Acylation of MLKL Impacts Its Function in Necroptosis.

Mixed lineage kinase domain-like (MLKL) is a key signaling protein of necroptosis. Upon activation by phosphorylation, MLKL translocates to the plasma membrane and induces membrane permeabilization, which contributes to the necroptosis-associated inflammation. Membrane binding of MLKL is initially initiated by electrostatic interactions between the protein and membrane phospholipids. We previously showed that MLKL and its phosphorylated form (pMLKL) are S-acylated during necroptosis. Here, we characterize the acylation sites of MLKL and identify multiple cysteines that can undergo acylation with an interesting promiscuity at play. Our results show that MLKL and pMLKL undergo acylation at a single cysteine, with C184, C269, and C286 as possible acylation sites. Using all-atom molecular dynamic simulations, we identify differences that the acylation of MLKL causes at the protein and membrane levels. Through investigations of the S-palmitoyltransferases that might acylate pMLKL in necroptosis, we showed that zDHHC21 activity has the strongest effect on pMLKL acylation, inactivation of which profoundly reduced the pMLKL levels in cells and improved membrane integrity. These results suggest that blocking the acylation of pMLKL destabilizes the protein at the membrane interface and causes its degradation, ameliorating the necroptotic activity. At a broader level, our findings shed light on the effect of S-acylation on MLKL functioning in necroptosis and MLKL-membrane interactions mediated by its acylation.

Perfluorooctane Sulfonate (PFOS) Negatively Impacts Prey Capture Capabilities in Larval Zebrafish.

Per- and polyfluoroalkyl substances (PFAS) are widely used in many industrial and domestic applications, which has resulted in unintentional human exposures and bioaccumulation in blood and other organs. Perfluorooctane sulfonate (PFOS) is among the most prevalent PFAS in the environment and has been postulated to affect brain functions in exposed organisms. However, the impacts of PFOS in early neural development have not been well described. We used zebrafish larvae to assess the effects of PFOS on two fundamental complex behaviors, prey capture and learning. Zebrafish exposed to PFOS concentrations ranging from 2 to 20 µM for differing 48-h periods were viable through early larval stages. In addition, PFOS uptake was unaffected by the presence of a chorion. We employed two different experimental paradigms; first we assessed the impacts of increasing organismal PFOS bioaccumulation on prey capture and learning, and second, we probed stage-specific sensitivity to PFOS by exposing zebrafish at different developmental stages (0-2 vs. 3-5 days post fertilization). Following both assays we measured the amount of PFOS present in each larva and found that PFOS levels varied in larvae from different groups within each experimental paradigm. Significant negative correlations were observed between larval PFOS accumulation and percentage of captured prey, whereas nonsignificant negative correlations were observed between PFOS accumulation and experienced-induced prey capture learning. These findings suggest that PFOS accumulation negatively affects larval zebrafish's ability to perform complicated multisensory behaviors and highlights the potential risks of PFOS exposure to animals in the wild, with implications for human health. Environ Toxicol Chem 2024;43:847-855. © 2023 SETAC.

Cellular Lipidome Changes during Retinoic Acid (RA)-Induced Differentiation in SH-SY5Y Cells: A Comprehensive In Vitro Model for Assessing Neurotoxicity of Contaminants.

The SH-SY5Y, neuroblastoma cell line, is a common in vitro model used to study physiological neuronal function and the neuronal response to different stimuli, including exposure to toxic chemicals. These cells can be differentiated to neuron-like cells by administration of various reagents, including retinoic acid or phorbol-12-myristate-13-acetate. Despite their common use, there is an incomplete understanding of the molecular changes that occur during differentiation. Therefore, there is a critical need to fully understand the molecular changes that occur during differentiation to properly study neurotoxicity in response to various environmental exposures. Previous studies have investigated the proteome and transcriptome during differentiation; however, the regulation of the cellular lipidome in this process is unexplored. In this work, we conducted liquid chromatography-mass spectrometry (LC-MS)-based untargeted lipidomics in undifferentiated and differentiated SH-SY5Y cells, induced by retinoic acid. We show that there are global differences between the cellular lipidomes of undifferentiated and differentiated cells. Out of thousands of features detected in positive and negative electrospray ionization modes, 44 species were identified that showed significant differences (p-value ≤0.05, fold change ≥2) in differentiated cells. Identification of these features combined with targeted lipidomics highlighted the accumulation of phospholipids, sterols, and sphingolipids during differentiation while triacylglycerols were depleted. These results provide important insights into lipid-related changes that occur during cellular differentiation of SH-5YSY cells and emphasize the need for the detailed characterization of biochemical differences that occur during differentiation while using this in vitro model for assessing ecological impacts of environmental pollutants.

