bluuebunny/crossref_metadata_2025_split huggingface dataset:
Examination of the Mechanism of Intersubunit Communication in Pyruvate Carboxylase Using Mixed Hybrid Tetramers
Authors: Westerhold Lauren E. , Zeczycki Tonya N. 
Published: November 2023

Pyruvate carboxylase (PC) is a biotin‐dependent, homotetrameric enzyme that is responsible for the MgATP‐dependent catalytic conversion of pyruvate to oxaloacetate in the presence of the essential allosteric activator, acetyl‐CoA. PC is a critical anaplerotic enzyme in glucose metabolism, serving to replenish oxaloacetate consumed by the TCA cycle. Increased production of acetyl‐CoA, due to high lipid supply, has been shown to upregulate PC activity in the liver, contributing to the development of obesity‐induced insulin resistance and Type 2 Diabetes. Previous crystallographic studies have identified four distinct domains that comprise the PC monomer, namely the biotin carboxylase (BC), biotin carboxyl carrier (BCCP), carboxyl transferase (CT), and allosteric domains. Each domain has been functionally characterized via steady‐state kinetics and site‐directed mutagenic studies of catalytically relevant residues. The tetramer is a dimer of dimers, whereby each face is composed of a dimer of monomers arranged antiparallel to each other.Pyruvate binding has been shown to trigger the movement of the BCCP domain to an opposing polypeptide chain, though it is not known whether this is facilitated through an inter‐or intramolecular “signal.” That is, does pyruvate binding on the same monomer or on the opposing polypeptide chain facilitate the intermolecular BCCP domain translocation? To determine whether BCCP domain movements are governed by intermolecular or intramolecular mechanisms, we have generated PC hybrid tetramers (HTs) consisting of two strategically chosen mutants: one with an inactive BC domain and wild‐type CT domain (E218A); and the other with a wild‐type BC domain and a mutant CT domain (T882S) exhibiting a 62‐fold decrease in kcat and 3‐fold increase in Km, pyruvate as compared to wild‐type. Steady‐state kinetics of the full‐forward reaction catalyzed by the T882S:E218A(1:1) HT demonstrate that the pyruvate concentration needed to facilitate BCCP translocation is significantly higher than that required for the carboxylation reaction. More importantly, our results indicate that the occupancy of the CT domain by pyruvate on an opposing polypeptide chain “triggers” the translocation of the BCCP domain from the BC domain on a monomer to the opposing CT domain. Further study of additional molecular interactions within the tetrameric structure, such as BC—BC domain interactions between faces of the enzyme, will be used to elucidate the nature of the mechanism by which PC controls BCCP translocation and the coordination of catalysis between two spatially distinct active sites.
Control of Enzyme function through loop dynamics
Authors: Chi Celestine N. , Vogeli Beat , Bibow Stefan 
Published: November 2023

Allosteric regulation has been revealed as an essential property of all proteins. Dynamic shift of structural equilibrium at one site can have structural and functional effect at other sites. However for small, single domain proteins these phenomena is difficult to access, albeit quantify easily. One way of shifting structural equilibrium distribution is by changing the the dynamics of flexible loops by amino acid substitution because large changes maybe achieved with minimal modification of protein. A ligand‐selective change of the binding affinity to the active‐site of the enzyme cyclophlin by tuning the dynamics of a highly flexible loop is presented.This system may serve as a platform to study the effects of various timescales of loop motion on protein function turned by mutations and opens the possibility for elucidating the role of time‐scale dependent allosterySupport or Funding InformationThis work was support in part by the Swedish research council Wenner‐Gren Stiftelsern WG‐13, Postdoctoral fellowship to C. N. Chi, the Swiss National Science Foundation with Grant 140214 and ETH Research Grant ETH‐04 13‐1 to B. Vögeli.
Regulation of N‐myristoyltransferase by Akt/PKB Mediated Phosphorylation
Authors: Kumar Sujeet , Sharma Rajendra K. 
Published: November 2023

