Biology subjects

Lomenick, B.

Publications and source records attributed to Lomenick, B..

6 recordsLinked to original sources

bioRxiv2023

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics

Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD to date. Despite its discovery twenty years ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change. Significance StatementFatty liver disease affects nearly a quarter of the worlds population and has both environmental and genetic risk factors. A mutation in the gene PNPLA3 that converts Ile 148 to Met is the strongest known genetic risk factor for developing fatty liver disease. Using a series of techniques to track endogenous PNPLA3 and PNPLA3-I148M biogenesis and localization, we reveal new insights into how the mutation changes cellular dynamics. Although previous reports focus on its role on lipid droplets, we reveal that PNPLA3-I148M also functions at the Golgi apparatus, an organelle critical for protein transport into and out of the cell and lipid signaling. PNPLA3-I148M causes altered Golgi morphology and drives changes reminiscent of liver disease.

cell biology↗

bioRxiv2023

Functional glycoproteomics by integrated network assembly and partitioning

The post-translational modification (PTM) of proteins by O-linked {beta}-N-acetyl-D-glucosamine (O-GlcNAcylation) is widespread across the proteome during the lifespan of all multicellular organisms. However, nearly all functional studies have focused on individual protein modifications, overlooking the multitude of simultaneous O-GlcNAcylation events that work together to coordinate cellular activities. Here, we describe Networking of Interactors and SubstratEs (NISE), a novel, systems-level approach to rapidly and comprehensively monitor O-GlcNAcylation across the proteome. Our method integrates affinity purification-mass spectrometry (AP-MS) and site-specific chemoproteomic technologies with network generation and unsupervised partitioning to connect potential upstream regulators with downstream targets of O-GlcNAcylation. The resulting network provides a data-rich framework that reveals both conserved activities of O-GlcNAcylation such as epigenetic regulation as well as tissue-specific functions like synaptic morphology. Beyond O-GlcNAc, this holistic and unbiased systems-level approach provides a broadly applicable framework to study PTMs and discover their diverse roles in specific cell types and biological states.

cell biology↗

bioRxiv2023

Staphylococcal secreted cytotoxins are competition sensing signals for Pseudomonas aeruginosa

Coinfection with two notorious opportunistic pathogens, the Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus, dominates chronic pulmonary infections. While coinfection is associated with poor patient outcomes, the interspecies interactions responsible for such decline remain unknown. Here, we dissected molecular mechanisms of interspecies sensing between P. aeruginosa and S. aureus. We discovered that P. aeruginosa senses S. aureus secreted peptides and, counterintuitively, moves towards these toxins. P. aeruginosa tolerates such a strategy through "competition sensing", whereby it preempts imminent danger/competition by arming cells with type six secretion (T6S) and iron acquisition systems. Intriguingly, while T6S is predominantly described as weaponry targeting Gram-negative and eukaryotic cells, we find that T6S is essential for full P. aeruginosa competition with S. aureus, a previously undescribed role for T6S. Importantly, competition sensing was activated during coinfection of bronchial epithelia, including T6S islands targeting human cells. This study reveals critical insight into both interspecies competition and how antagonism may cause collateral damage to the host environment.

microbiology↗

bioRxiv2022

The metabolite alpha-ketobutyrate increases health and life spans by activating AMPK

Aging is a complex process that is directly related to human health and disease. The extraordinary finding that aging is malleable, as shown in model organisms whose life and health spans are extended by specific gene mutations or dietary or pharmacological perturbations 1-3, has offered enormous hope for our understanding and treatment of aging and related diseases. Although many molecules have been identified that can extend the lifespan of model organisms, few have been shown to alleviate age-related symptoms or illness in mammals 4. Here we show that supplementation with the endogenous metabolite -ketobutyrate (-KB) increases the lifespan of adult C. elegans. Using Gelfree DARTS-PROTOMAP, we identified microtubule-actin cross-linking factor (MACF1) that was protected against proteolysis in the presence of -KB. MACF1 belongs to the spectraplakin family of giant, evolutionarily conserved proteins with versatile functions 5, but their link to longevity regulation has not been explored. -KBs longevity effect in C. elegans is abrogated by loss-of-function mutation in vab-10, encoding the worm ortholog of mammalian MACF1 6. Like -KB treatment, vab-10 knockdown activates AMP-activated protein kinase (AMPK), and AMPK is required for -KB effects on longevity. The findings suggest a model in which -KB increases longevity by activating AMPK via VAB-10/MACF1 modulation. -KB also delays aging in mammals, increasing the lifespan of aged male mice and the healthspan of both male and female animals. Targeting of broadly expressed scaffolding proteins in connection to cellular energy homeostasis seems to be a clever way that nature has devised for metabolite signals to impinge upon multiple organ and tissue systems, which may have utility for controlling aging and related diseases.

physiology↗

bioRxiv2022

MTCH2 is a mitochondrial outer membrane protein insertase

In the mitochondrial outer membrane, tail-anchored (TA) proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPRi screens, we identify factors involved in mitochondrial TA biogenesis in human cells. We show that MTCH2, and its paralog MTCH1, are required for insertion of biophysically diverse mitochondrial TAs, but not outer membrane {beta}-barrel proteins. In a reconstituted system, purified MTCH2 is sufficient to mediate insertion into proteoliposomes. Functional and mutational studies reveal that MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for outer membrane protein biogenesis, controlling mislocalization of TAs into the endoplasmic reticulum and the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction. One-Sentence SummaryMTCH2 is both necessary and sufficient for insertion of diverse -helical proteins into the mitochondrial outer membrane, and is the defining member of a family of insertases that have co-opted the SLC25 transporter fold.

cell biology↗

bioRxiv2022

Pervasive SUMOylation of heterochromatin and piRNA pathway proteins

Genome regulation involves complex and highly regulated protein interactions that are often mediated through post-translational modifications (PTMs). SUMOylation - the covalent attachment of the small ubiquitin-like modifier (SUMO) - is a conserved PTM in eukaryotes that has been implicated in a number of essential processes such as nuclear import, DNA damage repair, transcriptional control, and chromatin organization. In Drosophila, SUMO is essential for viability and its depletion from the female germline causes infertility associated with global loss of heterochromatin, and illicit upregulation of transposons and lineage-inappropriate genes. However, the specific targets of SUMO and its mechanistic role in different cellular pathways are still poorly understood. Here, we developed a proteomics-based strategy to characterize the SUMOylated proteome in Drosophila that allowed us to identify ~1500 SUMO sites in 843 proteins in the fly ovary. A high-confidence set of SUMOylated proteins is highly enriched in factors involved in heterochromatin regulation and several different aspects of the piRNA pathway that represses transposons, including piRNA biogenesis and function. Furthermore, we show that SUMOylation of several piRNA pathway proteins occurs in a Piwi-dependent manner, indicating a functional implication of this modification in the cellular response to transposon activity. Together, these data highlight the impact of SUMOylation on epigenetic regulation and reveal an unexpectedly broad role of the SUMO pathway in the cellular defense against genomic parasites. Finally, this work provides a valuable resource and a system that can be adapted to the study of SUMOylation in other Drosophila tissues.

molecular biology↗