Biochemical and Biophysical Research Communications

The chemokine-like peptide GPR15LG is a known agonist of G protein-coupled receptor 15 (GPR15), a ligand-receptor pair primarily implicated in mammalian mucosal immunity and lymphocyte homing. However, the evolutionary origin and phylogenetic distribution of this signaling system remain poorly understood due to the extreme sequence diversity of GPR15LG orthologs. In this study, we identified GPR15LG orthologs in several fish species for the first time according to their conserved gene synteny, genomic organization, and amino acid sequence features. A representative ortholog from the spotted gar (Lepisosteus oculatus), termed Lo-GPR15LG, was recombinantly prepared and functionally characterized using NanoLuc Binary Technology (NanoBiT)-based {beta}-arrestin recruitment assay and homogenous ligand-receptor binding assay. Our results demonstrated that Lo-GPR15LG directly binds to and efficiently activates its cognate receptor, Lo-GPR15, with a dissociation constant (Kd) of approximately 60 nM and an EC50 value of approximately 10 nM. Functional assays further revealed that receptor activation is critically dependent on the conserved C-terminal residues. Notably, human and fish orthologs exhibited no cross-species activity, consistent with their high sequence divergence. These findings reveal that the GPR15LG-GPR15 signaling system originated in ancient fish ancestors and has remained a conserved signaling axis throughout vertebrate evolution, suggesting a fundamental role in immunity across all vertebrate lineages.

The KCNE (KCNE1-6) proteins are single-pass transmembrane auxiliary subunits of the voltage-gated K+ channel KCNQ1. KCNQ1-KCNE complexes have been well studied in jawed vertebrates ranging from zebrafish to humans, but KCNE subunits from earlier-diverging vertebrates remain poorly characterized. Here, we functionally characterize a single KCNE-like gene in lamprey, a jawless vertebrate, and designate it kcne0 as an early-diverging member of the KCNE family. KCNE0 shows moderate amino acid sequence similarity to KCNE1-6 but is not particularly similar to any single isoform. Both kcnq1 and kcne0 transcripts were detected in multiple lamprey organs. When co-expressed with lamprey KCNQ1, KCNE0 produced a constitutively active current, similar to KCNE3. By contrast, KCNE0 modulated KCNQ1 from other species less effectively, suggesting species-specific tuning of KCNQ1-KCNE compatibility. Introducing into KCNE0 an intracellular tetra-leucine motif analogous to that in KCNE4 markedly reduced KCNQ1 current amplitude, conferring a KCNE4-like inhibitory effect. Overall, this work provides a functional reference for comparing KCNE-dependent modulation of KCNQ1 across vertebrates and suggests an underlying compatibility mechanism.

Cav1.2 is an L-type voltage-gated Ca2+ channel (VGCC) that supports Ca2+ influx in response to membrane depolarization. Ca2+ entering via Cav1.2 alters gene expression, activates Ca2+-dependent enzymes and has been implicated in synaptic plasticity. ORL-1 is a Gi/o-coupled G protein-coupled receptor (GPCR) that is expressed in the peripheral and central nervous systems. Both Cav1.2 and ORL-1 are expressed in the hippocampus, where they have been implicated in learning and memory. It is well-documented that ORL-1 interacts with another VGCC, Cav2.2. However, less is known about potential interactions between Cav1.2 and ORL-1. Here, we examine the interplay between Cav1.2 (Cav1c, Cav2{delta}-1, Cav{beta}1) and ORL-1 co-expressed in tsA-201 cells by using biochemical, electrophysiological and confocal imaging analysis. Co-immunoprecipitations revealed that ORL-1 independently interacts with Cav1c and Cav2{delta}-1 subunits of the Cav1.2 channel complex. Electrophysiological recordings revealed that co-expression with ORL-1 reduced Cav1.2 peak current density without altering its biophysical properties. Acute perfusion with the ORL-1 receptor agonist nociceptin (1 M) did not alter Cav1.2 current density. Confocal imaging experiments revealed that ORL-1 significantly decreases Cav1.2 plasma membrane expression by disrupting forward trafficking. Interestingly, ORL-1 did not affect Cav1.2 endocytosis. Overall, our results demonstrate a previously unrecognized interaction between ORL-1 and Cav1.2 that alters Cav1.2 membrane expression without affecting biophysical properties.

