Comparative Membrane Proteomics Reveals a Nonannotated <i>E. coli</i> Heat Shock Protein

Yuan, Peng; D'Lima, Nadia G.; Slavoff, Sarah A.

doi:10.1021/acs.biochem.7b00864

Cited by 29 publications

(41 citation statements)

References 25 publications

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“…An independent validation of the functional significance of one of the OOF 471 iTISs identified by Ribo-RET came from a recent study aimed at characterizing E. 472 coli proteins activated by heat-shock (Yuan et al, 2018). Among the tryptic 473 peptides, one mapped to the gnd gene, which encodes 6-phosphogluconate 474 dehydrogenase (6-PGD).…”

Section: '-Proximal Itis Gene May Generate Differentially-targeted Pmentioning

confidence: 99%

Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

Meydan

Marks

Klepacki

et al. 2019

Preprint

130

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The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes but, strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the alternative bacterial proteome. The internal start sites my also play regulatory roles in gene expression.

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Section: '-Proximal Itis Gene May Generate Differentially-targeted Pmentioning

confidence: 99%

Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

Meydan

Marks

Klepacki

et al. 2019

Preprint

130

View full text Add to dashboard Cite

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“…Improvements in small protein enrichment, LC‐MS/MS technology, and optimized database searching subsequently enabled mass spectrometric identification of many more hundreds of micropeptides across human cell lines and tissue samples (Ma et al., ; Slavoff et al., ; Vanderperre et al., ). Further advances have improved coverage of small proteins and to allow for semiquantitative comparison of various cellular conditions (D'Lima et al., ; Ma et al., ; Yuan, D'Lima, & Slavoff, ).…”

Section: Commentarymentioning

confidence: 99%

Proteomic Detection and Validation of Translated Small Open Reading Frames

Khitun

Slavoff

2019

CP Chemical Biology

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Small open reading frames (smORFs) encode previously unannotated polypeptides or short proteins that regulate translation in cis (eukaryotes) and/or are independently functional (prokaryotes and eukaryotes). Ongoing efforts for complete annotation and functional characterization of smORF‐encoded proteins have yielded novel regulators and therapeutic targets. However, because they are excluded from protein databases, initiate at non‐AUG start codons, and produce few unique tryptic peptides, unannotated small proteins cannot be detected with standard proteomic methods. Here,, we outline a procedure for mass spectrometry‐based detection of translated smORFs in cultured human cells from protein extraction, digestion, and LC‐MS/MS, to database preparation and data analysis. Following proteomic detection, translation from a unique smORF may be validated via siRNA‐based silencing or overexpression and epitope tagging. This is necessary to unambiguously assign a peptide to a smORF within a specific transcript isoform or genomic locus. Provided that sufficient starting material is available, this workflow can be applied to any cell type/organism and adjusted to study specific (patho)physiological contexts including, but not limited to, development, stress, and disease. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Protein extraction, size selection, and trypsin digestion Alternate Protocol 1: In‐solution C8 column size selection Support Protocol 1: Chloroform/methanol precipitation Support Protocol 2: Reduction, alkylation, and in‐solution protease digestion Support Protocol 3: Peptide de‐salting Basic Protocol 2: Two‐dimensional LC‐MS/MS with ERLIC fractionation Basic Protocol 3: Transcriptomic database construction Alternate Protocol 2: Transcriptomics database generation with gffread Basic Protocol 4: Non‐annotated peptide identification from LC‐MS/MS data Basic Protocol 5: Validation using isotopically labeled synthetic peptide standards and siRNA Basic Protocol 6: Transcript validation using transient overexpression

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“…However, not all heat shock proteins have been fully characterized, especially lowabundance, small, membrane-associated polypeptides. A recently developed approach for quantitative membrane proteomics identified a tryptic peptide apparently belonging to a previously unknown small heat shock protein named GndA (128). The identified tryptic peptide mapped to a short ORF located entirely within the gnd gene encoding 6-phosphogluconate dehydrogenase, but in the -1 frame (128).…”

Section: A Heat Shock Protein Is Encoded In An Alternative Reading Frmentioning

confidence: 99%

“…A recently developed approach for quantitative membrane proteomics identified a tryptic peptide apparently belonging to a previously unknown small heat shock protein named GndA (128). The identified tryptic peptide mapped to a short ORF located entirely within the gnd gene encoding 6-phosphogluconate dehydrogenase, but in the -1 frame (128). At what codon the translation of the small gndA ORF is initiated is unknown because two in-frame ATG codons are found upstream of the sequence encoding the mapped peptide (Table 1).…”

Section: A Heat Shock Protein Is Encoded In An Alternative Reading Frmentioning

confidence: 99%

“…This approach, however, has its intrinsic limitations because neither very short nor very long peptides can be easily detected by mass spectrometry and, in addition, low-abundance proteins (a common scenario for many of the internally initiated polypeptides) evade detection. A recently developed methodology combines bottom-up mass spectrometry with analysis of hypothetical polypeptides encoded in all six frames in the genome of interest (128). Applying this approach to membrane proteins helped to identify the novel OOF internal ORF gndA within the gnd gene of E. coli (128) that we described earlier.…”

Section: Figurementioning

confidence: 99%

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Genes within Genes in Bacterial Genomes

2018

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Genetic coding in bacteria largely operates via the "one gene-one protein" paradigm. However, the peculiarities of the mRNA structure, the versatility of the genetic code, and the dynamic nature of translation sometimes allow organisms to deviate from the standard rules of protein encoding. Bacteria can use several unorthodox modes of translation to express more than one protein from a single mRNA cistron. One such alternative path is the use of additional translation initiation sites within the gene. Proteins whose translation is initiated at different start sites within the same reading frame will differ in their N termini but will have identical C-terminal segments. On the other hand, alternative initiation of translation in a register different from the frame dictated by the primary start codon will yield a protein whose sequence is entirely different from the one encoded in the main frame. The use of internal mRNA codons as translation start sites is controlled by the nucleotide sequence and the mRNA folding. The proteins of the alternative proteome generated via the "genes-within-genes" strategy may carry important functions. In this review, we summarize the currently known examples of bacterial genes encoding more than one protein due to the utilization of additional translation start sites and discuss the known or proposed functions of the alternative polypeptides in relation to the main protein product of the gene. We also discuss recent proteome- and genome-wide approaches that will allow the discovery of novel translation initiation sites in a systematic fashion.

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Comparative Membrane Proteomics Reveals a Nonannotated E. coli Heat Shock Protein

Cited by 29 publications

References 25 publications

Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

Proteomic Detection and Validation of Translated Small Open Reading Frames

Genes within Genes in Bacterial Genomes

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