Noncanonical Roles of Lipids in Different Cellular Fates - American

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Non-canonical Roles of Lipids in Different Cellular Fates Darleny Y. Lizardo, Laura Parisi, Nasi Li, and G. Ekin Atilla-Gokcumen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00862 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 12, 2017

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Non-canonical Roles of Lipids in Different Cellular Fates Darleny Y. Lizardo†, Laura R. Parisi†, Nasi Li†, G. Ekin Atilla-Gokcumen* Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260, United States †

These authors contributed equally.

* Correspondence: [email protected]

Abstract Lipids are a diverse class of biomolecules. The biosynthesis and transport of these molecules are controlled by a considerable number of proteins, which facilitate spatio-temporal regulation of lipids during different fundamental cellular processes. Although lipids are traditionally considered as molecules for energy storage and as structural components of membranes, they are being increasingly recognized for their signaling roles. There is a growing appreciation of lipids’ chemical diversity, which approaches that of proteins. In this perspective, we discuss recent studies that suggest novel functions for distinct lipid species during different cellular processes. In particular, we discuss findings from our laboratory on the involvement of ceramides, polyunsaturated triacylglycerols, and very long chain fatty acids in different cellular fates. We also highlight recent innovative methods that have enabled the recognition of previously unknown lipid classes and/or roles of these molecules in different biological processes. We envision that advances in lipid identification, visualization, and perturbation will pave the way for broader investigations into this fascinating and influential class of biomolecules.

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1. Introduction Lipids are essential biomolecules. Traditionally recognized for their structural and energy storage roles, more recently, lipids are also being recognized as important signaling molecules. But do they really get the attention that they deserve? The mammalian lipidome is composed of thousands of unique molecules. Despite this, the chemical diversity of the lipidome is often underappreciated. Lipids can be divided into a number of categories based on their core structures. Several examples such as fatty acids, glycerolipids, glycerophospholipids, sphingolipids, and sterols are illustrated in Figure 1. Within each class of lipids, there is a plethora of further distinctions. Glycerolipids, for example, consist of one, two, or three fatty acyl chains bonded to a glycerol backbone. These lipids can further be modified to glycerophospholipids, whose phosphate moiety can bear a number of different head groups including serine, ethanolamine, inositol, choline, and glycerol (shown in blue in Figure 1). Individual acyl chains vary in length (commonly 16-24 carbons in mammalian cells), and number and position of double bonds, with precise structures facilitated by a network of elongases and desaturases. Additional processing of acyl chain and head groups can result in hydroxylation, esterification, and ether linkages. These structural differences greatly influence the physicochemical properties which determine lipid localization, intermolecular interactions, and ultimately their function in cells.1,2 Major technological developments have enabled recent discoveries of non-traditional roles of lipids in cellular processes. Liquid chromatography-mass spectrometry (LC-MS)-based untargeted lipidomics3 has emerged as an excellent discovery tool in this regard, allowing researchers to gain a better understanding of the lipidome as a whole. This approach has facilitated the identification of lipid species that are linked to various physiological and pathophysiological processes. For instance, a novel class of lipids, fatty acid esters of hydroxy fatty acids (FAHFAs), was recently discovered via an untargeted LC-MS-based lipidomics approach.4 Using synthetic analogs to elucidate absolute configuration, FAHFAs were discovered to have a signaling role to stimulate insulin production and increase glucose uptake, suggesting a new therapeutic target for diabetes.4,5 Lipidomics approaches are increasing in popularity due to technological advances which allow higher throughput analysis of lipid content. However, changes in the spatial regulation of lipids are as equally important as the compositional regulation. With the development of new probes and techniques, it is now possible to elucidate the trafficking of specific lipids between different cellular organelles. Biosynthesis of phosphatidic acid, a lipid second-messenger, has been directly visualized in cellular membranes via a chemoenzymatic strategy whereby phospholipase D utilizes an alkynol which can be subsequently tagged with a fluorophore using copper catalyzed azide-alkyne cycloaddition.6 Fluorophore-conjugated lipids have also been tracked in various organelles; a recent study imaged mitochondria and endoplasmic reticulum dynamics, where the hydrophobic nature of the lipid bilayer enabled high spatio-temporal resolution.7 Along these lines, biorthogonal structure-specific lipid probes, including azido-8 and alkyne-9,10 functionalized lipids, have been instrumental in determining location-dependent lipid functions. Desmosterol, a cholesterol precursor, was recently linked to Hepatitis C infection using a


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combination of untargeted lipidomics and Raman-based imaging of a deuterium-labeled desmosterol probe.11,12

Figure 1. Major classes of lipids in the mammalian lipidome. Representative structures of each class are shown here. Core structures of individual lipid classes are highlighted in red. Examples of possible head groups are highlighted in blue. Chemical diversity within each lipid class is achieved by different combinations of head groups and fatty acyl chains, which may vary in length and/or number of double bonds.

