Alessia Arcaro

Interaction of aldehydes derived from lipid peroxidation and membrane proteins.

A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA) and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE) is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation, and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.

Role of 4-Hydroxynonenal-Protein Adducts in Human Diseases

SIGNIFICANCE Oxidative stress provokes the peroxidation of polyunsaturated fatty acids in cellular membranes, leading to the formation of aldheydes that, due to their high chemical reactivity, are considered to act as second messengers of oxidative stress. Among the aldehydes formed during lipid peroxidation (LPO), 4-hydroxy-2-nonenal (HNE) is produced at a high level and easily reacts with both low-molecular-weight compounds and macromolecules, such as proteins and DNA. In particular, HNE-protein adducts have been extensively investigated in diseases characterized by the pathogenic contribution of oxidative stress, such as cancer, neurodegenerative, chronic inflammatory, and autoimmune diseases. RECENT ADVANCES In this review, we describe and discuss recent insights regarding the role played by covalent adducts of HNE with proteins in the development and evolution of those among the earlier mentioned disease conditions in which the functional consequences of their formation have been characterized. CRITICAL ISSUES Results obtained in recent years have shown that the generation of HNE-protein adducts can play important pathogenic roles in several diseases. However, in some cases, the generation of HNE-protein adducts can represent a contrast to the progression of disease or can promote adaptive cell responses, demonstrating that HNE is not only a toxic product of LPO but also a regulatory molecule that is involved in several biochemical pathways. FUTURE DIRECTIONS In the next few years, the refinement of proteomical techniques, allowing the individuation of novel cellular targets of HNE, will lead to a better understanding the role of HNE in human diseases.

Exposure of HL-60 human leukaemic cells to 4-hydroxynonenal promotes the formation of adduct(s) with α-enolase devoid of plasminogen binding activity

HNE (4-hydroxynonenal), the major product of lipoperoxidation, easily reacts with proteins through adduct formation between its three main functional groups and lysyl, histidyl and cysteinyl residues of proteins. HNE is considered to be an ultimate mediator of toxic effects elicited by oxidative stress. It can be detected in several patho-physiological conditions, in which it affects cellular processes by addition to functional proteins. We demonstrated in the present study, by MS and confirmed by immunoblotting experiments, the formation of HNE-alpha-enolase adduct(s) in HL-60 human leukaemic cells. Alpha-enolase is a multifunctional protein that acts as a glycolytic enzyme, transcription factor [MBP-1 (c-myc binding protein-1)] and plasminogen receptor. HNE did not affect alpha-enolase enzymatic activity, expression or intracellular localization, and did not change the expression and localization of MBP-1 either. Confocal and electronic microscopy results confirmed the plasma membrane, cytosolic and nuclear localization of alpha-enolase in HL-60 cells and demonstrated that HNE was colocalized with alpha-enolase at the surface of cells early after its addition. HNE caused a dose- and time-dependent reduction of the binding of plasminogen to alpha-enolase. As a consequence, HNE reduced adhesion of HL-60 cells to HUVECs (human umbilical vein endothelial cells). These results could suggest a new role for HNE in the control of tumour growth and invasion.

Mitochondrial Dysfunction in Cancer and Neurodegenerative Diseases: Spotlight on Fatty Acid Oxidation and Lipoperoxidation Products

In several human diseases, such as cancer and neurodegenerative diseases, the levels of reactive oxygen species (ROS), produced mainly by mitochondrial oxidative phosphorylation, is increased. In cancer cells, the increase of ROS production has been associated with mtDNA mutations that, in turn, seem to be functional in the alterations of the bioenergetics and the biosynthetic state of cancer cells. Moreover, ROS overproduction can enhance the peroxidation of fatty acids in mitochondrial membranes. In particular, the peroxidation of mitochondrial phospholipid cardiolipin leads to the formation of reactive aldehydes, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), which are able to react with proteins and DNA. Covalent modifications of mitochondrial proteins by the products of lipid peroxidation (LPO) in the course of oxidative cell stress are involved in the mitochondrial dysfunctions observed in cancer and neurodegenerative diseases. Such modifications appear to affect negatively mitochondrial integrity and function, in particular energy metabolism, adenosine triphosphate (ATP) production, antioxidant defenses and stress responses. In neurodegenerative diseases, indirect confirmation for the pathogenetic relevance of LPO-dependent modifications of mitochondrial proteins comes from the disease phenotypes associated with their genetic alterations.

