bluuebunny/crossref_metadata_2025_split huggingface dataset:
Forbidden Protein Angles: Triosephosphate Isomerase as a Model for Studying Peripheral Residue Contribution to Catalysis.
Authors: Allen Patrick 
Published: April 2020

Over the past 3.5 billion years of evolution, enzymes have adopted a seemingly infinite number of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be forbidden to all amino acids, save glycine, due to steric hindrance. Triosephosphate Isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site Lysine 13 (K13) with a dihedral within the forbidden zone (phi=69/psi=−139). Both the amino acid and the dihedral angle are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted β‐sheet dihedral values of phi=−135/psi=170 but experiments show a 1,000‐fold loss in activity. Based on these results, we hypothesized that adopting the forbidden torsion angle for K13 contributes to catalysis. Using a combination of computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine from single to quadruple mutants. In Silico Molecular Dynamic (MD) simulations of 5×200ns per mutant species were made using 2JK2 unliganded Human TIM as a starting structure. Heat Maps, containing 400,000 K13 dihedral values per run reveal full or partial loss of forbidden zone angles depending on mutant combination. Initial experimental results with E97A single mutant showed a three‐fold decrease in kcat with little change in the KM value suggesting a modest catalytic role for this residue. For this mutant the heatmaps revealed the K13 angle spends less than 30% of its time in the forbidden zone compared to over 50% occupation in the wildtype suggesting the rate decrease may be due to the mutation affecting the positioning of K13. In addition to ongoing In Vivo characterization of the remaining mutants, double mutant cycles will be used to evaluate their energetic connections with K13. As modern protein design relies on naturally occurring protein folds, understanding how to recreate forbidden angles may enhance catalytic rates in unnatural enzymes and achieve chemical reactions not yet envisioned by nature nor organic chemistry.Support or Funding InformationWe would like to thank California State University Long Beach and the Carl E. Riley Endowed STEM scholarship for their generous funding.Heat map of where K13 dihedral occupies space on Ramachandran Plot over 200ns of MD simulation using Wild Type Human Triosephosphate Isomerase (RCSB:2JK2).Figure 1
Role of Nuclear Receptor Condensates in Ligand Induced Co‐operative Gene Regulation
Authors: Nair Sreejith J , Yang Lu , Meluzzi Dario , Yang Feng , Oh Soohwan , Tan Yuliang , Gamliel Amir , Alshareedah Ibraheem , Wang Susan , Suter Tom , Friedman Meyer , Ma Qi , Jayani Ranveer , Zhang Jie , Aggarwal Aneel K , Banerjee Priya , Rosenfeld Michael G 
Published: April 2020

Enhancers are transferable DNA elements that regulate target genes within the confines of Topologically Associated Domains (TADs) through Cohesin‐dependent looping. However, the potential regulatory functions and mechanisms of long‐distance, inter‐TAD enhancer associations remain largely unexplored. We report that 17β‐estradiol (E2) rapidly increases the spatial proximity of a fraction of E2‐responsive enhancers located in widely separated TADs. These enhancers are characterized by the recruitment of mega‐dalton sized multi transcription factor complex and high enhancer RNA (eRNA) induction. Optimal activation of these enhancer targets requires their co‐localization with phase‐separated interchromatin granules (ICGs). Chemical disruption of phase‐separated structures abolishes enhancer complex assembly and, specifically deactivates the most robust signal induced enhancers. Surprisingly, the physical property of the enhancer complex alters with the duration of hormone stimulation. In support of the long distance enhancer cooperativity model, the deletion of the most robust E2 induced enhancers resulted in a quantifiable decrease in the transcriptional activity of homotypic enhancers in the same chromosome. These data reveal previously unappreciated mechanisms for co‐ordinated gene regulation: (i) The biophysical properties of enhancer complex is critical for transcriptional output (ii) Phase separation mediated long‐distance enhancer cooperativity is required for optimal transcriptional activation by acute signaling programs. These findings reveal a new, generalizable model for the acute spatial gene regulation.Support or Funding InformationResearch support for S.J.N include postdoctoral fellowship from American Heart Association (14POST19860025), T32 Fellowship (5T32DK007044), and KO1 career grant (1K01DK121871). D.M. was supported by NIH‐NIDDK Grant 5F32DK112682. MGR is supported by Howard Hughes Medical Institute.Model of E2‐induced changes in the chromatin architecture. E2 stimulation results in ERa dependent recruitment of MegaTrans complex, Condensin complex, and enhancer RNA as phase‐separated ribonucleoprotein condensates. Spatial enhancer association results from “megaloops” formation between the most robust enhancers (E.g. TFF1e1 and NRIP1e3). Maximal cooperative enhancer activation occurs with the co‐localization of the enhancers in the interchromatin granule (ICG).Figure 1
Detection of transformed endothelial cells associated with survived cardiomyocytes in sub‐endocardial region of infarct myocardium
Authors: Song Xin , Xiao Ying , Chu Qing , Meng Xia , Kang Y. James 
Published: April 2020

