Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5'-deoxy-5'-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function.

Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation, glucolipid metabolism, and inflammation. Thiazolidinediones are PPARγ full agonists with potent insulin-sensitizing effects, whereas their oral usage is restricted because of unwanted side effects, including obesity and cardiovascular risks. Here, via virtual screening, microscale thermophoresis analysis, and molecular confirmation, we demonstrate that diosmin, a natural compound of wide and long-term clinical use, is a selective PPARγ modulator that binds to PPARγ and blocks PPARγ phosphorylation with weak transcriptional activity. Local diosmin administration in subcutaneous fat (inguinal white adipose tissue [iWAT]) improved insulin sensitivity and attenuated obesity via enhancing browning of white fat and energy expenditure. Besides, diosmin ameliorated inflammation in WAT and liver and reduced hepatic steatosis. Of note, we determined that iWAT local administration of diosmin did not exhibit obvious side effects. Taken together, the present study demonstrated that iWAT local delivery of diosmin protected mice from diet-induced insulin resistance, obesity, and fatty liver by blocking PPARγ phosphorylation, without apparent side effects, making it a potential therapeutic agent for the treatment of metabolic diseases.

Nuclear factor kappa B (NF-κB) activity is regulated by various posttranslational modifications, of which Ser276 phosphorylation of RelA/p65 is particularly impacted by reactive oxygen species (ROS). This modification is responsible for selective upregulation of a subset of NF-κB targets; however, the precise mechanism remains elusive. ROS have the ability to modify cellular molecules including DNA. One of the most common oxidation products is 8-oxo-7,8-dihydroguanine (8-oxoGua), which is repaired by the 8-oxoguanine DNA glycosylase1 (OGG1)-initiated base excision repair pathway. Recently, a new function of OGG1 has been uncovered. OGG1 binds to 8-oxoGua, facilitating the occupancy of NF-κB at promoters and enhancing transcription of pro-inflammatory cytokines and chemokines. In the present study, we demonstrated that an interaction between DNA-bound OGG1 and mitogen-and stress-activated kinase 1 is crucial for RelA/p65 Ser276 phosphorylation. ROS scavenging or OGG1 depletion/inhibition hindered the interaction between mitogen-and stress-activated kinase 1 and RelA/p65, thereby decreasing the level of phospho-Ser276 and leading to significantly lowered expression of ROS-responsive cytokine/chemokine genes, but not that of Nfkbis. Blockade of OGG1 binding to DNA also prevented promoter recruitment of RelA/p65, Pol II, and p-RNAP II in a gene-specific manner. Collectively, the data presented offer new insights into how ROS signaling dictates NF-κB phosphorylation codes and how the promoter-situated substrate-bound OGG1 is exploited by aerobic mammalian cells for timely transcriptional activation of ROS-responsive genes.

African swine fever (ASF) is a viral hemorrhagic disease that affects domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). The ASFV virion contains a long double-stranded DNA genome, which encodes more than 150 proteins. However, the immune escape mechanism and pathogenesis of ASFV remain poorly understood. Here, we report that the pyroptosis execution protein gasdermin D (GSDMD) is a new binding partner of ASFV-encoded protein S273R (pS273R), which belongs to the SUMO-1 cysteine protease family. Further experiments demonstrated that ASFV pS273R-cleaved swine GSDMD in a manner dependent on its protease activity. ASFV pS273R specifically cleaved GSDMD at G107-A108 to produce a shorter N-terminal fragment of GSDMD consisting of residues 1 to 107 (GSDMD-N1-107). Interestingly, unlike the effect of GSDMD-N1-279 fragment produced by caspase-1-mediated cleavage, the assay of LDH release, cell viability, and virus replication showed that GSDMD-N1-107 did not trigger pyroptosis or inhibit ASFV replication. Our findings reveal a previously unrecognized mechanism involved in the inhibition of ASFV infection-induced pyroptosis, which highlights an important function of pS273R in inflammatory responses and ASFV replication.

Mutations in the myotubularin 1 (MTM1) gene can cause the fatal disease X-linked centronuclear myopathy (XLCNM), but the underlying mechanism is incompletely understood. In this report, using an Mtm1-/y disease model, we found that expression of the intragenic microRNA miR-199a-1 is up-regulated along with that of its host gene, dynamin 2 (Dnm2), in XLCNM skeletal muscle. To assess the role of miR-199a-1 in XLCNM, we crossed miR-199a-1-/- with Mtm1-/y mice and found that the resultant miR-199a-1-Mtm1 double-knockout mice display markers of improved health, as evidenced by lifespans prolonged by 30% and improved muscle strength and histology. Mechanistic analyses showed that miR-199a-1 directly targets nonmuscle myosin IIA (NM IIA) expression and, hence, inhibits muscle postnatal development as well as muscle maturation. Further analysis revealed that increased expression and phosphorylation of signal transducer and activator of transcription 3 (STAT3) up-regulates Dnm2/miR-199a-1 expression in XLCNM muscle. Our results suggest that miR-199a-1 has a critical role in XLCNM pathology and imply that this microRNA could be targeted in therapies to manage XLCNM.

