bluuebunny/crossref_metadata_2025 huggingface dataset:
General Amino Acid Control and 14‐3‐3 Proteins Bmh1/2 Are Required for Nitrogen Catabolite Repression‐Sensitive Regulation of Intracellular Gln3 and Gat1 Localization
Authors: Cooper Terrance G. , Tate Jennifer J. , Buford David , Rai Rajendra
Published: November 2023

Nitrogen Catabolite Repression (NCR), the ability of Saccharomyces cerevisiae to utilize good nitrogen sources in preference to poor ones, derives from nitrogen‐responsive regulation of the GATA family transcription activators Gln3 and Gat1. In nitrogen replete conditions the GATA factors are cytoplasmic and NCR‐sensitive transcription minimal. When only poor nitrogen sources are available, Gln3 is nuclear dramatically increasing GATA factor‐mediated transcription. In addition, to these two growth conditions, Gln3 localization responds to short‐ and long‐term nitrogen starvation as well as rapamycin and methionine sulfoximine (glutamine synthetase inhibitor) treatments. Originally these responses were all attributed to mTorC1‐mediated control of Gln3. We subsequently showed that this was not the case, but that there were at least three distinct regulatory pathways controlling Gln3 localization. Two regulatory systems act cumulatively to maintain cytoplasmic Gln3 sequestration; one is TorC1. The second regulatory system has remained elusive. A third regulatory pathway takes over once Gln3 enters the nucleus. This pathway is glutamine specific. When glutamine levels are low, Gln3 must bind to its DNA target promoter sequences (GATAA) before it can exit from the nucleus. On the other hand, when glutamine (or glutamine analogue) levels are high, Gln3 can exit from the nucleus in the absence of DNA binding. Present experiments identify Gcn2‐dependent general amino acid control as the regulatory system that has thus far escaped detection. We show that the uncharged tRNA‐activated protein kinase Gcn2 is required for NCR‐sensitive nuclear Gln3‐Myc13 localization but not for short‐ and long‐term nitrogen starvation or responses to Msx and rapamycin treatments. From epistasis experiments, it appears that Gcn2 functions upstream of Ure2. Ure2 sequesters Gln3 in the cytoplasm in nitrogen replete conditions. 14‐3‐3 proteins Bmh1/2 are also required for nuclear Gln3‐Myc13 localization and GATA factor‐mediated transcription. In contrast with Gcn2, however, Bmh1/2 appear to function downstream of Ure2. Overall Gln3 phosphorylation levels decrease upon loss of either Gcn2 or Bmh1/2. Our results add a new dimension to the cumulative participation of these three major pathways in the nitrogen‐responsive control of GATA transcription factor localization and NCR‐sensitive gene expression in yeast.

Support or Funding Information

NIH GM‐35642‐27
Signaling Pathways Involved in Tributyltin‐Induced Increases in Interleukin 6 Production by Lymphocytes
Authors: Hamza Nafisa , Brown Shyretha , Whalen Margaret
Published: November 2023

Tributyltin (TBT) is a toxic compound that has industrial uses, such as slime control on masonry, wood preservation, and disinfection of circulating industrial cooling water and is most frequently used in antifouling paints on the hulls of ships. As a result of TBT's lipophilic character, it absorbs readily into organisms and contaminates the environment and is found in human blood in concentrations as high as 261 nM. TBT decreases the lytic function and secretion of interferon gamma (IFNγ), tumor necrosis factor alpha and interleukin 1 beta (IL‐1β) from human lymphocytes. IL‐6 is a cytokine which regulates the function of many cells including tumor cells. Recent studies in our lab have shown that TBT alters production of IL‐6 by lymphocytes. The current study aims to determine whether TBT utilizes MAPK signaling pathways (ERK 1/2, p38) to cause alterations in IL‐6 production. TBT‐induced production of IL‐6 in human lymphocytes was measured in the presence and absence of ERK1/2 pathway (PD98059) and p38 (SB202190). Results indicated that the stimulation of IL‐6 production by TBT decreased in the presence of each of the MAPK pathway inhibitors. These data suggest that TBT may be causing increased IL‐6 production by activating ERK1/2 and p38 MAPKs.