We highlight these recent studies to illustrate the importance of using integrative methods to gain a better understanding of the roles of lipids in cellular processes. Our lab has focused on studying the roles of lipids in programmed cell death and senescence, two natural processes that terminate the proliferative life of a cell, and play central roles in normal aging, cancer, and other pathologies.13-15 These processes involve key alterations in cellular membranes, as shown in Figure 2. However, although their protein players are well known, the roles of lipids in these processes are not fully understood. In this perspective, we discuss recent findings from our own lab and others that advance our understanding of how lipids are involved in these critical cellular fates.


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Figure 2. Involvement of lipids in different cellular fates. Apoptosis (A), necroptosis (N), and senescence (S) are three cellular fates which are accompanied by distinct membrane-related phenotypes and changes in lipid composition. Apoptosis is characterized by DNA damage, cell shrinking, membrane blebbing, and formation of apoptotic bodies. Necroptosis results in cellular/organelle swelling and loss of plasma membrane integrity. Senescence is characterized by an increase in cell size and lysosomal content (orange), and a distinct secretory phenotype. Polyunsaturated fatty acyl chain containing TAGs (PUFA-TAGs) accumulate during apoptosis and senescence and may possibly protect cells from oxidative stress-induced membrane damage. Ceramides, a central sphingolipid, accumulate in all three processes. In apoptosis, ceramides may facilitate pore formation in the mitochondrial outer membrane; their roles in senescence and necroptosis are less well understood. During apoptosis, clearance of apoptotic bodies is facilitated by the translocation of phosphatidylserines (PSs) from the inner leaflet of the plasma membrane to the outer leaflet, where they act as a signaling lipid to stimulate phagocytosis. Very long chain fatty acids (VLCFAs) accumulate during necroptosis; their precise functional role has yet to be elucidated. Certain phosphatidylinositol phosphates (PIPs) are known to be involved in the recruitment of phosphorylated MLKL to the plasma membrane, which cause the hallmark plasma membrane permeabilization during necroptotic cell death.

2. Lipids & programmed cell death Programmed cell death (PCD) plays an important role in many physiological and pathophysiological conditions. For many years, apoptosis was considered as the only form of PCD; however, accumulating evidence has revealed a number of distinct and highly regulated forms of PCDs including ferroptosis, pyroptosis, and necroptosis, among others.16,17 These forms of PCDs are controlled by different molecular mechanisms.17 Apoptosis depends on the 4 ACS Paragon Plus Environment

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sequential activation of caspases, a family of proteolytic enzymes, and proceeds via two main pathways: the extrinsic pathway (triggered by death receptor activation) or the intrinsic pathway (mitochondria-dependent) (Figure 3).18 On the other hand, necroptosis is caspase-independent and is mediated by the receptor interacting protein kinases 1 and 3 (RIP1 and 3) and mixed lineage kinase domain-like protein (MLKL) (Figure 3).19

Figure 3. Simplified scheme of programmed cell death. Apoptosis and necroptosis are two forms of regulated cell death. Apoptosis can be induced by various stimuli including chemotherapeutic agents and radiotherapy (intrinsic pathway). These agents indirectly shift the balance between pro- and anti-apoptotic members of the Bcl-2 family of proteins. Pro-apoptotic Bcl-2 proteins induce mitochondrial outer membrane permeabilization (MOMP), which results in the release of cytochrome C and subsequent activation of caspase 9 (Casp9), which activates executioner caspases 3 and 7. Apoptosis can also be induced by death receptor activation (extrinsic pathway), whereby caspase 8 activates caspases 3 and 7 directly, and induces MOMP via activation of the Bcl-2 family protein BID to tBID. Necroptosis can also be initiated by death receptor activation. When death receptor pathways are activated and prosurvival pathways are repressed, caspase 8 acts as the molecular switch to determine cell fate. When caspase 8 is active, it cleaves RIP1, suppressing necroptotic signaling, and apoptosis proceeds. Necroptosis can be initiated when caspase 8 is inactive. Autophosphorylated RIP1 phosphorylates RIP3, which then phosphorylates Mixed Lineage Kinase Domain-like protein (MLKL). Phospho-MLKL (pMLKL) oligomers form pores in the plasma membrane which causes calcium influx, cell swelling, and release of intracellular contents including inflammatory damageassociated molecular patterns (DAMPs).

