Biology subjects

Kennelly, J. P.

Publications and source records attributed to Kennelly, J. P..

7 recordsLinked to original sources

bioRxiv2024

Dietary control of peripheral adipose storage capacity through membrane lipid remodelling

Complex genetic and dietary cues contribute to the development of obesity, but how these are integrated on a molecular level is incompletely understood. Here, we show that PPAR{gamma} supports hypertrophic expansion of adipose tissue via transcriptional control of LPCAT3, a membrane-bound O-acyltransferase that enriches diet-derived omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in the phospholipidome. In high-fat diet-fed mice, lowering membrane n-6 PUFA levels by adipocyte-specific Lpcat3 knockout (Lpcat3AKO) or by dietary lipid manipulation leads to dysfunctional triglyceride (TG) storage, ectopic fat deposition and insulin resistance. Aberrant lipolysis of stored TGs in Lpcat3AKO adipose tissues instigates a non-canonical adaptive response that engages a futile lipid cycle to increase energy expenditure and limit further body weight gain. Mechanistically, we find that adipocyte LPCAT3 activity promotes TG storage by selectively enriching n-6 arachidonoyl-phosphatidylethanolamine at the ER-lipid droplet interface, which in turn favours the budding of large droplets that exhibit greater resistance to ATGL-dependent hydrolysis. Thus, our study highlights the PPAR{gamma}-LPCAT3 pathway as a molecular link between dietary n-6 PUFA intake, adipose expandability and systemic energy balance.

physiology↗

bioRxiv2024

Cholesterol binding to VCAM-1 promotes vascular inflammation

Hypercholesterolemia has long been implicated in endothelial cell (EC) dysfunction, but the mechanisms by which excess cholesterol causes vascular pathology are incompletely understood. Here we used a cholesterol-mimetic probe to map cholesterol-protein interactions in primary human ECs and discovered that cholesterol binds to and stabilizes the adhesion molecule VCAM-1. We show that accessible plasma membrane (PM) cholesterol in ECs is acutely responsive to inflammatory stimuli and that the nonvesicular cholesterol transporter Aster-A regulates VCAM-1 stability in activated ECs by controlling the size of this pool. Deletion of Aster-A in ECs increases VCAM-1 protein, promotes immune cell recruitment to vessels, and impairs pulmonary immune homeostasis. Conversely, depleting cholesterol from the endothelium in vivo dampens VCAM-1 induction in response to inflammatory stimuli. These findings identify cholesterol binding to VCAM-1 as a key step during EC activation and provide a biochemical explanation for the ability of excess membrane cholesterol to promote immune cell recruitment to the endothelium.

physiology↗

bioRxiv2024

Lipid- and protein-directed photosensitizer proximity labeling captures the cholesterol interactome

The physical properties of cellular membranes, including fluidity and function, are influenced by protein and lipid interactions. In situ labeling chemistries, most notably proximity-labeling interactomics are well suited to characterize these dynamic and often fleeting interactions. Established methods require distinct chemistries for proteins and lipids, which limits the scope of such studies. Here we establish a singlet-oxygen-based photocatalytic proximity labeling platform (POCA) that reports intracellular interactomes for both proteins and lipids with tight spatiotemporal resolution using cell-penetrant photosensitizer reagents. Using both physiologically relevant lipoprotein-complexed probe delivery and genetic manipulation of cellular cholesterol handling machinery, cholesterol-directed POCA captured established and unprecedented cholesterol binding proteins, including protein complexes sensitive to intracellular cholesterol levels and proteins uniquely captured by lipoprotein uptake. Protein-directed POCA accurately mapped known intracellular membrane complexes, defined sterol-dependent changes to the non-vesicular cholesterol transport protein interactome, and captured state-dependent changes in the interactome of the cholesterol transport protein Aster-B. More broadly, we find that POCA is a versatile interactomics platform that is straightforward to implement, using the readily available HaloTag system, and fulfills unmet needs in intracellular singlet oxygen-based proximity labeling proteomics. Thus, we expect widespread utility for POCA across a range of interactome applications, spanning imaging to proteomics.

