Jared R. Auclair

A soluble α-synuclein construct forms a dynamic tetramer

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated “hairpin” structure of α-synuclein.

Mass spectrometry analysis of HIV-1 Vif reveals an increase in ordered structure upon oligomerization in regions necessary for viral infectivity

HIV‐1 Vif, an accessory protein in the viral genome, performs an important role in viral pathogenesis by facilitating the degradation of APOBEC3G, an endogenous cellular inhibitor of HIV‐1 replication. In this study, intrinsically disordered regions are predicted in HIV‐1 Vif using sequence‐based algorithms. Intrinsic disorder may explain why traditional structure determination of HIV‐1 Vif has been elusive, making structure‐based drug design impossible. To characterize HIV‐1 Vifs structural topology and to map the domains involved in oligomerization we used chemical cross‐linking, proteolysis, and mass spectrometry. Cross‐linking showed evidence of monomer, dimer, and trimer species via denaturing gel analysis and an additional tetramer via western blot analysis. We identified 47 unique linear peptides and 24 (13 intramolecular; 11 intermolecular) noncontiguous, cross‐linked peptides, among the noncross‐linked monomer, cross‐linked monomer, cross‐linked dimer, and cross‐linked trimer samples. Almost complete peptide coverage of the N‐terminus is observed in all samples analyzed, however reduced peptide coverage in the C‐terminal region is observed in the dimer and trimer samples. These differences in peptide coverage or “protections” between dimer and trimer indicate specific differences in packing between the two oligomeric forms. Intramolecular cross‐links within the monomer suggest that the N‐terminus is likely folded into a compact domain, while the C‐terminus remains intrinsically disordered. Upon oligomerization, as evidenced by the intermolecular cross‐links, the C‐terminus of one Vif protein becomes ordered by wrapping back on the N‐terminal domain of another. In addition, the majority of the intramolecular cross‐links map to regions that have been previously reported to be necessary for viral infectivity. Thus, this data suggests HIV‐1 Vif is in a dynamic equilibrium between the various oligomers potentially allowing it to interact with other binding partners. Proteins 2007.

Strategies for stabilizing superoxide dismutase (SOD1), the protein destabilized in the most common form of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3- to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches are needed to combat neurodegenerative disease. Some familial forms of ALS (fALS) have been linked to mutations in the Cu/Zn superoxide dismutase (SOD1). The dominant inheritance of mutant SOD1 and lack of symptoms in knockout mice suggest a “gain of toxic function” as opposed to a loss of function. A prevailing hypothesis for the mechanism of the toxicity of fALS-SOD1 variants, or the gain of toxic function, involves dimer destabilization and dissociation as an early step in SOD1 aggregation. Therefore, stabilizing the SOD1 dimer, thus preventing aggregation, is a potential therapeutic strategy. Here, we report a strategy in which we chemically cross-link the SOD1 dimer using two adjacent cysteine residues on each respective monomer (Cys111). Stabilization, measured as an increase in melting temperature, of ∼20 °C and ∼45 °C was observed for two mutants, G93A and G85R, respectively. This stabilization is the largest for SOD1, and to the best of our knowledge, for any disease-related protein. In addition, chemical cross-linking conferred activity upon G85R, an otherwise inactive mutant. These results demonstrate that targeting these cysteine residues is an important new strategy for development of ALS therapies.

Post-translational modification by cysteine protects Cu/Zn-superoxide dismutase from oxidative damage

Reactive oxygen species (ROS) are cytotoxic. To remove ROS, cells have developed ROS-specific defense mechanisms, including the enzyme Cu/Zn superoxide dismutase (SOD1), which catalyzes the disproportionation of superoxide anions into molecular oxygen and hydrogen peroxide. Although hydrogen peroxide is less reactive than superoxide, it is still capable of oxidizing, unfolding, and inactivating SOD1, at least in vitro. To explore the relevance of post-translational modification (PTM) of SOD1, including peroxide-related modifications, SOD1 was purified from postmortem human nervous tissue. As much as half of all purified SOD1 protein contained non-native post-translational modifications (PTMs), the most prevalent modifications being cysteinylation and peroxide-related oxidations. Many PTMs targeted a single reactive SOD1 cysteine, Cys111. An intriguing observation was that unlike native SOD1, cysteinylated SOD1 was not oxidized. To further characterize how cysteinylation may protect SOD1 from oxidation, cysteine-modified SOD1 was prepared in vitro and exposed to peroxide. Cysteinylation conferred nearly complete protection from peroxide-induced oxidation of SOD1. Moreover, SOD1 that has been cysteinylated and peroxide oxidized in vitro comprised a set of PTMs that bear a striking resemblance to the myriad of PTMs observed in SOD1 purified from human tissue.