Post‐translational modifications of proteins create highly dynamic relay system that regulates the core dynamic signalling pathways in response to the alterations in the cellular microenvironment. N‐myristoyltransferase (NMT) has emerged as a central node upstream of diverse signalling proteins regulating cellular survival. NMT modifies proteins in both co‐and post‐translational manner and modulates functioning of the signalling proteins by addition of the myristoyl moiety to an exposed N‐terminal glycine. The sequence feature of NMT reflects that it harbours an Akt/PKB kinase recognition motif within the aD‐ aE loop region. The in vitro kinase assay on the synthetic peptides corresponding to the aD‐aE loop followed by MALDI‐MS analysis established that the sequence motif is a valid substrate for the Akt/PKB kinase. The MS‐MS analysis of the phosphorylated peptide identified Thr319 within aD‐ aE loop region as the site of phosphorylation. To delineate the effects of phosphorylation, phosphomimetic mutant (Thr319Glu) and a null‐mutant (Thr319Ala) were engineered onto the catalytic domain the NMT. Enzymatic assays of the purified recombinant enzymes reflected a drastic loss in activity by mutations at the site Thr319. The analysis of NMT crystal structures shows that this loop region undergoes a conformational change upon binding of the co‐substrate myristoyl‐CoA (MYA). The binding of peptide substrate follows the MYA binding event and Thr319 in the aD‐ aE loop region lies in proximity to the peptide‐substrate binding site. In summary, we have established the site of phosphorylation and the regulation of NMT by the kinase Akt/PKB. These findings indicate that Akt/PKB kinase restricts NMT activity by modulating the peptide substrate affinity presumably by changing in the interaction network around the substrate‐binding site. Delineating of the functional consequences of this novel phosphorylation event is expected to improve our understanding of NMT regulations in diversified cellular needs.Support or Funding InformationThis work was supported by the Canadian Breast Cancer Foundation‐Prairies/NWT operating grant to RKS (Grant number ‐ 412572).
Organic synthesis of novel phosphatidylinositol 3‐kinase (PI3K) inhibitors activated by prostate specific antigen (PSA) for the treatment of androgen‐independent prostate cancer
Authors: Nelson Ronald Alvin 
Published: November 2023

The PI3K/Akt pathway is one of the most frequently dysregulated signaling pathways in cancer and remains a crucial target in drug development. Current therapeutic drugs are potent, but often lack the cellular specificity needed to lower toxicity effects. Here we show the design and synthesis of two types of novel PSA‐activated PI3K inhibitors for the treatment of androgen‐independent prostate cancer. Both literature and predictive molecular modeling studies suggest PI3K enzyme inhibition in the nanomolar/picomolar range. Type 1 PI3K Inhibitors contain : (1) a pyrimidine or triazine core, (2) a secondary amine ‐ usually a morpholine ring, and (3) an aromatic/heteroaromatic group capable of hydrogen bonding. Type 2 PI3K Inhibitors contain these same three moieties but with added electrophilic functional groups or electron withdrawing groups (EWG) on the secondary amine and the aromatic/heteroaromatic group. These functional groups will allow reversible covalent bond forming reactions to occur with nucleophilic amino acids in the active site of PI3K ‐ an approach that has not been attempted. Reversible covalent bond forming reactions are hypothesized to allow the inhibitor to stay in the active site longer and thus enhance it's potency towards the targeted enzyme. Additionally, the attachment of a peptide chain, with the embedded amino acid sequence HSSKLQ recognized and cleaved by prostate specific antigen (PSA), to both Type 1 and Type 2 PI3K inhibitors is hypothesized to significantly improve specificity towards prostate cells (based off our previously published work) and subsequently: (1) prevent further cellular proliferation of prostate cancer cells, (2) lower toxicity effects, and (3) induce apoptosis through Caspase activity. Testing our active Type 1 PI3K inhibitors via western blot analysis against the current clinical trial PI3K inhibitor ZSTK474 showed 10 fold improved inhibition. Predictive trends for our Type 2 PI3K inhibitors, based off of previous data, suggests 100 fold (or greater) improved inhibition when compared to ZSTK474.Support or Funding InformationThe author would like to thank Dr. Fred Salsbury for the molecular modeling and computational work, Dr. George Kulik's lab for previous drug screening data, Dr. Welker's Lab for prior organic synthesis developments, and Wake Forest University's Chemistry Department for providing lab resources and equipment. The author would also like to thank Wake Forest University's Center of Molecular Communication and Signaling for financially supporting this project.
In silico Selective Screening for Inhibitors of ABC Transporters that Minimizes Off Target Toxicity
Authors: McCormick James W. , Follit Courtney A. , Vogel Pia D. , Wise John G. 
Published: November 2023

