Kagan RM et al. (1995) Biochemistry "Protein L-isoaspartyl methyltransferase from the nematode Caenorhabditis elegans: genomic structure and substrate specificity."

Kagan RM, Clarke S

[Biochemistry, 1995]

We identified a protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) in the nematode worm Caenorhabditis elegans. The methylation of abnormal L-isoaspartyl residues by this enzyme can lead to their conversion to L-aspartyl residues and represents a protein repair step for polypeptides damaged by spontaneous reactions during the aging process. We show that the levels of this enzyme increase 2-fold in C. elegans in the dauer larval form, a developmental stage where the organism can survive for extended periods of time. Utilizing degenerate oligonucleotide primers derived from conserved amino acid sequences of mammalian, plant, and bacterial L-isoaspartyl methyltransferases and PCR amplification, we made DNA probes that allowed us to obtain cDNA and genomic DNA clones encoding this enzyme in the nematode. The deduced amino acid sequence is 53% identical to the human enzyme and 29% identical to the Escherichia coli enzyme. Overexpression of the cDNA for the C. elegans enzyme in E. coli gave an active product with micromolar Km values for L-isoaspartyl-containing peptide substrates and for the methyl donor S-adenosyl-L-methionine. No methylation of D-aspartyl-containing peptides was detected under conditions where the human enzyme catalyzed this reaction, suggesting that the ability to methylate D-aspartyl residues in addition to L-isoaspartyl residues was a later evolutionary adaptation of this enzyme. The C. elegans gene for the methyltransferase, designated pcm-1, was mapped to a single site in a 31 kb region in the central portion of chromosome V. The gene is 3.2 kb in length and includes six introns. Although much smaller, its genomic organization is similar to that of the corresponding mouse gene, with identically positioned intron--exon splice junctions at five of seven sites. We propose that this gene plays an important role in facilitating the long term survival of this organisms.

Kagan RM et al. (1997) Comp Biochem Physiol B Biochem Mol Biol "Molecular phylogenetics of a protein repair methyltransferase."

O'Connor C, McFadden HJ, Clarke S, Kagan RM, McFadden PN

[Comp Biochem Physiol B Biochem Mol Biol, 1997]

Protein-L-isoaspartyl (D-aspartyl) O-methyltransferase (E.C. 2.1.1.77) is a well-conserved and widely distributed protein repair enzyme that methylates isomerized or racemized aspartyl residues in age-damaged proteins. We exploited the availability of protein sequences from 10 diverse animal, plant and bacterial taxa to construct a phylogenetic tree and determine the rates of amino acid substitution for this enzyme. We used a likelihood ratio test to show that this enzyme fulfills the conditions for a molecular clock. We found that the rate of substitution is 0.39 amino acid substitutions per site per 10(9) years and remains relatively constant from bacteria to humans. We argue that this degree of sequence conservation may result from the functional constraints necessitated by the requirement to specifically recognize altered aspartyl but not normal aspartyl residues in proteins. Relative rate analysis of the Caenorhabditis elegans sequence suggests that the amino acid substitution rate in the nematode lineage may be higher than that in other lineages and that the divergence of nematodes may have been a more recent event than suggested by previous analysis.

Kagan RM et al. (1994) Arch Biochem Biophys "Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes."

Kagan RM, Clarke S

[Arch Biochem Biophys, 1994]

