Budhraja, Rohit

AbstractCongenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N‐glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N‐glycans from misfolded, retro‐translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated. We undertook a multiplexed tandem mass tags‐labeling‐based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1‐deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cell‐derived factor 1, collagen alpha‐1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin‐1. We further detected a total of 3255 N‐glycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N‐glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N‐glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient‐derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system‐level insights for understanding NGLY1‐CDDG.

AbstractPIK3CA is one of the most frequently mutated genes in human cancers, with the two most prevalent activating mutations being E545K and H1047R. Although the altered intracellular signaling pathways in these cells have been described, the effect of these mutations on their extracellular vesicles (EVs) has not yet been reported. To study altered cellular physiology and intercellular communication through proteomic analysis of EVs, MCF10A cells and their isogenic mutant versions (PIK3CA E545K and H1047R) were cultured and their EVs enriched by differential ultracentrifugation. Proteins were extracted, digested with trypsin and the peptides labeled with tandem mass tag (TMT) reagents and analyzed by liquid chromatography tandem mass spectrometry (LC‐MS/MS). Four thousand six hundred and fifty‐five peptides were identified from 579 proteins of which 522 proteins have been previously described in EVs. Relative quantitation revealed altered levels of EV proteins including several cell adhesion molecules. Mesothelin, E‐cadherin, and epithelial cell adhesion molecule were elevated in both mutant cell‐derived EVs. Markers of tumor invasion and progression like galectin‐3 and transforming growth factor beta induced protein were increased in both mutants. Overall, activating mutations in PIK3CA result in altered EV composition with characteristic changes associated with these hotspot mutations.