Budhraja, Rohit

Congenital disorders of glycosylation (CDG) and mitochondrial disorders are multisystem disorders with overlapping symptomatology. Pathogenic variants in the PMM2 gene lead to abnormal N-linked glycosylation. This disruption in glycosylation can induce endoplasmic reticulum stress, contributing to the disease pathology. Although impaired mitochondrial dysfunction has been reported in some CDG, cellular bioenergetics has never been evaluated in detail in PMM2-CDG. This prompted us to evaluate mitochondrial function and autophagy/mitophagy in vitro in PMM2 patient-derived fibroblast lines of differing genotypes from our natural history study. We found secondary mitochondrial dysfunction in PMM2-CDG. This dysfunction was evidenced by decreased mitochondrial maximal and ATP-linked respiration, as well as decreased complex I function of the mitochondrial electron transport chain. Our study also revealed altered autophagy in PMM2-CDG patient-derived fibroblast lines. This was marked by an increased abundance of the autophagosome marker LC3-II. Additionally, changes in the abundance and glycosylation of proteins in the autophagy and mitophagy pathways further indicated dysregulation of these cellular processes. Interestingly, serum sorbitol levels (a biomarker of disease severity) and the CDG severity score showed an inverse correlation with the abundance of the autophagosome marker LC3-II. This suggests that autophagy may act as a modulator of biochemical and clinical markers of disease severity in PMM2-CDG. Overall, our research sheds light on the complex interplay between glycosylation, mitochondrial function, and autophagy/mitophagy in PMM2-CDG. Manipulating mitochondrial dysfunction and alterations in autophagy/mitophagy pathways could offer therapeutic benefits when combined with existing treatments for PMM2-CDG.

Abstract A quadrupole time‐of‐flight mass spectrometer coupled with a trapped ion mobility spectrometry (timsTOF) operated in parallel accumulation‐serial fragmentation (PASEF) mode has recently emerged as a platform capable of providing four‐dimensional (4D) features comprising of elution time, collision cross section (CCS), mass‐to‐charge ratio, and intensity of peptides. The PASEF mode provides ∼100% ion sampling efficiency both in data‐dependent acquisition (DDA) and data‐independent acquisition (DIA) modes without sacrificing sensitivity. In addition, targeted measurements using PASEF integrated parallel reaction monitoring (PRM) mode have also been described. However, only limited number of studies have used timsTOF for analysis of clinical samples. Although Orbitrap mass spectrometers have been used for biomarker discovery from cerebrospinal fluid (CSF) in a variety of neurological diseases, these Orbitrap‐derived datasets cannot readily be applied for driving experiments on timsTOF mass spectrometers. We generated a catalog of peptides and proteins in human CSF in DDA mode on a timsTOF mass spectrometer and used these data to build a spectral library. This strategy allowed us to use elution times and ion mobility values from the spectral library to design PRM experiments for quantifying previously discovered biomarkers from CSF samples in Alzheimer's disease. When the same samples were analyzed using a DIA approach combined with a spectral library search, a higher number of proteins were identified than in a library‐free approach. Overall, we have established a spectral library of CSF as a resource and demonstrated its utility for PRM and DIA studies, which should facilitate studies of neurological disorders.

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.