bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolutionary and Comparative Genome Analysis of Phage NAD Metabolic Genes
Authors: Sorci Leonardo , Kazanov Marat D , Gerdes Svetlana
Published: November 2023

Bacteriophages typically carry an essential core of genes that enables their infection and replication. In addition to this “core” genome, they may carry auxiliary metabolic genes (AMG) which presumably provide a selective benefit to the phages’ biology, ultimately affecting their evolutionary success. The actual roles of these accessory phage genes, which are mostly acquired from host genomes, are not always well understood. A thorough comparative genomic analysis of completely sequenced viral genomes revealed that a statistically significant subset of T4‐like phages carry two genes, i.e. nadV and nadM that implement a two‐step synthesis of nicotinamide adenine dinucleotide (NAD) from Vitamin B3. NAD is an essential coenzyme that plays important roles in metabolic reactions and cell regulation in all organisms. Interestingly, the genomic reconstruction of NAD metabolic subsystem of the corresponding bacterial hosts highlighted that the infected bacteria use alternative routes, i.e., a different set of genes, for synthesizing NAD. This evidence suggests that NadV‐NadM metabolic shunt has been retained by phages in order to provide some sort of fitness to their biology. What could it be? This enhanced NAD synthesis in phage‐infected cells could be rationalized by the occurrence of phage‐encoded NAD‐consuming activities, such as those catalyzed by RNA polymerase‐ADP‐ribosyltransferase (gene alt), NAD‐dependent DNA ligases (gene ligA), and NAD‐dependent deacetylase (gene cobB) which often co‐occurr with nadM and nadV in T4‐like phage genomes. Sequence analysis and construction of 3D models of viral NadM and NadV indicated that all the key residues responsible for their enzymatic activities are present, and are properly oriented in the active sites. To conclude, we performed a phylogenetic study on the NadM and NadV family of enzymes from Bacteria, Archaea, Viruses, including the eukaryotic counterparts. The results offer a complex evolutionary scenario whereby some bacteriophages acquired the genetic capability to synthesize NAD, and also played a role in the spread of these functional roles across different organisms through horizontal gene transfer events.
GRNmap and GRNsight: open source software for dynamical systems modeling and visualization of medium‐scale gene regulatory networks
Authors: Dahlquist Kam D. , Fitzpatrick Ben G. , Dionisio John David N. , Anguiano Nicole A. , Carrillo Juan S. , Hong Monica V. , Horstmann Kristen M. , Jackson Kayla C. , Johnson K. Grace , Morris Tessa A. , Roque Trixie Anne M. , Samdarshi Mihir , Varshneya Anindita , Williams Natalie E. , Wyllie Kevin W.
Published: November 2023

A gene regulatory network (GRN) consists of genes, transcription factors, and the regulatory connections between them that govern the level of expression of mRNA and proteins from those genes. Our group has developed a MATLAB software package, called GRNmap, that uses ordinary differential equations to model the dynamics of medium‐scale GRNs. The program uses a penalized least squares approach (Dahlquist et al. 2015, DOI: 10.1007/s11538‐015‐0092‐6) to estimate production rates, expression thresholds, and regulatory weights for each transcription factor in the network based on gene expression data, and then performs a forward simulation of the dynamics of the network. GRNmap has options for using a sigmoidal or Michaelis‐Menten production function. Parameters for a series of related networks, ranging in size from 15 to 35 genes, were optimized against DNA microarray data measuring the transcriptional response to cold shock in wild type and five strains individually deleted for the transcription factors, Cin5, Gln3, Hap4, Hmo1, Zap1, of budding yeast, Saccharomyces cerevisiae BY4741. Model predictions fit the experimental data well, within the 95% confidence interval. Open source code and a compiled executable that can run without a MATLAB license are available from . GRNsight is an open source web application for visualizing such models of gene regulatory networks. GRNsight accepts GRNmap‐ or user‐generated spreadsheets containing an adjacency matrix representation of the GRN and automatically lays out the graph of the GRN model. The application colors the edges and adjusts their thicknesses based on the sign (activation or repression) and the strength (magnitude) of the regulatory relationship, respectively. Users can then modify the graph to define the best visual layout for the network. The GRNsight open source code and application are available from .

Support or Funding Information

This work was partially supported by NSF award 0921038 (K.D.D., B.G.F.), the Kadner‐Pitts Research Grant (K.D.D., M.V.H., K.G.J., N.E.W.) the Loyola Marymount University Summer Undergraduate Research Program 2015 (K.W.W., A.V.), the Loyola Marymount University Rains Research Assistant Program (N.A.A., T.A.M.), and an Loyola Marymount University Honors Summer Research Fellowship (M.V.H., K.G.J., N.E.W.).
Arc fluorescence in situ hybridization in cleared whole brains utilizing hybridization chain reaction amplification
Authors: Nguyen Minhkhoi , Chawla Monica K , Barnes Carol A
Published: November 2023