The ATP‐binding cassette (ABC) class of transporters is involved in many diseases. Chemotherapy resistant, recurrent cancers commonly express ABCB1 P‐glycoprotein transporter (P‐gp) and ABCG2 Breast Cancer Resistance Protein (BCRP). At high levels of expression, both transporters lower the intracellular concentration of chemotherapeutics to a subtherapeutic level. The prokaryotic transporter MsbA is an essential ABC lipid A flippase that is homologous to the human multidrug resistance proteins. MsbA serves both as a drug target as well as an indicator of possible off target interaction of P‐gp/BCRP inhibitors due to the similar structure and ease of assays in bacterial model systems. Currently used methods of computational screening for inhibitors lack discrimination against off target interactions and are dependent on the quality of the starting structure. These problems are compounded with ABC transporters since inhibitors have historically been substrates of the transporters and often are substrates for cytochrome P450 3A4, which is heavily involved in the oxidation and metabolism of many drugs. These problems, and the lack of any high resolution crystal structure for the human proteins, has made finding ABC transporter inhibitors suitable for human use elusive.Previous work identified multiple structures of P‐gp that we used for computational screening for potential inhibitors. To prevent inherent biases from single structures, simulations of a catalytic cycle using targeted molecular dynamics was performed (Wise 2012 Biochemistry 51(25), 5125–5141; McCormick et al. 2015 Biochemistry 54(28), 4374–4390.). Molecular dynamics simulations of MsbA and Cyt P450 3A4 were also used to generate screening targets. Targeting the ATP binding domains and drug binding domains of P‐gp, and related domains of MsbA and the active site of P450 allowed for selection of hit compounds that are specific to one region of the protein of interest and avoid off site interactions. To limit computational time, iterative screening on similarity subsets of the ZINC database was performed, with subsequence screens based on the diversity of hits found in initial screens. Top hit compounds were purchased and assayed using biochemical and cell culture methods as described in Brewer et al. 2014 Molecular pharmacology 86(6), 716–726 and Follit et al 2015 Pharmacology Research & Perspectives 3(5), e00170. The correlation between computational screening methods and results from biochemical and cell culture assays for inhibition of P‐glycoproteins is described.Support or Funding InformationThis work is supported by NIH NIGMS [Grants R15‐GM094771‐01A1 and R15‐GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzann Ruff of Dallas, Texas.
Neurodevelopmental Gene Expression of Cannabinoid Receptor 2 (CB2R) in C57Bl/6J and BTBRT+tf/J and the CB2R Expression in Transgenic knockout mice (MUBx, DAT, DATCI, SERT)
Authors: Dennis Eugene Kobina 
Published: November 2023

The endocannabinoid system (ECS) consists of receptors, ligands, and the enzymes that degrade them. Two G‐protein coupled receptors that have been identified, cannabinoid receptors 1 and 2 (CB1R and CB2R). CB1R was discovered first and has been extensively studied. It has been shown to regulate the stress response through the HPA‐axis and also has been implicated in addiction. CB2R has been shown to play a role in embryo neurodevelopment and growth. It was recently discovered that CB1R is expressed in macrophages, microglial cells, and also on neurons. CB2R has been implicated in a number of brain disorders, but its function and role in the brain is not fully understood. Part of the controversy is due the fact that CB2R is induced during inflammation. Previous studies done in our lab with whole brains showed that the autism spectrum BTBRT+tf/J mice expressed more CB2R than the wild type C57bl/6J. This experiment sought to establish a baseline for the CB2R expression in the brain at various stages in their development (D0, D7, D14, D21, D35 and D42). Brains were dissected into three main parts; the prefrontal cortex (PFC), the cerebellum (CER) and the remainder of the brain designated as the mid brain (MID). RNA was extracted from these brain samples, converted to cDNA and then using RT‐PCR the CB2R gene expression was determined using the delta‐delta CT method. Transgenic mice strains were used for the second part of this study were dopamine transporter knockout (DAT), Serotonin transporter knockout (SERT), Mu‐Opioid receptor knockout (MUBx) and the Dopamine transporter knockout cocaine insensitive (DAT‐CI). Comparisons were made between the wild type and mutants of the knockout species. However the DAT mutant and heterozygous were compared because the mutant species do not survive. The results show differences in the expression of the CB2R at different stages in development and also in the dissected brain parts. Overall the BTBR mice had more CB2R gene expression than the wild type C57. These results were consistent with whole brain studies. The transgenic study showed different levels of CB2R expression between the strains. The different expression levels in the transgenic mice indicate a possible interaction between the CB2R and the knockout pathways. Based on these results conditional knockout species are being bred for further research.Support or Funding InformationNIH grant DA032890
Superresolution Microscopy Studies of Stx3 Clustering Mechanisms in Hippocampal Neurons
Authors: Thomson Holly , Lowenstein Sarah , Lochner Janis 
Published: November 2023