Lipids are important regulators of programmed cell death. For instance, ferroptosis, an irondependent form of cell death20, is mediated by the dysregulation of proteins leading to the formation of lipid hydroperoxides.21,22 Apoptosis and necroptosis are of particular interest to our research group due to the substantially different membrane-related changes that occur during these processes (Figure 2). Apoptosis is characterized by cell shrinking, membrane blebbing, and formation of apoptotic bodies18, while necroptotic cell death results in cell swelling and loss of plasma membrane integrity (Figure 2).19 2.1 Lipids in apoptosis 2.1.1 Sphingolipids in apoptosis Several classes of lipids, including phospholipids and sphingolipids, have been associated with 5 ACS Paragon Plus Environment

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apoptosis. For instance, phosphatidylserine activates phagocytosis during apoptosis23 (Figure 2), while plasmalogens, ether-linked phospholipids, may be involved in suppressing neuronal apoptosis.24 Sphingolipids are membrane lipids with diverse roles during apoptosis.25-27 Sphingosine-1phosphate regulates cell growth and survival.25 Ceramides, on the other hand, promote apoptosis.27 It has been proposed that ceramides might act as pro-apoptotic messengers by increasing the permeability of the mitochondrial outer membrane, thereby contributing to the release of cytochrome c, which mediates downstream activation of caspases (Figure 2).27 Ceramides and dihydroceramides are composed of a fatty acyl chain linked to a sphingosine or sphinganine backbone, respectively. The variety of fatty acyl chains results in a diverse pool of lipids with potentially different functions based on their chemical structures.2 We recently showed that distinct ceramide species are differentially regulated as apoptosis progresses.28 For example, specific ceramides showed depletions during early apoptosis in a colorectal cancer cell line, followed by profound increases during a later apoptotic state. Notably, dihydroceramides also strongly accumulated in a time- and fatty acyl chain-dependent manner during apoptosis.28 Such precise and dynamic regulation of individual sphingolipid species was unexpected, indicating that distinct lipid species may have defined roles in apoptosis. Cancer cells undergo significant metabolic alterations, including changes in lipid metabolism, to provide necessary materials for accelerated cell division as well as biosynthesis of signaling molecules associated with oncogenic activity.29 These changes can result in alterations in the cellular lipidome, which might have profound effects on how cancerous and non-cancerous cells utilize these molecules differently under stress conditions. We investigated this in an experimental framework where we compared lipid regulation in a variety of cancerous and noncancerous cells, including one of each originating from colon tissue, during apoptosis. We found that sphingolipids are indeed regulated differently in cancerous vs. non-cancerous cells during apoptosis: sphingolipids in general, and ceramides in particular, showed profound accumulations in cancerous cells as compared to non-cancerous cells during apoptosis.28 We also showed that cancerous cells have lower basal levels of ceramides, potentially due to lower expression of specific ceramide synthases as compared to non-cancerous cells. Furthermore, exogenous addition of C16-ceramide induced higher levels of toxicity in cancerous cells compared to noncancerous cells, suggesting that cancerous cells are more sensitive to changes in ceramide levels.28 Collectively, our results suggested that downregulation of ceramide synthases, which results in lower ceramide levels might be a mechanism cancerous cells use to evade apoptosis. Overall, these findings illuminate the way toward future investigations on the involvement of the sphingolipid pathway in oncogenic transformations. 2.1.2. Non-membrane lipids in apoptosis The majority of lipids reside in cellular membranes and function as structural and/or signaling molecules.30 Triacylglycerols (TAGs) are one of the few groups of lipids that are mostly found outside of membrane structures. They are comprised of a glycerol backbone esterified with three fatty acyl chains and are primarily stored in lipid droplets, an endoplasmic reticulum-derived dynamic organelle consisting of a neutral lipid core surrounded by a monolayer of phospholipids 6 ACS Paragon Plus Environment

