Nrf2 sensitizes ferroptosis through l-2-hydroxyglutarate-mediated chromatin modifications in sickle cell disease.

Sickle cell disease (SCD) is a chronic hemolytic and systemic hypoxia condition with constant oxidative stress and significant metabolic alterations. However, little is known about the correlation between metabolic alterations and the pathophysiological symptoms. Here, we report that Nrf2, a master regulator of cellular antioxidant responses, regulates the production of the metabolite l-2-hydroxyglutarate (L2HG) to mediate epigenetic histone hypermethylation for gene expression involved in metabolic, oxidative, and ferroptotic stress responses in SCD. Mechanistically, Nrf2 was found to regulate the expression of L2HG dehydrogenase (L2hgdh) to mediate L2HG production under hypoxia. Gene expression profile analysis indicated that reactive oxygen species (ROS) and ferroptosis responses were the most significantly affected signaling pathways after Nrf2 ablation in SCD. Nrf2 silencing and L2HG supplementation sensitize human sickle erythroid cells to ROS and ferroptosis stress. The absence of Nrf2 and accumulation of L2HG significantly affect histone methylation for chromatin structure modification and reduce the assembly of transcription complexes on downstream target genes to regulate ROS and ferroptosis responses. Furthermore, pharmacological activation of Nrf2 was found to have protective effects against ROS and ferroptosis stress in SCD mice. Our data suggest a novel mechanism by which Nrf2 regulates L2HG levels to mediate SCD severity through ROS and ferroptosis stress responses, suggesting that targeting Nrf2 is a viable therapeutic strategy for ameliorating SCD symptoms.

Inhibition of the BTK-IDO-mTOR axis promotes differentiation of monocyte-lineage dendritic cells and enhances anti-tumor T cell immunity.

Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton's tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.

C-terminal modification is required for GABARAP-mediated GABA(A) receptor trafficking.

We investigated the ubiquitin-like modification of GABA(A) receptor-associated protein (GABARAP) and its function. A fusion protein of GABARAP with v5 in the N terminus and myc in the C terminus was expressed in rat cultured hippocampal neurons and PC12 cells. Western blotting with antibodies to v5 and myc revealed that the C terminus of GABARAP was cleaved off. Cleavage was blocked by mutating the C-terminal Gly116 to Ala, suggesting that G116 is required for the processing. Unlike ubiquitin, GABARAP was not incorporated covalently into higher-molecular-weight protein complexes. Nor was GABARAP degraded by ubiquitinylation through the proteasome, although GABARAP formed noncovalent dimers. Immunofluorescent confocal microscopy demonstrated that recombinantly expressed GABARAP was diffusely localized in PC12 cells. However, prevention of C-terminal processing in the mutant GABARAP(G116A) resulted in redistribution to the Golgi. In neurons, punctate cytoplasmic staining of GABARAP was seen in soma and processes, whereas GABARAP(G116A) was limited to soma. Compared with wild-type GABARAP, the colocalization and interaction of GABARAP(G116A) with GABA(A) receptors were significantly reduced, resulting in a reduction in expression of receptors in the plasma membrane. When alpha1beta2gamma2S-containing GABA(A) receptors were expressed in oocytes, the increased surface expression of GABA(A) receptors, as shown by increased GABA currents and surface-accessible GABA(A) receptor subunit polypeptides resulting from GABARAP coexpression, was prevented by mutation G116A. In addition, the distribution of NSF (N-ethylmaleimide-sensitive factor) was affected in GABARAP(G116A)-expressing neurons. These results suggest that glycine 116 is required for C-terminal processing of GABARAP and that processing is essential for the localization of GABARAP and its functions as a trafficking protein.

GABAA receptor-associated protein regulates GABAA receptor cell-surface number in Xenopus laevis oocytes.

GABA(A) receptor-associated protein (GABARAP) was isolated previously in a yeast two-hybrid screen using the intracellular loop of the gamma2 subunit of the GABA(A) receptor as bait. GABARAP has been shown to participate in the membrane-clustering and intracellular-trafficking of GABA(A) receptors, including a stimulation of the surface expression of GABA(A) receptors. To assess this quantitatively, we used Xenopus laevis oocytes expressing alpha1beta2gamma2S-containing GABA(A) receptors to demonstrate that coexpression of GABARAP increased net surface levels of GABA(A) receptors as shown by both increased GABA currents and surface-expressed protein. This GABARAP stimulation of GABA currents required the receptor gamma2 subunit and full-length GABARAP: deletion of the microtubule-binding domain (amino acids 1-22) or disrupting the polymerization of microtubules abolished the enhancement, indicating that the effect of GABARAP was derived from the interaction with microtubules. GABARAP coexpression did not alter the general properties of GABA(A) receptors such as sensitivity to GABA or benzodiazepines, but it increased surface levels of receptor protein in oocytes. Rather, it seems to supplement inadequate amounts of endogenous GABARAP to support optimum trafficking and/or stabilization of surface GABA(A) receptors.

