Biology subjects

Mashek, D. G.

Publications and source records attributed to Mashek, D. G..

6 recordsLinked to original sources

bioRxiv2026

Cell-type resolved prenylomics in mouse liver enabled by a low-input proteomics workflow

Protein prenylation is a lipid modification crucial for membrane association and cellular signaling, but its dysregulation is linked to cancer and neurodegenerative diseases. Typical "prenylomic" workflows rely on metabolic labeling with bio-orthogonal isoprenoid probes followed by streptavidin enrichment; however, they generally require milligram-scale protein inputs (15-25 million cells), preventing the study of scarce primary or sorted cell populations. To overcome this limitation, we developed a highly sensitive, low-input prenylomic workflow combining optimized streptavidin enrichment with library-free data-independent acquisition (DIA) mass spectrometry. This method enables deep prenylomic coverage from just 50 ug of total protein input, a 20-fold reduction compared to prior methods. In COS7 cells, this workflow identified 91 prenylated protein groups, outperforming TMT-DDA methods that yielded 81 groups from 2 mg of input, using the same bio-orthogonal probe for enrichment, while coefficient of variation (CV) analysis demonstrated superior quantitative precision and reproducibility. Applied in vivo in mice following subcutaneous probe administration, the low-input methodology overcame sample input limitations to profile cell-type-specific prenylomes from dissociated liver tissue, identifying 29 enriched prenylated proteins in hepatocytes and 18 in macrophages. Pathway enrichment analysis showed that the identified proteins are consistent with the biology of the respective cell-types. This robust methodology significantly lowers the sample threshold for lipid proteomics, expanding the accessibility of prenylomics to limited primary cells and sorted tissue populations to study cell-type-specific prenylation dynamics in disease contexts.

biochemistry↗

bioRxiv2026

Acetylated PLIN2 coordinates the innate immune system and oxylipin synthesis on lipid droplets to propagate inflammation

Research has implicated lipid droplets (LDs) and their associated proteins in inflammation and the innate immune response. The key LD protein Perilipin 2 (PLIN2) is tightly correlated with lipid storage and inflammation, but little is known about the mechanisms underlying its proinflammatory effects. Here, we show that PLIN2 is required for the inflammatory response to mitogens and the recruitment of pro-inflammatory proteins to the surface of LDs. PLIN2 directly binds arachidonic acid and interacts with key oxylipin synthetic enzymes such as PLA2 and COX2 to promote LD-localized oxylipin synthesis and propagate inflammatory signaling. Moreover, LPS induces PLIN2 hyperacetylation, and acetylation-dead PLIN2 mutants lose their proinflammatory properties in the presence of an inflammatory stimulus, as if PLIN2 were ablated. Thus, acetylated PLIN2 forms a key metabolon on LDs that is essential for the response and amplification of proinflammatory signaling.

immunology↗

bioRxiv2026

ATGL-catalyzed lipid catabolism promotes DNA repair

An imbalance of DNA damage over DNA repair contributes to the genomic instability that drives aging and numerous age-related diseases. While numerous DNA repair mechanisms have been elucidated over decades of study, little is known about the contribution of metabolism to genomic stability. We report that adipose triglyceride lipase (ATGL), a lipolytic enzyme, promotes DNA repair. We show that lipid droplets (LDs) accumulate in response to DNA damage and that inhibition of LD biogenesis before genotoxic stress increases the persistence of DNA damage. Overexpression of ATGL, which increases lipolysis, reduces DNA damage following etoposide and ionizing radiation, thereby promoting genomic stability. Further, ATGL expression prior to DNA damage attenuates the long-term consequences of DNA damage, reducing senescence and enhancing viability. Mechanistically, ATGL promotes double-strand break repair via NHEJ and HR to mitigate DNA damage. Overall, these studies reveal a novel role for LDs and their proteins in DNA damage and repair, thereby unveiling a mechanism by which lipid metabolism contributes to genomic stability.

