bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Epigenomic Status of the US OEF/OIF War Veterans: A Pilot Clinical Study
Authors: Chakraborty Nabarun , Muhie Seid , Yang Ruoting , Gautam Aarti , Donohue Duncan , Daigel Bernie , Zhang Yuanyang , Amara Duna Abu , Flory Jannie , Yehuda Rachel , Doyle Frank , Hammamieh Rasha , Marmar Charles , Jett Marti
Published: November 2023

Management of post‐traumatic stress disorder (PTSD) is complicated by the overlapping symptoms of its co morbidities and the diagnostic reliance on self‐report and time consuming psychological evaluation. A more comprehensive understanding of molecular pathophysiology of PTSD could facilitate an unbiased biomarker‐driven next‐generation intervention strategy. Herein, we cast light on the epigenomic consequences of combat elicited PTSD. In this study, hypermethylated genes were investigated as to the implications for behavior, immune response, nervous system development, and relevant PTSD co‐morbidities such as cardiac health and diabetes.

52 PTSD‐positive male veterans of US Operation Iraqi Freedom (OIF) and Operation Enduring Freedom (OEF) were matched to 52 controls by age and ethnicity. PTSD diagnosis was determined by a clinician‐administered PTSD scale (CAPS), score >40, while the control group demonstrated a CAPS <10. Methylation status of DNA extracted from whole blood was assayed using high density arrays (Agilent, Inc.). 5,000 probes were statistically differentially methylated (FDR < 0.1), representing approximately 3,600 unique genes.

Chromosome 4 and 18 imprints a significantly large portion of the methylated probes, including those which control emotional and cognition process, and glucocorticoid deficiency. Interestingly, a significant number of genes facilitating telomere maintenance and insulin reception were hypermethylated at both promoter and gene body sites; therefore the DNA methylation status in these genes could be prevailing. Nearly 85% of the differentially methylatedprobes were hypermethylated in PTSD patients. The majority of these probes encode the candidate proteins responsible for transcription regulation and enzymaticactions. Genes involved in memory consolidation, emotion/aggressive behavior, and perturbed circadian rhythm were preferentially hypermethylated. PTSD epigenetically perturbed both the cellular and humoral immune system; in addition the morphologies of two brain regions known to control PTSD symptoms, namely cerebral cortex and hippocampus were perturbed. Genes involved in several PTSD comorbidities, such as cardiomyopathy and poor insulin management, were also hypermethylated. Integration of the epigenomic observations with other omics outcomes is underway, as well as validation of these findings in an independent cohort.
Structural And Functional Analysis Of Acetylcholinesterase Complexes With “In Situ Click‐Chemistry” Inhibitors
Authors: MARCHOT Pascale , SHARPLESS K. Barry , TAYLOR Palmer , BOURNE Yves
Published: November 2023

Ligand binding sites on acetylcholinesterase (AChE) comprise the active center, buried at the base of a deep and narrow gorge lined by aromatic residues, and the peripheral anionic site, located at the gorge entry. These features propelled AChE as an initial reaction vessel suited for in situ click‐chemistry synthesis of high‐affinity syn‐TZ2PA6 and TZ2PA5 inhibitors, forming a central syn‐triazole upon cycloaddition within the gorge from alkyne and azide reactants bound to the active site and peripheral site, respectively [1,2]. Subsequent crystallographic analyses of mouse AChE or its Tyr337Ala mutant in complexes with the syn‐and anti‐TZ2PA6 regioisomers demonstrated that association of the syn product is accompanied by distinctive side chain positions, freezing‐in‐frame a conformation distinct from an unbound state or an anti complex [3,4]. To correlate the inhibitor dimensions with reactivity and explore whether cycloaddition can occur in a crystalline template, we developed crystal‐soaking procedures and solved structures of AChE complexes with the two TZ2PA5 regioisomers and their TZ2 and PA5 precursors [5]. These new structures, complementary to those of the preformed TZ2PP6 complexes, point to yet undescribed motions in the active site and at the gorge mouth associated with TZ2 binding and reveal coordinated side chain motions that guide the enzyme toward a state favoring syn‐triazole formation. We will present the rationale of the work, describe those molecular determinants that dictate, in a rate‐limiting step, proper alignment of the azide and alkyne reactants to form the triazole product within the gorge, and compare our data with those from other in situ click chemistry studies.

Support or Funding Information

Supported by grants RO‐1‐GM18360‐41 and UO1‐NS 058046 (to PT) and by CNRS grants “Action CNRS/USA” and “PICS” (to PM and YB)
Evolution of Phospholipases A2 in Catalysis and Allosteric Regulation by Membranes
Authors: Mouchlis Varnavas D , McCammon J. Andrew , Dennis Edward A
Published: November 2023

Defining the molecular details and consequences of the association of water‐soluble proteins with membranes is fundamental to understanding protein–lipid interactions and membrane functioning.1 Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that comprise the membrane bilayer, provide the ideal system for studying protein–lipidinteractions.2 Our current study focuses on understanding the catalyticcycle of two different recombinant human intracellular PLA2s: the GIVA cPLA2 and GVIA iPLA2, which are responsible forarachidonic acid release for eicosanoid signaling and for membrane phospholipidre modeling, respectively. Molecular dynamics (MD) simulations, guided by hydrogen/deuterium exchange mass spectrometric (DXMS)3 experimental data, were used to show that the channel to the active sites of these PLA2s are opened upon all ostericinteraction of the enzyme surface with the membrane to facilitate entry of the substrate phospholipid. This constitutes the first detailed study describing the binding and the interaction mechanism of intracellular PLA2s with the membrane bilayer as well as how they bind a single phospholipid molecule in the catalytic site. These enzymes are implicated in many diseases, and understanding their detailed mechanism of action will aid in the discovery of new therapeutics.

