Proteomic analysis of fatty-acylated proteins in mammalian cells with chemical reporters reveals S-acylation of histone H3 variants

Bioorthogonal chemical reporters are useful tools for visualizing and identifying post-translational modifications on proteins. Here we report the proteomic analysis of mammalian proteins targeted by a series of fatty acid chemical reporters ranging from myristic to stearic acid. The large-scale analysis of total cell lysates from fully solubilized Jurkat T cells identified known fatty-acylated proteins and many new candidates, including nuclear proteins and in particular histone H3 variants. We demonstrate that histones H3.1, H3.2, and H3.3 are modified with fatty acid chemical reporters and identify the conserved cysteine 110 as a new site of S-acylation on histone H3.2. This newly discovered modification of histone H3 could have implications for nuclear organization and chromatin regulation. The unbiased proteomic analysis of fatty-acylated proteins using chemical reporters has revealed a greater diversity of lipid-modified proteins in mammalian cells and identified a novel post-translational modification of histones.     

Fatty acid acylation of vaccinia virus proteins.

Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.     

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P?], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P? that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P? headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P? headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P?-binding proteins.     

Differential expression and glycative damage affect specific mitochondrial proteins with aging in rat liver

Aging is accompanied by the gradual deterioration of cell functions. Particularly, mitochondrial dysfunction, associated with an accumulation of damaged proteins, is of key importance due to the central role of these organelles in cellular metabolism. However, the detailed molecular mechanisms involved in such impairment have not been completely elucidated. In the present study, proteomic analyses looking at both changes at the expression level as well as to glycative modifications of the mitochondrial proteome were performed. Two-dimensional difference gel electrophoresis analysis revealed 16 differentially expressed proteins with aging. Thirteen exhibited a decreased expression and are crucial enzymes related to OXPHOS chain complex I/V components, TCA cycle or fatty acid β-oxidation reaction. On the other hand, 2 enzymes involved in fatty acid β-oxidation cycle were increased in aged mitochondria. Immunodetection and further identification of glycated proteins disclosed a set of advanced glycation endproduct-modified proteins, including 6 enzymes involved in the fatty acid β-oxidation process, and 2 enzymes of the TCA/urea cycles. A crucial antioxidant enzyme, catalase, was among the most strongly glycated proteins. In addition, several AGE-damaged enzymes (aldehyde dehydrogenase 2, medium chain acyl-CoA dehydrogenase and 3-ketoacyl-CoA dehydrogenase) exhibited a decreased activity with age. Taken together, these data suggest that liver mitochondria in old rats suffer from a decline in their capacity for energy production, due to (i) decreased expression of OXPHOS complex I/V components and (ii) glycative damage to key fatty acid β-oxidation and TCA/urea cycle enzymes.     

Quantification and Proteomic Characterization of β-Hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice

AMP-activated protein kinase alpha 2 (AMPKα2) regulates energy metabolism, protein synthesis, and glucolipid metabolism myocardial cells. Ketone bodies produced by fatty acid β-oxidation, especially β-hydroxybutyrate, are fatty energy–supplying substances for the heart, brain, and other organs during fasting and long-term exercise. They also regulate metabolic signaling for multiple cellular functions. Lysine β-hydroxybutyrylation (Kbhb) is a β-hydroxybutyrate–mediated protein posttranslational modification. Histone Kbhb has been identified in yeast, mouse, and human cells. However, whether AMPK regulates protein Kbhb is yet unclear. Hence, the present study explored the changes in proteomics and Kbhb modification omics in the hearts of AMPKα2 knockout mice using a comprehensive quantitative proteomic analysis. Based on mass spectrometry (LC-MS/MS) analysis, the number of 1181 Kbhb modified sites in 455 proteins were quantified between AMPKα2 knockout mice and wildtype mice; 244 Kbhb sites in 142 proteins decreased or increased after AMPKα2 knockout (fold change >1.5 or <1/1.5, p < 0.05). The regulation of Kbhb sites in 26 key enzymes of fatty acid degradation and tricarboxylic acid cycle was noted in AMPKα2 knockout mouse cardiomyocytes. These findings, for the first time, identified proteomic features and Kbhb modification of cardiomyocytes after AMPKα2 knockout, suggesting that AMPKα2 regulates energy metabolism by modifying protein Kbhb.     