SREBP activation contributes to fatty acid accumulations in necroptosis.

Necroptosis is a type of programmed cell death. It is characterized by membrane permeabilization and is associated with the release of intracellular components due to compromised membrane integrity which induces a strong inflammatory response. We recently showed that the accumulation of very long chain fatty acids (VLCFAs) contributes to membrane permeabilization during necroptosis. However, the mechanisms that result in the accumulation of these cytotoxic lipids remain unknown. Using comparative transcriptomics and digital PCR validations, we found that several target genes of sterol regulatory element-binding proteins (SREBPs) were upregulated during necroptosis, suggesting that they might be responsible for the accumulation of VLCFA in this process. We demonstrated that activation of SREBPs during necroptosis exacerbates the permeability of the plasma membrane and cell death. Consistent with these observations, targeting sterol regulatory element-binding protein cleavage-activating protein (SCAP), a protein involved in SREBP activation, reversed the accumulation of VLCFAs, and restored cell death and membrane permeabilization during necroptosis. Collectively, our results highlight a role for SREBP in regulating lipid changes during necroptosis and suggest SREBP-mediated lipid remodeling as a potential target for therapeutics to reduce membrane permeabilization during necroptosis.

Omics approaches to better understand the molecular mechanism of necroptosis and their translational implications.

Necroptosis is a type of programed cell death characterized by an inflammatory phenotype due to extensive membrane permeabilization and rupture. Initiation of necroptosis involves activation of tumor necrosis factor receptors by tumor necrosis factor alpha (TNFα) followed by coordinated activities of receptor-interacting protein kinases and mixed lineage kinase-like protein (MLKL). Subsequently, MLKL undergoes phosphorylation and translocates to the plasma membrane, leading to permeabilization. Such permeabilization results in the release of various cytokines and causes extensive inflammatory activity at the organismal level. This inflammatory activity is one of the major differences between apoptosis and necroptosis and links necroptosis to several human pathologies that exhibit inflammation, in addition to the ultimate cell death phenotype. Given the crosstalk between the activation of cell death pathway and inflammatory activity, approaches that provide insights on the regulation of transcripts, proteins and their processing at the global level have substantially improved our understanding of necroptosis and its involvement in different disease states. In this review, we highlight recent omic studies probing the transcriptome, proteome and lipidome which elucidate potential new mechanisms and signaling pathways during necroptosis and the necroptosis-associated inflammatory activity observed in various diseases. We specifically focus on studies investigating the transcriptome and intracellular and released proteome that contribute to inflammatory nature of necroptotic cells. We also highlight different lipids that have been implicated in necroptosis and lipidomic studies identifying lipid players in necroptosis. Finally, we review studies which suggest certain necroptosis-related genes as potential prognosis markers for different cancers and discuss their translational implications.

Emerging Roles of Ceramides in Breast Cancer Biology and Therapy.

One of the classic hallmarks of cancer is the imbalance between elevated cell proliferation and reduced cell death. Ceramide, a bioactive sphingolipid that can regulate this balance, has long been implicated in cancer. While the effects of ceramide on cell death and therapeutic efficacy are well established, emerging evidence indicates that ceramide turnover to downstream sphingolipids, such as sphingomyelin, hexosylceramides, sphingosine-1-phosphate, and ceramide-1-phosphate, is equally important in driving pro-tumorigenic phenotypes, such as proliferation, survival, migration, stemness, and therapy resistance. The complex and dynamic sphingolipid network has been extensively studied in several cancers, including breast cancer, to find key sphingolipidomic alterations that can be exploited to develop new therapeutic strategies to improve patient outcomes. Here, we review how the current literature shapes our understanding of how ceramide synthesis and turnover are altered in breast cancer and how these changes offer potential strategies to improve breast cancer therapy.

