Biology subjects

Hayes, M. A.

Publications and source records attributed to Hayes, M. A..

4 recordsLinked to original sources

bioRxiv2025

Dielectrophoresis Reveals Stimulus-Induced Remodeling of Insulin Granule Subpopulations

The pancreatic {beta}-cell contains several functional subpopulations of insulin secretory granules (ISGs). These subpopulations vary in maturity, age, and secretory capacity. Differences in protein and lipid composition of ISGs are correlated with disease but require further study to understand how ISG remodeling regulates normal biology. Due to limitations in traditional separation methods, the extent of these subpopulations, any overlap between them, and how they are affected by insulinotropic signals have not been determined. In this work, we adapted direct current insulator-based dielectrophoresis (DC-iDEP) to separate ISGs isolated from INS-1E cells, an immortalized rat insulinoma cell line model, according to their electrokinetic mobility ratio (EKMr). We were able to separate ISG subpopulations from unstimulated cells to determine a baseline distribution and identify characteristic profiles for immature, young, and old ISGs. We then analyzed distributions of subpopulations from cells stimulated with insulin secretion signals known to induce biophysical remodeling and maturation. We found significant changes in each subpopulation studied in response to stimulation, consistent with the increases in maturation, crystallization, and changes in size reported in the literature. This work provides new insights into how the cell controls ISG remodeling and may drive future development of more effective therapies. SIGNIFICANCEUnderstanding insulin secretory granule (ISG) heterogeneity and the functional role of subpopulations is a critical step towards unraveling the mechanisms of insulin secretion. We adapted direct current insulator-based dielectrophoresis (DC-iDEP) to resolve immature, young, and old ISG subpopulations based on their biophysical properties. We also explored the biophysical remodeling of ISGs under insulinotropic stimuli to further probe how the cell controls remodeling and secretion. Our work provides a new framework for quantifying granule heterogeneity by linking biophysical features to functional subtypes and assessing how environmental stimuli remodel ISGs. This methodology establishes a broadly applicable platform to interrogate organelle and vesicle diversity in complex biological systems.

biophysics↗

bioRxiv2024

Delineation of the Complete Reaction Cycle of a Natural Diels-Alderase

The Diels-Alder reaction is one of the most effective methods for the synthesis of substituted cyclohexenes. The development of protein catalysts for this reaction remains a major priority, affording new sustainable routes to high value target molecules. Whilst a small number of natural enzymes have been shown capable of catalysing [4+2] cycloadditions, there is a need for significant mechanistic understanding of how these prospective Diels-Alderases promote catalysis to underpin their development as biocatalysts for use in synthesis. Here we present a molecular description of the complete reaction cycle of the bona fide natural Diels-Alderase AbyU, which catalyses formation of the spirotetronate skeleton of the antibiotic abyssomicin C. This description is derived from X-ray crystallographic studies of AbyU in complex with a non-transformable synthetic substrate analogue, together with transient kinetic analyses of the AbyU catalysed reaction and computational reaction simulations. These studies reveal the mechanistic intricacies of this enzyme system and establish a foundation for the informed reengineering of AbyU and related biocatalysts.

biochemistry↗

bioRxiv2024

Ancestral versus Modern Substrate Scope in Family-1 Glycosidases

Experimental studies support that protein engineering based on ancestral sequence reconstruction often leads to variants with biotechnologically useful biomolecular properties. These may include high stability, enhanced conformational flexibility and a modified catalysis range. Carbohydrate-active enzymes have numerous applications related with the degradation and synthesis of carbohydrates and glycoconjugates. Here, we explore how ancestral reconstruction may impact substrate scope in glycosidases, highly diverse enzymes that catalyze the hydrolysis of glycosidic bonds in all living cells and find applications as catalysts of the reverse reaction. To this end, we screen a library of [~]500 potential glycosidase substrates for degradation by both, a modern family-1 glycosidase from Halothermothrix orenii and a putative ancestral family-1 glycosidase derived from sequence reconstruction at a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for most substrates. But the ancestral glycosidase is more efficient with flavonoid glycosides bearing large aglycon moieties. Analysis of the catalytic parameters for a selected set of substrates, alongside analysis of the library data using a supervised learning algorithm, support the hypothesis that the modern enzyme tends to become less catalytically efficient with increasing substrate size, while this trend is not observed for the ancestral glycosidase. Molecular simulations support that the ancestral catalysis pattern is linked to the existence of a highly flexible region of the ancestral structure and a cavity capable of accommodating large aglycons. Our results provide guidelines for the engineering of enzymes for the synthesis and hydrolysis of large glycoconjugates.

biochemistry↗

bioRxiv2021

iDEP-assisted isolation of insulin secretory vesicles

Organelle heterogeneity and inter-organelle associations within a single cell contribute to the limited sensitivity of current organelle separation techniques, thus hindering organelle subpopulation characterization. Here we use direct current insulator-based dielectrophoresis (DC-iDEP) as an unbiased separation method and demonstrate its capability by identifying distinct distribution patterns of insulin vesicles from pancreatic {beta}-cells. A multiple voltage DC-iDEP strategy with increased range and sensitivity has been applied, and a differentiation factor (ratio of electrokinetic to dielectrophoretic mobility) has been used to characterize features of insulin vesicle distribution patterns. We observed a significant difference in the distribution pattern of insulin vesicles isolated from glucose-stimulated cells relative to unstimulated cells, in accordance with functional maturation of vesicles upon glucose stimulation, and interpret this to be indicative of high-resolution separation of vesicle subpopulation. DC-iDEP provides a path for future characterization of subtle biochemical differences of organelle subpopulations within any biological system.

biophysics↗