Single Methionine Research Articles

A new species of alien land flatworm in the Southern United States.

Specimens of a flat and dark brown land planarian were found in a plant nursery in North Carolina, USA in 2020. On the basis of examination of photographs of the live specimens only, the specimens were considered as belonging to Obama nungara, a species originally from South America, which has now invaded a large part of Europe. Unexpectedly, a molecular analysis revealed that the specimens did not belong to this species, neither to the genus Obama. We then undertook its histological study, which finally confirmed that the species is a member of the genus Amaga: the species is herein described as a new species, Amaga pseudobama n. sp. The species has been found in three locations in North Carolina and some infested plants were from Georgia. We reinvestigated specimens collected in Florida in 2015 and found that they also belong to this species. Citizen science observations suggest its presence in other states. Therefore, it is likely that A. pseudobama has already invaded a part of south-east USA and that the invasion took place more than ten years ago. The complete 14,909 bp long mitochondrial genome was obtained. The mitogenome is colinear with those of other Geoplanidae and it was possible to find and annotate a tRNA-Thr, which has been reported missing in several geoplanids. Amaga pseudobama shares with other Geoplaninae the presence of alternative start codons in three protein-coding genes of its mitogenome. The availability of this new genome helped us to improve our annotations of the ND3 gene, for which an ATT start codon is now suggested. Also, the sequence of the ATP6 gene raised questions concerning the use of genetic code 9 to translate the protein-coding genes of Geoplanidae, as the whole translated protein would not contain a single methionine residue when using this code. Two maximum likelihood phylogenies were obtained from genomic data. The first one was based on concatenated alignments of the partial 28S, Elongation Factor 1-alpha (EF1) and cox1 genes. The second was obtained from a concatenated alignment of the mitochondrial proteins. Both strictly discriminate A. pseudobama from O. nungara and instead associate it with Amaga expatria. We note that the nine species currently accepted within Amaga can be separated into two groups, one with extrabulbar prostatic apparatus, including the type species A. amagensis, and one with intrabulbar prostatic apparatus, including the new species A. pseudobama. This suggests that species of the latter group should be separated from Amaga and constitute a new genus. This finding again illustrates the possible emergence of new invasive species in regions naturally devoid of large land planarians, such as North America. Amaga pseudobama thus deserves to be monitored in the USA, although its superficial resemblance to O. nungara and Geoplana arkalabamensis will complicate the use of photographs obtained from citizen science. Our molecular information provides tools for this monitoring.

Inter-species association mapping links splice site evolution to METTL16 and SNRNP27K.

Eukaryotic genes are interrupted by introns that are removed from transcribed RNAs by splicing. Patterns of splicing complexity differ between species, but it is unclear how these differences arise. We used inter-species association mapping with Saccharomycotina species to correlate splicing signal phenotypes with the presence or absence of splicing factors. Here, we show that variation in 5' splice site sequence preferences correlate with the presence of the U6 snRNA N6-methyladenosine methyltransferase METTL16 and the splicing factor SNRNP27K. The greatest variation in 5' splice site sequence occurred at the +4 position and involved a preference switch between adenosine and uridine. Loss of METTL16 and SNRNP27K orthologs, or a single SNRNP27K methionine residue, was associated with a preference for +4 U. These findings are consistent with splicing analyses of mutants defective in either METTL16 or SNRNP27K orthologs and models derived from spliceosome structures, demonstrating that inter-species association mapping is a powerful orthogonal approach to molecular studies. We identified variation between species in the occurrence of two major classes of 5' splice sites, defined by distinct interaction potentials with U5 and U6 snRNAs, that correlates with intron number. We conclude that variation in concerted processes of 5' splice site selection by U6 snRNA is associated with evolutionary changes in splicing signal phenotypes.

Site-Specific 68Ga Radiolabeling of Trastuzumab Fab via Methionine for ImmunoPET Imaging.

