Oxidative Stress and Mechanisms of Ochronosis in Alkaptonuria

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Free Radical Biology and Medicine

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Oxidative stress and mechanisms of ochronosis in alkaptonuria

Daniela Braconi, Lia Millucci, Giulia Bernardini, Annalisa Santucci n

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy article info abstract

Article history: Alkaptonuria (AKU) is a rare metabolic disease due to a deficient activity of the enzyme homogentisate Received 1 December 2014 1,2-dioxygenase (HGD), involved in Phe and Tyr catabolism. Due to such a deficiency, AKU patients Received in revised form undergo accumulation of the metabolite homogentisic acid (HGA), which is prone to oxidation/ 29 January 2015 polymerization reactions causing the production of a melanin-like pigment. Once the pigment is Accepted 19 February 2015 deposited onto connective tissues (mainly in joints, spine, and cardiac valves), a classical bluish-brown discoloration is imparted, leading to a phenomenon known as “ochronosis”, the hallmark of AKU. A Keywords: clarification of the molecular mechanisms for the production and deposition of the ochronotic pigment Amyloid in AKU started only recently with a range of in vitro and ex vivo human models used for the study of Homogentisic acid HGA-induced effects. Thanks to redox-proteomic analyses, it was found that HGA could induce Lipid peroxidation significant oxidation of a number of serum and chondrocyte proteins. Further investigations allowed Protein carbonyls highlighting how HGA-induced proteome alteration, lipid peroxidation, thiol depletion, and amyloid Proteomics Redox proteomics production could contribute to oxidative stress generation and protein oxidation in AKU. This review fl fi Serum amyloid A brie y summarizes the most recent ndings on HGA-induced oxidative stress in AKU, helping in the Thiol oxidation clarification of the molecular mechanisms of ochronosis and potentially providing the basis for its pharmacological treatment. Future work should be undertaken in order to validate in vivo the results so far obtained in in vitro AKU models. & 2015 Elsevier Inc. All rights reserved.

Introduction Globally, homogentisic aciduria, ochronosis, and arthritis-like damage constitute the temporally related triad characterizing AKU Alkaptonuria (AKU, OMIM: 203500) is an ultrarare disease [21]. After birth and early in the course of the disease, the (estimated incidence is 1:250,000–1,000,000 in most ethnic abnormal renal excretion of HGA due to HGD defects can be easily groups [1,2]) associated with a defective catabolic pathway of identified because HGA undergoes a spontaneous oxidation (on air Phe and Tyr due to low activity of the enzyme homogentisate 1,2- oxidation or alkalinization) with production of a dark-brown dioxygenase (HGD, EC.1.13.11.5) following HGD gene mutations. pigment. The blackening of urine is a pathognomonic sign of the HGD is expressed in liver, kidney, prostate, small intestine, colon, disease. The HGA that is not excreted undergoes oxidation and and human osteoarticular cells [3]; in healthy subjects, HGD polymerization in nonmineralized connective tissues of skeletal, converts homogentisic acid (HGA) to maleylacetoacetic acid. In integumentary, ocular, and cardiovascular systems, leading to a AKU patients, on the contrary, HGA is not further metabolized and pathological bluish-black discoloration, a phenomenon known as it is partly excreted with urine, partly accumulated in the body. “ochronosis” that is the hallmark of AKU. Ochronosis is visible to Several different HGD mutations have been identified so far in the naked eye as blue/black pigmentation of the eye and ear; patients from various populations [4–17], and most of them are microscopically, the ochronotic pigmented tissues appear firm and collected into a dedicated database [18]. However, a clear correla- inelastic [21]. In early life, AKU is mainly asymptomatic, with tion between genotype and residual HGA enzyme activity or severe symptoms occurring later when the third and clinically disease phenotype is still lacking, hindering also the development most relevant component of the triad appear [1,21]; the reasons of a disease severity scoring system, which now is only ques- for such a delay are still to be clarified. Usually, arthritic manifes- tionnaire based [19,20]. tations appear in AKU patients at an age significantly earlier

