Oxidation Of Methionine Residues Research Articles

Stability of Multi-Peptide Vaccines in Conditions Enabling Accessibility in Limited Resource Settings

We have previously shown that lyophilized mixtures of six- and twelve-melanoma peptide vaccines retain stability, purity, and amino acid sequence identity for up to five years when stored at -80 °C. However, this temperature requirement presents a significant obstacle to storage and distribution of vaccines for low-resource environments. Thus, we examined the stability of these peptides over a range of temperatures for varying durations of time. When stored at +4 °C or at room temperature for up to three months, 17 of the 18 peptides remained stable. The only change for the remaining peptide was an increase in an oxidized methionine residue. The results presented in this report support broadening the accessibility of these and other peptide-based vaccines to resources-limited communities.

Methionine Alkylation as an Approach to Quantify Methionine Oxidation Using Mass Spectrometry.

Post-translational oxidation of methionine residues can destabilize proteins or modify their functions. Although levels of methionine oxidation can provide important information regarding the structural integrity and regulation of proteins, their quantitation is often challenging as analytical procedures in and of themselves can artifactually oxidize methionines. Here, we develop a mass-spectrometry-based method called Methionine Oxidation by Blocking with Alkylation (MObBa) that quantifies methionine oxidation by selectively alkylating and blocking unoxidized methionines. Thus, alkylated methionines can be used as a stable proxy for unoxidized methionines. Using proof of concept experiments, we demonstrate that MObBa can be used to measure methionine oxidation levels within individual synthetic peptides and on proteome-wide scales. MObBa may provide a straightforward experimental strategy for mass spectrometric quantitation of methionine oxidation.

Methionine sulfoxide reductases and cholesterol transporter STARD3 constitute an efficient system for detoxification of cholesterol hydroperoxides

Methionine sulfoxide reductases (MSRs) are key enzymes in the cellular oxidative defense system. Reactive oxygen species oxidize methionine residues to methionine sulfoxide, and the methionine sulfoxide reductases catalyze their reduction back to methionine. We previously identified the cholesterol transport protein STARD3 as an invivo binding partner of MSRA (methionine sulfoxide reductase A), an enzyme that reduces methionine-S-sulfoxide back to methionine. We hypothesized that STARD3 would also bind the cytotoxic cholesterol hydroperoxides and that its two methionine residues, Met307 and Met427, could be oxidized, thus detoxifying cholesterol hydroperoxide. We now show that in addition to binding MSRA, STARD3 binds all three MSRB (methionine sulfoxide reductase B), enzymes that reduce methionine-R-sulfoxide back to methionine. Using pure 5, 6, and 7 positional isomers of cholesterol hydroperoxide, we found that both Met307 and Met427 on STARD3 are oxidized by 6α-hydroperoxy-3β-hydroxycholest-4-ene (cholesterol-6α-hydroperoxide) and 7α-hydroperoxy-3β-hydroxycholest-5-ene (cholesterol-7α-hydroperoxide). MSRs reduce the methionine sulfoxide back to methionine, restoring the ability of STARD3 to bind cholesterol. Thus, the cyclic oxidation and reduction of methionine residues in STARD3 provides a catalytically efficient mechanism to detoxify cholesterol hydroperoxide during cholesterol transport, protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Antioxidant role of methionine-containing intra- and extracellular proteins.

Significant evidence suggests that reversible oxidation of methionine residues provides a mechanism capable of scavenging reactive species, thus creating a cycle with catalytic efficiency to counteract or mitigate deleterious effects of ROS on other functionally important amino acid residues. Because of the absence of MSRs in the blood plasma, oxidation of methionines in extracellular proteins is effectively irreversible and, therefore, the ability of methionines to serve as interceptors of oxidant molecules without impairment of the structure and function of plasma proteins is still debatable. This review presents data on the oxidative modification of both intracellular and extracellular proteins that differ drastically in their spatial structures and functions indicating that the proteins contain antioxidant methionines/the oxidation of which does not affect (or has a minor effect) on their functional properties. The functional consequences of methionine oxidation in proteins have been mainly identified from studies in vitro and, to a very limited extent, in vivo. Hence, much of the functioning of plasma proteins constantly subjected to oxidative stress remains unclear and requires further research to understand the evolutionary role of methionine oxidation in proteins for the maintenance of homeostasis and risk factors affecting the development of ROS-related pathologies. Data presented in this review contribute to increased evidence of antioxidant role of surface-exposed methionines and can be useful for understanding a possible mechanism that supports or impairs structure-function relationships of proteins subjected to oxidative stress.