Fluorescence in situ hybridization (FISH) has been used to track the localization of Arc transcripts as a molecular method for quantifying behavior‐driven activity in the hippocampus (Guzowski et al., 1999). But with the development of several tissue clearing techniques like CUBIC (Susaki et al., 2014), CLARITY (Chung et al., 2013), and passive‐CLARITY (Tomer et al., 2014) the opportunity to use Arc FISH on transparent brains to map out intact neural networks is now feasible. Convention has been to use the full length Arc riboprobe (~3.1 kb; Lyford et al., 1995) for FISH in 20 micron thick slices. However, in whole cleared brains, the full length Arc cRNA probe cannot easily penetrate the brain due to its thickness. We sought a solution to the issue of penetration with hybridization chain reaction (HCR) (Choi et al., 2014). HCR is a technique that utilizes DNA probes instead of RNA riboprobes. Since DNA probes are inherently more stable, and HCR probes are less than 150 bases in length, this should drastically improve penetration. In addition, HCR fluorescence hairpins self‐polymerize off of the DNA probe and thus, amplify the fluorescence signal. F344 rats were given maximal electroconvulsive seizure (MECS) to induce transcription, anesthetized, and decapitated with a rodent guillotine. The brains were extracted, fixed in paraformaldehyde, and cleared using the CUBIC reagent (containing aminoalcohols) for 2–3 weeks. Using HCR amplified fluorescence in situ hybridization, we expect to visualize Arc positive cells in rat hippocampus using an advanced intravital multi‐photon microscope.

Support or Funding Information

McKnight Brain Research Foundation; NIH Grant AG048907
Easier elution of urinary proteins preserved on nitrocellulose membrane with heating
Authors: QIN WEIWEI
Published: November 2023

Biomarker is measurable changes associated with the disease. Urine collects changes of the body and it is a better source for biomarker research than blood which is kept almost constant by homeostatic mechanisms of the body. Urimem, adsorbing urinary proteins on a piece of membrane which can be dried and stored in a vacuum bag, was proposed in the lab. In general, this approach is simple, economical, environmentally friendly and space‐saving, which enables comprehensive and systematic collection of urine from all people. It can potentially be an important part of the medical record. The preservation of urinary proteins on a membrane plays a vital role in searching biomarker in urine, and the efficient elution of proteins preserved on the membrane help to lower the barrier for more applications. The original elution procedure relied mainly on vortexing for complete dissolve of nitrocellulose in organic solvent. In current procedure, we raised the temperature for easier nitrocellulose dissolve, and then we precipitated proteins with gentle rotating to keep nitrocellulose dissolved during the procedure. SDS‐PAGE and LC‐MS/MS analysis showed there was no degradation of proteins prepared by heat‐and‐elute procedure. Its overlapping rate of the proteins prepared in separate experiments was 92.6%. By semi‐quantitative spectral counting the quantities of 85.2% of identified proteins’ CV values were within 20%. And the heating elution procedure was much simpler and more efficient than the previous one. It helps more researchers to adopt the membrane preservation of urinary proteins.

Support or Funding Information

National Basic Research Program of China (973 Program)(Nos. 2012CB517605, 2013CB530805); 111 Project (No. B08007).
Modification of Urinary Metabolome in Young Women after Cranberry Juice Consumption Were Revealed Using UHPLC‐Q‐Orbitrap‐HRMS‐Based Global Metabolomics Approach
Authors: Liu Haiyan , Garrett Timothy J , Su Zhihua , Khoo Christina , Gu Liwei
Published: November 2023

The objective of this study was to investigate urinary metabolome modifications in young women following cranberry juice consumption using a global UHPLC‐Q‐Orbitrap‐HRMS approach. Seventeen female college students, between 21–29 years old, were given either cranberry juice or apple juice for three days using a cross‐over design. Urine samples were collected before and after juice consumption. Metabolome in urine were analyzed using UHPLC‐Q‐Orbitrap‐HRMS‐based metabolomics followed by multilevel partial least squares‐discriminant analyses (PLS‐DA). Variable importance projection (VIP) plot was used to identify discriminant metabolites. Validated multilevel PLS‐DA analyses showed that urinary metabolome in young women were altered following cranberry juice consumption. Compared to baseline condition or after apple juice consumption, cranberry juice consumption caused a greater urinary excretion of both exogenous and endogenous metabolites. Tentatively identified exogenous metabolites included quinic acid, phenol, catechol, catechol sulfate, methylcatechol, 2‐methoxycatechol‐O‐sulfate, diphenyl glucuronide, benzoyl glucuronide, 5‐(dihydroxyphenyl)‐ϒ‐valerolactone, 3, 4‐dihydroxyphenylacetic acid, 3‐(hydroxyphenyl) propionic acid, coumaric acid and caffeic acid‐O‐sulfate. Modifications of endogenous metabolites after cranberry juice consumption included increases in homocitric acid, hippuric acid, phenylglycine, 4‐hydroxyphenylacetylglycine, N‐acetyl‐L‐glutamate 5‐semialdehyde and a decrease of oxalosuccinic acid. These metabolites may serve as biomarkers of cranberry juice consumption and explain its health benefits against urinary tract infections.
A Metabolomics Investigation of the Physiological Component of the Innate Immune Response in RAW264.7 Macrophages Treated with Nitric Oxide Inhibitors
Authors: May Amanda , Wepy James , Marnett Lawrence
Published: November 2023

