Welch Foundation Research Articles

Chemistry in Texas

Last week took me to Houston to a reception and banquet to celebrate the winners of the 2019 Welch Award in Chemistry. I had heard of the award before and knew a little bit about the work of the Welch Foundation through reading past C&EN articles published on the occasion of the Welch Foundation’s 50th anniversary. The foundation was founded in 1954 (2 years after Robert Alonzo Welch’s death) in Houston, and it is one of the oldest private funding sources for basic chemistry research in the US. So far, it has contributed nearly $930 million to the advancement of chemistry through a variety of activities, including research and departmental grants, endowed chairs, and other chemistry-related programs, all in the state of Texas. There isn’t a lot of information out there about the man behind the foundation, but if you are interested, you can read more about him in the

Global phase diagram of a spin–orbit-coupled Kondo lattice model on the honeycomb lattice**Project supported by the Ministry of Science and Technology of China, the National Key R&D Program of China (Grant No. 2016YFA0300504), the National Natural Science Foundation of China (Grant No. 11674392), and the Research Funds of Remnin University of China (Grant No. 18XNLG24). Work at Rice was in

Motivated by the growing interest in the novel quantum phases in materials with strong electron correlations and spin–orbit coupling, we study the interplay among the spin–orbit coupling, Kondo interaction, and magnetic frustration of a Kondo lattice model on a two-dimensional honeycomb lattice. We calculate the renormalized electronic structure and correlation functions at the saddle point based on a fermionic representation of the spin operators. We find a global phase diagram of the model at half-filling, which contains a variety of phases due to the competing interactions. In addition to a Kondo insulator, there is a topological insulator with valence bond solid correlations in the spin sector, and two antiferromagnetic phases. Due to the competition between the spin–orbit coupling and Kondo interaction, the direction of the magnetic moments in the antiferromagnetic phases can be either within or perpendicular to the lattice plane. The latter antiferromagnetic state is topologically nontrivial for moderate and strong spin–orbit couplings.

OR23-3 Differential Effects of Chronic Exposure to Bisphenol-A on Ion Channel Activity and Expression in Mouse Pancreatic Beta-Cells

Many dairy products found in plastics, cosmetics and food containers disrupt human health due to their actions as endocrine-disrupting chemicals (EDCs). Studies in different cellular and animal models have reported that low doses of the EDC bisphenol A (BPA), modify pancreatic β-cell function, induce insulin resistance and other metabolic alterations. Here, we aimed to investigate whether administration of low doses of BPA could regulate ion channels expression and function in mice pancreatic islets. For this purpose, we used microarrays to analyze the global gene expression profile, focusing on the modulation of ion channels-related genes. For in vivo experiments, mice were subcutaneously injected with BPA (100 μg/kg/day during 4 days). Islets were isolated 12 h after the last injection and prepared for RNA extraction and hybridization on Affymetrix GeneChip Mouse Genome 430 2.0 Array. Array scanning and data analysis were performed using Affymetrix Expression Console and the Affymetrix Transcriptome Analysis Console (TAC) Softwares. Expression level analysis was performed using the normalization method based on the processing algorithm called robust multi-array average (RMA). Whole islets or dispersed islets from C57BL/6 or estrogen receptor beta knockout (βERKO) mice were treated in vitro with BPA or vehicle during 48 h. Whole-cell patch-clamp recordings were used to measure sodium and potassium currents. Microarray analysis of islets isolated from mice treated with BPA showed that some ion channels, such as sodium channel voltage-gated type IX, alpha (Scn9a), potassium large conductance calcium-activated channel alpha member 1 (Kcnma1), and Kv channel-interacting protein 1 (Kcnip1), were modulated. These results were confirmed by quantitative RT-PCR. Electrophysiological measurements showed a decrease in both sodium currents and total potassium currents. Additionally, the currents through the voltage-gated potassium channel subunits Kv2.1 and 2.2 as well as the KCa1.1 (encoded by Kcnma1 gene) channels were also reduced. Interestingly, beta-cells from βERKO mice did not present such reductions, suggesting that these effects occur mainly via ERβ. Our results show that, acting as a xenoestrogen, BPA modulates sodium and potassium currents as well as gene expression via ERβ. Funding: Ministerio de Economia y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), EU Grants SAF2014-58335-P, BFU2017-86579-R, BFU2016-77125-R and Generalitat Valenciana PROMETEO II/2015/016. LM holds a Juan de la Cierva fellowship from the Ministry of Economy, Industry and Competitiveness (IJCI-2015-24482). CIBERDEM is an initiative of the Instituto de Salud Carlos III. J-AG was supported by the Robert A. Welch Foundation (E-0004).