On the electrochemical oxidation of methionine residues of proteins

The oxidation of methionine side chain residues in proteins provides a great diversity of possible oxidation mechanisms dictated, among others, by the oxidizing species, solvent properties, and protein structure. The oxidation behavior of a series of short acetylated synthetic peptides, Ac-GMG, Ac-GGMGG and Ac-GGGMGGG, was investigated by differential pulse and square wave voltammetry, in a wide pH range, at glassy carbon electrode. It was found that always the first oxidation step represents the one-electron oxidation of thioether moiety with the formation of a radical cation. Following this, depending on the experimental conditions and side chain position of methionine, the radical is stabilized by the nucleophilic attack of water or by catalytic support of the neighboring carbonyl and amide groups in an intermediate structure, finally converted in methionine sulfoxide which can be further oxidized, at more positive potential, into methionine sulfone. For Ac-GGGMGGG, at pH 8.0, the amino and amide groups are active involved in the oxidation process and the electrode reaction takes place with proton transfer

A novel method for in silico assessment of Methionine oxidation risk in monoclonal antibodies: Improvement over the 2-shell model.

Over the past decade, therapeutic monoclonal antibodies (mAbs) have established their role as valuable agents in the treatment of various diseases ranging from cancers to infectious, cardiovascular and autoimmune diseases. Reactive groups of the amino acids within these proteins make them susceptible to many kinds of chemical modifications during manufacturing, storage and in vivo circulation. Among these reactions, the oxidation of methionine residues to their sulfoxide form is a commonly observed chemical modification in mAbs. When the oxidized methionine is in the complementarity-determining region (CDR), this modification can affect antigen binding and thus abrogate biological activity. For these reasons, it is essential to identify oxidation liabilities during the antibody discovery and development phases. Here, we present an in silico method, based on protein modeling and molecular dynamics simulations, to predict the oxidation-liable residues in the variable region of therapeutic antibodies. Previous studies have used the 2-shell water coordination number descriptor (WCN) to identify methionine residues susceptible to oxidation. Although the WCN descriptor successfully predicted oxidation liabilities when the residue was solvent exposed, the method was much less accurate for partially buried methionine residues. Consequently, we introduce a new descriptor, WCN-OH, that improves the accuracy of prediction of methionine oxidation susceptibility by extending the theoretical framework of the water coordination number to incorporate the effects of polar amino acids side chains in close proximity to the methionine of interest.

Differential expression of Mical2 and its activator (Sema3a) in Alzheimer’s disease mouse model

AbstractBackgroundMicals are actin regulatory enzymes which can enhance depolymerization of actin filaments (F‐Actin). Activated Micals catalyze the selective oxidation of methionine residues, Met44 and Met47 within the D‐loop of F‐actin. The oxidation of methionine residues disrupts the interaction between G‐actin monomers, thus facilitating F‐actin depolymerization. Dendritic spines are enriched with filamentous actin (F‐actin) where it regulates the formation, elimination and structural plasticity of spines. Early stage of AD is associated with loss of dendritic spines and F‐actin from synapses, leading to memory deficits. However, the potential mechanism involved in the loss of F‐actin remains largely unknown. Therefore, we aimed to elucidate the role of Micals and semaphorins in actin depolymerization at the synapse of AD mouse model.MethodsTotal RNA was isolated from the cortex, hippocampus and entorhinal cortex of adolescent, young, and middle aged wildtype and APP/PS1 mice as well as from the synaptosomes of respective age groups. The cDNA is synthesized and qRT‐PCR is performed using SYBR green reagents with specific primers to Micals and Semaphorins. Data was analyzed using two tailed non parametric test with a p value of <0.05 signifies the result.ResultsThe mRNA expression levels of Micals and Semaphorins in entorhinal cortex, hippocampus, and cortex of wildtype and APP/PS1 as well as in the synaptosomes of age matched animals were measured by qRT‐PCR. We found that Semaphorin3a showed differential mRNA expression levels in entorhinal cortex of adolescence and young adults of APP/PS1 mice in comparison to wildtype littermate controls. Further, we detected that the synapse specific mRNA levels of Mical 2 and Semaphorin3a are significantly decreased in adolescent APP/PS1 mice in comparison to WT mice. However, we did not detect any significant alteration in the mRNA expression levels of Mical1, Mical3 and Semaphorin3f in APP/PS1 mice at different age groups in comparison to WT littermate controls.ConclusionAltered expression levels of actin regulatory proteins like Micals and Semaphorins at the dendritic spines could play critical roles in Aβ mediated F‐actin depolymerization in Alzheimer's disease pathology.