Development of a Liquid Chromatography-Mass Spectrometry-Based In Vitro Assay to Assess Changes in Steroid Hormones Due to Exposure to Per- and Polyfluoroalkyl Substances.

Per- and poly-fluorinated substances (PFASs) are organic pollutants that have been linked to numerous health effects, including diabetes, cancers, and dysregulation of the endocrine system. This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to measure changes in 17 hormones in H295R cell line (a steroid producing adrenocortical cells) upon exposure to PFASs. Due to the challenges in the analysis of steroid hormones using electrospray ionization MS, a chemical derivatization method was employed to achieve 0.07-2 µg/L detection limits in LC-MS/MS. Furthermore, a 10-fold concentration factor through solid-phase extraction (SPE) allows for consistent sub-parts per billion detections. Optimization of the derivatization conditions showed doubly-derivatized products in some hormone analytes, including progesterone, corticosterone, and cortisol, and gave improved ionization efficiency up to 20-fold higher signal than the singly-derivatized product. The use of SPE for sample cleanup to analyze hormones from cellular media using weak anion exchange sorbent yielded 80-100% recovery for the 17 targeted hormones. The method was validated by exposing H295R cells to two known endocrine disruptors, forskolin and prochloraz, which showed expected changes in hormones. An initial exposure of H295R cells with various PFAS standards and their mixtures at 1 µM showed significant increases in progestogens with some PFAS treatments, which include PFBS, PFHxA, PFOS, PFDA, and PFDS. In addition, modest changes in hormone levels were observed in cells treated with other sulfonated or carboxylated headgroup PFASs. This sensitive LC-MS/MS method for hormone analysis in H295R cells will allow for the investigations of the alterations in the hormone production caused by exposure to various environmental insults in cell-based assays and other in vitro models.

Endocrine Therapy-Resistant Breast Cancer Cells Are More Sensitive to Ceramide Kinase Inhibition and Elevated Ceramide Levels Than Therapy-Sensitive Breast Cancer Cells.

ET resistance is a critical problem for estrogen receptor-positive (ER+) breast cancer. In this study, we have investigated how alterations in sphingolipids promote cell survival in ET-resistant breast cancer. We have performed LC-MS-based targeted sphingolipidomics of tamoxifen-sensitive and -resistant MCF-7 breast cancer cell lines. Follow-up studies included treatments of cell lines and patient-derived xenograft organoids (PDxO) with small molecule inhibitors; cytometric analyses to measure cell death, proliferation, and apoptosis; siRNA-mediated knockdown; RT-qPCR and Western blot for gene and protein expression; targeted lipid analysis; and lipid addback experiments. We found that tamoxifen-resistant cells have lower levels of ceramides and hexosylceramides compared to their tamoxifen-sensitive counterpart. Upon perturbing the sphingolipid pathway with small molecule inhibitors of key enzymes, we identified that CERK is essential for tamoxifen-resistant breast cancer cell survival, as well as a fulvestrant-resistant PDxO. CERK inhibition induces ceramide-mediated cell death in tamoxifen-resistant cells. Ceramide-1-phosphate (C1P) partially reverses CERK inhibition-induced cell death in tamoxifen-resistant cells, likely through lowering endogenous ceramide levels. Our findings suggest that ET-resistant breast cancer cells maintain lower ceramide levels as an essential pro-survival mechanism. Consequently, ET-resistant breast cancer models have a unique dependence on CERK as its activity can inhibit de novo ceramide production.

Cellular Interactions and Fatty Acid Transporter CD36-Mediated Uptake of Per- and Polyfluorinated Alkyl Substances (PFAS).

