bluuebunny/crossref_metadata_2025_split huggingface dataset:
The small GTPase Rab43 is a novel regulator of G protein‐coupled receptor trafficking
Authors: Li Chunman , Huang Wei , Wu Guangyu 
Published: November 2023

Ras‐like small Rab GTPases are master regulators of vesicle‐mediated membrane traffic. Recent studies have identified several Rab GTPases involved in the anterograde cell surface transport of newly synthesized G protein‐coupled receptors (GPCRs). Here, we have screened the Rab GTPase family using stable cells expressing α2B‐adrenergic receptor (α2B‐AR), a prototypic GPCR, and demonstrated that Rab43 is a novel regulator of GPCR trafficking. Attenuation of Rab43 function, via expression of its dominant‐negative mutants, siRNA‐mediated depletion, and expression of its GTPase‐activating protein RN‐tre, significantly attenuated the cell surface expression of α2B‐AR and induced an extensive accumulation of the receptor in the endoplasmic reticulum (ER). The studies using inducible α2B‐AR‐expressing cell lines and a previously characterized Golgi‐localized α2B‐AR mutant, in combination with bredeldin A treatment/washout assay, further showed that Rab43 controlled the ER export rate and content of nascent receptors. More interestingly, in addition to α2B‐AR, the cell surface transport of a number of other GPCRs, but not VSVG and EGF receptor, was regulated by Rab43. Furthermore, Rab43 was shown to directly interact with α2B‐AR, specifically its third intracellular loop, in an activation‐dependent fashion, but it was unable to associate with the tethering factor protein p115, a downstream effector of Rab1 GTPase which is well‐known to control the ER‐Golgi traffic. Altogether, these data demonstrate a novel Rab43‐mediated, as yet undefined pathway for members of the GPCR family to move forward from the ER to the Golgi.Support or Funding InformationR01GM118915
When the Tail Wags the Dog: Phosphodiesterase 4D7 Regulation of a cAMP‐signalosome Located at the Rear of Migrating Smooth Muscle Cells Regulates Localized RhoA/ROCK Signaling and Tail Retraction During Migration
Authors: Maurice Donald H , Truong Tammy , Wudwud Alie 
Published: November 2023

Inhibiting the maladaptive migration of human arterial smooth muscle cells (HASMCs) in injured blood vessels may help reduce the development of intimal lesions in vascular diseases, including atherosclerosis or restenosis. Agents that act to increase intracellular levels of cyclic adenosine monophosphate (cAMP) impact numerous individual events involved in coordinating HASMC migration. Signaling via cAMP is ubiquitous and dynamic, and requires control through signaling termination mechanisms. cAMP Phosphodiesterase (PDE) enzymes catalyze the hydrolysis of cAMP and are critical in the control of cAMP signaling, most specifically when they regulate cAMP levels within numerous discrete subcellular domains. Through spatial restriction, these enzymes are thought to allow cAMP‐elevating agents to selectively regulate numerous distinct responses. The PDE4D gene family plays a dominant role in cAMP hydrolysis in HASMCs. Considerable interest exists in the idea that the unique N‐terminal domains of the numerous distinct PDE4D isoform variants promotes their spatial restriction in cells, and that this is coordinated through selective interactions of these PDE4Ds with other proteins. This study examined the targeting of individual PDE4D isoforms in HASMCs through the overexpression of GFP constructs containing the unique N‐terminal domains of the PDE4D isoforms (NT‐PDE4D/GFP) known to be expressed endogenously in these cells (PDE4D8, 9, 5, 7). Although transient over‐expression of NT/PDE4D8‐GFP, NT/PDE4D9‐GFP or NT/PDE4D5‐GFP did not significantly impact HASMC morphology or migration character or speed, over‐expression of NT/PDE4D7‐GFP was observed to alter HASMC morphology of migrating HASMCs as well as their migratory velocity. By combining these observations with the effects of selective siRNA‐mediated knockdown of PDE4D7, we established that PDE4D7 is involved in controlling rear retraction in migrating HASMCs. We show that altering PDE4D7 targeting and expression in these cells affects rear retraction largely through its ability to impact RhoA‐ROCK signaling. We also report that PDE4D7 interacts with AKAP5, PKA and EPAC1 in migrating and immotile HASMCs. Our results suggest a functional role for localized PDE4D7 activity in regulating cAMP‐mediated rear retraction in migrating HASMCs, and identify PDE4D7 as a potential therapeutic target in controlling HASMC migration.Support or Funding InformationCanadian Institutes of Health Sciences
Role of MitoBK Channels in Renal Cold Preservation
Authors: Shrum Stephen , Parajuli Nirmala , Rusch Nancy J. , MacMillan‐Crow Lee‐Ann 
Published: November 2023

