Epilipidomics platform for holistic profiling of oxidized complex lipids in blood plasma of obese individuals

Lipids are a structurally diverse class of biomolecules which can undergo a variety of chemical modifications. Among them, lipid (per)oxidation attracts most of the attention due to its significance in regulation of inflammation, cell proliferation and death programs. Despite their apparent regulatory significance, the molecular repertoire of oxidized lipids remains largely elusive as accurate annotation of lipid modifications is challenged by their low abundance and largely unknown, biological context-dependent structural diversity. Here we provide a holistic workflow based on the combination of bioinformatics and LC-MS/MS technologies to support identification and relative quantification of oxidized complex lipids in a modification type- and position-specific manner. The developed methodology was used to identify epilipidomics signatures of lean and obese individuals with and without type II diabetes. Characteristic signature of lipid modifications in lean individuals, dominated by the presence of modified octadecanoid acyl chains in phospho- and neutral lipids, was drastically shifted towards lipid peroxidation-driven accumulation of oxidized eicosanoids, suggesting significant alteration of endocrine signalling by oxidized lipids in metabolic disorders.

Sonic hedgehog is basolaterally sorted from the TGN and transcytosed to the apical domain involving Dispatched-1 at Rab11-ARE

Hedgehog (Hh) secretion from apical and/or basolateral domains occurs in different epithelial cells impacting development and tissue homeostasis. Palmitoylation and cholestyrolation attach Hh proteins to membranes and Dispatched-1 (Disp-1) promotes their release. How these lipidated proteins are handled by the complex secretory and endocytic pathways of polarized epithelial cells remains unknown. We show that MDCK cells address newly synthesized sonic hedgehog (Shh) from the TGN to the basolateral cell surface and then to the apical domain through a transcytosis pathway that includes Rab11-apical recycling endosomes (Rab11-ARE). Both palmitoylation and cholestyrolation contribute to this sorting behavior, otherwise Shh lacking these lipid modifications is unpolarized. Disp-1 mediates first basolateral secretion from the TGN and then transcytosis from the Rab11-ARE. At steady state, Shh predominates apically and can be basolaterally transcytosed. This complex Shh trafficking provides several steps for regulation and variation in different epithelia, subordinating the apical to the basolateral secretion.

Changes in Key Mitochondrial Lipids Accompany Mitochondrial Dysfunction and Oxidative Stress in NAFLD

Nonalcoholic fatty liver disease (NAFLD) is a dysmetabolic hepatic damage of increasing severity: simple fat accumulation (steatosis), nonalcoholic steatohepatitis (NASH), and hepatic fibrosis. Oxidative stress is considered an important factor in producing hepatocyte injury associated with NAFLD progression. Studies also suggest a link between the accumulation of specific hepatic lipid species, mitochondrial dysfunction, and the progression of NAFLD. However, it is unclear whether mitochondrial lipid modifications are involved in NAFLD progression. To gain insight into the relationship between mitochondrial lipids and disease progression through different stages of NAFLD, we performed lipidomic analyses on mouse livers at different stages of western diet-induced NAFLD, with or without hepatic fibrosis. After organelle separation, we studied separately the mitochondrial and the “nonmitochondrial” hepatic lipidomes. We identified 719 lipid species from 16 lipid families. Remarkably, the western diet triggered time-dependent changes in the mitochondrial lipidome, whereas the “nonmitochondrial” lipidome showed little difference with levels of hepatic steatosis or the presence of fibrosis. In mitochondria, the changes in the lipidome preceded hepatic fibrosis. In particular, two critical phospholipids, phosphatidic acid (PA) and cardiolipin (CL), displayed opposite responses in mitochondria. Decrease in CL and increase in PA were concurrent with an increase of coenzyme Q. Electron paramagnetic resonance spectroscopy superoxide spin trapping and Cu2+ measurement showed the progressive increase in oxidative stress in the liver. Overall, these results suggest mitochondrial lipid modifications could act as an early event in mitochondrial dysfunction and NAFLD progression.

Protein Lipidation by Palmitoylation and Myristoylation in Cancer

Posttranslational modification of proteins with lipid moieties is known as protein lipidation. The attachment of a lipid molecule to proteins endows distinct properties, which affect their hydrophobicity, structural stability, localization, trafficking between membrane compartments, and influences its interaction with effectors. Lipids or lipid metabolites can serve as substrates for lipidation, and the availability of these lipid substrates are tightly regulated by cellular metabolism. Palmitoylation and myristoylation represent the two most common protein lipid modifications, and dysregulation of protein lipidation is strongly linked to various diseases such as metabolic syndromes and cancers. In this review, we present recent developments in our understanding on the roles of palmitoylation and myristoylation, and their significance in modulating cancer metabolism toward cancer initiation and progression.