In vivo acylation of Dictyostelium actin with palmitic acid

Cells of Dictyostelium discoideum were incubated with [³H]palmitic acid during development, and recovery of the fatty acid label in soluble and membrane-associated proteins was investigated. One of the major labeled proteins was found exclusively in the soluble fraction. This protein, with an apparent mol. wt. of 44 kd, was identified as actin based on its labeling with a monoclonal anti-actin antibody, its coincidence with the major [³⁵S]methionine-labeled protein after two-dimensional electrophoresis and its binding to a DNase I affinity column. The ³H-label was resistant to chloroform-methanol extraction and boiling in SDS-containing buffer. After partial purification by preparative SDS-polyacrylamide gel electrophoresis, the 44-kd protein was treated with KOH, the fatty acids released were derivatized to methyl esters and palmitic acid methylester was identified by gas-liquid chromatography.     

生物正交化学在活体标记及药物传递中的研究进展

生物正交化学反应是一类可以在生理条件下发生的化学反应,具有简单、高效、高特异性的特点,在生物医学的研究中被广泛应用.基于生物体天然生命过程的代谢工程,可对生物分子进行无损、高效的生物代谢修饰,是一种理想的生物修饰技术.通过生物代谢途径可有效地将各种化学报告基团引入靶标物的生物分子中,有利于携带配对基团的标记物与其发生生物正交反应,从而在活体系统中实现生物分子的标记示踪和药物递送.这种基于代谢工程与生物正交化学的标记策略因为具有两者之间的优势,在生物医学工程中的标记、成像示踪、诊断等领域展现出巨大的研究价值与应用潜力.本文介绍了生物正交和代谢工程的原理与生物医学研究进展,阐述了生物正交化学在分子成像和药物传递等方面的研究与应用.     

Acylation of cellular proteins with endogenously synthesized fatty acids

A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [3H]acetate, a general precursor of all fatty acids, using BC3H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [3H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemistry in living systems

Dissecting complex cellular processes requires the ability to track biomolecules as they function within their native habitat. Although genetically encoded tags such as GFP are widely used to monitor discrete proteins, they can cause significant perturbations to a protein's structure and have no direct extension to other classes of biomolecules such as glycans, lipids, nucleic acids and secondary metabolites. In recent years, an alternative tool for tagging biomolecules has emerged from the chemical biology community--the bioorthogonal chemical reporter. In a prototypical experiment, a unique chemical motif, often as small as a single functional group, is incorporated into the target biomolecule using the cell's own biosynthetic machinery. The chemical reporter is then covalently modified in a highly selective fashion with an exogenously delivered probe. This review highlights the development of bioorthogonal chemical reporters and reactions and their application in living systems.     

Identification of a Substrate-binding Site in a Peroxisomal β-Oxidation Enzyme by Photoaffinity Labeling with a Novel Palmitoyl Derivative