Previous studies have shown that cardiomyocytes in the sub‐endocardial region of infarct myocardium survive from ischemic injury. The present study was undertaken to explore a possible mechanism by which these cardiomyocytes survive in response to myocardial ischemia. Wild type mice (C57BL/6J, male, 8–10 weeks), or endothelial cell‐specific red fluorescent protein (RFP) expression transgenic mice (Tie2CRETdTomato) were subjected to permanent ligation of left anterior descending of coronary artery along with sham‐operated controls in which the same surgery procedure was applied with an exception of the artery ligation. Pimonidazole was injected intravenously to define the hypoxic region, and lectin was injected to trace blood infiltration before sacrifice in all of the mice. The hearts were harvested on day one after the surgery and cut into serial sections for detection of cardiomyocytes and endothelial cells. The staining of pimonidazole showed that most of the infarct myocardium was under hypoxic condition except the sub‐endocardial region in which survived cardiomyocytes were detected. The RFP positive endothelial cells in the sub‐endocardial region also survived but displayed a suppressed expression of PECAM‐1 and VEGFR2. Correspondently, lectin infiltration analysis suggested blood leakage in the sub‐endocardial region although there was no blood infiltration in other regions of the infarct area. The results suggest that blood infiltration through the transformed endothelial cells in the sub‐endocardial region of infarcted myocardium would provide a non‐hypoxic microenvironment, contributing to the survival of cardiomyocytes in this region.Support or Funding InformationWest China Hospital, Sichuan UniversityThe changes of hypoxic microenvironment in the infarct myocardium region.Hypoxic condition (partial pressure of O2 was less than 10 mmHg) was marked with hpi (pimonidazole) at 1st day after ischemic injury. The blood infiltrations were reflected by FITC‐conjugated WGA through tail vein at 1st, 4th, 7th day after LAD ligation. Hypoxic region stained by pimonidazole (red), and nuclear labeled by DAPI (blue). Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; MI, myocardial infarction.Figure 1The change of blood supplement in the sub‐endocardial region.The blood infiltrations were completely reflected by Lectin (FITC‐conjugated WGA) through tail vein at 1st day after LAD ligation. Cardiomyocytes stained by TNNI3 (red), Blood infiltration labeled by FITC‐conjugated WGA (green), and nuclear labeled by DAPI (blue). Abbreviations: WGA, wheat germ agglutinin; DAPI, 4′, 6‐diamidino‐2‐phenylindole; MI, myocardial infarction.Figure 2
Translational pharmacology of TD139, an inhaled small molecule galectin‐3 (Gal‐3) inhibitor for the treatment of idiopathic pulmonary fibrosis (IPF)
Authors: Slack R. J. , Hirani N. , Gibbons M. A. , Simpson A.J. , Ford P. , Leffler H. , Nilsson U. J. , Sethi T. , Pedersen A. , Schambye H. , Maher T.M. , MacKinnon A. C. 
Published: April 2020