Triple α -particle resonances in the decay of hot nuclear systems * * Supported by the National Natural Science Foundation of China (1176014, 11605097, 11421505,11865010), the US Department of Energy (DE-FG02-93ER40773, NNSA DE-NA0003841 (CENTAUR)), and the Robert A. Welch Foundation (A-1266). This work was also supported by the Chinese Academy of Sciences (CAS) President’s International Fellowship Initiative (2015VWA070), Strategic Priority

The Efimov (Thomas) trimers in excited 12C nuclei, for which no observation exists yet, are discussed by means of analyzing the experimental data of 70(64)Zn(64Ni) + 70(64)Zn(64Ni) reactions at the beam energy of E/A = 35 MeV/nucleon. In heavy ion collisions, -particles interact with each other and can form complex systems such as 8Be and 12C. For the 3 -particle systems, multi-resonance processes give rise to excited levels of 12C. The interaction between any two of the 3 -particles provides events with one, two or three 8Be. Their interfering levels are clearly seen in the minimum relative energy distributions. Events with the three -particle relative energies consistent with the ground state of 8Be are observed with the decrease of the instrumental error for the reconstructed 7.458 MeV excitation level in 12C, which was suggested as the Efimov (Thomas) state.

Hydralazine Improves Mitochondrial Function via SIRT1/SIRT5 Activation and Protects against Glucose Toxicity

Mitochondrial dysfunction is one of the hallmarks of aging. Improving mitochondrial activity has been a prime target for improving organismal fitness and longevity and in many cases a potential treatment for age‐related diseases. In this study, we demonstrate that hydralazine, an FDA approved antihypertensive drug, improves mitochondrial function in a SIRT1/SIRT5‐dependent manner. We show that hydralazine treatment alters central carbon metabolism, increases TCA cycle metabolites, and improves respiration inC. elegans. Our data suggest that hydralazine‐induced mitochondrial activation is essential for the drug's prolongevity effect in C. elegans. We demonstrate that under glucose stress, hydralazine treatment restores locomotor performance, mitochondrial health, and lifespan in C. elegans. These new insights into hydralazine's ability to alter the mitochondrial activity and metabolic homeostasis deepen our understanding of hydralazine's mechanism of action and its potential health benefits and side effects.Support or Funding InformationThis work was supported by the National Institutes of Health (grant R03AG045504 to H.M.), Robert A. Welch Foundation (grant I‐1850 to H.M.) and the Cancer Prevention and Research Institute of Texas (grant R1121 to H.M.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel Regulation of Lipid Metabolism by a Phosphatidylinositol Transfer Protein and a Phosphatidylinositol 4‐Kinase

Membrane contact sites (MCSs) are regions in cells where membranes of two organelles are in close apposition. MCSs are proposed to facilitate non‐vesicular trafficking of lipids. Lipid transfer proteins (LTPs) are one of the most commonly‐found components in the MCS system and are speculated to mediate lipid transfer between membranes. However, both the biological functions of MCSs and the functional role of LTP in MCSs remain largely elusive. Herein, we examine in detail a previously‐proposed MCSs model, the ER‐Golgi/endosome MCS facilitating the phosphatidylserine (PtdSer) decarboxylation 2 (Psd2) pathway, which converts PtdSer into phosphatidylethanolamine (PtdEtn) in Saccharomyces cerevisiae. Particularly, we investigate the biological role of a yeast LTP, one of the yeast Phosphatidylinositol (PtdIns)‐Transfer Proteins (PITPs), Sfh4, and a PtdIns 4‐OH kinase Stt4, in this Psd2‐MCS system.We found that Psd2, Sfh4, and Stt4 are the only essential components facilitating Psd2‐dependent PtdEtn synthesis while other previously proposed elements constituting the Psd2‐MCS (Scs2, Scs22, and Pbi1) are dispensable. Surprisingly, neither the PtdIns‐transfer activity of Sfh4 nor its capacity to activate Stt4 is required to stimulate the Psd2 pathway. Instead, Sfh4 activates the Psd2 pathway via an Sfh4‐Psd2 physical interaction involving the F175 residue on the surface of Sfh4. Stt4 also displays physical interaction with Psd2 in an Sfh4‐independent manner. Instead of directly activating Psd2, Stt4 regulates the substrate pool accessible to Psd2, thereby indirectly controlling the Psd2‐mediated synthesis of PtdEtn. These results demonstrate that the ER‐endosomal MCS model is an inaccurate description of the Psd2 system in yeast, and provide an outstanding example where PITP biological function is uncoupled from its ‘canonical’ activity as a PtdIns transfer protein. Additionally, our findings reveal a novel mechanism in which a PITP and a PtdIns 4‐OH kinase regulate cellular phospholipid homeostasis.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (GM44530) and the Robert A. Welch Foundation (BE‐0117) to VAB. The authors declare no financial conflicts.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The CYP4A/20‐HETE Axis Regulates Ischemia‐induced Neovascularization via Its Actions on Endothelial Progenitor and Preexisting Endothelial Cells

