bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of connexin43 by the tyrosine kinase Tyk2
Authors: Li Hanjun , Sorgen Paul
Published: November 2023

The identification of serine and tyrosine kinases that phosphorylate Connexin43 (Cx43) have been instrumental towards understanding the regulation of gap junction intercellular communication (GJIC). In this study, we performed an in vitro tyrosine phosphorylation screen using the purified carboxyl terminal domain (CT) of Cx43 and identified phosphorylation by tyrosine kinase 2 (Tyk2). This interaction was confirmed by using a GST‐tagged pull down assay involving the Cx43CT and MDA‐MB‐231 cell lysate (expresses Tyk2) as well as a co‐immunoprecipitation assay in Tyk2 transfected NRK cells (expresses Cx43). Both mass spectrometry analysis of the Cx43CT phosphorylated by Tyk2 in vitro and immunoblotting using phospho‐specific antibodies in constitutively active Tyk2 transfected NRK cells show that Tyk2 increased the level of Cx43 pY247 and pY265. The phosphorylation on Cx43 caused by Tyk2 is independent of Src which is known to phosphorylate Cx43 directly on Y247 and Y265. Immunofluorescence results combined with other molecular biological assays suggested that Tyk2 decreases the half‐life of the Cx43 gap junction plaque. Based upon the correlation between increased Angiotensin II (Ang II) levels leading to increased activation of Tyk2 and down regulation of ventricular Cx43 GJIC, we found that transfection of wild type Tyk2 in NRK cells enhanced Ang II induced tyrosine and serine phosphorylation of Cx43 (S279/282, S368, Y265, Y247), which explains the down regulation of Cx43 GJIC. Altogether, we identified a novel tyrosine kinase for Cx43 that leads to decreased GJIC and may be involved in Ang II mediated gap junction remodeling.
Single‐Molecule Studies of Eukaryotic DNA Replication
Authors: Bell Stephen P , Ticau Simina , Friedman Larry J , Gelles Jeff
Published: November 2023

The earliest step in eukaryotic DNA replication is the loading of the Mcm2–7 replicative DNA helicase onto origin DNA. This event marks all potential origins of replication and establishes the head‐to‐head configuration required for bidirectional replication initiation. Helicase loading requires the coordinate action of the Mcm2–7 helicase, and three helicase‐loading proteins: the origin‐recognition complex (ORC), Cdc6 and Cdt1. In an ATP‐hydrolysis‐dependent process, these proteins open the Mcm2–7 ring, insert the double stranded DNA into the central channel, and close the Mcm2–7 ring. The final outcome of this process is a head‐to‐head Mcm2–7 double hexamer.

We have developed a reconstituted single‐molecule helicase‐loading assay using Colocalization Single Molecule Spectroscopy (CoSMoS). We use co‐localization of the fluorescently‐labeled DNA and proteins as a real‐time assay for protein‐DNA binding and helicase loading. This assay recapitulates all the know requirements of Mcm2–7‐loading. Using this assay, we found that initial association and loading of the two Mcm2–7 complexes at the origin DNA occurs sequentially. Furthermore, each of the two Mcm2–7 loading events is driven by distinct Cdc6 and Cdt1 molecules that are sequentially recruited and released. In contrast to Cdc6 and Cdt1, a single ORC complex is sufficient to load both of the oppositely‐oriented helicases of the double hexamer. We have extended this assay by developing a FRET‐based method to monitor opening and closing of the Mcm2–7 ring during helicase loading. We are using this assay to determine when these events occurs during the loading reaction and what proteins mediate these changes. We will discuss the implications of our findings for the mechanism of Mcm2–7 loading.

Support or Funding Information

This work was supported by NIH grants GM52339 (S.P.B.) and R01 GM81648 (J.G.) and a grant from the G. Har‐ old and Leila Y. Mathers Foundation (J.G.). S.T. was supported in part by an NIH Pre‐Doctoral Training Grant (GM007287). S.P.B. is an investigator with the Howard Hughes Medical Institute.
Structural Mechanisms for Initiating DNA Replication
Authors: Berger James
Published: November 2023

RecA and AAA+ family ATPases play key roles in cellular events ranging from vesicle trafficking and proteolysis to chromatin remodeling and DNA replication/repair. A large subset of these enzymes form multimeric rings or spirals that respond to cycles of ATP binding and hydrolysis by undergoing changes in oligomeric and/or conformational state, and then transducing these physical rearrangements onto target macromolecules for controlling key biological processes. New structural and biochemical findings pertaining to how protein and DNA targets are moved or remodeled by RecA/AAA+ ATPases during the initiation of DNA replication will be discussed.