Ultra performance liquid chromatography‐high resolution mass spectrometry (UPLC‐HRMS) based metabolomics is capable of providing a global snapshot of alterations in the intracellular small molecule content (the metabolome) that can be used to probe the temporal changes in metabolism in response to perturbations. Using untargeted UPLC‐HRMS metabolomics (Anal. Chem., 2010, 82 (8), pp 3212–3221), the metabolome of RAW264.7 macrophages treated with Kdo‐Lipid2 (KLA), a chemically defined lipopolysaccharide, was measured and compared to a control metabolome from untreated cells. Initial biochemical studies were used to supplement the metabolomics data and have shown that treatment of RAW264.7 macrophages with KLA induces nitric oxide synthase (iNOS), which leads to the production of nitric oxide (NO) through the conversion of arginine to citrulline. While arginine concentrations remained unchanged in the two populations, other amino acids involved in nitrogen metabolism (glutamine and glutamate) and oxidative stress (glutathione), show decreases in pool size when cultures are treated with KLA after 8 hours. Metabolites involved in arginine and proline metabolism, the metabolic pathway encompassing NO synthesis, also show a decrease in fold change at 8 h. These are proline, sarcosine, L‐argininosuccinate, and aspartate. Macrophages were also co‐incubated with KLA and either a broad NOS inhibitor, L‐NAME (Methods Enzymol., 1996, 268, pp 375–392), or selective iNOS inhibitor 1400W (J Biol Chem., 1997, 272, pp 4959–4963), and the metabolomes and NO production from these cells were recorded. Global alterations in metabolism were noted, and the NO concentrations were diminished at 24 hours, as NO was not detected prior to then, when incubated with either inhibitor. The iNOS activity was also indirectly monitored by measuring citrulline via liquid chromatography‐tandem mass spectrometry. This concentration of this metabolite increased between the KLA treated and untreated populations, while the addition of each inhibitor subsequently diminished the increase at 24 hours. Herein, the temporal metabolic response of RAW264.7 macrophages either treated with KLA or not over 24 hours is reported to reveal differences in metabolism for cells that are experiencing an induction of the inflammatory response via KLA.

Support or Funding Information

Authors would like to acknowledge funding of this research through the NIEHS (2T32ES007028–40).
Identification of Apoptosis Inducing Signaling Pathways and Characterization of Sclerotium rolfsii Lectin (SRL) Binding Proteins from Human Colon Epithelial Cancer HT29 cells
Authors: Guha Nilanjan , Gudihal Ravindra , Barkeer Sreekanth , Deepak SA , Padmanaban Arunkumar , Inamdar Shashikala
Published: November 2023

Sclerotium rolfsii lectin (SRL) is isolated from fungus Sclerotium rolfsii and exhibits strong receptor mediated anti‐proliferative activity towards human colon, breast and ovarian cancer cells by inducing apoptosis. In this study, we investigated the signaling pathways that lead to apoptosis by combining transcriptomics data from gene expression and miRNA microarray analysis data and performed pathway analysis using features of GeneSpring 13.1 (GS 13.1). We also identified SRL binding membrane proteins from HT29 cells using proteomics experiments.

It was found that SRL treatment results in altered expression of several hundred molecules including MAPK and c‐JUN‐associated, apoptosis‐associated, cell cycle and DNA replication‐associated signaling molecules. Pathway analysis using GS 13.1 revealed that SRL treatment induces changes of MAPK and c‐JUN associated signaling pathways as early as 2h while changes of cell cycle, DNA replication and apoptosis pathways were significantly affected after 24h. A significant change of miRNA expression was also observed after 12h SRL treatment. These changes were further validated by qRT‐PCR and immunoblotting. The study suggests that the presence of SRL affects multiple signaling pathways in cancer cells with early effects on cell proliferation pathways associated with MAPK and c‐JUN, followed by miRNA‐associated cell activity and apoptosis.

Membrane proteins from HT29 cells were isolated by phase separation and purified by affinity chromatography using SRL‐Sepharose‐4B matrix. Affinity purified proteins were subjected to in‐gel and in‐solution trypsin digestion, analyzed by ESI‐Q‐TOF LC‐MS and Agilent Spectrum Mill software. Considering the specificity of SRL towards O‐glycans, the presence of O‐GalNAc sites in SRL interacting proteins were tested using NetOGlyc software. Western blotting was performed to validate the MS identified proteins. A major protein band around 25kDa following in‐gel trypsin digestion was identified as Keratin 1 by MS. In‐solution trypsin digestion followed by MS identified 25 SRL interacting proteins namely, keratins, heat shock proteins, tubulins, pyruvate kinase M2, peroxiredoxin‐1, ATP synthase subunit alpha, actin, annexin‐A2, prohibitin, ADP/ATP translocase‐2 and alpha enolase. NetOGlyc software analysis revealed 21 proteins positive for O‐glycosylation sites including keratins containing 27 to 50 O‐GalNAc sites. Keratin 1 identified and validated by western blotting as major SRL interacting protein showed 49 O‐GalNAc sites. Proteins identified in our proteomics studies contain O‐GalNAc sites and are known to be involved in cell survival, apoptosis and tumorigenesis. These findings demonstrate the signature profiles of SRL ‐induced gene expression which helps in understanding the lectin's mechanism of action.
Genomics of Gastrointestinal Disorders: a Familial Based Approach
Authors: Coble Joel L. , Sheldon Kathryn , Yue Feng , Schneper Lisa , Choi Christine , Poirier Kendra , Salameh Tarik , Harris Leonard , Deiling Sue , Ruggiero Francesca , Broach James R. , Koltun Walter A. , Gerhard Glenn S.
Published: November 2023

Background

Inflammatory Bowel Disease (IBD) and Diverticular Disease (DD) are both gastrointestinal disorders known to have a genetic component. IBD comprises both Ulcerative Colitis (UC) and Crohn's Disease (CD). The role of genetics is suggested to be 40–53% for DD and 75% for CD. However, less than 20% of the heritability of CD is accounted for by the known genomic loci. No causative genes have yet been implicated for DD. The purpose of our study is to identify novel genetic contributors to these diseases.

