Extending Comet for Global Amino Acid Variant and Post‐Translational Modification Analysis Using the PSI Extended FASTA Format

Post-translational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such function. Here, we present function and structure of NpmB1 whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single amino acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

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Single Amino Acid Variant Discovery in Small Numbers of Cells

Journal of Proteome Research ◽

10.1021/acs.jproteome.8b00694 ◽

2018 ◽

Cited By ~ 7

Author(s):

Zhijing Tan ◽

Xinpei Yi ◽

Nicholas J. Carruthers ◽

Paul M. Stemmer ◽

David M. Lubman

Keyword(s):

Amino Acid ◽

Single Amino Acid ◽

Variant Discovery ◽

Amino Acid Variant

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Structural and Functional Consequences of Age-Related Isomerization in α-Crystallins

10.1101/364497 ◽

2018 ◽

Cited By ~ 1

Author(s):

Yana A. Lyon ◽

Dylan L. Riggs ◽

Miranda P. Collier ◽

Matteo T. Degiacomi ◽

Justin L.P. Benesch ◽

...

Keyword(s):

Mass Spectrometry ◽

Molecular Dynamics ◽

Amino Acid ◽

Native Mass Spectrometry ◽

Salt Bridges ◽

Loss Of Function ◽

Post Translational Modification ◽

Age Related ◽

Αb Crystallin ◽

Functional Consequences

AbstractLong-lived proteins are subject to spontaneous degradation and may accumulate a range of modifications over time, including subtle alterations such as isomerization. Recently, tandem-mass spectrometry approaches have enabled the identification and detailed characterization of such peptide isomers, including those differing only in chirality. However, the structural and functional consequences of these perturbations remain largely unexplored. Here we examine the site-specific impact of isomerization of aspartic acid and epimerization of serine in human αA- and αB-crystallin. From a total of 81 sites of modification identified in aged eye lenses, four (αBSer59, αASer162, αBAsp62, αBAsp109) map to crucial oligomeric interfaces. To characterize the effect of isomerization on quaternary assembly, molecular dynamics calculations and native mass spectrometry experiments were performed on recombinant forms of αA- and αB-crystallin that incorporate, or mimic, isomerized residues. In all cases, oligomerization is significantly affected, with epimerization of a single serine residue (αASer162) sufficing to weaken inter-subunit binding dramatically. Furthermore, phosphorylation of αBSer59, known to play an important regulatory role in oligomerization, is severely inhibited by serine epimerization and altered by isomerization of nearby αBAsp62. Similarly, isomerization of αBAsp109 disrupts a vital salt-bridge with αBArg120, a loss previously shown to yield aberrant oligomerization and aggregation in several disease variants. Our results illustrate how isomerization of amino-acid residues, which may seem like a minor structural perturbation, can have profound consequences on protein assembly and activity by disrupting specific hydrogen bonds and salt bridges.Significance StatementProteins play numerous critical roles in our bodies but suffer damage with increasing age. For example, isomerization is a spontaneous post-translational modification that alters the three-dimensional connectivity of an amino acid, yet remains invisible to traditional proteomic experiments. Herein, radical-based fragmentation was used for isomer identification while molecular dynamics and native mass spectrometry were utilized to assess structural consequences. The results demonstrate that isomerization disrupts both oligomeric assembly and phosphorylation in the α-crystallins, which are long-lived proteins in the lens of the eye. The loss of function associated with these modifications is likely connected to age-related diseases such as cataract and neurodegenerative disorders, while the methodologies we present represent a framework for structure-function studies on other isomerized proteins.

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Faculty Opinions recommendation of A non-conserved amino acid variant regulates differential signalling between human and mouse CD28.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732850338.793546604 ◽

2018 ◽

Author(s):

Mandy McGeachy

Keyword(s):

Amino Acid ◽

Amino Acid Variant ◽

Human And Mouse

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Chitin-binding proteins in potato (Solanum tuberosum L.) tuber. Characterization, immunolocalization and effects of wounding

Biochemical Journal ◽

10.1042/bj2830813 ◽

1992 ◽

Vol 283 (3) ◽

pp. 813-821 ◽

Cited By ~ 16

Author(s):

D J Millar ◽

A K Allen ◽

C G Smith ◽

C Sidebottom ◽

A R Slabas ◽

...

Keyword(s):

Solanum Tuberosum ◽

Amino Acid ◽

Amino Acid Sequence ◽

Binding Proteins ◽

Solanum Tuberosum L ◽

Major Protein ◽

Post Translational Modification ◽

Terminal Amino Acid ◽

Terminal Amino ◽

Terminal Amino Acid Sequence

Tubers of potato (Solanum tuberosum L.) contain a number of chitin-binding proteins which have possible functions in defence against pathogens. A major protein of the tuber is the chitin-binding lectin which has been further characterized with respect to its antigenicity and N-terminal amino acid sequence. By using an antiserum monospecific for tuber lectin in unwounded potato the protein was found in the cytoplasm and vacuole, unusually for a hydroxyproline-rich glycoprotein, but consistent with its soluble nature in subcellular extracts. Little increased synthesis of the lectin precursor or the post-translationally modified form could be demonstrated in excised potato tuber discs. However, after wounding there is increased synthesis of another hydroxyproline-containing glycoprotein of Mr 57,000, which binds to chitin and shares common epitopes with the lectin. In comparison with the tuber lectin, this novel glycoprotein contains less hydroxyproline, but from its overall composition it is clearly not an underhydroxylated form of the tuber lectin. It differed in its N-terminal amino acid sequence and was much less glycosylated, although arabinose was still present. Synthesis of the Mr-57,000 polypeptide began after the initial burst of protein synthesis and increased, reaching a peak at 24 h after wounding. The protein was produced with its enzymes of post-translational modification, prolyl hydroxylase and arabinosyltransferase, concomitantly with the marker enzymes for wounding, phenylalanine ammonia-lyase and membrane-bound phenol oxidase and peroxidase.

