bluuebunny/crossref_metadata_2025 huggingface dataset:
Screening for novel long‐chain bacterial esterase activity
Authors: Jozwiakowski Jakob Leland , Johnson R. Jeremy
Published: June 2021

Hydrolases are ubiquitous cellular enzymes with more than 1200 different hydrolases, >1/3 of all enzyme structures in the PDB, and 13 different enzyme commission subclasses. Hydrolases are also fairly promiscuous and can also catalyze hydrolysis reactions on varied substrates including esters, amides, thioesters, phosphoric acid esters, and acid anhydrides. All of these reactions originate from the same α/β hydrolase protein fold and utilize the catalytic triad, suggesting great plasticity and generality in this reaction. Using a fluorogenic mimic of palmitic acid linked molecules, we performed a high‐throughput screen in Escherichia coli searching for previously unidentified depalmitoylase activity. Simultaneous analysis of forty‐five different hydrolase knockout strains with this fluorogenic probe identified a single uncharacterized esterase with high specificity for the probe. To confirm this reactivity, this hydrolase was heterologously expressed, purified to homogeneity, and characterized for its detailed substrate specificity using a library of fluorogenic esters. Our screen highlighted the potential novel hydrolase reactivity present within even model organisms like E. coli that could have applications in protein engineering or biological imaging.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Molecular Determinants of Heterocycle Formation in Hybrid Nonribosomal Peptides/Polyketides
Authors: Dowling Daniel P. , Kung Yan , Croft Anna , Taghizadeh Koli , Kelly Wendy , Walsh Christopher T. , Drennan Catherine L.
Published: June 2021

Many secondary metabolites from natural sources contain thiazole or oxazole derivatives that stem from the precursor amino acids cysteine, serine or threonine, and how Nature generates these azolines depends on the biosynthetic strategy used by that organism. Here, we explore azoline formation within hybrid nonribosomal peptide (NRP) and polyketide (PK) compounds, a class of molecules that are extremely beneficial for drug discovery. Epothilones are example hybrid NRP/PK products that demonstrate antitumor activity by stabilizing microtubules. They are biosynthesized through the action of EpoA‐F from Sorangium cellulosum, in which the EpoA and EpoB proteins act as the starter unit to generate the 2‐methylthiazole functionality. EpoB contains a cyclization (Cy) domain capable of catalyzing both chain extension and cyclization reactions to yield a heterocycle. We report crystal structures for a didomain of the EpoB Cy domain tethered to its N‐terminal docking domain, which have implications in locating carrier protein binding sites, identifying phosphopantetheine binding tunnels, and determining residues involved in catalysis. Site‐directed mutagenesis of active site residues have identified key amino acids required for catalysis. X‐ray crystal structures of the EpoB Cy domain and related systems provide an opportunity to analyze critical similarities and differences that may guide future bioengineering efforts for green chemistry applications.

Support or Funding Information

U.S. National Institutes of Health [1R15GM123425‐01 to D.P.D.]. C.L.D. is a Howard Hughes Medical Institute Investigator. Support for this research was provided by National Institute of Environmental Health Sciences, NIH Core Center Grant P30‐ES002109.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthetic biology, biosynthesis, enzymology, (meta)genomics, et al. Learning from Nature.
Authors: Agarwal Vinayak
Published: June 2021

Nature is an organic chemist. The language in which we interact with Nature is chemistry, with small molecule natural products being the alphabets of this language. Natural products range from antibiotics and pharmaceuticals that we use in the clinic, to toxins and pollutants that pervade our environment. Research in the Agarwal lab seeks to understand how natural products are produced, what are the biological forces driving their syntheses in Nature, and how we can leverage this knowledge to drive scientific progress and human well‐being. We employ a combination of chemical and biochemical approaches, including but not limited to microbial genetics and metagenomics, structural and biochemical enzymology, and synthetic and analytical chemistry.