Methods

Using exome sequencing and 2 DD and 1 CD affected families with strong phenotypes, we identified missense single nucleotide variants (SNVs) whose segregation matched disease presentation in the families. The SNVs lie within genes that therefore may contribute to the pathogenesis of each disease. Targeted sequencing of those genes in non‐familial but affected (sporadic) patients was performed to identify any patients with other SNVs in those genes that also have disease. To assess the functional effect of these SNVs, immunofluorescence was performed to identify changes in protein localization and abundance.

Results

We found two rare missense variants in the genes for basement membrane proteins, LAMB4 and COL1A1, which each segregated with disease in separate DD affected families. Decreased expression of LAMB4 was observed in the myenteric plexus of patients with DD and variants in LAMB4. This decreased expression may affect the development of the enteric nervous system, which can contribute to the pathogenesis of DD. We observed increased COL1A1 protein in the submucosa of patients with DD and with variants in COL1A1. This increased expression confirms the increased collagen expression observed in DD. Altered plasticity of the submucosa may contribute to the development of DD. We identified multiple missense SNVs that matched the pattern of inheritance of the CD affected family and collectively may contribute to the development of CD. These include NOD2 interacting protein DDX58, a regulator of autophagy ATG4B, and a member of a protein family previously associated with CD, ADAMTS16.

Conclusion

We have identified additional SNVs and genes that may account for some of the missing heritability in CD. Additionally, we have identified the first genes to be associated with DD, LAMB4 and COL1A1. Further research is needed to elucidate the genomic and molecular pathogenesis of these diseases.
Cytotoxicity of Calpain Inhibitor KR‐180
Authors: Alzahrani Roba , Baashirah Azzah , Myles Ebert , Donkor Isaac , Boadi William , Wang Xiaofei
Published: November 2023

Calpains are calcium dependent, non‐lysosomal cysteine proteases. They are expressed ubiquitously in mammals and many other organisms. Excessive activities of calpain have been associated with various diseases, including Alzheimer's disease (AD), cancer, neurological injury, ischemia/reperfusion injury, atherosclerosis, and diabetes and cataract formation. Thus calpain inhibitors are used to treat these diseases. A newly synthesized proprietary compound KR‐180 was investigated for its cytotoxicity in this preliminary study. We tested cell viability after treatment of cells for 24 h with KR‐180 at 1 mM 100 μM, 10 uM, 1 uM, 100 nM, 10 nM. Cell viability was measured by the alamarBlue colorimetric assay. For 3T3‐L1 cells, the KR‐180 treatment at 1 mM reduced cell viability by 80%, while treatment at lower KR‐180 concentrations did not affect 3T3‐L1 viability. The similar effect was observed for lung cancer cell line (A549) where a significant reduction (about 40%) in cell viability was seen at the 1 mM dose. For colon cancer cell line (SW620) and prostate cancer cell line (PC3), all tested KR‐180 concentrations did not cause any significant reduction in viability. In summary, preliminary results suggest that the compound has low cytotoxicity, since it could cause cell death at high concentration 100 μM or higher.
Ramachandran outliers in ligand‐induced protein structural changes
Authors: West Korbin H.J. , Novak Walter R.P.
Published: November 2023

Ramachandran outliers are amino acid residues that have unfavorable dihedral angles in proteins. Often found in protein structure analysis, the presence of these outliers may be simply errors in the structure; however, other times these outliers existence is supported by structural data. There has been little investigation into the role that these energetically unfavorable outliers have in protein structure. Of special interest is how ligand binding may affect protein structures with respect to Ramachandran outliers. These structural changes may drastically alter the properties of proteins: ligand affinities, protein‐protein interactions, or activity levels. In this study, we survey the Ramachandran outliers in pairs of proteins in the ligand‐bound and unliganded states available in the Protein Structural Change Database (http://idp1.force.cs.is.nagoya‐u.ac.jp/pscdb/). We also examine whether these outliers are supported by the structural data using PDB_REDO (http://xtal.nki.nl/PDB_REDO/).

Support or Funding Information

This research is supported by the Wabash College Haines Biochemistry Fund.
Conservative Substitutions of Buried Amino Acid Residues Distant from the Active Site of Horse Liver Alcohol Dehydrogenase Have Small Effects on Structure, Catalysis and Dynamics
Authors: Plapp Bryce V. , Shanmuganatham Karthik , Wallace Rachel S. , Lee Ann Ting‐I.
Published: November 2023

Global protein dynamics are proposed to participate in enzyme catalysis. Conformational changes and proton transfers, on a relatively slow (~ms) time scale, can be important to establish the scaffold for preorganizing the enzyme‐substrate complex. The present study tests the hypothesis that global dynamics, on the fast time (~ps) scale, can affect protein promoting vibrations and the reorganization involved in the hydride transfer step of alcohol dehydrogenase. The rationale is that distal substitutions of buried, conserved amino acid residues should alter the stability of the protein and affect the fast global dynamics if they are coupled to the chemistry of catalysis. The fast dynamics can be reflected in the X‐ray crystallographic temperature factors. A prerequisite for the present test is that the global structure of the substituted enzymes should not be altered, because allosteric effects could change the structure of the active site. Five different amino acid residues that are buried in hydrophobic clusters and the hinge region around which the global conformation changes when coenzyme binds were substituted as a result of partially random site‐directed mutagenesis. The proteins were purified and the structures were determined by X‐ray crystallography at high resolution. The G173A; V197I; I220V, L or F; V222I; and F322L enzymes have almost identical three‐dimensional structures, except for local perturbations at the site of substitution, as compared to wild‐type enzyme. These enzymes have very similar kinetic constants for oxidation of benzyl alcohol and reduction of benzaldehyde. The random mutagenesis also led to other single substitutions of these amino acid residues (G173V, E, K, or R; V197G, S, or T; I220G, S, T, or N; and V222G, S, or T), but expression of these proteins was too poor to allow biochemical studies. The F322I or F322V enzymes were poorly expressed. It appears that the five residues are in critical locations because the non‐conservative substitutions can affect protein stability. We conclude that the enzyme can tolerate conservative, distal substitutions that do not affect the scaffold of the protein. Furthermore, it appears that fast, global dynamics do not participate significantly in catalysis. In contrast, similar studies of enzymes with substitutions of amino acid residues in the active site can have significant effects on catalysis, either by altering reorganization or local dynamics.