Peroxisomes play an essential role in a number of important metabolic pathways including β-oxidation of fatty acids and their derivatives. Therefore, peroxisomes possess various β-oxidation enzymes and specialized fatty acid transport systems. However, the molecular mechanisms of these proteins, especially in terms of substrate binding, are still unknown. In this study, to identify the substrate-binding sites of these proteins, we synthesized a photoreactive palmitic acid analogue bearing a diazirine moiety as a photophore, and performed photoaffinity labeling of purified rat liver peroxisomes. As a result, an 80-kDa peroxisomal protein was specifically labeled by the photoaffinity ligand, and the labeling efficiency competitively decreased in the presence of palmitoyl-CoA. Mass spectrometric analysis identified the 80-kDa protein as peroxisomal multifunctional enzyme type 2 (MFE2), one of the peroxisomal β-oxidation enzymes. Recombinant rat MFE2 was also labeled by the photoaffinity ligand, and mass spectrometric analysis revealed that a fragment of rat MFE2 (residues Trp²⁴⁹ to Arg²⁵¹) was labeled by the ligand. MFE2 mutants bearing these residues, MFE2(W249A) and MFE2(R251A), exhibited decreased labeling efficiency. Furthermore, MFE2(W249G), which corresponds to one of the disease-causing mutations in human MFE2, also exhibited a decreased efficiency. Based on the crystal structure of rat MFE2, these residues are located on the top of a hydrophobic cavity leading to an active site of MFE2. These data suggest that MFE2 anchors its substrate around the region from Trp²⁴⁹ to Arg²⁵¹ and positions the substrate along the hydrophobic cavity in the proper direction toward the catalytic center.     

A bioorthogonal chemical reporter for fatty acid synthase–dependent protein acylation

Mammalian cells acquire fatty acids (FAs) from dietary sources or via de novo palmitate production by fatty acid synthase (FASN). Although most cells express FASN at low levels, it is upregulated in cancers of the breast, prostate, and liver, among others, and is required during the replication of many viruses, such as dengue virus, hepatitis C, HIV-1, hepatitis B, and severe acute respiratory syndrome coronavirus 2, among others. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo FA synthesis contributes to host or viral protein acylation has been traditionally difficult to study. Here, we describe a cell-permeable and click chemistry–compatible alkynyl acetate analog (alkynyl acetic acid or 5-hexynoic acid [Alk-4]) that functions as a reporter of FASN-dependent protein acylation. In an FASN-dependent manner, Alk-4 selectively labels the cellular protein interferon-induced transmembrane protein 3 at its known palmitoylation sites, a process that is essential for the antiviral activity of the protein, and the HIV-1 matrix protein at its known myristoylation site, a process that is required for membrane targeting and particle assembly. Alk-4 metabolic labeling also enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.     

过氧化物酶体脂肪酸β氧化

除线粒体外,过氧化物酶体也是真核细胞脂肪酸β氧化分解的重要部位．过氧化物酶体β氧化过程包括氧化、加水、脱氢和硫解4步反应,主要参与极长链、支链脂肪酸等的分解．近年关于过氧化物酶体β氧化的研究活跃,在代谢途径及功能等方面有了新的认识,尤其在对相关代谢酶的研究中取得了较大进展．本文就过氧化物酶体β氧化相关进展作一综述．     

Acylation of proteins by myristic acid in isolated mitochondria

Isolated and highly purified mitochondria from rat liver were incubated with [1-14C]myristate, solubilized in boiling sodium dodecyl sulfate, and analyzed by polyacrylamide gel electrophoresis and autoradiography. Six to eight protein bands were found to be radioactively labeled. If the mitochondria were heated for 5 min at 95 degrees C prior to incubation with this fatty acid, no labeling was observed. By preexposing the mitochondria to unlabeled fatty acids of varying chain lengths, the extent of labeling by [1-14C]myristate was reduced in a chain length-dependent manner, exhibiting maximal inhibition at lauric acid. Reversibility of the labeling was demonstrated by chasing the incorporated radioactivity with unlabeled fatty acids of varying chain length, resulting in a maximal displacement of the tracer again by lauric acid. Fractionation of the labeled mitochondria into mitochondrial matrix and inner mitochondrial membrane components before or after labeling showed that the modified proteins are located inside the inner mitochondrial membrane. In both cases, the pattern of labeling was different from the one observed with intact mitochondria. The labeled bands in the gel were sensitive to alkaline methanol or hydroxylamine treatment. The radioactivity recovered after this treatment co-migrated with myristic acid on thin layer chromatography plates. The chain length specificity and the rapid reversibility of the observed acylation argue for a new type of reaction, different from the acylation observed in whole cells. The possible involvement of the acylated proteins in the regulation of oxidative phosphorylation is discussed.     

