Biology subjects

Macedo-da-Silva, J.

Publications and source records attributed to Macedo-da-Silva, J..

3 recordsLinked to original sources

bioRxiv2021

Urine proteomics as a non-invasive approach to monitor exertional rhabdomyolysis during military training

Exertional rhabdomyolysis (ERM), a condition often associated with strenuous exercise, a common practice in the military activities, can be defined as the process of injury and rupture of muscle cell membranes, with leakage of its components into the blood stream. Creatine kinase (CK) has been extensively used for ERM diagnosis, albeit several studies reported the discrepancy between CK levels and clinical signs or symptoms. In this study, we analyzed the biochemical profile of the blood, and the urinary proteome of ten marine soldiers in a special training course. The samples were collected in two periods, M1 and M2, which correspond to the lowest and highest CK levels during training, respectively. Quantitative urinary proteome profile of M1 and M2 was determined showing changes with highest significance in immune system and cell adhesion-related pathways after strenuous physical exercise. Changes in the abundance of several proteins was observed in individuals carrying genetic polymorphisms related to greater risk for muscle damage. Remarkably, we identified a panel of proteins (CTSH, PIK3IP1, DEFB1, ITGB1, BCAN, and TNFRSF10C) that present high correlation with three classical blood biochemical markers of ERM and AGT MET235Thr and ACE I/D polymorphisms. These proteins represent potential urine markers of muscle damage due to intense physical conditions such as military training activities.

biochemistry↗

bioRxiv2021

Protein arginylation is regulated during SARS-CoV-2 infection

In 2019, the world witnessed the onset of an unprecedented pandemic. In September 2021, the infection by SARS-CoV-2 had already been responsible for the death of more than 4 million people worldwide. Recently, we and other groups discovered that SARS-CoV-2 infection induces ER-stress and activation of unfolded protein response (UPR) pathway. The degradation of misfolded/unfolded proteins is an essential element of proteostasis and occurs mainly in lysosomes or proteasomes. The N-terminal arginylation of proteins is characterized as an inducer of ubiquitination and proteasomal degradation by the N-end rule pathway. Here we present, for the first time, data on the role of arginylation during SARS-CoV-2 infection. We studied the modulation of protein arginylation in Vero CCL-81 and Calu-3 cells infected after 2h, 6h, 12h, 24h, and 48h. A reanalysis of in vivo and in vitro public omics data combined with immunoblotting was performed to measure the levels of ATE1 and arginylated proteins. This regulation is seen specifically during infections by coronaviruses. We demonstrate that during SARS-CoV-2 infection there is an increase in the expression of the ATE1 enzyme associated with regulated levels of specific arginylated proteins. On the other hand, infected macrophages showed no ATE1 regulation. An important finding revealed that modulation of the N-end rule pathway differs between different types of infected cells. We also confirmed the potential of tannic acid to reduce viral load, and furthermore, to modulate ATE1 levels during infection. In addition, the arginylation inhibitor merbromin (MER) is also capable of both reducing viral load and reducing ATE1 levels. Taken together, these data show the importance of arginylation during the progression of SARS-CoV-2 infection and open the door for future studies that may unravel the role of ATE1 and its inhibitors in pathogen infection.

cell biology↗

bioRxiv2021

Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay

A new method to probe the conformational changes of glycoproteins on a systems-wide scale, termed limited deglycosylation assay (LDA), is described. The method measures the differential rate of deglycosylation of N-glycans on natively folded proteins by the common peptide:N-glycosidase F (PNGase F) enzyme which in turn informs on their spatial presentation and solvent exposure on the protein surface hence ultimately the glycoprotein conformation. LDA involves 1) protein-level N-deglycosylation under native conditions, 2) trypsin digestion under denaturing conditions, 3) glycopeptide enrichment, 4) peptide-level N-deglycosylation and 5) quantitative MS-based analysis of the formerly N-glycosylated peptides. LDA was initially developed and the experimental conditions optimized using bovine RNase B and fetuin. The method was then applied to glycoprotein extracts from LLC-MK2 epithelial cells upon treatment with dithiothreitol to induce endoplasmic reticulum stress and promote protein misfolding. Data from the LDA and 3D structure analysis showed that glycoproteins predominantly undergo structural changes in loops/turns upon ER stress as exemplified with detailed analysis of ephrin-A5, GALNT10, PVR and BCAM. These results show that LDA accurately reports on systems-wide conformational changes of glycoproteins induced under controlled treatment regimes. Thus, LDA opens avenues to study glycoprotein structural changes in a range of other physiological and pathophysiological conditions relevant to acute and chronic diseases.

biochemistry↗