20‐hydroxyeicosatetraenoic acid (20‐HETE) was recently identified as a novel contributor of ischemia‐induced neovascularization based on the key observation that pharmacological interferences of the CYP4A/20‐HETE axis decrease ischemic neovascularization. The objective of the present study is to determine whether the underlying cellular mechanisms involve endothelial progenitor cells (EPC) and pre‐existing endothelial cells (EC).We found that ischemia leads to a time‐dependent increase in 20‐HETE that is endothelial in origin using LC/MS/MS analysis and immunofluorescent (IF) microscopy. This is accompanied by increases in SDF‐1a expressions, EPC mobilization from bone marrow, and subsequent homing to ischemic tissues. Pharmacological interferences of the CYP4A/20‐HETE axis with a 20‐HETE synthesis inhibitor Dibromo‐dodecenyl‐methylsulfimide(DDMS) or a 20‐HETE antagonist N‐(20‐hydroxyeicosa‐6(Z), 15(Z)‐dienoyl) glycine(6, 15‐20‐HEDGE) significantly attenuated these increases. Importantly, we also determined that 20‐HETE plays a novel role in maintaining EPC functions and increases in the expression of Oct4, Sox2, and Nanog expression that are indicative of increased progenitor cell stemness. Flow cytometric analysis revealed that pharmacological interferences of the CYP4A/20‐HETE axis decrease the EPC population in culture, whereas 20‐HETE increases the cultured EPC population. Furthermore, ischemia also markedly increased the proliferation, oxidative stress, and ICAM‐1 expression in preexisting EC in the hindlimb gracilis muscles. We found that these increases were markedly negated by DDMS and 6, 15‐20‐HEDGE.Taken together, the CYP4A/20‐HETE axis regulates ischemia‐induced compensatory neovascularization via its combined actions on promoting EPC and local preexisting EC responses that are associated with increased neovascularization.Support or Funding InformationAmerican Heart Association [Grant 11SDG6870004]; National Institutes of Health [Grants HL34300], [Grant DK38226]; Robert A. Welch Foundation [GL625910]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Establishing the Role of Rieske Protein Reduction Potential in the Formation of Reactive Oxygen Species in Complex III

The factors that modulate the reduction potential of complex III's catalytic Rieske metalloprotein subunit (RP) have been the subject of much study. During the bifurcated Q‐cycle, RP transports one electron from quinol to cytochrome c1. Quinol's other electron is passed from heme bL to heme bH, which reduces a quinone bound at the QN site. If the Q‐cycle is disrupted, electrons may interact with molecular oxygen to produce reactive oxygen species (ROS). For example, if RP's reduction potential is too low, it may never accept quinol's electron or do so inefficiently. The latter scenario may shift Q‐cycle equilibria against RP movement towards cytochrome c1, instead favoring RP electron release and interaction with molecular oxygen to produce ROS. Thus, we propose that a mismatch in reduction potential between RP and quinol may serve as a possible source of ROS generated from complex III. Understanding the sources of ROS in the electron transport chain may lead to better treatments for diseases that arise from high levels of ROS.We are investigating this hypothesis by (1) measuring the reduction potential of isolated Saccharomyces cerevisiae (yeast) RP expressed in Escherichia coli (E. coli) cells (ScRieske) and (2) measuring the ROS produced by intact yeast complex III expressed in yeast cells (ScComplexIII). We created a wild‐type ScComplexIII yeast strain (JPJ1‐RIP1) by restoring respiratory function to an anaerobic RP‐deletion strain (JPJ1) via complementation with a RP expression vector. Now, we are measuring JPJ1‐RIP1 and JPJ1 ROS production by fluorometry using dichlorofluorescein (DCF) as a ROS indicator. We have also expressed and purified wild‐type ScRieske in E. coli and have characterized its structure using circular dichroism (CD) and UV‐visible spectroscopy. Furthermore, we have probed the electronics of ScRieske's [2Fe‐2S] cluster via chemical modification experiments with diethyl pyrocarbonate (DEPC). Lastly, we have obtained E. coli and JPJ1‐RIP1 expression vectors for ScRieske mutants Y185F, S183A, and S183T. Ultimately, we will use our ROS assay to quantify ROS production of ScRieske mutants with modulated reduction potentials to study the relationship between RP reduction potential and ROS production in complex III.Support or Funding InformationTrinity Chemistry and Neuroscience Departments, Welch Foundation, The San Antonio Area Foundation, 2017 Murchison Summer Research Fellowship, 2017 ASBMB Undergraduate Research AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modeling the 20‐HETE Binding Site on GPR75

