bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNA‐mediated Signaling by Proteins with Iron‐Sulfur Clusters
Authors: Barton Jacqueline 
Published: November 2023

Many experiments have now shown that double helical DNA can serve as a conduit for efficient charge transport over long molecular distances. This chemistry is exquisitely sensitive to perturbations in the DNA base stack, such as arise with base mismatches, lesions, and protein binding. We have now been exploring how this chemistry may be used within the cell for long range signaling. Increasingly, 4Fe‐4S clusters are being found in DNA‐binding proteins involved in genome maintenance. These 4Fe‐4S clusters, common redox cofactors, have been found to be associated with a variety of DNA repair proteins and, more recently, also with DNA polymerases and primase. Studies are described to characterize DNA–mediated charge transport by these metalloproteins. Experiments indicate that this chemistry may provide a first step in how DNA repair proteins may localize in the vicinity of lesions. This redox chemistry at a distance, mediated by the DNA helix, offers a route for long range signaling and coordination of DNA repair and DNA‐processing proteins across the genome.Support or Funding InformationNational Institutes of Health, GMS
Phosphatidylcholine Transfer Protein (PC‐TP) Regulates Lipid Droplet Morphology in Experimental Non‐Alcoholic Steatohepatitis (NASH)
Authors: Nicholls Hayley T , Song Jiunn , Wu Jessica , Hornick Jason L , Cohen David E 
Published: November 2023

BackgroundCholine is essential for the biosynthesis of phosphatidylcholine (PC), which is the most abundant phospholipid in most mammalian membranes and in the surfactant monolayer that stabilizes lipid droplets within cells. Choline deficiency promotes hepatic steatosis, and when fed to mice, a methionine‐choline deficient (MCD) diet recapitulates key histopathologic features of non‐alcoholic steatohepatitis (NASH). Phosphatidylcholine transfer protein (PC‐TP; synonym StarD2) is a highly specific PC binding protein that is enriched in the liver. PC‐TP interacts with thioesterase superfamily member 2 (Them2) to suppress hepatic insulin signaling and promote lipogenesis. PC‐TP also catalyzes intermembrane PC transfer, suggesting the possibility of a direct role in the development of hepatic steatosis.AimsThis study was designed to investigate the contribution of PC‐TP to NASH induced by the MCD‐diet.MethodsPctp‐deficient and littermate control mice were fed a MCD‐diet for 5 weeks. NASH was assessed histologically. Hepatic lipids were determined via mass spectrometry and enzymatic assays, and oxidative stress in liver was quantified using a thiobarbituric acid reactive substances (TBARS) assay.ResultsThe absence of PC‐TP did not alter the severity of NASH; Pctp‐deficient mice exhibited similar levels of hepatic steatosis, inflammation and fibrosis as observed for controls. Hepatic PC, ceramide, diacylglycerol and triacylglycerol molecular species, as well as plasma triglycerides, free fatty acids and total cholesterol, were not different in Pctp‐deficient mice compared to controls. However, the absence of PC‐TP altered lipid droplet morphology so that livers of Pctp‐deficient mice accumulated more medium sized ‘enlarged’ lipid droplets and fewer smaller sized ‘initial’ lipid droplets. Whereas hepatic TBARS were similar in Pctp‐deficient and control mice, plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels were lower in the absence of PC‐TP expression.ConclusionsPC‐TP may regulate lipid droplet biogenesis in the development of hepatic steatosis, potentially by controlling the surface concentration of PCs. We speculate that PC‐TP may contribute to NASH severity by altering the intrahepatic distribution of triglycerides.Support or Funding InformationNIH DK056626 and DK048873.
Ribosome‐Based Quality Control Of Chemically Damaged mRNA
Authors: Zaher Hani , Simms Carrie L , Hudson Benjamin H 
Published: November 2023