Support or Funding Information

Supported by NIH grant GM078446
Transition Metal Cation Inhibition of Mycobacterium tuberculosis Esterase Rv0045c
Authors: Lancaster Benjamin , Hoops Geoffrey , Johnson R. Jeremy
Published: November 2023

The survival of Mycobacterium tuberculosis within its human host is highly dependent upon the lipid metabolism performed by the numerous serine hydrolase enzymes at the bacterium's disposal. Among these serine hydrolases, Rv0045c is a mycobacterial‐specific serine hydrolase that has been characterized for global substrate specificity by scanning the hydrolase against a library of fluorogenic hydrolase substrates. Rv0045c expresses strong selectivity for short, straight‐chained alkyl esters, but the relative enzyme kinetic rate is slow in comparison to a majority of bacterial hydrolases. Herein, we determined the transition metal ion dependence of Rv0045c on hydrolysis of the four‐carbon methoxyacetic fluorogenic substrate. The catalytic activity of Rv0045c was significantly inhibited by low concentrations of Ni+2, Cu+2, and Zn+2 metal cations while other 3d metal cations displayed no effects on the activity of the hydrolase. The low inhibition constants for these three metals on Rv0045c suggest a likely physiological relevance for the metal dependent inhibition of Rv0045c.

Support or Funding Information

Funding provided by a Butler University Holcomb research award and a Senior Research Grant from the Indiana Academy of Sciences.
Impact of a KDAC4 Active Site Residue on Activity and Specificity
Authors: Bryant Jenae R , Toro Tasha B , Watt Terry J
Published: November 2023

Acetylation of lysine is a common post‐translational modification to proteins. Lysine deacetylases (KDACs) react with acetylated lysines, catalyzing the hydrolysis of ɛ‐N‐acetyl lysine to generate an unmodified lysine residue and acetate. Perturbations in this system have been linked to specific diseases, including cancer. Thus, determining the mechanism of KDAC function and substrate specificity could ultimately lead to new therapeutics for these diseases. Recent studies suggest that a semi‐conserved active site residue appears to be important for enzymatic activity. We are evaluating the activity of several KDAC4 variants to determine how the active site residue affects enzyme function and substrate specificity. In this study, KDAC4 variants were expressed and tested for activity against a panel of peptide substrates. A gain‐of‐function variant had a specificity profile that was distinct from wild‐type KDAC4 and from other KDACs. The identified peptide substrates are the first biologically relevant substrates to have demonstrated reactivity with any KDAC4 variant, and provide insight into the substrate specificity and function of KDAC4.
Biochemical characterization of two evolutionary distant ten‐eleven translocation enzymes and their utility in 5‐methylcytosine sequencing in the genomes at single‐base resolution
Authors: Saleh Lana , Tamanaha Esta , Vaisvila Romualdas , Pais June E. , Dai Nan , Guan Shengxi , Corrêa Ivan R , Zheng Yu
Published: November 2023

The ten‐eleven translocation (TET) enzymes iteratively oxidize 5‐methylcytosine (5mC) on DNA to 5‐hydroxymethylcytosine, 5‐formylcytosine, and 5‐carboxycytosine. Here, we examine the in vitro biochemical activity of two evolutionary distant TETs, mTET1 from mouse and NgTET1 from the single‐celled protist Naegleria gruberi. We show that both of these enzymes are 5‐methylpyrimidine oxygenases with activity on both 5mC (major activity) and thymidine (T) (minor activity) and preference to 5mCpG and TpG dinucleotide sites. Intriguingly, NgTET1 displays higher T oxidation activity in vitro than mTET1 supporting a closer evolutionary relationship between NgTET1 and the base J binding proteins from trypanosomes. In fact, unprecedented evidence for the formation of two new bases, 5‐formyluridine and 5‐carboxyuridine, is shown for NgTET1 activity in vitro. Mutagenesis studies performed in NgTET1 reveal a delicate balance between choice of 5mC or T as the preferred substrate. Steady‐state kinetic analysis show that both mTET1 and NgTET1 are distributive in their oxidative chemistry with each oxidized species released from the enzyme upon formation. Furthermore, both enzymes are physically distributive in the recognition of their substrates on template DNA with no apparent preference to a specific 5mC oxidation intermediate. These data indicate a role for the TET enzymes in the maintenance of the three oxidized forms of 5mC and support the in vivo data implicating that these bases are not simply intermediates in a methylation cycle but represent additional epigenetic states in genomic DNA with regulatory functions to be explored. Finally, we demonstrate the utility of both enzymes in 5mC sequencing technologies and present examples for the mapping of 5mC in different genomes at single base resolution.
The role of Aspartate 141 in the regulation of the Agrobacterium tumefaciens ADP‐Glc Pyrophosphorylase
Authors: Patel Hiralben , Gonzalez‐Martin Laura , Renta Vincent , Ballicora Miguel
Published: November 2023