Patients with end‐stage renal failure require kidney transplantation to avoid life‐long hemodialysis associated with high mortality. Kidneys from brain‐dead or deceased donors are placed in cold storage (CS) preservation solutions until a recipient is identified. However, kidneys exposed to CS solutions are subjected to extended ischemia, and our laboratory reported that renal CS induces oxidative stress and subsequent mitochondrial damage. Thus, there is a critical need to develop strategies to protect donor kidneys from CS ‐induced damage to improve long‐term renal function in the transplant recipients. In this regard, the high‐conductance Ca2+‐activated K+ (BK) channel recently was discovered in the mitochondrial inner membrane (mitoBK) of cardiomyocytes and other cell types. When activated, the mitoBK channel reportedly protects against ischemia‐reperfusion injury in the heart by reducing mitochondrial damage and reactive oxygen species (ROS). We show, for the first time, that the mitoBK channel is expressed in the mitochondria of rat renal proximal tubular cells in vitro and in rat kidney in vivo. Our preliminary evidence suggests that CS reduces expression of the pore‐forming a subunit of the mitoBK channel in rat renal proximal tubular cell (n=3) and rat kidney (n=4) whole‐cell lysates. Thus, we hypothesize that: MitoBK channels down‐regulate in response to CS‐induced mitochondrial damage, contributing to donor kidney injury. We also propose that restoring mitoBK channel function will provide protection from CS ‐induced injury and improve transplanted kidney function. Our preliminary studies using High Resolution Respirometry (HRR) with fresh kidney biopsies show that Complex I, II, and III (but not Complex IV) activity was significantly impaired in rat kidneys exposed to CS for 18 hours. Twenty‐four hours after renal transplant, recipients of kidneys exposed to 18 hours of CS showed elevated serum creatinine (4.4 mg/dl, n=6) compared to rats having undergone only sham surgery (0.3 mg/dl, n=5). Blood urea nitrogen also was elevated in transplant recipients of CS kidneys (CS + transplant, ~140 mg/dl; sham, ~20 mg/dl). These indicators of impaired renal function correlated with loss of mitoBK protein in CS kidneys. Ongoing patch‐clamp studies are evaluating the properties and function of mitoBK channels in our cell and animal models of renal CS, and evaluating whether pharmacological activation of mitoBK channels during CS can attenuate ROS, limit mitochondrial injury, and improve the function of transplanted kidneys.Support or Funding InformationFunded by AHA Predoctoral Fellowship #16PRE30830010 and UAMS pilot grant G1‐51454‐99.
Muscle Nicotinic Acetylcholine Receptors containing α1‐V275 or α1‐Y233 Mutants found in a functional Screen reveal enhanced Agonist Potencies
Authors: Siehler Sandra , Ferrand Sandrine , Kelley Nicholas , Bill Anke , Freuler Felix , Imbert Pierre‐Eloi , Marelli Anthony , Gerwin Nicole , Sivilotti Lucia G , Miraglia Loren , Orth Anthony P , Oakeley Edward J , Schopfer Ulrich , Groot‐Kormelink Paul J 
Published: November 2023

The adult form of the human muscle nicotinicacetylcholine receptor (nAChR) consists of four different subunits forming apentamer around the channel pore, (α1)2β1δɛ. The two orthosteric binding sites are located at the α1‐ɛ and α1‐δ interfaces. A library of 2816α1 subunit mutants was created using high throughput mutagenesis by error‐prone PCR and sequences of all mutants were determined using single molecule real timesequencing. Each α1 mutant was transiently transfected with wildtype β1, δ, and ɛ subunits into HEK293 cells, and the channel function examined in an ion flux assay. Mutant hits were selected that responded to the nicotinic agonistepibatidine, but were resistant to the orthosteric blockers α‐bungarotoxin ortubocurarine. Eight α1 subunit mutant hits were discovered, six of which contained mutations at Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were functionally characterized, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations do not impair blocker binding, but rather evoke again‐of‐function phenotype, e.g. increased agonist sensitivity. Immunocytochemical staining showed that the surface expression of mutated α1subunits was comparable to the wildtype protein. Functional analysis of the mutant library containing alterations for each of the 457 amino acids of the human α1 subunit therefore yielded structure‐function insights for the nAChR.Support or Funding InformationThis work was supported by the Novartis Institutes for BioMedical Research (NIBR).
Computational approaches to understanding activation of the angiotensin II type 1 receptor
Authors: Anderson Shane D , Botello‐Smith Wesley M. , Luo Yun Lyna , Andresen Bradley T. 
Published: November 2023

