Transcriptomic and proteomic studies on longevity induced by over-expression of HSP22 in drosophila melanogaster

Le vieillissement est un processus complexe accompagné par une capacité diminuée des cellules à tolérer et répondre aux formes différentes de stress causant des dommages comme l'agrégation de protéine dans les différentes composantes de la cellule. Les chaperons sont des joueurs probablement importants dans le processus de vieillissement en prévenant la dénaturation et l'agrégation des protéines. Chez Drosophila melanogaster, une petite protéine de choc thermique, Hsp22, localisée dans la matrice mitochondriale montre une expression elevée pendant le vieillissement. Sa surexpression chez la mouche augmente la durée moyenne de vie ainsi que la résistance au stress. Bien que Hsp22 montre une activité de chaperon dans des essais in vitro, les mécanismes par lesquels Hsp22 permet d’accroitre la durée de vie in vivo sont toujours inconnus. Une analyse transcriptionelle de tout le génome par microarrays et une analyse comparative du protéome mitochondrial par MALDI-TOF a été entreprise pour dévoiler les différences d’expression entre les mouches surexprimant Hsp22 et les contrôles appropriés. La surexpression générale de Hsp22 en utilisant le système GAL4/UAS dans Drosophila résulte en une augmentation de ~ 30% dans la durée de vie moyenne. L'analyse du transcriptome suggère que Hsp22 joue un rôle dans la détermination de durée de vie en changeant le processus général de vieillissement normal. Effectivement, les mouches surexprimant Hsp22 affichent une surexpression de gènes dont l’expression baisse normalement durant le vieillissement. Les gènes sont impliqués dans la production d'énergie, la biosynthèse des protéines, le taux de renouvellement des protéines et le métabolisme lipidique. L'analyse du protéome mitochondrial soutient aussi un rôle de Hsp22 sur la détermination de la durée de vie en maintenant la fonction mitochondriale et en favorisant la protéolyse. Les présentes données suggèrent l'importance de la maintenance de l’homéostasie protéique durant le vieillissement et discutent des mécanismes potentiels d’extension de la longévitié chez les mouches surexprimant Hsp22.Aging is a complex process accompanied by a decreased capacity of cells to tolerate and respond to various forms of stresses leading to damages such as protein aggregation in various components of the cell. Chaperones are thus likely important players in the aging process by preventing protein denaturation and aggregation. In Drosophila melanogaster, a small heat shock protein Hsp22 localized in the mitochondrial matrix is preferentially up-regulated during aging. Its over-expression results in an extension of lifespan and an increased resistance to stress. Although Hsp22 has been shown to have a chaperone-like activity in vitro, the mechanisms by which it extends lifespan in vivo are still unknown. Genome-wide transcriptional analysis by microarray and comparative mitochondrial proteomic analysis by MALDI-TOF mass analysis have been performed to unveil differences in long-lived Hsp22 over-expressing flies and normal-lived control flies. Ubiquitous over-expression of Hsp22 using the GAL4/UAS system in Drosophila resulted in a ~ 30% increase in mean lifespan. The genomic analysis suggests that Hsp22 plays a role in lifespan determination by altering the regulation of the overall process of normal aging. Indeed, flies over-expressing Hsp22 display an up-regulation of genes normally down-regulated with age and involved in energy production, protein biosynthesis, protein turnover, and lipid metabolism. Mitochondrial proteomic analysis also supports a putative role of Hsp22 on lifespan determination by maintaining mitochondrial function and favoring proteolysis. The present data suggest the importance of the maintenance of protein homeostasis in aging and potential mechanisms of longevity in the Hsp22 over-expressing flies

Biochemical effects of protein glycation by methylglyoxal in Saccharomyces cerevisiae