Three regions of sequence similarity have been reported in several protein and small-molecule S-adenosylmethionine-dependent methyltransferases. Using multiple alignments, we have now identified these three regions in a much broader group of methyltransferases and have used these data to define a consensus for each region. Of the 84 non-DNA methyltransferase sequences in the GenBank, NBRF PIR, and Swissprot databases comprising 37 distinct enzymes, we have found 69 sequences possessing motif I. This motif is similar to a conserved region previously described in DNA adenine and cytosine methyltransferases. Motif II is found in 46 sequences, while motif III is found in 61 sequences. All three regions are found in 45 of these enzymes, and an additional 15 have motifs I and III. The motifs are always found in the same order on the polypeptide chain and are separated by comparable intervals. We suggest that these conserved regions contribute to the binding of the substrate S-adenosylmethionine and/or the product S-adenosylhomocysteine. These motifs can also be identified in certain nonmethyltransferases that utilize either S-adenosylmethionine or S-adenosylhomocysteine, including S-adenosylmethionine decarboxylase, S-adenosylmethionine synthetase, and S-adenosylhomocysteine hydrolase. In the latter two types of enzymes, motif I is similar to the conserved nucleotide binding motif of protein kinases and other nucleotide binding proteins. These motifs may be of use in predicting methyltransferases and related enzymes from the open reading frames generated by genomic sequencing projects.

Kagan RM et al. (1997) Arch Biochem Biophys "Targeted gene disruption of the Caenorhabditis elegans L-isoaspartyl protein repair methyltransferase impairs survival of dauer stage nematodes."

Niewmierzycka A, Kagan RM, Clarke S

[Arch Biochem Biophys, 1997]

The methylation of abnormal L-isoaspartyl residues by protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77) can lead to their conversion to L-aspartyl residues. For polypeptides damaged by spontaneous reactions that generate L-isoaspartyl residues, these steps represent a protein repair pathway that can limit the accumulation of potentially detrimental proteins in the aging process. We report here the construction and the characterization of an animal model deficient in this methyltransferase. We utilized Tc1-transposon-mediated mutagenesis in the nematode Caenorhabditis elegans to construct a homozygous excision mutant lacking exons 2-5 of the pcm-1 gene encoding this enzyme. Nematodes carrying this deletion exhibited no detectable L-isoaspartyl methyltransferase activity. These worms demonstrated normal morphology and behavior and adult mutant nematodes exhibited a normal lifespan. However, the survival of dauer-phase mutants was diminished by 3.5-fold relative to wild-type dauers after 50 days in the dauer phase. The fitness of the pcm-1 deletion nematodes was reduced by about 16% relative to that of wild-type nematodes as measured by the ability of these mutants to compete reproductively against a wild-type population. We found that the absence of the functional methyltransferase gene leads to a modest accumulation of altered protein substrates in aged dauer worms. However, in the viable fraction of these dauer worms, no differences were seen in the levels of altered substrate proteins in the parent and methyltransferase-deficient worms, suggesting that the enzyme in wild-type cells does not efficiently catalyze the repair of spontaneously damaged proteins.

Ma C et al. (2015) Molecules "Potent Activities of Roemerine against Candida albicans and the Underlying Mechanisms."

Yan L, Xu G, Wang C, He G, Du F, Jiang Y, Ma C, Rao G, He J

[Molecules, 2015]

Roemerine (RM) is an aporphine alkaloid isolated from the fresh rattan stem of Fibraurea recisa, and it has been demonstrated to have certain antifungal activity. This study aimed to investigate the antifungal activity of RM and the underlying mechanisms in Candida albicans (C. albicans). The in vitro antifungal activity of RM was evaluated by a series of experiments, including the XTT reduction assay, confocal laser scanning microscopy assay, scanning electron microscope assay. Results showed that 1 g/mL RM inhibited biofilm formation significantly (p < 0.01) both in Spider medium and Lee's medium. In addition, RM could inhibit yeast-to-hyphae transition of C. albicans in a dose-dependent manner. The biofilm-specific and hypha-specific genes such as YWP1, SAP5, SAP6, HWP1, ECE1 were up-regulated and EFG1 was down-regulated after 8 g/mL RM treatment. Furthermore, the toxicity of RM was investigated using C. elegans worms, three cancer cells and one normal cell. The date showed that RM had no significant toxicity. In conclusion, RM could inhibited the formation of C. albicans biofilm in vitro, but it had no fungicidal effect on planktonic C. albicans cells, and the anti-biofilm mechanism may be related to the cAMP pathway.

Gouveia GJ et al. (2021) Anal Chem "Long-Term Metabolomics Reference Material."