Support or Funding Information

Acknowledgements

This work was supportedby NIH Grant GM20501
Coevolution and Disease‐Causing Mutations in Glucose‐6‐Phosphate Dehydrogenase (G6PD)
Authors: Cunningham Anna , Colavin Alexandre , Huang KC , Mochly‐Rosen Daria
Published: November 2023

Glucose‐6‐phosphate dehydrogenase (G6PD) is essential for protection against oxidative stress. Over 160 different missense mutations in G6PD reduce the activity and/or stability of the enzyme, causing G6PD deficiency, which results in anemia and neonatal jaundice, and contributes to many other diseases. Severity of G6PD deficiency is categorized into four clinical classes based on G6PD activity in patient blood samples; however, the structural location and class of a mutation do not fully predict its biochemical effects. The goal of this work is to further understand how mutations affect different parameters of G6PD function.

Towards this goal, sequence coevolution analysis (SCA) was used to identify coevolving groups (sectors) of G6PD residues. Previous applications of SCA to protein domains have identified coevolving sectors that control distinct parameters of protein function. Application of a novel SCA algorithm to G6PD identified several sectors that were enriched in disease‐causing mutations. Overall, the sectors select for known G6PD‐deficiency‐causing mutations and exclude predicted non‐phenotypic mutations.

G6PD mutants from several sectors, as well as mutants not associated with any sectors, were biochemically characterized. Principal component analysis of the biochemical data showed that the mutants do not cluster by sector, but do cluster by clinical classification. We conclude that coevolution may be a good predictor of whether a G6PD mutation causes G6PD deficiency, but coevolving sectors do not correlate to specific biochemical parameters of G6PD.

Support or Funding Information

NSF Graduate Research Fellowship Program

Stanford Interdisciplinary Graduate Fellowship (SIGF)

Stanford Child Health Research Institute (CHRI)

NIH
Understanding the Ability of Heavily Contaminated Soil to Sustain Enzyme Function
Authors: LeRoux Danielle
Published: November 2023

Liberty State Park (LSP) in Jersey City, NJ was a flourishing industrial rail yard and as a result has various types of contamination, particularly high heavy metal concentrations. Now, the park sustains a flourishing and diverse ecosystem. LSP has successfully produced the enzymes that catalyze ecological reactions necessary for forest growth. In fact, preliminary data show that the site with the highest heavy metal concentrations at LSP has the highest enzyme activity compared to sites with little to no heavy metal contamination. This presents an interesting anomaly because research typically shows that high concentrations of heavy metals are detrimental to the health of organisms.

This project aims to understand the unprecedented ability of the contaminated soil to foster growth. Three locations were used in our analysis of these characteristics: LSP 14/16 which has the highest concentrations of metals, LSP 43 which has intermediate levels, and Hutcheson Memorial Forest (HMF), located in Franklin Township, NJ was used as the control site with no history of heavy metal contamination. All three sites have the same natural succession.

Properties of the soil such as the exchangeable metal concentrations, phosphatase activity, percent organic matter, and surface area were measured. These characteristics were analyzed with respect to distinct particle sizes: coarse sand (425 – 2000 μm), fine sand (75 – 425 μm), silt (2 – 75 μm) and clay (< 2 μm). Exchangeable metals are the metals that are loosely bound to the soil and the concentrations are determined with an ICP‐MS. Organic matter is essential for the health and productivity of the soil. It is measured by loss‐on‐ignition. Surface area is typically directly correlated with other characteristics and is measured using a particle size analyzer.

Exchangeable metals from this experiment show similar values to previous measurements, but they have decreased over the period of several years. The organic matter is at healthy levels in HMF but is nearly eight times as much in LSP. Finally, the surface area of the soil in HMF is larger than the surface area of the soil in LSP. The results have led us to conclude that the surface area does not play a role in the anomaly and more experiments need to be conducted on the organic matter.
Fluorophore Conjugation to Substrates Affects Lysine Deacetylase Activity and Specificity
Authors: Toro Tasha B , Watt Terry J
Published: November 2023

Analysis of the human proteome has identified thousands of unique protein sequences that contain acetylated lysine residues in vivo. Lysine deacetylases (KDACs) are enzymes that reverse this post‐translational modification, by catalyzing the hydrolysis of ɛ‐N‐acetyllysine residues in proteins via a conserved mechanism. KDAC activity has also been linked to numerous diseases, in particular chronic diseases such as asthma, cancers, muscular disorders, and diabetes. However, details regarding exactly how these enzymes work and KDAC substrate recognition are not well understood. Widely used in vitro systems for studying KDACs involve peptide substrates containing fluorescent dye molecules, such as coumarin. While experimentally convenient for studying enzyme function and testing potential KDAC inhibitors, the impact of these fluorophores on enzyme activity and specificity remain largely unknown. We recently developed a biologically relevant assay that allows us to quantitate deacetylase activity using unlabeled substrates. Using this assay, we determined that the fluorophore enhanced activity with metal‐dependent KDACs and reduced activity with NAD‐dependent KDACs compared to unlabeled substrate sequences. A hydrophobic residue in the C‐terminal position adjacent to the acetyllysine in place of the fluorophore does not mimic the effect of the fluorophore, indicating that the effect is specific to the dye used and is not biologically relevant. Furthermore, the fluorophore does not simply change binding affinity, as we observed changes in both catalytic turnover and substrate specificity. This suggests that the dye molecules affect the KDAC‐substrate interaction in a complex manner, which has significant implications for the interpretation of experiments using fluorophore‐labeled substrates and the biological relevance of results obtained using them.
Expression, Purification and Enzymatic Characterization of Brugia malayi Dihydrofolate Reductase
Authors: Perez‐Abraham Romy
Published: November 2023

