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F. Grimolizzi and L. Arranz et al.
mote deoxyribonucleic acid methylation through inhibi- tion of histone demethylases and the ten-eleven transloca- tion family proteins (Figure 1), and thereby to play a role in the cellular epigenetic landscape.21,22 Recently, a novel post- translational modification was associated to succinate accumulation that results in lysine succinylation.3,9 Succinylation implies dramatic structural changes in pro- teins and confers a negative charge at physiological pH.9,10 Many succinylation sites on histones have been identified, which may result in alterations in chromatin structure and, consequently, gene expression.8,9 Mechanisms that trigger lysine succinylation are still not clear and little is known about its potential role in immune modulation. In Escherichia coli, where succinylation was discovered, accu- mulation of succinate promotes succinyl coenzyme A syn- thetase activity, leading to higher production of succinyl coenzyme A that acts, in turn, as succinyl donor.23 In mice, succinylation is reverted by sirtuin 5, a member of the nicotinamide adenine dinucleotide-dependent family of deacetylases that exhibits desuccinylase activity.23
Succinate dehydrogenase complex mutations seem to relate to hematopoietic malignancies, particularly lym- phoid leukemias, in addition to endocrine cancers.19 Interestingly, human neutrophils isolated from patients with heterozygous germ line mutations in the enzyme SDHB, display enhanced cell survival and increased suc- cinylation.24 However, the functional consequences of SDHB defects for the inflammatory phenotype were not identified, and neither was any causal mechanism found linking SDHB mutations and succinylation of proteins in neutrophils. Mouse models have been used to further understand these processes, but many questions remain. Macrophages from mice lacking SDHB accumulate succi- nate.25 This strategy together with LPS stimulation leads to decreased pro-inflammatory gene expression along with enhanced expression of a variety of anti-inflammatory sol- uble mediators, including IL-1 receptor antagonist and IL- 10.25 Accordingly, inhibition of SDH with itaconate treat- ment, a metabolite structurally similar to succinate, abol- ishes inflammatory response of macrophages reducing myocardial infarct in both in vitro and in vivo models by lim- iting succinate oxidation.26 Thus, limiting the oxidation of succinate may have an anti-inflammatory effect. The prominent mechanisms mediating these effects seem to relate to mitochondrial metabolic reprogramming and inhi- bition of reactive oxygen species (ROS) production.25,26 Unfortunately, the potential role of succinylation in these processes has not been studied.
Conversely, macrophages from mice lacking sirtuin 5 become hypersensitive to LPS and display increased expression of IL-1b.27 One important limitation of this strategy is that it does not allow us to rule out possible deacetylation from desuccinylation activity of sirtuin 5. Nevertheless, the Authors elegantly showed a mechanism that at least partially explains these results.27 Succinylation is a modification that may occur to multiple proteins in addition to histones. This is the case of pyruvate kinase M2, whose succinylation at K311 promotes its transloca- tion into the nucleus. Once in the nucleus, pyruvate kinase M2 forms a transcriptional complex with HIF-1α that directly binds to the IL-1b promoter gene and activates its transcription.27 Furthermore, pyruvate kinase M2 hyper- succinylation in sirtuin 5-deficient mice renders the ani- mals more susceptible to experimental colitis through boosted IL-1b production.27
Thus, future study should examine the link between suc- cinylation and inflammatory pathways, its regulation in different blood cell subsets, and its consequences in terms of gene expression and immune cell functions in both healthy and clinical conditions.
Succinate as intercellular communicator
Activation of succinate receptor 1 (SUCNR1), or G pro- tein-coupled receptor 91 (GPR91), was recently recognized as a regulatory mediator on a variety of cell subsets, includ- ing cardiomyocytes, adipocytes, renal and blood cells.28-30 Half-maximal effective response for GPR91 is obtained with succinate concentrations of 28-56 mM, indicative of GPR91 activation at succinate concentrations higher than the steady-state levels.30 Thus, succinate-GPR91 axis may func- tion as an early sensor of homeostasis perturbations. In this context, selection of appropriate experimental conditions is essential to prevent biased results. For example, use of car- bon dioxide as a method of euthanasia in rodents should be avoided since it rapidly raises blood succinate levels.14
G protein-coupled receptors are typically classified based on the G protein subfamily that they activate, e.g. Gαi, Gαq, Gs or G12/13. Given the enriched expression of GPR91 in kidney, human embryonic kidney cells 293 are often used to examine its mechanisms of signal transduc- tion. However, attempts to characterize downstream path- ways have led to controversial results. Some studies show that GPR91 activates both Gαi and Gαq signaling, leading to inhibition of cyclic adenosine monophosphate produc- tion and deployment of calcium mobilization, respectively.30 In contrast, others have shown that Gαq is not required for succinate-induced activation of GPR91 and Gαi alone is sufficient to quench cyclic adenosine monophosphate and mobilize calcium.29 Despite these controversies, in vitro studies in kidney cell lines seem to agree that GPR91 signaling is mediated by mobilization of intracellular calcium stores rather than being dependent on influx of extracellular calcium.29 However, stimulation of platelets with succinate failed to induce intracellular calci- um mobilization, indicating the potential cell type-specific signaling transduction pathways of GPR91.29 Clues as to the cell-wide plasticity of GPR91 can further be seen from mesenchymal cells in the liver, where GPR91 activation neither increases cytosolic calcium nor inhibits adenosine monophosphate production.31
Furthermore, distinct mechanisms are responsible for GPR91 switch off in a cell-type dependent manner. Unlike human embryonic kidney cells 293, which undergo inter- nalization of GPR91 in presence of succinate,32 Madin- Darby canine kidney cells instead show a temporal desen- sitization of GPR91 following succinate binding.32 Thus, cell-type specific responses of GPR91 machinery and on/off dynamics may confer great plasticity to this regula- tory pathway and may also underlie seemingly controver- sial results. Therefore, characterizing GPR91 signaling in cells of interest appears to be essential to assess the impact on functional activity and to identify specific drug targets upon pathogenesis.
GPR91 in inflammatory responses
Early observations on blood cells show that GPR91 acti- vation boosts pro-inflammatory responses, but recent
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haematologica | 2018; 103(10)