Biology subjects

Savitski, M.

Publications and source records attributed to Savitski, M..

4 recordsLinked to original sources

bioRxiv2023

Dextromethorphan inhibits collagen transport in the endoplasmic reticulum eliciting an anti-fibrotic response in ex-vivo and in vitro models of pulmonary fibrosis

Excessive deposition of fibrillar collagen in the interstitial extracellular matrix (ECM) of human lung tissue causes fibrosis, which can ultimately lead to organ failure. Despite our understanding of the molecular mechanisms underlying the disease, a cure for pulmonary fibrosis has not yet been found. In this study, we screened an FDA-approved drug library containing 712 drugs and found that Dextromethorphan (DXM), a cough expectorant, significantly reduces the amount of excess fibrillar collagen deposited in the ECM in in-vitro cultured primary human lung fibroblasts (NHLF) and ex-vivo cultured human precision-cut lung slice (hPCLS) models of lung fibrosis. Reduced extracellular fibrillar collagen levels in the ECM upon DXM treatment are due to a reversible trafficking inhibition of collagen type I (COL1) in the endoplasmic reticulum (ER) in TANGO1 and HSP47 positive structures. Mass spectrometric analysis shows that DXM causes hyper-hydroxylation of proline and lysine residues on Collagen (COL1, COL3, COL4, COL5, COL7, COL12) and Latent-transforming growth factor beta-binding protein (LTBP1 and LTBP2) peptides coinciding with their secretion block. In addition, thermal proteome profiling of cells treated with DXM shows increased thermal stability of prolyl-hydroxylases such as P3H2, P3H3, P3H4, P4HA1 and P4HA2, suggesting a change in activity. Transcriptome analysis of pro-fibrotic stimulated NHLFs and hPCLS upon DXM treatment showed activation of an anti-fibrotic program via regulation of pathways such as those involved in the MMP-ADAMTS axis, WNT, and fibroblast-to-myofibroblast differentiation. Taken together, the data obtained from both in-vitro and ex-vivo models of fibrogenesis show that Dextromethorphan has potent anti-fibrotic activity by efficient inhibition of COL1 membrane trafficking in the ER.

cell biology↗

bioRxiv2022

Transient glycolytic complexation of arsenate enhances resistance in the enteropathogen Vibrio cholerae

The ubiquitous presence of toxic arsenate (AsV) in the environment has virtually raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that V. cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress- related systems to counter ROS production caused by intracellular AsV. Characterization of the var mutants suggested these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions.

microbiology↗

bioRxiv2022

Deep thermal proteome profiling for detection of proteoforms and drug sensitivity biomarkers

The complexity of the functional proteome extends significantly beyond the protein coding genome resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases, but challenging to perform systematically. Here, we apply thermal proteome profiling with deep peptide coverage to detect functional proteoforms in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detect 15,846 proteoforms, capturing differently spliced, post-translationally modified, and cleaved proteins expressed from 9,290 genes. We identify differential coaggregation of proteoform pairs and establish links to disease biology. Moreover, we systematically make use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach thus provides a powerful and unique tool for systematic detection and functional annotation of proteoforms.

biochemistry↗

bioRxiv2019

Systematic localization of Gram-negative bacterial membrane proteins

The molecular architecture and function of the Gram-negative bacterial cell envelope is dictated by protein composition and localization. Proteins that localize to the inner (IM) and outer (OM) membranes of Gram-negative bacteria play critical and distinct roles in cellular physiology, however, approaches to systematically interrogate their distribution across both membranes and the soluble cell fraction are lacking. We employed multiplexed quantitative mass spectrometry to assess membrane protein localization in a proteome-wide fashion by separating IM and OM vesicles from exponentially growing E. coli K-12 cells on a sucrose density gradient. The migration patterns for >1600 proteins were classified in an unbiased manner, accurately recapitulating decades of knowledge in membrane protein localization in E. coli. For 559 proteins that are currently annotated as peripherally associated to the IM (Orfanoudaki and Economou, 2014) and display potential for dual localization to either the IM or cytoplasm, we could allocate 110 to the IM and 206 as soluble based on their fractionation patterns. In addition, we uncovered 63 cases, in which our data disagreed with current localization annotation in protein databases. For 42 of them, we were able to find supportive evidence for our localization findings in literature. We anticipate our systems-level analysis of the E. coli membrane proteome will serve as a useful reference dataset to query membrane protein localization, as well as provide a novel methodology to rapidly and systematically map membrane protein localization in more poorly characterized Gram-negative species.

microbiology↗