Dihydrofolate reductase (DHFR) is an essential enzyme that plays an important role in the production of cofactors that are required in deoxyribonucleic acid (DNA) synthesis. The literature suggests that DHFR is a potential drug target for the elimination of the parasitic worm, Brugia malayi. Brugia malayi is one of the causative parasites of lymphatic filariasis, a globally neglected tropical disease. In this study, we expressed and purified B. malayi DHFR. Expression was carried out in E. coli with a His6‐tagged construct, and purification was achieved through affinity chromatography using LOBSTR E. coli cells. The resulting purified and active enzyme was used for steady‐state kinetics characterization and Michaelis‐Menten inhibition studies. The catalytic activity, kcat, was found to be 1.4 ± 0.1s−1, the Michaelis Menten constant, KM, 14.7 ± 3.6μM for dihydrofolate, and the equilibrium dissociation constant, KD, 22 ±0.01μM. B. malayi DHFR was compared to other DHFR homologs in terms of ligand specificity determining residues; L. major, T. cruzi, T. bruci, and T. gondii exhibited the highest homology. Known DHFR inhibitors of these DHFR homologs were assayed with B. malayi DHFR; IC50 values were determined to be 0.003 ± 0.36 μM for methotrexate, 90.2 ± 7.9 μM for pyrimethamine, 31.8 ± 5.4 μM for trimethoprim, 770.6 ± 534 μM for cycloguanil, >20,000 μM for 2,4‐diaminopyrimidine, and 192.9 ± 88.0 μM for 2,4‐diaminoquinazoline. These results provide the basis for the development of more potent compounds that can inhibit B. malayi DHFR without affecting human DHFR, and help cure lymphatic filariasis.
Dynamic flexibility of p97 AAA ATPase is important for cofactor regulation
Authors: CHOU TSUI‐FEN
Published: November 2023

The p97 protein is a highly conserved, abundant, functionally diverse, and structurally dynamic AAA ATPase. We have previously shown that regulation of p97's ATPase activity by its cofactors p37 and p47 is important for its normal cellular role. The conserved linker residue Leu‐464 has been reported to be critical for degradation of one p97 substrate in cells; structural data suggests that Leu‐464 controls interprotomer transmission. Here, we demonstrate that Leu‐464 controls communication of cofactor binding from the N‐domain to the D1 and D2 ATPase domains by affecting the regulation of three p97 cofactors: p37, p47, and the Npl4‐Ufd1 heterodimer (NU). Leu‐464 mutants abolished p47's ATPase‐inhibitory effect and produced activation at higher p47 concentrations. Both p37‐ and NU‐mediated activation decreased slightly for L464A and L464E, but increased for L464P. Using ND1L protein, we demonstrate that the flexible linker region is essential for the D1 ATPase activity: a point mutation, L464P, reduced ND1L activity to 2%. We demonstrate that all three cofactors decrease the Km of ATP to ND1L. NU inhibited D1 activity (kcat) for WT‐, L464E‐, and L464P‐ND1L proteins. Neither p37 nor p47 affected the kcat values for WT‐ and L464E‐ND1L, but both raised the kcat of L464P‐ND1L (14‐fold and 7‐fold, respectively). This significant increase suggests that p37 or p47 binding induces conformational changes in the linker region to correct the defect caused by the Pro residue. Most importantly, we found that L464E blocked the activation of the disease variant R155H and also completely disrupted the p47‐mediated inhibition of R155H. The linker region not only assists D1 in maintaining a competent conformation, but also transmits the binding effect of p97 cofactors from the N‐domain (44.5 Å away) to the D1 and D2 domains.
Phosphorylation site specificity for 14‐3‐3 protein binding to tyrosine hydroxylase studied by chemical cross‐linking
Authors: Daubner Susan Colette , Vogt Ellysa J , Mallet Sarah
Published: November 2023