Nucleic acids are constantly under assault from both endogenous and exogenous agents that can affect their chemical properties and hence their function. These assaults include reactive oxygen species (ROS), ultraviolet light and alkylating agents. ROS are present under normal conditions as byproducts from metabolic reactions. Furthermore, under oxidative stress, ROS levels increase significantly. In contrast to DNA, damaged RNA has received little attention presumably due to its transient nature. My laboratory is currently attempting to expand our understanding of the quality control processes that are responsible for recognizing damaged RNAs and their impact on cellular fitness. We have evidence that argues for an active role for the ribosome in some of the pathways in which the recognition process initiates in the decoding center of the ribosome. Changes in decoding during tRNA selection are likely to trigger a signaling cascade taking advantage of existent quality control processes to target the mRNA for degradation. To this end, we have recently explored the effect of oxidized and alkylated bases on decoding by the ribosome through the use of a well‐defined high‐resolution in vitro system. We had anticipated that, similar to DNA polymerization, the oxidized base 8‐oxoG would cause miscoding due to its ability to pair with A. However, the presence of 8‐oxoG in the A‐site codon regardless of its position stalled the ribosome; we measured rates of peptide‐bond formation for cognate aa‐tRNA that were greater than 3 orders of magnitude slower on the oxidized message relative to its intact cousin. In contrast, the alkylative adduct O6‐meG appears to affect the decoding process in a completely different fashion, where we observe differential effects on decoding depending on the position of the adduct within the codon. We are also investigating how these RNAs are subsequently targeted for degradation and whether the ribosome plays an active role in the process. In particular we have recently shown that 8‐oxoG mRNAs accumulate in the absence of no‐go decay factors in yeast and associate with polyribosomes. These observations provide compelling evidence that mRNA‐surveillance mechanisms have evolved to deal with damaged mRNA.Support or Funding InformationThis work was supported by the National Institutes of Health (R01GM112641) and Searle Scholars award to HSZ
Structural studies of soluble guanylate cyclase
Authors: Childers Kenneth , Garcin Elsa 
Published: November 2023

Cardiovascular diseases such as hypertension, atherosclerosis and myocardial infarction contribute to the highest mortality rates in the United States, cumulatively responsible for 1 in 3 deaths per year. The multidomain enzyme, soluble guanylate cyclase (sGC), is an established drug target for the treatment of pulmonary hypertension. sGC catalyzes the conversion of guanosine triphosphate (GTP) to cyclic GMP (cGMP), which acts on downstream kinases to inhibit platelet aggregation and stimulate vasodilation, maintaining cardiovascular health. Nitric oxide (NO) binds to the sGC N‐terminal heme prosthetic group, and enhances sGC activity 100 to 200 fold. Dysfunction in the NO‐sGC‐cGMP pathway including low NO bioavailability and sGC heme oxidation lead to lower cGMP levels, and contribute to the etiology of cardiovascular diseases. How NO binding to the N‐terminus of sGC increases catalytic activity at the sGC C‐terminus is still unknown. Elucidation of the structural switch from basal to NO‐stimulated sGC will provide the basis for the rational design of improved drugs that target sGC for the treatment of cardiovascular diseases.Our lab has solved the first x‐ray structure of the wild‐type human guanylate cyclase (GC) catalytic domains in the inactive apo form1. Activity assay measurements revealed a largely inactive αβGC heterodimer, showing ~0.01% of full‐length sGC basal activity. We proposed that residues located at the heterodimeric interface act as key regulators of the orientation‐dependent activation of the catalytic subunits. We aim to elucidate the structural transitions required between the inactive and active states of the catalytic domains. To accomplish this goal, we have designed mutations in the catalytic domains that have been shown to activate full‐length sGC. Mutants and wild‐type enzymes have been expressed and purified in E. coli. Structural and biochemical studies of these activating mutants are currently underway. Ultimately, these studies will allow us to understand the molecular mechanisms regulating sGC activity and will pave the way to the design of novel sGC activators.Support or Funding Information
Proteomic Identification of Novel LMF1 Partners: Defining the LPL Maturation Complex
Authors: Neher Saskia , Babilonia‐Rosa Melissa , Broadwell Lindsey 
Published: November 2023

Lipoprotein Lipase (LPL) is a secreted lipase responsible for clearing triglycerides from the blood. If LPL levels are insufficient, individuals suffer from severe hypertriglyceridemia. LPL requires a specialized chaperone, Lipase Maturation Factor 1 (LMF1), for proper folding and exit from the endoplasmic reticulum (ER). When LMF1 is absent, LPL forms disulfide‐bonded aggregates in the ER, resulting in a phenotype similar to LPL deficiency. We aimed to understand how LMF1 assists with LPL folding. To this end, we combined site‐specific incorporation of an unnatural, photocrosslinkable amino acid into LMF1 with proteomics to identify novel LMF1‐interacting proteins. We also found the known LMF1 interacting partner, SEL1L. For certain candidates including protein‐disulfide isomerases and chaperones we confirmed complex formation with LMF1. We further established the role of our candidate proteins in LPL folding and secretion using RNAi knockdowns in cultured cells. Reduction in levels of the novel LMF1 partners reduced LPL secretion. Secretion of the sequence‐related, but LMF1‐independent lipase Pancreatic Lipase (PL) was not affected. Thus, we have identified multiple novel LMF1‐binding partners that are necessary for proper LPL folding and secretion.Support or Funding InformationThis work was funded by grants from the NIH (1ROOHL098277) and the Pew Charitable Trusts to SBN and by a UNC dissertation completion fellowship to MAB.
LRP‐1 Deficiency in Type II Pneumocytes Decreased Surfactant but Increased Intracellular Lipids
Authors: Garcia‐Arcos Itsaso , Goldberg Ira J. , Foronjy Robert F. 
Published: November 2023

