Limited Evidence for Protein Products of Noncoding Transcripts in the HEK293T Cellular Cytosol

Bogaert, Annelies; Fijałkowska, Daria; Staes, An; Steene, Tessa Van de; Demol, Hans; Gevaert, Kris

doi:10.1016/j.mcpro.2022.100264

Cited by 11 publications

(26 citation statements)

References 87 publications

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“…We evaluated how efficient N-terminal COFRADIC enriched for N-terminal peptides and found enrichments up to 60%, which is similar as previously reported [43] (see Supplementary Figure S1.C ). Similar as reported in [45], the enrichment efficiency for chymotrypsin-digested samples is lower.…”

Section: Resultssupporting

confidence: 88%

“…To study the interactome of N-terminal proteoforms, we first constructed a catalogue of Nt-proteoforms of cytosolic proteins in HEK293T cells as Virotrap currently only functions in these cells and favors cytosolic proteins as baits. Such a decrease in proteome sample complexity also increases the possibility of identifying Nt-proteoforms [45]. Both Western blot and Gene Ontology Cellular Component data analysis indicated that we strongly enriched for cytosolic proteins (see Supplementary Figure S1.A and B ).…”

Section: Resultsmentioning

confidence: 99%

“…During this merge, all information was retained (e.g. all listed accessions are retained in the isoform column) and, after merging, the workflow was followed as outlined in [45] to filter for N-terminal peptides stemming from translation events.…”

Section: Methodsmentioning

confidence: 99%

“…All experimental details on the sample preparation and LC-MS/MS analysis can be found in Supplementary Materials and Methods. From the set of identified peptides, N-terminal peptides were selected and classified as described in [45]. Different here is that data from two database searches are combined.…”

Section: Methodsmentioning

confidence: 99%

“…Further bioinformatics data curation and analysis, facilitating the selection of N-terminal proteoform pairs for interactome analysis, is summarized in Figure 2A. First, we applied stringent filtering on the identified peptides to retain N-terminal peptides originating from translation, as previously described [45] (see By this filtering strategy, we only retained confident N-terminal peptides pointing to databaseannotated protein starts or N-terminal proteoforms, and assigned a confidence level to these peptides. As a last step, Nt-peptides identified by different proteases were merged into a final dataset (see Figure 2) of 3,306 unique Nt-peptides (see Table 1 and Supplementary Table S2).…”

Section: Construction Of An N-terminal Proteoform Catalogue Of the He...mentioning

confidence: 99%

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N-terminal proteoforms may engage in different protein complexes

Bogaert

Fijałkowska

Staes

et al. 2023

Preprint

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Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared to their canonical counterparts. Such proteoforms can have altered localizations, stabilities and functions. While proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. Additionally, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues as well as tissue-specific expression, highlighting their biological relevance. Protein-protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared to their canonical counterpart, thus further expanding the functional diversity of proteomes.

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Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Construction Of An N-terminal Proteoform Catalogue Of the He...mentioning

confidence: 99%

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N-terminal proteoforms may engage in different protein complexes

Bogaert

Fijałkowska

Staes

et al. 2023

Preprint

Self Cite

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Microproteins—Discovery, structure, and function

Mohsen,

Martel,

Slavoff

2023

Proteomics

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Advances in proteogenomic technologies have revealed hundreds to thousands of translated small open reading frames (sORFs) that encode microproteins in genomes across evolutionary space. While many microproteins have now been shown to play critical roles in biology and human disease, a majority of recently identified microproteins have little or no experimental evidence regarding their functionality. Computational tools have some limitations for analysis of short, poorly conserved microprotein sequences, so additional approaches are needed to determine the role of each member of this recently discovered polypeptide class. A currently underexplored avenue in the study of microproteins is structure prediction and determination, which delivers a depth of functional information. In this review, we provide a brief overview of microprotein discovery methods, then examine examples of microprotein structures (and, conversely, intrinsic disorder) that have been experimentally determined using crystallography, cryo‐electron microscopy, and NMR, which provide insight into their molecular functions and mechanisms. Additionally, we discuss examples of predicted microprotein structures that have provided insight or context regarding their function. Analysis of microprotein structure at the angstrom level, and confirmation of predicted structures, therefore, has potential to identify translated microproteins that are of biological importance and to provide molecular mechanism for their in vivo roles.

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