IntroductionGal‐3 is a pro‐fibrotic β‐galactoside‐binding lectin highly expressed in the lungs of IPF patients
1. TD139 is an inhaled, small molecule Gal‐3 inhibitor in development for IPF[2]. In this study the translational pharmacology of TD139 from pre‐clinical to clinical studies are presented.MethodsTD139 was evaluated in pre‐clinical studies investigating Gal‐3 in vitro and in vivo in naïve and bleomycin‐treated mice (male C57/Bl6 mice)
1,
2. A randomized, double‐blind, multi‐centre, placebo‐controlled, phase IIa study was completed to assess safety, tolerability, and PK/PD of TD139 in 24 patients with IPF. Inhaled TD139 was delivered at 0.3 mg, 3 mg or 10 mg QD to IPF patients (8 patients/cohort, 5:3 ratio (active:placebo)) for 14 days. Patients underwent bronchoalveolar lavage (BAL) prior to dosing and after 14 days. TD139 drug concentration was measured in the BAL fluid, BAL macrophages and plasma. Gal‐3 expression on BAL macrophages was measured by flow cytometry and systemic biomarkers of Gal‐3 inhibition and fibrosis measured in plasma.ResultsTD139 demonstrated high affinity (KD = 2.1 ± 0.1 nM (mean ± SD, n=4)) in vitro and reduced Gal‐3 expression ex vivo on IPF macrophages (IC50 = 361 ± 108 nM (mean ± SD, n=3 patients)). TD139 in vivo attenuated mechanistic PD (BAL Gal‐3) and fibrotic endpoints (total lung collagen/histology) in bleomycin‐treated mice with duration driven by lung retention. Clinically, inhaled TD139 was well tolerated at all doses with concentrations in BAL macrophages >567‐fold higher than systemic exposure. Gal‐3 expression on BAL macrophages was significantly lower in the 3 mg (% change −52.52%, 95% CI −85.43% to −4.74%, P=0.023) and 10 mg (% change −78.60%, 95% CI −110.48% to −29.78%, P<0.0001) dose groups compared to placebo, with a concentration‐dependent inhibition. Reduced Gal‐3 expression on BAL macrophages was associated with lower plasma biomarkers relevant to IPF pathobiology (PAI‐1, YKL‐40 and PDGF‐BB).ConclusionTD139 is safe and well tolerated in man and demonstrates low systemic exposure coupled with high lung concentrations resulting in suppression of Gal‐3 on BAL macrophages and decreased plasma biomarkers associated with IPF progression. Pre‐clinical in vitro and in vivo studies have shown to be clinically translatable. This study supported the progression of TD139 into a phase IIb IPF study
3.Support or Funding InformationAll studies were funded by Galecto Biotech AB.
Sympathetic β‐Adrenergic Stimulation Evokes Less Positive Inotropic Response But More Ventricular Arrhythmias in Heart Failure
Authors: Greco Lisa V. , Li Ying , Zhang Youhua 
Published: April 2020

IntroductionIt is well‐known that heart failure (HF) results in impaired sympathetic autonomic signaling in the heart, known as “β‐adrenergic receptor downregulation or desensitization”. This results in reduced positive inotropic effect under β‐adrenergic stimulation in HF. Our recent data indicate that HF significantly alters sympathetic stimulation induced atrial fibrillation (AF) inducibility. In contrast to normal hearts, failing hears are more vulnerable to sympathetic stimulation induced AF. This study is to investigate whether ventricular arrhythmia inducibility is altered in HF by sympathetic β‐adrenergic stimulation.MethodsHF was induced in 7 rats 2 months after myocardial infarction surgery with left coronary artery ligation, and 6 animals with sham surgery served as normal controls. Left ventricular pressure was recorded and the adrenergic β1‐agonist dobutamine (25μg/kg, iv) was administered to evoke positive inotropic effect. Ventricular arrhythmia inducibility test was performed with caffeine plus dobutamine challenge (caffeine 50mg/kg, ip, followed by dobutamine 25μg/kg, ip) and surface ECG as well as intracardiac right atrial electrocardiograms were recorded.ResultsDobutamine induced positive inotropic effects were significantly less in HF rats than in sham‐controls (+dp/dt increased 49±13% in control versus 14±6% in HF, P<0.001). Caffeine and dobutamine challenge induced severe ventricular tachyarrhythmias (frequent premature ventricular contractions and ventricular tachycardia, figure) in 6/7 HF rats while no ventricular arrhythmia was induced in the controls (p<0.01).ConclusionCompared with normal hearts, failing hearts are much more vulnerable to develop severe ventricular tachyarrhythmia during sympathetic β‐adrenergic stimulation with dobutamine and caffeine challenge, despite β‐adrenergic receptor downregulation in HF, as reflected by the less positive inotropic effects induced by β‐adrenergic stimulation.Example of induced premature ventricular contractions (PVC) and ventricular tachycardia (VT)Figure 1
Myocardial Ischemia‐Reperfusion Injury: Does Disruption of Mitochondrial Cristae Integrity Play a Requisite Mechanistic Role?
Authors: Kulek Andrew , Raghunayakula Sarita , Anzell Anthony , Undyala Vishnu , Sanderson Thomas , Przyklenk Karin 
Published: April 2020

