bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanisms of G Protein‐Selectivity in Muscarinic Acetylcholine Receptor Family
Authors: Santiago Luis Jaimes , Abrol Ravinder 
Published: June 2021

Muscarinic acetylcholine receptors are integral membrane proteins that regulate physiological activity. Acetylcholine is a neurotransmitter that binds to the extracellular side of these G protein coupled receptors (GPCRs) activating the Gα subunit of the trimeric G protein inside the cell. There are 5 different muscarinic receptor subtypes (M1–M5), where the same ligand acetylcholine causes M2 and M4 receptors to preferentially couple to the Gαi subunit and the receptors M1, M3, and M5 to preferentially couple to the Gαq subunit. This selectivity of Gα subunits mediates different secondary messenger pathways in the cell: Gαi being responsible for deactivating adenyl cyclase activity and Gαq being responsible for activating phospholipase C. We are using computational biophysical methods to probe the mechanisms of G protein selectivity of this muscarinic receptor family. We have built structural models for the M1 and M2 receptors coupled to Gαi or Gαq proteins and used molecular dynamics simulations to obtain relaxed structures of these complexes, so that the thermodynamic analysis of the receptor G protein interactions can be conducted. The analyses are beginning to show that thermodynamically, some complexes are more stable than others in agreement with corresponding biochemical experiments. In addition, some receptor ‐ G protein interactions may be kinetically controlled. Overall, these studies are beginning to provide us with the missing mechanistic insight into the G protein selectivity of the muscarinic receptors.Support or Funding InformationWe acknowledge the CSUPERB New Investigator Grant. We thank the RISE (GM063787) program for their student support.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Systematic Disruption of the Nonpolar β‐Helix Core of Hemolysin A and its Site‐Specific Effect on Protein Structure, Function, and Secretion
Authors: Schlimgen Roman , Grilley Dan , Weaver Todd 
Published: June 2021

Gram‐negative bacteria utilize a two‐partner secretion (TPS) pathway to export disease causing virulence factors. This pathway is a member of the Type Vb secretion systems which are thought to utilize the energy of protein folding to accomplish secretion across the outer membrane. In the TPS pathway, the secreted protein (TpsA) is produced along with a specific outer membrane protein (TpsB), which uses POTRA domains to recognize nonpolar segments on the TpsA. In our current model, this recognition leads to transport and folding of the TpsA protein via a Brownian ratchet mechanism. Utilizing this TPS pathway, Hemolysin A (HpmA), a hemolytic protein from Proteus mirabilis, is able to cross the bacterial outer membrane. A version of this protein, comprising a conserved TPS domain and truncated at position 265 (HpmA265), serves as a model for secretion and folding. HpmA265 adopts a β‐helical fold that encompasses three subdomains. Folded HpmA265 can function in trans as a template to fold and activate unfolded full‐length HpmA into its hemolytically active form (Template Assisted Hemolytic Activity – TAHA). This templating is thought to mimic the Brownian ratchet mechanism of secretion‐coupled folding of the full‐length protein. Previous work suggests that destabilization of the lowest stability subdomain enhances templating while destabilization of the second subdomain reduces templating ability. To identify the positional dependence of this effect, we have systematically replaced nonpolar amino acids with polar amino acids in the nonpolar core of the protein. Using CD spectroscopy to quantify the destabilization caused by each of the introduced variants, we have correlated the folding free energies of each variant with measured values of templating ability determined via TAHA assays. These measurements have allowed us to refine our model for how HpmA265 templates the folding of full‐length HpmA. In vivo, the nonpolar amino acids that were targeted may also play a role in the POTRA domain recognition. Previous data has shown that introduction of polar residues can increase secretion, suggesting that any destabilization of the folded form is offset by changes to POTRA domain interactions. To more fully map this effect, we have quantitatively determined the levels of secretion for each variant protein. Together, these results have allowed us to refine our model of the driving mechanisms in the TPS pathway, down to an amino acid level.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Basis of Activation and Sustained Signaling by the PTH Receptor
Authors: Sutkeviciute Ieva , Zhao Li‐Hua , Zhang Yan , Xu Eric , Vilardaga Jean‐Pierre 
Published: June 2021