Bioconjugates of antibodies and their derivatives radiolabeled with β+-emitting radionuclides can be utilized for diagnostic PET imaging. Site-specific attachment of radioactive cargo to antibody delivery vectors provides homogeneous, well-defined immunoconjugates. Recent studies have demonstrated the utility of oxaziridine chemistry for site-specific labeling of methionine residues. Herein, we applied this approach to site-specifically radiolabel trastuzumab-derived Fab immunoconjugates with 68Ga, which can be used for in vivo PET imaging of HER2-positive breast cancer tumors. Initially, a reactive azide was introduced to a single solvent-accessible methionine residue in both the wild-type Fab and an engineered derivative containing methionine residue M74, utilizing the principles of oxaziridine chemistry. Subsequently, these conjugates were functionalized with a modified DFO chelator incorporating dibenzocyclooctyne. The resulting DFO-WT and DFO-M74 conjugates were radiolabeled with generator-produced [68Ga]Ga3+, to yield the novel PET radiotracers, [68Ga]Ga-DFO-WT and [68Ga]Ga-DFO-M74. In vitro and in vivo studies demonstrated that [68Ga]Ga-DFO-M74 exhibited a higher affinity for HER2 receptors. Biodistribution studies in mice bearing orthotopic HER2-positive breast tumors revealed a higher uptake of [68Ga]Ga-DFO-M74 in the tumor tissue, accompanied by rapid renal clearance, enabling clear delineation of tumors using PET imaging. Conversely, [68Ga]Ga-DFO-WT exhibited lower uptake and inferior image contrast compared to [68Ga]Ga-DFO-M74. Overall, the results demonstrate that the highly facile methionine-oxaziridine modification approach can be simply applied to the synthesis of stable and site-specifically modified radiolabeled antibody-chelator conjugates with favorable pharmacokinetics for PET imaging.

Facile synthesis of mesoporous carbon materials with a three-dimensional ordered mesostructure and rich FeNX/C-S-C sites for efficient electrocatalytic oxygen reduction

Incorporation of sulfur (S) atoms into iron/nitrogen co-doped (FeNX) carbon materials is attractive for developing efficient electrocatalysts for the oxygen reduction reaction (ORR). Synthesis of such materials with suitable pore structures and rich active sites are important to improve the ORR performance. In this study, we demonstrate the facile synthesis of novel N/S co-doped ordered mesoporous carbon materials with a three-dimensional (3D) ordered bicontinuous cubic mesopore structure and rich FeNX and carbon-sulfur-carbon (C-S-C) sites (denoted as FeNX@NSOMCs) using the solvent-free nanocasting method. The 3D ordered bicontinuous cubic mesopore structure is replicated in the FeNX@NSOMCs from the mesoporous silica hard template KIT-6. The uniform N/S dopants in the carbon matrix is derived from the confined pyrolysis of a single amino acid methionine (Met). The creation of the FeNX sites is enabled by using anhydrous FeCl3 as the precursor. The absence of solvent and the coordination between FeCl3 and Met can reduce metal hydrolysis and aggregation, reduce N loss, and thus promote the formation of FeNX sites. The FeNX@NSOMCs possess high surface areas (830–938 m2/g), large pore volumes (0.97–1.36 cm3/g), disordered macropores and ordered mesopores (3.5–3.6 nm), and well-graphitized carbon walls. They are promising for ORR. The represent sample shows superior performance under alkaline conditions, achieving a high half-wave potential of 0.9 V, a high kinetic current density of 4.2 mA/cm2, a low Tafel slope of 55.3 mV/dec, a near four-electron process with a low H2O2 yield and a high stability. A comparative study using various control samples reveals that the FeNX sites, the S doping and the ordered mesopore structure are crucial to achieve the high ORR performance.

Abstract B46: CATACOMB: An endogenous inducible gene that antagonizes H3K27 methylation activity of Polycomb Repressive complex 2 via a H3K27M-like mechanism

Abstract Dysregulation of Polycomb Repressive Complex 2 (PRC2) function is a common feature of many cancer types, including both solid and hematologic malignancies. A number of chromosomal translocations involving Polycomb group proteins have been identified; however, the molecular function of these fusion proteins remains largely unexplored. Here we characterize two endometrial stromal sarcoma (ESS) associated fusion proteins: JAZF1-SUZ12 and MBTD1-CXORF67. We identify JAZF1 as a previously unreported subunit of the NuA4 complex, which when fused to SUZ12 creates a bridge between the NuA4 and PRC2 complexes. Intriguingly, the MBTD1-CXORF67 fusion binds to PRC2 and leads to strongly reduced levels of the PRC2 catalytic products H3K27me2/3. We demonstrate that this inhibitory function is mediated by the CXORF67 half of the fusion protein. Because of this striking property, we propose a new gene name: CATACOMB (CATalytic Antagosnist of polyCOMB; official gene name: EZHIP). We map CATACOMB’s inhibitory function to a short, highly conserved region and identify a single methionine residue essential for diminution of H3K27me2/3 levels. Remarkably, the amino acid sequence surrounding this critical methionine resembles that of the oncogenic histone H3 lysine-27-to-methionine (H3K27M) mutation found in high-grade pediatric gliomas. Finally, we show CATACOMB expression is silenced through DNA methylation and upon treatment with DNA demethylating agents, CATACOMB is expressed, binds to PRC2, and antagonizes its catalytic activity. In conclusion, we have identified an endogenous inducible gene, CATACOMB, which can regulate the catalytic activity of PRC2 and propose that such mechanism of regulation of histone modifications through the expression of antagonistic subunits may also exist for other histone-modifying enzyme complexes. Citation Format: Andrea Piunti, Edwin Smith, Marc Morgan, Michal Ugarenko, Natalia Khaltyan, Kathryn Helmin, Caila Ryan, David Murray, Ryan Rickels, Bahar Yilmaz, Emily Rendleman, Jeffrey Savas, Benjamin Singer, Serdar Bulun, Ali Shilatifard. CATACOMB: An endogenous inducible gene that antagonizes H3K27 methylation activity of Polycomb Repressive complex 2 via a H3K27M-like mechanism [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B46.