Effects of postdecapitation ischemia on the metabolism of [14C]arachidonic acid and [14C]palmitic acid in the mouse brain

The effect of postdecapitation ischemia on the labeling of the free fatty acid pool and their incorporation in lipids was examined during the first 10 min after decapitation in mouse brain that had been injected intracerebrally with either [1-14C]arachidonic acid or [1-14C]palmitic acid. One min after decapitation, animals injected with labeled arachidonic acid exhibited a greatly reduced incorporation of label in brain phospholipids, diglycerides, and triglycerides. When radioactive palmitic acid was used, brain lipids exhibited considerably less inhibition of label. However, a similar degree of inhibition was observed 10 min after decapitation with both fatty acids. At this time, free arachidonic acid had decreased 84% as compared to the 24% decrease observed in the controls, and about 77% of the free palmitic acid remained in the free fatty acid fraction as compared with 30% in the controls. This decreased labeling may reflect ATP shortage that affects the fatty acid activation-reacylation reactions or the enzymes involved. Alternatively, the enhanced endogenous free arachidonic acid may compete with the radiolabeled arachidonic acid resulting in an inhibition of lipid labeling. Inhibition of label may have been greater in radiolabeled arachidonic acid than palmitic because of the larger accumulation of the former endogenous fatty acid during early ischemia.     

Labeling of adipocyte membranes by sulfo-N-succinimidyl derivatives of long-chain fatty acids: Inhibition of fatty acid transport

Summary Sulfo-N-succinimidyl derivatives of the long-chain fatty acids, oleic and myristic, were synthesized and covalently reacted with isolated rat adipocytes. The plasma membrane proteins labeled by these compounds and the effect of labeling on the transport of long-chain fatty acids were investigated. Sulfo-N-succinimidyl oleate (SSO) and myristate (SSM) inhibited the transport of fatty acids (by about 70%). Inhibition of fatty acid transport was not a result of alterations in cell integrity, as intracellular water volume was not changed. It did not reflect effects on fatty acid metabolism, since it was observed under conditions where greater than 90% of the fatty acid taken up was recovered in the free form. The inhibitory effect was specific to the fatty acid transport system, as the transport of glucose and the permeation of retinoic acid, a substance with structural similarities to long-chain fatty acids, were unaffected. Sulfosuccinimidyl oleate reacted exclusively with a plasma membrane protein with an apparent size of 85 kDa while sulfosuccinimidyl myristate also labeled a 75-kDa while sulfosuccinimidyl myristate also labeled a 75-kDa protein. These proteins were among the ones labeled by diisothiocyanodisulfonic acid (DIDS) which also inhibits fatty acid transport irreversibly. The data suggest that the 85-kDa protein, which is the only one labeled by all three inhibitors is involved in facilitating membrane permeation of long-chain fatty acids.     

Photoaffinity labeling and fatty acid permeation in 3T3-L1 adipocytes

Long chain fatty acid uptake was investigated in 3T3-L1 cells. Differentiation of these cells from fibroblasts to adipocytes was accompanied by an 8.5-fold increase in the rate of oleate uptake. This was saturable in adipocytes with apparent Kt and Vmax values of 78 nM and 16 nmol/min/mg cell protein, respectively. A number of proteins in various subcellular fractions of differentiated cells were labeled with the photoreactive fatty acid 11-m-diazirinophenoxy[11-3H]undecanoate. A 15-kDa cytoplasmic protein was induced upon differentiation to adipocytes. This protein was labeled with the photoreactive fatty acid in cytoplasm isolated from differentiated adipocytes, but not in cytoplasm from undifferentiated, fibroblastic cells. Furthermore, a high affinity fatty acid binding protein of 22 kDa was identified in plasma membranes of undifferentiated cells, and its level of labeling increased 2-fold upon differentiation. These results indicate the usefulness of the photoreactive fatty acid in identifying cellular fatty acid binding proteins, and its potential to elucidate the spatial and temporal distribution of fatty acids in intact cells.     