Parathyroid hormone (PTH) receptor (PTHR) is a medically important family B G Protein Coupled Receptor (GPCR) that primarily couples to Gs/cAMP and Gq/Ca2+ signaling pathways and has a central role in regulating Ca2+ homeostasis and bone turnover. PTHR is activated by two endogenous hormones, endocrine PTH and paracrine PTH related protein (PTHrP), but only PTH mediates prolonged activation of the receptor and sustained cAMP responses after receptor internalization to the endosomes. To investigate the structural basis of prolonged activation of PTHR, we employed cryo‐EM and solved the high resolution (3Å) structure of PTHR coupled to Gs and activated by long‐acting PTH (LA‐PTH), an analog of PTH that remarkably sustains endosomal PTHR signaling and induces significantly prolonged elevation in blood calcium in mice and monkeys. LA‐PTH binds the receptor as a single extended α‐helix with its N‐terminus inserted deep into the transmembrane domain (TMD) of the receptor, while positioning the extracellular domain (ECD) of PTHR perpendicular to the cell membrane. The receptor activating N‐terminal half of LA‐PTH forms a dense network of hydrophobic and polar interactions with receptor's TMD resulting in a tight binding and giving structural basis for remarkably sustained activation of PTHR by LA‐PTH. Binding of LA‐PTH activates the receptor by inducing partial unwinding of the C‐terminus of transmembrane helix 6 (TM6) and a sharp kink at the middle of this helix, a hallmark of family B GPCR activation. This results in a pronounced outward movement of TM6, opening the cytosolic cavity of the receptor, which allows subsequent G protein coupling. Additionally, our cryo‐EM data revealed structural dynamics of PTHR demonstrated by the ability of receptor ECD to adopt multiple conformational states. Together, these results reveal the structural determinants of PTHR signaling and provide a molecular framework for structure‐based design of novel therapeutics for osteoporosis and hypocalcaemia treatment.Support or Funding InformationNIH R01 DK116780, DK102495 and DK111427 awarded to JPV; the Jay and Betty Van Andel Foundation and NIH GM127710 grants awarded to EXThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Examining the Metabolic Role of the Pseudomonas aeruginosa MDO Operon
Authors: Tombrello Katie , Ellis Holly R. , DeSantis Anita , Zeczycki Tonya N. 
Published: June 2021

Mammalian cysteine dioxygenase (CDO) is a mononuclear iron enzyme that catalyzes the oxidation of L‐cysteine to L‐cysteine sulfinic acid. It contains an amino acid‐derived cofactor formed between residues Cys93‐Tyr157. Putative CDO enzymes have been identified in several bacteria; however, all known bacterial CDO enzyme homologs lack the Cys‐Tyr amino acid‐derived cofactor. 1Although the MDO enzyme has been proposed to convert 3‐mercaptopropionate (3‐MPA) to 3‐sulfinopropionate (3‐SPA), the physiological function of either 3‐MPA or 3‐SPA remains unclear. 2,3In numerous bacteria, the MDO gene is on the same operon as an annotated sulfurtransferase, and the operon is expressed during bacterial sulfur limitation. The sulfurtransferase has amino acid sequence similarity to mercaptopyruvate sulfurtransferase enzymes.Different thiol‐containing substrates were evaluated to support a defined metabolic pathway involving both MDO and the sulfurtransferase. MDO gave similar catalytic parameters with both 3‐MPA and mercaptopyruvate, suggesting that both are viable substrates for the enzyme. However, the specificity of MDO for mercaptopyruvate correlates with the classification of the coexpressed sulfurtransferase. Therefore, MDO and the sulfurtransferase enzymes may be metabolically linked, and optimal catalytic function could involve protein‐protein interactions between the two enzymes. Potential protein‐protein interactions between MDO and the sulfurtransferase were identified by surface plasmon resonance, and hydrogen‐deuterium exchange was performed to further identify where the interaction sites of MDO and the sulfurtransferase are located. Coupling of MDO with the sulfurtransferase was also investigated to understand the functional role of the enzymes in bacteria. The proposed overall mechanism for both enzymes would result in sulfite release that could be utilized in the sulfur assimilation pathway. Based on these findings, the MDO/sulfurtransferase system represents an additional pathway for bacteria to adapt to sulfur limiting conditions that had not been previously identified.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein‐Protein and Protein‐Detergent Interactions of the Burkholderia Type III Secretion System Protein BipC
Authors: Wilkinson Mason Christopher , Dey Supratim , Yadava Sanjay , De Guzman Roberto 
Published: June 2021