Recently, the cytochrome P450‐derived eicosanoid 20‐Hydroxyeicosatetraenoic acid (20‐HETE) was shown to activate the orphan receptor GPR75. The pairing of 20‐HETE and GPR75 activates a signaling pathway that elicits increases in endothelial angiotensin converting enzyme (ACE) and promotes the development of hypertension. Little is known about the critical binding sites/pockets across GPR75 involved in interacting with 20‐HETE that ultimately leads to receptor activation. Computational docking analysis of GPR75 uncovered a putative ligand binding site located between transmembranes five and six on GPR75 (SiteScore: 1.182 and Dscore: 1.303). Independent analyses of ligand‐receptor docking using 20‐HETE placed the lipid in close proximity to the putative binding pocket (Docking Score: −3.229, Glide Emodel: −29.045). Additional modeling of GPR75 alongside another proposed ligand, the chemokine CCL5 (RANTES), revealed a potential protein‐protein interaction model occurring at the top (extracellular face) between the first and seventh transmembranes of GPR75. This distant site suggests the possibility of distinct ligand‐receptor interactions that promote unique signaling cascades. In order to establish a functional assay to further investigate the significance of the proposed 20‐HETE binding site/pocket, we have adapted the PRESTO‐Tango system. This system allows for the quantification of ligand‐dependent β‐arresstin2 recruitment, a common hallmark of G‐protein coupled receptor activation, biased agonism and receptor inactivation/internalization. Preliminary studies using the GPR75‐Tango construct demonstrated a robust signal wherein 20‐HETE stimulates the recruitment of β‐arresstin2 after GPR75 activation (EC50=3μM). Further studies will be conducted to assess the significance of residues located within the putative binding pocket with respect to 20‐HETE‐mediated activation of GPR75.Support or Funding InformationThis study was supported by National Institute of Health grants HL39793(M.L. Schwartzman), HL39793‐01S1 (V. Garcia) and the Robert A. Welch Foundation (I‐0011, J.R. Falck).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional Analysis and Characterization of the Metal Bound Sco Protein from Thermus thermophilus

Cytochrome c oxidase (CcO) is the final complex of the respiratory electron transport chain, which is a set of complexes that participate in the production of adenosine triphosphate (ATP), the main form of energy used by cells. CuA is the initial electron acceptor of CcO and consists of two copper ions bridged by two cysteines and ligated by two histidines, a methionine and the carbonyl backbone of a nearby glutamine. Sco proteins have been implicated in the assembly of the CuA, site but the exact role that it plays is the subject of much debate and study. Previous work has found that Sco proteins share a fold similar to thioredoxins, pointing toward the possibility of thiol‐disulfide oxidoreductase activity as a function of the protein. The ability of Thermus thermophilus Sco (TtSco) to act as a thiol‐disulfide oxidoreductase has been probed using an experiment to trap the protein‐protein intermediate between TtSco and its target, Thermus thermophilus CuA (TtCuA). The effect of nearby amino acids and metal binding prior to reaction have been determined for the formation of the protein‐protein intermediate, the mixed disulfide intermediate (MDI). The formation of MDI was monitored with either UV‐Visible spectroscopy by observing an absorbance increase at 412 nm as a chromophore tag, TNB, was released upon reaction between TtSco and TtCuA or by non‐reducing SDS‐PAGE gels. Altering Arg 48, which is adjacent to the attacking cysteine to an Asp has a very large effect on MDI production. TtSco also has metal binding ability, found to bind Cu(I), Cu(II), Ni(II), Cd(II) and Co(II) with varying relative affinities, in addition to disulfide reductase activity. To further characterize MDI formation with TtSco, the impact of conserved residues involved in metal binding on MDI formation were probed and showed that changing the conserved histidine did not affect MDI formation. However, binding of metal to Sco prior to incubating with TtCuA abrogated MDI formation. In characterizing the metal bound protein, preliminary studies to determine the binding affinity of Cu(II) to TtSco have been completed. The impact of conserved cysteines on metal binding has also been studied and the results indicate that one cysteine binds much more tightly to the Cu(II) than the other. While this work allows for a better understanding of TtSco function, much work remains to be done to understand the impacts of metal binding and to further characterize the Sco family of proteins.Support or Funding InformationResearch Corporation (Cottrell College Science Award 7963), the Welch Foundation (W‐0031 [Trinity Chemistry Department]), FASTER grant SURF‐ National Science Foundation DUE S‐STEM Award 1153796 and the Arnold and Mabel Beckman Foundation Beckman Scholars AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Reassessment of Phosphatidylinositol Transfer Protein Alpha's Growth Factor Signaling Role