Per- and polyfluorinated alkyl substances (PFAS) are a class of widely used compounds in an array of commercial and industrial applications. Due to their extensive use and chemical stability, PFAS persist in the environment and bioaccumulate in humans and wildlife. PFAS exposure have been linked to several negative health effects, including the formation of various cancers, disruption of the endocrine system, and obesity. However, there is a major gap in understanding how structural differences in PFAS impact their interactions within a biological system. In this study, we examined the toxicity of PFAS with differences in chain length, head group, and degree of fluorination in human retinal epithelial cells. We focused on fluorotelomeric and fully fluorinated sulfonates and carboxylates and measured their uptake. Our results showed that sulfonates are taken up at higher levels as compared to their fluorotelomer and carboxylate counterparts. Furthermore, PFAS with 8 and 10 carbons (C8 and C10) are taken up at a higher level compared to those with six carbons (C6). We also investigated the role of the fatty acid transporter CD36 in PFAS uptake and found that increased CD36 levels result in higher levels of PFAS in cells. Overall, our results suggest that the head group structure of PFAS impacts toxicity, with sulfonates inducing a higher decrease in cell viability (∼50%) than carboxylates. Our results also link the activity of CD36 to PFAS uptake into cells.

Ceramide-1-Phosphate Is Involved in Therapy-Induced Senescence.

Sphingolipids are key signaling lipids and their dysregulation has been associated with various cellular processes. We have previously shown significant changes in sphingolipids in therapy-induced senescence, a state of cell cycle arrest as a response to chemotherapy, including the accumulation of ceramides, and provided evidence suggesting that ceramide processing is important for this process. Herein, we conducted a focused small molecule inhibitor screen targeting the sphingolipid pathway, which highlighted a new lipid regulator of therapy-induced senescence. Among the inhibitors tested, the inhibition of ceramide kinase by NVP-231 reduced the levels of senescent cells. Ceramide kinase knockdown exhibited similar effects, strongly supporting the involvement of ceramide kinase during this process. We showed that ceramide-1-phosphate was upregulated in therapy-induced senescence and that NVP-231 reduced ceramide-1-phosphate levels in different cell line models of therapy-induced senescence. Finally, ceramide-1-phosphate addition to NVP-231-treated cells reversed the effects of NVP-231 during senescence. Overall, our results identify a previously unknown lipid player in therapy-induced senescence and highlight a potential targetable enzyme to reduce the levels of therapy-induced senescent cells.

Modeling the molecular fingerprint of protein-lipid interactions of MLKL on complex bilayers.

Lipids, the structural part of membranes, play important roles in biological functions. However, our understanding of their implication in key cellular processes such as cell division and protein-lipid interaction is just emerging. This is the case for molecular interactions in mechanisms of cell death, where the role of lipids for protein localization and subsequent membrane permeabilization is key. For example, during the last stage of necroptosis, the mixed lineage kinase domain-like (MLKL) protein translocates and, eventually, permeabilizes the plasma membrane (PM). This process results in the leakage of cellular content, inducing an inflammatory response in the microenvironment that is conducive to oncogenesis and metastasis, among other pathologies that exhibit inflammatory activity. This work presents insights from long all-atom molecular dynamics (MD) simulations of complex membrane models for the PM of mammalian cells with an MLKL protein monomer. Our results show that the binding of the protein is initially driven by the electrostatic interactions of positively charged residues. The protein bound conformation modulates lipid recruitment to the binding site, which changes the local lipid environment recruiting PIP lipids and cholesterol, generating a unique fingerprint. These results increase our knowledge of protein-lipid interactions at the membrane interface in the context of molecular mechanisms of the necroptotic pathway, currently under investigation as a potential treatment target in cancer and inflamatory diseases.

Sex-specific phenotypic effects and evolutionary history of an ancient polymorphic deletion of the human growth hormone receptor.

The common deletion of the third exon of the growth hormone receptor gene (GHRd3) in humans is associated with birth weight, growth after birth, and time of puberty. However, its evolutionary history and the molecular mechanisms through which it affects phenotypes remain unresolved. We present evidence that this deletion was nearly fixed in the ancestral population of anatomically modern humans and Neanderthals but underwent a recent adaptive reduction in frequency in East Asia. We documented that GHRd3 is associated with protection from severe malnutrition. Using a novel mouse model, we found that, under calorie restriction, Ghrd3 leads to the female-like gene expression in male livers and the disappearance of sexual dimorphism in weight. The sex- and diet-dependent effects of GHRd3 in our mouse model are consistent with a model in which the allele frequency of GHRd3 varies throughout human evolution as a response to fluctuations in resource availability.

Solving the enigma: Mass spectrometry and small molecule probes to study sphingolipid function.

