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Enrichment of Newly Synthesized Proteins following treatment of C2C12 Myotubes with Endotoxin and Interferon-γ

Abstract

Inflammation in muscle induces the synthesis of mediators that can impair protein synthesis and enhance proteolysis, and when sustained lead to muscle atrophy. Furthermore, muscle-derived mediators that are secreted may participate in disrupting the function of other peripheral organs. Selective identification of newly synthesized proteins can provide insight on biological processes that depend on the continued synthesis of specific proteins to maintain homeostasis as well as those proteins that are up- or down-regulated in response to inflammation. We used puromycin-associated nascent chain proteomics (PUNCH-P) to characterize new protein synthesis in C2C12 myotubes and changes resulting from their exposure to the inflammatory mediators lipopolysaccharide (LPS) and interferon (IFN)-γ for either a short (4 h) or prolonged (16 h) time period. We identified sequences of nascent polypeptide chains belonging to a total of 1523 proteins and report their detection from three independent samples of each condition at each time point. The identified nascent proteins correspond to approximately 15% of presently known proteins in C2C12 myotubes and are enriched in specific cellular components and pathways. A subset of these proteins was identified only in treated samples and has functional characteristics consistent with the synthesis of specific new proteins in response to LPS/IFNγ. Thus, the identification of proteins from their nascent polypeptide chains provides a resource to analyze the role of new synthesis of proteins in both protein homeostasis and in proteome responses to stimuli in C2C12 myotubes. Our results reveal a profile of actively translating proteins for specific cellular components and biological processes in normal C2C12 myotubes and a different enrichment of proteins in response to LPS/IFNγ. Collectively, our data disclose a highly interconnected network that integrates the regulation of cellular proteostasis and reveal a diverse immune response to inflammation in muscle which may underlie the concomitantly observed atrophy and be important in inter-organ communication.

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Availability of Data and Materials

The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomexchange.org) via the PRIDE partner repository with the dataset identifier < PXD003144 > . The following files are available free of charge via the Internet at http://pubs.acs.org. The data used to support the finding of this study are available from the corresponding author upon request.

Abbreviations

PUNCH-P:

Puromycin-associated nascent chain proteomics, LC–MS/MS, liquid chromatography-tandem mass spectrometry

FDR:

False discovery rate

LPS:

Lipopolysaccharide

IFNγ:

Interferon-gamma

NO:

Nitric oxide

IL-6:

Interleukin-6

TFA:

Trifluoroacetic acid

FA:

Formic acid

TCA:

Tricarboxylic acid

HRP:

Horseradish peroxidase

ECL:

Enhanced chemiluminescence

GO:

Gene Ontology

DAVID:

Database for Annotation, Visualization and Integrated Discovery

CC:

Cellular Component

BP:

Biological Process

%NSP:

Percent newly synthesizing proteins

EF:

Enrichment factor

Csf1:

Macrophage colony-stimulating factor 1

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Acknowledgements

We would like to thank Vincent Chau for helpful discussions and suggestions, and Anne Stanley of the Penn State College of Medicine Mass Spectrometry and Proteomics Facility (RRID:SCR_017831).

Funding

This research was supported by a grant from the National Institutes of Health, R37 AA11290.

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Authors

Contributions

Dr. Coleman contributed to the manuscript as follows: conceptualization of project, data curation and formal analysis, investigation, methodology, software, validation, visualization, and writing portions of the original draft, and reviewing and editing both the original and revised manuscript. Dr. Stanley contributed to the manuscript as follows: conceptualization of project, data curation and formal analysis, investigation, methodology, software, resources, validation, and writing portions of the original draft, and reviewing and editing both the original and revised manuscript. Dr. Lang contributed to the manuscript as follows: conceptualization of project, funding acquisition, investigation, methodology, project administration, resources, visualization, and writing portions of the original draft, and reviewing and editing both the original and revised manuscript.

Corresponding author

Correspondence to Charles H. Lang.

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Supplementary Information

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10753_2022_1622_MOESM1_ESM.xlsx

Supplementary file1 (XLSX 125 KB) Compilation of all proteins identified in PUNCH-P analysis. A list of 1523 proteins identified by their nascent polypeptide chains was obtained by merging proteins from untreated, 4 h and 16 h treatments (Supplementary Tables S2, S3 and S4, respectively). References to specific protein isoforms have been removed from protein names and UniprotKB accession and gene symbol corresponding to the “canonical” sequence of proteins have been added to each entry. Proteins in clusters are assigned with multiple names with corresponding accession entries and gene symbols. The Total number of peptides refers to the number of peptides found for each identified protein while Distinct peptides (shown in parenthesis), refer to peptides that are not shared by other proteins or by proteins not in the same cluster. Sample frequency reports the number of replicate samples in which each protein was observed. Entries for peptides and sample frequency from Untreated, 4 h- and 16 h-treated myotube samples are separated by semicolons in the respective order. The 424 proteins and clusters found only in myotubes treated with LPS/IFNγ are listed first, otherwise, the proteins are listed in alphabetical order based on their name.

10753_2022_1622_MOESM2_ESM.xlsx

Supplementary file2 (XLSX 197 KB ) Compilation of all proteins identified in PUNCH-P analysis in untreated C2C12 myotubes.

10753_2022_1622_MOESM3_ESM.xlsx

Supplementary file3 (XLSX 178 KB) Compilation of all proteins identified in PUNCH-P analysis in C2C12 myotubes identified after LPS/IFNγ treatment for 4 h.

10753_2022_1622_MOESM4_ESM.xlsx

Supplementary file4 (XLSX 210 KB) Compilation of all proteins identified in PUNCH-P analysis in C2C12 myotubes identified after LPS/IFNγ treatment for 16 h.

10753_2022_1622_MOESM5_ESM.xlsx

Supplementary file5 (XLSX 92 KB) Table listing candidate proteins induced by LPS/IFNγ. A filtered list of 132 proteins was generated after clustering of the 424 proteins found only under LPS/IFNγ treatment conditions (Input-1 from Table 4) to the following Biological Process GO terms: response to interferon-gamma (GO: 0034341); response to stress (GO:0006950); defense response (GO:0006952); innate immune response (GO:0045087); defense response to bacterium (GO:0042742); adhesion of symbiont to host (GO:0044406); defense response to other organism (GO: 0098542); response to cytokine (GO:0034097); cellular response to interferon-beta (GO:0035458); regulation of leukocyte mediated cytotoxicity (GO:0001910); immune system process (GO: 0002376). The additional 57 proteins listed were identified by analysis using Input-4 from Table 4 as described in the Results.

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Coleman, C.S., Stanley, B.A. & Lang, C.H. Enrichment of Newly Synthesized Proteins following treatment of C2C12 Myotubes with Endotoxin and Interferon-γ. Inflammation 45, 1313–1331 (2022). https://doi.org/10.1007/s10753-022-01622-3

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  • DOI: https://doi.org/10.1007/s10753-022-01622-3

KEY WORDS

  • Muscle
  • Proteome
  • LPS
  • Inflammation
  • Immune response