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and proteins (Figure 4).31 TAGs are traditionally considered as energy storage molecules and very little is known about their structural or signaling roles.31,32 Using an untargeted lipidomics approach, we recently discovered that levels of specific TAGs change during apoptosis. Polyunsaturated fatty acyl chain containing TAGs (PUFA-TAGs) accumulated, while PUFA-containing species from other lipid families remained the same during this process.33 Intrigued by this specificity, we investigated three key enzymes that maintain cellular TAG levels: diacylglycerol acyltransferase (DGAT) 1 and 2, and adipose triglyceride lipase (ATGL), which catalyze the rate-determining steps of TAG biosynthesis and breakdown, respectively (Figure 4). We found that DGATs were activated at the transcriptional and enzymatic levels, suggesting that activated TAG biosynthesis is responsible for the observed TAG accumulation.33 We also found that mRNA levels of long chain acyl-coenzyme A synthetase 1 (ACSL1), which catalyzes the activation of long-chain fatty acids to fatty acylCoAs to be used in the synthesis of complex lipids34, were elevated during apoptosis.33 The simultaneous activation of DGATs and ACSL1 strongly supported the role of activated TAG biosynthesis as the key mechanism responsible for the accumulation of TAGs and, consequently, lipid droplets we observed during apoptosis. Previous studies have suggested that cells sequester excess fatty acids by incorporating them into TAGs which are stored inside lipid droplets.31 In Atgl-/- mice macrophages, TAG accumulation has been shown to induce mitochondrial-dependent apoptosis.35 Perhaps more interestingly, a recent study in Drosophila larvae showed that PUFAs, which are prone to lipid peroxidation36, are stored as TAGs in lipid droplets, which prevent lipid peroxidation-induced membrane damage under oxidative stress in glial cells.37 In our study, we observed a concurrent accumulation of lipid droplets and PUFA-TAGs.33 Thus, it is possible that PUFA-TAGs are stored in lipid droplets to protect cells from potential membrane damage induced by oxidative stress during apoptosis (Figure 4). Overall, our results suggested a new functional role for TAGs and lipid droplets during apoptosis. Current studies in our laboratory are underway investigating the content of lipid droplets during apoptosis and the functional consequences of perturbing TAG biosynthesis during this process.


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Figure 4. Potential role of PUFA-containing lipids under oxidative stress. FA, fatty acid; PUFA, polyunsaturated fatty acid; ACSLs, long chain acyl-coenzyme A synthetases (key enzymes in free fatty acid activation); DAG, diacylglycerol; TAG, triacylglycerol; DGAT 1&2, diacylglycerol acyltransferase 1 & 2 (key enzymes involved in TAG biosynthesis); ATGL, adipose triglyceride lipase (involved in TAG breakdown); ROS, reactive oxygen species. Both de novo lipogenesis and FA uptake contribute to the cellular FA pool. FAs are activated and can then be modified (ex. addition of double bonds to form PUFA) and/or used for the synthesis of downstream lipids. PUFAs (highlighted in blue) can be used to synthesize membrane lipids (namely phospholipids), or glycerolipids such as TAGs, which are sequestered in lipid droplets. In the presence of ROS, PUFA-containing membrane lipids undergo lipid peroxidation and form lipid hydroperoxides (example highlighted in red), which change membrane permeability and induce membrane damage.

2.2. Lipids in necroptosis In recent years, there has been increasing evidence that lipids play a role in other types of regulated cell death, which are also important to human health and disease.16 Our group has been particularly intrigued by the role of lipids during necroptosis, a form of regulated necrotic cell death which involves distinct membrane-related phenotypes compared to apoptosis.38 Necroptotic cell death is ultimately caused by pore formation in the plasma membrane, which results in the release of cytoplasmic material to the extracellular environment (Figures 2 and 3).19 This can cause inflammation at the organismal level14; as such, necroptosis is involved in a number of inflammatory disease states including ischemia/reperfusion injuries and neurodegenerative diseases.14,38 Much research has been devoted to study the proteins that are involved in necroptosis. Briefly, death receptor activation results in the formation of a cytosolic complex containing phosphorylated RIP1, RIP3 and MLKL proteins under certain conditions where caspase activity is low. Phosphorylated MLKL (pMLKL) oligomers translocate to the plasma membrane, where they are thought to form cation channels (Figure 3).39,40 This process is lipid-dependent. Phosphatidylinositol phosphates (PIPs, Figure 2) can interact with pMLKL via electrostatic 8 ACS Paragon Plus Environment

