The effects of p53 gene inactivation on mutant proteome expression in a human melanoma cell model.

BACKGROUND: The identification of mutated proteins in human cancer cells-termed proteogenomics, requires several technologically independent research methodologies including DNA variant identification, RNA sequencing, and mass spectrometry. Any one of these methodologies are not optimized for identifying potential mutated proteins and any one output fails to cover completely a specific landscape. METHODS: An isogenic melanoma cell with a p53-null genotype was created by CRISPR/CAS9 system to determine how p53 gene inactivation affects mutant proteome expression. A mutant peptide reference database was developed by comparing two distinct DNA and RNA variant detection platforms using these isogenic cells. Chemically fractionated tryptic peptides from lysates were processed using a TripleTOF 5600+ mass spectrometer and their spectra were identified against this mutant reference database. RESULTS: Approximately 190 mutated peptides were enriched in wt-p53 cells, 187 mutant peptides were enriched in p53-null cells, with an overlap of 147 mutated peptides. STRING analysis highlighted that the wt-p53 cell line was enriched for mutant protein pathways such as CDC5L and POLR1B, whilst the p53-null cell line was enriched for mutated proteins comprising EGF/YES, Ubiquitination, and RPL26/5 nodes. CONCLUSION: Our study produces a well annotated p53-dependent and p53-independent mutant proteome of a common melanoma cell line model. Coupled to the application of an integrated DNA and RNA variant detection platform (CLCbio) and software for identification of proteins (ProteinPilot), this pipeline can be used to detect high confident mutant proteins in cells. GENERAL SIGNIFICANCE: This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.

Resolvin D1 attenuates lipopolysaccharide induced acute lung injury through CXCL-12/CXCR4 pathway.

BACKGROUND: The recruitment of neutrophils plays an important role in the progress of acute lung injury (ALI). Excessive neutrophils released from bone marrow accumulate in lung, release proinflammatory factors, and cause tissue damage. CXCL-12/CXCR4 is an important signaling pathway, which regulates the migration of bone marrow hematopoietic cells out of bone marrow and involves in neutrophil accumulation and retention in the inflammatory site. Resolvin D1 (RvD1) is a kind of lipid mediators, which can alleviate many inflammatory diseases. We hypothesized that RvD1 can alleviate lipopolysaccharide (LPS)-induced ALI through regulating CXCL-12/CXCR4 pathway. METHODS: We randomized mice into five groups: control group, RvD1 group, LPS group, LPS plus RvD1 group, and LPS plus AMD3100 group. ALI was established by intratracheal instillation of LPS. After 24 and 72 h, mice were sacrificed, and lung tissues were harvested for histologic analysis, wet-to-dry ratio, myeloperoxidase activity, and CXCL-12 expression. Bronchoalveolar fluid was collected for protein analysis, cytokines assay, and flow cytometry analysis. RESULTS: Histologic findings as well as wet-to-dry ratio, protein concentration, cytokines assay, neutrophil number, and myeloperoxidase activity confirmed that RvD1 and AMD3100 alleviated LPS-induced ALI. RvD1 decreased CXCL-12 messenger RNA expression in lung. However, RvD1 promoted CXCR4 expression in neutrophils in the initial stage of inflammation and reduced its level in the later stage. CONCLUSIONS: RvD1 protects LPS-induced ALI partially through regulating CXCL-12/CXCR4 pathway.