N‐ethylmaleimide sensitive factor attachment protein receptor proteins (SNAREs) are the universal molecular machinery for membrane fusion. The presynaptic membrane‐bound hippocampal SNARE syntaxin 1 (Stx1) mediates synaptic vesicle release. Syntaxin 3 (Stx3) is a postsynaptic hippocampal syntaxin variant that facilitates insertion of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors (AMPARs) into the membrane during long‐term potentiation (LTP). We implemented photoactivated localization microscopy (PALM), a superresolution fluorescence microscopy technique, to elucidate Stx1 and Stx3 distribution in cultured rat hippocampal neurons. Stx1 and Stx3 each form clusters of nonuniform density in hippocampal processes with the greatest density at cluster cores. Stx1 clusters are generally larger than Stx3 clusters, and the largest Stx1 clusters contain multiple density gradients. Several mechanisms have been implicated in Stx1 cluster formation. Electrostatic interactions between lipid headgroups and a conserved syntaxin polybasic region adjacent to the transmembrane domain (TMD) facilitate Stx1 cluster formation. Additionally, the discrepancy between TMD length and lipid bilayer width, termed hydrophobic mismatch, contributes to Stx1 clustering. Hydrophobic mismatch produces an energy penalty for protein association with the membrane that may be minimized by protein clustering. To determine whether these mechanisms also drive Stx3 clustering, we have generated an array of Stx3 mutants. The distribution of each Stx3 mutant will be characterized using PALM.Support or Funding InformationThis work is supported by National Institutes of Health grant 2 R15 NS40425‐03.
Spatiotemporal Dynamics and Regulation of cAMP and PKA in Early Stage Hippocampal Neurons
Authors: Gorshkov Kirill , Ramamurthy Santosh , Mehta Sohum , Ronnett Gabrielle V. , Zhou Fengquan , Zhang Jin 
Published: November 2023

Cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) are critical axon determinants that regulate neuronal polarity and axon formation, but the spatiotemporal dynamics of cAMP and PKA in developing neuronal subcompartments are poorly understood. Here, we use genetically encoded fluorescent biosensors to understand the compartmentalized dynamics of cAMP and PKA signaling in developing hippocampal neurons in vitro. In five‐day‐old hippocampal neurons, we detected an axon‐directed cAMP gradient dependent on phosphodiesterase activity and an axon‐directed PKA gradient dependent on the association of PKA with A‐kinase anchoring proteins (AKAPs). Furthermore, disrupting PKA anchoring to AKAPs in early hippocampal neurons leads to enhanced axon elongation. This effect is correlated with an enhancement of the stimulated axon‐directed cAMP and PKA gradients. We propose that a negative feedback loop in the cAMP/PKA/AKAP signaling microdomain plays an essential role in controlling the developmental program of hippocampal neurons.Support or Funding InformationThis work was supported by NIH RO1 DK073368 (to J.Z.), DGIST Convergence Science Center grant 11‐BD‐04 (to G.V.R), NSF GRF 1232825 (to K.G.).
Sphingosine‐1‐Phosphate Signaling in Non‐Alcoholic Steatohepatitis
Authors: Cowart Lauren Ashley 
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) is a rampant health problem, but mechanisms underlying its progression are not completely understood. Because targeting pathways that mediate inflammation and fibrosis in NAFLD could serve as novel therapeutic strategies, research in this area is of major clinical interest. NAFLD occurs in the context of metabolic disease, of which a component is lipid oversupply. Much experimental evidence has linked lipid oversupply to endoplasmic reticulum (ER) stress and inflammation, which corresponds to progression of NAFLD to non‐alcoholic steatohepatitis (NASH). Our laboratory has recently found that Sphingosine Kinase 1 is induced under conditions of lipid oversupply both in primary hepatocytes and in vivo in human NASH and a mouse NASH model. Our data suggest that this occurs via activation of ER stress by fatty acids. The bioactive lipid product of sphingosine kinase 1, sphingosine‐1‐phosphate, led to proinflammatory cytokine production in hepatocytes and, in the mouse model of NASH, bona fide inflammation as indicated by production of proinflammatory cytokines as well as macrophage and neutrophil infiltration. In isolated hepatocytes, sphingosine‐1‐phosphate activated S1P receptors and promoted activation of NfkB. Palmitate also activated NfkB and cytokine production, but this did not occur in hepatocytes lacking SphK1. These data support a hypothesis by which lipid oversupply, through activation of ER stress, increases Sphingosine Kinase 1/Sphingosine‐1‐phosphate, and S1P receptor‐mediated signaling activates NfkB‐dependent inflammation.NASH serves as a ‘gateway’ to hepatic fibrosis, cirrhosis, and hepatocellular carcinoma; however, mechanisms mediating this progression are unknown. We addressed by co‐culture experiments. Co‐culture of palmitate‐treated hepatocytes with hepatic stellate cells led to activation of TGF‐b‐mediated fibrogenic programs in the stellate cells, and this did not occur in stellate cells cultured with palmitate treated hepatocytes lacking SphK1. These data raise the possibility that sphingosine‐1‐phosphate generated in hepatocytes in response to lipid oversupply is transferred to stellate cells, thereby linking inflammation to fibrosis. In sum, our data suggest that ER stress links lipid oversupply to inflammation through induction of Sphingosine Kinase 1 and synthesis of sphingosine‐1‐phosphate., and that hepatocyte‐generated sphingosine‐1‐phosphate mediates cross‐talk with stellate cells to activate pro‐fibrotic programs. These findings may provide rationale for pursuit of novel therapeutic agents targeting sphingosine‐1‐phosphate signaling in NAFLD.Support or Funding InformationThis work was supported by a VA Merit Award, NIH grant 1R01HL117233, and NIH grant 5P30GM103339‐03.
Redefining caveolae as signalling platforms: specificity of interactions on endocytic caveolae in response to stress
Authors: GAMBIN Yann , SIERECKI Emma , Jung WooRam , Ariotti Nicholas , Giles Nichole , Bastiani Michele , Parton Robert 
Published: November 2023

Caveolae are small invaginations of the plasma membrane, which can expand upon stimulation and be internalized as endocytic vesicles. In addition to their role in clathrin‐independent endocytosis, caveolae play a role in many different signalling pathways.The major caveolar membrane protein, caveolin‐1 (CAV1) forms a dense coat of approx. 150 monomers assembled at the surface of the caveolae. CAV1 is believed to possess a “scaffolding domain” capable of interacting directly with numerous signalling proteins; its high oligomeric state amplifies its potency as a signalling platform.Here we have conducted a comprehensive in vitro screen of potential interacting partners to verify this “caveolin signalling” hypothesis. We have developped a binding assay using co‐expression of interactors and CAV1 in a cell‐free system which mimics the folding and insertion of caveolin in native membranes. Electron tomography shows that CAV1 generates membrane domains of uniform size and curvature and that we can re‐create small decorated vesicles that closely resemble endocytic caveolae.We measured binding of 40 potential interactors on these “reconstituted” caveolae using single‐molecule fluorescence and screened for interaction at the monomer level (non‐inserted CAV1) using a binding assay (AlphaScreen). We reveal exquisite selectivity of the assembled caveolae compared to monomeric CAV1. CAV1 phosphorylation, as found in cellular stress, showed amplification of binding to a subset of interactors. We further characterized binding sites by testing different mutations and truncations that alter the structure and oligomerisation state of CAV1. The results were confirmed in cells by proximity ligation assay, validating protein‐protein interactions and the effect of protein posttranslational modifications.Taken together, our findings create a novel picture of the role of caveolae in cell signalling and stress‐response.