Role of Carbonyl Modifications on Aging-Associated Protein Aggregation.
ABSTRACT: Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis.
Project description:Different molecular signaling pathways, biological processes, and intercellular communication mechanisms control longevity and are affected during cellular senescence. Recent data have suggested that organelle communication, as well as genomic and metabolic dysfunctions, contribute to this phenomenon. Oxidative stress plays a critical role by inducing structural modifications to biological molecules while affecting their function and catabolism and eventually contributing to the onset of age-related dysfunctions. In this scenario, proteins are not adequately degraded and accumulate in the cell cytoplasm as toxic aggregates, increasing cell senescence progression. In particular, carbonylation, defined as a chemical reaction that covalently and irreversibly modifies proteins with carbonyl groups, is considered to be a significant indicator of protein oxidative stress and aging. Here, we emphasize the role and dysregulation of the molecular pathways controlling cell metabolism and proteostasis, the complexity of the mechanisms that occur during aging, and their association with various age-related disorders. The last segment of the review details current knowledge on protein carbonylation as a biomarker of cellular senescence in the development of diagnostics and therapeutics for age-related dysfunctions.
Project description:Motivation:Oxidative stress and protein damage have been associated with over 200 human ailments including cancer, stroke, neuro-degenerative diseases and aging. Protein carbonylation, a chemically diverse oxidative post-translational modification, is widely considered as the biomarker for oxidative stress and protein damage. Despite their importance and extensive studies, no database/resource on carbonylated proteins/sites exists. As such information is very useful to research in biology/medicine, we have manually curated a data-resource (CarbonylDB) of experimentally-confirmed carbonylated proteins/sites. Results:The CarbonylDB currently contains 1495 carbonylated proteins and 3781 sites from 21 species, with human, rat and yeast as the top three species. We have made further analyses of these carbonylated proteins/sites and presented their occurrence and occupancy patterns. Carbonylation site data on serum albumin, in particular, provides a fine model system to understand the dynamics of oxidative protein modifications/damage. Availability and implementation:The CarbonylDB is available as a web-resource and for download at http://digbio.missouri.edu/CarbonylDB/. Supplementary information:Supplementary data are available at Bioinformatics online.
Project description:Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including proteins that undergo multiple modifications such as fragmentation, cross-linking, and carbonylation that result in protein unfolding and aggregation. Because the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmic reticulum (ER), aggravated ER stress, and potentiated cytotoxicity toward tumor cells. We observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response. Pretreatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132, and PSI, gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60% to 100% complete responses was observed in vivo in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether, these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application because bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors.
Project description:Nonenzymatic post-translational modification (PTM) of proteins is a fundamental molecular process of aging. The combination of various modifications and their accumulation with age not only affects function, but leads to crosslinking and protein aggregation. In this study, aged human lens proteins were examined using HPLC-tandem mass spectrometry and a blind PTM search strategy. Multiple thioether modifications of Ser and Thr residues by glutathione (GSH) and its metabolites were unambiguously identified. Thirty-four of 36 sites identified on 15 proteins were found on known phosphorylation sites, supporting a mechanism involving dehydroalanine (DHA) and dehydrobutyrine (DHB) formation through ?-elimination of phosphoric acid from phosphoserine and phosphothreonine with subsequent nucleophilic attack by GSH. In vitro incubations of phosphopeptides demonstrated that this process can occur spontaneously under physiological conditions. Evidence that this mechanism can also lead to protein-protein crosslinks within cells is provided where five crosslinked peptides were detected in a human cataractous lens. Nondisulfide crosslinks were identified for the first time in lens tissue between ?B2- & ?B2-, ?A4- & ?A3-, ?S- & ?B1-, and ?A4- & ?A4-crystallins and provide detailed structural information on in vivo crystallin complexes. These data suggest that phosphoserine and phosphothreonine residues represent susceptible sites for spontaneous breakdown in long-lived proteins and that DHA- and DHB-mediated protein crosslinking may be the source of the long-sought after nondisulfide protein aggregates believed to scatter light in cataractous lenses. Furthermore, this mechanism may be a common aging process that occurs in long-lived proteins of other tissues leading to protein aggregation diseases.