Tyrosine hydroxylase (TyrH) is the rate‐limiting enzyme of catecholamine synthesis and is regulated by phosphorylation at multiple serines in its N‐terminal domain. Binding by the chaperone 14‐3‐3 proteins is reported to activate TyrH when phosphorylated at serine19, but conflicting reports cite serine40 phosphorylation as necessary also. There are seven isoforms of rat brain 14‐3‐3 protein. We are studying whether some 14‐3‐3 isoforms have more specificity for TyrH than others; we have started with the zeta and eta isoforms, since zeta is found at high levels in the adrenal gland, and eta is not. We have made TyrHA17R and TyrHA17RS40A variants that can be phosphorylated at both serines (A17R) or only serine19 (A17RS40A) by PKA (native TyrH is phosphorylated at serine40 by PKA). We have made variants of the 14‐3‐3 protein that can be chemically cross‐linked to TyrH: two arginine residues have been mutated to lysine to enhance bisimidate crosslinking (etaR57K/zetaR56K and etaR132K/zetaR127K), and variants with cysteine substitutions to enable crosslinking with bismaleimides (etaG54C/zetaG53C and etaS46C/zetaS45C). The latter are cross‐linked to TyrH variants with cysteines substituted into the N‐terminus at positions 10, 20, 30, 41, and 50. The TyrH variants have been grown in E. coli, purified by traditional methods and are fully active. The etaR132K/zetaR127K and etaS46C/zetaS45C variants have been grown and purified. Preliminary experiments with wild‐type 14‐3‐3 and the 14‐3‐3 variants etaR132K/zetaR127K and dimethylsuberimidate suggest that complexes are made between 14‐3‐3 and TyrH if serine19 is phosphorylated.
Regulation of Tyrosine Hydroxylase Studied by Fluorescence Anistotropy and Fluorescent Resonance Energy Transfer
Authors: Daubner Susan Colette , Teran Leonel , Rodriguez Augustin , Cruz Eduardo
Published: November 2023

Tyrosine hydroxylase (TyrH) is regulated by feedback inhibition and activation by multiple phosphorylation at three different serine residues in its N‐terminal regulatory (R) domain. TyrH phosphorylated at serine19 and 40 may be stabilized by 14‐3‐3 protein binding. We have cloned the rat brain 14‐3‐3 proteins, and have variants of TyrH that allow cAMP‐dependent protein kinase (PKA) to phosphorylate both serine19 and ‐40 residues (TyrHA17R) or just serine19 (TyrHA17R‐S40A). Wild‐type TyrH is phosphorylated only at serine40 by PKA. These variants are fully active and can be fully phosphorylated at the targeted serine residues using PKA. Using fluorescence anisotropy we are studying the interaction between several of the seven isoforms of 14‐3‐3 and TyrH in its different phosphorylation states. After tagging the 14‐3‐3 proteins with DANSYL‐Cl, titration is performed with unlabeled, once‐phosphorylated, and doubly phosphorylated TyrH. This protocol gives increases in anisotropy as complexation occurs. Preliminary experiments including dansylated zeta 14‐3‐3 and TyrHA17RpS19pS40 gave a saturation curve with a Kd value of 3 μM.

To study the movements of the N‐terminal regulatory (R) domain of TyrH we are using Fluorescent Resonance Energy Transfer, FRET, to measure changing distances between the catalytic (C) and R domains. A TyrH variant with all but one tryptophan mutated to phenylalanine, leaving tryptophan233 in the C domain, and all the three exposed cysteine residues mutated to alanine, has been made and called TyrHW2FC3A. Using TyrHW2FC3A as template, a series of variants was made with new cysteine residues in the N‐terminus (Q10C, E20C, T30C, I35C, and A45C). These cysteine residues are tagged with IAEDANS, and energy transfer between tryptophan233 and the new cysteines is measured. These variants have been overexpressed and purified and are active. KM values for tetrahydropterin for the TyrHE20C‐, TyrHT30C‐, and TyrHI35CW2FC3A variants are 60, 90, and 30 μM, comparable to the value for wild‐type TyrH of 40 μM.
Structural Basis for Weight Loss in Patients with Chronic Obstructive Pulmonary Disease: Modeling of Gly80 and Ser80 Variants of Human Group IID Phospholipase A2 and their Receptor Complexes
Authors: Hariprasad Gururao , Ethayathulla Abdul Samarth , Khan Mohd Imran
Published: November 2023

Weight loss is a well known systemic manifestation of Chronic Obstructive Pulmonary Disease. a Gly80Ser mutation on human group IID secretory phospholipase A2 enhances expression of cytokines that are responsible for weight loss. In this study, we seek to establish the structural correlation of wild type and foresaid mutation with its function. Secretory phospholipase A2 with glycine and serine at eightieth positions and M‐type receptor were modeled. The enzymes were docked to the receptor and molecular dynamics was carried out to 40ns. Structural analysis shows the enzymes to be comprising of three helices (HI‐H3), two short helices (SH1 and SH2), five loops including a calcium binding loop (L1‐L5), and stabilized by seven disulfide bonds. The overall back bone folds of the two models are very similar with main chain RMSD of less than 1Å. Active site within the substrate binding channel shows catalytic triad of water‐His67‐ Asp112 which show hydrogen bonded network. The major structural differences between wild and mutant enzymes are: (1) loop‐L3 between H2 and H3 that bears the residue Gly80 in the wild type is flexible, being completely exposed to solvent and forms three hydrogen bonded interactions: (a) main chain nitrogen of Gly80 with hydroxyl group of Tyr86; (b) Oδ2 of Asp84 with amide nitrogen of Tyr82, and (c) polar contact between main chain oxygen atom of Gly80 and water molecule. Whereas, in the mutant enzyme the loop region between residues Ser80 and Arg87 adopts a relatively closed conformation with respect to the channel opening, where it is more rigid and stabilized by five hydrogen bonded interactions and two salt bridges. This along with the loss of hydrogen bond between Ser80 and Tyr86 provides flexibility to the loop; (2) conformation of loop in sPLA2‐Ser80 makes interfacial binding surface more planar thereby increasing compactness of the enzyme as indicated by decrease of volume by 2nm3, and (3) interfacial binding surface contact area and quality of interactions of the mutant enzyme is better with the receptor as compared to the wild type. Therefore, the structural differences delineated in this study are potential biophysical factors that determine the potency of wild and mutant enzymes with macrophage receptors, for respective functions of cytokine release resulting in clinical scenario of cachexia.