In mammals, the START‐like phosphatidylinositol transfer proteins (PITPs) remain an understudied group of proteins implicated in human health and disease. Phosphatidylinositol transfer protein alpha (PITPa) is linked to human epidermal growth factor receptor (HER2)‐positive metastatic breast cancer drug resistance, and muscular dystrophy, but its precise mechanism of action is unclear. Current dogma states that PITPa is essential for growth factor signaling through the epidermal growth factor (EGFR) and netrin signaling pathways, but little data has emerged supporting these claims. Data from our laboratory, from multiple experimental systems, strongly dispute PITPa requirement in these pathways. Strikingly, mouse and human cell lines lacking PITPa sense and chemotax towards EGF, and signaling responses downstream of EGFR‐stimulation are unimpaired, including phosphatidylinositol (3,4,5)P3 generation, and phosphorylation of protein kinase B (Akt). Surprisingly, lack of PITPa in human cells results in potentiation of Akt phosphorylation (threonine 308) following EGFR stimulation, indicating that contrary to current thinking, PITPa may actually repress this pathway. The dispensability of PITPa for EGF‐signaling explains the remarkable absence of EGF‐signaling associated phenotypes in PITPa nullizygous (Pitpa0/0) mice. We have previously reported that Pitpa0/0 animals exhibit a complex phenotype including spinocerebellar neurodegeneration characterized by gliosis and neuronal apoptosis of cerebellum, and hindbrain, and that neuronal specific eviction of embryonic PITPa and PITPb results in progeny born lacking a forebrain. Axon guidance and mouse forebrain development require netrin‐1 signaling through the netrin receptor (DCC), a process reported to require PITPa. We demonstrate that while compromise of netrin‐signaling results in structural brain derangements, these defects do not result from absence of PITPa. Furthermore, we fail to reproduce the previously reported physical interaction between the netrin receptor (DCC) and PITPa. In light of our findings the ‘essential’ role of PITPa in growth factor signaling must be reevaluated.Support or Funding InformationThis work was supported by the Robert A. Welch Foundation (BE0017), and a National Institutes of Health grant (GM44530) awarded to Vytas A. BankaitisThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Methocinnamox: Sustained Antagonism of the Antinociceptive Effects of Morphine and Not Spiradoline in Rats

The opioid epidemic continues in the US. Currently available pharmacotherapies for opioid abuse and overdose include mu opioid receptor agonists (methadone and buprenorphine) and antagonists (naltrexone and naloxone), and while each treatment can be effective, they have limitations. A novel mu opioid receptor antagonist, methocinnamox (MCAM), has a long duration of action in blocking the abuse‐related and toxic effects of opioids, and its antagonist effects are not surmounted by subsequent administration of mu opioid receptor agonists. Consequently, MCAM might overcome the limitations of other antagonists, although other problems could emerge with long‐term blockade of mu opioid receptors (e.g. prescription opioids would be ineffective for treating pain). The current study measured antinociception using a warm water tail withdrawal procedure in rats and examined the persistence, surmountability, and selectivity of antagonism by MCAM. In 1 group of 8 rats, morphine dose‐effect curves were determined 1 day after MCAM administration and periodically thereafter, and in a different group of 8 rats, a dose‐effect curve for the kappa opioid receptor agonist spiradoline was determined 1 day after MCAM administration with morphine dose‐effect curves determined periodically thereafter. Morphine (1.78–17.8 mg/kg) and spiradoline (1–10 mg/kg) dose‐dependently increased the latency for rats to remove their tails from water maintained at 50°C. MCAM (1–10 mg/kg) attenuated the antinociceptive effects of morphine in a dose‐ and time‐dependent manner, with 10 mg/kg MCAM producing a 30‐fold rightward shift in the morphine dose‐effect curve 1 day after it was administered. For at least 37 days after administration of the largest dose of MCAM, doses of 178–560 mg/kg of morphine were needed to produce a maximal antinociceptive effect. In contrast, the spiradoline dose‐effect curve was not changed 1 day after administration of 10 mg/kg MCAM, although in the same rats the antinociceptive effects of morphine were attenuated 4 days later (i.e. 5 days after MCAM administration). Thus, MCAM produces a sustained and selective antagonism of the antinociceptive effects of mu opioid receptor agonists. Under these conditions, antagonism by MCAM was surmounted by very large doses of morphine, although it remains to be determined whether the antinociceptive effects of those doses of morphine are mediated by mu opioid receptors. Nevertheless, the selectivity of MCAM in antagonizing the effects of mu opioid receptor agonists suggests that pain could be treated through mechanisms other than mu opioid receptors in patients that are taking MCAM for opioid abuse or overdose.Support or Funding InformationSupported by NIH (R01DA005018; R01DA07315) and the Welch Foundation (AQ‐0039).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cryo‐EM analysis of full‐length receptor tyrosine kinase