Support or Funding Information

Supported by NIH grant 5U54CA163066
Role of MAPKs and NFκB in Tributyltin‐stimulated Interleukin 1 Beta Secretion and Production from Human Immune Cells
Authors: Boules Mariam , Brown Shyretha , Whalen Margaret
Published: November 2023

Tributyltin (TBT) contaminates the environment due to its many uses and has been found in human blood samples at levels as high as 261 nM. TBT interferes with the lytic function of human natural killer lymphocytes and alters the secretion of interleukin 1 beta (IL‐1β), Interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) from human leukocytes. IL‐1β regulates cellular growth and immune responsiveness. Elevated levels of IL‐1β have been shown to increase the invasiveness of tumors. Our recent studies showed that TBT exposures of 5–25 nM (levels that are seen in human blood samples) caused increased secretion of IL‐1β from monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs). In this study we examine the roles of the MAPKs, (ERK1/2, p38, and JNK) and NFκB in TBT‐induced increases in IL‐1β secretion and intracellular levels to assess their role in TBT‐induced production of IL‐1β. MD‐PBMCs were treated with the MEK1/2 inhibitor PD98059, p38 inhibitor SB202190, JNK inhibitor BI78D3 or NFκB inhibitor BAY 11‐7085 for 1h prior to exposure to TBT concentrations of 25, 10, and 5 nM for 24 h. Secretion of IL‐1β was measured using ELISA and intracellular protein levels were monitored using western blot. Results indicated that the TBT‐induced secretion of IL‐1β requires the ERK1/2 and p38 pathways. But does not require the NFκB or JNK pathways. The increases in secretion are accompanied by increased protein levels in TBT‐exposed cells and this increase in protein production also utilized the ERK1/2 and p38 pathways. These data suggest that increased protein production accompanies the increases in secretion of IL‐1β seen in human immune cells exposed to TBT and these increases are dependent on MAPK pathway activation.

Support or Funding Information

Supported by NIH grant U54CA163066.
Involvement of MAPK Signaling Pathways in Tributyltin‐induced Increases of Interleukin 1 beta and Interleukin 6 mRNA in Human Lymphocytes
Authors: Brown Shyretha D , Boules Mariam , Hamza Nafisa , Whalen Margaret
Published: November 2023

IL‐1β and IL‐6 are pro‐inflammatory cytokines produced by monocytes, macrophages, and lymphocytes. Both are responsible for immune response regulation and cellular growth. Increased levels of these two cytokines are associated with several pathologies, including rheumatoid arthritis, multiple sclerosis, and specific cancers (such as breast, lung, and pancreatic). Tributyltin (TBT) is an environmental contaminant which is found in human blood and other tissues, and it has been shown to increase incidences of tumors in mammals. Previous ex vivo studies have shown that when TBT is introduced as a stress inducer to the cellular environment, the production (secretion and/or intracellular levels) of IL‐1β and IL‐6 was increased in human lymphocytes, and ERK1/2 and p38 MAPK pathways were shown to be necessary for this TBT effect. The current study examines whether this increased production (protein levels) of IL‐1β and IL‐6 is due to TBT‐induced increases in the transcription of IL‐1β and IL‐6 mRNA. Additionally, this study will examine the roles of MAPKs (ERK1/2 and p38) on any demonstrated TBT‐induced increases in expression of IL‐1β and IL‐6 mRNA in lymphocytes (monocyte‐depleted (MD) peripheral blood mononuclear cells (PBMCs)). MD‐PBMCs were exposed to 0–200 nM TBT followed by isolation of RNA. The IL‐1β and IL‐6 mRNA levels were monitored using semi‐quantitative RT‐PCR. mRNA levels of each cytokine were also monitored in the presence and absence of ERK1/2 (PD98059) and p38 (SB202190) pathway inhibitors. Results indicated that exposure to 50–200 nM TBT caused increased levels of both IL‐1β and IL‐6 mRNA. Initial results suggest that the MAPK pathways, although involved in the TBT‐induced increased in cytokine production, may not have a role in the TBT‐induced increases on mRNA expression. These data suggest that MAPK pathway activation is needed for TBT to cause increased production for both IL‐1β and IL‐6.