The angiotensin II type 1 receptor (AT1R) is a major player in the cardiovascular system and is involved in volume and salt homeostasis. Multiple models of the AT1R have been made to predict molecular function; however, the previous models were invalidated by the crystallization of the AT1R bound to ZD 7155 (4ZUD) as well as the inverse agonist olmesartan (4YAY). Molecular dynamics models of receptors have a benefit as they allow dynamic interrogation of structure‐activity relationships and aid in designing novel therapeutics. Therefore, the aim of this project is to generate models of the empty and active human AT1R to determine structural changes involved in receptor activation. To produce a model of the empty AT1R, olmesartan and BRIL were removed from 4YAY, and the missing loops and the amino‐terminus were added to the structure. This model was inserted into an 87% POPC and 13% cholesterol membrane and simulated using AMBER16 for 150 ns. Atomic fluctuation measurements from the 150ns simulation revealed extensive movements within the helices as well as the loops; generally, the empty AT1R relaxed resulting in increased distances between helixes. Residue alpha carbons were measured between 4YAY and the stable empty AT1R structure. There were multiple changes including mean changes at Tyr351.39 (1.9 Å), Arg167ECL2 (2.1 Å), Ser189ECL2 (3.7 Å), Val2646.59 (4.4 Å), Gln2676.62 (4.6 Å), Iso2884.38 (1.9 Å), and Phe3047.55 (4.7 Å) and Phe3138.54 (9.2 Å). These changes are presumed to reflect the induced fit structure of olmesartan‐bound AT1R. A constitutively active AT1R (CA‐AT1R) was constructed from the stable empty AT1R by generating N111G AT1R followed by a 300 ns MD simulation. Major changes between the empty and CA‐AT1R occurred with residues Tyr351.39 (1.4 Å), Trp2195.62 (2.7 Å), Leu2977.48 (2.9 Å), and Phe3138.54 (9.5 Å). Intramolecular alpha carbon measurements between the models resulted in displaying changes specific to olmesartan binding such as Ser189ECL2 and Ala1634.61 to Gln2676.62: 6.0 Å and 18.2 Å, respectively, for olmesartan bound compared to 10.7 Å and 14.5 Å for the empty AT1R and 10.4 Å and 14.6 Å for the CA‐AT1R. Conversely, there are changes unique to CA‐AT1R structure deeper into the AT1R such as Ser2526.47 to Iso2907.40 and the salt bridge between NH1 Arg1263.50 and O− Arg2346.29: 8.6 Å and 10.4 Å for CA‐AT1R, respectively, compared to 6.7 Å and 8.6 Å for olmesartan bound and 6.7 Å and 2.7 Å for the empty AT1R. Future directions of this project include docking angiotensin II to the empty and CA AT1R as well as the biased agonist [Sar1‐Ile4‐Ile8]‐angiotensin II to the empty AT1R and comparing the conformational changes.Support or Funding InformationThis project was funded by internal WesternU funds.
A novel regulatory role of RGS4 in δ‐opioid receptor mediated neuronal outgrowth and differentiation
Authors: Georgoussi Zafiroula‐Iro , Pallaki Paschalina , Serafimidis Ioannis , Symeonof Alexandra , Sofia Koutloglou , Papakonstantinou Maria , Georganta Eirini , Thomaidou Dimitra , Gaitanou Maria 
Published: November 2023