Tese de doutoramento em Bioquímica (Regulação Bioquímica), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2008Protein glycation, the non-enzymatic and irreversible modification of amino groups by carbonyl compounds, is assuming an important role in the context of a widerange of human pathologies, including diabetes mellitus, age-related disorders andneurodegenerative diseases of amyloid type. Hence, there is a growing interest in thispost-translational modification and, ultimately, the quest for inhibitors of proteinglycation. However, despite extensive research, mainly by in vitro glycation studies withrelevant or model proteins, the role of glycation in pathological conditions is stillunknown. Therefore, cellular research models are required to investigate proteinglycation in vivo, the resulting biochemical effects and its role in human diseases.In the work presented in this thesis, a novel approach was developed toinvestigate protein glycation in vivo by methylglyoxal and its biochemical effects.Moreover, the role of glycation by methylglyoxal in protein amyloidogenesis was alsoinvestigated. It was found that protein glycation occurs in yeast and defined glycationphenotypes were identified. Although protein glycation was primarily associated withcomplex organisms and long-lived proteins, this post-translational modification alsoaffects short-lived organisms like yeast. A direct relationship between methylglyoxalformation rate and protein glycation was observed. A kinetic model of methylglyoxalmetabolism was developed to investigate the relative importance of the glyoxalasepathway, aldose reductase and methylglyoxal formation rate on the methylglyoxalsteady-state concentration and their relationship with protein glycation in vivo. It wasfound that the glyoxalase system and aldose reductase enzyme are equally important askey anti-glycation defenses in yeast. A higher methylglyoxal input leads to a directincrease in methylglyoxal concentration even in the presence of the glyoxalase systemand aldose reductase. In fact, challenging non-growing yeast cells with a high D-glucosemedium, consequently increasing methylglyoxal formation rate, causes methylglyoxalderivedprotein glycation even in the reference strain with all enzymatic defenses againstmethylglyoxal. Therefore, there is a subtle balance between methylglyoxal metabolismand the accumulation of MAGE-modified proteins, in which cells can prevent MAGEformation only until anti-glycation defenses are overcome.SummaryxviiiInterestingly, glycation in yeast appears to be a targeted process, whereby only afew proteins are modified. Protein identification, MAGE assignment and location weredetermined by mass spectrometry. The heat shock proteins Hsp71/72 and Hsp26 werefound to be glycated in vivo. Hsp26, a critical element in the unfolding stress response,was only detected in the soluble form upon glycation. This finding shows that Hsp26 ismost likely activated in glycation conditions, similarly to what happens in thermal stress.Three glycolytic enzymes were also glycated in yeast, namely phosphoglycerate mutase,aldolase and enolase, being the latter the main glycation target. This discovery prompteda deeper study of the biochemical implications of enolase glycation in vivo. Despite theobserved glycation-dependent activity loss of enolase, glycolysis and cell viabilityremained unchanged, hinting that yeast cells evolved to cope with high glycation levels.To investigate the effects of glycation on enolase structure and enzymatic activity, theprotein was purified from yeast cells under native and in vivo glycation conditions. Forthe first time, a protein was studied while enduring glycation in physiological conditions,allowing a direct comparison between in vivo and in vitro protein glycation. Significantdifferences were found between these distinct experimental conditions. In vivo glycationappears to be a specific process with only a few amino acid residues consistentlymodified with the same MAGE. Structural changes, evaluated by circular dichroismspectroscopy and thermal denaturation, showed that glycation mainly decreases α-helicalcontent and increase unordered structure while enolase structural rigidity increases. Invitro, a greater molecular heterogeneity was observed with different MAGE occurring atthe same molecular locations. α-Helical content also decreased, but in vitro glycationmarkedly increases ß-sheet content and structural rigidity was further enhanced. It wasalso observed that glycation causes enolase unfolding and dimer dissociation.Based on the identification of MAGE location by mass spectrometry, a molecularmodel for enolase inactivation upon glycation was developed. Glycation occurs at R414,a critical residue for dimer stability. Modification of R414 disrupts electrostaticinteractions with E20 on the other enolase chain that stabilize the enolase dimer, leadingto its dissociation and consequent formation of inactive monomers.The molecular location of MAGE in enolase suggests that the tri-dimensionalstructure may directly influence glycation reaction. The modified-arginine residues wereSummaryxixmainly found in an arginine-rich crevice located at the dimer interface, but solventaccessible. This arginine-rich cave could create a favourable environment formethylglyoxal-derived glycation reactions, sequestering free methylglyoxal that evadedfrom its catabolic routes. Hence, the high enolase reactivity towards methylglyoxalinducedprotein glycation could scavenge methylglyoxal, preventing changes in thebiochemical functionalities of other proteins. Upon glycation, enolase unfolds but cellsactivate the refolding chaperone pathway to counteract enolase misfolding and to limitthe harmful effects associated with extensive protein misfolding and aggregation.Yeast cells emerged as a living test tube to investigate the biochemical effects ofglycation on protein structure and function in vivo. So, taking advantage of this finding,the link between protein glycation and amyloid disorders, was investigated. Using animproved procedure for the extraction of amyloid fibrils from FAP patients, we providedthe first unequivocal evidence that methylglyoxal-derived advanced glycation endproductsare present in transthyretin amyloid deposits of FAP patients. Thus, we studiedthe effect of protein glycation in transthyretin amyloid fibril formation in yeast. For thispurpose, TTR variants with different amyloidogenic potentials (TTR-wt, TTR-L55P andTTRd-D) were expressed in Saccharomyces cerevisiae and amyloid deposits weredetected by fluorescence microscopy. It was observed that TTR is glycated when yeastcells are exposed to glycation conditions. Furthermore, the formation of transthyretinamyloidaggregates in cells expressing the amyloidogenic TTR-L55P variant is induced.These results provide the first direct evidence that glycation causes protein aggregation invivo in the context of human amyloid disorders.The presented results and conclusions are of great value not only to increase ourknowledge about protein glycation and its biochemical effects in vivo, but also to assign aclear role of this non-enzymatic process in the development of amyloid disorders. In thiscontext, yeast cells will certainly be useful as a eukaryotic model to study these processesat a cellular level. This is of vital importance in the design of novel or improved therapeutic strategies to inhibit protein glycation and counteract its harmful effects.A glicação de proteínas é uma modificação pos-traducional, tendo como consequência a modificação irreversível de grupos amina por compostos contendo grupos carbonilo. Esta modificação tem sido implicada em diversas patologias humanas, tais como diabetes mellitus, doenças relacionadas com o envelhecimento e doenças neurodegenerativas do tipo amilóide. Neste contexto, a glicação de proteínas tem sido alvo de interesse na comunidade científica, com o objectivo final de desenvolver estratégias para inibir esta modificação pos-traducional. No entanto, apesar de intensivamente