Abbreviations: AKU, alkaptonuria; BQA, 1,4-benzoquinone-2-acetic acid; HGA, homogentisic acid; HGD, homogentisate 1,2-dioxygenase; HSP, heat shock protein; LPO, lipid peroxidation; OA, osteoarthritis; PTM, posttranslational modiﬁcation; SAA, serum amyloid A n Correspondence to: via Aldo Moro 2, 53100 Siena (SI) Italy. Fax: þ39 0577 234254. E-mail address: [email protected] (A. Santucci). http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.021 0891-5849/& 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Braconi, D; et al. Oxidative stress and mechanisms of ochronosis in alkaptonuria. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.021i 2 D. Braconi et al. / Free Radical Biology and Medicine ∎ (∎∎∎∎) ∎∎∎–∎∎∎ compared to the general population (namely, around the fourth human serum-, cell-, and tissue-based human AKU models where decade of life) and mainly affect [21]: the effects of HGA could be investigated [26–39] (Table 1). These models unequivocally pointed out that oxidative stress was one of i. Joints and spine, where a premature severe disabling the most important factors for explaining HGA toxicity. Though it osteoarthritis-like damage develops, increasing the risk of was originally thought that HGA may act as a chemical irritant fractures and ruptures of ligaments and tendons and signifi- inducing tissue degeneration through inflammation, it was found cantly reducing patients' quality of life [22]. that HGA could first undergo spontaneous oxidation into 1,4- ii. The cardiovascular system, due to deposition of HGA-induced benzoquinone-2-acetic acid (BQA) (Fig. 1) and then polymerization ochronotic pigment in cardiac valves, increasing the risk of devel- into a compound of as yet undefined composition and properties oping aortic valve disease and coronary artery disease [23]. (first hypothesized structure is reported in Fig. 1A) [40–42]. The conversion of HGA into BQA is also concomitant with the produc- – tion of oxygen radicals such as superoxide anion (O2 ), hydroxyl For AKU, similar to many other rare conditions, gaining insights radical (OH ), and hydrogen peroxide (H2O2) [43] and of a into the molecular mechanisms by which the metabolic defect fluorescent, melanin-like ochronotic pigment [44,45]. Free radicals leads to the pathological manifestations has been hindered by the generated by the conversion of HGA into BQA were hypothesized rarity of the disease itself. The difficulty of collecting ochronotic to be responsible for the alterations of connective tissues observed samples (requiring very invasive techniques) should also be taken in AKU, similar to that found in other rheumatic diseases [46] and into account for AKU, together with the fact that ochronosis often disorders of Tyr metabolism [47–49]. In particular, it was sug- causes severe damage to tissues so that the collection of suitable gested that hydroxyl radicals could damage hyaluronic acid found samples from biopsies might be more problematic. Globally, all in synovial fluid of joints [45]. The interactions between HGA or these issues also hindered finding a dedicated cure for AKU and BQA and collagen have been analysed in vitro as well. It has been ochronosis. Current treatments are only palliative and do not shown in vitro that the interaction between HGA and collagen is address the intrinsic causes of AKU; surgery for total joint and mainly a reversible and pH-dependent phenomenon unlikely to heart valves replacement is often necessary. Two clinical trials for have significant effects in vivo [50]. Conversely, the interaction the use of the drug nitisinone in AKU have been undertaken with the oxidized (BQA)/polymerized forms seemed to involve a recently (http://clinicaltrials.gov/show/NCT01828463, http://clini- direct irreversible binding to collagen molecules occurring in vitro caltrials.gov/show/NCT01916382) [24]. However, since nitisinone quite irrespective of pH and allowing us to hypothesize that a is a competitive inhibitor of the enzyme that precedes HGD in the tanning phenomenon is likely responsible in vivo for the morpho- catabolic pathway [25], it is associated with Tyr buildup and logical changes observed in AKU [21,50]. BQA, through: related side effects. Furthermore, nitisinone can at best stop or delay the disease progression without modifying the underlying i. Salt linkages between the carboxyl group of its polymerized molecular HGD defect. form and the collagen ionic groups, Today, evidence is growing that AKU can be considered as a ii. coordinate bonds between hydroxyl groups and/or adjacent model for other more common rheumatic diseases such as activated ¼CH- groups of its polymerized form and keto-imide osteoarthritis (OA) and rheumatoid arthritis [26]. Although the bonds of collagen, disease is rare, the social and economic impact of studying AKU could be much wider than what was originally expected and this should boost related scientific research. was found to significantly increase collagen cross-linking, To overcome the difficulties related to the study of rare diseases stabilizing the collagen lattice structure. This in turn could like AKU, designing appropriate models to reproduce and study increase the cohesion of adjacent polypeptide chains in collagen, in vitro the pathological condition appears fundamental. Though displacing water molecules and so increasing the resistance of AKU is a well-described condition from a clinical point of view, the tanned collagen to degradation [21]. molecular bases of the disease are still quite obscure and only Similarities between AKU and neurodegenerative diseases may also recently underwent deeper biochemical investigations. This was be found. Although multiple factors are known to contribute to possible thanks to the setup and characterization of a range of oxidative stress observed in neurodegenerative diseases (including,