Sphingolipids are highly bioactive lipids. Sphingolipid metabolism produces key membrane components (e.g. sphingomyelin) and a variety of signaling lipids with different biological functions (e.g. ceramide, sphingosine-1-phosphate). The coordinated activity of tens of different enzymes maintains proper levels and localization of these lipids with key roles in cellular processes. In this review, we highlight the signaling roles of sphingolipids in cell death and survival. We discuss recent findings on the role of specific sphingolipids during these processes, enabled by the use of lipidomics to study compositional and spatial regulation of these lipids and synthetic sphingolipid probes to study subcellular localization and interaction partners of sphingolipids to understand the function of these lipids.

A Single-Organelle Optical Omics Platform for Cell Science and Biomarker Discovery.

Research in fundamental cell biology and pathology could be revolutionized by developing the capacity for quantitative molecular analysis of subcellular structures. To that end, we introduce the Ramanomics platform, based on confocal Raman microspectrometry coupled to a biomolecular component analysis algorithm, which together enable us to molecularly profile single organelles in a live-cell environment. This emerging omics approach categorizes the entire molecular makeup of a sample into about a dozen of general classes and subclasses of biomolecules and quantifies their amounts in submicrometer volumes. A major contribution of our study is an attempt to bridge Raman spectrometry with big-data analysis in order to identify complex patterns of biomolecules in a single cellular organelle and leverage discovery of disease biomarkers. Our data reveal significant variations in organellar composition between different cell lines. We also demonstrate the merits of Ramanomics for identifying diseased cells by using prostate cancer as an example. We report large-scale molecular transformations in the mitochondria, Golgi apparatus, and endoplasmic reticulum that accompany the development of prostate cancer. Based on these findings, we propose that Ramanomics datasets in distinct organelles constitute signatures of cellular metabolism in healthy and diseased states.

Protein acylation by saturated very long chain fatty acids and endocytosis are involved in necroptosis.

Necroptosis is a form of cell death characterized by receptor-interacting protein kinase activity and plasma membrane permeabilization via mixed-lineage kinase-like protein (MLKL). This permeabilization is responsible for the inflammatory properties of necroptosis. We previously showed that very long chain fatty acids (VLCFAs) are functionally involved in necroptosis, potentially through protein fatty acylation. Here, we define the scope of protein acylation by saturated VLCFAs during necroptosis. We show that MLKL and phosphoMLKL, key for membrane permeabilization, are exclusively acylated during necroptosis. Reducing the levels of VLCFAs decreases their membrane recruitment, suggesting that acylation by VLCFAs contributes to their membrane localization. Acylation of phosphoMLKL occurs downstream of phosphorylation and oligomerization and appears to be, in part, mediated by ZDHHC5 (a palmitoyl transferase). We also show that disruption of endosomal trafficking increases cell viability during necroptosis, possibly by preventing recruitment, or removal, of phosphoMLKL from the plasma membrane.

Short Photoswitchable Ceramides Enable Optical Control of Apoptosis.

We report short ceramide analogs that can be activated with light and further functionalized using azide-alkyne click chemistry. These molecules, termed scaCers, exhibit increased cell permeability compared to their long-chain analogs as demonstrated using mass spectrometry and imaging. Notably, scaCers enable optical control of apoptosis, which is not observed with long-chain variants. Additionally, they function as photoswitchable substrates for sphingomyelin synthase 2 (SMS2), exhibiting inverted light-dependence compared to their extended analogs.

Lipid Players of Cellular Senescence.

Lipids are emerging as key players of senescence. Here, we review the exciting new findings on the diverse roles of lipids in cellular senescence, most of which are enabled by the advancements in omics approaches. Senescence is a cellular process in which the cell undergoes growth arrest while retaining metabolic activity. At the organismal level, senescence contributes to organismal aging and has been linked to numerous diseases. Current research has documented that senescent cells exhibit global alterations in lipid composition, leading to extensive morphological changes through membrane remodeling. Moreover, senescent cells adopt a secretory phenotype, releasing various components to their environment that can affect the surrounding tissue and induce an inflammatory response. All of these changes are membrane and, thus, lipid related. Our work, and that of others, has revealed that fatty acids, sphingolipids, and glycerolipids are involved in the initiation and maintenance of senescence and its associated inflammatory components. These studies opened up an exciting frontier to investigate the deeper mechanistic understanding of the regulation and function of these lipids in senescence. In this review, we will provide a comprehensive snapshot of the current state of the field and share our enthusiasm for the prospect of potential lipid-related protein targets for small-molecule therapy in pathologies involving senescence and its related inflammatory phenotypes.