biochemistry↗

bioRxiv2025

Ketone body driven lipid remodeling supports thermogenic adaptation to fasting

Metabolic adaptation to fasting may have conferred survival advantage to early humans and predicts weight gain caused by overnutrition in modern societies. Fasting suppresses brown adipose tissue (BAT) thermogenesis; however, it is unclear how BAT rewires cellular metabolism to balance between energy conservation and heat generation. Here, we report that BAT in mice under fasting and cold challenge consumed ketone bodies, specifically acetoacetate (AcAc). Ablating liver ketogenesis decreased, while enhancing hepatic AcAc output defended, body temperature in mice facing the dual challenge. Using stable isotope tracing in brown adipocytes in vitro combined with quantitative analysis of metabolic fluxes and lipidomics in BAT from genetic mouse models, we disentangled the two metabolic fates of AcAc - terminal oxidation in the mitochondria and lipid biosynthesis in the cytosol. Notably, AcAc-sourced carbon preferentially supported polyunsaturated fatty acid synthesis in BAT, linking to the positive impact of intermittent fasting on lipid profiles in both mice and humans. Therefore, ketone body utilization by thermogenic adipocytes contributes to metabolic resilience of mammals and can be targeted to optimize benefits of dietary regimens.

physiology↗

bioRxiv2024

Sorbate Induces Lysine Sorbylation Through Non-Canonical Activities of Class I HDACs to Regulate the Expression of Inflammation Genes

Metabolic and environmental factors may impact gene expression through the production of active metabolites and epigenetic modifications of histones and transcription factors. In this study, we discovered that cellular uptake of sorbate, an FDA-approved and widely used food preservative, can induce lysine sorbylation (Ksor), a new posttranslational modification and epigenetic mark. We identified over 40 Ksor sites on core histones from mammalian cells and tissue upon sorbate uptake and further showed that the dynamics of histone Ksor could be regulated by the non-canonical activities of Class I histone deacetylases (HDAC1-3). We demonstrated that Class I HDACs catalyzed sorbylation upon sorbate uptake and desorbylation in the absence of sorbate both in vitro and in vivo. Sorbate uptake in mice livers led to a significant increase in histone Ksor without affecting overall histone acetylation, which correlated with the decreased expression of genes in inflammation signaling pathways. Accordingly, sorbate treatment in macrophage RAW264.7 cells upon LPS stimulation dose-dependently downregulated the expression of proinflammatory genes and production of nitric oxide. Global proteomic profiling revealed widespread lysine sorbylation substrates in diverse metabolic and signaling pathways and identified RelA (p65), a component of the NF-B complex, and its interacting proteins as bona fide Ksor targets. Sorbate treatment significantly decreased NF-B transcriptional activities in response to LPS stimulation in RAW264.7 cells. Taken together, our study demonstrated a non-canonical mechanism of sorbate uptake in regulating epigenetic histone modifications and inflammatory gene expression.

biochemistry↗

bioRxiv2019

Mitochondrial PE potentiates respiratory enzymes to amplify skeletal muscle aerobic capacity

Exercise capacity is a strong predictor of all-cause mortality. Skeletal muscle mitochondrial respiratory capacity, its biggest contributor, adapts robustly to changes in energy demands induced by contractile activity. While transcriptional regulation of mitochondrial enzymes has been extensively studied, there is limited information on how mitochondrial membrane lipids are regulated. Herein, we show that exercise training or muscle disuse alters mitochondrial membrane phospholipids including phosphatidylethanolamine (PE). Addition of PE promoted, whereas removal of PE diminished, mitochondrial respiratory capacity. Surprisingly, skeletal muscle-specific inhibition of mitochondrial-autonomous synthesis of PE caused a respiratory failure due to metabolic insults in the diaphragm muscle. While mitochondrial PE deficiency coincided with increased oxidative stress, neutralization of the latter did not rescue lethality. These findings highlight the previously underappreciated role of mitochondrial membrane phospholipids in dynamically controlling skeletal muscle energetics and function.

physiology↗