010a,b; Tinti et al, 2010, 2011b). AKU cells experience significant protein oxidation and aggregation which might help, in turn, the production of ochronotic pigments.
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