Table 1 Use of in vitro and ex vivo human AKU models to reveal HGA-related oxidative stress phenomena.

Finding In vitro AKU models Ex vivo AKU samples

Protein carbonyls C20 chondrocytic cell line [31] Chondrocytes [34] Chondrocytes [39] Serum [35,38] Protein thiol oxidation serum [35,38] Proteome alterations Changes in protein expression due to HGA addition Changes in protein expression observed in two subpopulations of AKU chondrocytes vs vs untreated control control chondrocytes from healthy donors C20 chondrocytic cell line [31] Serum [35] Chondrocytes [34] LPO Chondrocytes [33] Cardiac valve [28,29] Serum [35] Cartilage [30] Amyloid Cartilage and chondrocytes [26] Cardiac valve [28,29] Chondrocytes [33] Cartilage [30] Cartilage, synovia, periumbelical fat, salivary gland [26] Fat pad aspirates, labial salivary gland, cartilage and synovia specimens [27] Efﬁcacy of antioxidants C20 chondrocytic cell line [31] – Chondrocytes [33,39] Serum [38]

Fig. 1. Structures of HGA, BQA, and possible structures of the BQA-derived polymer according to [42] (A) and [56] (B). but not limited to, neuroinflammation, dysregulated metal homeostasis, instance, a direct comparison with what was observed for catechol and mitochondrial dysfunctions) [51,52], an interesting correlation and resorcinol derivatives with respect to hydroquinone deriva- between autoxidation of the neurotransmitter dopamine and oxidative tives such as HGA and BQA [56]. The issue was addressed in the stress observed in Parkinson's and Alzheimer's diseases has been work by Eslami and co-workers, who studied the electrochemical highlighted. The pigmentation of dopaminergic neurons is due to the behavior of HGA in aqueous and nonaqueous solutions. These autoxidation of dopamine to black neuromelanin; Parkinson's is authors found that HGA is oxidized by a one-step, two-electron– characterized by a progressive degeneration and depigmentation of two-proton redox reaction [57]. A further coupled chemical reac- such neurons through pathological processes involving aberrant oxidation of dimerization, dependent on HGA concentration and pH, tion of dopamine and generation of reactive quinone species. Analo- was also highlighted in acetonitrile–water solution [56] (Fig. 1B). gously, in AKU a form of ochronotic-like melanin pigment is formed In recent years, several HGA-related oxidative effects were from excess HGA undergoing oxidation and polymerization; further- demonstrated in a wide range of AKU models, such as oxidation more, a quinone compound (BQA) is generated. The cytotoxicity of of chondrocytic proteins [31,34,38,39]; production of a fluorescent quinones is mediated by their reactivity toward the nucleophilic side melanin-like ochronotic pigment in human serum, which could be chain of many amino acid residues and it has been ascribed to the partially counteracted by the use of antioxidants [35,38] (Table 2); derivatization of amino acid residues (mainly Cys) in proteins through enhanced lipid peroxidation (LPO) [26,29,33,35] and inflamma- the formation of covalent bonds with the aromatic rings [53].Mod- tion, as assessed by the analysis of released pro-inflammatory ification of proteins through their linkage to quinone can induce cytokines [29,33]; depletion of thiols and oxidation of protein thiol structural and functional alterations, promoting their pathological groups [35,38] (Table 1). Thus, several lines of evidence demon- aggregation and precipitation. In a recent work by Nicolis and co- strated that HGA is the first responsible, with BQA as the main workers, catecholamine-derived reactive species were generated for effector, for the oxidative cell and tissue damage observed in AKU. dopamine. Combined studies of tandem mass spectrometry and protein However, besides the accumulation of HGA, the potential role and unfolding indicated the presence of quinone-induced modifications of presence of other proteins have been hypothesized as additional neuroglobin, suggesting that the catecholamine-oxidation products can factors involved in ochronosis [30,58]. Future work is hence extensively modify proteins affecting their stability [53]. Similarly, it was mandatory to understand how BQA can interact with biological found in vitro that BQA can bind to several human serum proteins molecules in order to clarify the exact molecular mechanisms while propagating oxidative stress [35,38].Althoughotherfactorscan underlying ochronosis. certainly account for the oxidative derangement observed in AKU, such In this review, the most recent findings on HGA-induced a finding suggested that in vivo an extensive BQA-induced protein oxidative stress in AKU will be briefly summarized and discussed. posttranslational modification (PTM) may occur. This phenomenon, ultimately affecting protein function, stability, and propensity to aggregation, may possibly contribute to the production of the alkaptonuric HGA-induced oxidative posttranslational modification of ochronotic pigment. proteins in AKU As for the chemical reactivity of HGA and especially BQA, much still remain to be clarified. The possibility of oxidizing in vitro HGA The presence of excess HGA in biological fluids such as urine into its quinone metabolite, BQA, by Na2Cr2O7 was reported [54] and blood is already adopted as a diagnostic biomarker for AKU. and similar studies investigated the oxidation of HGA by dissolved However, like many other rare and common diseases, AKU still oxygen in aqueous solution [43,55]. Despite the obvious relevance lacks biomarkers with prognostic and predictive value that could of the subject for AKU, however, deeper investigations to clarify be used, for instance, to monitor disease progression or to evaluate the chemical reactivity of HGA were undertaken only recently. This the efficacy of pharmacological interventions. also in the light of the different reactivity imparted by the position For the biomarker discovery process, the identification and of hydroxyl functional groups in benzenediols, not allowing, for quantification of proteins in complex biological mixtures together