Pulmonary surfactant is synthesized by type II pneumocytes and is mainly composed of phospholipids and minor amounts of cholesterol and specific proteins.Surfactant allows the lungs to inflate by decreasing alveolar surface tension and the work needed for inspiration in each breathing cycle. A delicate balance between synthesis, secretion, recycling and degradation of the lipids is required to maintain surfactant function and lung performance.Single nucleotide polymorphisms in the LDL receptor associated protein 1 (LRP‐1) are associated with decreased lung function in patients with chronic obstructive pulmonary disease (COPD). But the function of LRP1 in the lung is not known. Amongst other roles, LRP‐1 participates in liver lipoprotein metabolism, in adipocyte cell differentiation and in macrophage inflammatory cascades.We deleted LRP‐1 in type II pneumocyte‐derived A549 cells using stable transfection. LRP1 shRNA‐treated cells and scrambled shRNA‐treated cells were cultured in collagen coated transwell inserts at the air‐liquid interphase, exposing the cellular basal side to the culture media and the apical side to the air.LRP‐1 shRNA decreased surfactant phospholipid, cholesterol and cholesteryl esters on the apical surface of the cells. LRP‐1 shRNA cells showed reduced gene expression of the rate limiting enzymes for fatty acid synthesis FAS, ACAT1 and SCD1, and of phosphatidylcholine synthesis enzymes choline transferase and choline kinase. Surprisingly, triglycerides and cholesteryl esters accumulated in intracellular lipid droplets in LRP‐1 shRNA cells, while phospholipids and unesterified cholesterol were not changed when compared to scrambled shRNA cells. LRP‐1 shRNA cells showed >3 fold overexpression of the fatty acid transporter CD36 and reduced gene expression of acyl‐CoA oxidase. After an overnight incubation with fresh media in the basal side, LRP‐1 shRNA cells showed higher media depletion of phospholipid, cholesterol esters and unesterifed cholesterol. Uptake of LDL‐derived phosphatidylcholine was not altered by LRP‐1 deletion, but the gene expression of phospholipid and cholesterol exporters ABCA1 and ABCG1 was reduced. Insulin signaling was affected by LRP‐1 deletion. LRP‐1 shRNA cells showed decreased protein expression of insulin receptor substrates 1 and 2 (IRS‐1 and IRS‐2) and increased phospho‐serine IRS‐1/total IRS‐1 ratio. After IGF‐1 treatment, LRP‐1 shRNA cells failed to Tyr‐phosphorylate the IGF‐1 receptor b, and phosphorylation of the downstream target Akt was lower than in scrambled shRNA cellsThese data suggest that LRP‐1 in type II pneumocytes is involved in lipid export, intracellular surfactant lipid metabolism and insulin signaling.Support or Funding InformationFAMRI Young Clinical Scientist Award (to I.G.A); AHA postdoctoral fellowship (to I.G.A), and NHLBI (to R.F.F).
KCNMB1−/− Mice as a Model of Pulmonary Arterial Hypertension
Authors: Barnes Elizabeth , Chen Chihhsin , Barnes Shayna , Kim Francis , Lee Lori , Alvira Cristina , Cornfield David 
Published: November 2023