Metabolic coessentiality mapping identifies C12orf49 as a regulator of SREBP processing and cholesterol metabolism.

Coessentiality mapping has been useful to systematically cluster genes into biological pathways and identify gene functions1-3. Here, using the debiased sparse partial correlation (DSPC) method3, we construct a functional coessentiality map for cellular metabolic processes across human cancer cell lines. This analysis reveals 35 modules associated with known metabolic pathways and further assigns metabolic functions to unknown genes. In particular, we identify C12orf49 as an essential regulator of cholesterol and fatty acid metabolism in mammalian cells. Mechanistically, C12orf49 localizes to the Golgi, binds membrane-bound transcription factor peptidase, site 1 (MBTPS1, site 1 protease) and is necessary for the cleavage of its substrates, including sterol regulatory element binding protein (SREBP) transcription factors. This function depends on the evolutionarily conserved uncharacterized domain (DUF2054) and promotes cell proliferation under cholesterol depletion. Notably, c12orf49 depletion in zebrafish blocks dietary lipid clearance in vivo, mimicking the phenotype of mbtps1 mutants. Finally, in an electronic health record (EHR)-linked DNA biobank, C12orf49 is associated with hyperlipidaemia through phenome analysis. Altogether, our findings reveal a conserved role for C12orf49 in cholesterol and lipid homeostasis and provide a platform to identify unknown components of other metabolic pathways.

Flux Balance Analysis for Media Optimization and Genetic Targets to Improve Heterologous Siderophore Production.

Siderophores are small molecule metal chelators secreted in sparse quantities by their native microbial hosts but can be engineered for enhanced production from heterologous hosts like Escherichia coli. These molecules have been proved to be capable of binding heavy metals of commercial and/or environmental interest. In this work, we incorporated, as needed, the appropriate pathways required to produce several siderophores (anguibactin, vibriobactin, bacillibactin, pyoverdine, and enterobactin) into the base E. coli K-12 MG1655 metabolic network model to computationally predict, via flux balance analysis methodologies, gene knockout targets, gene over-expression targets, and media modifications capable of improving siderophore reaction flux. E. coli metabolism proved supportive for the underlying production mechanisms of various siderophores. Within such a framework, the gene deletion and over-expression targets identified, coupled with complementary insights from medium optimization predictions, portend experimental implementation to both enable and improve heterologous siderophore production. Successful production of siderophores would then spur novel metal-binding applications.

Promotion of plasmalogen biosynthesis reverse lipid changes in a Barth Syndrome cell model.

In Barth syndrome (BTHS) mutations in tafazzin leads to changes in both the quantities and the molecular species of cardiolipin (CL), which are the hallmarks of BTHS. Contrary to the well-established alterations in CL associated with BTHS; recently a marked decrease in the plasmalogen levels in Barth specimens has been identified. To restore the plasmalogen levels, the present study reports the effect of promotion of plasmalogen biosynthesis on the lipidome of lymphoblasts derived from Barth patients as well as on cell viability, mitochondria biogenesis, and mitochondrial membrane potential. High resolution 31P NMR phospholipidomic analysis showed an increase in the levels of plasmenylethanolamine (the major plasmalogen in lymphoblasts), which reached values comparable to the control and a compensatory decrease in the levels of its diacyl-PE counterpart. Importantly, 31P NMR showed a significant increase in the levels of CL, while not altering the levels of monolysocardiolipin. Mass spectrometry measurements showed that the promotion of plasmalogen biosynthesis did not change the molecular species profile of targeted phospholipids. In addition, promotion of plasmalogen biosynthesis did not impact on cellular viability, although it significantly decrease mitochondria copy number and restored mitochondrial membrane potential. Overall, the results showed the efficacy of the promotion of plasmalogen biosynthesis on increasing the CL levels in a BTHS cell model and highlight the potential beneficial effect of a diet supplemented with plasmalogen precursors to BTHS patients.