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Screening of OXA-244 producers, a difficult-to-detect and emerging OXA-48 variant?

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa155 ◽

2020 ◽

Cited By ~ 1

Author(s):

Cecile Emeraud ◽

Laura Biez ◽

Delphine Girlich ◽

Agnès B Jousset ◽

Thierry Naas ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Hydrolytic Activity ◽

Single Amino Acid ◽

Broth Microdilution ◽

Mlst Analysis ◽

E Coli ◽

Amino Acid Variant ◽

High Prevalence ◽

High Concentrations

Abstract Background OXA-244, a single amino acid variant of OXA-48, demonstrates weaker hydrolytic activity towards carbapenems and temocillin compared with OXA-48. Of note, these antimicrobials are present in high concentrations in several carbapenemase-producing Enterobacterales (CPE) screening media. As a result, some screening media fail to grow OXA-244-producing isolates, while the prevalence of OXA-244 producers is constantly increasing in France. Methods Here, we evaluate the performance of three commercially available CPE screening media [ChromID® CARBA SMART (bioMérieux), Brilliance™ CRE (Thermo Fisher) and mSuperCARBA™ (MAST Diagnostic)] for their ability to detect OXA-244 producers (n = 101). As OXA-244 producers may also express an ESBL, two additional ESBL screening media were tested (Brilliance™ ESBL and ChromID® BLSE). MICs of temocillin and imipenem were determined by broth microdilution. The clonality of OXA-244-producing Escherichia coli isolates (n = 97) was assessed by MLST. Results Overall, the sensitivity of the ChromID® CARBA SMART, Brilliance™ CRE and mSuperCARBA™ media were 14% (95% CI = 8.1%–22.5%), 54% (95% CI = 43.3%–63.4%) and 99% (95% CI = 93.8%–100%), respectively, for the detection of OXA-244 producers. Among the 101 OXA-244-producing isolates, 96% were E. coli and 77%–78% grew on ESBL screening media. MLST analysis identified five main STs among OXA-244-producing E. coli isolates: ST38 (n = 37), ST361 (n = 17), ST69 (n = 12), ST167 (n = 11) and ST10 (n = 8). Conclusions Our results demonstrated that the mSuperCARBA™ medium is very efficient in the detection of OXA-244 producers, unlike the ChromID® CARBA SMART medium. The high prevalence of ESBLs among OXA-244 producers allowed detection of 77%–78% of them using ESBL-specific screening media.

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iDPGK: characterization and identification of lysine phosphoglycerylation sites based on sequence-based features

BMC Bioinformatics ◽

10.1186/s12859-020-03916-5 ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Kai-Yao Huang ◽

Fang-Yu Hung ◽

Hui-Ju Kao ◽

Hui-Hsuan Lau ◽

Shun-Long Weng

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Training Dataset ◽

Post Translational Modification ◽

Web Based ◽

Charged Amino Acids ◽

Pair Composition ◽

Weighted Matrix ◽

Svm Model ◽

Effective Performance

Abstract Background Protein phosphoglycerylation, the addition of a 1,3-bisphosphoglyceric acid (1,3-BPG) to a lysine residue of a protein and thus to form a 3-phosphoglyceryl-lysine, is a reversible and non-enzymatic post-translational modification (PTM) and plays a regulatory role in glucose metabolism and glycolytic process. As the number of experimentally verified phosphoglycerylated sites has increased significantly, statistical or machine learning methods are imperative for investigating the characteristics of phosphoglycerylation sites. Currently, research into phosphoglycerylation is very limited, and only a few resources are available for the computational identification of phosphoglycerylation sites. Result We present a bioinformatics investigation of phosphoglycerylation sites based on sequence-based features. The TwoSampleLogo analysis reveals that the regions surrounding the phosphoglycerylation sites contain a high relatively of positively charged amino acids, especially in the upstream flanking region. Additionally, the non-polar and aliphatic amino acids are more abundant surrounding phosphoglycerylated lysine following the results of PTM-Logo, which may play a functional role in discriminating between phosphoglycerylation and non-phosphoglycerylation sites. Many types of features were adopted to build the prediction model on the training dataset, including amino acid composition, amino acid pair composition, positional weighted matrix and position-specific scoring matrix. Further, to improve the predictive power, numerous top features ranked by F-score were considered as the final combination for classification, and thus the predictive models were trained using DT, RF and SVM classifiers. Evaluation by five-fold cross-validation showed that the selected features was most effective in discriminating between phosphoglycerylated and non-phosphoglycerylated sites. Conclusion The SVM model trained with the selected sequence-based features performed well, with a sensitivity of 77.5%, a specificity of 73.6%, an accuracy of 74.9%, and a Matthews Correlation Coefficient value of 0.49. Furthermore, the model also consistently provides the effective performance in independent testing set, yielding sensitivity of 75.7% and specificity of 64.9%. Finally, the model has been implemented as a web-based system, namely iDPGK, which is now freely available at http://mer.hc.mmh.org.tw/iDPGK/.

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