UniProt: the universal protein knowledgebase

,

doi:10.1093/nar/gkw1099

Cited by 4,035 publications

(2,490 citation statements)

References 29 publications

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“…These mapped to the protected, intraluminal surface and, as expected, were still detectable following proteinase K treatment (Supplementary Figure 5). Next, we assessed peptides for the tetraspanins, CD9 and CD81, both of which were only identified by mass spectrometry in the absence of proteinase K. Consistent with our hypothesis, in the absence of proteinase K, detected peptides for CD9 and CD81 were exclusively localised to domains annotated as external to the outer EV membrane [27], and proteinase K treatment resulted in loss of these peptides and failure to detect either of these proteins (Supplementary Figure 5). Finally, we examined CD63, which was enriched following proteinase K treatment.…”

Section: Resultssupporting

confidence: 54%

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Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography

Foers

Chatfield

Dagley

et al. 2018

J of Extracellular Vesicle

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As a complex biological fluid, human synovial fluid (SF) presents challenges for extracellular vesicle (EV) enrichment using standard methods. In this study of human SF, a size exclusion chromatography (SEC)-based method of EV enrichment is shown to deplete contaminants that remain after standard ultracentrifugation-based enrichment methods. Specifically, considerable levels of serum albumin, the high-density lipoprotein marker, apolipoprotein A-I, fibronectin and other extracellular proteins and debris are present in EVs prepared by differential ultracentrifugation. While the addition of a sucrose density gradient purification step improved purification quality, some contamination remained. In contrast, using a SEC-based approach, SF EVs were efficiently separated from serum albumin, apolipoprotein A-I and additional contaminating proteins that co-purified with high-speed centrifugation. Finally, using high-resolution mass spectrometry analysis, we found that residual contaminants which remain after SEC, such as fibronectin and other extracellular proteins, can be successfully depleted by proteinase K. Taken together, our results highlight the limitations of ultracentrifugation-based methods of EV isolation from complex biological fluids and suggest that SEC can be used to obtain higher purity EV samples. In this way, SEC-based methods are likely to be useful for identifying EV-enriched components and improving understanding of EV function in disease.

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Section: Resultssupporting

confidence: 54%

“…Gene ontology was investigated with FunRich v3.1.3 using the Gene Ontology Database [24,25]. The peptides identified by mass spectrometry were visualised using Protter [26] with membrane orientations as specified in UniProt annotations [27]. Data has been uploaded to EVpedia [28].…”

Section: Methodsmentioning

confidence: 99%

Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography

Foers

Chatfield

Dagley

et al. 2018

J of Extracellular Vesicle

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“…We constructed alignments for RodA (33670 sequences) and PBP2 (40764 sequences) as described above for RodA alone, using the April 2017 Uniprot release 42 for clarity on species identifiers. We concatenated RodA and PBP2A sequences from each species when they were within 10,000 nucleotides of each other based on European Nucleotide Archive (ENA) data downloaded in February 2017 43 , and evolutionary couplings were computed on the complex alignment as previously described 44 .…”

Section: Methodsmentioning

confidence: 99%

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

Sjodt

Brock

Dobihal

et al. 2018

Nature

120

139

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The Shape, Elongation, Division, and Sporulation (“SEDS”) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was long enigmatic, but recent work1–3 has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase – a role previously attributed exclusively to members of the penicillin binding protein family4. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis for SEDS activity, and no structural data are available for RodA or any homolog thereof. Here, we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a novel ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating it is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.

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“…To construct a robust benchmark dataset, experimentally validated protein sequences with lysine succinylation site details were collected from SwissProt/UniProt (retrieved on 21 May 2017) [17] and Compendium of Protein Lysine Modifications database curated by CUCKOO Workgroup [18]. Initially, 897 proteins with 2523 verified succinylation site (i.e.…”

Section: Datasetsmentioning

confidence: 99%

DV-iSucLys: Decision Voting to Improve Protein Lysine Succinylation Site Identification from Sequence Data

Islam¹

2017

AJBLS

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Protein Post Translation Modification identification is one of the important steps in conducting disease-associated mutation studies. Though multiple chemical alterations happen in a protein after translation, the addition of succinyl group to lysine residue plays a vital role in regulating cellular metabolism and thus disease. Use of a classification algorithm on some features, driven either from protein structural, physicochemical or even biochemical information becomes a common approach that can yield a satisfactory result up to a certain level. Although, researchers already developed many computational methods to identify whether a lysine residue modified with succinyl group after translation, most of them focused on the improvement either on a single decision using a single method or feature enrichment or even development of a benchmark dataset. Therefore, there still exists scope for further improvement to characterise lysine residues of a protein sequence by considering multiple predictors at a time. In this study, an ensemble based approach called DV-iSucLys has been designed to characterise the lysine residue by adapting three well known and conceptually different classifiers and ensembling their decisions. Also, a benchmark succinylation dataset was extracted from existing benchmark datasets and recently updated succinylation data from UniProt consortium to investigate the performance of the proposed approach as well as contribute to further research. Analysing rigorous cross-validation results show that DV-iSucLys can characterise succinyl lysine residue better than the existing predictors.

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UniProt: the universal protein knowledgebase

Cited by 4,035 publications

References 29 publications

Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography

Enrichment of extracellular vesicles from human synovial fluid using size exclusion chromatography

Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis

DV-iSucLys: Decision Voting to Improve Protein Lysine Succinylation Site Identification from Sequence Data

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