Identification of a misfolded region in superoxide dismutase 1 that is exposed in amyotrophic lateral sclerosis.

Background: Misfolded SOD1 is associated with sporadic and familial ALS. Results: The epitope of C4F6, an antibody specific for misfolded SOD1, has been defined. Loops IV and VII in SOD1 modulate exposure of this epitope. Conclusion: Exposure of the C4F6 epitope correlates with heightened SOD1-mediated toxicity. Significance: Concealing the C4F6 epitope by stabilizing SOD1 loops IV and VII has therapeutic potential for ALS. Mutations and aberrant post-translational modifications within Cu,Zn-superoxide dismutase (SOD1) cause this otherwise protective enzyme to misfold, leading to amyotrophic lateral sclerosis (ALS). The C4F6 antibody selectively binds misfolded SOD1 in spinal cord tissues from postmortem human ALS cases, as well as from an ALS-SOD1 mouse model, suggesting that the C4F6 epitope reports on a pathogenic conformation that is common to misfolded SOD1 variants. To date, the residues and structural elements that comprise this epitope have not been elucidated. Using a chemical cross-linking and mass spectrometry approach, we identified the C4F6 epitope within several ALS-linked SOD1 variants, as well as an oxidized form of WT SOD1, supporting the notion that a similar misfolded conformation is shared among pathological SOD1 proteins. Exposure of the C4F6 epitope was modulated by the SOD1 electrostatic (loop VII) and zinc binding (loop IV) loops and correlated with SOD1-induced toxicity in a primary microglia activation assay. Site-directed mutagenesis revealed Asp92 and Asp96 as key residues within the C4F6 epitope required for the SOD1-C4F6 binding interaction. We propose that stabilizing the functional loops within SOD1 and/or obscuring the C4F6 epitope are viable therapeutic strategies for treating SOD1-mediated ALS.

Structural consequences of cysteinylation of cu/zn-superoxide dismutase.

The metalloenzyme Cu/Zn-superoxide dismutase (SOD1) catalyzes the reduction of superoxide anions into molecular oxygen and hydrogen peroxide. Hydrogen peroxide can oxidize SOD1, resulting in aberrant protein conformational changes, disruption of SOD1 function, and DNA damage. Cells may have evolved mechanisms of regulation that prevent such oxidation. We observed that cysteinylation of cysteine 111 (Cys111) of SOD1 prevents oxidation by peroxide (DOI 10.1021/bi4006122 ). In this article, we characterize cysteinylated SOD1 using differential scanning fluorometry and X-ray crystallography. The stoichiometry of binding was one cysteine per SOD1 dimer, and there does not appear to be free volume for a second cysteine without disrupting the dimer interface. Much of the three-dimensional structure of SOD1 is unaffected by cysteinylation. However, local conformational changes are observed in the cysteinylated monomer that include changes in conformation of the electrostatic loop (loop VII; residues 133-144) and the dimer interface (loop VI; residues 102-115). In addition, our data shows how cysteinylation precludes oxidation of cysteine 111 and suggests possible cross-talk between the dimer interface and the electrostatic loop.

Mildly acidic conditions eliminate deamidation artifact during proteolysis: digestion with endoprotease Glu-C at pH 4.5.

Common yet often overlooked, deamidation of peptidyl asparagine (Asn or N) generates aspartic acid (Asp or D) or isoaspartic acid (isoAsp or isoD). Being a spontaneous, non-enzymatic protein post-translational modification, deamidation artifact can be easily introduced during sample preparation, especially proteolysis where higher-order structures are removed. This artifact not only complicates the analysis of bona fide deamidation but also affects a wide range of chemical and enzymatic processes; for instance, the newly generated Asp and isoAsp residues may block or introduce new proteolytic sites, and also convert one Asn peptide into multiple species that affect quantification. While the neutral to mildly basic conditions for common proteolysis favor deamidation, mildly acidic conditions markedly slow down the process. Unlike other commonly used endoproteases, Glu-C remains active under mildly acid conditions. As such, as demonstrated herein, deamidation artifact during proteolysis was effectively eliminated by simply performing Glu-C digestion at pH 4.5 in ammonium acetate, a volatile buffer that is compatible with mass spectrometry. Moreover, nearly identical sequence specificity was observed at both pH’s (8.0 for ammonium bicarbonate), rendering Glu-C as effective at pH 4.5. In summary, this method is generally applicable for protein analysis as it requires minimal sample preparation and uses the readily available Glu-C protease.