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interactions with a positively charged patch of amino acids located in its N-terminal helical bundle domain.39 It has been shown that this helical domain interacts with PIP-containing liposomes, which results in the permeabilization of the membrane. This permeabilization can be prevented by replacing PIPs with phosphatidylinositols.39 Although perturbing the biosynthesis of PIPs prevented TNF-induced necroptosis39, the precise contribution of individual PIP species remains to be elucidated. In addition to these studies, which suggest a crucial functional role of PIPs during necroptosis, changes in sphingolipid composition have also been reported to occur during necroptosis.41 However, a great deal of work remains to fully understand the role of these and other lipids in this process. One of the focuses of our laboratory is to understand the role of lipids in the distinct membranerelated phenotypes of necroptosis, such as the permeabilization of the plasma membrane (Figure 2). We recently conducted the first global lipidomics investigation of necroptosis and identified lipids whose levels are highly regulated during this process. We found that very long chain fatty acids (fatty acids containing ≥ 22 carbons42) accumulate during necroptosis.43 These species had not been previously associated with necroptotic cell death. Furthermore, inactivation of fatty acid biosynthesis, which prevented very long chain fatty acid accumulation during necroptosis, prevented cell death and restored plasma membrane integrity. These results strongly support a functional role for very long chain fatty acids during necroptosis.43 We envision that these lipids, which are quite hydrophobic, may disrupt cellular membranes such as the mitochondrial or plasma membrane, and thus play a role in the damage that occurs to these membranes during necroptosis, either as small molecules or as a part of larger biomolecules such as lipidated proteins. 3. Lipids & cellular senescence Our cells age and stop dividing, and we do not exactly know how. Cellular senescence, the ceasing of cell division, is accompanied by progressive and diverse phenotypic alterations.44 A key component of organismal aging, cellular senescence is driven by the p53/p21 and/or the p16/pRB pathways.44 Although their growth is arrested, senescent cells are metabolically active and may contribute to age-related diseases, cancer progression and wound repair.13 Research efforts over the last couple of decades have primarily focused on elucidating pivotal proteins and key characteristics of cellular senescence. Senescent cells are characterized by gene alterations, a distinct secretory phenotype, and an increase in size and lysosomal content (Figure 2).44-46 Given the membrane-related alterations associated with cellular senescence, the role of lipids in replicative senescence is an exciting research direction which largely remains to be explored. Previous studies have associated numerous membrane-, and thus, lipid-related phenotypes with this process.46 At the organismal level, changes in certain lipid families have been associated with aging. For instance, fatty acid levels have been shown to be altered as different organisms age (reviewed in47,48). It has been suggested that elevated levels of unsaturated fatty acids may play a role in protecting against oxidative stress and damage, thereby extending lifespan.48,49 It has also been shown that ceramides accumulate during replicative senescence in human lung fibroblast cells, and chronic addition of C6-ceramide to young fibroblast cells induced a senescent-like phenotype.50 Collectively, these findings suggest that lipids are involved in senescence. 9 ACS Paragon Plus Environment