Andersen EC, Morse AM, Garcia BM, Edison AS, Shaver AO, Gouveia GJ, McIntyre LM

[Anal Chem, 2021]

The use of quality control samples in metabolomics ensures data quality, reproducibility, and comparability between studies, analytical platforms, and laboratories. Long-term, stable, and sustainable reference materials (RMs) are a critical component of the quality assurance/quality control (QA/QC) system; however, the limited selection of currently available matrix-matched RMs reduces their applicability for widespread use. To produce an RM in any context, for any matrix that is robust to changes over the course of time, we developed iterative batch averaging method (IBAT). To illustrate this method, we generated 11 independently grown <i>Escherichia coli</i> batches and made an RM over the course of 10 IBAT iterations. We measured the variance of these materials by nuclear magnetic resonance (NMR) and showed that IBAT produces a stable and sustainable RM over time. This <i>E. coli</i> RM was then used as a food source to produce a <i>Caenorhabditis elegans</i> RM for a metabolomics experiment. The metabolite extraction of this material, alongside 41 independently grown individual <i>C. elegans</i> samples of the same genotype, allowed us to estimate the proportion of sample variation in preanalytical steps. From the NMR data, we found that 40% of the metabolite variance is due to the metabolite extraction process and analysis and 60% is due to sample-to-sample variance. The availability of RMs in untargeted metabolomics is one of the predominant needs of the metabolomics community that reach beyond quality control practices. IBAT addresses this need by facilitating the production of biologically relevant RMs and increasing their widespread use.

Lawal TA et al. (2020) Orphanet J Rare Dis "Preclinical model systems of ryanodine receptor 1-related myopathies and malignant hyperthermia: a comprehensive scoping review of works published 1990-2019."

Dowling JJ, Terry NL, Todd JJ, Wires ES, Lawal TA

[Orphanet J Rare Dis, 2020]

BACKGROUND: Pathogenic variations in the gene encoding the skeletal muscle ryanodine receptor (RyR1) are associated with malignant hyperthermia (MH) susceptibility, a life-threatening hypermetabolic condition and RYR1-related myopathies (RYR1-RM), a spectrum of rare neuromuscular disorders. In RYR1-RM, intracellular calcium dysregulation, post-translational modifications, and decreased protein expression lead to a heterogenous clinical presentation including proximal muscle weakness, contractures, scoliosis, respiratory insufficiency, and ophthalmoplegia. Preclinical model systems of RYR1-RM and MH have been developed to better understand underlying pathomechanisms and test potential therapeutics. METHODS: We conducted a comprehensive scoping review of scientific literature pertaining to RYR1-RM and MH preclinical model systems in accordance with the PRISMA Scoping Reviews Checklist and the framework proposed by Arksey and O'Malley. Two major electronic databases (PubMed and EMBASE) were searched without language restriction for articles and abstracts published between January 1, 1990 and July 3, 2019. RESULTS: Our search yielded 5049 publications from which 262 were included in this review. A majority of variants tested in RYR1 preclinical models were localized to established MH/central core disease (MH/CCD) hot spots. A total of 250 unique RYR1 variations were reported in human/rodent/porcine models with 95% being missense substitutions. The most frequently reported RYR1 variant was R614C/R615C (human/porcine total n=39), followed by Y523S/Y524S (rabbit/mouse total n=30), I4898T/I4897T/I4895T (human/rabbit/mouse total n=20), and R163C/R165C (human/mouse total n=18). The dyspedic mouse was utilized by 47% of publications in the rodent category and its RyR1-null (1B5) myotubes were transfected in 23% of publications in the cellular model category. In studies of transfected HEK-293 cells, 57% of RYR1 variations affected the RyR1 channel and activation core domain. A total of 15 RYR1 mutant mouse strains were identified of which ten were heterozygous, three were compound heterozygous, and a further two were knockout. Porcine, avian, zebrafish, C. elegans, canine, equine, and drosophila model systems were also reported. CONCLUSIONS: Over the past 30years, there were 262 publications on MH and RYR1-RM preclinical model systems featuring more than 200 unique RYR1 variations tested in a broad range of species. Findings from these studies have set the foundation for therapeutic development for MH and RYR1-RM.