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the "dark kinome". Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma.

The voltage-gated potassium (Kv) 1.3 channel plays a crucial role in the immune responsiveness of T-lymphocytes and macrophages, presenting a potential target for treatment of immune- and inflammation related-diseases. FS48, a protein from the rodent flea Xenopsylla cheopis, shares the three disulfide bond feature of scorpion toxins. However, its three-dimensional structure and biological function are still unclear. In the present study, the structure of FS48 was evaluated by circular dichroism and homology modeling. We also described its in vitro ion channel activity using patch clamp recording and investigated its anti-inflammatory activity in LPS-induced Raw 264.7 macrophage cells and carrageenan-induced paw edema in mice. FS48 was found to adopt a common αββ structure and contain an atypical dyad motif. It dose-dependently exhibited the Kv1.3 channel in Raw 264.7 and HEK 293T cells, and its ability to block the channel pore was demonstrated by the kinetics of activation and competition binding with tetraethylammonium. FS48 also downregulated the secretion of proinflammatory molecules NO, IL-1β, TNF-α, and IL-6 by Raw 264.7 cells in a manner dependent on Kv1.3 channel blockage and the subsequent inactivation of the MAPK/NF-κB pathways. Finally, we observed that FS48 inhibited the paw edema formation, tissue myeloperoxidase activity, and inflammatory cell infiltrations in carrageenan-treated mice. We therefore conclude that FS48 identified from the flea saliva is a novel potassium channel inhibitor displaying anti-inflammatory activity. This discovery will promote understanding of the bloodsucking mechanism of the flea and provide a new template molecule for the design of Kv1.3 channel blockers.

Pharmacological inhibition of protein kinases induces adaptive reprogramming of tumor cell regulatory networks by altering expression of genes that regulate signaling, including protein kinases. Adaptive responses are dependent on transcriptional changes resulting from remodeling of enhancer and promoter landscapes. Enhancer and promoter remodeling in response to targeted kinase inhibition is controlled by changes in open chromatin state and by activity of specific transcription factors, such as c-MYC. This review focuses on the dynamic plasticity of protein kinase expression of the tumor cell kinome and the resulting adaptive resistance to targeted kinase inhibition. Plasticity of the functional kinome has been shown in patient window trials where triple-negative and human epidermal growth factor receptor 2-positive breast cancer patient tumors were characterized by RNAseq after biopsies before and after 1 week of therapy. The expressed kinome changed dramatically during drug treatment, and these changes in kinase expression were shown in cell lines and xenografts in mice to be correlated with adaptive tumor cell drug resistance. The dynamic transcriptional nature of the kinome also differs for inhibitors targeting different kinase signaling pathways (e.g., BRAF-MEK-ERK versus PI3K-AKT) that are commonly activated in cancers. Heterogeneity arising from differences in gene regulation and mutations represents a challenge to therapeutic durability and prevention of clinical drug resistance with drug-tolerant tumor cell populations developing and persisting through treatment. We conclude that understanding the heterogeneity of kinase expression at baseline and in response to therapy is imperative for development of combinations and timing intervals of therapies making interventions durable.

The Kv1.3 channel has been widely demonstrated to play crucial roles in the activation and proliferation of T cells, which suggests that selective blockers could serve as potential therapeutics for autoimmune diseases mediated by T cells. We previously described that the toxin mimic FS48 from salivary gland of Xenopsylla cheopis downregulates the secretion of proinflammatory factors by Raw 264.7 cells by blocking the Kv1.3 channel and the subsequent inactivation of the proinflammatory MAPK/NF-κB pathways. However, the effects of FS48 on human T cells and autoimmune diseases are unclear. Here, we described its immunomodulatory effects on human T cells derived from suppression of Kv1.3 channel. Kv1.3 currents in Jurkat T cells were recorded by whole-cell patch-clamp, and Ca2+ influx, cell proliferation, and TNF-α and IL-2 secretion were measured using Fluo-4, CCK-8, and ELISA assays, respectively. The in vivo immunosuppressive activity of FS48 was evaluated with a rat DTH model. We found that FS48 reduced Kv1.3 currents in Jurkat T cells in a concentration-dependent manner with an IC50 value of about 1.42 μM. FS48 also significantly suppressed Kv1.3 protein expression, Ca2+ influx, MAPK/NF-κB/NFATc1 pathway activation, and TNF-α and IL-2 production in activated Jurkat T cells. Finally, we show that FS48 relieved the DTH response in rats. We therefore conclude that FS48 can block the Kv1.3 channel and inhibit human T cell activation, which most likely contributes to its immunomodulatory actions and highlights the great potential of this evolutionary-guided peptide as a drug template in future studies.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs are regulated by different stimulators including viral infection, which is involved in the antiviral activity of host cells to limit viral propagation. To survive, several viruses have been reported to execute various strategies, such as modulating SG formation, to create optimal surroundings for viral replication. African swine fever virus (ASFV) is one of the most notorious pathogens in the global pig industry. However, the interplay between ASFV infection and SG formation remains largely unknown. In this study, we found that ASFV infection inhibited SG formation. Through SG inhibitory screening, we found that several ASFV-encoded proteins are involved in inhibition of SG formation. Among them, an ASFV S273R protein (pS273R), the only cysteine protease encoded by the ASFV genome, significantly affected SG formation. ASFV pS273R interacted with G3BP1 (Ras-GTPase-activating protein [SH3 domain] binding protein 1), a vital nucleating protein of SG formation. Furthermore, we found that ASFV pS273R cleaved G3BP1 at the G140-F141 to produce two fragments (G3BP1-N1-140 and G3BP1-C141-456). Interestingly, both the pS273R-cleaved fragments of G3BP1 lost the ability to induce SG formation and antiviral activity. Taken together, our finding reveals that the proteolytic cleavage of G3BP1 by ASFV pS273R is a novel mechanism by which ASFV counteracts host stress and innate antiviral responses.