Support or Funding Information

This research was supported by Department of Science and Technology, Govt. of India: SR/FT/18.
Structural Insights into Novel Esterase
Authors: RHEE SANGKEE , KIM SANGHOON
Published: November 2023

Chloramphenicol (Cm) and florfenicol (Ff) are broad‐spectrum antibiotics that inhibit protein synthesis. Both antibiotics are a bacteriostat but differ in their chemical structure in that a fluoro group is attached to C3 of Ff. Specifically, both antibiotics prevent a formation of the peptide bond by irreversibly binding to a receptor site on the 50S subunit of the bacterial ribosome. It is well established that chloramphenicol acetyltransferase inactivates Cm by first recognizing the hydroxyl group of C3 in Cm and then specifically acetylating the hydroxyl group. Due to this specificity, Ff cannot serve as a substrate of chloramphenicol acetyltransferase. Interestingly, a novel esterase, which was recently characterized by metagenome screening, inactivates both antibiotics possibly by hydrolysis of those antibiotics, suggesting that this esterase could be a novel enzyme in inactivating both Cm and Ff. Sequence analysis indicates that its sequence is highly similar to those of microbial hormone senstive lipase. Consistent with this comparison, a newly identified esterase contains Ser156 in the GxSxG motif and Asp252 and His282, the catalytic triad conserved in the family of microbial hormone sensitive lipase. In order to understand this novel enzymatic feature, we are carrying out X‐ray crystallographic analysis. Our studies will provide structural insights into antibiotics hydrolysis.

Support or Funding Information

This work was supported by a grant from the Next‐Generation BioGreen 21 Program, Rural Development Administration, KOREA.
Correlating Gene Expression Profiles to Lipid Induction in the Microalga Chlorella protothecoides
Authors: Maggard Rachel Susanne Jolly , Waikel Rebekah L.
Published: November 2023

Microalgae are a promising source for sustainable biofuel production, but large‐scale production can be cost‐prohibitive. An understanding of the relationship between algal genome and metabolic pathways could serve to decrease cost and increase commercial viability. In this study, we examine the relationship between gene expression and lipid induction in Chlorella protothecoides. Using NextGen sequencing, we identified candidate genes, which were differentially expressed in algae grown in nitrogen sufficient (low lipid production) and nitrogen deficient (high lipid production) conditions. Using TaqMan real‐time PCR, differential expression was confirmed and quantified for each candidate gene in algae grown in media of varying nitrogen concentrations, and patterns of expression were defined. Systematic alterations were then made in pH, light, and micronutrients, and new expression profiles were generated. These expression profiles were correlated with cellular concentrations of lipids measured by lipid stain and flow cytometry to establish a distinct gene expression profile for lipid producing C. protothecoides. These expression profiles provide insight into C. protothecoides specific lipid production pathways. In addition, these lipid induction profiles are currently being used to rapidly assess the impact of environmental factors on lipid production days before detectable lipid production would be quantified.

Support or Funding Information

Research Scholars Program, Kentucky NSF EPScOR
Tissue Engineering: Simple Versus Complex Organs
Authors: Taylor Doris
Published: November 2023

To date only “simple” bioengineered tissues have been FDA approved and are currently on the market. These tissues typically lack either vasculature or nerve innervation and are simple tubular or balloon‐like structures. In order to move forward with bioengineered organs, the challenges of developing sustainable vasculature and/or innervation still have to be overcome.

Current success include engineered bladder, trachea, esophagus, and urethra. I will discuss the engineering of these “simpler” organs versus that of a complex tissue such as heart, one of the most complex organs to be bioengineered.

Our approach involves the use of biologic matrices. Cardiac extracellular matrix (ECM) is a complex architecturally‐organized mixture of proteins glycoamino‐, and proteo‐glycans that contains an embedded vascular network. In fact, during cardiac development, cell‐matrix interactions give rise to regional cell fate specifications. We hypothesize that ECM plays a critical role in stem cell fate, localization and function after tissue injury.

We will show that decellularized ECM (dECM) composition and architecture provides physical, mechanical and biochemical cues that direct stem or progenitor cell fate – and that age, sex, and disease state can alter those biologic cues.
Docking into Homology Models in tandem with Structure Activity Relationships: Bradykinin B2 Receptor Complexed with Non‐opioid Dynorphin A Analogues
Authors: Chan Christopher , Lee Yeon Sun , Hruby Victor
Published: November 2023