Synthetic 6-Br-Q0C10 has been shown to have a partial electron transfer activity of native coenzyme Q in the isolated mitochondria. It reduces energy coupling efficiency by 30 %, suggesting that it may be useful in the management of obesity. The effect of 6-Br-Q0C10 on cell growth has been confirmed by several cell lines. Whether or not it behaves in the same way in the animal, however, has not yet been tested. Recently, we investigated the effect of 6-Br-Q0C10 on growth of rats. When 6-Br-Q0C10 was dissolved in different media, such as carboxymethyl cellulose, ethanol, and mixture of oil and butter and then feed to rats It shows no toxicity and little negative effects on growth as measured body weight gains over a period of time. When higher concentration (0.5 mg) of 6-Br-Q0C10 was given to each rat in 0.3 mL of oil/butter (70%/30%) mixture via intragastric injection daily for a period, a significant reduction in body weight gains was observed. These results validate the earlier observation that 6-Br-Q0C10 reduces the growth (30-60%) of all cell lines tested, in a time- and concentration dependent manner. These results strengthen the idea of using 6-Br-Q0C10 to manage obesity. It is also implying that 6-BrQ0C10 may slow the growth rate of cancer cells and thus prolong life. (This study was partially funded by NIH grants AA030221 and DA046258 to S.L.Chang.)

The mucosal chemoattractant C-X-C motif chemokine ligand 17 (CXCL17) was recently identified as a ligand for the orphan G protein-coupled receptor 25 (GPR25). Although CXCL17 orthologs have been identified in fishes, amphibians, and mammals, their presence in reptiles and birds remains unclear. In this study, we employed bioinformatic searches based on gene synteny and sequence features to identify CXCL17 orthologs in public databases. We identified functional CXCL17 orthologs in 46 reptilian species, including lizards, snakes, turtles, and alligators. In contrast, we found only non-functional gene relics in 22 bird species, suggesting the avian lineage lost functional CXCL17 during evolution. A recombinant reptilian CXCL17 from the loggerhead turtle (Caretta caretta), termed Cc-CXCL17, directly bound to and efficiently activated its corresponding receptor, Cc-GPR25, in a C-terminal fragment-dependent manner. Activation of Cc-GPR25 by Cc-CXCL17 also induced chemotactic movement of transfected human embryonic kidney (HEK) 293T cells. In cross-species activity assays, CXCL17s from human and tropical clawed frog could activate Cc-GPR25 albeit with lower potency, but fish orthologs lacked this activity; all tested CXCL17s had no detectable activity towards chicken GPR25, but Cc-CXCL17 had low activity towards mallard GPR25. Our findings demonstrate the presence of functional CXCL17 orthologs in extant reptiles and provide evidence for their evolutionary loss in birds, offering new insights into the phylogenetic distribution of the newly identified CXCL17-GPR25 signaling system.

Voltage-gated potassium channels (Kv) are a large family of potassium channels composed of 40 members across 12 subtypes. The KCNH genes encode 3 subfamilies of voltage-gated potassium channels: Kv10 (EAG, ether a go go), Kv11 (ERG, EAG-related gene), and Kv12 (ELK, EAG-like K). Kv channels play prominent roles in the neuronal and cardiovascular systems. Mutations in Kv channels have been linked to many human diseases, such as epilepsy, heart arrhythmias, and cancers. Significant progress has been made in understanding protein structures, physiological functions, and the pharmacological modifiers. However, the evolutionary history and gene expression of vertebrate KCNH genes during embryonic development remain largely unknown. We systematically identified and cloned 14 kcnh genes in zebrafish. Then, we examined vertebrate KCNH channel evolution by phylogenetic and syntenic analyses. Our data revealed that the three subtypes of the KCNH gene family have already evolved in invertebrates, long before the emergence of vertebrates. The number of vertebrate KCNH genes increased, most likely due to whole-genome duplications (WGDs). In addition, we examined zebrafish kcnh gene expression during early embryogenesis by in situ hybridization. Each subgroups genes showed similar but distinct gene expression domains with some exceptions. Most of them were expressed in neural tissues. Notably, kcnh6a showed robust expression in the developing heart, consistent with its conserved role in cardiac repolarization. Additionally, a few kcnh genes were transiently expressed in nonneural tissues, such as somites and the notochord, suggesting they may have a unique role in embryonic development. Our phylogenetic and developmental analyses of KCNH channels shed light on their evolutionary history and potential roles during embryogenesis, in line with their physiological functions and human channelopathies.

