Quantification of the immunometabolite protein modifications S-2-succinocysteine and 2,3-dicarboxypropylcysteine.

The tricarboxylic acid (TCA) cycle metabolite fumarate nonenzymatically reacts with the amino acid cysteine to form S-(2-succino)cysteine (2SC), referred to as protein succination. The immunometabolite itaconate accumulates during lipopolysaccharide (LPS) stimulation of macrophages and microglia. Itaconate nonenzymatically reacts with cysteine residues to generate 2,3-dicarboxypropylcysteine (2,3-DCP), referred to as protein dicarboxypropylation. Since fumarate and itaconate levels dynamically change in activated immune cells, the levels of both 2SC and 2,3-DCP reflect the abundance of these metabolites and their capacity to modify protein thiols. We generated ethyl esters of 2SC and 2,3-DCP from protein hydrolysates and used stable isotope dilution mass spectrometry to determine the abundance of these in LPS-stimulated Highly Aggressively Proliferating Immortalized (HAPI) microglia. To quantify the stoichiometry of the succination and dicarboxypropylation, reduced cysteines were alkylated with iodoacetic acid to form S-carboxymethylcysteine (CMC), which was then esterified. Itaconate-derived 2,3-DCP, but not fumarate-derived 2SC, increased in LPS-treated HAPI microglia. Stoichiometric measurements demonstrated that 2,3-DCP increased from 1.57% to 9.07% of total cysteines upon LPS stimulation. This methodology to simultaneously distinguish and quantify both 2SC and 2,3-DCP will have broad applications in the physiology of metabolic diseases. In addition, we find that available anti-2SC antibodies also detect the structurally similar 2,3-DCP, therefore "succinate moiety" may better describe the antigen recognized.NEW & NOTEWORTHY Itaconate and fumarate have roles as immunometabolites modulating the macrophage response to inflammation. Both immunometabolites chemically modify protein cysteine residues to modulate the immune response. Itaconate and fumarate levels change dynamically, whereas their stable protein modifications can be quantified by mass spectrometry. This method distinguishes itaconate and fumarate-derived protein modifications and will allow researchers to quantify their contributions in isolated cell types and tissues across a range of metabolic diseases.

Defective function of α-ketoglutarate dehydrogenase exacerbates mitochondrial ATP deficits during complex I deficiency.

The NDUFS4 knockout (KO) mouse phenotype resembles the human Complex I deficiency Leigh Syndrome. The irreversible succination of protein thiols by fumarate is increased in select regions of the NDUFS4 KO brain affected by neurodegeneration. We report that dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate dehydrogenase complex (KGDHC) of the tricarboxylic acid (TCA) cycle, is succinated in the affected regions of the NDUFS4 KO brain. Succination of DLST reduced KGDHC activity in the brainstem (BS) and olfactory bulb (OB) of KO mice. The defective production of KGDHC derived succinyl-CoA resulted in decreased mitochondrial substrate level phosphorylation (SLP), further aggravating the existing oxidative phosphorylation (OXPHOS) ATP deficit. Protein succinylation, an acylation modification that requires succinyl-CoA, was reduced in the KO mice. Modeling succination of a cysteine in the spatial vicinity of the DLST active site or introduction of succinomimetic mutations recapitulates these metabolic deficits. Our data demonstrate that the biochemical deficit extends beyond impaired Complex I assembly and OXPHOS deficiency, functionally impairing select components of the TCA cycle to drive metabolic perturbations in affected neurons.

Extracts of select endemic plants from the Republic of Mauritius exhibiting anti-cancer and immunomodulatory properties.