Oxidative stress–induced autonomous activation of the calcium/calmodulin-dependent kinase II involves disulfide formation in the regulatory domain

Calcium/calmodulin-dependent protein kinase II δ (CaMKIIδ) has a pivotal role in cardiac signaling. Constitutive and deleterious CaMKII "autonomous" activation is induced by oxidative stress, and the previously reported mechanism involves oxidation of methionine residues in the regulatory domain. Here, we demonstrate that covalent oxidation leads to a disulfide bond with Cys273 in the regulatory domain causing autonomous activity. Autonomous activation was induced by treating CaMKII with diamide or histamine chloramine, two thiol-oxidizing agents. Autonomy was reversed when the protein was incubated with DTT or thioredoxin to reduce disulfide bonds. Tryptic mapping of the activated CaMKII revealed formation of a disulfide between Cys273 and Cys290 in the regulatory domain. We determined the apparent pKa of those Cys and found that Cys273 had a low pKa while that of Cys290 was elevated. The low pKa of Cys273 facilitates oxidation of its thiol to the sulfenic acid at physiological pH. The reactive sulfenic acid then attacks the thiol of Cys290 to form the disulfide. The previously reported CaMKII mutant in which methionine residues 281 and 282 were mutated to valine (MMVV) protects mice and flies from cardiac decompensation induced by oxidative stress. Our initial hypothesis was that the MMVV mutant underwent a conformational change that prevented disulfide formation and autonomous activation. However, we found that the thiol-oxidizing agents induced autonomy in the MMVV mutant and that the mutant undergoes rapid degradation by the cell, potentially preventing accumulation of the injurious autonomous form. Together, our results highlight additional mechanistic details of CaMKII autonomous activation.

Mechanisms of chlorate toxicity and resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that often encounters hypoxic/anoxic environments within the host, which increases its tolerance to many conventional antibiotics. Toward identifying novel treatments, we explored the therapeutic potential of chlorate, a pro-drug that kills hypoxic/anoxic, antibiotic-tolerant P. aeruginosa populations. While chlorate itself is relatively nontoxic, it is enzymatically reduced to the toxic oxidizing agent, chlorite, by hypoxically induced nitrate reductase. To better assess chlorate's therapeutic potential, we investigated mechanisms of chlorate toxicity and resistance in P. aeruginosa. We used transposon mutagenesis to identify genes that alter P. aeruginosa fitness during chlorate treatment, finding that methionine sulfoxide reductases (Msr), which repair oxidized methionine residues, support survival during chlorate stress. Chlorate treatment leads to proteome-wide methionine oxidation, which is exacerbated in a ∆msrA∆msrB strain. In response to chlorate, P. aeruginosa upregulates proteins involved in a wide range of functions, including metabolism, DNA replication/repair, protein repair, transcription, and translation, and these newly synthesized proteins are particularly vulnerable to methionine oxidation. The addition of exogenous methionine partially rescues P. aeruginosa survival during chlorate treatment, suggesting that widespread methionine oxidation contributes to death. Finally, we found that mutations that decrease nitrate reductase activity are a common mechanism of chlorate resistance.

Mass-spectrometric identification of oxidative modifications in plasma-purified plasminogen: Association with hypofibrinolysis in patients with acute pulmonary embolism