BackgroundKATNIP (Katanin-interacting protein), also known as KIAA0556, is one of the human genes with pathogenic variants linked to Joubert syndrome, an archetypal neurodevelopmental ciliopathy. KATNIP is a scaffolding protein with a critical role in ciliogenesis. In this study, we characterized the ciliopathy phenotypes due to KATNIP gene deletion. ResultsWe produced a Katnip null mouse model using CRISPR-Cas12a (Cpf1). The null heterozygotes appeared normal while the homozygotes died around postnatal day 9, showing severe hydrocephalus and deficiency in neuroprogenitor cell proliferation. Katnip-deficient cells in the brain have a higher rate of cilia formation and longer cilia than wild type cells. ConclusionKATNIP loss of function gives rise to hydrocephalus found in Joubert syndrome. The results indicate that KATNIP restricts ciliogenesis and cilia extension and supports proliferation of neuroprogenitor cells in the brain.

The influenza virus poses a significant global health threat due to its continuous evolution, immune evasion, and zoonotic spillover. The rise of drug resistance, reduced susceptibility to existing antiviral medications, and the limited effectiveness of annual vaccines underscore the need for new antiviral strategies. To infect, the influenza virus binds to sialic acid (SA)-containing molecules on host cell membranes through hemagglutinin (HA). Blocking this interaction represents a promising antiviral approach. Herein, we report that SA containing plasma membrane-derived vesicles (PMV) efficiently inhibits in vitro Influenza A virus (IAV) infection. Using orthogonal methods, we demonstrate that PMV derived from A549, MDCK, and HEK cells competitively bind to H1N1 (WSN) and H3N2 (X-31) IAV strains, block entry and infection in human respiratory epithelial cells in a dose-dependent manner, without causing significant toxicity. When the size of the vesicles was reduced through extrusion, the antiviral activity was enhanced, and this was found to be correlated with a size-dependent increase in hemagglutination inhibition and reduced IAV internalisation. Plasma membrane-derived vesicles may serve as a novel antiviral strategy against influenza virus infections due to their simple production method and conserved SA binding site on HA.

The {beta}-cardiac myosin (MYH7) mutation E525K was first identified in 2012 in a patient with dilated cardiomyopathy (DCM). Work using engineered myosin constructs has shown that this mutation causes hypocontractility by stabilizing the interacting heads motif (IHM) of myosin despite the mutant E525K motor head exhibiting increased ATPase activity. However, no measurements have been made in myofilaments or cardiomyocytes to determine how this mutation affects contractile function. Here, we present force and contractile kinetics measurements from induced pluripotent stem cell (iPSC)-derived cardiomyocytes engineered for heterozygous expression of E525K. Contraction of E525K single cells decreased by 65%, and isometric twitch force in engineered heart tissues (EHTs) decreased by 39%. In contrast, maximal isometric force in isolated myofibrils increased by 45%. Structural analysis revealed reduced myofibril content (13.7% decrease) and organization (increased z-disk dispersion angle) in E525K cells. We confirmed that E525K S1 myosin has higher actin affinity than WT S1 and elevated ATPase activity. However, no change was observed in the rate of ADP release. Importantly, there was no change in the rate of force development or relaxation in myofibrils, cells, or EHTs. These findings suggest that myosin crossbridge cycling is not altered under load by E525K. Decreased force generation in EHTs and shortening in cardiomyocytes arise from reduced sarcomere number and myofibrillar disorganization. Additional force deficits likely result from stabilization of the IHM, as recently reported by others. This study demonstrates the value of multi-scale analysis for determining the functional profile of cardiomyocytes containing disease-related sarcomere protein mutations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/733270v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@5f85f5org.highwire.dtl.DTLVardef@153b3b6org.highwire.dtl.DTLVardef@3b8f21org.highwire.dtl.DTLVardef@31d323_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract: A Model for how the E525K mutation impacts contracting myofibrils Here we have shown that the E525K mutation impacts contraction in three ways: (1) Decreased sarcomere organization in cells and tissues drives decreased force generation. (2) Increased binding affinity of E525K myosin for actin contributes to increased force generation in isolated myofibrils. (3) Increased IHM stability. (4) The rate limiting step of loaded crossbridge cycling, ADP release, is unchanged by the E525K mutation and the rate of loaded contraction and relaxation is unchanged at all scales of contraction measured here. C_FIG