Regulators of G proteins signaling (RGS) despite their initial role as GTPase activating proteins, serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription to tumorgenesis. Evidence suggests the cytoplasmic/nuclear distribution and translocation of RGS proteins is mediated by interactions with other proteins which mediate cross talk between G proteins and other cellular processes. RGS4 a member of the B/R4 family of RGS proteins has been found to be a universal negative regulator of μ, δ and κ‐opioid receptor signaling that confers selectivity for G protein coupling to these receptors by directly interacting with them (Georgoussi et al., 2006, 2012; Leontiadis et al. 2009; Papakonstantinou et al., 2015). It was also shown that δ‐opioid receptor (δ‐OR) forms a multicomponent signaling complex, consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription STAT5B, that leads to neurite outgrowth and neuronal differentiation via a phosphoryalted‐STAT5B‐Gαi/o pathway (Georganta et al., 2010, 2013). Knowing that RGS4 is a multitask protein we questioned whether RGS4 could be implicated in neuronal differentiation and neurite outgrowth upon δ‐OR activation through a similar signaling pathway. We demonstrate that RGS4 directly interacts with STAT5B in vitro and in living cells independently of δ‐OR presence. This interaction involves the N‐terminal portion of RGS4 and the DNA‐binding‐SH3 domain of STAT5B. Expression of RGS4 in HEK293 cells expressing δ‐OR and/or erythropoietin receptor results in inhibition of [D‐Ser2, Leu5, Thr6]‐enkephalin (DSLET) and erythropoietin‐dependent STAT5B phosphorylation and subsequent transcriptional activation. Measurements of DSLET‐dependent neurite outgrowth of neuroblastoma cells is also blocked by RGS4 expression, whereas primary cortical cultures of RGS4 knock out mice (RGS4−/−) exhibit enhanced neuronal sprouting after δ‐OR activation. Finally, neuronal progenitor cultures from RGS4−/− mice exhibit enhanced proliferation with concomitant increases in the mRNA levels of the anti‐apoptotic genes Bcl2 and Bcl‐xl STAT5B target genes. These observations suggest that RGS4 plays a significant regulatory role in opioid dependent neuronal differentiation and neurite outgrowth via a “non‐canonical” signaling pathway involving STAT5B‐directed responses.Support or Funding InformationThis work was supported by the Excellence II‐3722, (“NO‐ALGOS”) of the Hellenic General Secretariat of Research and Technology and was carried out in facilities of the OPENSCREEN‐GR National (Greece) Research Infrastructure. Z.G participates in the EU COST Action CM1207 (GLISTEN).
The Role of Bitter Taste Receptors (T2Rs) in Cystic Fibrosis
Authors: Jaggupilli Appalaraju , Singh Nisha , Robert Renaud Sylvain , Hanrahan John , Duan Kangmin , Chelikani Prashen 
Published: November 2023

IntroductionBitter taste receptors (T2Rs) belong to the G protein‐coupled receptor superfamily. In humans, 25 T2Rs perform a chemosensory function, however very little is known regarding their expression and function in extraoral tissues. Recent studies showed that T2Rs recognize quorum‐sensing molecules (QSMs) secreted by bacteria. Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The opportunistic pathogen Pseudomonas aeruginosa colonizes CF airways and secretes QSMs, which increase intracellular calcium and modulate innate immune responses through unidentified cell surface receptors. We hypothesized that T2Rs are expressed in epithelial cells of human airways, where they mediate QSM responses and modulate immune responses in CF.Methods and ResultsWe monitored the expression of T2Rs in primary CF and non‐CF bronchial epithelial cells. Cells were studied at both the mRNA and protein levels using qPCR and FACS analysis, respectively. We found similar expression of T2Rs in non‐CF and CF cells. Next we treated these cells with a number of QSMs and measured the intracellular calcium mobilized. We found that the calcium mobilized was sensitive to the PLC inhibitor U‐73122 suggesting that the signaling in these cells occurs predominantly through the QSM‐T2R‐Gαβγ‐PLC pathway. To further characterize the candidate T2Rs that may recognize specific QSMs and modulate signaling in CF, a heterologous expression system was used to stably express selected T2Rs and study the effects of QSMs. We found that a single QSM (for example, 3‐oxo‐C12‐AHL) can activate more than one T2R, and that multiple QSMs can activate the same T2R.ConclusionsT2Rs are expressed in bronchial epithelial cells and are functional. Although expression was not altered in CF cells, these results suggest there is a synergistic effect of QSMs on T2R signaling in CF airways and provides a rationale for further research on the pathophysiological function of T2Rs in extraoral tissues. These mechanistic studies should improve our understanding of host response mechanisms and may lead to new therapeutic approaches for CF that target T2Rs.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)
Inhibition of soluble and membrane‐bound neutrophil elastase by inhibitor POL6014
Authors: Korkmaz Brice , Hamon Yveline , Guarino Carla , Epinette Christophe , Gauthier Francis , Sellier‐Kessler Odile , Chevalier Eric 
Published: November 2023

