Biology subjects

Willoughby, M. M.

Publications and source records attributed to Willoughby, M. M..

3 recordsLinked to original sources

bioRxiv2025

The ESCRT-0 protein HRS regulates hepatocellular lipid droplet catabolism

Lipid droplets (LDs) are dynamic organelles that regulate lipid storage and metabolism pathways central to metabolic liver disease. LD turnover occurs in part through lysosomal catabolism (i.e. lipophagy) whereby LDs are thought to follow two distinct trafficking pathways: autophagosome-dependent macrolipophagy and the autophagosome-independent microlipophagy. However, the molecular machinery that regulates these two distinct pathways, especially that of microlipophagy in mammalian cells, is poorly understood. In yeast, microlipophagy has been shown to rely on a protein family known as the endosomal sorting complex required for transport (ESCRT). Here, we used an ESCRT-specific RNAi library in hepatocytes which identified the ESCRT-0 protein hepatocyte growth factor receptor substrate (HRS) as a critical regulator of LD homeostasis. HRS depletion leads to significant LD accumulation which is not due to increased LD formation but from impaired LD catabolism. HRS-deficient cells retain lipolysis activity; however, they exhibit decreased LD targeting via microlipophagy, accompanied by compensatory increases in autophagosome targeting to LDs. In agreement with these findings, HRS knockdown suppressed mTOR signaling, boosted autophagosome formation, and reduced the degradation of autophagic cargo. Despite maintaining lysosome numbers, HRS knockdown raised lysosomal pH causing decreased autophagic degradative capacity and contributing to LD accumulation. Overall, these findings identify HRS as a modulator of LD turnover in mammalian cells, regulating lipophagy through lysosomal function. Significance StatementO_LIThe regulatory molecular mechanisms of lipophagy are not clearly defined. This study identifies novel ESCRT proteins as regulators of LD homeostasis in several cell lines. C_LIO_LIIn hepatocytes, we identified HRS specifically regulates LD catabolism, whereby HRS-dependent regulation of LDs is dual-faceted, affecting LD-lysosomal targeting and lysosomal function. C_LIO_LIOur findings are significant because they provide mechanistic insights into the role of ESCRT proteins in LD metabolism. Elucidating ESCRT-mediated lipophagy can potentially aid in developing novel targets to prevent aberrant lipid trafficking and utilization, particularly in the liver where LDs can accumulate and cause irreversible liver damage. C_LI

biochemistry↗

bioRxiv2021

Mitochondrial Contact Site and Cristae Organizing System (MICOS) Machinery Supports Heme Biosynthesis by Enabling Optimal Performance of Ferrochelatase

Heme is an essential cofactor required for a plethora of cellular processes in eukaryotes. In metazoans the heme biosynthetic pathway is typically partitioned between the cytosol and mitochondria, with the first and final steps taking place in the mitochondrion. The pathway has been extensively studied, and all the biosynthetic enzymes have been structurally characterized to varying extents. Nevertheless, our understanding of the regulation of heme synthesis and factors that influence this process in metazoans remains incomplete. Herein we investigate the molecular organization as well as the catalytic and structural features of the terminal pathway enzyme, ferrochelatase (Hem15), in the yeast Saccharomyces cerevisiae. Biochemical and genetic analyses reveal dynamic association of Hem15 with Mic60, a core component of the mitochondrial contact site and cristae organizing system (MICOS). Loss of MICOS negatively impacts Hem15 activity and results in accumulation of highly reactive and potentially toxic tetrapyrrole precursors that may result in oxidative damage. Restoring intermembrane connectivity in MICOS-deficient cells mitigates these cytotoxic effects. Our data provide new insights into how heme biosynthetic machinery is organized and regulated, linking mitochondrial architecture-organizing factors to heme homeostasis.

biochemistry↗

bioRxiv2019

The heme biosynthetic enzyme, 5-aminolevulinic acid synthase (ALAS), and GTPases in control of mitochondrial dynamics and ER contact sites, regulate heme mobilization to the nucleus.

Heme is an iron-containing cofactor and signaling molecule that is essential for much of aerobic life. All heme-dependent processes in eukaryotes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins located throughout the cell. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. Herein, using genetically encoded fluorescent heme sensors, we developed a live cell assay to monitor heme distribution dynamics between the mitochondrial inner-membrane, where heme is synthesized, and the mitochondrial matrix, cytosol, and nucleus. We found that heme distribution occurs simultaneously via parallel pathways. In fact, surprisingly, we find that trafficking to the nucleus is [~]25% faster than to the cytosol or mitochondrial matrix. Moreover, we discovered that the heme biosynthetic enzyme, 5-aminolevulinic acid synthase (ALAS), and GTPases in control of the mitochondrial dynamics machinery, Mgm1 and Dnm1, and ER contact sites, Gem1, regulate the flow of heme between the mitochondria and nucleus. Altogether, our results indicate that the nucleus acquires heme faster than the cytosol or mitochondrial matrix, presumably for mitochondrial-nuclear retrograde signaling, and that GTPases that regulate mitochondrial dynamics and ER contact sites are hard-wired to cellular heme distribution systems.\n\nSummary StatementThe factors that govern the trafficking of heme, an essential but potentially cytotoxic cofactor and signaling molecule, are poorly understood. Herein, we developed a live-cell assay to monitor heme distribution kinetics and identified the first enzyme in the heme synthesis pathway and GTPases in control of mitochondrial-ER contact sites and dynamics as being critical modulators of heme trafficking.

cell biology↗