Mauritius Island possesses unique plant biodiversity with a potential reservoir of biologically active compounds of pharmacological interest. In the current study, we investigated Mauritius endemic plant families Asteraceae, Ebenaceae, Sapotaceae, and Erythroxylaceae, for anti-cancer properties on T cell lymphoma and B16F10 Melanoma cells and immunomodulatory properties on primary T and B cells. The cytotoxicity of methanolic plant extracts at 1, 10, 25 µg/ml was determined. The most active plant species were evaluated for their apoptosis-inducing effects. The immunomodulatory properties of the plants were also studied, and preliminary phytochemical screening of selected plants was done by LC-MS analysis. Psiadia lithospermifolia (Lam.) Cordem (Asteraceae) at 25 µg/ml was the most cytotoxic on both EL4 and B16 cells and triggered apoptosis by the death receptor pathway, and at least in part, by the mitochondrial pathway. Most plant species from Asteraceae, Ebenaceae, Erythroxylaceae, and Sapotaceae inhibited the proliferation of activated T and B cells, although some promoted T cell proliferation. LC-MS profile of Asteraceae plants showed the presence of terpenes, terpenoids, fatty acids, and phenolic. Flavonoids and phenolic acid were also detected from Ebenaceae and Sapotaceae plants. Together, our study demonstrated that Mauritius endemic flora exhibit potential anti-cancer and anti-inflammatory properties worthy of further in-depth studies.

Cannabinoid Receptor Activation on Haematopoietic Cells and Enterocytes Protects against Colitis.

BACKGROUND AND AIMS: Cannabinoid receptor [CB] activation can attenuate inflammatory bowel disease [IBD] in experimental models and human cohorts. However, the roles of the microbiome, metabolome, and the respective contributions of haematopoietic and non-haematopoietic cells in the anti-colitic effects of cannabinoids have yet to be determined. METHODS: Female C57BL/6 mice were treated with either cannabidiol [CBD], Δ 9-tetrahydrocannabinol [THC], a combination of CBD and THC, or vehicle, in several models of chemically induced colitis. Clinical parameters of colitis were assessed by colonoscopy, histology, flow cytometry, and detection of serum biomarkers; single-cell RNA sequencing and qRT-PCR were used to evaluate the effects of cannabinoids on enterocytes. Immune cell transfer from CB2 knockout mice was used to evaluate the contribution of haematopoietic and non-haematopoietic cells to colitis protection. RESULTS: We found that THC prevented colitis and that CBD, at the dose tested, provided little benefit to the amelioration of colitis, nor when added synergistically with THC. THC increased colonic barrier integrity by stimulating mucus and tight junction and antimicrobial peptide production, and these effects were specific to the large intestine. THC increased colonic Gram-negative bacteria, but the anti-colitic effects of THC were independent of the microbiome. THC acted both on immune cells via CB2 and on enterocytes, to attenuate colitis. CONCLUSIONS: Our findings demonstrate how cannabinoid receptor activation on both immune cells and colonocytes is critical to prevent colonic inflammation. These studies also suggest how cannabinoid receptor activation can be used as a preventive and therapeutic modality against colitis.

Fumarate and oxidative stress synergize to promote stability of C/EBP homologous protein in the adipocyte.

C/EBP homologous protein (CHOP) is a transcription factor that is elevated in adipose tissue across many models of diabetes and metabolic stress. Although increased CHOP levels are associated with the terminal response to endoplasmic reticulum stress and apoptosis, there is no evidence for CHOP mediated apoptosis in the adipose tissue during diabetes. CHOP protein levels increase in parallel with protein succination, a fumarate derived cysteine modification, in the adipocyte during metabolic stress. We investigated the factors contributing to sustained CHOP proteins levels in the adipocyte, with an emphasis on the regulation of CHOP protein turnover by metabolite-driven modification of Keap1 cysteines. CHOP protein stability was investigated in conditions of nutrient stress due to high glucose or elevated fumarate (fumarase knockdown model); where cysteine succination is specifically elevated. CHOP protein turnover is significantly reduced in models of elevated glucose and fumarate with a ~30% increase in CHOP stability (p > 0.01), in part due to decreased CHOP phosphorylation. Sustained CHOP levels occur in parallel with elevated heme-oxygenase-1, a production of increased Nrf2 transcriptional activity and Keap1 modification. While Keap1 is directly succinated in the presence of excess fumarate derived from genetic knockdown of fumarase (fumarate levels are elevated >20-fold), it is the oxidative modification of Keap1 that predominates in adipocytes matured in high glucose (fumarate increases 4-5 fold). Elevated fumarate indirectly regulates CHOP stability through the induction of oxidative stress. The antioxidant N-acetylcysteine (NAC) reduces fumarate levels, protein succination and CHOP levels in adipocytes matured in high glucose. Elevated CHOP does not contribute elevated apoptosis in adipocytes, but plays a redox-dependent role in decreasing the adipocyte secretion of interleukin-13, an anti-inflammatory chemokine. NAC treatment restores adipocyte IL-13 secretion, confirming the redox-dependent regulation of a potent anti-inflammatory eotaxin. This study demonstrates that physiological increases in the metabolite fumarate during high glucose exposure contributes to the presence of oxidative stress and sustained CHOP levels in the adipocyte during diabetes. The results reveal a novel metabolic link between mitochondrial metabolic stress and reduced anti-inflammatory adipocyte signaling as a consequence of reduced CHOP protein turnover.