Support or Funding Information

NIH grant U54CA163066
Expression of E3‐Ubiquitin Ligase genes in P. patens during sexual reproduction
Authors: Abrash Elizabeth Whitney , Hicks Karen A
Published: November 2023

Bryophytes and flowering plants diverged from a common ancestor 450 million years ago, yet both are induced to sexual reproduction as a result of seasonal cues such as day length and ambient temperature. While these pathways have been studied well in flowering plants, it is unknown whether this mechanism evolved before or after the divergence of bryophytes. In order to gain insight into the evolution of these pathways, our lab studies the moss Physcomitrella patens. Here we study the expression of two E3‐Ubiquitin‐Ligase genes that are differentially expressed in response to temperature cues based on RNA Sequencing analysis. E3‐Ubiquitin‐Ligases are part of the process known as ubiquitination, in which ubiquitin is added to a protein leading to degradation. Ubiquitin‐Ligase proteins have been shown to regulate photoperiod and temperature signaling in flowering plants. We hypothesize that regulation of sexual reproduction evolved prior to the divergence of bryophytes, and as such E3‐Ubiquitin‐Ligases will play a similar role in P. patens to that in flowering plants. These results will confirm RNA Seq analysis and identify potential knock out genes for future study.
Characterizing of the Novel Gene At4g33666 in the Abiotic Stress Response in Arabidopsis
Authors: Clark Amber Elizabeth Miller , Wershoven Nicole , Palmer Christine
Published: November 2023

Expanding populations and environmental changes can leave the plants we depend upon to meet our food and energy needs facing increasingly difficult growing conditions. Enhancing our understanding of the methods that plants naturally employ to overcome abiotic factors such as heat, drought, high salinity, or poor light will enable us to better select for plants that can succeed under challenging conditions. Here we examine At4g33666, a novel gene in Arabidopsis thaliana, which is thought to play a role in perceiving or overcoming abiotic stressors. In order to begin to understand the role of this gene in the stress response, we have examined expression at both an RNA and protein level. We extracted RNA from plants exposed to abiotic stress and performed quantitative PCR. To visualize phenotypic changes caused by the loss or overexpression of the gene, we measured the rate of germination of different genotypes under osmotic stress. This protein is hypothesized to be localized to the chloroplast, and to learn more about post translational modifications that might occur, we used immunoprecipitation to isolate protein for analysis via mass spectrometry. By studying the role of At4g33666, we can improve our understanding of plant stress responses and optimize success of the plants we depend upon.

Support or Funding Information

This research was supported by a Castleton University student research grant.
Controlled BMP2 Release from Keratin‐based Hydrogels Modulates Osteoinduction
Authors: Olson Lucas C. , Hyzy Sharon L. , Saul Justin M. , Cohen David J. , Kajan Illya , Schwartz Zvi , Boyan Barbara D.
Published: November 2023

Keratin biomaterials have potential for tissue engineering applications. When processed keratin is naturally non‐immunogenic, contains cell attachment sites, and has a tunable degradation rate, resulting in an effective scaffolding to simulate extracellular matrix (ECM). Keratose (oxidatively derived keratin) degrades in a period of weeks whereas kerateine (reductively derived keratin) degrades on an order of months to years, due to higher cysteine cross linking in kerateine. Clinically type 1 collagen sponge is soaked in bone morphogenetic protein (BMP2), which is a potent osteoinductive factor, in order to promote bone healing in orthopedic and craniofacial sites. The sponge, however, does not modulate BMP2 release in vivo, resulting in supraphysiological levels of the osteoinductive factor. Harmful ectopic bone formation and inflammation can result. In contrast, keratin degradation and BMP2 release rate are correlated. Modulation of BMP2 release by keratin, coupled with its inherent properties as an ECM substitute, could result in a suitable construct for osteoinduction via controlled osteogenic signal release, inflammatory mitigation, and cellular infiltration. The aim of this study was to examine the ability of keratin‐derived materials containing BMP2 to induce bone formation in vivo.