There have been multiple studies validating the use of homology modeling in tandem with ligand docking software in developing novel drugs for G protein‐coupled receptor (GPCR) systems. However, homology modeling is a viable, albeit less accurate, alternative to crystal structures in binding site determination via in silico systems. Bradykinin B2 receptor (B2R) is implicated, following nerve injury, in promoting neuropathic pain. Until recently, bradykinin and kallidin were thought to be the only endogenous ligands for B2R. Thus, B2R activation by dynorphin A (Dyn A) – despite being structurally dissimilar to bradykinin– during chronic pain has garnered recent interest in developing a drug to modulate hyperalgesia. Coupled with recent structure‐activity relationships (SAR) studies, ligand‐receptor docking computations are refined and restrained to produce a structural model for future drug design. Default settings were used for Glide SP and Glide SP‐Peptide ligand receptor docking near the second extracellular loop (ECL2). We correlate binding affinities of Dyn A analogues to their binding energies (performed by Glide), pose RMSD, and Generally Applicable Replacement rmsD (GARD) scores to determine a homology model of BDKR2. Future progress would involve refining the results found by incorporating known residue interactions with the minimum pharmacophore.
Antiproliferative Effects of Hinute‐AM in Human Breast Cancer Cell Lines
Authors: Mukherjee Shyamali , Mitra Piyali , Ratna Anuradha , Maebuchi Motohiro , Furuta Hitoshi , Das Salil K
Published: November 2023

Breast cancer is a debilitating disease that affects women worldwide. In the United States, breast cancer is the second leading cause of cancer‐related deaths in women. Diet is an important factor in the development of numerous diseases, including breast cancer. We reported earlier that the consumption of soy protein controlled not only the proliferation of dimethylbenz [a] anthracene (DMBA)‐induced breast cancer cells in rats but also its aggressiveness. Soy protein is hydrolyzed in the gut to small peptides which are easily absorbed and are involved in various cellular functions. Fuji Oil Company, Osaka, Japan, has commercially marketed a soy protein hydrolysate, termed Hinute‐AM, composed of primarily di‐ and tripeptides, to be used as a health food. This product was found to have anti‐inflammatory property. It is possible that the antiproliferative effects of soy protein on breast cancer in rats may be mediated by its hydrolysis in the small intestine to small peptides. Hence, we hypothesized that the beneficial effect of soy protein in breast cancer development may be mediated by its hydrolysis product as peptides. Several normal (MCF 10A, 12A, and 184 A1), triple negative (MDA–MB‐231, BT‐549 and HCC‐1806) and steroid receptor positive (T47D and MCH 7) breast cancer cell lines were treated with different concentrations of Hinute‐AM for 24 h. Hinute‐AM at both low and high concentrations did not have any significant effect on proliferation of normal cells. However, while it inhibited proliferation of all breast cancer cell lines at low concentration (0.02–0.10 mg/ml), at high concentration (0.5–2.0 mg/ml), it had no effect. Furthermore, while Hinute‐AM had no effect on the levels of Ki‐67 and cyclin D1 levels in normal cells, it significantly decreased their levels in all breast cancer cell lines. The mechanism by which Hinute‐AM inhibits proliferation of breast cancer cell lines is under investigation.

Support or Funding Information

(Supported by grants from Fuji Oil Company, Osaka, Japan and NIH 5U54MD007593)
Biophysical and Biochemical Analyses of Cellulase‐Polymer Bioconjugates for Biofuel Applications
Authors: Makaroff Katherine , Lucius Melissa , Wright Thai , Stewart Jamie , Williams Cameron , Berberich Jason , Konkolewicz Dominik , Page Richard C
Published: November 2023

In recent years, due to high demand, protein‐polymer conjugates have become a more common and important biomaterial. Through specific derivatization strategies, polymers of different lengths and types are conjugated onto proteins. The conjugation of polymers onto proteins has been observed to stabilize proteins, and shift the optimal temperature and pH of proteins. To meet increasing demands for biofuels, the production of cellulosic ethanol will need to be increased greater than 600‐fold by 2022. This has lead to the call for more stable thermophilic cellulases, which catalyze the conversion of cellulose to glucose, a precursor for ethanol. Reversible addition‐fragmentation chain transfer (RAFT) is a common derivatization strategy used to produce protein polymer conjugates with increased stability. We initially optimized the RAFT technique for production of lysozyme‐polymer bioconjugates. A modified version of RAFT that involves grafting‐to and grafting‐from techniques was found to produce active lysozyme‐polymer bioconjugates with increased stability. The modified approach allows for fine control over the type and number of polymers conjugated onto the protein. The derivatization strategy and conditions used in the conjugation of lysozyme, are now being applied to the thermophilic cellulase FnCel5a. Results of the RAFT‐polymerization strategy with FnCel5a will be discussed with respect to the end goal of producing a cellulase that is more stable under the extreme industrial conditions utilized during biofuel production.
Analysis of Vascular Endothelial Growth Factor Receptor 2 (VEGFR‐2) Glycosylation and its Role in Angiogenesis
Authors: Chandler Kevin B , Khatri Kshitij , Leon Deborah R , Meyer Rosana D , Rahimi Nader , Costello Catherine E
Published: November 2023

Angiogenesis, the formation of new blood vessels from pre‐existing vessels, is required for tumor growth and metastasis. Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is one of the most important receptor tyrosine kinases (RTKs) among the VEGF receptor subfamily, and activation of VEGFR‐2 is essential for tumor angiogenesis. The extracellular domain of VEGFR‐2 contains seven immunoglobulin‐like (Ig) domains, each with multiple potential N‐glycosylation sites, and N‐glycosylation is thought to play a central role in receptor stability, ligand binding and trafficking. However, to date the N‐glycosylation sites and their putative role(s) in VEGFR‐2 function remain largely unknown. The objective of this study is to characterize VEGFR‐2 N‐glycosylation sites using tandem mass spectrometry (MS/MS) and investigate the functional importance of these modifications on VEGFR‐2 angiogenic signaling.