biochemistry↗

bioRxiv2024

Flow-Mediated Modulation of the Endothelial Cell Lipidome

The luminal surface of the endothelium is exposed to dynamic blood flow patterns that are known to affect endothelial cell phenotype. While many studies have documented the phenotypic changes by gene or protein expression, less is known about the role of blood flow pattern on the endothelial cell (EC) lipidome. In this study, shotgun lipidomics was conducted on human aortic ECs (HAECs) exposed to unidirectional laminar flow (UF), disturbed flow (DF), or static conditions for 48 hrs. A total of 520 individual lipid species from 17 lipid subclasses were detected. Total lipid abundance was significantly increased for HAECs exposed to DF compared to UF conditions. Despite the increase in the total lipid abundance, HAECs maintained equivalent composition of each lipid subclass (% of total lipid) under both DF and UF. However, by lipid composition (% of total subclass), 28 lipid species were significantly altered between DF and UF. Complimentary RNA sequencing of HAECs exposed to UF or DF revealed changes in transcripts involved in lipid metabolism. Shotgun lipidomics was also performed on HAECs exposed to pro-inflammatory agonists lipopolysaccharide (LPS) or Pam3CSK4 (Pam3) for 48 hrs. Exposure to LPS or Pam3 reshaped the EC lipidome in both unique and overlapping ways. In conclusion, exposure to flow alters the EC lipidome and ECs undergo stimulus-specific lipid reprogramming in response to pro-inflammatory agonist exposure. Ultimately, this work provides a resource to profile the transcriptional and lipidomic changes that occur in response to applied flow that can be accessed by the vascular biology community to further dissect and extend our understanding of endothelial lipid biology.

cell biology↗

bioRxiv2024

T cell cholesterol transport is a metabolic checkpoint that links intestinal immune responses to dietary lipid absorption

The intrinsic pathways that control membrane organization in immune cells and the impact of such pathways on cellular function are not well defined. Here we report that the non-vesicular cholesterol transporter Aster-A links plasma membrane (PM) cholesterol availability in T cells to immune signaling and systemic metabolism. Aster-A is recruited to the PM during T-cell receptor (TCR) activation, where it facilitates the removal of newly generated "accessible" membrane cholesterol. Loss of Aster-A leads to excess PM cholesterol accumulation, resulting in enhanced TCR nano-clustering and signaling, and Th17 cytokine production. Finally, we show that the mucosal Th17 response is restrained by PM cholesterol remodeling. Ablation of Aster-A in T cells leads to enhanced IL-22 production, reduced intestinal fatty acid absorption, and resistance to diet-induced obesity. These findings delineate a multi-tiered regulatory scheme linking immune cell lipid flux to nutrient absorption and systemic physiology.

physiology↗

bioRxiv2023

Aster-dependent non-vesicular transport facilitates dietary cholesterol uptake

Intestinal cholesterol absorption is an important contributor to systemic cholesterol homeostasis. Niemann-Pick C1 Like 1 (NPC1L1), the target of the drug ezetimibe (EZ), assists in the initial step of dietary cholesterol uptake. However, how cholesterol moves downstream of NPC1L1 is unknown. Here we show that Aster-B and Aster-C are critical for non-vesicular cholesterol movement in enterocytes, bridging NPC1L1 at the plasma membrane (PM) and ACAT2 in the endoplasmic reticulum (ER). Loss of NPC1L1 diminishes accessible PM cholesterol in enterocytes and abolishes Aster recruitment to the intestinal brush border. Enterocytes lacking Asters accumulate cholesterol at the PM and display evidence of ER cholesterol depletion, including decreased cholesterol ester stores and activation of the SREBP-2 transcriptional pathway. Aster-deficient mice have impaired cholesterol absorption and are protected against diet-induced hypercholesterolemia. Finally, we show that the Aster pathway can be targeted with a small molecule inhibitor to manipulate dietary cholesterol uptake. These findings identify the Aster pathway as a physiologically important and pharmacologically tractable node in dietary lipid absorption. One-Sentence SummaryIdentification of a targetable pathway for regulation of dietary cholesterol absorption

physiology↗

bioRxiv2023

Polarized Mechanosensitive Signaling Domains Protect Arterial Endothelial Cells Against Inflammation

Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, which supports an anti-inflammatory phenotype that protects them from atherosclerosis. High laminar shear stress also supports flow-aligned cell elongation and front-rear polarity, but whether this is required for athero-protective signaling is unclear. Here, we show that Caveolin-1-rich microdomains become polarized at the downstream end of ECs exposed to continuous high laminar flow. These microdomains are characterized by higher membrane rigidity, filamentous actin (F-actin) and lipid accumulation. Transient receptor potential vanilloid-type 4 (Trpv4) ion channels, while ubiquitously expressed, mediate localized Ca2+ entry at these microdomains where they physically interact with clustered Caveolin-1. The resultant focal bursts in Ca2+ activate the anti-inflammatory factor endothelial nitric oxide synthase (eNOS) within the confines of these domains. Importantly, we find that signaling at these domains requires both cell body elongation and sustained flow. Finally, Trpv4 signaling at these domains is necessary and sufficient to suppress inflammatory gene expression. Our work reveals a novel polarized mechanosensitive signaling hub that induces an anti-inflammatory response in arterial ECs exposed to high laminar shear stress.

cell biology↗