T cells are important components of the adaptive immune system and develop through a selection process regulated by signaling through the T-cell receptor (TCR). Thymocyte-Expressed Molecule Expressed in Selection (THEMIS) is a TCR-proximal protein that modulates the activity of Shp1 phosphatase to influence TCR signaling during development. THEMIS has been shown to both activate and inhibit Shp1, but the molecular mechanisms of these functions are poorly understood. THEMIS contains two rare Cysteine All-Beta In THEMIS (CABIT) domains, the N-terminal of which interacts with Shp1 and is likely responsible for modulation of its phosphatase activity. Herein, we report the first crystal structure of the THEMIS CABIT1 domain. While a portion of the CABIT1 domain is poorly resolved, it appears to share the same overall fold observed in our recent CABIT2 crystal structure and AlphaFold predictions. We show that phosphorylation of the CABIT1 domain by LCK is required for association with SHP1 and that phosphorylated CABIT1 can protect Shp1 from oxidation and inhibition by reactive oxygen species (ROS), which may serve as a mechanism by which THEMIS enhances Shp1 activity.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

The inositol triphosphate-associated, ER transmembrane proteins IRAG and LRMP are isoform specific regulators of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, while IRAG mimics the effect of cAMP on the channel. We previously showed that regulation by LRMP requires both the N-terminus of HCN4 and a unique orientation of the HCN4 cAMP transduction center, which is comprised of the N-terminal HCN domain, the C-linker, and the S4-S5 linker. However, it remains unknown if the homologous IRAG requires similar structural features to mimic cAMP-dependent potentiation, or if the site and mechanism of action are different between the two regulators. Using patch clamp electrophysiology, we determined that the initial 43 amino acids of IRAG are necessary and sufficient to confer regulation of HCN4. Similar to LRMP, IRAG also requires a portion of the N-terminus of HCN4 to confer its regulatory effects. Also similar to LRMP, two point mutations in the C-linker region, which are the only sequence differences in that region between HCN4 and the other HCN isoforms, were able to eliminate the effect of IRAG suggesting the unique orientation of the cAMP transduction center in HCN4 is likely important for IRAG function. Taken together, these findings suggest a model whereby IRAG and LRMP interact with the channel in similar regions, although potentially in unique ways, and act on the cAMP transduction center with LRMP inhibiting the coupling of this region to gating and IRAG strengthening it. SUMMARYThe ER transmembrane protein IRAG binds to and potentiates HCN4 channels. This study demonstrates that IRAG regulation of HCN4 requires only the first 43 amino acids of IRAG and involves contributions from the N-terminus and cAMP transduction center of HCN4.

G-protein-coupled receptor 75 (GPR75) has emerged as an important mediator in diet-induced obesity (DIO) and a promising therapeutic target for anti-obesity drugs. However, the anatomical location of GPR75 in the brain remains unclear, hindering the understanding of GPR75 biology in DIO. Here, we generated a new GPR75-GFP-Ires-Cre knockin mouse strain, in which the Cre expression is driven by the endogenous GPR75 promoter and the GFP is fused with the C-terminal of the GPR75 protein. Both Cre and GFP were confirmed to be colocalized with the endogenous GPR75 expression. In addition, the GPR75-GFP fusion protein remains functionally normal with unaltered susceptibility to DIO. Moreover, using this mouse strain, we found that GPR75 is broadly expressed throughout the brain and mainly localized to the cytoplasm of brain neurons. This new genetic tool can therefore be used to study the neural basis for GPR75 in mediating DIO.

TAT (Twin-Arginine Translocation) is a preprotein translocation system dedicated to membrane translocation of prefolded proteins in plants and bacteria. The TAT translocon, consisting of the TatABC subunits, drives translocation using proton motive force. However, there have been no reports on the successful reconstitution of the TAT system. In this report, we show that MPIase, a glycolipid that has known to catalyze membrane protein integration, is essential for the TAT system. Our findings in recombinant Escherichia coli demonstrate that overproducing TatABC increases MPIase levels and that depleting MPIase results in TAT precursor accumulation in the cytosol. Furthermore, co-reconstitution of MPIase with TatABC revealed the translocation activities of TAT substrates in a proton motive force-dependent manner. This is the first successful reconstitution of the TAT system and will be advantageous for understanding its mechanisms.