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In an effort to understand how the lipidome changes during replicative senescence, our group has integrated transcriptomics and untargeted lipidomics analyses in human fibroblast cells.51 We cultured fibroblast cells from an early population doubling until they naturally reached the senescent state. Given that senescence is a complex process regulated by multiple pathways, we envisioned that investigating the changes at the transcriptional level would provide valuable information. Thus, we carried out transcriptomics analysis in early population and senescent cells and found that lipid-related genes showed significantly greater differences in expression levels between proliferating and senescent cells as compared to the rest of the transcriptome.51 A closer look at the lipid-related transcripts revealed that lipid biosynthetic pathways were regulated differentially as cells senesce. For instance, expression levels of genes related to fatty acid biosynthesis remained the same, while expression levels of certain fatty acid desaturases and elongases changed as the cells became senescent.51 These findings suggest that control of lipid-related pathways at the RNA level is essential to the progression of replicative senescence. Investigation of changes in lipids at the metabolite level via LC-MS-based untargeted lipidomics revealed an unexpected finding: among hundreds of species we analyzed, PUFA-TAGs constituted the majority of the changes between dividing and senescent cells, most likely due to activated fatty acid uptake via CD36, a cell surface free fatty acid transporter, and increased levels of ACSL4 resulting in the storage of these species as TAGs (Figure 4). Intriguingly, analysis of other PUFA-containing species in the lipidome of senescent cells obtained from multiple cell lines confirmed that the accumulation of these species was specific to TAGs. Previous studies in plants have shown an overall accumulation of TAGs as they reached their growth capacity, which has been linked to nutrient limitation.52,53 However, to the best of our knowledge, we demonstrated for the first time that lipids are highly regulated at the transcriptional and metabolite level during senescence, and that PUFA-TAGs accumulate in mammalian cells, suggesting that these species could be functionally involved in this process.51 As we discussed above, TAGs are generally non-membrane lipids with major roles in energy storage. However, we observed their upregulation in mammalian and plant cells under stress conditions.33,51,52 Both apoptosis and replicative senescence are accompanied by increased oxidative stress54,55, and both processes are accompanied by an increase in specific lipids containing PUFA species, which are particularly prone to peroxidation.36 Based on these insights, we raise the following question: Could accumulation of PUFA-TAGs be involved in a protective mechanism against oxidative stress? We hypothesize that sequestering PUFAs as TAGs, which are isolated within lipid droplets, protects cells from membrane damage resulting from lipid peroxidation under oxidative stress (Figure 4). Our findings support this hypothesis, at least in the context of apoptosis and senescence. Though further studies are needed to investigate the generality of this phenomenon during different cellular processes, we hypothesize that inhibition of TAG biosynthesis which will result in the incorporation of PUFAs to phospholipids could enhance toxicity during these processes. This, in turn, could be a novel approach to modulate apoptosis and/or senescence in different human diseases and pathologies. 4. Emerging non-traditional roles of lipids in cellular fates


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It has always been clear that lipids are the fundamental structural component of cellular membranes, and we all know that “fats” are used to store energy. It is only recently appreciated, however, that the thousands of unique lipids have many diverse roles in fundamental cellular processes such as cell division and cell death.32 Two major key technological advancements have facilitated recent discoveries which have promoted this view. First, advancements in -omics era instrumentation and data processing have boosted our ability to detect and analyze lipids at high sensitivity. In parallel, databases such as LIPID MAPS56 and METLIN57 help to disseminate knowledge and resources that would otherwise require years of expertise. Second, the expansion of lipid probes has enabled subcellular, spatio-temporal investigations of lipid trafficking7 and their interactions10 with other biomolecules. These approaches have facilitated exciting discoveries of many different roles of bioactive lipid species. Collectively, we are now beginning to understand that a plethora of different structures in the lipidome have unique functions. Untargeted lipidomics has had an extensive impact on the research questions formulated by our laboratory and others. In this perspective, we focused on three different lipid families that were prioritized based on our untargeted lipidomics results. We have shown that different ceramide species accumulate in a structure-specific manner in cancerous cells and proposed de novo ceramide biosynthesis as a potential pathway in the oncogenic transformations in colon tissue. We also demonstrated that PUFA-TAGs are actively regulated in processes involving oxidative stress, where we believe that their accumulation could be a way for the cell to prevent lipid hydroperoxide-induced toxicity. Most recently, we made a functional link between very long chain fatty acids and necroptosis, and proposed that these species could be involved in the permeabilization of cellular membranes during this process. Our work, along with others (only a few of which we were able to cover in this perspective), has brought to light the many possible functions of lipids in different cellular processes. Much work remains in developing new tools and technologies to understand the numerous roles that lipids play, and we envision that there are many more roles yet to be discovered. We hope that we have inspired other researchers to join us and take up the challenge. Acknowledgements. The studies from the authors’ laboratory described in the perspective were supported by start-up funds to G.E.A.-G. (Research Foundation, SUNY) and the National Science Foundation (Award No: CBET-1438172).


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For Table of Contents Use Only Non-canonical Roles of Lipids in Different Cellular Fates Darleny Y. Lizardo†, Laura R. Parisi†, Nasi Li†, G. Ekin Atilla-Gokcumen* Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260, United States †

These authors contributed equally.