Natural product biosynthesis is a treasure trove of novel enzymology. We will describe a total enzymatic reconstitution of the biosynthesis of a polybrominated marine natural product. Crystal structures of biosynthetic enzymes provide deep insights into mechanisms of key enzymatic transformations and allow for structure guided engineering of catalysts. These findings set the stage for the development of methods for mining complex metagenomic datasets to tackle the immense biological complexity of natural product sources.

Support or Funding Information

NIH R00ES026620

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improved Synthesis of N‐methyl Cadaverine
Authors: Anderson Kayla Nicole , Moaven Shiva , Cozzolino Anthony , D'Auria John C.
Published: June 2021

Polyamines are abundant in nature, and the methylation of polyamines is a common enzymatic step in plants, especially plants containing alkaloids. This includes the family of compounds called granatane alkaloids. Pomegranate (Punica granatum) is one of the most commonly known granatane alkaloid‐producing plants. Pomegranate extracts are widely used for their antioxidant, anti‐inflammatory, and anticarcinogenic effects. [1] One of the proposed enzymes in the hypothetical pathway for the formation of granatane alkaloids is cadaverine N‐methyl transferase.[2] It is important to find a method of synthesis for N‐methyl cadaverine (N‐methylpentane‐1,5‐diamine) and other N‐methyl polyamines, because they can be utilized as a standard for quantification purposes as well as providing a positive control when evaluating enzymatic activities of putative alkaloid producing N‐methyl transferases. The synthesis of N‐methyl cadaverine was employed by utilizing a substitution reaction between N‐methylbenzylamine and 5‐bromo‐valeronitrile, removing the protecting group (or benzyl ring) attached to the methylated amine, and reduction of the nitrile. [3]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Initial Characterization of DszA, a Monooxygenase from the Rhodococcus Biodesulfurization Pathway
Authors: Truong Sharon , Sanchez Misael
Published: June 2021

Sulfur dioxide is a pollutant found in our atmosphere that affects our health, environment, and economy. The most common source of sulfur dioxide pollution is the combustion of fossil fuels. To control pollution, it is important to remove sulfur from these fuels. The current sulfur removal method, hydrodesulfurization, is inefficient because it cannot remove sulfur from refractory organosulfur compounds. There are multiple bacterial species, including Rhodococcus erythropolis, that can remove sulfur from aromatic compounds, such as dibenzothiophene (DBT). DBT is a major organosulfur compound found in crude petroleum. The biodesulfurization pathway in Rhodococcus erythropolis can successfully remove sulfur from DBT to produce hydroxybiphenyl and sulfite. Our study focuses on an enzyme in this biodesulfurization pathway, the DBT sulfone monooxygenase, DszA that catalyzes the conversion of DBT sulfone to 2′‐hydroxybiphenyl 2‐sulfinate. Our goal was to obtain pure, active, and soluble protein for initial characterization of DszA. Thus far, we have determined the optimal parameters for expression of DszA by various expression and pH trials. The optimal temperature and pH for enzyme expression were 18°C and 7.5, respectively. DszA was successfully overexpressed in Escherichia coli and will be ready to purify by nickel affinity chromatography to take advantage of the His tag found in our protein. Our future steps are to apply steady‐state and transient‐kinetics to determine the kinetic parameters and study the mechanism of this important reaction step in the Rhodococcus biodesulfurization pathway. We will also analyze the substrate and product by high‐performance liquid chromatography to determine substrate specificity. These initial studies will provide a greater understanding towards the biodesulfurization mechanism for industrial purposes and pollution control.