Pheromone receptors (PRs) recognize specific pheromone compounds to guide the behavioral outputs of insects, which are the most diverse group of animals on earth. The activation of PRs is known to couple to the calcium permeability of their coreceptor (Orco) or putatively with G proteins; however, the underlying mechanisms of this process are not yet fully understood. Moreover, whether this transverse seven transmembrane domain (7TM)-containing receptor is able to couple to arrestin, a common effector for many conventional 7TM receptors, is unknown. Herein, using the PR BmOR3 from the silk moth Bombyx mori and its coreceptor BmOrco as a template, we revealed that an agonist-induced conformational change of BmOR3 was transmitted to BmOrco through transmembrane segment 7 from both receptors, resulting in the activation of BmOrco. Key interactions, including an ionic lock and a hydrophobic zipper, are essential in mediating the functional coupling between BmOR3 and BmOrco. BmOR3 also selectively coupled with Gi proteins, which was dispensable for BmOrco coupling. Moreover, we demonstrated that trans-7TM BmOR3 recruited arrestin in an agonist-dependent manner, which indicates an important role for BmOR3-BmOrco complex formation in ionotropic functions. Collectively, our study identified the coupling of G protein and arrestin to a prototype trans-7TM PR, BmOR3, and provided important mechanistic insights into the coupling of active PRs to their downstream effectors, including coreceptors, G proteins, and arrestin.

The thymus is the central immune organ, but it is known to progressively degenerate with age. As thymus degeneration is paralleled by the wasting of aging skeletal muscle, we speculated that the thymus may play a role in muscle wasting. Here, using thymectomized mice, we show that the thymus is necessary for skeletal muscle regeneration, a process tightly associated with muscle aging. Compared to control mice, the thymectomized mice displayed comparable growth of muscle mass, but decreased muscle regeneration in response to injury, as evidenced by small and sparse regenerative myofibers along with inhibited expression of regeneration-associated genes myh3, myod, and myogenin. Using paired box 7 (Pax7)-immunofluorescence staining and 5-Bromo-2'-deoxyuridine-incorporation assay, we determined that the decreased regeneration capacity was caused by a limited satellite cell pool. Interestingly, the conditioned culture medium of isolated thymocytes had a potent capacity to directly stimulate satellite cell expansion in vitro. These expanded cells were enriched in subpopulations of quiescent satellite cells (Pax7highMyoDlowEdUpos) and activated satellite cells (Pax7highMyoDhighEdUpos), which were efficiently incorporated into the regenerative myofibers. We thus propose that the thymus plays an essential role in muscle regeneration by directly promoting satellite cell expansion and may function profoundly in the muscle aging process.

Metaflammation is a primary inflammatory complication of metabolic disorders characterized by altered production of many inflammatory cytokines, adipokines, and lipid mediators. Whereas multiple inflammation networks have been identified, the mechanisms by which metaflammation is initiated have long been controversial. As the mevalonate pathway (MVA) produces abundant bioactive isoprenoids and abnormal MVA has a phenotypic association with inflammation/immunity, we speculate that isoprenoids from the MVA may provide a causal link between metaflammation and metabolic disorders. Using a line with the MVA isoprenoid producer geranylgeranyl diphosphate synthase (GGPPS) deleted, we find that geranylgeranyl pyrophosphate (GGPP) depletion causes an apparent metaflammation as evidenced by abnormal accumulation of fatty acids, eicosanoid intermediates, and proinflammatory cytokines. We also find that GGPP prenylate cytochrome b5 reductase 3 (CYB5R3) and the prenylated CYB5R3 then translocate from the mitochondrial to the endoplasmic reticulum (ER) pool. As CYB5R3 is a critical NADH-dependent reductase necessary for eicosanoid metabolism in ER, we thus suggest that GGPP-mediated CYB5R3 prenylation is necessary for metabolism. In addition, we observe that pharmacological inhibition of the MVA pathway by simvastatin is sufficient to inhibit CYB5R3 translocation and induces smooth muscle death. Therefore, we conclude that the dysregulation of MVA intermediates is an essential mechanism for metaflammation initiation, in which the imbalanced production of eicosanoid intermediates in the ER serve as an important pathogenic factor. Moreover, the interplay of MVA and eicosanoid metabolism as we reported here illustrates a model for the coordinating regulation among metabolite pathways.