Table 2 Antioxidants tested in several in vitro AKU models to counteract HGA-induced effects.

Antioxidanta In vitro AKU model Output Reference

ASC C20 chondrocytic cell line, The evaluation of ASC-induced changes in terms of protein expression levels (comparative proteomics) and [31] treated or not with HGA protein carbonylation (comparative redox proteomics) revealed a pro-oxidant action of ASCþHGA ASC HGA-treated human serum Assessment of the ability of NACþASC, PHY, TAU, LIP, and FER to prevent or delay the production of HGA- [38] NAC induced melanin-like pigment (SDS-PAGE and quantitation of melanin-like fluorescence) and to reduce NACþASC oxidative protein PTMs (carbonyls and oxidized thiols, evaluated by SDS-PAGE and Western blot) PHY TAU FER LIP DMSO NAC HGA-treated chondrocytes Assessment of the ability of NAC in counteracting HGA-induced apoptosis (tested by Annexin V binding and [39] ASC flow cytometry), growth reduction (tested by MTT and DNA quantitation assays), and inhibition of NACþASC proteoglycan release (tested by an immunoenzymatic method) Assessment of the ability of NAC and NACþASC in reducing HGA-induced oxidative protein carbonylation (evaluated by SDS-PAGE and Western blot) PHY HGA-treated chondrocytes Assessment of the antiamyloid capacity (evaluated by Congo Red staining) of all the tested antioxidants [33] LIP TAU NACþASC Assessment of the ability of NAC and NACþASC in reducing SAA release (tested by ELISA assay) from HGA- treated chondrocytes Assessment of the ability of NACþASC in reducing the release of several pro-inflammatory cytokines (tested by a Multiplex assay) from HGA-treated chondrocytes

a Abbreviations used: ASC, ascorbic acid; DMSO, dimethyl sulfoxide; FER, ferulic acid; LIP, lipoic acid; NAC, N-acetylcysteine; PHY, phytic acid; TAU, taurine.