The main regulatory step of the glycogen and starch metabolic pathways takes place at the level of the synthesis of ADP‐glucose (ADP‐Glc), which is the first committed step and it is catalyzed by ADP‐Glc Pyrophosphorylase (ADP‐Glc PPase). This is an allosterically regulated enzyme, which catalyzes the conversion of ATP and glucose‐1‐phosphate to ADP‐Glc and inorganic pyrophosphate in the presence of Mg2. In previous studies, it was observed that an inter‐subunit conformational change in the potato tuber ADP‐Glc PPase may play a role in the allosteric regulation of the enzyme. Removal of a disulfide bond involving Cys12 in the small subunit, either by mutation or by reduction resulted in a more active enzyme (Ballicora et al, 2000, J. Biol. Chem. 275:1315–1320), indicating that the inter‐subunit interaction between α subunits is an important part of the allosteric mechanism (Jin, X. et al, 2005, EMBO J. 24:694–704). In the structure of the Agrobacterium tumefaciens ADP‐Glc PPase, we found that Asp141 is a candidate to interact with Arg11 of the opposing subunit (Cupp‐Vickery, et al. 2008, Biochemistry 47:12795–801). The extreme N‐terminal Arg5 and Arg11 are quite clearly involved in activation by pyruvate (Pyr) as it has been demonstrated before (Gomez‐Casati et al. Biochem, 40(34),2001, pp. 10169–10178). To evaluate the hypothesis that of Asp 141 is involved in regulation, we mutated it to Ala. D141A was successfully expressed and purified to near homogeneity. This mutant enzyme exhibited reduced Vmax values in the absence of activators (Pyr and Fru6P), and lower apparent affinity for ATP and Fru6P. In the presence of Fru6P, D141A enzyme exhibited only ~ 4.3% of the wild‐type activity, which indicates that it virtually insensitive to Fru6P. This mutant enzyme also displayed 2.5‐fold reduced apparent affinity to the activator Fru6P when compared to the wild‐type enzyme. Our results suggest a unique functional role of D141 in regulation, possibly by interacting with R11 in the opposing subunit.

Support or Funding Information

National Science Foundation (NSF)
Characterization of a Novel Flavin‐dependent Monooxygenase in Bacillus niacini
Authors: Dasovich Morgan A , Snider Mark J , Peterson Michael T
Published: November 2023

Nicotinate metabolism is a model system for the degradation of N‐heterocyclic molecules, a common class of environmental contaminants. A putative monooxygenase in gram‐positive Bacillus niacini is hypothesized to carry out a decarboxylation‐hydroxylation reaction in a degradation pathway for nicotinate that is relatively uncharacterized. B. niacini is of particular interest because it exhibits a novel method of nicotinic acid degradation divergent from previously characterized pathways in Pseudomonas and Bordetella. Whole‐genome sequencing of B. niacini has identified a putative nicotinate‐degrading (nic) cluster and the proteins in this cluster have been expressed and purified, including the protein of interest. The putative monooxygenase contains a Rossman fold and is flavin‐dependent, utilizing NAD(P)H as a cofactor. It shows sequence homology with the 6‐hydroxynicotinate‐3‐monooxygenase (NicC) enzyme of P. fluorescens and B. bronchiseptica. NicC catalyzes a similar decarboxylation and hydroxylation reaction with 6‐hydroxynicotinate, thus it is hypothesized that the B. niacini homologue functions by catalyzing the decarboxylation‐hydroxylation of 2,6‐dihydroxynicotinic acid to 2,3,6‐trihydroxypyridine. To investigate this theory, 2,6‐dihydroxynicotinic acid and 2,3,6‐trihydroxypyridine were synthesized from commercially available 2,6‐dimethoxypyridine‐3‐carboxaldehyde. Compound purity has been assessed with NMR and mass spectrometry. UV spectrophotometry was used to determine the rate constants of the enzymatic reaction with NAD(P)H, and 2,6‐dihydroxynicotinic acid.

Support or Funding Information

Support and funding from the Department of Chemistry at the College of Wooster made this study possible.
Characterization and Identification of Metabolites in the Oxidation of Nicotinic Acid by Bacillus Niacini
Authors: Lesner Nicholas P. , Peterson Michael T. , Snider Mark J.
Published: November 2023

N‐Heterocylic compounds are of environmental concern due to their ubiquitous contamination in groundwater and soil. Nicotinic acid is a simple N‐heterocylic compound that is known to be degraded by a select group of gram‐positive bacteria and can serve as a useful model compound for understanding biological degradation. Nicotinic acid's biological degradation has been well characterized in Pseudomonas, but a similar yet distinct pathway known in Bacillus niacini has yet to be fully characterized. Utilizing the recently sequenced genome of B. niacini, in which a gene cluster was hypothesized to code for the enzymes that catalyze the degradation of nicotinic acid, a enzyme hypothesized to catalyze the oxidative ring‐opening step in the pathway was characterized. Protein sequence analysis revealed identity with NicX in Pseudomonas, but does not reveal mechanistic insight concerning ring opening. Here we describe the synthesis of the hypothesized substrate 2,3,6‐trihydroxypyridine, confirm the enzyme catalyzed product, and report results of a kinetic assay developed to examine mechanistic proposals. The results provide insight to the chemistries involved in the degradation of N‐heterocylic compounds by aerobic bacteria found in the environment.

