bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of constitutively active Gsα by molecules targeted at R231
Authors: Schumacher Morgan , Carroll Julie , Anderson Shane D , Santos Priscilla , Thu Yin Win , Nguyen Hannah , Bohlen Kade , Poch Mark , Andresen Bradley T , Rylaarsdam Robin Pals 
Published: November 2023

Activating mutations in Gsα are responsible for a variety of human pathologies, including polyostotic fibrous dysplasia, McCune‐Albright Syndrome, and a variety of endocrine tumors. We hypothesized that designing a molecule that preferentially binds to the mutated form of Gsα could provide the starting point for drug development and better therapy for these diseases. Previous work in our lab identified the region near R231 as a potential druggable target site on the mutated version of the protein, as mutations at this residue reduced the constitutive activity associated with mutations at R201. Utilizing GNASE2−/E2− cells, R201H/R231C Gsα was found to have similar basal activity and adenylyl cyclase coupling (forskolin‐mediated activity) as wild‐type Gsα, and significantly reduced isoproterenol‐mediated cAMP production. In silico docking analysis provided a slate of candidate molecules to test for inhibition of the MAS protein's constitutive activation of adenylyl cyclase. Two molecules, 6‐oxo‐N‐{[2‐(pyrrolidin‐1‐yl)pyridin‐4‐yl]methyl}‐1,6‐dihydropyridazine‐3‐carboxamide and N‐{[3‐(3‐methylphenyl)phenyl]methyl}‐6‐oxo‐1,6‐dihydropyridazine‐3‐carboxamide were able to significantly and in a dose‐dependent manner reduce the elevated basal cAMP levels in HEK cells transiently transfected with the constitutively active Gs‐R201H allele. Neither compound changed forskolin‐driven cAMP production, indicating that the effects are not downstream of the G‐protein. Maximum levels of cAMP production in response to luteinizing hormone receptor activation were also unchanged; however, a 10‐fold increase in the EC50 for hormone was measured in the presence of both drugs.Support or Funding InformationThis work was supported in part by NIH grant 1R15ED020190‐01
The Molecular Mechanisms of Prostaglandin E2 Receptor 3 and its associated G protein, Gz, in the Pancreatic β‐cell
Authors: Schaid Michael D , Wisinski Jackie , Laudre Erin , Kimple Michelle 
Published: November 2023

Signaling mediated by the G protein‐coupled receptor (GPCR), Prostaglandin E2 Receptor 3 (EP3), and the unique inhibitory G protein alpha subunit, Gαz, is emerging as a key player in the pathogenesis of beta‐cell dysfunction and loss in diabetes. β‐cell EP3 expression, as well as production of its endogenous ligand, PGE2, are up‐regulated in the context of diabetes and can be specifically targeted to improve insulin secretion from diabetic islets. Genetic ablation of Gαz protects against hyperglycemia in both Type 1 and Type 2 diabetes models by allowing for either a significant expansion in β‐cell mass or enhanced glucose stimulated insulin secretion, or both. In the rat, mouse, and human genomes, the Ptger3 gene encodes for multiple EP3 receptor splice variants that vary only in their cytoplasmic C‐terminal tail: a critical determinant for G‐protein coupling; beta‐arrestin‐mediated receptor desensitization, endocytosis and intracellular vesicular signaling; and ability to signal through other non‐canonical pathways. The mouse genome encodes for three EP3 splice variants: EP3α, EP3 β, and EP3γ. Little is known about how these splice variants differ with regards to Gαz coupling or their involvement in other signal transduction mechanisms, particularly in the beta‐cell. The overall goal of our research program is to fully characterize beta‐cell signaling mediated by the PGE2/EP3/Gαz, signaling pathway that is inhibitory towards insulin secretion, with the long‐term goal of being able to rationally target this pathway for preventative or therapeutic purposes. In this project, we focused on characterizing signaling mediated by EP3α and EP3γ, which both have human homologs. We demonstrate that EP3γ is constitutively active and coupled solely to Gαz in the beta‐cell. The ability of EP3γ to reduce insulin secretion is lost in Gαz‐null islets and is not influenced by addition of either receptor agonist or antagonist. EP3a also couples to Gαz but can be further activated by sulprostone, an EP3‐selective agonist, and may couple to other G proteins in the absence of Gαz. In beta‐cell lines and islets, sulprostone mediates both cAMP‐dependent and cAMP‐independent signaling, the latter of which we suspect is mediated by the ability of activated Gαz to recruit Rap1GAP from the cytosol to the membrane, where it can bind and inactivate Rap1b. We show that Rap1GAP inhibits insulin secretion stimulated by glucose, KCl, or sulfonylureas, and that this inhibitory effect can be further potentiated by addition of sulprostone. Thus, we postulate that EP3 isoforms direct Gαz and other effectors to mediate a variety of signaling pathways in the pancreatic β‐cell, all of which converge to reduce insulin secretion.Support or Funding Information• R01 DK102598/DK/NIDDK NIH HHS/United States
DAPLE links heterotrimeric G proteins to Wnt signaling during vertebrate development
Authors: Marvin Arthur , DiGiacomo Vincent , Leyme Anthony , Garcia‐Marcos Mikel 
Published: November 2023