with the analysis of their PTM are fundamental [59]. PTMs are of machinery, generating a vicious cycle [60].Furthermore,carbonyla- paramount importance in determining the structure, function, and tion can often cause loss or alteration of protein function [65]. fate of each protein. Since more than two hundreds human Several biochemical and analytical methods are available for diseases have been linked to oxidative stress, which is defined as identifying and quantifying protein carbonyls [60] including: an unbalanced production of ROS/RNS that cannot be fully counteracted by antioxidant systems [60], the involvement of i. quantitative spectrophotometric and chromatographic assays, redox-related PTMs in many human diseases is not surprising. ii. biochemical and immunological assays providing global infor- For instance, redox-related modifications are involved in arthritic mation on modified proteins and oxidation levels, disorders, whereas other PTMs can be considered unique bio- iii. mass spectrometry techniques to identify both the modified chemical markers related to alterations of extracellular matrix protein and, potentially, the exact modification site. remodeling [61]. Redox proteomics collectively studies all the components of a proteome undergoing reversible or irreversible oxidation. A rever- If possible, these assays should also be corroborated by func- sible oxidation process is common for Cys, Met, and Sec, whereas tional assays to test whether carbonylation has altered protein Trp, Tyr, Arg, and protein backbone react irreversibly with oxidant biological activity. molecules [62]. As a central focus in biomedical research, the Human AKU models and ex vivo AKU samples were investi- redox proteome attracted the attention of researchers aimed at gated for protein carbonyls through a redox-proteomic approach. investigating the link between redox chemistry and protein In a chondrocytic cell line, the treatment with HGA, ascorbic acid structure/function [62]. In this context, proteomic and redox- (ASC), and ASCþHGA induced a marked increase of carbonylated proteomic studies were undertaken to assess if also HGA could proteins [31,38]. Major targets of carbonylation were proteins with induce oxidative PTMs of proteins in terms of carbonylation and structural functions (e.g., actin, vimentin, annexin 2), involved in thiol oxidation. protein folding, maturation, and transport (e.g., calreticulin, 75 kDa glucose-regulated protein, serpin H1, heat shock cognate 71 kDa protein, protein disulfide isomerase A1), and proteins of the HGA-induced protein carbonylation stress response [e.g., catalase and 60 kDa heat shock protein (HSP)]. A common protein “carbonylation signature” was found A wide range of ROS-derived protein PTMs can be induced, but a for ASC and HGA treatment, since the same proteins were found to large body of evidence pointed out that protein carbonylation could be oxidized in both treatments, though specifically carbonylated be considered a major hallmark of oxidative stress [63].Protein proteins could also be found. Interestingly, high molecular weight carbonylation is an irreversible PTM introducing a reactive carbonyl protein aggregates were found, and this was a very marked group in a protein following metal-catalyzed oxidation, LPO, or phenomenon in ASCþHGA-treated cells, confirming the pro- glycoxidation, though the relative contribution of each of the above oxidant and pro-aggregating effects of the combination of HGA noted mechanisms is still to be clarified [60]. Regardless of the with ASC [31]. The pro-oxidant behavior of ASC under specific mechanism by which they are produced, carbonylated proteins circumstances has been already hypothesized [66,67]. In particu- cannot be repaired and are more prone to aggregation due to their lar, referring to AKU, ASC can be cooxidized to dehydroascorbate increased hydrophobicity and disruption of protein stabilizing ele- in vitro during HGA oxidation at pH 7.4 yielding an increased H2O2 ments [64]. If carbonylated proteins are not properly eliminated by production that could be partially counteracted by the addition of proteasomal systems, they can lead to cell death [60]; nevertheless, catalase [43]. The H2O2 formed was attributed to reactions invol- the carbonylation itself or the carbonylation-induced protein aggre- ving ASC, ascorbyl radical, HGA and its semiquinone with mole- – – gation make the proteins less recognizable by the chaperone cular oxygen or O2 , and to dismutation of O2 [43].

