Succinate acts as an extracellular mediator signaling through the G protein-coupled receptor GPR91. Here we show that dendritic cells had high expression of GPR91. In these cells, succinate triggered intracellular calcium mobilization, induced migratory responses and acted in synergy with Toll-like receptor ligands for the production of proinflammatory cytokines. Succinate also enhanced antigen-specific activation of human and mouse helper T cells. GPR91-deficient mice had less migration of Langerhans cells to draining lymph nodes and impaired tetanus toxoid-specific recall T cell responses. Furthermore, GPR91-deficient allografts elicited weaker transplant rejection than did the corresponding grafts from wild-type mice. Our results suggest that the succinate receptor GPR91 is involved in sensing immunological danger, which establishes a link between immunity and a metabolite of cellular respiration.

In protein interaction analysis, one promising method to identify the involved proteins and to characterize interacting sites at the same time is the mass spectrometric analysis of enzymatic hydrolysates of covalently cross-linked complexes. While protein identification can be accomplished by the methodology developed for proteome analysis, the unequivocal detection and characterization of cross-linked sites remained involved without selection criteria for linked peptides in addition to mass. To provide such criteria, we incorporated cross-links with a distinct isotope pattern into the microtubule-destabilizing protein Op18/stathmin (Op18) and into complexes formed by Op18 with tubulin. The deuterium-labeled cross-linking reagents bis(sulfosuccinimidyl)-glutarate-d4, -pimelate-d4, and -sebacate-d4 were prepared together with their undeuterated counterparts and applied as a 1:1 mixture of the respective d0 and d4 isotopomers. The resulting d0/d4 isotope tags allowed a straightforward mass spectrometric detection of peptides carrying the linker even in complex enzymatic protein hydrolysates. In the structure elucidation of the linked peptides by MS/MS, the assignment of the linked amino acids was again greatly facilitated by the d0/d4 tag. By applying two cross-linkers with similar reactivity but different spacer length in parallel, even doublets with very low intensity could be assigned with high confidence in MS and MS/MS spectra. Since in the Op18-tubulin complexes only a limited number of peptides carried the linker, the identification of the involved proteins per se was not impeded, thus accomplishing both protein identification and characterization of interacting sites in the same experiment. This novel methodology allowed us to significantly refine the current view of the complex between Op18 and tubulin corroborating the tubulin "capping" activity of the N-terminal domain of Op18.

The anti-cancer agent Indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which Indisulam mediates DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-Indisulam-RBM39(RRM2) complex structure to 2.3 Å. DCAF15 has a novel topology which embraces the RBM39(RRM2) domain largely via nonpolar interactions, and Indisulam binds between DCAF15 and RBM39(RRM2) and coordinates additional interactions between the two proteins. Studies with RBM39 point mutants and Indisulam analogs validated the structural model and defined the RBM39 alpha-helical degron motif. The degron is found only in RBM23 and RBM39 and only these proteins were detectably downregulated in Indisulam-treated HCT116 cells. This work further explains how Indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade novel targets.

SH3 and multiple ankyrin repeat domains 3 (SHANK3) haploinsufficiency is causative for the neurological features of Phelan-McDermid syndrome (PMDS), including a high risk of autism spectrum disorder (ASD). We used unbiased, quantitative proteomics to identify changes in the phosphoproteome of Shank3-deficient neurons. Down-regulation of protein kinase B (PKB/Akt)-mammalian target of rapamycin complex 1 (mTORC1) signaling resulted from enhanced phosphorylation and activation of serine/threonine protein phosphatase 2A (PP2A) regulatory subunit, B56β, due to increased steady-state levels of its kinase, Cdc2-like kinase 2 (CLK2). Pharmacological and genetic activation of Akt or inhibition of CLK2 relieved synaptic deficits in Shank3-deficient and PMDS patient-derived neurons. CLK2 inhibition also restored normal sociability in a Shank3-deficient mouse model. Our study thereby provides a novel mechanistic and potentially therapeutic understanding of deregulated signaling downstream of Shank3 deficiency.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G) 20 ]. Chemical modification of the initial "hit" improved the compound potency to an IC 50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 M. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC 50 of 5 M). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC 50 s of >100 M against hepatitis C virus RdRp and human DNA polymerase ␣ and ␤. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.