investigada in vitro com proteínas modelo ou clinicamente relevantes, o papel da glicação de proteínas no desenvolvimento de diversas condições patológicas não é ainda conhecido. Assim, é de extrema importância desenvolver modelos celulares para investigar a glicação de proteínas in vivo, os seus efeitos bioquímicos e finalmente a suaimportância nas diversas doenças humanas em que está envolvida.Neste trabalho, foi desenvolvida uma nova abordagem para investigar a glicaçãoin vivo pelo metilglioxal e os seus efeitos bioquímicos. Para além disso, foi tambéminvestigado o papel da glicação de proteínas pelo metilglioxal na formação de fibrasamilóides derivadas de uma proteína amiloidogénica. Observou-se a acumulação deproteínas glicadas pelo metilglioxal na levedura Saccharomyces cerevisiae, comdiferentes fenótipos de glicação. Apesar da glicação de proteínas ter sido associada aorganismos complexos e proteínas com baixo turnover, esta modificação pos-traducionaltambém afecta microrganismos como a levedura. Foi observado que existe uma relaçãodirecta entre a velocidade de formação de metilglioxal e a ocorrência de glicação. Ummodelo cinético do metabolismo do metilglioxal em S. cerevisiae foi construído parainvestigar a importância da via dos glioxalases, do aldose reductase e da velocidade deformação de metilglioxal na concentração em estado estacionário deste α-oxoaldeído.Com esta análise, verificou-se que o sistema dos glioxalases e o aldose reductase sãoigualmente importantes como defesas anti-glicação pelo metilglioxal. Observou-setambém que existe uma relação directa entre a velocidade de formação de metilglioxal e asua concentração em estado estacionário, mesmo na presença do sistema dos glioxalasesResumoxxiie do aldose reductase. De facto, um aumento da formação de metilglioxal, através daexposição de células a uma concentração elevada de D-glucose, provoca a glicação deproteínas pelo metilglioxal na estirpe de referência que apresenta todas as defesasenzimáticas contra o metilglioxal. Em condições fisiológicas, as células previnem aacumulação de proteínas modificadas pelo metilglioxal, mas só até as suas defesas seremultrapassadas.Na levedura S. cerevisiae, a glicação é um processo específico, onde apenasalgumas proteínas são modificadas. Estas proteínas foram identificadas porespectrometria de massa, assim como a natureza e localização dos produtos avançados deglicação derivados do metilglioxal (MAGE, Methylglyoxal Advanced Glycation Endproducts).As proteínas de choque térmico Hsp71/72 e Hsp26 foram identificados eapresentam modificações pos-traducionais derivadas da glicação pelo metilglioxal. AHsp26, um elemento crítico na resposta celular a condições de stress de unfolding, foiapenas detectada na forma solúvel após glicação. Esta observação indicia que a Hsp26 éactivada em condições de glicação, tal como se verifica em condições de stress térmico.Para além destas proteínas, três enzimas glicolíticos estão também glicados in vivo: ofosfoglicerato mutase, o aldose e o enolase, sendo este último o principal alvo deglicação. Um estudo detalhado das implicações bioquímicas da glicação in vivo doenolase foi realizado. Apesar de ter sido detectada uma diminuição da actividade desteenzima devido à glicação, a via glicolítica e a viabilidade celular permaneceraminalteradas, sugerindo que a levedura evoluiu de forma suportar elevados níveis deglicação. Para averiguar o efeito da glicação na estrutura e actividade enzimática doenolase, esta proteína foi purificada em condições nativas e em condições de glicação.Pela primeira vez, os efeitos da glicação in vivo na estrutura e função de uma proteína,foram investigados. Para além disso, este estudo permitiu efectuar uma comparaçãodirecta entre a glicação de proteínas in vivo e in vitro, tendo sido encontradas diferençassignificativas. In vivo, a glicação aparenta ser um processo específico, em que apenasalguns resíduos de aminoácidos são consistentemente modificados pelo mesmo MAGE.Nestas condições, a glicação induz alterações estruturais, observadas por dicroismocircular, com uma diminuição do conteúdo em hélice α e um aumento da estruturadesordenada e da rigidez estrutural. In vitro, foi observado uma elevada heterogeneidadeResumoxxiiimolecular, com o mesmo resíduo de aminoácido modificado por diferentes MAGE emdiferentes moléculas de proteína. Foi também detectada uma diminuição do conteúdo emhélice α, mas a glicação in vitro induz um aumento significativo de folha ß. Em ambas ascondições experimentais, a glicação causa a desnaturação do enolase, com a consequentedissociação do dímero, a forma activa do enzima.Com base na identificação da localização molecular dos MAGE porespectrometria de massa, foi proposto um modelo para a inactivação do enolase pelaglicação. Esta modificação pos-traducional ocorre em R414, essencial para a estabilidadeda forma dimérica. A formação de uma hidroimidazolona neste resíduo de argininadestrói as interacções electrostáticas com E20 da outra cadeia que estabilizam o dímero,levando à sua dissociação e consequente formação de monómeros inactivos.A localização molecular dos MAGE no enolase sugere que a estruturatridimensional da proteína influencia as reacções de glicação. As modificações ocorremmaioritariamente numa cavidade rica em resíduos de arginina localizada na interface dodímero. Este local pode criar um ambiente favorável a reacções de glicação pelometilglioxal, eliminando assim o metilglioxal não catabolisado pelos sistemasenzimáticos. Assim, a elevada reactividade do enolase para reacções de glicaçãoderivadas do metilglioxal pode diminuir a concentração intracelular deste α-oxoaldeído,prevenindo assim a alteração da função de outras proteínas celulares. Após glicação, oenolase sofre alterações estruturais resultando na desnaturação da proteína e a célulaactiva a via de refolding para neutralizar os efeitos nocivos associados a processos demisfolding proteico e agregação.Os estudos apresentados nesta tese revelaram que a levedura S. cerevisiaeconstitui um excelente modelo para investigar in vivo os efeitos bioquímicos da glicaçãona estrutura e função de proteínas. Assim, o papel da glicação de proteínas em doençasamilóides foi estudado na levedura S. cerevisiae como modelo celular. Utilizando ummétodo aperfeiçoado de extracção de fibras amilóides de pacientes com polineuropatiaamiloidótica familiar (FAP, Familial Amyloidotic Polyneuropathy), foi detectadainequivocamente a presença da argipirimidina (um produto avançado de glicaçãoderivado do metilglioxal) nos depósitos amilóides de transtirretina (TTR). Estaobservação indica que a glicação de proteínas deverá estar envolvida nesta doençaResumoxxivamilóide. Para investigar o papel da glicação na transtirretina e consequente formação defibras amilóides in vivo, variantes da TTR com diferentes potenciais amilóidogénicos(TTR-wt, TTR-L55P and TTRd-D) foram expressos em S. cerevisiae e os depósitosamilóides foram detectados por microscopia de fluorescência. Foi observada glicação invivo da TTR quando as células foram expostas a condições favoráveis à ocorrência desteprocesso. Nestas condições experimentais, foi observada uma indução da formação dedepósitos amilóides derivados da variante amiloidogénica TTR-L55P. Esta observaçãoconstitui a primeira evidência experimental de que a glicação de proteínas induz aagregação e formação de fibras amilóides in vivo.No seu conjunto, os resultados apresentados nesta tese e as conclusões inerentessão bastante importantes não apenas para compreender os mecanismos envolvidos naglicação de proteínas e consequentes efeitos bioquímicos in vivo, mas também paraclarificar o papel deste processo não enzimático no desenvolvimento de diversaspatologias humanas, como as doenças do tipo amilóide. Neste contexto, a leveduraSaccharomyces cerevisiae constitui um excelente modelo para investigar estes processosa nível celular. Estes estudos são de vital importância para desenvolver novas abordagensterapêuticas para inibir a glicação de proteínas e minimizar os seus efeitos prejudiciais