Repurposing erectile dysfunction drugs tadalafil and vardenafil to increase bone mass

We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine β-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.

A novel stable isotope tracer method to simultaneously quantify skeletal muscle protein synthesis and breakdown

Background/aimsMethodological challenges have been associated with the dynamic measurement of muscle protein breakdown (MPB), as have the measurement of both muscle protein synthesis (MPS) and MPB within the same experiment. Our aim was to use the transmethylation properties of methionine as proof-of-concept to measure rates of MPB via its methylation of histidine within skeletal muscle myofibrillar proteins, whilst simultaneously utilising methionine incorporation into bound protein to measure MPS. ResultsDuring the synthesis measurement period, incorporation of methyl[D3]-13C-methionine into cellular protein in C2C12 myotubes was observed (representative of MPS), alongside an increase in the appearance of methyl[D3]-methylhistidine into the media following methylation of histidine (representative of MPB). For further validation of this approach, fractional synthetic rates (FSR) of muscle protein were increased following treatment of the cells with the anabolic factors insulin-like growth factor-1 (IGF-1) and insulin, while dexamethasone expectedly reduced MPS. Conversely, rates of MPB were reduced with IGF-1 and insulin treatments, whereas dexamethasone accelerated MPB. ConclusionsThis is a novel stable isotope tracer approach that permits the dual assessment of muscle cellular protein synthesis and breakdown rates, through the provision of a single methionine amino acid tracer that could be utilised in a wide range of biological settings.

Essential Amino Acid Profile in Parenteral Nutrition Mixtures: Does It Meet Needs?

Background and Aims: The study compares the essential amino acid (EAA) composition of different parenteral nutrition (PN) mixtures with whey protein EAA profile and the theoretical daily EAA requirements (set by WHO/FAO/UNU or IAAO method). According to the individual EAA profile, the potential effect of several PN mixtures was evaluated on the skeletal muscle mass (SMM) of patients on home PN. Methods: Eight AA solutions and fifteen complete PN mixtures were considered. Twenty-nine clinically stable patients with short bowel syndrome on home total PN were retrospectively evaluated. SMM was estimated by bioelectrical impedance analysis. Results: The prescribed doses of EAA that showed a significant increase in home PN patients muscle mass were considerably greater than the theoretical ones, showing an EAA profile similar to whey protein. At the daily dose of 1 g of total AA s/kg body weight (BW), the considered PN mixtures mostly failed to improve SMM. Only prescribed doses which included more than 0.25 g/kg BW of total BCAA with at least 0.10 g/kg BW leucine, 0.08 g/kg BW isoleucine, and 0.06 g/kg BW methionine showed a significant increase in SMM. Conclusions: The theoretical daily requirement for each EAA was met by all considered PN solutions when the prescribed daily dose of total AAs was set at 1 g/kg BW. Nevertheless, our data suggest that only an increase in total BCAA, also richer in single AA leucine, isoleucine, and methionine, is associated with the maintenance and/or increase of SMM. According to these preliminary observations, we support the prescription of an EAA composition of PN mixtures close to that of whey protein for the preservation of SMM in patients on long-term total PN.

Amyloid-like Structures Formed by Single Amino Acid Self-Assemblies of Cysteine and Methionine.