CaMKII{delta} is the dominant isozyme of Ca2+/calmodulin-dependent protein kinase II in the heart. Under certain pathological conditions, it can be oxidized, causing a constitutive activation that can lead to cardiac failure. We recently showed that, in purified CaMKII{delta} exposed to oxidative conditions, a disulfide link formed between Cys273 and Cys290 causes this autonomous activation. Cys273 has a low pKa that facilitates the oxidation of its thiol to a sulfenic acid at physiological pH. Does this matter in vivo? To answer that question, we created a transgenic mouse with Cys273 mutated to serine (CaMKII{delta}C273S) to prevent disulfide formation. We conducted a detailed assessment of cardiac function at rest and in a dobutamine stress test. We found that the CaMKII{delta} Cys273Ser mutation does not have deleterious effects on cardiac physiology. Then, we assessed whether the mutation would protect the heart from ischemia-reperfusion in the Langendorff model. The CaMKII{delta}C273S mouse had improved cardiac function and decreased infarct size compared to the wild-type mouse. We conclude that blocking disulfide formation at Cys273 protects the heart against ischemia-reperfusion injury. Drugs that specifically target Cys 273 may be therapeutic in human cardiac disease.

Total body irradiation (TBI) can impair the bone marrow (BM) microenvironment and dysregulate the fates of BM-residing cells by overproducing reactive oxygen species (ROS) and inflammatory mediators. This study aims to investigate the potency and mechanism of Deinococcus radiodurans-derived deinoxanthin (DEIX) in mitigating TBI-mediated injuries in the BM microenvironment and BM-resident cells. C57BL/6 mice were divided into control, TBI, TBI+DEIX, and/or DEIX groups, in which the mice were exposed to sub-lethal TBI (5 Gy) or in combination with oral DEIX supplementation (25 mg/kg body weight). While the DEIXs effect on BM and BM-resident cells was determined after five weeks of TBI, RNA sequence profiling on the mouse group-derived BM cells was performed after two weeks of TBI. Supplementation with DEIX protected mice against TBI-mediated decrease in bone mineral density of trabecular bones. Supplemental DEIX suppressed BM microenvironmental impairment and the induction of oxidative stress and senescence in BM cells of TBI-exposed mice. That suppression was orchestrated by the DEIX-induced restoration of TBI-stimulated disorders in osteogenic, osteoclastogenic, and adipogenic activation in the BM. Ex vivo assays using BM cells supported the notion that DEIX restores TBI-mediated defects in BM cell function, including colony formation, migration, and differentiation. RNA sequence profiling demonstrated DEIXs potency to modulate the expression of genes that regulate cellular and systemic immune responses, cell proliferation and differentiation, and bone metabolism. Collectively, our results highlight the roles and associated mechanisms of DEIX in mitigating TBI-mediated microenvironmental impairment and in regulating BM-resident cells.

Mammal tails have long been recognized for their diversity of morphological form and function, however, there remains a substantial gap between the motivation to understand and emulate the various performance functions of the tail and what is known about tail anatomy. In this study, we were motivated to discover the anatomical foundations of the fast, whipping motions of the tail of the lesser Egyptian jerboa (Jaculus jaculus), which may aid in the quick changes of direction as the animal escapes from predators using ricochetal bipedal hopping. We employed microCT scans, dissections, and museum data to describe the musculoskeletal anatomy of the jerboa in comparison with the laboratory mouse (Mus musculus) and rat (Rattus norvegicus). While many aspects of tail anatomy are conserved across these species, the jerboa does possess unique characteristics such as an extremely long tail arising from caudal vertebral elongation, development of extensive dorsal musculature differentiated into lateral and medial components to increase points of skeletal attachment, and a novel anatomical feature - the bi-lobed cranial transverse process - which serves as a supernumerary dorsal tendon attachment site and possible brace to protect the ventral tendons and intrinsic muscles for a section of caudal vertebrae which likely experiences high mechanical stress.

Glucose has important roles in the development of the hematopoietic stem cells and the brain in vertebrate embryos; however, in most oviparous animals, the amount of glucose in the yolk is scarce. In zebrafish, gluconeogenesis takes place in the yolk syncytial layer (YSL), an extraembryonic tissue that surrounds the yolk. Gluconeogenic activity have also been observed in extraembryonic YSL-like tissue or endoderm-derived tissues in cloudy catshark, sterlet, and western clawed frog during development. However, it remains unclear when such ability was acquired or how it changed over the evolution of vertebrates. In this study, we used the Arctic lamprey, a cyclostome sister group of jawed vertebrates, to compare changes in metabolite levels and gluconeogenic gene expression patterns during development. Also, gluconeogenic activity was assessed using 13C-labeled substrates. Our metabolite analysis revealed that glucose levels increased during development and that glycerol was actively metabolized to produce glucose. In addition, many gluconeogenic genes were expressed in the muscle, notochord, and epithelium, making a striking contrast to previous observations in the above-mentioned vertebrates. Genomic DNA sequence motif analysis using HOMER and MEME identified common transcription factors binding motifs in the upstream regions of g6pc1/2 and fbp1 across vertebrate lineages. Among them, interestingly, the binding motif for HNF4A was not detected in g6pc1/2 and fbp1 genes of cyclostomes, suggesting distinct transcriptional regulation of gluconeogenesis in cyclostomes. These results indicate that gluconeogenesis is an essential process during development across vertebrate lineages, including cyclostomes, although the tissues and regulatory mechanisms for this function vary among lineages.