Support or Funding Information

The College of Wooster Department of Chemistry
Decoupling the Allosteric Activation of Ubiquitin Specific Protease 7 by Its Ubiquitin‐like Domains for the Development of Small Molecule Inhibitors
Authors: Hjortland Nicole M , Pepe Antonella , Molland Katrina , Mesecar Andrew
Published: November 2023

Ubiquitin specific protease 7, USP7/HAUSP, regulates cellular levels of E3 ligase‐MDM2 and tumor suppressor p53 by the deubiquitination and subsequent stabilization of MDM2. The up‐regulation of USP7 in many types of cancer make it an ideal target for the development of anti‐cancer therapeutics. It is well known that the catalytic domain of USP7 has 100‐fold less activity than that of the full length enzyme. Recent work by Fasen et. al. (2011) reported that the catalytic function of USP7 is activated through the C‐terminal HAUSP ubiquitin‐like (HUBL) domain interaction with the switching loop of the catalytic domain. Our studies, utilizing a fluorescence based kinetic assay, define the kinetic parameters that surround the coupling of the activation of the catalytic domain by the HUBL domains. A small molecule inhibitor only capable of inhibiting the full length enzyme was identified by high‐throughput screening of a 20,000 compound library at Purdue University. Analogues of this compound were synthesized and significant structure activity relationships were defined. By kinetic studies, we probed the mode of inhibition of the best analogues and identified the need of specific HUBL domains for the inhibition of the enzyme by these compounds. These compounds can therefore serve as molecular probes in decoupling the allosteric activation of the HUBL domains for further studies of the intra‐molecular interactions of USP7.

Support or Funding Information

This work is funded by a grant from the Walther Cancer Foundation
The Agrobacterium tumefaciens ADP‐Glucose Pyrophosphorylase G329D Mutant Exists in a Pre‐Activated State
Authors: hill benjamin luke , Mascarenhas Romila , Koenig Amanda M , Huerta Fidel , Khan Samia , Iglesias Alberto A. , Liu Dali , Ballicora Miguel
Published: November 2023

Allosteric regulation of ADP‐glucose (ADP‐Glc PPase, EC 2.7.7.27) is critical for determining the amount of glycogen and starch that gets synthesized in bacteria and plants, respectively [1]. This enzyme combines ATP and glucose‐1‐phosphate to form ADP‐glucose, which serves as the glucose moiety donor for polyglucan synthesis. ADP‐Glc PPases are regulated by intermediates of the major carbon assimilatory pathway that exists within a given species. To date, all identified regulators have a negative charge. The ADP‐Glc PPase from Agrobacterium tumefaciens is strongly activated by pyruvate and fructose‐6‐phosphate (Fru‐6P). Since ADP‐Glc PPase serves as a major control point for the production of glycogen and starch, which are renewable and biodegradable carbon sources, these enzymes have been attractive targets for protein engineering [1,2,3].

A growing body of evidence suggests that the interface between the enzyme's distinct C‐ter and N‐ter domains is important for allosteric regulation [2,4,5]. Here, we report on the aspartate mutant of Gly329 in the A. tumefaciens enzyme, which, based on the crystal structure [2], is a residue that residues near this cleft. When this enzyme was characterized, it was found to be in a pre‐activated state, meaning that it did not need allosteric activators present in order to achieve levels of activity similar to the activated wild‐type enzyme. We suspect that the positioning of an aspartate group in this region mimics the action of negatively charged activators like pyruvate. Furthermore, these findings could help pinpoint the exact location of the allosteric binding site. This work could provide important information into the allosteric regulation of ADP‐Glc PPases.

Support or Funding Information

National Science Foundation Grant MCB‐1024945(MAB)
Determining the Structure of NRPS‐Independent Siderophore (NIS) Synthetase DesD Using X‐ray Crystallography
Authors: Karimi Kimya L , Hoffmann Katherine M
Published: November 2023

A lack of iron leads to secretion of siderophores (small molecule metal chelators) by bacteria. Siderophores have high affinity for iron outcompeting host chelators, and making them virulence factors in infection. Some siderophores (of particularly virulent pathogens) are assembled via an NRPS‐independent siderophore (NIS) pathway. Understanding the structure of NIS siderophore biosynthesis enzymes may lead to the design of specific antibiotics to fight pathogenic bacteria.

The Hoffmann lab is using x‐ray crystallography to determine the protein structure of NIS synthetase DesD, from Streptomyces coelicolor. DesD makes the siderophore desferrioxamine B, requiring three iterative bonds and a macrocyclization event. DesD is one of the best biochemically characterized NIS synthetases so far, and we are solving this structure as a model for the rest. The goal of this project is to solve the x‐ray structure of DesD bound to substrate and ATP, as well as apo (empty of substrate) structures, in order to characterize the conformational changes that may occur with binding. DesD crystalizes in 20% PED 8,000, 15% glycerol, and 15% ethylene glycol conditions, and crystals diffract to 2.48 Å with good statistics. We are solving the structure using molecular replacement and are pursuing substrate‐bound crystals. This structural information may allow us to design an inhibitor that will prevent the binding of substrate in future work.
Human alpha3 beta1 isoform of Na+/K+‐ATPase: expression in yeast, mutations and their relation to neurological disorders
Authors: Geletičová Jaroslava , Kubala Martin , Sorensen David , Pedersen Per Amstrup , Nissen Poul
Published: November 2023

The Na+/K+‐ATPase (NKA) is a crucial membrane protein responsible for maintaining the membrane potential by actively transporting sodium and potassium ions. The proper function of this pump is essential for all cells (e. g. in homeostasis, membrane potential). The human body contains four isoforms of this enzyme, which are spread over whole organism. One of the most important localizations of NKA is the brain. Medical research of Heinzen et al. (2014) suggests that there is a possible connection between NKA alpha3 isoform mutations (mainly D801N, E815K, D923N, D923Y and G947R) and neurological disorders. Detailed studies of patient's genome revealed that all these mutations are closely linked to Rapid‐Onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood.