53rd National Meeting of the Italian Society of Biochemistryand Molecular Biology (SIB)andNational Meeting of Chemistry of Biological Systems – Italian Chemical Society (SCI - Section CSB)

The 53rd National Congress of the Italian Society of Biochemistry and Molecular Biology (SIB), which will be held in Riccione from 23 to 26 September, is characterised by the elevated scientific level and interdisciplinary interest of the numerous sessions in which it is organised. The Scientific Programme comprises three joint Symposia of the SIB and the Chemistry of Biological Systems section of the Italian Chemistry Society (SCI) on Molecular Systems Biology, Chemistry of Nucleic Acids, Protein and Drug Structure, and Environmental Biotechnology. These Symposia address groundbreaking arguments, making the joint interest of the two societies particularly fascinating; the joint organisation of these events in fact signals the shared intention to proceed along the path of scientific exchange. The topics of the other sessions have been chosen by the Scientific Committee on the basis of their scientific relevance and topicality, with particular attention paid to the selection of the speakers. The SIB sessions will range from Signal Transduction and Biomolecular Targets, Protein Misfolding and its Relationship with Disease, Emerging Techniques in Biochemistry, Gene Silencing, Redox Signalling and Oxidative Stress, Lipids in Cell Communication and Signal Transduction, Mitochondrial Function and Dysfunction

