R4 and R12 subfamily RGS proteins: structures, functions, and emerging chemical biology

It is essential that cells respond to their extracellular environment with appropriate intracellular changes. Many environmental cues are received at the cell membrane, by a family of G-protein coupled receptors (GPCRs) and their heterotrimeric G-proteins, composed of Gα, Gβ and Gγ subunits. Upon binding of a hormone, neurotransmitter, tastant, or small molecule agonist at the membrane-bound GPCR, the receptor catalyzes the exchange of GDP for GTP on the heterotrimeric Gα subunit. This change results in the release of Gβγ from the Gα subunit. The dissociated Gα and Gβγ dimer can each signal to downstream effectors until the Gα hydrolyzes GTP, resulting in the reassociation of the Gαβγ heterotrimer. The duration of effector activation is therefore controlled by the duration of the Gα subunit in its GTP-bound state. The state of Gα as a GTP-bound protein is short-lived, however, given that the protein has an intrinsic ability to hydrolyze GTP to GDP and inorganic phosphate - an activity that can be greatly accelerated by Regulator of G-protein Signaling (RGS) proteins, which are known to act as GTPase-accelerating proteins (GAPs) for Gα subunits. The work described herein represents series of studies aimed at furthering our understanding of the molecular determinants of RGS protein/Gα interaction specificity, facilitating the identification of small molecule modulators of RGS protein activity, and understanding the biochemical function and physiological roles of RGS21. Toward the first aim, I performed mutagenesis on residues predicted to change the Gα specificity of RGS2 and extensively characterized these mutants using GTP hydrolysis assays and Gα interaction assays employing surface plasmon resonance and in vitro FRET. To comprehensively understand the role that each mutation was playing in allowing RGS2 to bind to a non-native Gα binding partner, I solved a crystal structure of a mutant RGS2 in complex with Gαi. Toward the second aim, facilitating the identification of small molecule modulators of RGS protein function, I used a variety of biophysical tools to determine the mechanism of action of the first commercially available RGS protein inhibitor - which was ultimately determined to be a non-specific, thiol-reactive compound. In order to identify new small molecule modulators of RGS protein function, I developed and validated a high-throughput screen for the RGS12/Gαi1 interaction. This screen was run against several compound libraries, both locally and at the NIH Chemical Genomics Center (NCGC); however, no hits were subsequently validated as in vivo inhibitors of the RGS12/Gαi1 interaction. Given these setbacks, we rethought how we were screening for RGS protein inhibitors and developed a completely novel, enzymatic-based assay that can be used for high-throughput screening. Toward the final aim, we confirmed the disputed report by von Buchholtz et al. that RGS21 is expressed only in chemosensory cells; however, we were also able to identify RGS21 transcripts in sensory digestive and pulmonary epithelia. Using biochemical methods, we demonstrated that RGS21 exhibits high affinity binding toward a variety of Gα substrates and that it can accelerate their GTP hydrolysis in vitro. We also present data that endogenous RGS21 expression serves to negatively regulate tastant receptor signal transduction in a cellular model of gustation

Reducing nasal morbidity after skull base reconstruction with the nasoseptal flap: Free middle turbinate mucosal grafts

The nasoseptal flap provides hearty, vascularized tissue for reconstruction of Expanded Endonasal Approaches (EEA); however, it produces donor site morbidity due to exposed cartilage. Mucosalization of the septum requires 12 weeks, multiple debridements, and frequent saline rinses. This study addresses the reduction of nasal morbidity by grafting middle turbinate mucosa onto the exposed septum

A P-loop Mutation in Gα Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis

Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gαi1(G42R) binding to GDP·AlF4− or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gαq(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants

Structural Determinants of RGS-RhoGEF Signaling Critical to Entamoeba histolytica Pathogenesis

SummaryG protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G protein α subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and guanosine triphosphatase-accelerating protein, EhRGS-RhoGEF, in a nucleotide state-selective fashion. Coexpression of EhRGS-RhoGEF with constitutively active EhGα1 and EhRacC leads to Rac-dependent spreading in Drosophila S2 cells. EhRGS-RhoGEF overexpression in E. histolytica trophozoites leads to reduced migration toward serum and lower cysteine protease activity, as well as reduced attachment to, and killing of, host cells. A 2.3 Å crystal structure of the full-length EhRGS-RhoGEF reveals a putative inhibitory helix engaging the Dbl homology domain Rho-binding surface and the pleckstrin homology domain. Mutational analysis of the EhGα1/EhRGS-RhoGEF interface confirms a canonical “regulator of G protein signaling” domain rather than a RhoGEF-RGS (“rgRGS”) domain, suggesting a convergent evolution toward heterotrimeric and small G protein cross-talk

Heterotrimeric G-protein Signaling Is Critical to Pathogenic Processes in Entamoeba histolytica

Heterotrimeric G-protein signaling pathways are vital components of physiology, and many are amenable to pharmacologic manipulation. Here, we identify functional heterotrimeric G-protein subunits in Entamoeba histolytica, the causative agent of amoebic colitis. The E. histolytica Gα subunit EhGα1 exhibits conventional nucleotide cycling properties and is seen to interact with EhGβγ dimers and a candidate effector, EhRGS-RhoGEF, in typical, nucleotide-state-selective fashions. In contrast, a crystal structure of EhGα1 highlights unique features and classification outside of conventional mammalian Gα subfamilies. E. histolytica trophozoites overexpressing wildtype EhGα1 in an inducible manner exhibit an enhanced ability to kill host cells that may be wholly or partially due to enhanced host cell attachment. EhGα1-overexpressing trophozoites also display enhanced transmigration across a Matrigel barrier, an effect that may result from altered baseline migration. Inducible expression of a dominant negative EhGα1 variant engenders the converse phenotypes. Transcriptomic studies reveal that modulation of pathogenesis-related trophozoite behaviors by perturbed heterotrimeric G-protein expression includes transcriptional regulation of virulence factors and altered trafficking of cysteine proteases. Collectively, our studies suggest that E. histolytica possesses a divergent heterotrimeric G-protein signaling axis that modulates key aspects of cellular processes related to the pathogenesis of this infectious organism