Ex vivo AKU chondrocytes were seen to possess a similar revealed an HGA-induced decreased activity of the enzyme glu- pattern of carbonylated proteins, including proteins belonging to tathione peroxidase and massive depletion of total thiol groups cell organization (e.g., vimentin) and protection from stress (e.g., [35]. Thanks to labeling of free thiols with biotinylated iodoaceta- catalase and mitochondrial 60 kDa HSP). Also in this model, mide coupled with 2D-PAGE and Western blot, the comparison carbonylated high molecular weight protein aggregates stacked with an untreated serum control sample revealed that protein on the top of the SDS–PAGE gel were found [34]. thiol oxidation was mainly directed against the same functional Similarly, human serum showed an increased protein carbony- classes of proteins already found to be carbonylated, namely lation on in vitro treatment with HGA [38], a phenomenon that serum proteins with carrier/metal binding functions, including could be partially counteracted in vitro by the addition of anti- the previously discussed serum transferrin [35]. oxidants (Table 2). In a further redox-proteomic characterization of Blood is vital in maintaining a proper redox balance, but with this model, specific molecular targets of carbonylation could be aging blood thiols become increasingly oxidized, in turn providing identified as well, revealing mainly the oxidation of proteins with a lower antioxidant capacity and an increased risk of adverse carrier/metal binding functions [35]. Among them, albumin, hap- effects on oxidative stress. Due to the circulating excess HGA, AKU toglobin, ceruloplasmin, and transferrin were identified. All of sufferers experience oxidative stress throughout their life; when these proteins are considered as major contributors to the anti- this condition becomes concomitant with the reduced antioxidant oxidant capacity of serum and play critical roles for the preserva- defenses of elderly ages, one could possibly better explain the late tion of a correct redox balance. In particular, referring to metal onset of AKU manifestations at the articular/cardiac level. In this homeostasis, haptoglobin combines with free plasma hemoglobin, context, a protein thiolation index was recently proposed to be preventing loss of iron and avoiding it from interacting with other adopted for AKU [72]. components to yield free radicals through oxidative reactions [68]. Ceruloplasmin catalyzes the oxidation of ferrous iron into ferric How oxidative posttranslational modification of proteins can be iron, which is generally regarded to as a “safe” reaction because its originated in AKU? by-products are innocuous (see reaction below). Based on the most recent findings on HGA-induced stress in 4Fe2 þ þ 4Hþ þ O -4Fe3 þ þ2HO 2 2 in vitro and ex vivo AKU samples, several mechanisms could thus be proposed to explain how oxidative protein PTMs could be Once iron has been transformed, it then binds to transferrin by originated in vivo in AKU sufferers and contribute to the onset and which it is transported to storage and utilization sites. progression of ochronosis. These will be briefly discussed in the Ceruloplasmin-catalyzed iron oxidation and removal from circula- following paragraphs. tion by transferrin are very important processes since they reduce the amount of ferrous iron available for participation in Fenton's Altered expression of proteins with a role in the oxidative stress reactions, whose by-products are, on the contrary, highly reactive/ response toxic oxygen radicals. Overall, thus, the ceruloplasmin-transferrin antioxidant system accounts for the capacity of blood to sequester Thanks to the proteomic analyses undertaken in in vitro and exchangeable metals (copper, iron), avoiding them to take part in ex vivo AKU chondrocyte models to clarify the effects of HGA at reactions generating and promoting oxidative stress [69,70]. the molecular level, it was possible to highlight how HGA could Evidence of a subtle alteration in the handling of transition metals, induce important alterations in the abundance of proteins including a decreased ceruloplasmin oxidase activity, has been involved in the oxidative stress response [31,34]. In particular, recently highlighted in plasma and serum in Alzheimer's disease chondrocytes from AKU donors showed low levels of mitochon- [71]. Moreover, in AKU, a further modification was also noted for drial 60 kDa HSP, alpha-crystallin B chain, endoplasmin, glu- serum transferrin and haptoglobin, as they both were found to tathione S-transferase omega-1, glutathione S-transferase P, 70 bind in vitro BQA molecules generated by treatment with HGA kDa HSP 4 (HSP74), and mitochondrial superoxide dismutase [34]. [35]. Overall, it could thus be speculated that HGA-mediated The simultaneous deficiency of these proteins suggested the protein carbonylation, by enhancing protein oxidation and aggre- hypothesis that AKU cells cannot adequately control ROS- and gation (possibly also through BQA binding), could significantly BQA-mediated toxicity, possibly leading to an HGA-induced contribute to ochronosis in AKU. enhanced production of oxidatively modified proteins in AKU. The analysis of HGA-induced proteomic alterations in a human HGA-induced protein thiol oxidation serum model revealed also that BQA could bind to several proteins involved in metal ion homeostasis and proteins with carrier Thiol is likely the most important functional group involved in functions, such as haptoglobin and transferrin. As quinolation maintaining a proper redox balance: its reversible/irreversible may alter protein structure/function, it was hypothesized that an oxidative PTM affects enzyme catalytic activity, protein structure, unbalanced metal homeostasis could occur, in turn enhancing and binding capacity, ultimately tuning cell signaling, prolifera- protein oxidation and aggregation concomitantly with the production, differentiation, and death. However, challenging technical tion of the ochronotic pigment [35]. Such a finding was also difficulties characterize the study of thiols redox state. This is due corroborated by the aberrant expression of proteins related to to an extreme reactivity, the transient/permanent nature of the folding and amyloidogenesis, which represented another indirect modification, the delicate balance of switching between physiolo- confirmation of HGA-mediated protein aggregation in AKU con- gical modification and functional alteration following excess nected to the existence of an alkaptonuria-related amyloidosis, as oxidation. discussed in the following paragraph [26]. In light of the reactivity of quinones compounds (such as BQA) toward thiol groups [40], the above-described AKU model based Amyloidosis on HGA-treated human serum was also successfully used to investigate HGA-induced protein thiol oxidation. When challenged Oxidative stress, together with chronic inflammation, plays a with HGA, human serum showed an increased protein thiol central role for amyloid fibril formation and deposition, as oxida- oxidation that could be partially counteracted by addition of tive stress is an integral component of amyloidotic tissues regard- antioxidants [38] (Table 2). Further analyses on the same model less of the chemical nature of the amyloid deposit [73].