The Coupling Between Folding, Zinc Binding, and Disulfide Bond Status of Human Cu, Zn Superoxide Dismutase: A Dissertation

Cu, Zn superoxide dismutase (SOD1) is a dimeric, β-sandwich, metalloenzyme responsible for the dismutation of superoxide. Mutations covering nearly 50% of the amino acid sequence of SOD1 have been found to acquire a toxic gain-of-function leading to amyotrophic lateral sclerosis. A hallmark of this disease is the presence of insoluble aggregates containing SOD1 found in the brain and spinal cord. While it is unclear how these aggregates or smaller, precursor oligomeric species may be the source of the toxicity, mutations leading to increased populations of unstable, partially folded species along the folding pathway of SOD1 may be responsible for seeding and propagating aggregation. In an effort to determine the responsible species, we have systematically characterized the stability and folding kinetics of five well studied ALS variants: A4V, L38V, G93A, L106V and S134N. The effect of the amino acid substitutions was determined on a variety of different constructs characterizing the various post-translational maturation steps of SOD1: folding, disulfide bond formation and Zn binding. Zn was found to bind progressively tighter along the folding pathway of SOD1, minimizing populations of monomeric species. In contrast, ALS variants were found to have the greatest perturbation in the equilibrium populations of the folded and unfolded state for the most immature, disulfide-reduced metal-free SOD1. In this species, at physiological temperature, four out of five ALS variants were \u3e50% unfolded. Finally the energetic barriers in the folding and unfolding reaction were studied to investigate the unusually slow folding of SOD1. These results reveal that both unfolding and refolding are dominated by enthalpic barriers which may be explained by the desolvation of the chain and provide insights into the role of sequence in governing the folding pathway and rate