Lethal myocardial ischemia‐reperfusion (IR) injury (i.e., cardiomyocyte death resulting in ‘heart attack’ or myocardial infarction, MI) is the leading global cause of death and disability. Despite improvements in the clinical management of MI, the molecular mechanisms responsible for lethal IR injury are incompletely understood. Data from our group and others have revealed that cardiomyocytes subjected to IR display a loss in integrity of optic atrophy protein (OPA1), the master‐regulator of fusion at the inner mitochondrial membrane and putative gate‐keeper of cristae junction integrity. Our goal in the current study was to establish whether disruption of OPA1 integrity plays a causal role in determining cardiomyocyte fate. To test this concept, HL‐1 cardiomyocytes were subjected to 2.5 hours of simulated ischemia + reperfusion. In
Protocol 1, the cleavage and subsequent release of OPA1 from mitochondria into the cytosol was assessed by immunoblotting at 30 min post‐R. As expected, IR was associated with degradation of OPA1 (in particular, the long (L) forms of the protein critical for inner mitochondrial membrane architecture), and a >15‐fold increase in cytosolic expression of the lower molecular weight short (S) forms (data not shown). In
Protocol 2, to demonstrate cause‐and‐effect, HL‐1 cells were transfected with either siRNA targeting OPA1 or scrambled peptide before undergoing 2.5 hours I + 24 hours R or a time‐matched normoxic period. Additional non‐transfected cells served as controls. Using 50 nM siRNA, near‐total knockdown (KD) of OPA1 (most notably, the critical L‐forms) was achieved (Figure
). In the normoxic cohorts, cardiomyocyte viability (quantified using the MTT assay) was modestly but significantly reduced in siRNA‐transfected cells (Figure
– left: *p<0.05 versus normoxic non‐transfected cells). However, contrary to the premise of our study, OPA1 knockdown had no effect on viability in cells subjected to IR: i.e., did not exacerbate IR‐induced cardiomyocyte death (Figure
– right). These data suggest that maintenance of OPA1 integrity does not have a requisite, causal role in mediating lethal myocardial ischemia‐reperfusion injury.Support or Funding InformationDetroit Cardiovascular Training Grant T32 (NIH 2T32HL120822) through the National Heart, Lung, and Blood Institute ‐ Wayne State UniversityOPA1 Knockdown with siRNAFigure 1HL‐1 Cell viability with OPA1 KnockdownFigure 2
Generation of the Configurational Ensemble of an Intrinsically Disordered Protein
Authors: Petridis Loukas 
Published: April 2020

A major challenge in biology is characterizing the structural flexibility of intrinsically disordered proteins (IDPs). Ensemble‐averaged experimental data do not provide the underlying protein structures. Here, we performed independently small‐angle neutron and X‐ray scattering experiments and unbiased molecular dynamics simulations to probe the solution structure of an IDP. We report that enhancing the sampling of the simulations can generate an ensemble of IDP structures in quantitative agreement with scattering and NMR, without the need for biasing the simulation or reweighting the results. The demonstration of established simulation technology that produces accurate physical models of flexible biosystems may pave the way to relating conformational flexibility to biological function.Support or Funding InformationThis work is supported by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, and by project ERKP300 funded by the Office of Biological & Environmental Research in the Department of Energy Office of Science.Configurational ensemble of c‐Src kinaseFigure 1
Development and Characterization of a Novel Small Animal Model of Heart Failure with Preserved Ejection Fraction
Authors: LaPenna Kyle B , Hidalgo Hunter A , Gao Juan , Haydel Amelia , Kapusta Daniel , Goodchild Traci , Lefer David J 
Published: April 2020