Support or Funding Information

NIGMS RO1 GM‐071747
Effects of Divalent Metal Ions on the Structure and Stability of SIKE
Authors: Kim Seung‐Hwan , Bell Ellis , Bell Jessica
Published: November 2023

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure of SIKE. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. A Three dimensional model of SIKE was made using the Phyre2 server using a variety of template molecules and ab initio modelling. The optimal model (with 97% of the sequence modelled at >90% confidence) was submitted to the 3DLigandSite server to predict potential binding sites. The model was predicted to contain a divalent metal ion binding site comprised of Aspartate 143, Leucine 173 and Glutamate 176 residues. His tagged SIKE was expressed, extracted using a Guanidine Hydrochloride solubilization procedure and purified using Nickel NTA affinity chromatography after renaturation on the resin. The purified protein was used in Fluorescence based Thermal Shift assays to in the presence and absence of a variety of divalent metal ions to screen for binding. Preliminary analysis of the results suggest that divalent metal ions including zinc do in fact affect the thermal stability of SIKE suggesting the presence of a metal binding site consistent with the optimal model.

Support or Funding Information

This work was supported by NIH grant R21A1 107447 to JKB
Biophysical Studies of STAT3‐SH2 Interactions with Ligands and Inhibitors
Authors: VanPelt Jamie , Williams Cameron , Cottingim Kelsey , George Kathleen , Poth Robert , Lin Jiayuh , Wang Hong , Page Richard C
Published: November 2023

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that plays a key role in many biological processes including cell proliferation, apoptosis, and cell differentiation. STAT3 is activated upon the phosphorylation of the STAT3‐Tyr705, forming a homodimer that translocates to the nucleus. In the nucleus, the STAT3 dimer promotes transcription of targeted genes by binding to specific regions of DNA. In various cancer cell lines, STAT3 is overexpressed leading to cell proliferation and inhibition of apoptosis. In this way, STAT3 activation is a viable target for treating cancer. XZH‐5, a small molecule inhibitor of STAT3, works on the principle of interrupting homodimerization by disrupting the interactions between the pTyr705 peptide and its associated pocket on the SH2 domain. In an attempt to optimize the binding of the inhibitor to the pTyr705 pocket, we have developed a family of XZH‐5 derivatives. Computational studies were performed to investigate the structural aspects of the binding of various XZH‐5 analogues to the pTyr705 pocket of the STAT3‐SH2 domain. The docking results were compared to in vitro activity assays against multiple human cancer cells lines with the goal of identifying optimal XZH‐5 analogues. Obtaining structural data of the interaction between the STAT3‐SH2 domain and XZH‐5 analogues would be adventitious for elucidating the ideal XZH‐5 analogue, however, purified wild type SH2 domain is highly unstable. Recently it has been suggested that Src homology 2 (SH2) domains are highly susceptible to beta‐sheet aggregation and a proposed TANGO algorithm can be utilized to determine the likelihood of this aggregation. Taking advantage of the TANGO algorithm, a mutant SH2 domain plasmid has been generated with the goal of developing a stable construct. Currently, the expression and purification of the mutant SH2 domain is being optimized. Kinetic studies will be used to confirm that the mutant SH2 domain maintains its dimerization function.
1Arg188 is Indispensible for RhoC Membrane Localization
Authors: Williams‐Perez Sophia , Shirar Sarah , Feller Gillian , Feller Samantha , Rooney Patrick , Ellerbroek Shawn Michael
Published: November 2023

RhoA and RhoC GTPases are 92% identical and possess unique cellular functions. Phosphorylation of Ser188 has widely been reported to inhibit RhoA activity. RhoC retains a canonical PKA recognition sequence (KRR) at positions 185–187, but possesses Arg188 in place of Ser188. We report here that transiently expressed RhoC was more membrane associated than RhoA. Further, RhoC‐R188S was a PKA substrate in vitro and exhibited less GTP loading compared to wild‐type RhoC when expressed in cells. RhoC‐R188S was less membrane associated than RhoC regardless of GTP‐binding status (Q63L). Surprisingly, RhoC‐R188A shared a similar membrane fractionation profile as RhoC‐R188S. In accordance, RhoA‐S188R was more membrane associated than either RhoA or RhoA‐S188A regardless of GTP‐binding status (Q63L). Altogether, these data suggest that expansion of the polybasic carboxyl tract through divergent appearance of Arg188 contributes more to increased RhoC membrane stabilization than loss of phospho‐regulation.