Tyrosine hydroxylase and HSPB8 interact with the prion protein

The prion protein (PrP) is currently one of the most studied molecules in the neurosciences as it causes a group of neurological diseases collectively named transmissible spongiform encephalophaties (TSEs). The TSEs are characterized by a variety of motor and/or cognitive symptoms distinct from those of Parkinson and Alzheimer diseases and severely affect both humans and a variety of mammals. A great effort has thus been made to understand the molecular basis of PrP activity, both in physiological and pathological terms. In this context, the identification of neuronally-relevant interactors of PrP, capable of governing or interfering with its activity, cellular localization and/or expression, plays a crucial role. Through the expression of the proteins of interest in recombinant form in E.coli cells and the analysis of their interaction by Western blot and dot-blot, we identified two specific and neurologically relevant interactions involving the prion protein and on the one hand tyrosine hydroxylase, the enzyme that catalyzes the initial and rate-limiting reaction of the biosynthesis of catecholamines dopamine, norepinephrine and epinephrine; on the other hand the heat shock protein B8, member of the small heat shock protein family that appears to play an important role in those diseases that, like transmissible spongiform encephalopathies, are characterized by the accumulation of misfolded proteins. The association/dissociation constants of the complexes have been calculated using surface plasmon resonance and interactions were confirmed by immunohistochemistry. The data obtained show a specific and high affinity interaction (KD in the nano molar range) between the TH N-terminal regulatory domain (1-152) and the C-terminal structured domain (90-230) of PrP. The co-expression of the two proteins causes a shift in prion protein expression from a prevalent membrane-associated expression to a greater cytoplasmic localization, and also a down-regulation of the levels of expression of the prion protein without, however, affecting the topology and expression of tyrosine hydroxylase. The link between PrP and HSPB8 involves the C-terminal domain of the prion protein and the co-expression of the two proteins leads to a down-regulation of both prion protein and HSPB8. The latter, moreover, protects the prion protein from degradation by Proteinase K, while it seems to accelerate the thermal denaturation. The mutants K141E and K141N of the heat shock protein, associated with the development of neuropathy, show minor binding affinity with respect to the wild type protein and they are less effective in regulating the levels of expression, localization and degradation of the prion protein. Our results, in particular if confirmed in pathological patterns, can help to understand the physiological and pathological mechanisms of action of the prion protein and suggest tyrosine hydroxylase and heat shock protein B8 as prion protein modulators, thus as potential targets for therapeutic applications

Transglutaminase in the life and death of the pancreatic β-cell

Tissue transglutaminase (TG2) is a ubiquitous enzyme that catalyses both the Ca2+-dependent formation of protein cross-links via intermolecular isopeptide bonds, and the Ca2+-independent hydrolysis of GTP. The multifunctional nature of the TG2 protein has been reported in numerous intracellular mechanisms, cell-surface associations, and extracellular matrix (ECM) interactions. In the pancreas, the expression of TG may be fundamental to the insulin-secretion function of β-cells, and associated diabetic disorders. The functional roles of TG2 in the pancreas were investigated in the present study using in vitro models of rat pancreatic insulinoma β-cells (BRIN-BD11), and ex vivo islet of Langerhans models from human, rat, TG2(-/-) knockout mice and their wild-type counterparts. The importance of ECM-associated TG2 in the maintenance of β-cell survival and function was also investigated using an in vitro human urinary bladder carcinoma support matrix (5637 cells). Biochemical analysis of both clonal BRIN-BD11 and islet β-cells showed a thiol-dependent TG2 activity mechanism that was reciprocally regulated by Ca2+ and GTP

REDOX PROTEOMICS IDENTIFICATION OF OXIDATIVELY MODIFIED PROTEINS AND THEIR PHARMACOLOGICAL MODULATION: INSIGHT INTO OXIDATIVE STRESS IN BRAIN AGING, AGE-RELATED COGNITIVE IMPAIRMENT