Labeling of the proteins at the surface of the pancreatic zymogen granule using diazotized [125I]iodosulfanilic acid

Purified pig and rat pancreatic zymogen granules have been covalently labeled with the membrane impermeant agent diazotized [125I]iodosulfanilic acid. Following alkaline lysis, the radioactivity was almost entirely (92%) recovered in a dense protein pellet designated as the 1 M sucrose pellet. The rest (8%) of the label was recovered in the membrane fraction. The specificity of this procedure in labeling the cytoplasmic aspect of the granule is demonstrated by the absence of label from granule content proteins and by the removal of iodinatable proteins following protease treatment of intact granules. No characteristic integral membrane proteins were labeled. In the pig, four major protein bands were labeled in both subfractions at Mr of 15,000, 33,000, 35,000 and 38,000. In the rat, a similar set of protein bands was labeled except for that of 15,000 Mr which was poorly labeled. Due to their location, it is suggested that these proteins may play an important role in the recognition between the granule membrane and the cell membrane and thereby the control of the exocytosis process.     

Identification of high affinity membrane-bound fatty acid-binding proteins using a photoreactive fatty acid

A photoaffinity labeling method was developed to identify and characterize high affinity fatty acid-binding proteins in membranes. The specific labeling of these sites requires the use of low concentrations (nanomolar) of the photoreactive fatty acid 11-m-diazirinophenoxy-[11-³H]undecanoate. It was delivered as a bovine serum albumin (BSA) complex which serves as a reservoir for fatty acid and thus allows precise control of unbound fatty acid concentrations. ThefadL protein ofE. coli, which is required for fatty acid permeation of its outer membrane, was labeled by the photoreactive fatty acid neither specifically nor saturably when the probe was added in the absence of BSA; however when a nanomolar concentration of the uncomplexed probe was maintained in the presence of BSA, the labeling of thefadL protein was highly specific and saturable. This photoaffinity labeling method was also used to characterize a 22 kDa, high affinity fatty acid-binding protein which we have recently identified in the plasma membrane of 3T3-L1 adipocytes. This protein bound the probe with a K_d of 216 nM. The approach described is easily capable of identifying membrane-bound fatty acid-binding proteins and can distinguish between those of high and low affinities for fatty acids. It represents a general method for the identification and characterization of fatty acid-binding proteins.Abbreviations BSA Bovine Serum Albumin - DAP m-Diazirinophenoxy - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis     

Dynamic proteomic and metabonomic analysis reveal dysfunction and subclinical injury in rat liver during restraint stress

Stress is a risk factor for many diseases. In this study, we used fluorescence difference gel electrophoresis combined MALDI-TOF/TOF and ¹H-NMR to monitor the intracellular processes in rat liver at proteomic and metabonomic levels when a rat was treated with restraint stress for 8 weeks. Dynamic changes in 42 proteins and 32 chemical groups were monitored and identified. These proteins and chemical groups were implicated in glycolysis, the tricarboxylic acid cycle, fatty acid oxidation, and the urea cycle. To verify the DIGE result, three proteins including DJ-1, Blvrb and AdoHycase were validated by Western blot. Furthermore, some metabolites related to diseases such as lactate, fatty acid, glucose and homocysteine, were observed to be increasing during 8 weeks of restraint stress. Our data indicated that subclinical hepatic injury occurs during restraint stress, including inhibition of glycolysis and gluconeogenesis in the liver, and dysfunction of fatty acid β-oxidation. The results suggest a comprehensive map that addresses how functional proteins act on metabolites to produce energy and process materials in rat liver as it responds to restraint stress. Further functional study on these dynamic change proteins and metabolites may lead to better understanding of the mechanisms of stress-induced diseases.