NPalmitoylDeep-PseAAC: A Predictor for N-Palmitoylation sites in Proteins using Deep Representations of Proteins and PseAAC via modified 5-steps rule

Background: Among all the major Post-translational modification, lipid modifications possess special significance due to their widespread functional importance in eukaryotic cells. There exist multiple types of lipid modifications and Palmitoylation, among them, is one of the broader types of modification, having three different types. The N-Palmitoylation is carried out by attachment of palmitic acid to an N-terminal cysteine. Due to the association of N-Palmitoylation with various biological functions and diseases such as Alzheimer’s and other neurodegenerative diseases, carrying out important processes in the life cycle of various pathogens, its identification is very important. Objective: The in vitro, ex vivo and in vivo identification of Palmitoylation is laborious, time-taking and costly. There is a dire need of an efficient and accurate computational model to help researchers and biologists identifying these sites, in an easy manner. Herein, we propose a novel prediction model for identification of N-Palmitoylation sites in proteins. Method: Proposed prediction model is developed by combining the Chou’s Pseudo Amino Acid Composition (PseAAC) with deep neural networks. We used well-known deep neural networks (DNNs) for both the tasks of learning a feature representation of peptide sequences and developing prediction model to perform classification. Results: Among different DNNs, Gated Recurrent Unit (GRU) based RNN model showed highest scores in terms of accuracy, and all other computed measures, and outperforms all the previously reported predictors. Conclusion: The proposed GRU based RNN model can help identifying N-Palmitoylation in a very efficient and accurate manner which can help scientists understand the mechanism of this modification in proteins.

Characteristics, immobilization, and application of Candida rugosa lipase: a review

Candida rugosa lipase (CRL) is one of the lipases widely used in various food industries and studies, especially in linkage with the modification of lipids. This review discusses CRL, including CRL features (molecular biology, the structure of the enzyme protein, the flap/ lid), catalytic mechanisms and substrates specificity, CRL immobilization technologies, and various applications in lipid modifications. CRL has five isoenzymes, namely LIP1 - LIP5, then develops again into LIP1 - LIP8. However, LIP1 is the most commonly found isoenzymes. CRL has a structure similar to that of Geotricum candidum lipase, has a flap/ lid, which is an active side cover in the form of α-helix, which is relatively shorter than other lipases. The active site of the CRL consists of triads ser-209, His-449, and Glu-341, while the catalytic mechanism of the CRL is the same as the other lipases by the nucleophilic attack. CRL catalyzes triacylglycerol at all positions randomly and has hydrolysis and synthesis activities that are strongly affected by the presence of water in the reaction system. CRL can be used for various lipid modifications through hydrolysis, esterification, interesterification/transesterification, and alcoholysis/ glycerolysis reactions.

Protein expression of prenyltransferase subunits in postmortem schizophrenia dorsolateral prefrontal cortex

The pathophysiology of schizophrenia includes altered neurotransmission, dysregulated intracellular signaling pathway activity, and abnormal dendritic morphology that contribute to deficits of synaptic plasticity in the disorder. These processes all require dynamic protein–protein interactions at cell membranes. Lipid modifications target proteins to membranes by increasing substrate hydrophobicity by the addition of a fatty acid or isoprenyl moiety, and recent evidence suggests that dysregulated posttranslational lipid modifications may play a role in multiple neuropsychiatric disorders, including schizophrenia. Consistent with these emerging findings, we have recently reported decreased protein S-palmitoylation in schizophrenia. Protein prenylation is a lipid modification that occurs upstream of S-palmitoylation on many protein substrates, facilitating membrane localization and activity of key intracellular signaling proteins. Accordingly, we hypothesized that, in addition to palmitoylation, protein prenylation may be abnormal in schizophrenia. To test this, we assayed protein expression of the five prenyltransferase subunits (FNTA, FNTB, PGGT1B, RABGGTA, and RABGGTB) in postmortem dorsolateral prefrontal cortex from patients with schizophrenia and paired comparison subjects (n = 13 pairs). We found decreased levels of FNTA (14%), PGGT1B (13%), and RABGGTB (8%) in schizophrenia. To determine whether upstream or downstream factors may be driving these changes, we also assayed protein expression of the isoprenoid synthases FDPS and GGPS1 and prenylation-dependent processing enzymes RCE and ICMT. We found these upstream and downstream enzymes to have normal protein expression. To rule out effects from chronic antipsychotic treatment, we assayed FNTA, PGGT1B, and RABGGTB in the cortex from rats treated long-term with haloperidol decanoate and found no change in the expression of these proteins. Given the role prenylation plays in localization of key signaling proteins found at the synapse, these data offer a potential mechanism underlying abnormal protein–protein interactions and protein localization in schizophrenia.

Palmitoylation of BMPR1a regulates neural stem cell fate

Neural stem cells (NSCs) generate neurons and glial cells throughout embryonic and postnatal brain development. The role of S-palmitoylation (also referred to as S-acylation), a reversible posttranslational lipid modification of proteins, in regulating the fate and activity of NSCs remains largely unknown. We used an unbiased screening approach to identify proteins that are S-acylated in mouse NSCs and showed that bone morphogenic protein receptor 1a (BMPR1a), a core mediator of BMP signaling, is palmitoylated. Genetic manipulation of S-acylated sites affects the localization and trafficking of BMPR1a and leads to altered BMP signaling. Strikingly, defective palmitoylation of BMPR1a modulates NSC function within the mouse brain, resulting in enhanced oligodendrogenesis. Thus, we identified a mechanism regulating the behavior of NSCs and provided the framework to characterize dynamic posttranslational lipid modifications of proteins in the context of NSC biology.