RationaleThe BK channel is a calcium‐sensitive ootassium (K+) channel fundamental to the control of smooth muscle cell (SMC) tone. The pore‐forming α subunit is encoded by the KCNMA1 gene and its activity can be modified by the β1 subunit, encoded by the KCNMB1 gene. With an increase in cytosolic calcium concentration, the BK channel is activated, K+ passively flows out of the channel and membrane hyperpolarization and vasorelaxation results. Loss of BK channel function can lead to membrane depolarization and vasoconstriction.ObjectivePulmonary arterial hypertension (PAH) is a syndrome wherein there is an increase in pulmonary vascular resistance resulting in right ventricular heart failure. To assess the contribution of the BK channel β1 subunit in the development of PAH, the following hypothesis was tested: the expression of the BK channel β1 subunit modulates the pulmonary vascular response to chronic hypoxia.MethodsAdult wild‐type (WT) and KCNMB1−/− mice were exposed to normoxia at 21% O2, chronic hypoxia for 3 weeks at 10% O2 or acute hypoxia for 5 minutes at 10% O2. Right ventricular systolic pressure (RVSP) and the weight ratio of right ventricle over left ventricle plus septum (RV/(LV+S)) were evaluated. Blood was collected by direct heart venipuncture and assayed for hematocrit levels. Peripheral pulmonary artery (PA) wall thickness was measured in vessels less than 100μm in diameter. Formalin‐fixed lung sections from mice exposed to chronic hypoxia were examined for β1 subunit and α‐SMA expression in PA. Peripheral pulmonary artery smooth muscle cells (PASMC) were isolated from WT and KCNMB1−/− mice and assayed for β1, HIF‐1α, and phosphorylated myosin light chain (pMLC) expression.ResultsUnder control conditions, there was no difference in RVSP between WT and KCNMB1−/− mice. However, KCNMB1−/− mice exhibited significantly higher RVSP compared to WT littermates with exposure to either acute or chronic hypoxia in the absence of histologic evidence of accentuated vascular remodeling. Both groups demonstrated similar increases in hematocrit, decreases in body weight, and maintained cardiac function after chronic hypoxic exposure. In lung tissue, β1 subunit expression was limited to PA less than 100μm in diameter. Moreover, pMLC, a determinant of SMC tone, was higher in SMC isolated from peripheral PA compared to those derived from larger PA.ConclusionThe absence of the β1 subunit of the BK channel potentiates the pulmonary vascular response to both acute and chronic hypoxia. Functionally, in PASMC derived from the microvasculature, the β1 subunit plays a physiologically significant role in limiting the pulmonary vascular response to hypoxia. Compromised PASMC β1 expression may contribute to the pathogenesis of PAH.
Metabolomic Signatures Of Antimycobacterial Drug Mechanisms Of Action
Authors: Rhee Kyu 
Published: November 2023

Antibiotic development remains hindered by limitations of existing knowledge and tools. Comparative systems level (omic) profiling has emerged as a powerful modality to expand our understanding and annotate the phenotypic activity of chemical compounds. Here, we report the ability of metabolomic profiling to accurately annotate and classify antimycobacterial compounds according to their target and/or mode‐of‐action. We further report that this ability can be resolved into key discriminatory subsets. These studies not only identify novel biomarkers with which to classify antitubercular compounds but more broadly serve to expand our understanding of antibiotic activity.Support or Funding InformationBill & Melinda Gates Foundation, TB Drug AcceleratorNIH/NIAID TBRU AI111143
Phosphatase Activity of the Fungal Adenylate Cyclase Cyr1, the Yeast Homologue of PHLPP
Authors: Grzechnik Agnieszka Teresa , Petty Emily , Pillus Lorraine , Newton Alexandra C 
Published: November 2023

PH domain Leucine Rich Repeat Protein Phosphatases (PHLPP) suppress survival signaling by at least two mechanisms: dephosphorylating AGC kinases, such as Akt, and regulating the epigenome by suppressing histone phosphorylation. Interestingly, the only adenylate cyclase encoded in the Saccharomyces cerevisiae genome, CYR1, has a previously uncharacterized amino‐terminal segment homologous to PHLPP. Here we use biochemical, molecular, and genetic techniques to characterize the PHLPP‐associated phosphatase activity in yeast. Specifically, we show that overexpression of Cyr1 results in reduced phosphorylation of substrates such as Ser10 on Histone H3. The segment of Cyr1 that is homologous to PHLPP comprises a Leucine Rich Repeat (LRR) segment and a Serine/Threonine PP2C phosphatase domain, but lacks the PH domain and C‐terminal PDZ ligand of mammalian PHLPP. Thus, the ability of Cyr1 to suppress histone phosphorylation supports studies with mammalian PHLPP showing that the LRR segment is critical for its epigenetic function. Our study establishes that Cyr1 contains a previously undescribed phosphatase function, and suggests a functional link between cAMP‐dependent signaling and PHLPP.Support or Funding InformationA.T.G. was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences, T32 GM007752. Additionally, this work was supported by NIH Grant GM067946 (to A.C.N.)
Prevention of collar rot disease in germinating peanut seedlings by Azotobacter sp. RA2
Authors: Parel Ravikumar R. , Thakkar Vasudev R. , Bhatt Khevna , Prajapati Maitry R , Subramanian Ramalingam B 
Published: November 2023