The Type III Secretion System (T3SS) is an essential component of pathogenesis in many Gramnegative bacteria, including Burkholderia pseudomallei, the causative agent of melioidosis. T3SS structural components assemble into a needle apparatus that facilitates injection of virulence factors into a host. Four main parts comprise this needle: a base that spans both bacterial membranes, a needle that protrudes from the base, a needle tip that contacts the host, and a translocon pore that forms in the host cell membrane. Minor translocon protein BipC from B. pseudomallei is believed to be an integral membrane protein that interacts with major translocon protein BipB, tip protein BipD, and chaperone BicA. However, interactions between BipC, its binding partners, and the membrane itself remain uncharacterized. Here we characterized interactions of BipC with detergent micelles and BicA/BipD using circular dichroism spectroscopy (CD) and transverse relaxation‐optimized spectroscopy (TROSY) respectively. Far‐UV CD spectra indicate that BipC normally exists in a partially folded state in solution, but adopts a distinctly alpha‐helical conformation upon titration with various detergents, suggesting that BipC becomes embedded in detergent micelles. [15N,1H]‐TROSY spectra of BipC titrated with either BicA or BipD revealed sites of interaction predominantly restricted to N‐terminal domain of BipC. In conclusion, our results suggest that BipC inserts into membranes and primarily interacts with its binding partners via its N‐terminal domain. These findings will help elucidate the mechanism of T3SS translocon assembly and the structural changes that occur within its components.Support or Funding InformationWe would like to thank the other members of De Guzman Lab, as well as Dr. JustinDouglas and Dr. Minli Xing from the KU NMR facility for their support and insight. Research reported here was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, as well as by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers R01AI074856 and P20GM103418 respectively. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases, the National Institute of General Medical Sciences, or the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Plasticity of a Human G Protein‐Coupled Receptor Studied by NMR Spectroscopy
Authors: Eddy Matthew 
Published: June 2021

The human genome encodes for 826 G protein‐coupled receptors (GPCRs), which are involved in nearly every physiological process and comprise the largest class of “druggable” proteins. Using nuclear magnetic resonance (NMR) in solution, we provide a global view of structural plasticity underlying the function of the human A2Aadenosine receptor (A2AAR), a representative class A human GPCR. To apply NMR to study a human GPCR we developed a robust molecular biology pipeline for production and sample preparation of stable‐isotope labeled receptors, yielding reproducible high‐quality NMR data. From these data, assignment of endogenous residues provided numerous probes distributed over the receptor intracellular surface, extracellular surface, and transmembrane domain, enabling a global characterization of A2AAR responses to variable efficacy of bound drugs. The NMR probes also permitted novel insights into changes in the local conformation of conserved GPCR activation motifs. In addition, we observed multiple local efficacy‐related conformers at the intracellular surface, which we relate directly to G protein signaling. The distributed NMR probes provide novel information on the role of a highly conserved GPCR allosteric center, permitting us to study the effect of this center on signaling pathways in the receptor. Amino acid replacement of the allosteric center caused striking changes in conformational polymorphisms at the intracellular surface but did not alter response to drug efficacy at the extracellular surface. We correlated changes at the intracellular surface to structural rearrangements at the toggle switch tryptophan and a conserved activation motif, providing novel insights into a widespread signaling mechanism in class A GPCRs. The presented work opens the door to exciting future studies of GPCR function, including studies of GPCRs in native environments.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Porphyromonas Gingivalis Infections Underline Association of Periodontitis with Systemic Diseases
Authors: Potempa Jan 
Published: June 2021