The studies presented in this work were completed with the goal ofgaining greater insight into the roles of protein oxidation in brain aging and age-relatedcognitive impairment. Aging is associated with the impairment of physiological systemssuch as the central nervous system (CNS), homeostatic system, immune system, etc.Functional impairments of the CNS is associated with increased susceptibility to developmany neurodegenerative diseases such as Alzheimer\u27s diseases (AD), Parkinson\u27s disease(PD), and amyotrophic lateral sclerosis (ALS). One of the most noticeable functionalimpairments of the CNS is manifested by cognitive decline. In the past three decades, thefree radical theory of aging has gained relatively strong support in this area. Excessiveproduction reactive oxygen species (ROS) was demonstrated as a contributing factor inage-related memory and synaptic plasticity dysfunction. This dissertation use proteomicsto identify the proteins that are oxidatively modified and post-translationally altered inaged brain with cognitive impairment and normal aging brain.Ongoing research is being pursued for development of regime to preventoxidative damage by age-related oxidative stress. Among which are those that scavengefree radicals by antioxidants, i.e. ??-lipoic acid (LA), and protecting the brains byreducing production of neurotoxic substance, i.e. reducing production of amyloid ??(A??).Therefore, proteomics were also used to identify the alteration of specific proteins in agedbrain treated with LA and antisense oligonucleotides again amyloid protein precursor.This dissertation provides evidences that certain proteins are less oxidatively modifiedand post-translationally altered in cognitively impaired aged brain treated with LA andantisense oligonucleotides against the A?? region of amyloid precursor protein (APP)(AO).Together, the studies in this dissertation demonstrated that increased oxidativestress in brain play a significant role in age-related cognitive impairment. Moreover, suchincreased oxidative stress leads to specific protein oxidation in the brain of cognitiveimpaired subject, thereby leading to cognitive function impairment. Moreover, thefunctional alterations of the proteins identified by proteomics in this dissertation mayleads to impaired metabolism, decline antioxidant system, and damaged synapticcommunication. Ultimately, impairment of these processes lead to neuronal damages andcognitive decline. This dissertation also show that several of the up-regulated andoxidized proteins in the brains of normal aging mice identified are known to be oxidizedin neurodegenerative diseases as well, suggesting that the expression levels of certainproteins may increase as a compensatory response to oxidative stress. This compensationwould allow for the maintenance of proper molecular functions in normal aging brainsand protection against neurodegeneration