We report for the very first time the discovery of amyloid-like self-assemblies formed by the nonaromatic single amino acids cysteine (Cys) and methionine (Met) under neutral aqueous conditions. The structure formation was assessed and characterized by various microscopic and spectroscopic techniques such as optical microscopy, phase contrast microscopy, scanning electron microscopy, and transmission electron microscopy. The mechanism of self-assembly and the role of hydrogen bonding and thiol interactions of Cys and Met were assessed by Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction, and solid state NMR along with various control experiments. In addition, molecular dynamics simulations were carried out to gain insight into assembly initiation. Further, Thioflavin T and Congo red binding assays with Cys and Met structures indicated that these single amino acid assemblies may have amyloid-like characteristics. To understand the biological significance of the Cys and Met structures, cytotoxicity assays of the assemblies were performed on human neuroblastoma IMR-32 cells and monkey kidney cells (COS-7). The results revealed that both Cys and Met fibers were cytotoxic. The cell viability assay further supported the hypothesis that aggregation of single amino acid may contribute to the etiology of metabolic disorders like cystinuria and hypermethioninemia. The results presented in this study are striking, and to the best of our knowledge this is the first report which demonstrates that nonaromatic amino acids like Cys and Met can undergo spontaneous self-assembly to form amyloidogenic aggregates. The results presented are also consistent with the established generic amyloid hypothesis and support a new paradigm for the study of the etiology of single amino acid initiated metabolic disorders in amyloid related diseases.

Reductive power of the archaea right-handed coiled coil nanotube (RHCC-NT) and incorporation of mercury clusters inside protein cages

Coiled coils are well described as powerful oligomerization motifs and exhibit a large diversity of functions, including gene regulation, cell division, membrane fusion and drug extrusion. The archaea S-layer originated right-handed coiled coil -RHCC-NT- is characterized by extreme stability and is free of cysteine and histidine moieties. In the current study, we have followed a multidisciplinary approach to investigate the capacity of RHCC-NT to bind a variety of ionic complex metal ions. At the outside of the RHCC-NT, one mercury ion forms an electrostatic interaction with the S-methyl moiety of the single methionine residue present in each coil. We demonstrate that RHCC-NT is reducing and incorporating metallic mercury in the large-sized interior cavities which are lined up along the tetrameric channel.

Analysis of Homeostatic Mechanisms in Biochemical Networks.

Cell metabolism is an extremely complicated dynamical system that maintains important cellular functions despite large changes in inputs. This "homeostasis" does not mean that the dynamical system is rigid and fixed. Typically, large changes in external variables cause large changes in some internal variables so that, through various regulatory mechanisms, certain other internal variables (concentrations or velocities) remain approximately constant over a finite range of inputs. Outside that range, the mechanisms cease to function and concentrations change rapidly with changes in inputs. In this paper we analyze four different common biochemical homeostatic mechanisms: feedforward excitation, feedback inhibition, kinetic homeostasis, and parallel inhibition. We show that all four mechanisms can occur in a single biological network, using folate and methionine metabolism as an example. Golubitsky and Stewart have proposed a method to find homeostatic nodes in networks. We show that their method works for two of these mechanisms but not the other two. We discuss the many interesting mathematical and biological questions that emerge from this analysis, and we explain why understanding homeostatic control is crucial for precision medicine.

DealingwithanUnconventionalGeneticCodein Mitochondria:TheBiogenesisandPathogenic Defectsofthe5-FormylcytosineModificationin MitochondrialtRNAMet.

Human mitochondria contain their own genome, which uses an unconventional genetic code. In addition to the standard AUG methionine codon, the single mitochondrial tRNA Methionine (mt-tRNAMet) also recognises AUA during translation initiation and elongation. Post-transcriptional modifications of tRNAs are important for structure, stability, correct folding and aminoacylation as well as decoding. The unique 5-formylcytosine (f5C) modification of position 34 in mt-tRNAMet has been long postulated to be crucial for decoding of unconventional methionine codons and efficient mitochondrial translation. However, the enzymes responsible for the formation of mitochondrial f5C have been identified only recently. The first step of the f5C pathway consists of methylation of cytosine by NSUN3. This is followed by further oxidation by ABH1. Here, we review the role of f5C, the latest breakthroughs in our understanding of the biogenesis of this unique mitochondrial tRNA modification and its involvement in human disease.