Precise control of heterotrimeric G protein signaling is essential for various physiological processes, including embryonic development, and failure in this process causes multiple diseases. We have previously identified new players in the activation cycle of heterotrimeric G proteins that bind and activate Gα subunits via an evolutionary conserved motif. More specifically, we have found that some non‐receptor proteins mimic the action of G Protein‐coupled receptors (GPCRs) by working as Guanine nucleotide Exchange Factors (GEFs). One of these proteins, namely DAPLE, is a previously described regulator of Wnt signaling that binds to a key adaptor of the Wnt signaling machinery, i.e., Dishevelled (Dvl). Despite the known pivotal role of Wnt signaling in early steps of the embryonic development, the role of DAPLE in this process is unknown. Using Xenopus laevis as a model organism, we observed that DAPLE regulates Wnt signaling during vertebrate development and that it does so through coupling to heterotrimeric G proteins. These findings provide in vivo evidence for the functionality of this group of non‐receptor GEFs characterized by a defined Gα binding and activating motif and delineate a novel molecular mechanism linking Wnt signaling and G proteins during vertebrate embryonic development.Support or Funding InformationNIH R01GM112631 and the Hartwell Foundation
Translocation of the non‐receptor protein GIV/Girdin to the plasma membrane activates heterotrimeric G proteins
Authors: Leyme Anthony , Parag‐Sharma Kshitij , DiGiacomo Vincent , Marivin Arthur , Broselid Stefan , Garcia‐Marcos Mikel 
Published: November 2023

Receptor tyrosine kinases (RTKs) and integrins have been traditionally considered to trigger signaling cascades different from those activated by G protein‐coupled receptors (GPCRs). However, GIV (aka Girdin) is a Guanine nucleotide Exchange Factor (GEF) that activates heterotrimeric G protein signaling downstream of RTKs and integrins. GIV is recruited to the cytoplasmic tail of RTKs and integrins upon stimulation, but the mechanism of activation of its GEF function is not well understood. We combined assays in yeast and BRET‐based biosensors in mammalian cells to investigate if association of GIV with the plasma membrane was sufficient to drive G protein activation. We found that GIV does not co‐fractionate with its substrate G protein Gαi3 on cell membranes of unstimulated cells. Anchoring of GIV to the plasma membrane of yeast cells in a constitutive manner or its rapid translocation to the plasma membrane of mammalian cells via chemically‐induced dimerization (CID) leads to robust G protein activation. GIV's Gα‐Binding and Activating (GBA) motif is necessary and sufficient for G protein activation upon membrane translocation. Furthermore, we engineered a synthetic protein to show that recruitment of GIV's GBA motif to membranes via association with active RTKs upon stimulation with a natural ligand, instead of via CID, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a mayor mechanism responsible for the activation of its G protein regulatory function.Support or Funding InformationThis work was supported by NIH grants (R01GM108733, R01GM112631) and American Cancer Society (RSG‐13‐362‐01‐TBE)
Structural and Functional Studies of the Metastatic Factors P‐Rex1 and P‐Rex2: Toward Small Molecule Inhibitor Development
Authors: Sharma Prateek , Davis Ellen M. , Cash Jennifer , Tesmer John J. G. 
Published: November 2023