Receptor tyrosine kinases (RTKs) represent a family of cell surface receptors that have been shown not only to play a key role in regulating normal cellular processes, but also to be critical in the development and progression of many types of human cancer. To date, many crystal structures of individual domains of RTKs are available; however, the high resolution structural information for full‐length RTK proteins either in their apo‐state or in ligand‐bound states remains elusive. In addition, there are no structural detail about how the active full‐length receptor can bind the effector protein for the activation of downstream signaling pathway. Lacking a complete structure of full‐length receptor signaling complex hinder our understanding of how this important protein functions. We determine the structures of full‐length RTK proteins, in complex with their ligands and downstream proteins, by single particle cryo‐EM, as key to understand the critical role that this protein family plays in the physiology of the cell. Epidermal growth factor receptor (EGFR), RET, cMET and Insulin receptor (IR), the most studied members in the RTK family, are chosen as the targets for structure determination. These pathways comprise a complex and diverse signaling network, and play an essential role in normal cell proliferation, differentiation, development and motility. Aberrant activation can lead to both tumor growth and metastatic progression of cancer cells, which makes them promising candidate for cancer therapy. Determination of full‐length RTKs in apo‐ and ligand‐bound states shed light on its mechanisms of kinase activation, enhance understanding of the interaction between receptor and their ligand and inhibitors, and facilitate development of selective anti‐cancer drugs. Reconstitution of fully assembled signaling complex reveal the mechanism by which the active receptors initialize the downstream signaling transudation, and provide direct structural evidence for how the plasma membrane plays roles in this processing. Solving the structures of different members of the RTK family reveal similarities and differences in the structure‐function relationships within this receptor family, which can potentially lead to new strategies to selectively inhibit/modulate individual pathways for cancer therapy or concurrently target various pathways, an approach that has been suggested to improve therapeutic effectiveness.Support or Funding Informationthe Welch foundation, and CPRIT foundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating the Interaction of V. parahaemolyticus Effector VopR and its Putative Chaperone VP1684

Vibrio parahaemolyticus (V. para) is a Gram‐negative, halophilic bacteria that causes acute gastroenteritis in humans upon consumption of contaminated seafood. The V. para life cycle is facilitated by an array of virulence factors, including two type III secretion systems (T3SS). T3SS‐mediated translocation of effectors into host cells is dependent on delivery of each effector to the base of the T3SS needle by a chaperone. VopR is an effector secreted by the first V. para T3SS (T3SS1) that uses an N‐terminal PIP2 –binding domain (BPD) to localize to the plasma membrane and induces host cell rounding through an unknown catalytic mechanism. We hypothesize that VP1684, encoded by a gene directly upstream of vopR in the V. para genome, is the cognate chaperone of VopR, and that the BPD also constitutes the chaperone‐binding domain. Through affinity column purification coupled with FPLC filtration we demonstrate that VP1684 forms a homodimer in solution much like other T3SS effector chaperones, which corroborates bioinformatics analyses indicating that VP1684 shares conserved biochemical properties with T3SS effector chaperones. We also show that VP1684 forms a stable complex with VopR. Moreover, we reveal through confocal microscopy of HeLa cells infected with V. para that the VopR‐mediated cell rounding phenotype is VP1684‐dependent. Finally, we show via increases in host cell cAMP levels by translocated cyaA‐tagged effectors that the translocation of VopR into the host cell by T3SS1 also depends on the presence of VP1684. Taken together, these data suggest that VP1684 is acting as the previously unidentified cognate chaperone for VopR, and as a necessary factor in effector translocation plays a significant role in V. para pathogenicity.Support or Funding InformationThis work was funded by the HHMI, National Institutes of Health (NIH) grant R01‐AI056404, NIH grant 5T32AI007520‐20, the Welch Foundation grant I‐1561, and Once Upon a Time&[hellip]Foundation. STARS program funding provided by the state of Texas.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neural Stem Cell Fatty Acid Beta‐Oxidation and Autism Spectrum Disease

Inborn errors of metabolism occur with high incidence in human populations, and inborn deficiencies in fatty acid b‐oxidation (FAO) are clinically associated with neurological deficits such as speech weakness, motor delay, and autism. Using in vivo assays for analyzing neural stem cells (NSCs) in the developing mammalian forebrain, we find that neural stem cell (NSC)‐autonomous deficiencies in long‐chain FAO deplete neural stem cell (NSC) pools from mouse embryonic neocortex. Acute interference with activity of TMLHE (catalyzes the first step in carnitine biosynthesis), or with CPT1A (the rate‐limiting enzyme for carnitine‐dependent transport of long‐chain fatty acids into mitochondria), results in impaired NSC self‐renewal. Moreover, dorsal forebrain‐specific eviction of the Cpt1a gene resulted in reduced thickness of the murine neocortex at birth. Importantly, NSC deficits induced by TMLHE insufficiencies are rescued by exogenous carnitine in both cultured explants and by supplementation of the maternal diet. Taken together, the data argue for a direct role for FAO in controlling NSC self‐renewal vs differentiation in mammalian embryonic brain. The data further argue that deranged stem cell homeostasis is a significant mechanism for linking IEMs with human cognitive disorders such as autism. The implications of these findings for new strategies for management of autism risk will be discussed.Support or Funding InformationThis work was supported by the Robert A. Welch Foundation Award BE0017 and grant RO1GM112591 from the National Institute of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel Contribution of Myeloperoxidase‐derived Hypochlorous Acid to 20‐HETE Production Post‐Ischemia