Chronic periodontitis (CPD) is arguably the most prevalent bacterial‐infection driven chronic inflammatory disease of the humankind. If morbidity caused by tooth loss due to advanced PD is not enough, the mounting evidence suggests a strong link between PD and life‐threatening systemic diseases. Here, I will present data showing that major periodontal pathogen, Porphyromonas gingivalis, can be directly implicated in development and severity of aspiration pneumonia and rheumatoid arthritis as well as it can add to pathology of esophagus cancer and Alzheimer disease. Furthermore, I will present mechanistic models of plausible engagement of specific P. gingivalis virulence factors, gingipains and peptidyl arginine deiminase (PPAD) in pathogenesis of first two diseases. Finally, the importance of hijacking host enzymes and signaling pathways by P. gingivalis will be discussed in the context of CPD and associated systemic diseases.Support or Funding InformationNIH/NIDCR R01 DE 022597This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational studies on HdeA and its pH‐Dependent Activation
Authors: Pacheco Sayuri , Ravinder Abrol , Crowhurst Karin 
Published: June 2021

HdeA is a chaperone protein found in pathogenic gram‐negative bacteria such as Escherichia coli that forms homodimers, provides bacterial stress response and acid resistance. HdeA is inactive in its dimeric form and becomes activated in low pH environments such as the mammalian stomach. In low pH environments, it binds to other periplasmic proteins protecting them from aggregation and denaturation. Computational biophysical methods such as constant pH molecular dynamics (cpHMD) in explicit solvent have been utilized to couple protein dynamics and protonation states in order to reveal a plausible unfolding mechanism for HdeA. Our computational studies are providing insight into the unfolding mechanisms for HdeA. CpHMD was used to measure the 16 pKa values of the acidic GLU and ASP residues. Solvent Accessible Surface Area (SASA) analysis has shown that pH 7.0 has the highest amount of buried area while pH 2.0 has the least amount of buried area. This is in agreement with experimental findings which show that at pH 7.0 HdeA remains a well‐folded homodimer, while at pH 2.0 HdeA unfolds into monomers (Garrison and Crowhurst, 2013). Interestingly, analysis of individual residue fluctuations has shown that some dimer interface core residues fluctuate more when HdeA is in its active state while other core residues become more static at lower pHs. Both monomer and dimer simulations show that residue GlN4 gets protected at low pH consistent with deuterium exchange experiments. These findings are creating a complex but detailed view of HdeA's transition from its inactive dimeric state to active monomeric state.Support or Funding Information8TL4GM118977‐02NIH BUILD PODERThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
4‐Phenylbutyrate rescues folding‐deficient creatine transporter‐1 variants linked to the creatine transporter deficiency syndrome
Authors: Sucic Sonja , El‐Kasaby Ali , Freissmuth Michael 
Published: June 2021