Phosphatidylinositol 3,4,5‐trisphosphate (PIP3)‐dependent Rac exchanger 1 (P‐Rex1) is a Rho guanine‐nucleotide exchange factor (RhoGEF) that regulates cytoskeletal rearrangement and cell motility and is linked to tumor metastasis. The P‐Rex family includes P‐Rex1 and P‐Rex2, which are activated by both the lipid PIP3, generated by activated receptor tyrosine kinases, and free Gβγ subunits, generated by activated GPCRs. However, the structural basis behind functional regulation with P‐Rex is poorly understood. My objective is to determine the structure of P‐Rex2 PH and characterize the interaction of small molecules with different PH domains through qualitative analysis using differential scanning fluorimetry (DSF) and detailed assessment through X‐ray crystallography. Rosetta (DE3) pLysS E. coli (Novagen) cells were used to overexpress P‐Rex2 PH constructs and recombinant protein was purified using Ni‐NTA resin and size exclusion chromatography. A DSF assay was used to analyze the binding interaction between compounds and PH domains. Crystallographic studies led to a high‐resolution structure (1.90 Å) of the independent P‐Rex2 PH domain. A comparison between the P‐Rex1 PH and P‐Rex2 PH structures shows divergence in the β3/β4 loop, shown by our lab to play a role in non‐specific membrane localization, and β5/β6 loop, a likely protein‐protein interaction site. Using DSF, we identified six small molecules from a medium‐throughput screen that bind the P‐Rex1 PH domain. P‐Rex2 and Akt PH (a kinase PH domain which has been successfully targeted with small molecule inhibitors) were used to further assess compound binding to a variety of PH domains. IP4, a soluble analog of PIP3, resulted in a 12.3 °C shift in Tm for P‐Rex1 PH and a 15.4 °C shift for P‐Rex2. Compound #13 (20 μM) was most notable in causing a 15.5 °C shift for P‐Rex1, 22.5 °C for P‐Rex2, and 13.5 °C for Akt PH. We are currently co‐crystallizing P‐Rex1 PH with these small molecules to verify that they target the PIP3‐binding site. As PIP3 binding is necessary for P‐Rex activation, small molecules can be developed to perturb this interaction, resulting in therapeutics with anti‐metastatic properties.Support or Funding InformationThis work was supported by an American Cancer Society – Michigan Cancer Research Fund Postdoctoral Fellowship (PF‐14‐224‐01‐DMC) to J.C. and an American Heart Association Undergraduate Student Research Program Award (14UFEL20510027) to E.D.
Structure of a GEM‐complexed Gai Protein Provides Novel Insights into the Emerging Human GEMiome
Authors: Kufareva Irina , Kalogriopoulos Nicholas , Rees Steven D , Sun Nina , Aznar Nicolas , Chang Geoffrey , Ghosh Pradipta 
Published: November 2023

Heterotrimeric G proteins are major signaling hubs and essential components of the signal gating machinery in eukaryotic cells. In their best‐studied capacity, G proteins transmit signals from integral membrane G protein coupled receptors (GPCRs) to intracellular effectors. Recently, a rapidly emerging paradigm revealed the existence of a novel class of G protein modulators: cytosolic GEMs (Guanine nucleotide Exchange Modulators). GEMs can fuel pathological networks by aberrantly coupling G protein signaling to a broad variety of cell surface receptors (growth factor RTKs, integrins, Wnt/Frizzled receptors, cytokine receptors, and others) and by amplifying this signaling in an unrestricted manner. They act pleiotropically via a conserved motif that activates cAMP‐inhibitory (Gαi) but inhibits cAMP‐stimulatory (Gαs) G proteins, thus helping to integrate downstream responses.The Ga‐interacting motif of the prototypical GEM GIV (Ga‐interacting Vesicle associated) was identified [1] based on distant homology with a bifunctional synthetic peptide KB‐752 [2]. Following the discovery of several additional GEMs with homologous G protein binding motifs [3, 4], it is now clear that the size of the human GEMiome and its footprint on diverse cellular processes are larger than initially expected.We recently solved an X‐ray structure of a Gai protein in complex with a GEM motif of GIV [5]. The structure demonstrated that GEMs act through a mechanism that is both unexpected and different from that employed by canonical G protein triggers, GPCRs. Using molecular modeling, we elucidated the structural basis of action of two additional GEMs, Daple and NUCB1. All studied GEMs appear to allosterically induce a low GDP affinity conformation of the nucleotide pocket by binding at the conformationally variable, nucleotide sensitive switch II region of the Gai molecule. Such binding is achieved through a precise set of hydrophobic and polar interactions involving residue positions conserved across all known GEMs. The obtained atomic resolution understanding of GEM action enabled prediction of novel members of the human GEMiome through structure‐ and model‐guided genome‐wide sequence searches.The GEMiome is an untapped source of revelations in the field of cancer biology as well as promising targets for anti‐cancer therapeutic discovery. The insights obtained through this study will help reveal novel cancer targets and enable rational design of truly effective therapeutics against them.Support or Funding InformationSupported by NIH grants R01 AI118985 and R01 GM117424.
Dissecting the Roles of ARHGEFs in Eosinophil Polarization
Authors: Botros Fady , Turton Keren , Mosher Deane 
Published: November 2023