Ectopic bone formation from collagen sponge, keratose gel, kerateine gel, and keratose: kerateine blends (30:70, 50:50, 70:30) ± 0.67μg BMP2/mg material was examined in a well‐known mouse model. Samples were placed in gelatin capsules and bilaterally implanted into pockets created in the gastrocnemius of 7w‐old male athymic nude mice (n=8 implants/variable). After 5w, new bone formation was ascertained by histology, microCT and histomorphometry. (Statistical significance: ANOVA with post‐hoc Bonferroni‐corrected t‐test).

All +BMP2 samples produced new bone formation on microCT and histology, while there was no new bone in‐BMP2. On microCT, the most new bone was seen in 50:50+BMP2. 30:70+BMP2 had more new bone volume than collagen+BMP2 or keratose+BMP2. Histologically, 30:70+BMP2 contained greater total bone area than collagen+BMP2 and keratose+BMP2. 70:30+BMP2 had more cortical‐type bone and bone marrow than keratose+BMP2, but 30:70+BMP2 had more material still present than collagen+BMP2, keratose+BMP2, or 50:50+BMP2. Keratose (±BMP2) degraded completely. In‐BMP2 samples, remaining material increased with increasing kerateine. In conclusion, keratose/kerateine carriers facilitate greater BMP2‐induced bone than standard collagen or either biomaterial separately, suggesting their suitability as slow‐release BMP2 carriers for clinical bone regeneration.

Support or Funding Information

NIH 5R01AR061391
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 Information

This work was supported in part by NIH grant 1R15ED020190‐01
Investigation of TCL/RhoJ Palmitoylation and its Effects on Cellular Localization
Authors: Franzen Avery , Dague Collin , Tader Brooke , Florke Rebecca R , Hamann Michael J
Published: November 2023

The Rho‐family GTPase TCL/RhoJ has been shown to enhance pro‐angiogenic and pro‐metastatic cellular processes; however, less is known about the inherent biochemistry of the protein. Recently, TCL nucleotide exchange and localization to the plasma membrane was found to depend on a unique 20 amino acid N‐terminal sequence not found in other Rho‐family GTPases. Deletion of this sequence shifts TCL localization to cellular vesicles. Interestingly, a similar re‐localization was observed for full‐length TCL after treating cells with 2‐bromopalmitate, suggesting plasma membrane localization may depend on palmitoylation of an N‐terminal cysteine. To investigate this possibility, N‐terminal cysteines of TCL were mutated to alanine in varying combinations and expression plasmids harboring the mutants were transfected into HeLa cells. A presumably palmitoylated form of full‐length TCL produced a second, shifted band on non‐reducing western blots. The second band disappeared when samples were treated with beta‐mercaptoethanol. Mutating Cys‐3 of TCL also caused the second band to disappear as well, suggesting it is a potential site of palmitoylation. These findings will be corroborated with BMCC‐biotin assays and the effect on cellular location will be investigated with fluorescent microscopy. Together, these results will show a novel facet of TCL biochemistry.
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
Investigating the Influence of the Rho‐family GTPase TCL/RhoJ on Vesicular Trafficking
Authors: Olson TamiJo , Taylor Stephen , Hamann Michael J
Published: November 2023

