RESUMO
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.
Assuntos
Necroptose , Proteínas Quinases , Proteínas Quinases/metabolismo , Fosforilação , Membrana Celular/metabolismo , ApoptoseRESUMO
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 the 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 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, C184, C269 and C286 are the possible acylation sites. Using all atom molecular dynamic simulations, we identify differences that the acylation of MLKL causes at the protein and membrane level. Through systematic investigations of the S-palmitoyltransferases that might acylate MLKL 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 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.
RESUMO
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.
Assuntos
Necroptose , Proteoma , Humanos , Necroptose/genética , Proteoma/metabolismo , Proteínas Quinases/genética , Apoptose/genética , FosforilaçãoRESUMO
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.
RESUMO
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.
RESUMO
Liposomes containing small amounts of porphyrin-phospholipid (PoP) have been shown to encapsulate small molecular weight cargos and then release them upon exposure to red light. A putative mechanism involves transient pore formation in the bilayer induced by PoP-mediated photo-oxidation of unsaturated lipids. However, little is known about the properties of such pores. This study assesses whether large carbohydrate and protein molecules could be released from PoP liposomes upon red light exposure. A small fluorophore with â¼0.5 kDa in molecular weight, fluorescently labeled dextrans of â¼5 and â¼500 kDa, and a â¼240 kDa fluorescent protein were passively entrapped in PoP liposomes. When exposed to 665 nm irradiation, liposomes containing PoP, but not liposomes lacking it, released all these cargos in a size-dependent manner that occurred with oxidation of unsaturated lipids included in the bilayer. Thus, this study demonstrates the feasibility of light-triggered release of large biomacromolecules from liposomes.
Assuntos
Lipossomos , Porfirinas , Corantes Fluorescentes , FosfolipídeosRESUMO
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.