Novel Perspectives in Redox Biology and Pathophysiology of Failing Myocytes: Modulation of the Intramyocardial Redox Milieu for Therapeutic Interventions—A Review Article from the Working Group of Cardiac Cell Biology, Italian Society of Cardiology

The prevalence of heart failure (HF) is still increasing worldwide, with enormous human, social, and economic costs, in spite of huge efforts in understanding pathogenetic mechanisms and in developing effective therapies that have transformed this syndrome into a chronic disease. Myocardial redox imbalance is a hallmark of this syndrome, since excessive reactive oxygen and nitrogen species can behave as signaling molecules in the pathogenesis of hypertrophy and heart failure, leading to dysregulation of cellular calcium handling, of the contractile machinery, of myocardial energetics and metabolism, and of extracellular matrix deposition. Recently, following new interesting advances in understanding myocardial ROS and RNS signaling pathways, new promising therapeutical approaches with antioxidant properties are being developed, keeping in mind that scavenging ROS and RNS tout court is detrimental as well, since these molecules also play a role in physiological myocardial homeostasis.

A single chondroitin 6-sulfate oligosaccharide unit at Ser-2730 of human thyroglobulin enhances hormone formation and limits proteolytic accessibility at the carboxyl terminus : Potential insights into thyroid homeostasis and autoimmunity

We localized the site of type D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg). hTg was chromatographically separated into chondroitin 6-sulfate-containing (hTg-CS) and chondroitin 6-sulfate-devoid (hTg-CS0) molecules on the basis of their d-glucuronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6%. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondroitin 6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxyl-terminal region, starting at residue 2514. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as LTAGXGLRE (residues 2726-2734, X being Ser2730 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5 ′-triiodothyronine (T3) (171%) and 3,5,3′,5′-tetraiodothyronine (T4) (134%) than hTg-CS0. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T4 and, in particular, T3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Lys2714-Gly2715 from proteolysis, during the limited digestion of hTg-CS with trypsin. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T4/T3 ratio in hTg. The potential implications in the ability of hTg to function as an autoantigen and into the pathogenesis of thyroidal and extra-thyroidal manifestations of autoimmune thyroid disease are discussed.

Hormonogenic donor Tyr2522 of bovine thyroglobulin. Insight into preferential T3 formation at thyroglobulin carboxyl terminus at low iodination level

A tryptic fragment (b5TR,NR), encompassing residues 2515-2750, was isolated from a low-iodine (0.26% by mass) bovine thyroglobulin, by limited proteolysis with trypsin and preparative, continuous-elution SDS-PAGE. The fragment was digested with Asp-N endoproteinase and analyzed by reverse-phase HPLC electrospray ionization quadrupole time-of-flight mass spectrometry, revealing the formation of: 3-monoiodotyrosine and dehydroalanine from Tyr2522; 3-monoiodotyrosine from Tyr2555 and Tyr2569; 3-monoiodotyrosine and 3,5-diiodotyrosine from Tyr2748. The data presented document, by direct mass spectrometric identifications, efficient iodophenoxyl ring transfer from monoiodinated hormonogenic donor Tyr2522 and efficient mono- and diiodination of hormonogenic acceptor Tyr2748, under conditions which permitted only limited iodination of Tyr2555 and Tyr2569, in low-iodine bovine thyroglobulin. The present study thereby provides: (1) a rationale for the preferential synthesis of T3 at the carboxy-terminal end of thyroglobulin, at low iodination level; (2) confirmation for the presence of an interspecifically conserved hormonogenic donor site in the carboxy-terminal domain of thyroglobulin; (3) solution for a previous uncertainty, concerning the precise location of such donor site in bovine thyroglobulin.