ObjectivesMechanisms behind disturbed fibrinolysis in pulmonary embolism (PE) are poorly understood. We hypothesized that oxidative stress-induced changes in plasminogen contribute to impaired fibrinolysis in patients with acute PE. MethodsOxidative and other modifications were investigated using mass-spectrometry in plasminogen purified from pooled plasma of 5 acute PE patients on admission and after 3 months of anticoagulant treatment, along with plasma clot lysis time, a measure of global efficiency of fibrinolysis, and a stable oxidative stress marker, plasma 8-isoprostane. ResultsTwenty sites of oxidation, 3 sites of carbonylation and 4 sites of S-nitrosylation were identified in plasminogen. The intensity of peptides oxidized at cysteine residues with respect to unmodified peptides decreased after 3 months of anticoagulation (p = 0.018). This was not observed for oxidized methionine residues (p = 0.9). Oxidized tryptophan (n = 4) and proline (n = 2), as well as carbonylation at 3 threonine residues were selectively identified in acute PE episode, not after 3 months. This was accompanied by 12.8% decrease in clot lysis time (p = 0.043). Deamidation occurred at the arginine, previously identified to undergo the cleavage by plasminogen activator. Methylated were two lysine-binding sites important for an interaction of plasminogen with fibrin. Other identified modifications involved: glycation, acetylation, phosphorylation, homocysteinylation, carbamylation and dichlorination (88 modifications at 162 sites). ConclusionsData suggest that oxidative stress-induced changes in plasminogen molecules may contribute to less effective global fibrinolysis in patients with acute PE. The comprehensive library of posttranslational modifications in plasminogen molecules was provided, including modifications of sites reported to be involved in important biological functions.

Oxidation Promotes Distinct Huntingtin Aggregates in the Presence and Absence of Membranes.

Expansion of a polyglutamine (polyQ) domain within the first exon of the huntingtin (htt) protein is the underlying cause of Huntington's disease, a genetic neurodegenerative disorder. PolyQ expansion triggers htt aggregation into oligomers, fibrils, and inclusions. The 17 N-terminal amino acids (Nt17) of htt-exon1, which directly precede the polyQ domain enhances polyQ fibrillization and functions as a lipid-binding domain. A variety of post-translational modifications occur within Nt17, including oxidation of two methionine residues. Here, the impact of oxidation within Nt17 on htt aggregation both in the presence and absence of lipid membranes was investigated. Treatment with hydrogen peroxide (H2O2) reduced fibril formation in a dose-dependent manner, resulting in shorter fibrils and an increased oligomer population. With excessive H2O2 treatments, fibrils developed a unique morphological feature around their periphery. In the presence of total brain lipid vesicles, H2O2 impacted fibrillization in a similar manner. That is, oligomerization was promoted at the expense of fibril elongation. The interaction of unoxidized and oxidized htt with supported lipid bilayers was directly observed using in situ atomic force microscopy. Without oxidation, granular htt aggregates developed on the bilayer surface. However, in the presence of H2O2, distinct plateau-like regions initially developed on the bilayer surface that gave way to rougher patches containing granular aggregates. Collectively, these observations suggest that oxidation of methionine residues within Nt17 plays a crucial role in both the aggregation of htt and its ability to interact with lipid surfaces.

Methionine oxidation activates pyruvate kinase M2 to promote pancreatic cancer metastasis

Cancer mortality is primarily a consequence of its metastatic spread. Here, we report that methionine sulfoxide reductase A (MSRA), which can reduce oxidized methionine residues, acts as a suppressor of pancreatic ductal adenocarcinoma (PDA) metastasis. MSRA expression is decreased in the metastatic tumors of PDA patients, whereas MSRA loss in primary PDA cells promotes migration and invasion. Chemoproteomic profiling of pancreatic organoids revealed that MSRA loss results in the selective oxidation of a methionine residue (M239) in pyruvate kinase M2 (PKM2). Moreover, M239 oxidation sustains PKM2 in an active tetrameric state to promote respiration, migration, and metastasis, whereas pharmacological activation of PKM2 increases cell migration and metastasis invivo. These results demonstrate that methionine residues can act as reversible redox switches governing distinct signaling outcomes and that the MSRA-PKM2 axis serves as a regulatory nexus between redox biology and cancer metabolism to control tumor metastasis.

Direct formation and site-selective elaboration of methionine sulfoximine in polypeptides.

Sulfoximines are emerging moieties for medicinal and biological chemistry, due in part to their efficacy in selective inhibition of amide-forming enzymes such as γ-glutamylcysteine synthetase. While small-molecule sulfoximines such as methionine sulfoximine (MSO) and its derivatives are well studied, structures with methionine sulfoximine residues within complex polypeptides have been generally inaccessible. This paper describes a straightforward means of late-stage one-step oxidation of methionine residues within polypeptides to afford NH-sulfoximines. We also present chemoselective subsequent elaboration, most notably by copper(ii)-mediated N-H cross-coupling at methionine sulfoximine residues with arylboronic acid reagents. This development serves as a strategy to incorporate diverse sulfoximine structures within natural polypeptides, and also identifies the methionine sulfoximine residue as a new site for bioorthogonal, chemoselective bioconjugation.