Volpatti JR et al. (2020) Elife "Identification of drug modifiers for RYR1 related myopathy using a multi-species discovery pipeline."

Groom L, Endo Y, Volpatti JR, Zuercher WJ, Brennan S, Roy P, Dirksen RT, Dowling JJ, Knox J, Noche R

[Elife, 2020]

Ryanodine receptor type I-related myopathies (RYR1-RMs) are a common group of childhood muscle diseases associated with severe disabilities and early mortality for which there are no available treatments. The goal of this study is to identify new therapeutic targets for RYR1-RMs. To accomplish this, we developed a discovery pipeline using nematode, zebrafish, and mammalian cell models. We first performed large-scale drug screens in <i>C. elegans</i> which uncovered 74 hits. Targeted testing in zebrafish yielded positive results for two p38 inhibitors. Using mouse myotubes, we found that either pharmacological inhibition or siRNA silencing of p38 impaired caffeine-induced Ca²⁺ release from wild type cells while promoting intracellular Ca²⁺ release in <i>Ryr1</i> knockout cells. Lastly, we demonstrated that p38 inhibition blunts the aberrant temperature-dependent increase in resting Ca²⁺ in myotubes from an RYR1-RM mouse model. This unique platform for RYR1-RM therapy development is potentially applicable to a broad range of neuromuscular disorders.

Yu Z et al. (2011) J Environ Sci (China) "Behavior toxicity to Caenorhabditis elegans transferred to the progeny after exposure to sulfamethoxazole at environmentally relevant concentrations."

Yu Z, Jiang L, Yin D

[J Environ Sci (China), 2011]

Sulfamethoxazole (SMX) is one of the most common detected antibiotics in the environment. In order to study whether SMX can affect behavior and growth and whether these effects could be transferred to the progeny, Caenorhabditis elegans was exposed at environmentally relevant concentrations for 24, 48, 72 and 96 hr, respectively. After exposure, the exposed parent generation (P0) was measured for behavior and growth indicators, which were presented as percentage of controls (POC). Then their corresponding unexposed progeny (F1) was separated and measured for the same indicators. The lowest POC for P0 after 96 hr-exposure at 100 mg/L were 37.8%, 12.7%, 45.8% and 70.1% for body bending frequency (BBF), reversal movement (RM), Omega turns (OT) and body length (BL), respectively. And F1 suffered defects with the lowest POC as 55.8%, 24.1%, 48.5% and 60.7% for BBF, RM, OT and BL, respectively. Defects in both P0 and F1 showed a time- and concentration-dependent fashion and behavior indicators showed better sensitivity than growth indicator. The observed effects on F1 demonstrated the transferable properties of SMX. Defects of SMX at environmental concentrations suggested that it is necessary to perform further systematical studies on its ecological risk in actual conditions.

Zaridah MZ et al. (2001) J Ethnopharmacol "In vitro antifilarial effects of three plant species against adult worms of subperiodic Brugia malayi."

Omar AW, Idid SZ, Zaridah MZ, Khozirah S

[J Ethnopharmacol, 2001]

Five aqueous extracts from three plant species, i.e., dried husks (HX), dried seeds (SX) and dried leaves (LX) of Xylocarpus granatum (Meliaceae), dried stems (ST) of Tinospora crispa (Menispermaceae) and dried leaves (LA) of Andrographis paniculata (Acanthaceae) were tested in vitro against adult worms of subperiodic Brugia malayi. The relative movability (RM) value of the adult worms over the 24-h observation period was used as a measure of the antifilarial activity of the aqueous extracts. SX extract of X. granatum demonstrated the strongest activity, followed by the LA extract of A. paniculata, ST extract of T. crispa, HX extract and LX extract of X. granatum.