* Correspondence: [email protected]


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Biochemistry

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C., Bertrand, M. J. M., and Vandenabeele, P. (2014) MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates, Cell Rep. 7, 971-981. [40] Xia, B., Fang, S., Chen, X., Hu, H., Chen, P., Wang, H., and Gao, Z. (2016) MLKL forms cation channels, Cell Res. 26, 517-528. [41] Sawai, H., Ogiso, H., and Okazaki, T. (2015) Differential changes in sphingolipids between TNFinduced necroptosis and apoptosis in U937 cells and necroptosis-resistant sublines, Leuk. Res. 39, 964-970. [42] Kihara, A. (2012) Very long-chain fatty acids: elongation, physiology and related disorders, J. Biochem. 152, 387-395. [43] Parisi, L. R., Li, N., and Atilla-Gokcumen, G. E. (2017) Very long chain fatty acids are functionally involved in necroptosis, Cell Chem. Biol., http://dx.doi.org/10.1016/j.chembiol.2017.08.026. [44] Rodier, F., and Campisi, J. (2011) Four faces of cellular senescence, J. Cell Biol. 192, 547-556. [45] Hayflick, L., and Moorhead, P. S. (1961) The serial cultivation of human diploid cell strains, Exp. Cell Res. 25, 585-621. [46] Dimri, G. P., and Campisi, J. (1994) Molecular and cell biology of replicative senescence, Cold Spring Harb. Symp. Quant. Biol. 59, 67-73. [47] Schroeder, E. A., and Brunet, A. (2015) Lipid profiles and signals for long life, Trends Endocrinol. Metab. 26, 589-592. [48] Ademowo, O. S., Dias, H. K. I., Burton, D. G. A., and Griffiths, H. R. (2017) Lipid (per) oxidation in mitochondria: an emerging target in the ageing process?, Biogerontology. https://doi.org/10.1007/s10522-017-9710-z. [49] Pararasa, C., Ikwuobe, J., Shigdar, S., Boukouvalas, A., Nabney, I. T., Brown, J. E., Devitt, A., Bailey, C. J., Bennett, S. J., and Griffiths, H. R. (2016) Age-associated changes in long-chain fatty acid profile during healthy aging promote pro-inflammatory monocyte polarization via PPARgamma, Aging Cell 15, 128-139. [50] Venable, M. E., Lee, J. Y., Smyth, M. J., Bielawska, A., and Obeid, L. M. (1995) Role of ceramide in cellular senescence, J. Biol. Chem. 270, 30701-30708. [51] Lizardo, D. Y., Lin, Y. L., Gokcumen, O., and Atilla-Gokcumen, G. E. (2017) Regulation of lipids is central to replicative senescence, Mol. Biosyst. 13, 498-509. [52] Matich, E., Butry, D., Ghafari, M., del Solar, V., Camgoz, E., Pferifer, B. A., Aga, D. S., Haznedaroglu, B. Z., and Atilla-Gokcumen, G. E. (2016) Mass spectrometry-based metabolomics of value-added biochemicals from Ettlia oleoabundans, Algal Res. 19, 146-154. [53] Brocard, L., Immel, F., Coulon, D., Esnay, N., Tuphile, K., Pascal, S., Claverol, S., Fouillen, L., Bessoule, J. J., and Brehelin, C. (2017) Proteomic analysis of lipid droplets from arabidopsis aging leaves brings new insight into their biogenesis and functions, Front. Plant Sci. 8, 1-23. [54] Liu, D., and Xu, Y. (2011) p53, oxidative stress, and aging, Antioxid. Redox Signal. 15, 1669-1678. [55] Childs, B. G., Baker, D. J., Kirkland, J. L., Campisi, J., and van Deursen, J. M. (2014) Senescence and apoptosis: dueling or complementary cell fates?, EMBO Rep. 15, 1139-1153. [56] Sud, M., Fahy, E., Cotter, D., Dennis, E. A., and Subramaniam, S. (2012) LIPID MAPS-Nature lipidomics gateway: an online resource for students and educators interested in lipids, J. Chem. Educ. 89, 291-292. [57] Tautenhahn, R., Cho, K., Uritboonthai, W., Zhu, Z., Patti, G. J., and Siuzdak, G. (2012) An accelerated workflow for untargeted metabolomics using the METLIN database, Nat. Biotechnol. 30, 826-828.