VEGFR‐2 isolated from porcine aortic endothelial cell (PAEC) lysate was immunoprecipitated with a polyclonal anti‐VEGFR2 antibody. Evaluation of the purity and site occupancy of VEGFR‐2 were carried out by gel electrophoresis, followed by protease digestion, PNGaseF/H218O treatment, and MS/MS analysis. MS/MS data were processed using Proteome Discoverer 1.4. To obtain site‐specific glycosylation information we performed proteolysis of VEGFR‐2, glycopeptide enrichment via hydrophilic interaction liquid chromatography (HILIC) and subsequent analysis of glycopeptides via an Agilent 6550 Quadrupole Time‐of‐Flight (Q‐TOF) MS using collision‐induced dissociation.

MS/MS analysis of the VEGFR‐2 tryptic digest achieved 56% sequence coverage. We detected candidate N‐glycosylated peptides in the extracellular domain of VEGFR‐2 after treatment with PNGaseF/H218O, including N98, N160, N247, N297, N521, N578, N702 and N719. MS/MS spectra were used to define the locations of the glycosylation sites. These results are the first evidence for which potential VEGFR‐2 N‐glycosylation sites are occupied by N‐linked glycans. In addition, VEGFR‐2 glycopeptide MS/MS data provide information on the glycoform distributions at each occupied site. All seven VEGFR‐2 immunoglobulin domains contain at least one occupied N‐glycosylation site. Observed glycosylation on Ig domain 2, involved in VEGFR‐2 ligand binding, and on Ig domain 7, involved in receptor dimerization and activation, could have important implications for protein folding, ligand binding and receptor dimerization. These analyses are essential for determination of the role of glycosylation in VEGFR‐2 function and trafficking.

Support or Funding Information

This research is supported by NIH grants P41 GM104603, R01 CA191970, and F32 CA196157.
Functional Reconstitution of P‐glycoprotein into Nanodiscs: Biochemical and Biophysical Evaluation
Authors: Chen Gang , Oliveira Maisa , Wise John G. , Vogel Pia D.
Published: November 2023

Multidrug resistance (MDR) proteins protect normal cells from xenobiotic attack by pumping a large range of toxins out of the cell. Unfortunately, over‐expression in cancer cells increases efflux of therapeutic drugs rendering them ineffective. In order to re‐sensitize multidrug resistance cancers to chemotherapy, we have found inhibitors of P‐gp by in silico screening methods (Brewer, F. K., Follit, C. A., Vogel, P. D., & Wise, J. G. (2014) Molecular Pharmacology, 86(6), 716–726). To further investigate the mechanism underlying the inhibition, we have employed biochemical assays and electron spin resonance spectroscopy (ESR), and have established nanodisc technologies that provide native‐like phospholipid bilayer environments to incorporate and stabilize P‐gp for our drug finding and mechanistic studies. A cysteine‐less mutant of the mouse MDR3 P‐gp (MDR3CL) has been incorporated into nanodiscs and showed several fold enhanced ATPase activities. The stability of P‐gp in nanodiscs was also dramatically increased. Biophysical studies using ESR showed that P‐gp in nanodiscs as compared to micelles retains the maximum ATP binding value of 2.2 mole/mole, while the dissociation constant Kd was decreased to 40 μM. Enzymatic activity assays showed similar inhibition of P‐gp in nanodiscs by potential inhibitors that were identified using P‐gp in micelles. These results indicate that the performance of P‐gp in nanodiscs was superior to that of micelles in terms of activity, stability and ATP binding. These nanodisc technologies are currently being established as a uniform platform to study different mutants of P‐pg including human MDR1, which was previously assumed to be too unstable to be used in these biochemical, biophysical and drug discovery studies.

Support or Funding Information

This work is supported by NIH NIGMS [Grants R15‐GM094771‐01A1 and R15‐GM094771‐02] to PDV and JGW, SMU University Research Council, 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.
Altered Zinc Ion Levels and Transporter Expression Indicate That High Liver Copper Disrupts Zinc Metabolism in the Atp7b−/− Mouse Model of Wilson Disease
Authors: Meacham Kelsey , Burkhead Jason
Published: November 2023

Wilson disease is an autosomal recessive disorder caused by a mutation in the ATP7B gene, which encodes for a copper‐transporting P‐type ATPase. This mutation impacts regulation of copper metabolism primarily in the liver, but in the kidneys and brain as well. Impaired function of the transporter ultimately results in a hyper‐accumulation of copper in these organs with subsequent toxicity. While Wilson disease is traditionally viewed as a copper‐related disorder, significantly elevated hepatic zinc levels (p <0.01) were observed via inductively‐coupled plasma mass spectroscopy in the Atp7b−/− mouse model used to study Wilson disease. In order to determine the mechanisms of this observed hepatic hyperzincemia, relative transcription levels of hepatic specific zinc transporters belonging to the ZIP and ZnT families were observed through the use of RT‐qPCR. The results obtained showed differences in mRNA levels of zinc transporters in the Atp7b−/− mice. Specifically, in 6 week old animals there was an increase in the relative amount of mRNA for the transporters ZIP9 and ZnT10. Further, differences in relative mRNA abundance were observed between 6 week old and greater than 20 week old animals, including decreased expression of ZIP7, ZIP9, ZnT4, ZnT6, and ZnT10 in the greater than 20 week old animals. This suggests there is indeed some connection between transcriptional regulation of hepatic zinc transporters and the abnormal zinc concentrations as a result of elevated hepatic copper levels.