Members of the Rho Guanine nucleotide exchange factor (ARHGEF) family regulate GTPase activity by enhancement of release of GDP to allow GTP binding. Eosinophils undergo a rapid morphological change, with polarized cells becoming acorn‐shaped. The nucleus is propelled into the cone (nucleopod), and the granules move to the other pole of the cell (granulomeric region). RhoA GTPase regulates ROCK2, a kinase crucial for re‐organization of the eosinophil cytoskeleton following cytokine‐induced activation. Of the over 40 known ARHGEFs, three that are known to activate RhoA‐‐ARHGEF1, ARHGEF2, and ARHGEF18‐‐are highly expressed in eosinophils as determined by published RNA‐Seq and quantitative proteomic analyses. For the RhoGEF domain (comprised of pleckstrin homology and Dbl homology modules) present in all three, ARHGEF2 and ARHGEF18 have 50 % sequence identity, whereas ARHGEF1's sequence identity with ARHGEF18 is 31 %. Using predictive bioinformatics tools, including Integrated Genome Viewer and Proteomics DB, to integrate transcriptomic and proteomic data, we determined that eosinophils express the X5 ARHGEF18 proteoform of 1361 residues rather than the 1015‐residue canonical X6 proteoform expressed in non‐blood cells. Immunoblotting with antibodies to the X6 proteoform (expected 114 kDa) yielded only a band of the size of the X5 proteoform (150 kDa) whereas PLB985 and Jurkat contain bands the sizes of both proteoforms. We performed immunofluorescent staining of eosinophils at five minutes and one hour after cytokine activation. We observed that initially ARHGEF2 and ARHGEF18 localized to the nucleopod, whereas ARHGEF1 localized to the granulomeric region, as did a large proportion of RhoA GTPase. ARHGEF18 distribution appeared to change over time, with more ARHGEF18 localizing to the granulomeric region after one hour. Differences in ARHGEF spatial and temporal distribution could underlie distinct albeit overlapping actin regulation pathways. Future work will include assessing the impact on protein localization when the cells are treated with cytochalasin B and nocodazole, inhibitors of actin and tubulin polymerization respectively; as well as elucidation of ARHGEF co‐distribution with filamentous actin and microtubules. We conclude that X5 is the major proteoform of ARHGEF18 in eosinophils, and that this protein co‐localizes with ARHGEF2 after initial activation, and ARHGEF1 after prolonged activation.Support or Funding Information1RO1AI125390‐01 Proteomics of Eosinophil Activation From NIHP01 HL088594 Role of Eosinophils in Airway Inflammation and Remodeling From NIH
Characterization and Partial Purification of an Inhibitory Factor Secreted by Bacillus anthracis and Aeromonas jandaei
Authors: McCartney Julia , Nesemeier Jacob , Peterson Nancy 
Published: November 2023