Biochemistry

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Figure 1. Major classes of lipids in the mammalian lipidome. Representative structures of each class are shown here. Core structures of individual lipid classes are highlighted in red. Examples of possible head groups are highlighted in blue. Chemical diversity within each lipid class is achieved by different combinations of head groups and fatty acyl chains, which may vary in length and/or number of double bonds. 129x155mm (300 x 300 DPI)

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Biochemistry

Figure 2. Involvement of lipids in different cellular fates. Apoptosis (A), necroptosis (N), and senescence (S) are three cellular fates which are accompanied by distinct membrane-related phenotypes and changes in lipid composition. Apoptosis is characterized by DNA damage, cell shrinking, membrane blebbing, and formation of apoptotic bodies. Necroptosis results in cellular/organelle swelling and loss of plasma membrane integrity. Senescence is characterized by an increase in cell size and lysosomal content (orange), and a distinct secretory phenotype. Polyunsaturated fatty acyl chain containing TAGs (PUFA-TAGs) accumulate during apoptosis and senescence and may possibly protect cells from oxidative stress-induced membrane damage. Ceramides, a central sphingolipid, accumulate in all three processes. In apoptosis, ceramides may facilitate pore formation in the mitochondrial outer membrane; their roles in senescence and necroptosis are less well understood. During apoptosis, clearance of apoptotic bodies is facilitated by the translocation of phosphatidylserines (PSs) from the inner leaflet of the plasma membrane to the outer leaflet, where they act as a signaling lipid to stimulate phagocytosis. Very long chain fatty acids (VLCFAs) accumulate during necroptosis; their precise functional role has yet to be elucidated. SpecificCertain


Biochemistry

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phosphatidylinositol phosphates (PIPs) are known to recruit phosphorylated MLKL to the plasma membrane, which cause the hallmark plasma membrane permeabilization during necroptotic cell death. 128x159mm (300 x 300 DPI)


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Biochemistry

Figure 3. Simplified scheme of programmed cell death. Apoptosis and necroptosis are two forms of regulated cell death. Apoptosis can be induced by various stimuli including chemotherapeutic agents and radiotherapy (intrinsic pathway). These agents indirectly shift the balance between pro- and anti-apoptotic members of the Bcl-2 family of proteins. Pro-apoptotic Bcl-2 proteins induce mitochondrial outer membrane permeabilization (MOMP), which results in the release of cytochrome C and subsequent activation of caspase 9 (Casp9), which activates executioner caspases 3 and 7. Apoptosis can also be induced by death receptor activation (extrinsic pathway), whereby caspase 8 activates caspases 3 and 7 directly, and induces MOMP via activation of the Bcl-2 family protein BID to tBID. Necroptosis can also be initiated by death receptor activation. When death receptor pathways are activated and pro-survival pathways are repressed, caspase 8 acts as the molecular switch to determine cell fate. When caspase 8 is active, it cleaves RIP1, suppressing necroptotic signaling, and apoptosis proceeds. Necroptosis can be initiated when caspase 8 is inactive. Autophosphorylated RIP1 phosphorylates RIP3, which then phosphorylates Mixed Lineage Kinase Domainlike protein (MLKL). Phospho-MLKL (pMLKL) oligomers form pores in the plasma membrane which causes calcium influx, cell swelling, and release of intracellular contents including inflammatory damage-associated molecular patterns (DAMPs). 69x58mm (600 x 600 DPI)


Biochemistry

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Figure 4. Potential role of PUFA-containing lipids under oxidative stress. FA, fatty acid; PUFA, polyunsaturated fatty acid; ACSLs, long chain acyl-coenzyme A synthetases (key enzymes in free fatty acid activation); DAG, diacylglycerol; TAG, triacylglycerol; DGAT 1&2, diacylglycerol acyltransferase 1 & 2 (key enzymes involved in TAG biosynthesis); ATGL, adipose triglyceride lipase (involved in TAG breakdown); ROS, reactive oxygen species. Both de novo lipogenesis and FA uptake contribute to the cellular FA pool. FAs are activated and can then be modified (ex. addition of double bonds to form PUFA) and/or used for the synthesis of downstream lipids. PUFAs (highlighted in blue) can be used to synthesize membrane lipids (namely phospholipids), or glycerolipids such as TAGs, which are sequestered in lipid droplets. In the presence of ROS, PUFA-containing membrane lipids undergo lipid peroxidation and form lipid hydroperoxides (example highlighted in red), which change membrane permeability and induce membrane damage. 91x70mm (300 x 300 DPI)


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Biochemistry

Table of Contents Graphic 88x44mm (300 x 300 DPI)


Noncanonical Roles of Lipids in Different Cellular Fates - American

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