Support or Funding Information

Alaska INBRE: NIH Grant Number P20GM103395; Alaska Heart Institute Fellowship
Hepatic Nuclear Factor 4 Alpha (HNF4α) Regulates Transcriptional Control of Amino Acid Catabolism
Authors: Tobon Sandra , Velazquez‐Villegas Laura A , Ortiz Victor , Torres Nimbe , Tovar Armando R
Published: November 2023

The liver is the main organ involved in the metabolism of amino acids (AA), which are oxidized by amino acid catabolizing enzymes (AACE). There is evidence that peroxisome proliferator‐activated receptor α (PPARα) can modulate hepatic amino acids oxidation, and it function as a physiological switch to preserve body proteins during the shift of the type of energy fuel. PPARα activates fatty acid oxidation and suppresses amino acid oxidation. However, it has been postulated by microarray analysis, that PPARα does not perform this effect directly. Our data suggest that PPARa regulates the expression of genes associated with amino acid oxidation via HNF4α. Therefore, the aim of the study was to demonstrate that HNF4α regulates the transcription of amino acid‐catabolizing enzymes (AACE) genes using the enzyme serine dehydratase (SDH) as a model of study.

Database MatInspector and Promoter database were consulted to analyze the potential HNF4α elements in the promoter sequences of the AACE including serine dehydratase. The analysis was done with a core of similarity above 0.7. The functionality of the putative response element in the promoter of serine dehydratase was tested overexpressing HNF4α and using the luciferase gene reporter assay. Also the silencing of HNF4α using shRNA HNF4α was assessed. Additionally, the binding specificity of the response element HNF4α was examined with electrophoretic mobility shift assays (EMSA) and Chromatin Inmunoprecipitation (ChIP).

After in silico analysis of the promoter sequences for the enzymes of amino acid catabolism, it was found that over 70% of the analyzed potential sites for enzymes HNF4α had a core of similarity higher than 0.8. In the SDH promoter, the potential HNF4α site turned out to be functionally active as detected with the luciferase reporter assays. The presence of specific binding of HNF4α to the response element of the SDH promoter was corroborated by EMSA and ChIP indicating the HNF4a is capable to activate SDH transcription. Finally, with the silencing of HNF4a it was possible to demonstrate the importance of this nuclear receptor in the regulatory mechanism of gene transcription of SDH, since the expression of SDH only reached basal levels.

The results indicated that HNF4α upregulates the transcription of SDH. Additionally, in silico analysis revealed the presence of HNF4α‐response element in promoter of enzymes involved in amino acid catabolism, suggesting that this transcription factor could function as a global regulator of the amino acid catabolism.

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CONACYT (grant ART)
Inhibition of Angiotensin Converting Enzyme Induces Beige Adipocytes through Angiotensin‐(1–7) in 3T1‐L1 Adipocytes and Obese Mice
Authors: Vargas‐Castillo Ariana E , Del Valle‐Mondragon Leonardo , Torres Nimbe , Hong Enrique , Tovar Armando R
Published: November 2023

The renin angiotensin system (RAS) has recently been recognized as an important factor in the regulation of energy balance. Adipocytes express all the components of renin angiotensin system (RAS), including Angiotensin‐(1–7) [Ang‐(1–7)] and Mas receptors. Ang‐(1–7), through Mas receptors, induces PPARy expression in 3T3‐L1 adipocytes and releases nitric oxide (NO) in diverse cells. White adipose tissue differentiates to beige adipose tissue through PPARy and NO stimulates mitochondrial biogenesis in brown adipose tissue (BAT). In recent years, beige adipocytes have been found, and they have some similar characteristics of BAT including a high thermogenic capacity due to a higher content of mitochondria and uncoupling protein 1 (UCP1). An increase of beige adipocytes has been associated with beneficial effects in health preventing obesity. The aim of this work is to determine if Ang‐(1–7) induces beige adipocytes in 3T3‐L1 cells and in high fat diet (HFD) mice. Captopril was used as a pharmacological strategy to increase Ang‐(1–7) concentrations. 3T3.L1 adipocytes were incubated with 3.16 μM of captopril during the maturation phase and then brown‐like genes and beige genes were measured by real‐time PCR. C57BL/6 mice were fed with a HFD for 24 weeks and captopril was administrated during the last four weeks in drinking water (5 mg/kg/day). Body weight, body composition, glucose tolerance test, cold resistance and energy expenditure were measured. Plasma Ang‐(1–7) was quantified with capilar electrophoresis and nitric oxide thorugh Griess reaction. Captopril increased the expression of UCP1 and beige marker genes in 3T3‐L1 adipocytes. In obese HFD‐mice, treatment with captopril reduced weight gain and decreased fat mass continuosly during the four weeks, although food intake was reduced only the first week, there was no change in remaining weeks. Moreover, HFD captopril‐treated group had improved glucose tolerance, increased energy expenditure and had an increase resistance to cold temperatures compared with the HFD group. On the other hand, HFD captopril‐treated group had increased plasma concentrations of Ang(1–7) and nitric oxide. In conclution, Ang‐(1–7) may be a new factor that induces browning process and partly explains the beneficial effects of ACE inhibitors in glucose and energy homeostasis.

Support or Funding Information

CONACYT