Support or Funding Information

This work was supported by a research grant from the McElroy Trust Foundation
Lipid Raft Coalescence Links HIV‐1 gp120 Induced Oxidative Stress to Neurodegeneration
Authors: Smith Lisa K , Walsh Keifer P , Whittington Lindsey G , Shaw Alisa E , Minamide Laurie S , Bamburg James R , Kuhn Thomas B
Published: November 2023

Host response to infection is a key determinant to disease pathogenesis. Hence, molecular understanding of host‐pathogen interactions provides a means of identifying novel therapeutic strategies in disease intervention. A number of pathogens, including the human immunodeficiency virus (HIV), utilize lipid rafts as their principal host entry portal. HIV1‐lipid raft interactions in the CNS are intimately linked to progressive neurodegeneration in a subset of infected individuals. Lipid rafts also play a critical role in other neurodegenerative diseases including Alzheimer disease. A signaling pathway common to amyloid β, cytokines, as well as HIV viral surface glycoprotein gp120 links oxidative stress and the formation of cofilin‐actin bundles (rods) to defects in synaptic function. Enhanced lipid raft coalescence mediated by the cellular prion protein (PrPC) and associated activation of NADPH oxidase are key to this pathway. Application of gp120 to SH‐SY5Y human neuroblastoma cells induced a time and dose‐dependent increase in lipid raft density reflected by a coalescence of lipid rafts into large macrodomains. Primary rat hippocampal neurons exposed to gp120 responded with a dose‐dependent formation of rods and increased lipid raft density. A rapid gp120‐induced production of reactive oxygen species (ROS) in hippocampal neurons was observed with the ROS‐sensitive fluorescence indicator DCF. NADPH oxidase activation in response to gp120 was causal to the formation of rods in hippocampal neurons. Blocking NADPH oxidase using either pharmacological inhibitors or by overexpressing dominant negative p22phox (a small membrane subunit of NOX) negated gp120‐mediated rod formation. More recent studies have identified a number of natural products that potently reduce viral infectivity. We demonstrate that a non‐polar fraction obtain from crude extracts of Alaskan wild bog blueberries (Vaccinium uliginosum) disrupt lipid raft coalescence in neurons exposed to neurotoxic stresses including gp120 and significantly decrease rod formation. In this scenario, lipid rafts serve as a signaling platform to progressive neurodegeneration associated with viral (AIDS dementia) as well as chronic degenerative CNS disorders. Natural products with the capability of modulating neurotoxic lipid raft signaling pathways could provide a novel approach to blunt progression of neurodegenerative processes.

Support or Funding Information

Supported in part by NIH grants AG044812, AG049668, Camille & Henry Dreyfus Sr. Scientist Mentor Award, and CSU Core Infrastructure Grant Microscope Imaging (JRB), and P20GM103395 and the Alzheimer's Resources of Alaska (TBK)
Sex‐Specific Differences of Transforming Growth Factor β Expression in Cardiac Hypertrophy and Heart Failure
Authors: James Maya
Published: November 2023

Transforming Growth Factor β (TGFβ) ligands and receptors are known to be pro‐hypertrophic and pro‐fibrotic factors in the heart, which could contribute to the development of cardiac hypertrophy and heart failure. It is well established that premenopausal females possess a lower incidence of these pathologies. To better understand the sex‐specific roles of TGFb family members in cardiac hypertrophy and heart failure, we analyzed gene expression in a rodent model of hypertrophy/heart failure and in human heart specimens. Gene expression analysis of sex‐specific rat neonatal cardiomyocyte hypertrophy model demonstrated that TGFβ ligands and receptors are expressed in a sex‐specific manner both in the presence and absence of pro‐hypertrophy agent phenylephrine. Sex differential expression of TGFb was also observed in a transaortic constriction model of cardiac hypertrophy in which males develop more severe hypertrophy. In contradiction, an angiotensin II (AngII) eluding pump mouse model did not result in sex differences and expression of TGFβ family members were more similar. This suggests that AngII, a known activator of TGFβ, may circumvent the cardioprotection afforded to females potentially through the direct activation of TGFβ. Preliminary analysis of human hearts reveals some sex differential gene regulation in both non‐failing and failing hearts. For example, TGFβ1 ligand is expressed at a lower level in non‐failing females hearts as compared to non‐failing males with levels increasing only in the failing hearts of females. Taken together these results provide evidence that sex specific differences in the development of cardiac hypertrophy and heart failure are mediated in part by TGFβ signaling.