Oxidation sensitizes TRPV2 to chemical and heat stimuli, but not mechanical stimulation

The transient receptor potential vanilloid 2 (TRPV2) ion channel is activated by a chemical ligand (2-aminoethoxydiphenyl borate; 2-APB), noxious heat and mechanical stimulation. In a heterologous mammalian cell expression system, the oxidant chloramine T (ChT) sensitizes TRPV2 activation in response to 2-APB and heat by oxidation of methionine residues at positions 528 and 607 in rat TRPV2. Here, we used a Xenopus oocyte expression system to determine whether ChT-mediated oxidation can also sensitize TRPV2 to mechanical stimulation. In this system, we confirmed that ChT sensitized TRPV2 activation in response to 2-APB and heat, but we detected no sensitization to mechanical stimulation. This result suggests that the activation mechanism of TRPV2 by a chemical ligand and heat differs from that for mechanical stimulation. Further, we demonstrated that two-electrode voltage clamp recording in the Xenopus oocyte expression system is an excellent format for high throughput analysis of oxidization of redox-sensitive TRP channels.

Hypoxia Tolerance Declines with Age in the Absence of Methionine Sulfoxide Reductase (MSR) in Drosophila melanogaster.

Unlike the mammalian brain, Drosophila melanogaster can tolerate several hours of hypoxia without any tissue injury by entering a protective coma known as spreading depression. However, when oxygen is reintroduced, there is an increased production of reactive oxygen species (ROS) that causes oxidative damage. Methionine sulfoxide reductase (MSR) acts to restore functionality to oxidized methionine residues. In the present study, we have characterized in vivo effects of MSR deficiency on hypoxia tolerance throughout the lifespan of Drosophila. Flies subjected to sudden hypoxia that lacked MSR activity exhibited a longer recovery time and a reduced ability to survive hypoxic/re-oxygenation stress as they approached senescence. However, when hypoxia was induced slowly, MSR deficient flies recovered significantly quicker throughout their entire adult lifespan. In addition, the wildtype and MSR deficient flies had nearly 100% survival rates throughout their lifespan. Neuroprotective signaling mediated by decreased apoptotic pathway activation, as well as gene reprogramming and metabolic downregulation are possible reasons for why MSR deficient flies have faster recovery time and a higher survival rate upon slow induction of spreading depression. Our data are the first to suggest important roles of MSR and longevity pathways in hypoxia tolerance exhibited by Drosophila.

Glycation of albumin and its implication in Diabetes: A comprehensive analysis using mass spectrometry

AimTo understand the mechanism of glycation of albumin and effects on cysteinylation and methionine oxidation. MethodsThe in vitro glycation of HSA and BSA was studied with varying concentrations of glucose. Clinical blood samples of diabetic subjects with varying HbA1c values, were analyzed to assess in vivo glycation. All samples and their tryptic digests were analyzed using liquid chromatography/mass spectrometry. Glycation sites were mapped on to the three-dimensional structure of the HSA and BSA. ResultsA total thirty-one sites for glycation and eight sites of Nε-carboxymethyl-lysine (CML) modification were identified on albumin. The site selectivity of glycation was correlated with the environment of the reactive residue in the three-dimensional structure. ConclusionsThe maximum percentage glycation under extreme conditions was in the range of ~55 to 88% in four weeks. Two major glycation sites K-233 and K-525 were identified, which together accounted for 40–50% of total glycation. A correlation was observed between glycation and oxidation of methionine residues in samples glycated in vitro. The role of spatially proximate residues in facilitating the glycation process is evident. The tri- and tetra-glycated isoforms of albumin can serve as biomarkers for the severe uncontrolled diabetic state.

Profiling Dopamine-Induced Oxidized Proteoforms of β-synuclein by Top-Down Mass Spectrometry.