Mutations in the coding sequence of the hCRT‐1 gene (SLC6A8) have been associated with the creatine transporter deficiency (CTD) syndrome. CTD encompasses a range of moderate to severe conditions, from epilepsy, mental retardation, autism, development delay, behavioural problems and motor dysfunction to gastrointestinal symptoms. Diseases arising from misfolding of other SLC6 transporters have been reported over recent years; e.g. folding‐deficient mutants of the dopamine transporter (hDAT) cause infantile/juvenile Parkinsonism dystonia in children, while orthostatic intolerance arises from misfolding of the human noradrenaline transporter (hNET). In hCRT‐1, dozens of naturally occurring variants have been linked to CTD. Remarkably, some of these mutations occur at highly conserved residues across the SLC6 family; e.g. a single point mutation inducing a sleepless phenotype in Drosophila DAT (dDAT‐G108Q), leads to severe mental retardation in children when mutated at the equivalent glycine residue in hCRT‐1 (hCRT‐1‐G132V). Similarly, the P554L mutation causes Parkinsonism/dystonia in hDAT, but triggers drug‐resistant epilepsy in children harbouring the same mutation in hCRT‐1. Here, we examined 16 clinically relevant hCRT‐1 variants to assess the molecular basis of CTD. Confocal microscopy imaging revealed that the YFP‐tagged mutant CRTs are trapped in the endoplasmic reticulum (ER), co‐localised with an ER‐resident chaperone calnexin, whereas the wild type hCRT‐1 reached the plasma membrane. The chemical chaperone 4‐phenylbutyrate (4‐PBA) proved to be effective in functionally rescuing several of the folding‐deficient CRT‐1 mutants. Radiolabeled creatine uptake assays were also verified by assessing the surface expression of the variant transporters by cell surface biotinylation and immunoblotting of detergent lysates (i.e. the mature fully‐glycosylated band appeared in response to positive chaperone action). In addition, the rescue action of 4‐PBA was also recapped in primary hippocampal neurons expressing the wild type transporter and CTD‐triggering mutations. Overall, this work is clinically relevant and ought to confer pristine therapeutic prospects awaited for by many CTD patients.Support or Funding InformationThis work was supported by the Austrian Science Fund (FWF) project no P31255‐B27 to Sonja Sucic.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In silico and in vitro Studies of Human 5α‐reductase Type II Reveal New Loss of Function Variants
Authors: Katharopoulos Efstathios , Sauter Kay‐Sara , Pandey Amit V , Flück Christa E 
Published: June 2021

BackgroundAndrogens are steroid hormones necessary for human sex development. Testosterone (T) and the more potent dihydrotestosterone (DHT) are maybe the best‐known androgens, which exert their effect by binding and activating the androgen receptor. The 5α steroid reductases (SRD5As) catalyze the conversion of T to DHT in the classical androgen production pathway, or from 17‐hydroxyprogesterone to 17OH‐dihydroprogesterone, and androstenedione to androstanedione in alternate pathways leading to DHT. There are two enzymes with differential expression, of which SRD5A2 is expressed in reproductive organs and liver, and catalyzes the reaction of T to DHT more efficiently than the SRD5A1. In humans, SRD5A2 loss‐of‐function mutations are known, and cause severe 46XY undervirilization, while gain‐of‐function variants have been suggested in androgen excess syndromes such as premature adrenarche, the polycystic ovary syndrome or prostate tumors, but these have not been identified so far.AimWe aimed to search for potential gain‐of‐function mutations in the human SRD5A2 gene.MethodsWe searched sequence databases for genetic variants and performed bioinformatic and functional tests on a set of selected variants. After the amino acid conservation analysis of SRD5A2, a novel 3D protein model was constructed to identify the exact position of amino acids in the tertiary structure and predict their effect on protein function and substrate interaction. We then collected 116 coding SNPs in SRD5A2 from OMIM, dbSNP, Pubmed, Clinvar, HGMD, and UniProt databases. These SNPs were ranked according to their association with phenotypes, physical location in our 3D model, and molecular dynamics simulation studies. Finally, we selected nine coding SNPs for in vitro studies. These SNPs were located within or close to highly conserved areas that form the binding cavities for substrates or the cofactor NADPH. The SRD5A2 variants were expressed in HEK293 cells, and enzyme activity was assessed by conversion of testosterone (T), progesterone (Prog) and androstenedione (Δ4A) to their 5α‐reduced metabolites.ResultVariants R50A and P173S showed partial activity with substrates T (34% and 28%) and Δ4A (37% and 22%). With Prog as substrate variants P106L, P106A, L167S, and R168C, showed partial activity (15% to 64%). Functional testing of all other variants showed loss‐of‐function. As predicted in our in silico analysis, all coding SNPs affected enzyme activity. However, none of them showed gain‐of‐functionConclusionWe provide a novel protein model for studies of SRD5A2. We have characterized seven human SRD5A2 variants, which although did not show any gain of function, might be of clinical relevance for their loss of enzyme activity. It is possible that individuals carrying these SNPs show a minor, yet unidentified phenotype.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.