TCL/RhoJ is a Rho family GTPase that was recently shown to have a novel N‐terminal sequence capable of regulating its localization to the cellular plasma membrane and vesicular compartments, as well as its ability to load with nucleotide. Additionally the protein has been shown to have a role in endosomal recycling pathways. To better understand how the N‐terminus of TCL may contribute to vesicular trafficking, WM‐115 cells will be used in transferrin uptake and recycling assays. WM‐115 cells are a continuous cell‐line derived from a human melanoma tumor and are believed to overexpress TCL. In order to manipulate TCL levels, the WM‐115 cells were initially tested for their transfection efficiency using DNA containing Venus fluorescent protein and were found to have a 60% transfection efficiency. To study vesicular trafficking, WM‐115 cells will be transfected with YFP‐TCL constructs corresponding to the wild‐type protein, an N‐terminal deletion mutant, as well as constitutively activating and dominant negative mutants of TCL. Transfected cells will be tested for transferrin uptake and recycling using Alexa‐647 transferrin and flow cytometry with gating on YFP‐positive cells. These experiments will help determine how transferrin recycling rates are affected by TCL and better illuminate its role in vesicular trafficking.
GTP‐loading Activity of TC10/TCL Chimeras Underscores Important Allosteric Regulatory Regions of TCL
Authors: Young Grace , Florke Rebecca R , Hamann Michael J
Published: November 2023

TCL/RhoJ is a Rho family GTPase that functions as a regulator of angiogenesis and may also contribute to tumor growth and metastasis in some melanomas and gastric cancers. Despite these interesting functions, the biochemistry of TCL is poorly understood. Recently our lab has investigated primary sequence differences between TCL and its closest homologs TC10 and Cdc42. TCL has an additional 20 amino acids in its N‐terminus compared to Cdc42, and deletion or mutation of amino acids 17–20 inhibit the ability of TCL to exchange guanine nucleotides. However, a similar N‐terminal deletion (ΔN) within its closest homologue, TC10, does not have a similar affect. Chimeras of TCL and TC10 identified amino acids 121–129 (designated as region R2c) of TCL as a distal sequence within TCL that influences the ability of the N‐terminus to regulate nucleotide loading. To further investigate this mode of allosteric regulation, chimeras were produced that replaced the R2c sequence of a TC10 ΔN construct, and mutation of this region was found to be sufficient to change the nucleotide binding characteristics of TC10 ΔN so it mimicked TCL ΔN, further supporting the importance of the R2c region for TCL GTP‐loading. Additional chimeras will be produced to test whether adding the N‐terminus of TCL to the TCL/TC10 R2c chimera restores nucleotide binding. Overall, the results demonstrate TCL is a unique GTPase in regard to its nucleotide exchange mechanism and may be a selectively targetable GTPase for chemotherapeutic intervention.
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 Information

NIH 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 Information

This 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 Information

This 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 Information

Supported 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 Information

1RO1AI125390‐01 Proteomics of Eosinophil Activation From NIH

P01 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 Information

We 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 Information

This 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 Information

This research was supported by The W.M. Keck Foundation, the National Institutes of Health GM63789, and the Chemistry‐Biology Interface Training Program T32GM070421.
Subversion of host vesicular trafficking by phosphoinositide‐binding bacterial proteins
Authors: Pike Colleen , Lein Sean , Neunuebel Ramona
Published: November 2023

Intracellular pathogens have evolved intricate strategies to adapt and survive within host cells. Replicating within host cells and evading host detection are major obstacles for intracellular bacteria to overcome. Legionella pneumophila, the causative agent of the severe pneumonia known as Legionnaires' Disease, successfully avoids these barriers by preventing lysosomal degradation concurrently with establishing a replication permissive plasma membrane‐derived compartment called the Legionella‐containing vacuole (LCV). This is accomplished by subverting host cell pathways via the translocation of effector proteins into the host cell throughout the course of infection. Multiple Legionella effectors hijack host vesicles away from their normal destinations and instead redirect them towards the LCV. Prior work has shown several L. pneumophila effectors anchor to these hijacked vesicles by specifically recognizing phosphoinositides (PIPs). PIPs are mammalian glycerophospholipids important for organelle identity, vesicle trafficking and cell signaling. We hypothesized that L. pneumophila has many more PIP binding effector proteins that have not yet been identified. A preliminary screen showed that as many as 89 novel L. pneumophila effector proteins display similarity to known eukaryotic PIP binding domains. Here we report the identification of eleven novel L. pneumophila effectors that interact with PIPs. These effectors target multiple species of host PIPs that may be critical for infection. Furthermore, we have identified the specific PIP binding regions of these effectors by generating protein truncations. Each protein fragment was purified and used in protein‐lipid overlay assays. In parallel, we validated PIP binding ability in vivo by performing co‐localization studies with known PIP binding markers. We expect that these novel PIP binding effectors have the potential to play important roles during infection and to be developed into highly specific PIP biosensors that can be used to track vesicle movement in live cells.
Collagen Mimetic Peptides as Probes for Bacterial Infection
Authors: Ellison Aubrey , Dempwolff Felix , Kearns Daniel , Raines Ronald
Published: November 2023