Biochemical Characterization of Pathogenic Mutations in Human Mitochondrial Methionyl-tRNA Formyltransferase

N-Formylation of initiator methionyl-tRNA (Met-tRNA(Met)) by methionyl-tRNA formyltransferase (MTF) is important for translation initiation in bacteria, mitochondria, and chloroplasts. Unlike all other translation systems, the metazoan mitochondrial system is unique in using a single methionine tRNA (tRNA(Met)) for both initiation and elongation. A portion of Met-tRNA(Met) is formylated for initiation, whereas the remainder is used for elongation. Recently, we showed that compound heterozygous mutations within the nuclear gene encoding human mitochondrial MTF (mt-MTF) significantly reduced mitochondrial translation efficiency, leading to combined oxidative phosphorylation deficiency and Leigh syndrome in two unrelated patients. Patient P1 has a stop codon mutation in one of the MTF genes and an S209L mutation in the other MTF gene. P2 has a S125L mutation in one of the MTF genes and the same S209L mutation as P1 in the other MTF gene. Here, we have investigated the effect of mutations at Ser-125 and Ser-209 on activities of human mt-MTF and of the corresponding mutations, Ala-89 or Ala-172, respectively, on activities of Escherichia coli MTF. The S125L mutant has 653-fold lower activity, whereas the S209L mutant has 36-fold lower activity. Thus, both patients depend upon residual activity of the S209L mutant to support low levels of mitochondrial protein synthesis. We discuss the implications of these and other results for whether the effect of the S209L mutation on mitochondrial translational efficiency is due to reduced activity of the mutant mt-MTF and/or reduced levels of the mutant mt-MTF.

Redox-sensitive residue in the actin-binding interface of myosin

We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress.

Structural Effects of Methionine Oxidation on Isolated Subdomains of Human Fibrin D and αC Regions

Oxidation of key methionine residues on fibrin leads to altered fibrin polymerization producing severely altered fibrin gel structure and function. This is important because fibrinogen and its modification by oxidative stress have been implicated as key contributors to both pathological thrombotic and hemorrhagic diseases ranging from cardiovascular thrombosis to the acute coagulopathy of trauma. However, how oxidation leads to altered fibrin polymerization remains poorly understood at the molecular level. We have applied a powerful and novel well-tempered ensemble parallel tempering (PT-WTE) technique along with conventional molecular dynamics (MD) simulation to investigate the molecular-level consequences of selective methionine oxidation of fibrinogen. We offer new insights into molecular mechanisms of oxidation-induced changes in fibrin polymerization, while focusing on the D region knob ‘B’ and hole ‘b’ interaction and αC-domain interactions, both of which are hypothesized to contribute to the lateral aggregation mechanism of fibrin fibrils. Methionine oxidation did not alter the native state or the stability of a bound knob ‘B’ surrogate when interacting with hole ‘b’ in the D region. However, applying PT-WTE simulation to a human homology model of the bovine N-terminal subdomain fragment from the αC-domain revealed that methionine oxidation altered the conformation of the hairpin-linking region to favor open rather than closed hairpin structures. We attribute this alteration to the disruption of the hairpin-linking region's conformation, with oxidation increasing the radius of gyration for this segment. This result is in agreement with experimental data demonstrating decreased fibrin protofibril lateral aggregation when methionine oxidation is present in the same αC-domain fragment. Therefore, single methionine oxidation within the αC-domain is a likely molecular mechanism.

Characterization of the Oligomerization and Aggregation of Human Serum Amyloid A

The fibrillation of Serum Amyloid A (SAA) – a major acute phase protein – is believed to play a role in the disease Amyloid A (AA) Amyloidosis. To better understand the amyloid formation pathway of SAA, we characterized the oligomerization, misfolding, and aggregation of a disease-associated isoform of human SAA – human SAA1.1 (hSAA1.1) – using techniques ranging from circular dichroism spectroscopy to atomic force microscopy, fluorescence spectroscopy, immunoblot studies, solubility measurements, and seeding experiments. We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers in vitro on refolding and cross-beta-rich aggregates following incubation at 37°C. Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway. A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.

Leishmania major Methionine Sulfoxide Reductase A Is Required for Resistance to Oxidative Stress and Efficient Replication in Macrophages

Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA) that could have a role in reducing oxidized free and proteinogenic methionine residues. A GFP-fusion of L. major MsrA was shown to have a cytoplasmic localization by immunofluorescence microscopy and subcellular fractionation. An L. major msrA null mutant, generated by targeted replacement of both chromosomal allelles, was viable in rich medium but was unable to reduce exogenous methionine sulfoxide when cultivated in the presence of this amino acid, indicating that msrA encodes a functional MsrA. The ΔmsrA mutant exhibited increased sensitivity to H2O2 compared to wild type parasites and was unable to proliferate normally in macrophages. Wild type sensitivity to H2O2 and infectivity in macrophages was restored by complementation of the mutant with a plasmid encoding MsrA. Unexpectedly, the ΔmsrA mutant was able to induce normal lesions in susceptible BALB/c indicating that this protein is not essential for pathogenesis in vivo. Our results suggest that Leishmania MsrA contributes to the anti-oxidative defences of these parasites, but that complementary oxidative defence mechansims are up-regulated in lesion amastigotes.

