Chlorolipids are produced during the neutrophil respiratory burst as a result of myeloperoxidase (MPO)-generated hypochlorous acid (HOCl) targeting the vinyl ether bond of plasmalogen phospholipids. The initial products of this reaction are 2-chlorofatty aldehydes (2-ClFALDs), which are subsequently oxidized to 2-chlorofatty acids (2-ClFAs). 2-Chlorohexadecanoic acid (2-ClHA) is the 16-carbon 2-ClFA species, and previous studies have shown that increased levels of plasma 2-ClHA associate with acute respiratory distress syndrome (ARDS)-caused mortality in human sepsis. 2-ClHA causes endothelial barrier dysfunction and increases neutrophil and platelet adherence to the endothelium. In this study, click chemistry analogs of 2-ClHA and hexadecanoic acid (HA) were used to identify proteins covalently modified by 2-ClHA and HA in human lung microvascular endothelial cells (HLMVECs). Eleven proteins were specifically modified by 2-ClHA, and an additional one hundred and ninety-four proteins were modified by both 2-ClHA and HA. STRING analysis of 2-ClHA-modified proteins revealed a network of proteins with RhoA as a hub. RhoA is one of the proteins specifically modified by 2-ClHA and not HA. The RhoA inhibitors, Rhosin and C3, inhibited both 2-ClHA-elicited HLMVEC barrier dysfunction and angiopoietin-2 (Ang-2) release from HLMVEC. Further studies showed 2-ClHA activates HLMVEC RhoA activity. The specificity of the 2-ClHA-RhoA pathway for endothelial activation was further confirmed since HA did not cause HLMVEC barrier dysfunction, Ang-2 release and RhoA activation. Collectively, these studies have identified multiple proteins modified exclusively by 2-ClHA in HLMVECs, including RhoA. These proteomics studies led to the key finding that RhoA is an important mediator of 2-ClHA-caused endothelial barrier dysfunction.

Lipid metabolism plays a critical role in lymphatic endothelial cell (LEC) development and vessel maintenance. Altered lipid metabolism is associated with loss of lymphatic vessel integrity, which compromises organ function, protective immunity, and metabolic health. Thus, understanding how lipid metabolism affects LECs is critical for uncovering the mechanisms underlying lymphatic dysfunction. Protein palmitoylation, a lipid-based post-translational modification, has emerged as a critical regulator of protein function, stability, and interaction networks. Insulin, a master regulator of systemic lipid metabolism, also regulates protein palmitoylation. However, the role of insulin-driven palmitoylation in LEC biology remains unexplored. To examine the role of palmitoylation in LEC function, we generated the first palmitoylation proteomics profile in human LECs, validated insulin-regulated targets, and determined the role of palmitoylation in LEC barrier function. In unstimulated conditions, palmitoylation occurred primarily on proteins involved in vesicular and membrane trafficking, and in translation initiation. Insulin treatment, instead, enriched palmitoylation of proteins involved in LEC integrity, namely junctional proteins such as claudin 5, along with small GTPases and ubiquitination enzymes. We also investigated the role of the long-chain fatty acid transporter CD36, a major mediator of palmitate uptake into cells, in regulating optimal lymphatic protein palmitoylation. CD36 silencing in LECs increased by 2-fold palmitoylation of proteins involved in inflammation and immune cell activation. Overall, our findings provide novel insights into the intricate relationship between lipid modification and LEC function, suggesting that insulin and palmitoylation play a critical role in lymphatic endothelial function.

2-Chloropalmitic acid (2-ClPA) and 2-bromopalmitic acid (2-BrPa) increase in inflammatory lung disease associated with formation of hypochlorous or hypobromous acid, and exposure to halogen gases. Moreover, these lipids may elicit cell responses that contribute to lung injury, but the mechanisms remain unclear. Here, we tested the hypothesis that 2-ClPA and 2-BrPA induce metabolic defects in airway epithelial cells by targeting mitochondria. H441 or primary human airway epithelial cells were treated with 2-ClPA or 2-BrPA and bioenergetics measured using oxygen consumption rates and extracellular acidification rates, as well as respiratory complex activities. Relative to vehicle or palmitic acid, both 2-halofatty acids inhibited ATP-linked oxygen consumption and reserve capacity, suggestive of increased proton leak. However, neither 2-ClPA nor 2-BrPA altered mitochondrial membrane potential, suggesting proton leak does not underlie inhibited ATP-linked oxygen consumption. Interestingly, complex II activity was significantly inhibited which may contribute to diminished reserve capacity, but activity of complexes I, III and IV remain unchanged. Taken together, the presented data highlight the potential of 2-halofatty acids to disrupt bioenergetics and in turn cause cellular dysfunction.

One of the hallmarks of cancer is metabolic reprogramming which controls cellular homeostasis and therapy resistance. Here, we investigated the effect of momordicine-I (M-I), a key bioactive compound from Momordica charantia (bitter melon), on metabolic pathways in human head and neck cancer (HNC) cells and a mouse HNC tumorigenicity model. We found that M-I treatment on HNC cells significantly reduced the expression of key glycolytic molecules, SLC2A1 (GLUT-1), HK1, PFKP, PDK3, PKM, and LDHA at the mRNA and protein levels. We further observed reduced lactate accumulation, suggesting glycolysis was perturbed in M-I treated HNC cells. Metabolomic analyses confirmed a marked reduction in glycolytic and TCA cycle metabolites in M-I-treated cells. M-I treatment significantly downregulated mRNA and protein expression of essential enzymes involved in de novo lipogenesis, including ACLY, ACC1, FASN, SREBP1, and SCD1. Using shotgun lipidomics, we found a significant increase in lysophosphatidylcholine and phosphatidylcholine loss in M-I treated cells. Subsequently, we observed dysregulation of mitochondrial membrane potential and significant reduction of mitochondrial oxygen consumption after M-I treatment. We further observed M-I treatment induced autophagy, activated AMPK and inhibited mTOR and Akt signaling pathways and leading to apoptosis. However, blocking autophagy did not rescue the M-I-mediated alterations in lipogenesis, suggesting an independent mechanism of action. M-I treated mouse HNC MOC2 cell tumors displayed reduced Hk1, Pdk3, Fasn, and Acly expression. In conclusion, our study revealed that M-I inhibits glycolysis, lipid metabolism, induces autophagy in HNC cells and reduces tumor volume in mice. Therefore, M-I-mediated metabolic reprogramming of HNC has the potential for important therapeutic implications.