Two Gα i1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity

RGS proteins are critical modulators of G protein-coupled receptor (GPCR) signaling given their ability to deactivate Gα subunits via “GTPase-accelerating protein” (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow GDP release from Gα and lack of solution-phase assays for detecting free GDP in the presence of excess GTP. To overcome these hurdles, we developed a Gαi1 mutant with increased GDP dissociation and decreased GTP hydrolysis, enabling detection of GAP activity using steady-state GTP hydrolysis. Gαi1(R178M/A326S) GTPase activity was stimulated 6~12 fold by RGS proteins known to act on Gαi subunits, and not affected by those unable to act on Gαi, demonstrating that the Gα/RGS domain interaction selectivity was not altered by mutation. Gαi1(R178M/A326S) interacted with RGS proteins with expected binding specificity and affinities. To enable non-radioactive, homogenous detection of RGS protein effects on Gαi1(R178M/A326S), we developed a Transcreener® fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Gαi1(R178M/A326S) with a homogenous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling

The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Gα-interaction face

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and are thus crucial to the timing of G protein-coupled receptor (GPCR) signaling. Small molecule inhibition of RGS proteins is an attractive therapeutic approach to diseases involving dysregulated GPCR signaling. Methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986) was reported as a selective RGS4 inhibitor, but with an unknown mechanism of action (Roman et al., 2007 Mol Pharmacol. 71:169−75). Here, we describe its mechanism of action as covalent modification of RGS4. Mutant RGS4 proteins devoid of surface-exposed cysteine residues were characterized using surface plasmon resonance and FRET assays of Gα binding, as well as single-turnover GTP hydrolysis assays of RGS4 GAP activity, demonstrating that cysteine-132 within RGS4 is required for sensitivity to CCG-4986 inhibition. Sensitivity to CCG-4986 can be engendered within RGS8 by replacing the wildtype residue found in this position to cysteine. Mass spectrometry analysis identified a 153 dalton fragment of CCG-4986 as being covalently attached to the surface-exposed cysteines of the RGS4 RGS domain. We conclude that the mechanism of action of the RGS protein inhibitor CCG-4986 is via covalent modification of Cys-132 of RGS4, likely causing steric hinderance with the all-helical domain of the Gα substrate

HPV-Associated Head and Neck Cancer: Molecular and Nano-Scale Markers for Prognosis and Therapeutic Stratification

Over the last 10 years, it has become clear that patients with head and neck cancer can be stratified into two distinct subgroups on the basis of the etiology of their disease. Patients with human papillomavirus-related cancers have significantly better survival rates and may necessitate different therapeutic approaches than those with tobacco and/or alcohol related cancers. This review discusses the various biomarkers currently in use for identification of patients with HPV-positive cancers with a focus on the advantages and limitations of molecular and nano-scale markers

Extramedullary Hematopoiesis in the Sinonasal Cavity: A Case Report and Review of the Literature

Approximately 1 in 600 African-Americans are homozygous for the sickle cell gene.1 This commonly inherited hematologic disorder causes sickling of red blood cells (RBCs), prompting rapid hemolysis. A common clinical manifestation of sickle cell disease (SCD) is chronic anemia. The body responds by increasing hematopoiesis. RBC production classically occurs in the bone marrow of the long bones, pelvis, spine, and sternum. With chronically elevated erythropoietin levels, organs such as the spleen and liver help augment the body’s RBC supply. These organs are areas of fetal erythropoiesis that do not typically contribute to physiologic RBC production in adults. Other, less commonly involved organs that have been documented as sites of extramedullary hematoposesis (EMH) include lymph nodes, paravertebral regions, intra-spinal canal, pre-sacral region, nasopharynx, and paranasal sinuses

COVID-19 related olfactory dysfunction prevalence and natural history in ambulatory patients

Background: Evidence regarding prevalence of COVID-19 related Olfactory dysfunction (OD) among ambulatory patients is highly variable due to heterogeneity in study population and measurement methods. Relatively few studies have longitudinally investigated OD in ambulatory patients with objective methods. Methods: We performed a longitudinal study to investigate OD among COVID-19 ambulatory patients compared to symptomatic controls who test negative. Out of 81 patients enrolled, 45 COVID-19 positive patients and an age- and sex-matched symptomatic control group completed the BSIT and a questionnaire about smell, taste and nasal symptoms. These were repeated at 1 month for all COVID-19 positive patients, and again at 3 months for those who exhibited persistent OD. Analysis was performed by mixed-effects linear and logistic regression. Results: 46.7% of COVID-19 patients compared to 3.8% of symptomatic controls exhibited OD at 1-week post diagnosis (p<0.001). At 1 month, 16.7%, (6 of 36), of COVID-19 patients had persistent OD. Mean improvement in BSIT score in COVID-19 patients between 1-week BSIT and 1 month follow-up was 2.0 (95% CI 1.00 – 3.00, p<0.001). OD did not correlate with nasal congestion (r= −0.25, 95% CI, −0.52 to 0.06, p=0.12). Conclusions: Ambulatory COVID-19 patients exhibited OD significantly more frequently than symptomatic controls. Most patients regained normal olfaction by 1 month. The BSIT is a simple validated and objective test to investigate the prevalence of OD in ambulatory patients. OD did not correlate with nasal congestion which suggests a congestion-independent mechanism of OD