Do Proteomics Analyses Provide Insights into Reduced Oxidative Stress in the Brain of an Alzheimer Disease Transgenic Mouse Model with an M631L Amyloid Precursor Protein Substitution and Thereby the Importance of Amyloid-Beta-Resident Methionine 35 in Alzheimer Disease Pathogenesis?

The single methionine (Met/M) residue of amyloid-beta (Aβ) peptide, at position 35 of the 42-mer, has important relevance for Aβ-induced oxidative stress and neurotoxicity. Recent in vivo brain studies in a transgenic (Tg) Alzheimer disease (AD) mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) (referred to as the J20 Tg mouse) demonstrated increased levels of oxidative stress. However, the substitution of the Met631 residue of APP to leucine (Leu/L) (M631L in human APP numbering, referred to as M631L Tg and corresponding to residue 35 of Aβ1-42) resulted in no significant in vivo oxidative stress levels, thereby supporting the hypothesis that Met-35 of Aβ contributes to oxidative insult in the AD brain. It is conceivable that oxidative stress mediated by Met-35 of Aβ is important in regulating numerous downstream effects, leading to differential levels of relevant biochemical pathways in AD. Therefore, in the current study using proteomics, we tested the hypothesis that several brain proteins involved in pathways such as energy and metabolism, antioxidant activity, proteasome degradation, and pH regulation are altered in J20Tg versus M631L Tg AD mice.

Methionine Scanning as an NMR Tool for Detecting and Analyzing Biomolecular Interaction Surfaces

Methyl NMR spectroscopy is a powerful tool for studying protein structure, dynamics, and interactions. Yet difficulties with resonance assignment and the low abundance of methyl groups can preclude detailed NMR studies, particularly the determination of continuous interaction surfaces. Here we present a straightforward strategy that overcomes these problems. We systematically substituted solvent-exposed residues with reporter methionines in the expected binding site and performed chemical shift perturbation (CSP) experiments using methyl-TROSY spectra. We demonstrate the utility of this approach for the interaction between the HECT domain of the Rsp5p ubiquitin ligase and its cognate E2, Ubc4. Using these mutants, we could instantaneously assign all newly arising reporter methyl signals, determine the Ubc4 interaction surface on a per-residue basis, and investigate the importance of each individual mutation for ligand binding. Our data show that methionine scanning significantly extends the applicability, information content, and spatial resolution of methyl CSP experiments.

Structural and Functional Impact of Site-Directed Methionine Oxidation in Myosin

We have examined the structural and functional effects of site-directed methionine oxidation in Dictyostelium (Dicty) myosin II using mutagenesis, in vitro oxidation, and site-directed spin-labeling for electron paramagnetic resonance (EPR). Protein oxidation by reactive oxygen and nitrogen species is critical for normal cellular function, but oxidative stress has been implicated in disease progression and biological aging. Our goal is to bridge understanding of protein oxidation and muscle dysfunction with molecular-level insights into actomyosin interaction. In order to focus on methionine oxidation and to facilitate site-directed spectroscopy, we started with a Cys-lite version of Dicty myosin II. For Dicty myosin containing native methionines, peroxide treatment decreased actin-activated myosin ATPase activity, consistent with the decline in actomyosin function previously observed in biologically aged or peroxide-treated muscle. Methionine-to-leucine mutations, used to protect specific sites from oxidation, identified a single methionine that is functionally sensitive to oxidation: M394, near the myosin cardiomyopathy loop in the actin-binding interface. Previously characterized myosin labeling sites for spectroscopy in the force-producing region and actin-binding cleft were examined; spin-label mobility and distance measurements in the actin-binding cleft were sensitive to oxidation, but particularly in the presence of actin. Overall secondary structure and thermal stability were unaffected by oxidation. We conclude that the oxidation-induced structural change in myosin includes a redistribution of existing structural states of the actin-binding cleft. These results will be applicable to the many biological and therapeutic contexts in which a detailed understanding of protein oxidation as well as function and structure relationships is sought.