The central nervous system transcriptome of the weakly electric brown ghost knifefish (Apteronotus leptorhynchus): de novo assembly, annotation, and proteomics validation

BackgroundThe brown ghost knifefish (Apteronotus leptorhynchus) is a weakly electric teleost fish of particular interest as a versatile model system for a variety of research areas in neuroscience and biology. The comprehensive information available on the neurophysiology and neuroanatomy of this organism has enabled significant advances in such areas as the study of the neural basis of behavior, the development of adult-born neurons in the central nervous system and their involvement in the regeneration of nervous tissue, as well as brain aging and senescence. Despite substantial scientific interest in this species, no genomic resources are currently available.ResultsHere, we report the de novo assembly and annotation of the A. leptorhynchus transcriptome. After evaluating several trimming and transcript reconstruction strategies, de novo assembly using Trinity uncovered 42,459 unique contigs containing at least a partial protein-coding sequence based on alignment to a reference set of known Actinopterygii sequences. As many as 11,847 of these contigs contained full or near-full length protein sequences, providing broad coverage of the proteome. A variety of non-coding RNA sequences were also identified and annotated, including conserved long intergenic non-coding RNA and other long non-coding RNA observed previously to be expressed in adult zebrafish (Danio rerio) brain, as well as a variety of miRNA, snRNA, and snoRNA. Shotgun proteomics confirmed translation of open reading frames from over 2,000 transcripts, including alternative splice variants. Assignment of tandem mass spectra was greatly improved by use of the assembly compared to databases of sequences from closely related organisms. The assembly and raw reads have been deposited at DDBJ/EMBL/GenBank under the accession number GBKR00000000. Tandem mass spectrometry data is available via ProteomeXchange with identifier PXD001285.ConclusionsPresented here is the first release of an annotated de novo transcriptome assembly from Apteronotus leptorhynchus, providing a broad overview of RNA expressed in central nervous system tissue. The assembly, which includes substantial coverage of a wide variety of both protein coding and non-coding transcripts, will allow the development of better tools to understand the mechanisms underlying unique characteristics of the knifefish model system, such as their tremendous regenerative capacity and negligible brain senescence.

Artifacts to avoid while taking advantage of top-down mass spectrometry based detection of protein S-thiolation.

Bottom‐up MS studies typically employ a reduction and alkylation step that eliminates a class of PTM, S‐thiolation. Given that molecular oxygen can mediate S‐thiolation from reduced thiols, which are abundant in the reducing intracellular milieu, we investigated the possibility that some S‐thiolation modifications are artifacts of protein preparation. Cu/Zn‐superoxide dismutase (SOD1) was chosen for this case study as it has a reactive surface cysteine residue, which is readily cysteinylated in vitro. The ability of oxygen to generate S‐thiolation artifacts was tested by comparing purification of SOD1 from postmortem human cerebral cortex under aerobic and anaerobic conditions. S‐thiolation was ∼50% higher in aerobically processed preparations, consistent with oxygen‐dependent artifactual S‐thiolation. The ability of endogenous small molecule disulfides (e.g. cystine) to participate in artifactual S‐thiolation was tested by blocking reactive protein cysteine residues during anaerobic homogenization. A 50‐fold reduction in S‐thiolation occurred indicating that the majority of S‐thiolation observed aerobically was artifact. Tissue‐specific artifacts were explored by comparing brain‐ and blood‐derived protein, with remarkably more artifacts observed in brain‐derived SOD1. Given the potential for such artifacts, rules of thumb for sample preparation are provided. This study demonstrates that without taking extraordinary precaution, artifactual S‐thiolation of highly reactive, surface‐exposed, cysteine residues can result.

Regulatory Convergence for Biologics through Capacity Building and Training

Several regulatory convergence efforts for biologics are underway globally, with the goal of ensuring global standards are applied consistently across regulatory agencies. Training and capacity building will ensure convergence through fostering international collaborations between agencies and ensure harmonized standards are applied, which will bring products to market faster and cheaper.