Biology subjects

Sargeant, J.

Publications and source records attributed to Sargeant, J..

3 recordsLinked to original sources

bioRxiv2025

Voxelotor (Oxbryta) Binds Multiple Hemoglobin Sites and Influences Protein Structure

Voxelotor (Oxbryta, GBT440) is a first-in-class drug, FDA-approved to treat sickle cell disease in 2019 but withdrawn from market in 2024. This drug acts as an allosteric modulator, designed to shift the equilibrium to the oxygenated R conformation. The drug was shown to both limit the accumulation of deoxygenated T-conformation sickle cell Hb fibers and increase Hb oxygen affinity. X-ray crystallography previously showed one-to-one Voxelotor binding stoichiometry for Hb, with the drug molecule bound to N-terminus of an alpha subunit. Here we use NMR spectroscopy to assess the structure of Voxelotor-bound hemoglobin in solution and mass spectrometry (MS) to determine stoichiometry and sites of binding. We find that the structure and stoichiometry of binding are far more heterogeneous than previously described. The addition of Voxelotor to R-conformation Hb induces an NMR signal found in the T-conformation of Hb. In addition, MS shows that the drug binds Hb at multiple sites, including the N-terminus of the beta subunit. The properties of Hb with Voxelotor bound at secondary sites have not been explored but should be considered at high doses. Heterogeneous binding should be assessed in other drugs of this class including GBT(021)601 currently in clinical trial.

biophysics↗

bioRxiv2020

STEADY-STATE REGULATION OF SECRETORY CARGO EXPORT BY INOSITOL TRISPHOSPHATE RECEPTORS AND PENTA EF HAND PROTEINS

Recently, we demonstrated that agonist-stimulated Ca2+ signaling involving IP3 receptors modulates ER export rates through activation of the penta-EF Hand (PEF) proteins apoptosis-linked gene-2 (ALG-2) and peflin. It is unknown, however, whether IP3Rs and PEF proteins regulate ER export rates at steady state. Here we tested this idea in normal rat kidney (NRK) epithelial cells by manipulation of IP3R isoform expression. Under standard growth conditions, spontaneous cytosolic Ca2+ oscillations occurred simultaneously in successive groups of contiguous cells, generating intercellular Ca2+ waves (ICWs) that moved across the monolayer periodically. Depletion of IP3R-3, typically the least promiscuous IP3R isoform, caused increased cell participation in ICWs in unstimulated cells. The increased spontaneous signaling was sufficient to cause increased ALG-2 and Sec31A, and decreased peflin localization at ER exit sites (ERES), resulting in increased ER-to-Golgi transport of the COPII client cargo VSV-G. The elevated ER-to-Golgi transport caused greater concentration of VSV-G at ERES and had reciprocal effects on transport of VSV-G and a bulk-flow cargo, though both cargos equally required Sec31A. Inactivation of client cargo sorting using 4-phenylbutyrate (4-PBA) had opposing reciprocal effects on client and bulk-flow cargo and neutralized any effect of ALG-2 activation on transport. This work extends our knowledge of ALG-2 mechanisms and indicates that in NRK cells, IP3R isoforms regulate homeostatic Ca2+ signaling that helps determine the basal secretion rate and stringency of COPII-dependent cargo sorting.

cell biology↗

bioRxiv2020

Calcium Sensors ALG-2 And Peflin Bind ER Exit Sites In Alternate States To Modulate Secretion In Response To Calcium Signaling

ER-to-Golgi transport is the first step in the constitutive secretory pathway which, unlike regulated secretion, is believed to proceed non-stop regardless of Ca2+ flux. Rowever, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca2+ signaling by adjusting the ER export rate of COPII-sorted cargos up or down by ~50%. At steady-state Ca2+, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. During Ca2+ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration-phenomena that could contribute to cellular growth and intercellular communication, following secretory increases, or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca2+-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca2+ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Within the NRK cell model, distinct Ca2+ signaling patterns can produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased COPTT outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPTT outer shell and decreased peflin at ERES. PEF protein complexes represent a true regulator of transport as they are dispensable for secretion yet adjust the secretion rate to physiological conditions. Their dynamics affects secretion of important physiological cargoes such as collagen T and significantly impacts ER stress.

cell biology↗