Neutrophil elastase (NE) is involved in the degradation of extracellular matrix molecules making it an attractive target for the development of anti‐inflammatory compounds in neutrophilic pulmonary diseases. It is mainly secreted extracellularly by activated neutrophils at inflammatory sites but a minor fraction is translocated to the membrane where it remains proteolytically active. We investigated the properties of the novel Protein Epitope Mimetic (PEM) POL6014, a medium sized fully synthetic macrocycle, towards soluble and membrane‐bound NE. Circulating human neutrophils from healthy volunteers were purified and used within 3 hours after activation from Calcium ionophore (A23187) as source of membrane bound NE. The enzymatic reaction using the Fluorescence Resonance Energy Transfer (FRET) Technology was started by addition of a specific human NE substrate (ABZ‐APEEIMRRQ‐EDDnp, 13 μM final) 5 minutes after incubation of POL6014 with human NE. We found that POL6014 inhibits membrane‐bound human NE and soluble human NE with similar Ki values in the sub‐nanomolar range (0.69 ± 0.31 nM (n=7) and 0.53 ± 0.37 nM (n=3), respectively). This means that POL6014 could inhibit human NE stoichiometrically in pathophysiological conditions so that no molar excess of inhibitor would be required to control NE activity. In addition we evaluated POL6014 activity on rat and non‐human primate (NHP) NE in order to qualify these species for in vivo testing. Lysates of circulating neutrophils from both species were used as source of NE. Species selective FRET substrates (ABZ‐QPMAVVQSVPQ‐Yno2 for rodent, ABZ‐APQQIMDDQ‐EDDnp for primate) were used to determine the inhibitory potency of POL6014. We found that POL6014 is also a potent low nanomolar inhibitor with IC50 = 1.0 ± 0.5 nM (n=4) against rat NE and IC50 = 2.2 ± 1.4 nM (n=3) against Cynomolgus (Macaca fascicularis) NHP NE, which makes these species appropriate to test the in vivo efficacy and toxicology of POL6014. In conclusion, POL6014 showed potent inhibition of soluble and membrane‐bound human NE and similar potency across human, rat and NHP.Support or Funding InformationThis work was supported by Polyphor Ltd.
Investigating novel small compounds targeting the Laminin Receptor for their effect on modifying immune and stem cell viability and differentiation
Authors: Loperena‐Medina Sue Heidi , Neto Manoel Figueiredo , Umbaugh Charles Samuel , Haffner Abigail , Figueiredo Marxa Leao 
Published: November 2023

Osteoarthritis affects over 27 million Americans age 25 and older and is a debilitating disease in which articular cartilage degradation leads to inflammation and pain in the joint. Medical management has not been very successful, as cartilage is avascular and has a difficult time regenerating itself. In joints, there are laminin receptors (LR) which bind to PEDF (pigment epithelial derived factor). When PEDF is bound to a LR, it has anti‐inflammatory, pro‐chondrogenic and anti‐angiogenic characteristics. However, the relatively large size and low stability of PEDF at 25°C indicates that smaller, more stable molecules could be an alternative for therapeutic purposes. We developed seven small compounds that bind to the PEDF docking zone at the LR, with the goal to mimic the effects of PEDF. In order to test our drugs in a pro‐inflammatory environment, THP‐1 macrophage cultures were stimulated with lipopolysaccharide. Quantitative real‐time polymerase chain reaction (qPCR) was used to assess up‐ and down‐regulation of inflammatory genes, including IL‐1β. Compound 3 (C3) appears to be the best PEDF mimic as it reduced IL‐1β expression by ~8‐fold. A cell pellet chondrogenesis assay using adipose‐derived stem cells (ASC) was used to examine the effect of compounds on upregulating cartilage‐specific genes at day 8 of differentiation. In the future, ASC pellets will be harvested at differentiation days 7, 14 and 21 to determine the timing of cartilage‐specific gene expression following treatment with the small compounds. From our results, C3 appears to be the most promising drug for future development since it is able to reduce expression of pro‐inflammatory gene IL‐1β and promote ASC to express cartilage‐specific genes. Additionally, C3 also appears to alter phenotype of cancer cells towards antitumor activity in several assays including scratch, endothelial tube, and proteomic assays. Future studies will continue to investigate C3 and a series of small molecule analogs designed and synthesized in collaboration with a medicinal chemistry laboratory.Support or Funding InformationCTSI Core Facility Award, Showalter Research Foundation Full Proposal Award, Merial Veterinary Scholars Program, and start up funds from the BMS department.