Project description:The role of ethanol-inducible cytochrome P450-2E1 (CYP2E1) in promoting aging-dependent hepatic disease is unknown and thus was investigated in this study. Young (7 weeks) and aged female (16 months old) wild-type (WT) and Cyp2e1-null mice were used in this study to evaluate age-dependent changes in liver histology, steatosis, apoptosis, fibrosis and many nitroxidative stress parameters. Liver histology showed that aged WT mice exhibited markedly elevated hepatocyte vacuolation, ballooning degeneration, and inflammatory cell infiltration compared to all other groups. These changes were accompanied with significantly higher hepatic triglyceride and serum cholesterol in aged WT mice although serum ALT and insulin resistance were not significantly altered. Aged WT mice showed the highest rates of hepatocyte apoptosis and hepatic fibrosis. Further, the highest levels of hepatic hydrogen peroxide, lipid peroxidation, protein carbonylation, nitration, and oxidative DNA damage were observed in aged WT mice. These increases in the aged WT mice were accompanied by increased levels of mitochondrial nitroxidative stress and alteration of mitochondrial complex III and IV proteins in aged WT mice, although hepatic ATP levels seems to be unchanged. In contrast, the aging-related nitroxidative changes were very low in aged Cyp2e1-null mice. These results suggest that CYP2E1 is important in causing aging-dependent hepatic steatosis, apoptosis and fibrosis possibly through increasing nitroxidative stress and that CYP2E1 could be a potential target for translational research in preventing aging-related liver disease.
Project description:Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.
Project description:Aging is characterized by the progressive decline of biochemical and physiological function in an individual. Consequently, aging is a major risk factor for diseases like cancer, obesity, and type 2 diabetes. The cellular and molecular mechanisms of aging are not well understood, nor is the relationship between aging and the onset of diseases. One of the hallmarks of aging is a decrease in cellular proteome homeostasis, allowing abnormal proteins to accumulate. This phenomenon is observed in both eukaryotes and prokaryotes, suggesting that the underlying molecular processes are evolutionarily conserved. Similar protein aggregation occurs in the pathogenesis of diseases like Alzheimer's and Parkinson's. Further, protein posttranslational modifications (PTMs), either spontaneous or physiological/pathological, are emerging as important markers of aging and aging-related diseases, though clear causality has not yet been firmly established. This review presents an overview of the interplay of PTMs in aging-associated molecular processes in eukaryotic aging models. Understanding PTM roles in aging could facilitate targeted therapies or interventions for age-related diseases. In addition, the study of PTMs in prokaryotes is highlighted, revealing the potential of simple prokaryotic models to uncover complex aging-associated molecular processes in the emerging field of microbiogerontology.
Project description:Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the K(d). This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nm rather than 212 nm at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation.
Project description:Mitochondrial oxidative stress and damage have been implicated in the etiology of temporal lobe epilepsy, but whether or not they have a functional impact on mitochondrial processes during epilepsy development (epileptogenesis) is unknown. One consequence of increased steady-state mitochondrial reactive oxygen species levels is protein post-translational modification (PTM). We hypothesize that complex I (CI), a protein complex of the mitochondrial electron transport chain, is a target for oxidant-induced PTMs, such as carbonylation, leading to impaired function during epileptogenesis. The goal of this study was to determine whether oxidative modifications occur and what impact they have on CI enzymatic activity in the rat hippocampus in response to kainate (KA)-induced epileptogenesis. Rats were injected with a single high dose of KA or vehicle and evidence for CI modifications was measured during the acute, latent, and chronic stages of epilepsy. Mitochondrial-specific carbonylation was increased acutely (48 h) and chronically (6 week), coincident with decreased CI activity. Mass spectrometry analysis of immunocaptured CI identified specific metal catalyzed carbonylation to Arg76 within the 75 kDa subunit concomitant with inhibition of CI activity during epileptogenesis. Computational-based molecular modeling studies revealed that Arg76 is in close proximity to the active site of CI and carbonylation of the residue is predicted to induce substantial structural alterations to the protein complex. These data provide evidence for the occurrence of a specific and irreversible oxidative modification of an important mitochondrial enzyme complex critical for cellular bioenergetics during the process of epileptogenesis.
Project description:Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ?1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.