Support or Funding Information

Eastern Kentucky University Department of Biological Sciences NIH 1R15HL 108192‐01NIH 1 R25 GM102776‐01
Developmental Factors Create a Niche for Cardiac Regeneration
Authors: Perrera Alec Bennett , Hoffman Kristen D , Camenisch Todd D
Published: November 2023

Heart disease is the number one cause of death in the United States. The adult mammalian heart has limited ability to repair itself, which is not sufficient to regenerate myocardial tissue injured by myocardial infarction (MI). Previous research has established that cardiac fibroblasts can be reprogrammed to adult cardiomyocyte‐like cells in vitro by administration of Gata4, Mef2c, and Tbx5 (GMT) as well as thymosin β4 (Tβ4). We have reported previously that hyaluronan (HA) supports a hematopoietic niche in the bone marrow as well as serving as a vital biomolecule for endocardial cushion and epicardial development during mouse embryogenesis. Epicardial cells are also implicated in myocardial regeneration, however the mechanisms of how this occurs in mammalian cells is not clear. TGFβ2 and HA are important for the induction of epicardial cell differentiation and invasion through regulation of epithelial to mesenchymal transformation (EMT). Thus, we hypothesize that TGFβ2 and HA alongside Tβ4 create a regenerative niche potentiating effective reprogramming of cells into cardiomyocytes. These factors should promote cellular differentiation without driving proliferation. We therefore tested whether alone or together HA, TGFβ2 and Tβ4 induces epicardial cell proliferation. Additional experiments are defining the differentiation pathway triggered by the regenerative cocktail of HA, TGFβ2 and Tβ4. These data are vital for understanding how upstream factors can promote GMT‐induced cardiac regeneration.

Support or Funding Information

NIEHS R25ES025494
Induction of Fatty Acid Synthase is Required for Profibrotic TGFβ Signaling
Authors: Jung Mi‐Yeon , Kang Jeong‐Han , Wang Youli , Hernandez Danielle , Lupu Ruth , Leof Edward B.
Published: November 2023

Alterations in cellular metabolism are known to play critical roles in a variety of normal as well as pathological processes, including (but not limited to) cancer, hepatic steatosis, and cell differentiation. While TGFβ is routinely considered a pleotropic cytokine due to its ability to impact numerous diseases and biological phenotypes, the role of metabolism in profibrotic TGFβ signaling is relatively unexplored/unknown. Fatty Acid Synthase (FASN) is the primary anabolic enzyme responsible for the de novo synthesis of fatty acids. Renewed interest in FASN has been sparked by the recognition of the critical inter‐relation of metabolic dysregulation and tumor progression with the findings that (i) FASN levels are significantly increased in numerous human tumors including breast, prostate, colorectal, and ovarian; and (ii) the efficacy of ErbB receptor inhibitors is enhanced by inhibiting FASN activity. In the current study we show that FASN expression is regulated by TGFβ in multiple murine and human fibroblasts, required for profibrotic TGFβ signaling, and, most importantly, treatment with C75 ((trans‐4‐carboxy‐5‐octyl‐3‐methylenebutyrolactone, a novel cerulenin‐derived semi‐synthetic FASN inhibitor) abrogates pathologic and physiologic changes in a murine model of lung fibrosis.

Support or Funding Information

This work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.
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 Information

National Institutes of Health, GMS
Exploring the Mechanistic Diversity of Non‐Heme‐Iron Enzymes by Freeze‐Quench Mössbauer Spectroscopy
Authors: Krebs Carsten , Bollinger J. Martin
Published: November 2023

Enzymes that activate dioxygen at a mononuclear or dinuclear non‐heme‐iron cofactor catalyze a wide variety of chemically difficult and biologically important oxidation reactions.(1–3) Our joint group is particularly interested in non‐heme‐iron enzymes that cleave aliphatic C‐H bonds. We study their reaction mechanisms by a combination of biochemical, kinetic, and spectroscopic methods.(4) This approach led to the identification of new cofactors,(5, 6) trapping and characterization of many reactive intermediates,(7, 8) and discovery of unexpected reactivities. The mechanistic diversity of these enzymes will be presented. Specifically, the role of 57Fe‐Mössbauer spectroscopy in these studies will be highlighted.
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

Background

Choline 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.

Aims

This study was designed to investigate the contribution of PC‐TP to NASH induced by the MCD‐diet.

Methods

Pctp‐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.

Results

The 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.

Conclusions

PC‐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 Information

NIH 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 Information

This 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 Information

This 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 cells

These data suggest that LRP‐1 in type II pneumocytes is involved in lipid export, intracellular surfactant lipid metabolism and insulin signaling.

