Biology subjects

Minard, A. Y.

Publications and source records attributed to Minard, A. Y..

5 recordsLinked to original sources

bioRxiv2025

K48-ubiquitin-activated proteases cut-up post-ER proteins

Polyubiquitin chains, linked via K48 or K63 of ubiquitin, target membrane proteins in the secretory system to degradative pathways. However its unclear whether these linkage isomers are functionally interchangeable. Here we show that for post-endoplasmic reticulum (ER) proteins, K63-linked polyubiquitination induces multivesicular bodies (MVBs) sorting and lysosomal degradation. In contrast, K48-linked polyubiquitination induces shearing from the membrane. Substrates are cleaved by the proteasome and by two ubiquitin-activated proteases: Ddi1, a conserved cytosolic ubiquilin that generates cytosolic fragments, and Rbd2, an intramembrane rhomboid protease that produces lumenal fragments. Rbd2 localizes to Golgi/endosomes but also acts on ubiquitinated substrates at the vacuolar membrane. Ddi1s catalytic core, the HDD-RVP domain is sufficient for ubiquitin-dependent proteolysis. It binds ubiquitin directly and its activity is amplified by auxiliary ubiquitin binding domains: an atypical UBL domain and a UBA domain. These findings demonstrate that polyubiquitin chains linked by different residues encode distinct degradative fates for post-ER proteins, and reveals two proteases that target ubiquitinated integral membrane cargo.

cell biology↗

bioRxiv2024

Insulated piggyBac and FRT vectors for engineering transgenic homozygous and heterozygous eHAP cells

Transgene expression in eHAP cells, a haploid cell line popularly used to generate gene knockouts, is difficult owing to its low transfection efficiency and propensity for silencing integrated transgenes. To simplify transgene expression, we engineered insulated integrating plasmids that sustain high levels of transgene expression in eHAP cells, and that can be used in other cell lines. These vectors are compatible with FLP-FRT and piggyBac integration, they flank a gene-of interest bilaterally with tandem cHS4 core insulators, and co-express nuclear-localized blue fluorescent protein for identification of high expressing cells. We further demonstrate that transgenic haploid eHAP cells can be fused to form transgenic heterozygous diploid cells. This method creates diploid cells carrying the transgenic material of the haploid progenitors and could also be used to create heterozygous cells of defined genotypes. These tools expand the repertoire of experiments that can be performed in eHAP cells and other cultured cells.

cell biology↗

bioRxiv2023

Beta-subunit-eliminated eHAP expression (BeHAPe) cells reveal new properties of the cardiac voltage-gated sodium channel

Voltage-gated sodium (NaV) channels drive the upstroke of the action potential and are comprised of a pore-forming -subunit and regulatory {beta}-subunits. The {beta}-subunits modulate the gating, trafficking, and pharmacology of the -subunit. These functions are routinely assessed by ectopic expression in heterologous cells. However, currently available expression systems may not capture the full range of these effects since they contain endogenous {beta}-subunits. To better reveal {beta}-subunit functions, we engineered a human cell line devoid of endogenous NaV {beta}-subunits and their immediate phylogenetic relatives. This new cell line, {beta}-subunit-eliminated eHAP expression cells (BeHAPe), were derived from haploid eHAP cells by engineering inactivating mutations in SCN1B, SCN2B, SCN3B, SCN4B, MPZL1, MPZL2, MPZL3, MPZ and JAML. In diploid BeHAPe cells, the cardiac NaV -subunit, NaV1.5, was highly sensitive to {beta}-subunit modulation and revealed that each {beta}-subunit and even MPZ imparted unique gating properties. Furthermore, combining {beta}1 and {beta}2 with NaV1.5 generated a sodium channel with hybrid properties, distinct from the effects of the individual subunits. Thus, this approach revealed an expanded ability of {beta}-subunits to regulate NaV1.5 activity and can be used to improve the characterization of other /{beta} NaV complexes.

biophysics↗

bioRxiv2021

ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals.

Ubiquitin (Ub) serves as a signal for clathrin-mediated endocytosis (CME) by engaging Ub-binding proteins with the internalization apparatus. Ub is a versatile internalization signal because it can be added to a wide variety of membrane proteins, expanding the capacity of cells to use a variety of regulatory mechanisms to specify the conditions under which a particular protein will be internalized. Several candidate adaptors that can recognize ubiquitinated membrane proteins have been identified that work in endocytic processes that are both clathrin-dependent and independent. These include Epsin and Eps15, which bind and help sort Ub-cargo into internalization sites. Here we identify additional components of the endocytosis apparatus that bind Ub. The N-terminal ANTH domains found in CALM, AP180, HIP1R and yeast Sla2 all bind monoubiquitin with {micro}M affinity. ANTH domains belong to a larger superfamily of domains including ENTH and VHS domains, many of which have Ub-binding regions outside of their VHS/ENTH/ANTH domains that enable them to mediate Ub-dependent sorting events throughout the cell. Solution NMR studies combined with a crystal structure of the CALM ANTH domain in a complex with Ub show that Ub binds to a C-terminal region of the ANTH domain that is not present in ENTH domains. Combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the internalization apparatus of yeast caused defects in the Ub-dependent internalization of the GPCR Ste2 but had no effect on internalization of Ste2 via other internalization signals. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

cell biology↗

bioRxiv2019

Genome-wide analysis of diet and gene interactions in Drosophila uncovers the glucose transporter, CG4607, as a diet-responsive gene.

Genetic and environmental factors play a major role in metabolic health. However, they do not act in isolation, as a change in an environmental factor such as diet may exert different effects based on an individuals genotype. Here, we sought to understand how such gene-diet interactions influenced nutrient storage and utilisation, a major determinant of metabolic disease. We subjected the Drosophila Genetic Reference Panel (DGRP), comprising 200 genetically divergent inbred fly strains, to diets varying in sugar, fat and protein. We assessed starvation resistance, a holistic phenotype of nutrient storage and utilisation that can be robustly measured. Diet influenced the starvation resistance of each strain, but this effect varied markedly between strains. This demonstrates that genetics plays a major role in the response to diet. Furthermore, heritability analysis revealed that the greatest variability arose from diets either high in sugar or high in protein. To uncover the genetic underpinnings of this variation, we mapped 1,239 diet-responsive SNPs in 534 genes, 325 of which have human orthologues. Using whole-body knockdown, we confirmed that 30 candidate genes were required for glucose tolerance, storage and utilization. In particular, we characterised CG4607, a GLUT6/GLUT8 homolog, as a key protein involved in sugar tolerance. Overall, this provides strong evidence that genetics is a major contributor to how individuals respond to diets of varying nutrient composition. It is likely that a similar principle may be applied to metabolic disease in higher organisms thus supporting the case for nutrigenomics as an important health strategy.

genetics↗