Collagen is the most abundant protein in humans and comprises a major structural component of the extracellular matrix (ECM). In wounds, collagen triple helices suffer damage. As collagen prefers to form a triple helix, we can take advantage of the damaged collagen and introduce collagen mimetic peptides (CMPs) that will adhere to the wound surface. These invasive CMPs can be used to deliver a payload to the collagen surface of a wound. Nature may also use this invasive strand mechanism as a form of host infection. Invasion of host tissue can be mediated by the interaction of bacterial surface proteins and the ECM. Group A Streptococcus (GAS) displays cell‐surface proteins that resemble human collagen. We hypothesize that the collagen‐like strands of GAS form triple helices with denatured collagen in a wound bed. The ensuing adherence could be a basis for infection by GAS. To test this hypothesis, we are using fluorophore‐labeled CMPs to detect collagen strands on the surface of live GAS cells. We are also evaluating knock‐in expression of collagen‐like strands, Scl 1 and Scl 2, on the surface of B. subtilis to transfer the ability to anneal to our CMPs. With these tools we hope to understand and ultimately antagonize infections by GAS.

Support or Funding Information

This work is supported by Grant R01 AR044276 (NIH).
Photosynthetic Apparatus and Biochemical Parameters in Tomato and Squash Crops Influenced by Cucumber Mosaic Virus (CMV) Infection
Authors: Montasser Magdy S. , Nayef Nourah Y , Afzal Mohammed
Published: November 2023

This study was aimed to compare the influence of two cucumber mosaic virus (CMV) strains (CMV‐KU1 and CMV‐16) on photosynthetic apparatus and different biochemical parameters with emphasis on carotenoids, fatty acids and phenolic compounds in both squash and tomato plants. Squash plants, infected with CMV‐16, showed a decrease in β‐carotene content with a concomitant increase in lutein by which the ratio of lutein/β‐carotene significantly increased by 62% (p<0.05) compared with the control plants. However, the change in lutein/β‐carotene ratio was less pronounced in tomato plants. In response to CMV‐16 infection, the desaturation index indicated by unsaturated/saturated fatty acids (UFA/SFA) increased in both squash and tomato plants, indicating that stress in test plants. Phenolic content in squash plants were significantly changed in response to CMV‐16 inoculation. In CMV‐16 infected squash plants, phenolic accumulation increased sharply indicating that plant tolerance for the viral infection. CMV‐16 in tomato had weaker impact on phenolic content compared with squash plants. This might be due to a possible involvement of symptom development and defense mechanisms due to plant‐virus interactions. All biochemical parameters studied were not significantly influenced in test plants infected with CMV‐KU1 compared with the severe CMV‐16. Satellite RNA in the CMV‐KU1 may have protected plants against infection caused by virulent strains. Our results indicated that CMV‐KU1 is benign to crops compared to the severe viral strain of CMV‐16.

This research work was funded by the Research Administration (Research Project # YS01/14)and supported by the College of Graduate Studies, Univ. of Kuwait, Kuwait.

Support or Funding Information

Research Project # YS01/14, University of Kuwait, Kuwait College of Graduate studies, University of Kuwait