Ischemia‐induced angiogenesis occurs as a mechanism to minimize the tissue damage caused by atherosclerotic diseases but is often insufficient to maintain adequate tissue perfusion and results in critical limb ischemia and amputation. The discovery of novel factors that increase angiogenesis in response to ischemia is therefore of clinical relevance.Recently, we discovered that 20‐hydroxy‐eicosatetraenoic acid (20‐HETE), an arachidonic acid metabolite produced by a cytochrome P450 (CYP) 4A/F ω‐hydroxylases, is a novel contributor of ischemia‐induced angiogenesis based on the following two major findings: 1) pharmacological interference of the CYP4A/20‐HETE axis attenuates ischemic‐induced angiogenesis; and 2) the expression of cyp4a12a and its product, 20‐HETE are both elevated in ischemic tissues of mice models. The resulting increase in 20‐HETE is endothelial in origin. However, the factors that cause endothelial cells (EC) to produce 20‐HETE following ischemia are largely unknown. The aim of these studies was to investigate the mechanism of post‐ischemic endothelial 20‐HETE increases. Inflammatory cells such as macrophages and neutrophils are the first responders to ischemia injury. Thus, we hypothesized that inflammatory cells contribute to post‐ischemic endothelial increases of 20‐HETE.We first examined the potential contribution of inflammatory response to hindlimb ischemia‐induced angiogenesis using immune‐deficient SCID mice. Following a femoral artery ligation and comparing SCID mice to Balb/C control mice, SCID mice exhibited impaired blood perfusion (55 ± 9% vs 89 ± 10%), decreased micro‐vessel counts (capillary:fiber ratio of 1.5 ± .25 vs 2.5 ± 3.75), as well as an inability to produce 20‐HETE (7.5 ± 2.5 vs 29 ± 5 pg/mg of protein).Furthermore, immune‐competent Balb/c mice depleted of macrophages or neutrophils showed that ischemia failed to induce 20‐HETE production in Balb/c depleted of neutrophils (13 ± 1.5 vs 35 ± 5 pg/mg of protein), accompanied with compromised blood perfusion (58 ± 10% vs 82 ± 9%), but not those depleted of macrophages. Neutrophils are known to release Myeloperoxidase (MPO) at sites of inflammation. The possibility that MPO contributes to ischemia‐induced 20‐HETE production was investigated using MPO−/− mice. Remarkably, the ischemic hind limb tissues of MPO−/− mice produce negligible amount of 20‐HETE (~2 ± .25 vs 35 ± 5 pg/mg of protein). MPO is known to produce hypochlorous acid (HOCl). Therefore, we further studied the role of MPO‐derived HOCl in 20‐HETE production in cultured human EC. The addition of physiological amounts of either MPO or HOCl markedly stimulated the expression of 20‐HETE synthase CYP4A11 and production of 20‐HETE (40 ± 12 vs 8 ± 5 pg/mg of protein); potentially via a HIF1‐a dependent mechanism.These observations suggest for the first time that neutrophil‐derived MPO critically contributes to the 20‐HETE increases post‐ischemia via a mechanism involving the generation of HOCl and activation of the HIF pathway in endothelium.Support or Funding InformationAHA [11SDG6870004(AMG), 17GRNT33430003(AMG)]NIH [HL34300(MLS), DK38226(JRF)]RA Welch Foundation [GL625910(JRF)]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Energetics and Interfacial Interactions of Spliceosomal Protein Dib1 Predicted with MD Simulations

The spliceosome is the highly dynamic macromolecular complex responsible for catalyzing the excision of introns from pre‐messenger RNA. The spliceosome consists of five small nuclear ribonucleoproteins as well as several additional proteins. As part of a complex assembly pathway, the essential protein Dib1 must leave the spliceosome. Experimental analysis has found a temperature sensitive phenotype of Dib1 (TS‐Dib1, F85A) that hinders splicing activity. This phenotype has also been hypothesized to perturb spliceosome assembly. Our current work uses atomistic molecular dynamic (MD) simulations to study the molecular interactions in the region of the spliceosome around Dib1 in order to determine what energetic and structural changes result from this point mutation.Starting with recently published cryo‐EM structures, the spliceosome was reduced to only include components in close proximity to Dib1 (either native or F85A). The reduced Dib1 structure consists of three snRNAs (U4, U5, and U6) and four proteins (Prp6, Prp8, Brr2, Prp31). Both systems, containing either native Dib1 or mutant Dib1, were simulated using atomistic MD simulations to identify energetic differences between native Dib1 and mutant TS‐Dib1. The simulation conditions mimicked the biological conditions of previous bulk splicing assays, with an aqueous solvent, 0.1 M salt, and temperature differences that have experimentally shown TS‐Dib1 inactivation. The MD simulations revealed a lower global binding energy in the TS‐Dib1 system compared to the native Dib1 system. Additionally, interfacial hydrogen bonding patterns displayed increased hydrogen bonding among Prp6, Prp8, and Dib1 in the native Dib1 system that were not present in the TS‐Dib1 system, which could contribute to the lower global binding energy. The results from the MD simulations support the experimentally observed temperature sensitivity in the TS‐Dib1 system and could help explain the role of Dib1 in spliceosome assembly.Support or Funding InformationNIH R15GM120720, Robert A Welch Foundation Grant W‐1905This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Kinetic Basis for Improved Specificity of CRISPR/Cas9 High Fidelity Variants