The amyloid nature of the HGA-related ochronotic pigment has may be an endeavor to mitigate melanin's cytotoxicity and/or been well documented in the most recent work on AKU [26– function as a scaffold or template for its synthesis, analogous to 29,33], allowing classification of AKU as a secondary amyloidosis what occurs physiologically in melanogenesis. due to the deposition of serum amyloid A (SAA) (namely, AA Altogether, these findings suggested that AKU is a complicating amyloidosis) [26] (Fig. 2) similar to other slowly progressing inflammatory multisystemic disease, involving many different rheumatic diseases with a lag phase before the onset of clinical organs where any body district expressing HGD may be affected signs. The finding of high SAA levels in AKU patients [26] by ochronosis and related AA amyloidosis [74]. The presence of concomitantly with amyloid deposition may hence be related to HGD in human articular cells has been documented only recently the HGA-induced oxidative stress; furthermore, the colocalization [3], suggesting that chondrocytes, synoviocytes, and osteoblasts of amyloid and ochronotic pigment is worth noting. Globally, a can produce the ochronotic pigment in loco and contribute to vicious cycle can thus be generated: HGA induces ROS production induction of ochronotic arthropathy. Other less classical manifes- while converted into BQA toward the production of the ochronotic tations of ochronosis were also detected in the central nervous pigment; the oxidative insult is cytotoxic and promotes inflamma- system, since ochronosis was found in the brain of AKU patients tion and degeneration of joints. A chronic inflammatory status [75,76], in dura mater and dural sinuses [77], and pineal gland paralleled by inadequate antioxidant defenses promotes the aber- pituitary body [78]. Neurological manifestations have been rant production of amyloidogenic proteins, ultimately leading to described in AKU as well, such as astrocytoma concomitant with secondary amyloid deposition. Amyloid itself is cytotoxic, and it pituitary adenoma [79], neuroblastoma [80], migraine headaches,

Fig. 2. SAA deposition in amyloid deposits in AKU cartilage and human cartilage challenged in vitro with HGA and its colocalization with HGA-induced melanin-like ochronotic pigment. (A) SAA deposition revealed by immunoﬂuorescence; bar is 75 mm. (B) AKU cartilage section showing the superimposition of amyloid deposits containing SAA and melanin ﬂuorescence; bar is 150 mm. DIC: differential interference contrast. Adapted with permission from [26].