Identification of Novel Protein Targets of Dimethyl Fumarate Modification in Neurons and Astrocytes Reveals Actions Independent of Nrf2 Stabilization.

The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.

Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome.

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys(77) and Cys(48) were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.

GSK-3ß Phosphorylation of Cytoplasmic Dynein Reduces Ndel1 Binding to Intermediate Chains and Alters Dynein Motility.

Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3ß in both neurons and non-neuronal cells. GSK-3ß coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within an Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK-3ß, (ii) an insulin-sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK-3ß inactivation. Thus, our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition.

Identification of protein succination as a novel modification of tubulin.

Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate 2SC [S-(2-succino)cysteine]. We demonstrate that both α- and ß-tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db/db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound DMF (dimethylfumarate, 500 µM) inhibited polymerization up to 35% and 59% respectively. Using MS we identified Cys347α, Cys376α, Cys12ß and Cys303ß as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteine residues increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose compared with normal glucose also had reduced reactivity with the anti-α-tubulin antibody; suggesting that succination may interfere with tubulin-protein interactions. DMF reacted rapidly with 11 of the 20 cysteine residues in the αß-tubulin dimer, decreased the number of free thiols and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggest that inhibition of tubulin polymerization is an important undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics.

Reducing the dietary omega-6:omega-3 utilizing α-linolenic acid; not a sufficient therapy for attenuating high-fat-diet-induced obesity development nor related detrimental metabolic and adipose tissue inflammatory outcomes.

AIMS: To examine the effect of manipulating the omega-6:omega-3 (1∶1, 5∶1, 10∶1, and 20∶1) utilizing only α-linolenic and linoleic acid within a clinically-relevant high-fat diet (HFD) composed of up to seven sources of fat and designed to be similar to the standard American diet (MUFA∶PUFA of 2∶1, 12% and 40% of calories from saturated and total fat, respectively) on body composition, macrophage polarization, inflammation, and metabolic dysfunction in mice. METHODS: Diets were administered for 20 weeks. Body composition and metabolism (HOMA index and lipid profile) were examined monthly. GC-MS was utilized to determine the eicosapentaenoic acid (EPA):arachidonic acid (AA) and the docosahexaenoic acid (DHA):AA in AT phospholipids. Adipose tissue (AT) mRNA expression of chemokines (MCP-1, Fetuin-A, CXCL14), marker genes for M1 and M2 macrophages (CD11c and CD206, respectively) and inflammatory markers (TNF-α, IL-6, IL-1ß, TLR-2, TLR-4, IL-10, GPR120) were measured along with activation of NFκB, JNK, and STAT-3. Macrophage infiltration into AT was examined using F4/80 immunohistochemistry. RESULTS: Any therapeutic benefit produced by reducing the omega-6:omega-3 was evident only when comparing the 1∶1 to 20∶1 HFD; the 1∶1 HFD resulted in a lower TC:HDL-C and decreased AT CXCL14 gene expression and AT macrophage infiltration, which was linked to a higher EPA:AA and DHA:AA in AT phospholipids. However, despite these effects, and independent of the omega-6:omega-3, all HFDs, in general, led to similar levels of adiposity, insulin resistance, and AT inflammation. CONCLUSION: Reducing the omega-6:omega-3 using α-linolenic acid is not an effective therapy for attenuating obesity and type II diabetes mellitus development.

Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress.

Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased > or =5-fold during adipogenesis in medium containing 30 mm glucose, producing a > or =10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one- and two-dimensional electrophoresis and approximately 60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.

Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systemin-mediated defense response against herbivorous insects.

Systemin is a wound-signaling peptide that mediates defenses of tomato plants against herbivorous insects. Perception of systemin by the membrane-bound receptor SR160 results in activation of MAPKs, synthesis of jasmonic acid (JA), and expression of defense genes. To test the function of MAPKs in the response to systemin, we used virus-induced gene silencing (VIGS) in plants that overexpress the systemin precursor prosystemin (35S::prosys plants). These transgenic plants accumulate high levels of defense proteins and exhibit increased resistance to herbivorous insects. Cosilencing of the MAPKs MPK1 and MPK2 reduced MPK1/2 kinase activity, JA biosynthesis, and expression of JA-dependent defense genes. Application of methyl-JA restored the full defense response. These data show that MPK1 and MPK2 are essential components of the systemin signaling pathway and most likely function upstream of JA biosynthesis. MPK1 and MPK2 are 95% identical at the amino acid level. Specific VIGS of only MPK1 or MPK2 resulted in the same reduction of defense gene expression as cosilencing of MPK1 and MPK2, indicating that gene dosage effects may be important for MPK signaling. In addition, VIGS of the closely related MPK3 also reduced systemin-induced defense responses. The function of MPK1/2 and orthologs in pathogen-induced defenses is well established. Here we show that cosilencing of MPK1 and MPK2 compromised prosystemin-mediated resistance to Manduca sexta (Lepidoptera) herbivory, demonstrating that MPK1 and MPK2 are also required for successful defenses against herbivorous insects.

S-(2-Succinyl)cysteine: a novel chemical modification of tissue proteins by a Krebs cycle intermediate.

S-(2-Succinyl)cysteine (2SC) has been identified as a chemical modification in plasma proteins, in the non-mercaptalbumin fraction of human plasma albumin, in human skin collagen, and in rat skeletal muscle proteins and urine. 2SC increases in human skin collagen with age and is increased in muscle protein of diabetic vs. control rats. The concentration of 2SC in skin collagen and muscle protein correlated strongly with that of the advanced glycation/lipoxidation end-product (AGE/ALE), N(epsilon)-(carboxymethyl)lysine (CML). 2SC is formed by a Michael addition reaction of cysteine sulfhydryl groups with fumarate at physiological pH. Fumarate, but not succinate, inactivates the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase in vitro, in concert with formation of 2SC. 2SC is the first example of spontaneous chemical modification of protein by a metabolic intermediate in the Krebs cycle. These observations identify fumarate as an endogenous electrophile and suggest a role for fumarate in regulation of metabolism.

A novel method for the measurement of in vitro fatty acid 2-hydroxylase activity by gas chromatography-mass spectrometry.

Fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is an enzyme responsible for the de novo synthesis of sphingolipids containing 2-hydroxy fatty acids. 2-Hydroxy sphingolipids are highly abundant in the brain, as major myelin galactolipids (galactosylceramide and sulfatide) contain a uniquely high proportion ( approximately 50%) of 2-hydroxy fatty acids. Other tissues, such as epidermis, epithelia of the digestive tract, and certain cancers, also contain 2-hydroxy sphingolipids. The physiological significance of the 2-hydroxylation on N-acyl chains of subsets of sphingolipids is poorly understood. To study the roles of FA2H and 2-hydroxy sphingolipids in various tissues, we developed a highly sensitive in vitro FA2H assay. FA2H-dependent fatty acid 2-hydroxylation requires an electron transfer system, which was reconstituted in vitro with an NADPH regeneration system and purified NADPH:cytochrome P-450 reductase. A substrate [3,3,5,5-D(4)]tetracosanoic acid was solubilized in alpha-cyclodextrin solution, and the 2-hydroxylated product was quantified by gas chromatography-mass spectrometry after conversion to a trimethylsilyl ether derivative. When the microsomes of FA2H-transfected COS7 cells were incubated with the electron transfer system and deuterated tetracosanoic acid, deuterated 2-hydroxy tetracosanoic acid was formed in a time- and protein-dependent manner. With this method, FA2H activities were reproducibly measured in murine brains and tissue culture cell lines.