Stability and Aggregation Studies of Immature Superoxide Dismutase

Amyotrophic Lateral Sclerosis is a devastating neurological disease with no known cure. In 1993, a genetic link was established between ALS and mutant forms of Cu, Zn-superoxide dismutase (SOD1), an antioxidant enzyme that catalyzes the dismutation of the damaging free radical superoxide anion (O2-) to hydrogen peroxide (H2O2) and dioxygen (O2). The inheritable form of ALS (fALS) accounts for ~10% of all ALS cases and so SOD1 mutations comprise ~1.5-2% of all ALS cases, but nevertheless represent a major known cause of the disease. Furthermore, the clinical symptoms of fALS and sALS are similar, although fALS patients with SOD1 mutations have an earlier age of disease onset than sALS (by ~10 years). A major hypothesis in the field of ALS research is that mutations decrease the stability and increase the aggregation propensity of SOD1, causing motor neuron degeneration. Attempts to identify relationships between the effects of the mutations and ALS characteristics have shown that these effects are highly complex and not correlated with disease characteristics in a simple way. SOD1 undergoes various in vivo modifications (notably disulfide bond formation and metal binding) and the form of SOD1 that is relevant to ALS toxicity is unknown. Recently, attention has focused on the immature forms of SOD1, which lack metal and/or disulfide bonds, because these forms are more destabilized by ALS-associated mutations compared to the mature, metal-bound, disulfide oxidize (holoSS) form. A powerful approach to uncovering the mechanisms of SOD1 misfolding and aggregation is to investigate the how mutations affect the global and local stability of SOD1 under physiological conditions. Here, the stability of disulfide-oxidized (SS), metal free (apo) SOD1 has been investigated by combining isothermal titration and differential scanning calorimetry techniques (ITC and DSC, respectively) to break down changes in global stability into dimer interface and monomer stability components. First, ITC was used to assess the thermodynamics of dimer dissociation for pWT and 13 ALS-associated mutants and the results were confirmed using size exclusion chromatography (SEC). Together these experiments reveal that all mutations investigated, even those far removed from the interface, promote dissociation. Furthermore, apo SOD1 dissociation is characterized by large ∆Cp, ∆H and ∆S changes, far larger than expected based on theoretical calculations of surface area changes estimated from the crystal structure. This finding suggests that large conformation changes accompany dissociation and that monomeric apo SOD1 is fairly malleable, a finding that suggests dimerization may play an important role in the maturation of SOD1, by preventing the buildup of partially unfolded, aggregation-protein species. Subsequent to these studies, total unfolding (ie. folded dimer to unfolded monomer) for the same set of mutants was characterized using DSC, and the data fit to a 3-state folding mechanism with monomer intermediate. Due to the complexity of this model, numerous DSC experiments for the same mutant were globally fit, with the energetics of the first transition fixed to values obtained by ITC, thus reducing the uncertainty in the fitted parameters that define monomer unfolding. The results from this approach reveal that mutations have variable effects on apoSS monomer stability. In most cases, apoSS monomers are only marginally stable; accordingly, mutations greatly elevate the levels of unfolded protein under physiological conditions. In contrast, two mutations, while decreasing dimer interface stability, actually increase monomer stability. These experiments show that mutations have markedly different effects on the populations of folded and unfolded monomers in vivo and disclose important implications for disease-relevant aggregation. We have also characterized the stability of the most immature form of SOD1 (apoSH SOD1), which lacks both disulfide bonds and metals. This form of the protein is mainly monomeric, with marginal stability that is greatly affected by ALS-associated mutations. Surprisingly however, we find that this form of SOD1 is remarkably resistant to aggregation under physiological-like conditions. Static and dynamic light scattering (SLS and DLS, respectively) as well as analytical ultracentrifugation (AUC) reveal higher-order interactions are present, but of the 12 mutants invested, only one showed evidence of aggregate formation. Increased protein concentrations or the addition of salt promote aggregation of some SOD1 mutants and the mechanism(s) of aggregation have been characterized using light scattering, atomic force microscopy (AFM), and Thioflavin-T (ThT) binding. Under conditions that enhance aggregation, DLS and AFM experiments reveal that some mutants form small fibrils ranging from ~20-100 nm in length, or ~2-100 monomers. Other mutants aggregate less, but the aggregates that do form are longer (greater than 1000 nm). Furthermore, ThT binding experiments suggest that the aggregates contain different degrees of β-structure. Mutations appear to have complex effects on the energy landscape of apoSH SOD1, promoting different aggregation pathways. This complexity may help explain the different disease phenotypes associated with different mutants. By characterizing both folding and aggregation of different forms of immature SOD1, we have employed a powerful approach to untangling the role of toxic aggregation in the syndrome of ALS.1 yea

53rd National Meeting of the Italian Society of Biochemistry and Molecular Biology (SIB) and National Meeting of Chemistry of Biological Systems – Italian Chemical Society (SCI - Section CSB)

Il 53° Congresso Nazionale della Società Italiana di Biochimica e Biologia Molecolare che si tiene a Riccione dal 23 al 26 Settembre si distingue per l'alto livello scientifico e l'interesse interdisciplinare delle numerose sessioni nelle quali è strutturato. Il Programma scientifico vede tre Simposi congiunti della SIB con la Sezione della Chimica dei Sistemi Biologici della Società Italiana di Chimica (SCI) su Molecular Systems Biology, Chemistry of Nucleic Acids, Protein and Drug Structure, Environmental Biotechnology. Questi Simposi, riguardano argomenti di avanguardia per i quali fa piacere l'interesse condiviso delle due Società, che per la prima volta organizzano dei Simposi congiunti a significare l'intento di procedere insieme negli scambi scientifici. Gli argomenti delle altre sessioni sono stati scelti dal comitato scientifico in base alla loro rilevanza e attualità scientifica, con particolare cura nella individuazione dei relatori. Le sessioni SIB spazieranno da Signal Transduction and Biomolecular Targets, Protein Misfolding and its Relationship with Diseases, Emerging Techniques in Biochemistry, Gene Silencing, Redox Signalling and Oxidative Stress, Lipids in Cell Communication and Signal Transduction, Mitochondrial Function and Dysfunction