Support or Funding Information

FAMRI 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

Rationale

The 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.

Objective

Pulmonary 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.

Methods

Adult 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.

Results

Under 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.

Conclusion

The 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 Information

Bill & 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 Information

A.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 Information

Authors 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 Information

This 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 Information

National Institutes of Health
Perspectives of Wife‐Husband Pairs on Their Roles in Infant and Young Child Feeding Decision‐Making in the Context of Malawi's Prevention of Mother‐to‐Child HIV Transmission Program
Authors: Chintalapudi Nainisha , Hamela Gloria , Mofolo Innocent , Hosseinipour Mina , Hoffman Irving , Bentley Margaret , Maman Suzanne , Flax Valerie
Published: November 2023

Studies have examined the role of male involvement in HIV counseling and testing within prevention of mother‐to‐child transmission (PMTCT) programs, but there is no corresponding data on male involvement in infant and young child feeding (IYCF) decision‐making and its relation to adherence to HIV and infant feeding guidelines. Adherence to IYCF guidelines remains important for limiting HIV transmission through breast milk and improving child survival. We explored wives’ and husbands’ perceptions of husbands’ roles in feeding decision‐making and practice in the context of Malawi's Option B+ PMTCT program through in‐depth interviews conducted in the catchment areas of 2 urban and 2 rural government clinics in Lilongwe District. Participants were 15 wife‐husband pairs (interviewed separately), where the wife was ≥ 18 years, HIV‐positive, and had a child < 24 months. The interviews were analyzed using thematic content analysis. Husbands were generally less well‐informed about IYCF recommendations than their wives, who were advised on IYCF during antenatal and PMTCT clinic visits. Most husbands and wives agreed that the wife was largely responsible for IYCF decision‐making and few husbands were involved in decisions about complementary feeding. Some husbands participated in decisions about when to stop breastfeeding the child because of their fears about the child becoming HIV infected, even if the wife was on antiretroviral therapy. In couples where the husband advised the wife on feeding, the wife usually said that she followed his wishes. Both wives and husbands wanted to avoid disagreements about IYCF because they were worried that such disagreements would harm the child's health and well being. For wives and husbands, the most important role of husbands in relation to IYCF was to purchase food or provide money for food for the child. Wives appreciated it when their husbands offered verbal support to encourage them to continue with the recommended IYCF practices. Wives whose husbands were supportive found it easier to follow breastfeeding recommendations, but adequate complementary feeding remained a challenge in the face of household food insecurity. In conclusion, Malawian husbands and wives reported highly gendered roles in relation to IYCF decision‐making in the context of HIV, but some women flagged their interest in having verbal, in addition to monetary, support from their husbands for IYCF practices. To address this gap, strategies should be tested for getting messages on IYCF to husbands through male‐friendly radio and community‐based programs or by encouraging them to participate in PMTCT visits with their wives.

Support or Funding Information

NIH BIRCWH K12 program, UNC CFAR developmental award
Nutrient adequacy among illegal drug users in the Greater Accra Region of Ghana
Authors: Setorglo Jacob Yaw , Gavor‐Kwashie Cyril , Pereko Kingsley Kwadwo Asare , Klevor Moses Kwashie , Steiner‐Asiedu Matilda
Published: November 2023

Background

Dietary diversity (DD) is a proxy for determining quality and nutrient adequacy of diet. Illegal drug user populations are believed to have poor dietary patterns which may compromise their nutritional health. The study assessed the diversity of the diets of illegal drug users in the Greater Accra Region of Ghana.

Design

A snowball sampling technique was used to select the sample whilst dietary data was collected using the 24‐hour recall method and background data through a questionnaire. Data were analysed using SPSS software (Version 16, Chicago Illinois). Foods consumed were grouped into 9 groups, and mean counts of groups were scored. Consumption of foods from less than 3 of the 9 food groups was considered as low DD whilst middle DD was classified as consuming foods from 4 to 5 different food groups. High DD was classified as consuming foods from more than 5 food groups. Ethical approval was provided by the Institutional Review Board of the University of Cape Coast

Results

Those aged less than 20 years had higher DD scores compared with those aged 20 years and above (P= 0.03). The mean sample DD score was 4.1. Both males and females had high scores for starchy staples and the least scores for milk and milk products. Marijuana users had higher mean DD scores compared with cocaine users (P= 0.03)

Conclusion

The sample studied had middle level nutrient adequacy. Younger drug users had better nutrient adequacy.