Modern biotechnological methods, especially the production of a heterologous protein in expression systems, enable deeper study of a wide range of non‐abundant proteins. The preparation of alpha3 NKA proteins (wild type and mutants) was performed according to Pedersen et al. (2006) with some modifications. In general, changes in the level of protein expression and determination of the relative enzyme activity could be the first step towards better understanding of the relation between NKA function and neurological disorders.

Support or Funding Information

This project was supported by grant No. L01204 (Sustainable development of research in the Centre of the Region Haná) from the National Program of Sustainability I, MEYS and by “Palacky University Foundation 2015”. The project was awarded by EMBO Short‐Term fellowship ASTF 243‐2015.
Molecular Cloning and Expression of an Oleate Hydratase From Nocardia Cholesterolicum NRRL 5767
Authors: Vanderway David R , Alhmadi Hekmat B , Diaz Joshua D , Wen Lisa , Huang Jenq‐Kuen
Published: November 2023

The production of household cooking oils in an industrial setting yields soapstock as a byproduct during the refining process. Soapstock contains a significant amount of oleic acid and due to soapstocks very low price, it serves as a cheap source of oleic acid. Oleate hydratase (Ohase) converts oleic acid to the industrially useful product 10(R)‐hydroxystearic acid (10‐HSA), in a regioselective manner. Classically, 10‐HSA is utilized to thicken grease, but it has potential to be used in the production of plastics, lubricants and other various industrially relevant materials. Barring the use of biocatalytic microorganisms containing Ohase, this reaction would take place under unfavorable conditions involving the use of high temperatures/pressures, strong acids or metal catalysts, and all of these methods lead to little regioselectivity in the final product. Therefore, the use of a biocatalyst that catalyzes the conversion of oleate hydratase to 10‐HSA is extremely desirable for industrial applications. The bacterium Nocardia Cholesterolicum species NRRL 5767 is able to convert 95% of oleic acid added into the bacterial culture to 10‐HSA. Ohase has never been isolated or characterized at either the protein level or DNA level from NRRL 5767. This work is the first report of molecular cloning of the Ohase gene from NRRL 5767 genomic DNA into the pET28a expression vector. Functional recombinant Ohase enzyme has been expressed and purified. Enzyme activity of Ohase was determined by the conversion of oleic acid to 10‐HSA or by spectrophotometrically through a coupling reaction.

Support or Funding Information

Western Illinois University Foundation and the Department of Chemistry.
Molecular Cloning and Expression of a Secondary Alcohol Dehydrogenase From Nocardia Cholesterolicum NRRL 5767
Authors: Diaz Joshua D , Vanderway David R , Flores Micah , Wen Lisa , Huang Jenq‐Kuen
Published: November 2023

Norcardia cholesterolicum NRRL 5767 (N. cholesterolicum NRRL 5767) is a well‐known bacterium capable of transforming 95% of added oleic acid to a more valuable product, 10‐hydroxystearic acid (10‐HSA) (Appl Microbiol Biotechnol 32, 299–304, 1989). This reaction is catalyzed by oleate hydratase. However, a small amount of the 10‐HSA is subsequently converted to 10‐ketostearic acid (10‐KSA) by secondary alcohol dehydrogenase (2º‐ADH). Since 10‐HSA is more valuable than 10‐KSA in industry; 10‐KSA is considered a side product which complicates downstream separation and purification of 10‐HSA. It is attractive to hinder the production of 10‐KSA through bacterium strain improvement by genetic engineering to block the production of 2º‐ADH. The objective of this research is to clone, overexpress, and characterize functional recombinant 2º‐ADH. An annotated 2º‐ADH gene has been successfully amplified by PCR from genomic DNA of N. cholesterolicum NRRL 5767and sub‐cloned into pET28a expression vector. The 2º‐ADH protein has been overexpressed by IPTG induction and purified to homogeneity by Ni‐NTA affinity column. Enzyme activity of the 2º‐ADH was determined by monitoring the formation of NADH spectrophotometrically.

Support or Funding Information

Western Illinois University Foundation and the Department of Chemistry.
Strategies to Enable Practical Electrochemical Diagnostics for Proteins and Small Molecules
Authors: Bonham Andrew J , Bulow Aviva , Fetter Lisa C , Richards Jonathan , Daniel Jessica , McCoy Michael
Published: November 2023

Despite decades of research into biosensors ‐ sensors based on biological interactions ‐ few have made the transition from the academic lab to practical diagnostic use in industry or the clinic. One common biosensor class utilizes electrochemical detection of changes in oligonucleotide structure upon binding to a target protein or small molecule. To make widespread adoption of this large class of biosensors more feasible, we have pursued multiple approaches to address typical shortcomings of biosensor design and performance. These include: the design and implementation of an oligonucleotide folding evaluation webserver, Fealden, which can identify sequences and folding patterns likely to yield significant signal response; investigations of surface passivation and hydrogel filtration to enable functional analysis of binding interactions in unprocessed whole blood; and automated signal analysis software to enable rapid turnaround from raw biosensor signal to reportable quantification of target. As proof of concept, we have applied these tools to design, generate, and test biosensors directed against the protein toxins in botulism and ricin poisoning, which are currently typically detected only with slow, cumbersome, and/or error‐prone diagnostics. This work thus provides insights into a multifaceted approach to utilize the wide pool of existing biosensors in practical applications ranging across food safety, environmental monitoring, and clinical diagnostics.