Fig. 3. (A) 4-HNE detection in AKU, OA, and normal cartilage. (a) Healthy cartilage, as a negative control; (b–g) AKU cartilage; (h) OA cartilage, as a positive and reference control. Immunofluorescence to 4-HNE was relevant in all AKU cartilage specimens (b–g). Immuno-specificity of the staining was assessed by the absence of fluorescence in the superficial zones of AKU cartilage (e, f, g) evidenced only by DAPI fluorescence (blue). The deep zone of cartilage surface, the pericellular matrix, the intracellular matrix, and the matrix around the cells, all showed positive staining, which suggested the presence of strong lipid peroxidation in these areas. Bar is 100 μm. (B) 4-HNE detection in AKU and normal chondrocytes. 4HNE-positive staining was found in AKU chondrocyte cytoplasm as assessed by confocal microscopy. Adapted with permission from [30].

Fig. 4. (A) Aortic valve showing ochronotic pigmentation. (B) TEM observation of a myelin ﬁgure perfectly superimposing an ochronotic pigmented patch. (C) The presence of 4-HNE was uniformly diffused and superimposing to ochronotic pigmented areas (magniﬁcation 20 ). Adapted with permission from [29].

Fig. 5. HGA-induced oxidative stress and the mechanisms proposed to contribute to protein oxidation and ochronosis in AKU. has been documented together with production of ochronotic impairment, and specific disease-related endpoints [93]. It is well pigment and cell death [29]. known that the development and validation of suitable biomar- Among other biomarkers related to LPO and apoptosis, myelin kers of oxidative stress are challenging and time-consuming tasks, figures can be cited [90]. These structures appear as concentric especially for their validation in epidemiologic studies. This issue membranous lamellar formations encapsulating thin bands of is obviously much more complicated for rare diseases such as AKU. cytoplasm separated by elongated vacuolar spaces. Their presence Nevertheless, we believe that identifying a range of oxidative was detected in an AKU aortic valve section by TEM analysis, biomarkers for AKU may be essential for a better understanding of confirming the presence of LPO and membrane degeneration the disease itself and for the development of appropriate pharma- (Fig. 4). Most importantly, due to their perfect superimposition cological treatments [93]. In this light, future work is mandatory in to pigmented ochronotic patches, this finding also suggested a order to validate in vivo the results so far obtained in in vitro and strict correlation between the cytotoxicity of ochronotic pigment ex vivo AKU models, determining, for instance, levels of protein and LPO [30]. carbonyls, free thiols, or other oxidative stress biomarkers such as F(2)-isoprostanes in AKU patients' body fluids. On the one hand, this will provide further support to the evidence so far produced Conclusions on the role of oxidative stress in the progression of AKU and ochronosis; on the other, it will help the design of new therapeutic In recent years, it has become clearer that studying rare approaches for AKU and ochronosis, which currently lack a diseases can provide insights into many research areas to an dedicated therapy. This fits well with the ambitious aim of the extent that is far greater than what one can expect simply International Rare Disease Research Consortium, a joint effort by considering the number of cases [91]. Rare diseases can contribute the US NIH and the European Commission, of developing new indeed to the study of more common diseases not only providing tools for the diagnosis and pharmacological treatments of all the new knowledge but also increasing the number of those who will known rare diseases by the year 2020 [94]. benefit from the newly obtained results [92]. For instance, OA and rheumatoid arthritis patients can be hypothesized in the case of AKU. Conflict of interests In this review, we have provided useful data to demonstrate how the study of oxidative stress by means of classical biochemical All the authors declare that no actual or potential conflict of assays, proteomics, and redox proteomics could help in the interest exists that could inappropriately influence, or be per- clarification of the molecular mechanisms of ochronosis in AKU ceived to influence, their work. and the identification of potential protein biomarkers for the disease with prognostic and predictive value. Though oxidative stress biomarkers identified in AKU models are not disease Acknowledgments specific, as they represent common modifications found in many other diseases [93], their presence confirms the role of HGA as a The authors thank aimAKU (Associazione Italiana Malati di pro-oxidant compound. 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Please cite this article as: Braconi, D; et al. Oxidative stress and mechanisms of ochronosis in alkaptonuria. Free Radic. Biol. Med. (2015), http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.021i