GABAA receptor-associated protein traffics GABAA receptors to the plasma membrane in neurons.

The trafficking of GABA(A) receptors is an important component of the pathway that regulates plasticity of inhibitory synapses. The 17 kDa GABA(A) receptor-associated protein (GABARAP) has been implicated in the trafficking of GABA(A) receptors because of its ability to interact not only with the gamma2 subunit of the receptor but also with microtubules and the N-ethylmaleimide-sensitive factor (NSF). To elucidate the role of GABARAP in the trafficking of GABA(A) receptors, we have constructed a yellow fluorescent protein (YFP) fusion protein of GABARAP and expressed it in neurons using adenovirus, so that its function may be examined. YFP-GABARAP colocalized with gamma2 subunit-containing GABA(A) receptors and NSF to the perinuclear cytoplasm in cultured hippocampal neurons and to the proximal regions of dendrites that are making synaptic contact. Expression of YFP-GABARAP in Cos7 cells and cultured hippocampal neurons was able to increase the level of GABA(A) receptors detected at the plasma membrane, even at low levels of YFP-GABARAP expression. This effect is specific to the function of GABARAP on GABA(A) receptor trafficking, because point mutations in the gamma2-binding domain of YFP-GABARAP interfered with the ability of YFP-GABARAP to increase GABA(A) receptor surface levels. These mutations also disrupted the colocalization of YFP-GABARAP with the gamma2 subunit and with NSF in hippocampal neurons. The results of this study show for the first time that GABARAP has a functional effect on the trafficking of GABA(A) receptors and provide decisive evidence for the role of GABARAP in transporting GABA(A) receptors to the plasma membrane in neurons.

Fishing for allosteric sites on GABA(A) receptors.

GABA(A) receptors have structural and functional homology with a super-family of cys-loop ligand-gated ion channel receptors including the nicotinic acetylcholine receptors. Amino acid residues involved in ligand-binding pockets are homologous among super-family members, leading to the multiple-loop model of binding sites situated at subunit interfaces, validated by structural studies on the nicotinic acetylcholine receptor and water-soluble snail acetylcholine binding protein. This article will briefly review the literature on the agonist binding sites on the receptor super-family, and then describe the current situation for attempts to identify sites for allosteric modulators on the GABA(A) receptors. A combination of mutagenesis and photoaffinity labeling with anesthetic ligands has given some leads in this endeavor. Current work by others and ourselves focuses on three putative sites for modulators: (1) within the ion channel domain TM2, near the extracellular end; (2) the agonist binding sites and homologous pockets at other subunit interfaces of the pentameric receptor; and (3) on the linker region stretching from the agonist site loop C to the top of the TM1 region. It is likely that concrete structural information will be forthcoming soon.

A single M1 residue in the beta2 subunit alters channel gating of GABAA receptor in anesthetic modulation and direct activation.

General anesthetics allosterically modulate the activity of neuronal gamma-aminobutyric acid, type A (GABAA), receptors. Previous mutational studies from our laboratory and others have shown that the regions in transmembrane domain 1 (M1) and pre-M1 of alpha and beta subunits in GABA receptors are essential for positive modulation of GABA binding and function by the intravenous (IV) general anesthetics. Mutation of beta2Gly-219 to Phe corresponded in rho nearly eliminated the modulatory effect of IV anesthetics in alpha1/beta2/gamma2S combination. However, the general anesthetics retained the ability to directly open the channel of mutant G219F, and the apparent affinity for GABA was increased, and desensitization rate was reduced. In this study, we made additional single mutations such as 219 Ser, Cys, Ile, Asp, Arg, Tyr, and Trp. The larger side chains of the replacement residues produced the greatest reduction in enhancement of GABA currents by IV anesthetics at clinical concentrations (Trp > Tyr = Phe > Arg > Asp > Ile > Cys > Ser = wild type). Compared with a 2-3-fold response in wild type, pentobarbital and propofol enhanced less than 0.5-fold; etomidate and alphaxalone modulation was reduced from more than 4- to 1-fold in G219F, G219Y, and G219W. A linear correlation was observed between the volume of the residue at position 219 and the loss of modulation. An identical correlation was found for the effect of modulation on left-shift in the GABA EC50 value; furthermore, the same rank order of residues, related to size, was found for reduction in the maximal direct channel-gating by pentobarbital (1 mm) and etomidate (100 mum) and for increased apparent affinity for direct gating by the IV anesthetics.