Use of plant growth promoting rhizobacteria (PGPR) to increase the production and decrease disease occurrence is a useful, ecofriendly method in agriculture. A rhizospheric bacterial culture named RA2 was isolated from peanut rhizosphere from Junagadh region of Gujarat, India and showed different direct and indirect plant growth promoting activity like indole acetic acid, gibberellic acid, siderophore, hydrogen cyanide, Ammonia and (1‐Aminocyclopropane‐1‐Carboxylate) deaminase production, N2 fixation, phosphate and potassium solubilization in vitro. RA2 was able to protect peanut germinating seedling from A. niger infection and reduce collar rot disease incidence from 72–60% to 41–35% and increase germination percentage from 75–70% to 97–82% in two varieties GG20 and GG2 of peanut. RA2 was found to induce resistance in A. hypogaea L. seedlings via induction of different defense related enzyme like phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, lipoxygenase and pathogenesis related protein like chitinase and β– 1, 3‐ glucanase. Jasmonic acid, one of the major signaling molecules during induce systemic resistance, was also found to induce due to RA2 treatments. RA2 bacterium also promoted peanut growth and reduced A. niger infection in pot studies. 16S rDNA sequence of RA2 showed 99 % homology to Azotobacter species.Support or Funding InformationAuthors are thankful to BRD School of Biosciences, Sardar Patel University for providing infrastructure and all necessary facilities. Authors are also grateful to University Grant commission (UGC Major Research Project F.No. 39‐247/ 2012 (SR)) for providing financial aid.
Structural and Dynamical Basis of the Cross Talk between cAMP and cGMP Signaling Pathways
Authors: Melacini Giuseppe , VanSchouwen Bryan 
Published: November 2023

Protein kinases A and G (PKA and PKG) are respectively activated by the cAMP and cGMP second messengers and regulate signaling pathways that are often distinct. Nevertheless, due to the typically higher intracellular concentrations of cAMP vs. cGMP, cAMP contributes to the partial activation of PKG and to the modulation of the cGMP‐dependence of PKG activity, resulting in cAMP vs. cGMP signaling cross talk. However, the structural and dynamical basis for such cAMP vs. cGMP cross regulation is only limitedly understood. While it is known that the partial activation of PKG by cAMP arises from a combination of reduced cAMP vs. cGMP affinity (potency) and activation (efficacy) [1], the contributions of different functional groups in the cyclic nucleotide bases to such variations in potency and efficacy have not been fully dissected. Here, we show how the distinct contributions to potency and efficacy of the carbonyl at position 6 and the amino at position 2 of the guanine base are dissected by mapping through NMR [2–8] the interactions of a library of cyclic‐nucleotides with the C‐terminal cyclic nucleotide‐binding domain (CNB) of PKG, which is primarily responsible for the selective activation of PKG by cGMP. A model is proposed to explain the non‐additive contributions of these two functional groups to the potency and efficacy of PKG activation, providing insight into the molecular basis of cAMP vs. cGMP signaling cross talk.Support or Funding InformationThis work was supported by Grant (MOP‐68897) from the Canadian Institutes of Health Research (to G. M.). The authors declare that they have no conflicts of interest with the contents of this article.
Controlling Product Stereochemistry in Class II Diterpene Cyclases
Authors: Schulte Sam , Peters Reuben J 
Published: November 2023

Class II diterpene cyclases (DTCs) catalyze the committed step in production of the large class of labdane‐related diterpenoid natural products, many of which function as important primary and secondary metabolites in plants. Mechanistic studies of DTCs, which bicyclize geranylgeranyl diphosphate (20‐C), have revealed the importance of a variable set of two amino acids comprising a catalytic base dyad. Here, the catalytic base dyads of the closely related DTCs DsCPS (Danshen copalyl diphosphate synthase) and MvPPS (Marrubium vulgare peregrinol diphosphate synthase) were probed to further elucidate the evolution and mechanisms of these enzymes. Such studies were completed using site‐directed mutagenesis, with the mutated DTCs expressed in an Escherichia coli metabolic engineering system. DsCPS ordinarily produces copalyl diphosphate (CPP), which adopts the chair‐chair conformation. However, upon mutating the catalytic base dyad residues (along with one other neighboring residue) to the amino acids of the catalytic base dyad in MvPPS, the DsCPS mutant produces terpentedienyl diphosphate (TPP). Intriguingly, TPP adopts the chair‐boat conformation, corresponding to the stereochemistry of the MvPPS product. Thus, simply introducing the catalytic base dyad of one DTC into another is sufficient to change product stereochemistry in this instance, illustrating the critical role of these two amino acids. This research provides valuable insight into how specific mutations in closely related DTCs have created the capacity to produce such a wide array of labdane‐related diterpenoids.Support or Funding InformationNational Institutes of Health