Heart Failure with Preserved Ejection Fraction (HFpEF) is a complex heterogeneous disease that currently affects approximately 3 million in the US and accounts for 60% of all heart failure hospitalizations. HFpEF is associated with significant morbidity, mortality and healthcare expenditures. At present, there are no FDA approved therapies for reducing mortality and hospitalization in HFpEF patients. The lack of approved therapies is due in part to a lack of preclinical models that faithfully mimic the clinical presentation of HFpEF.The obese ZSF‐1 rat is genetically predisposed to the development of hypertension, obesity, dyslipidemia, and diabetes, all of which contribute to the pathogenesis of HFpEF. We investigated the suitability of the obese ZSF‐1 rat as a model of HFpEF. Both obese and lean control ZSF‐1 rats (n = 4–6 per group) were utilized for these studies. The rats were studied from 16 weeks to 24 weeks of age. We measured several clinical parameters including cardiac structure and function (2‐D echocardiography), left ventricular (LV) pressures (LV Millar catheter), cardiac fibrosis, conscious blood pressure and heart rate (DSI radiotelemeters), vascular function, exercise capacity (rodent treadmill), and serum lipid and triglyceride levels (standard blood ELISA assays).Obese ZSF‐1 rats exhibited a significant increase in body weight and systemic blood pressure combined with hyperglycemia and insulin insensitivity. Echocardiographic evaluation of the left ventricle (LV) revealed highly significant increases in both the E/A and E/E′ ratios. LV end‐diastolic pressure (LVEDP) and LV relaxation (Tau) were decreased in the obese ZSF‐1 rats. Histological evaluation of the hearts revealed significant increases in cardiac fibrosis and cardiac myocyte injury in the obese vs. lean ZSF‐1. Furthermore, vascular reactivity and circulating nitric oxide levels were reduced in the obese ZSF‐1 rats. Exercise capacity testing revealed that exercise capacity was very significantly reduced in the ZSF‐1 obese rat throughout the 8 week experimental protocol. All these findings are consistent with the HFpEF phenotype observed in human HFpEF patients.This preclinical model of HFpEF, will provide novel insights into the pathobiology of HFpEF and provide a critically important platform for testing HFpEF therapeutics. Our future studies will be aimed at the identification of novel therapeutic interventions to treat HFpEF.Support or Funding InformationNational Institutes of General Medical Sciences (NIGMS) COBRE grant no. P30GM106392 to Dr. KapustaFigure 1
Protein Kinase A (PKA) Catalytic Subunits a and b Have Non‐Redundant Functions in Collecting Duct Cells
Authors: Salhadar Karim , Raghuram V. , Yang Chin-Rang , Limbutara Kavee , Knepper Mark 
Published: April 2020

Vasopressin regulates osmotic water transport in the renal collecting duct by PKA‐mediated control of the water channel aquaporin‐2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA‐Cα [Gene symbol: Prkaca]) and PKA catalytic β (PKA‐Cβ [Gene symbol: Prkacb]). To identify the relative roles of these two proteins, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA‐Cα (n=3) or PKA‐Cβ (n=3) was deleted using CRISPR‐Cas9‐based genome editing (generated in PMID 28973931). Cells were grown on permeable supports in the presence of the vasopressin analog dDAVP (0.1nM). Controls were cells carried through the genome editing procedure, but without deletion of PKA. Quantification used TMT mass tags in an Orbitrap Fusion Lumos ETD mass spectrometer. Preliminary measures of total protein amounts showed that PKA‐Cα deletion resulted in a near disappearance of AQP2 protein, while PKA‐Cβ deletion was associated with an
increase in AQP2. A total of 4635 phosphopeptides were quantified (Figure
1). The red data points represent phosphopeptides that correspond to previously identified PKA target sites. Stringent statistical criteria (Pmod < 0.01 and |log2(KO/control)| > 0.503) revealed that 67 phosphopeptides were significantly altered with PKA‐Cα deletion, while 21 phosphopeptides were significantly altered with PKA‐Cβ deletion. Only four sites were changed in both. We used Gene Ontology (GO‐CC) Cellular Component terms to identify sets of target phosphoproteins localized in different regions of the cell. The target proteins identified in PKA‐Cα‐null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA‐Cβ‐null cells were largely associated with the actin cytoskeleton and cell junctions. In summary, PKA‐Cα and PKA‐Cβ appear to serve substantially different functions in renal collecting duct cells. The differences in the phosphorylation targets of PKA‐Cα and PKA‐Cβ suggest differences in the cellular localization of the two PKA subunits. These findings in addition to the dramatically different effect of PKA‐Cα and PKA‐Cβ deletion on AQP2 protein abundance support the conclusion that PKA‐Cα and PKA‐Cβ have non‐redundant functions in collecting duct cells.Support or Funding InformationK.S. was supported by the Biomedical Engineering Summer Internship Program at NIH and carried out all of the mass spectrometry experiments. Work was supported by NHLBI Project ZIA‐HL001285.Volcano plots showing effect of PKA‐Cα deletion (left) and PKA‐Cβ deletion (right) on phosphoproteome of mouse mpkCCD cells. Three biological replicates for each.Figure 1