DNA damage by lipid peroxidation products: implications in cancer, inflammation and autoimmunity

Oxidative stress and lipid peroxidation (LPO) induced by inflammation, excess metal storage and excess caloric intake cause generalized DNA damage, producing genotoxic and mutagenic effects. The consequent deregulation of cell homeostasis is implicated in the pathogenesis of a number of malignancies and degenerative diseases. Reactive aldehydes produced by LPO, such as malondialdehyde, acrolein, crotonaldehyde and 4-hydroxy-2-nonenal, react with DNA bases, generating promutagenic exocyclic DNA adducts, which likely contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. However, reactive aldehydes, when added to tumor cells, can exert an anticancerous effect. They act, analogously to other chemotherapeutic drugs, by forming DNA adducts and, in this way, they drive the tumor cells toward apoptosis. The aldehyde-DNA adducts, which can be observed during inflammation, play an important role by inducing epigenetic changes which, in turn, can modulate the inflammatory process. The pathogenic role of the adducts formed by the products of LPO with biological macromolecules in the breaking of immunological tolerance to self antigens and in the development of autoimmunity has been supported by a wealth of evidence. The instrumental role of the adducts of reactive LPO products with self protein antigens in the sensitization of autoreactive cells to the respective unmodified proteins and in the intermolecular spreading of the autoimmune responses to aldehyde-modified and native DNA is well documented. In contrast, further investigation is required in order to establish whether the formation of adducts of LPO products with DNA might incite substantial immune responsivity and might be instrumental for the spreading of the immunological responses from aldehyde-modified DNA to native DNA and similarly modified, unmodified and/or structurally analogous self protein antigens, thus leading to autoimmunity.

Generation of Adducts of 4-Hydroxy-2-nonenal with Heat Shock 60 kDa Protein 1 in Human Promyelocytic HL-60 and Monocytic THP-1 Cell Lines

Heat shock 60 kDa protein 1 (HSP60) is a chaperone and stress response protein responsible for protein folding and delivery of endogenous peptides to antigen-presenting cells and also a target of autoimmunity implicated in the pathogenesis of atherosclerosis. By two-dimensional electrophoresis and mass spectrometry, we found that exposure of human promyelocytic HL-60 cells to a nontoxic concentration (10 μM) of 4-hydroxy-2-nonenal (HNE) yielded a HSP60 modified with HNE. We also detected adducts of HNE with putative uncharacterized protein CXorf49, the product of an open reading frame identified in various cell and tissue proteomes. Moreover, exposure of human monocytic THP-1 cells differentiated with phorbol 12-myristate 13-acetate to 10 μM HNE, and to light density lipoprotein modified with HNE (HNE-LDL) or by copper-catalyzed oxidation (oxLDL), but not to native LDL, stimulated the formation of HNE adducts with HSP60, as detected by immunoprecipitation and western blot, well over basal levels. The identification of HNE-HSP60 adducts outlines a framework of mutually reinforcing interactions between endothelial cell stressors, like oxLDL and HSP60, whose possible outcomes, such as the amplification of endothelial dysfunction, the spreading of lipoxidative damage to other proteins, such as CXorf49, the activation of antigen-presenting cells, and the breaking of tolerance to HSP60 are discussed.

Lipid Peroxidation-Derived Aldehydes, 4-Hydroxynonenal and Malondialdehyde in Aging-Related Disorders

Among the various mechanisms involved in aging, it was proposed long ago that a prominent role is played by oxidative stress. A major way by which the latter can provoke structural damage to biological macromolecules, such as DNA, lipids, and proteins, is by fueling the peroxidation of membrane lipids, leading to the production of several reactive aldehydes. Lipid peroxidation-derived aldehydes can not only modify biological macromolecules, by forming covalent electrophilic addition products with them, but also act as second messengers of oxidative stress, having relatively extended lifespans. Their effects might be further enhanced with aging, as their concentrations in cells and biological fluids increase with age. Since the involvement and the role of lipid peroxidation-derived aldehydes, particularly of 4-hydroxynonenal (HNE), in neurodegenerations, inflammation, and cancer, has been discussed in several excellent recent reviews, in the present one we focus on the involvement of reactive aldehydes in other age-related disorders: osteopenia, sarcopenia, immunosenescence and myelodysplastic syndromes. In these aging-related disorders, characterized by increases of oxidative stress, both HNE and malondialdehyde (MDA) play important pathogenic roles. These aldehydes, and HNE in particular, can form adducts with circulating or cellular proteins of critical functional importance, such as the proteins involved in apoptosis in muscle cells, thus leading to their functional decay and acceleration of their molecular turnover and functionality. We suggest that a major fraction of the toxic effects observed in age-related disorders could depend on the formation of aldehyde-protein adducts. New redox proteomic approaches, pinpointing the modifications of distinct cell proteins by the aldehydes generated in the course of oxidative stress, should be extended to these age-associated disorders, to pave the way to targeted therapeutic strategies, aiming to alleviate the burden of morbidity and mortality associated with these disturbances.