Biology subjects

Robinson, A. E.

Publications and source records attributed to Robinson, A. E..

4 recordsLinked to original sources

bioRxiv2025

Quantitative Substrate Kinetics Screening: One-Pot LC/MS Based Approach to Map Protease Specificity

Proteases play critical roles in many biological processes and diseases. Proteases tend to have limited and overlapping specificities, which hinders our understanding of their roles in physiology. We designed a naive protease substrate library with the aim of fully utilizing modern mass spectrometry proteomics tools to quantitatively map protease substrate specificity. A library of approximately 200,000 peptides was efficiently produced in E. coli with sufficient library diversity to identify cleavage efficiency of thousands of substrates for most proteases in a single assay. Each experiment results in ample substrate kinetics data to build a positional specificity matrix that quantitatively maps protease specificity. We apply this method to two different proteases, GluC commonly used as a proteomics tool, and Fibroblast Activation Protein Alpha that is overexpressed in activated fibroblasts of epithelial cancers and fibrotic diseases. This research quantitatively maps substrate specificity for GluC and FAP in a single assay and can be applied to map substrate specificity for most proteases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/680748v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1496d11org.highwire.dtl.DTLVardef@1979fc1org.highwire.dtl.DTLVardef@17967e8org.highwire.dtl.DTLVardef@194b741_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Protective role of the Atg8 1 homologue Gabarapl1 in regulating cardiomyocyte glycophagy in diabetic heart disease

Diabetic heart disease is highly prevalent and characterized by diastolic dysfunction. The mechanisms of diabetic heart disease are poorly understood and no targeted therapies are available. Here we show that the diabetic myocardium (type 1 and type 2) is characterized by marked glycogen elevation and ectopic cellular localization - a paradoxical metabolic pathology given suppressed cardiomyocyte glucose uptake in diabetes. We demonstrate involvement of a glycogen-selective autophagy pathway ( glycophagy) defect in mediating this pathology. Genetically manipulated deficiency of Gabarapl1, an Atg8 autophagy homologue, induces cardiac glycogen accumulation and diastolic dysfunction. Stbd1, the Gabarapl1 cognate autophagosome partner is identified as a unique component of the early glycoproteome response to hyperglycemia in cardiac, but not skeletal muscle. Cardiac-targeted in vivo Gabarapl1 gene delivery normalizes glycogen levels, diastolic function and cardiomyocyte mechanics. These findings reveal that cardiac glycophagy is a key metabolic homeostatic process perturbed in diabetes that can be remediated by Gabarapl1 intervention.

molecular biology↗

bioRxiv2020

A Simultaneous Methylome and Proteome Quantitative Workflow for Complex Samples

Proteoforms containing post-translational modifications (PTMs) represent a degree of functional diversity only harnessed through analytically precise simultaneous quantification of multiple PTMs. Here we present a method to accurately differentiate an unmodified peptide from its PTM-containing counterpart through data-independent acquisition-mass spectrometry, leveraging small precursor mass windows to physically separate modified peptidoforms from each other during MS2 acquisition. We utilize a lysine and arginine PTM-enriched peptide assay library and site localization algorithm to simultaneously localize and quantify seven PTMs including mono-, di-, and tri-methylation, acetylation, and succinylation in addition to total protein quantification in a single MS run without the need to enrich experimental samples. To evaluate biological relevance, this method was applied to liver lysate from differentially methylated non-alcoholic steatohepatitis (NASH) mouse models. We report altered methylation and acetylation together with total protein changes drive the novel hypothesis of a regulatory function of PTMs in protein synthesis and mRNA stability in NASH. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

systems biology↗

bioRxiv2017

A kinetically-driven free exchange mechanism of EmrE antiport sacrifices coupling efficiency in favor of promiscuity

EmrE is a small multidrug resistance transporter found in E. coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: Not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a new kinetically-driven free exchange model for EmrE antiport that is consistent with these results and recapitulates {Delta}pH-driven concentrative drug uptake. Our results suggest that EmrE sacrifices coupling efficiency for initial transport speed and multidrug specificity.\n\nSIGNIFICANCEEmrE facilitates E. coli multidrug resistance by coupling drug efflux to proton import. This antiport mechanism has been thought to occur via a pure exchange model which achieves coupled antiport by restricting when the single binding pocket can alternate access between opposite sides of the membrane. We test this model using NMR titrations and transport assays and find it cannot account for EmrE antiport activity. We propose a new kinetically-driven free exchange model of antiport with fewer restrictions that better accounts for the highly promiscuous nature of EmrE drug efflux. This model expands our understanding of coupled antiport and has implications for transporter design and drug development.

biophysics↗