The formation of multiple proteoforms by post-translational modifications (PTMs) enables a single protein to acquire distinct functional roles in its biological context. Oxidation of methionine residues (Met) is a common PTM, involved in physiological (e.g., signaling) and pathological (e.g., oxidative stress) states. This PTM typically maps at multiple protein sites, generating a heterogeneous population of proteoforms with specific biophysical and biochemical properties. The identification and quantitation of the variety of oxidized proteoforms originated under a given condition is required to assess the exact molecular nature of the species responsible for the process under investigation. In this work, the binding and oxidation of human β-synuclein (BS) by dopamine (DA) has been explored. Native mass spectrometry (MS) has been employed to analyze the interaction of BS with DA. In a second step, top-down fragmentation of the intact protein from denaturing conditions has been performed to identify and quantify the distinct proteoforms generated by DA-induced oxidation. The analysis of isobaric proteoforms is approached by a combination of electron-transfer dissociation (ETD) at each extent of modification, quantitation of methionine-containing fragments and combinatorial analysis of the fragmentation products by multiple linear regression. This procedure represents a promising approach to systematic assessment of proteoforms variety and their relative abundance. The method can be adapted, in principle, to any protein containing any number of methionine residues, allowing for a full structural characterization of the protein oxidation states.

Chemical and biological properties of anti-wrinkle peptide Argireline

Argireline, a peptide with the sequence: Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2, also known as Acetyl Hexapeptide-8, reduces facial lines and wrinkles by destabilization of the formation of the SNARE complex (SNAP Receptor, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor), thus preventing muscle contraction. It is a biosafe cosmetic alternative to the botulinum toxin. The method of choice in bioactive peptide analysis is reversed-phase high performance liquid chromatography coupled with mass spectrometry (LC-MS). The aim of this work as to present the properties of Argireline and the analysis of cosmetic products containing this peptide. Previous reports on possible Argireline transformations in cosmetic formulations have not confirmed deacetylation, whereas the oxidation of methionine residue was detected by our team. As the biological activity of the oxidized Argireline is not known, further biological studies, as well as efficient analytical procedures for transformation monitoring and quality control in cosmetic products, are necessary.

Staphylococcus aureus Peptide Methionine Sulfoxide Reductases Protect from Human Whole-Blood Killing.

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.

Oxidation of Huntingtin Modulates its Aggregation and Interaction with Membranes

Huntington's disease is an autosomal dominant disease caused by the expansion of a polyglutamine (polyQ) domain within the first exon of the huntingtin (htt) protein. PolyQ expansion promotes htt aggregation into oligomers, b-sheet rich fibrils, and other amorphous species. Htt aggregation is mediated by the first 17 N-terminal amino acids (N17) that directly precede the polyQ domain. In addition, N17 is a membrane binding domain. Both the ability of N17 to enhance htt aggregation and bind lipid membranes are attributed to its ability to form an amphipathic α-helix. N17 also contains several sites for potential posttranslational modifications (PTMs), including oxidation of two methionine residues. Here, the impact of oxidation of methionine residues within N17 on htt aggregation in the presence or absence of lipid membranes comprised of total brain lipid extract (TBLE) was investigated. Two experimental systems were used, a synthetic N-terminal peptide fragment of htt (N17-Q35-P10) and full-length htt-exon1 with 46 repeat glutamines expressed as a GST-fusion protein. For the second system, cleavage of the GST moiety initiates aggregation. To oxidize the methionine residues in N17, the experimental htt systems were treated with hydrogen peroxide. Based on ThT assays, hydrogen peroxide treatment reduced N17-Q35-P10 fibril formation in a dose-dependent manner. Fibrillization of full-length htt-exon1 appeared inhibited with similar hydrogen peroxide treatments as assessed by ex situ atomic force microscopy (AFM) assays. That is, increasing peroxide treatments led to the emergence of shorter and unbranched fibrils while promoting oligomerization. The addition of lipid vesicles generally reduced fibrilization of both experimental systems with and without hydrogen peroxide treatment. However, similar to the trend observed in the absence of lipid, increasing oxidant concentration favored the formation of oligomers. In addition, the aggregation of unoxidized and oxidized htt-exon1 was directly tracked on supported TBLE lipid bilayers using in situ AFM. Both oxidized and unoxidized lipid bilayers were investigated. In general, oxidation of either htt-exon1 alone or both htt and TBLE resulted in reduced membrane damage and disruption. Unoxidized htt deposited quickly and aggressively on TBLE bilayers, forming lumpy amorphous aggregates. With oxidized lipid membranes, htt-exon1 deposited less aggressively on the bilayer, forming a mix of amorphous and smaller grainy oligomeric aggregates over a longer period. In contrast, when both htt-exon1 and TBLE were oxidized, the aggregates formed were mostly grainy oligomeric aggregates. Collectively, these observations suggest that oxidation plays an important role in both the aggregation of htt and its ability to bind lipid membranes, which may ultimately be related to the location of the methionine residues within the amphipathic α-helix of N17.