Over the past 40 years, there has been a dramatic increase in the number of amphibious species that have become extinct or endangered. One cause of this increase is due to parasitic colonization of water mold which has been shown to be a significant cause of frog embryo mortality. Bacteria found on frog egg masses appear to have the ability to inhibit water mold growth. We tested two bacteria species, Bacillus anthracis and Aeromonas jandaei, for their ability to inhibit water mold growth. A narrow ring of bacteria was streaked on LB agar at the periphery of an 8′ petri dish and a 9 mm plug of water mold was placed at the center of the dish. The diameter of water mold growth was measured daily. B. anthracis and A. jandaei were plated 0, 1, and 2 days before adding the water mold plug; B. anthracis inhibited growth 11%, 38% and 93% respectively and A. jandaei inhibited water mold growth 20%, 47% and 91% respectively. We found the inhibition to be nutrient dependent; higher inhibition was observed when assayed on high protein compared to high sugar medium. Growth of bacteria on high protein medium resulted in an increase in the pH over time. However, when we grew water mold on pH‐buffered medium we showed that pH difference could not account for the all inhibition seen in our bioassays.After confirming inhibition, partial purification of the inhibitory factor was investigated. Bacteria were grown for 3–6 days in LB broth. After centrifugation to remove bacteria, the conditioned growth medium was tested for inhibitory factor using a similar bioassay described above. The conditioned media was also tested for heat resistance, showing stability up to 64°C for 30 minutes. The inhibitory factor was then extracted into ethyl acetate and separated using a silica column. Fractions were tested by bioassay and the active fractions were analyzed by NMR. We were able to detect low levels of contaminant plasticizer, Di‐octylphthalate, in both bioactive and inactive fractions. However, we were not able to detect the inhibitory factor itself. We are currently working to scale up the amount of conditioned media for the isolation of the active component.Taken together, these data show that the inhibitory factor works at a distance from the water mold indicating that it is secreted and diffusible from the bacteria. This suggests that populations of bacteria living in egg masses could serve to inhibit water mold colonization and increase survivorship of frog embryos. Further, the concentration of inhibitory molecule needed to affect growth is small, indicating that future practical application would allow small scale use.Support or Funding InformationWe would like to thank Dr. Ruthig, Dr. Boaz, and Kelsey LaMartina for technical assistance. Funding for this work was kindly provided by the North Central College Summer Grants and the North Central College Student Governing Association.
Relationship of Vpx and APOBEC3A
Authors: Rachuy Jacob , Moen Rebecca , Land Allison 
Published: November 2023

APOBEC3A is a catalytically active DNA cytosine deaminase expressed in monocyte immune cells. This function allows APOBEC3A to mutate and restrict viruses, potentially including HIV. HIV‐1, the causative agent of the major HIV/AIDS pandemic, is incapable of infecting monocytes. HIV‐2, a less common variant, is capable of infecting monocytes. The unique protein Vpx, produced by HIV‐2, but not HIV‐1, is thought to be responsible for allowing HIV‐2 infection in this immune cell. The objective of this study is to determine the sensitivity of human APOBEC3A to HIV‐2 Vpx. We hypothesize that HIV‐2 Vpx will be capable of mediating degradation of APOBEC3A and limiting its mutagenic capabilities, thus allowing HIV‐2 to infect monocytes. To test this hypothesis, a mutant Vpx protein, called H82A, was constructed using mutagenic primers. This mutant lacks the ability to bind to APOBEC3A. Once created, the plasmid containing Vpx‐H82A was transfected into 293T cells along with APOBEC3A. Vpx without the mutation, along with ABOBEC3A, was also expressed in 293T cells. Immunoblotting was utilized to visualize these results and determine if Vpx mediated degradation of APOBEC3A. We determined that neither wild type HIV‐2 Vpx nor Vpx‐H82A altered APOBEC3A levels. Currently, we are exploring the sensitivity of APOBEC3A to Vpx from other lentiviruses such as SIVmac, a simian immunodeficiency virus affecting Rhesus macaques. The same methods will be employed and an expression plasmid for the Vpx‐H82A has been made. This project will contribute to our understanding of the innate immune response to lentiviral infection.Support or Funding InformationThis work was supported by a Minnesota Foundation Grant awarded to JR; an MSU Faculty Research Grant awarded to AL and AL's start‐up funds.
Escherichia coli tRNA Induces Mammalian Cell Migration and Socialization
Authors: Buechler Andrew K. , Zhao Hanchao , Lieu D'Feau J. , Blanke Steven R. , Martinis Susan A. 
Published: November 2023

Housekeeping factors for protein synthesis, such as the aminoacyl‐tRNA synthetases, ribosomal proteins, and tRNA molecules, have selectively adapted throughout evolution to play other essential roles outside of translation. These roles include critical cell signaling events and can involve translocation out of the cytoplasm, where protein synthesis typically occurs. We fractionated E. coli cells and determined that leucyl‐tRNA synthetase and its co‐factor tRNALeu were selectively secreted under nutritional stresses. Because E. coli resides in the colon of its host, we separately incubated leucyl‐tRNA synthetase and tRNA molecules with human colon carcinoma cells. Surprisingly, the RNA molecules triggered cell migration to form very tightly packed assemblies of cells. Cell “socialization” that was triggered by exogenously added tRNA appeared to be dependent on nutritional deprivation. It was also reversible. We hypothesize that tRNA serves as a mediator in the complex interplay between bacteria and their human hosts.Support or Funding InformationThis research was supported by The W.M. Keck Foundation, the National Institutes of Health GM63789, and the Chemistry‐Biology Interface Training Program T32GM070421.