CRISPR/Cas9-mediated is a powerful, programmable genome editing tool that has been widely used for many applications. While engineered Cas9s have been reported to minimize the off-target cleavage efficiency compared with wild type Cas9 in vivo, a rational mechanism for enhanced specificity has not been put forward. Here, we provide kinetic characterization of the Hyper-accurate Cas9 variant (HypaCas9) to provide a basis for improved discrimination against off-target cleavage. We show that chemistry is the rate-limiting step for off-target cleavage in HypaCas9 and is much slower than on-target cleavage in WTCas9, where DNA unwinding is the rate-limiting step. In addition, the intrinsic cleavage rate and non-productive DNA binding state has also greatly been affected in engineered Cas9s. We find that HypaCas9 gains discrimination mainly through slowing down chemistry and rebalancing kinetic partitioning to favor release rather than cleavage of the bound substrate. Support or Funding Information This work was supported in part by the Welch Foundation grants (F-1604) (to K.A.J.) and (F-1938) (to D.W.T.). Free energy profile for WTCas9 and HypaCas9 for on-target and off-target cleavage (A)kobs, HNH cleavage rates for on-target and off-target cleavage by WTCas9 and HypaCas9. (B)The two-steps for R-loop formation rates for on-target and off-target cleavage of WTCas9 and HypaCas9 were estimated by stopped-flow fluorescence measurements. (C)The free energy profile was calculated using transition state theory: ΔG‡ = RT[ln(kT/h) − ln(kobs)] kcal/mol using the equilibrium constants from globally fitting. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Using a Fluorescent Unnatural Amino Acid to Characterize the Role of Conformational Dynamics in High Fidelity DNA Replication

The DNA polymerase of bacteriophage T7 is a model enzyme for understanding high fidelity DNA replication. Crystal structures of the open, binary (E‐DNA) complex and the closed, nucleotide bound ternary (E‐DNA‐dNTP) complex reveal large structural changes as nucleotide binds and the fingers domain closes around the correct base pair. However, the contribution of this structural transition to DNA replication fidelity has been greatly disputed, and its measurement has been further complicated by the difficulty in site specific fluorescent labeling of the T7 DNA polymerase using traditional cysteine – maleimide coupling chemistry. The work presented here determines the contribution of the motions of the fingers domain of the T7 DNA polymerase to its high DNA replication fidelity, through direct measurement using a fluorescent unnatural amino acid incorporated site‐specifically in the fingers domain of the enzyme. Each step in the pathway of correct nucleotide incorporation was measured using stopped flow and quench flow techniques, and data from all experiments were globally fit in Kintek Explorer to derive rate constants for each step in the pathway. Our data support a model where the conformational change preceding chemistry is not rate‐limiting but is still the major determinant of fidelity, and provides new information on post‐chemistry conformational dynamics of the enzyme and their contribution towards DNA replication fidelity.Support or Funding InformationThe Welch Foundation (F‐1684)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Ellis van Creveld protein 1 binds and hydrolyzes GTP to a mixture of GMP and GDP and possesses the characteristics of large GTPases

Mutations in the genes that code for EVC1 and EVC2 proteins cause Ellis van Creveld (EvC) syndrome and its milder form, Weyers acrofacial dysostosis (WAD). EVC1 physically binds to EVC2 and together, they are tethered to the base of the primary cilium by the EFCAB7/IQCE protein complex. This multi‐protein assembly positively regulates the Sonic and Indian hedgehog (Hh) signaling pathways by promoting downstream processes after activation of the receptor, smoothened (smo), by the hedgehog proteins. Bioinformatic analysis revealed the presence of several structural motifs in EVC1 sequences including P‐loop, leucine zipper, transmembrane and nuclear localization signal. Using a fragment of EVC1 from mice and humans that contains P‐loop and leucine zipper regions, we present experimental evidence to show that EVC1 protein possesses a high intrinsic GTPase activity with a kcat of ~10 min−1. In addition, dimerization of EVC1 via its leucine zipper causes the protein to hydrolyze GTP to a mixture of GDP and GMP at unequal ratios. The protein binds GTP at milli to micro molar concentrations, indicating low substrate affinity. These kinetic parameters are similar to those displayed by large GTPases such as Dynamin. We propose that the GTP‐hydrolyzing activity of EVC1 protein is an important mechanistic step in the regulation of the hedgehog signaling pathways.Support or Funding InformationWelch Foundation Grant # AN‐0008This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.