BackgroundShort-chain fatty acids (SCFAs) are produced by the gut microbiota as secondary metabolites during fermentation process of dietary fibers. Although SCFAs are beneficial for immuno-related diseases because they regulate the gene expression and functions of myeloid cells, the effects of SCFAs on the development of DCs remain unclear. MethodsWe analyzed the effect of SCFAs on the expression levels of surface proteins and mRNAs, and histone modification in Flt3L-induced bone marrow-derived DCs. ResultsSCFAs, particularly butyrate, regulated the expression of surface molecules on mouse bone marrow-derived dendritic cells (DCs): increases in MHCII, CD86, CD11b, and LPAM-1 (4{beta}7) levels and the ratio of CD11c+/PDCA-1-/B220- conventional DCs (cDCs) to CD11c+/PDCA-1+/B220+ plasmacytoid DCs (pDCs). Experiments using inhibitors of histone deacetylase (HDAC) and Gi proteins, and GPR109A deficient mice indicated that butyrate regulated DCs by suppression of HDACs and not through a stimulatory effect on G protein-coupled receptors. Butyrate and the HDAC inhibitor, trichostatin A (TSA), increased the cDC/pDC ratio, surface LPAM-1 and Itga4 mRNA, while the mRNA level of Itgb7 was not affected by butyrate and was reduced by TSA. ChIP assays showed that butyrate and TSA increased histone acetylation in the Itga4 and Spi1 genes. Furthermore, the butyrate treatment increased the levels of Spi1 mRNA and PU.1 protein and decreased those of Spib/SpiB in DCs. In knockdown (KD) experiments using siRNAs, the gene expression of Itga4 was decreased by KD of Spi1 or Irf8, and cDC/pDC ratio decreased by Spi1 KD. ConclusionsButyrate controls the gene expression and development of DCs through epigenetic regulation and DC-related transcription factors.

Regulator of G protein Signaling 6 (RGS6), heavily implicated in neurological and neuropsychiatric disorders, is enriched in mouse and human brain. Our initial cloning effort identified 36 RGS6 mRNAs in human brain. However, we recently identified an additional RGS6 protein isoform that is larger ([~]69kDa) than the ubiquitously expressed [~]56kDa RGS6L(+GGL) isoforms. Notably, this isoform, named "RGS6B" for "brain-specific", is selectively expressed in the nervous system of mice and humans. Here, we report the cloning of a new RGS6-encoding mRNA, which resembles the RGS6L1(+GGL) transcript identified in our initial cloning effort but includes a highly conserved novel exon (Alternative 3, A3) that alters the reading frame of terminal exon resulting in an extension of the protein C-terminus. When expressed in cells, RGS6LA31(+GGL) co-migrates with RGS6B, and, importantly, interfering RNA targeting exon A3 results in selective depletion of RGS6B in isolated primary cortical astrocytes. RGS6B is capable of stabilizing RGS6 binding partners R7BP and G{beta}5 and, in fact, exhibits an increased protein half-life relative to RGS6L. Both RGS6L and RGS6B are downregulated in human gliomas and share the ability to kill U87MG glioblastoma cells when overexpressed indicating conservation of non-canonical cytotoxic activity between RGS6L and RGS6B species. However, RGS6B lacks the ability to counteract Gi/o-dependent suppression of cAMP signaling, indicating a lack of functional GTPase activating protein (GAP) activity. Instead, RGS6B functions in a dominant negative manner to block Gi/o regulation by RGS6L. RGSB is the first identified RGS protein member that functions to promote, rather than inhibit, G protein signaling. The discovery of the molecular identity of RGS6B will now allow for delineation of unique functions for RGS6 protein isoforms in both physiological and pathophysiological brain states.