Support or Funding Information

This research was supported by the Research Corporation (CCSA 22672) and by the National Institutes of Health (NIH) Grant 5SC2AI109500.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of the Iterative Methylations by a Cobalamin‐Dependent Radical SAM Methyltransferase
Authors: Wang Yuanyou , Begley Tadhg P.
Published: June 2021

Natural products with branched alkoxy groups have been used widely in the development of bioactive compounds. However, the biosynthesis of branched alkoxy groups like the unique t‐butyl group is still poorly understood. The cystobactamids are a novel class of isopropyl substituted antibacterial compounds produced by myxobacteria. In the biosynthesis gene cluster of cystobactamid, CysS is assigned as a cobalamin‐dependent radical S‐adenosylmethionine (SAM) enzyme, which is proposed to catalyze the iterative methylations on the 3‐methoxy‐4‐aminobenzoic acid moieties of cystobactamids. To reconstitute CysS, different substrates analogs were synthesized. When the pantetheinyl substrate analogs were used, multiple methylations were observed to form ethyl, isopropyl, t‐butyl and sec‐butyl substituted products from a substrate methyl ether by CysS. These cobalamin dependent methylations also produce 5′‐deoxyadenosine(5′dA) and S‐adenosylhomocysteine (SAH) in equal amounts. The isotope labeling experiments suggest that the methyl group from SAM is first transferred to cobalamin followed by the attack of the substrate radical to give methylated products. A combinatorial antibiotic library of 25 cystobactamid analogs could potentially be built using CysS by Cystobacter sp. Different substituted substrate analogs were designed to trap the organic radical species, cobalamin intermediates and probe the radical substitution step. Here, we have showed the first in vitro reconstitution of a cobalamin‐dependent radical SAM enzyme catalyzing the conversion of a methyl group to a t‐butyl group by sequential methylations. The detailed mechanistic studies help us to unravel how CysS uses SAM, [4Fe–4S] cluster and cobalamin to perform iterative methylations on sp3‐hybridized carbon centers and better understand the mechanism of other widespread cobalamin‐dependent radical SAM methyltransferases.

Support or Funding Information

This work was supported by NIH (DK44083) and by the Robert A. Welch Foundation (A‐0034).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selective inhibition ATP Synthase in combating microbial infections
Authors: Ahmad Zulfiqar
Published: June 2021

Background

ATP synthase is main source of cellular energy for almost all organisms. Malfunction or inhibition of ATP synthase may cause cell death. A wide variety of inhibitors including phytochemicals and peptides are known to bind and inhibit ATP synthase. These phytochemicals and peptides bind to the specific binding pockets on ATP synthase. These binding pockets are flanked by variable amino acids in different species. Our lab is exploring the possibility of using ATP synthase as a selective molecular drug target to combat the antibiotic resistant microbial infections using Escherichia coli as a model organism.

Method

Wild type, null, and mutant E. coli growth properties were tested on fermentable glucose and non‐fermentable succinate carbon sources. Wild type and mutant enzymes were isolated by harvesting cells in the minimal media. Inhibitory studies were performed on membrane bound F1Fo ATP synthase. Structural modifications of inhibitors were made through replacement or re‐positioning of the functional groups on phytochemicals or addition of positive charges on the peptides. Wild type and mutant cell growth assays were tested in presence and absence of inhibitors along with null control.

Results

Phytochemicals and peptides caused variable degree of inhibition. Extent of inhibition was augmented by modification of inhibitors. For phytochemicals, re‐positioning and addition of new functional groups and for peptides, addition of a c‐terminal NH2 group enhanced the inhibitory potency on molar scale. We also found that incremental addition of positively charged residues in peptides can augment the inhibitory effects of peptides by about 100‐fold. Cell growth assays in presence and absence inhibitors suggest that ATP synthase is a potent molecular drug target to combat microbial infections. Currently, we are studying the synergistic inhibitory effects of phytochemicals and peptides on ATP synthase.

Conclusions

We conclude that phytochemicals and peptides bind and inhibit E. coli by interacting at the specific sites on F1 sector of ATP synthase and ATP synthase is a potential molecular drug target.

Support or Funding Information

KCOM‐ATSU Biomedical Sciences Graduate Program Grant Award # 850‐611, 850‐619, and 851‐026

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IDH1 mutation‐inspired α‐ketoglutaric acid mimics for oxidative therapy of higher grade gliomas through α‐ketoglutarate dehydrogenase inhibition
Authors: Madala Hanumantha Rao , Punganuru Surendra Reddy , Srivenugopal Kalkunte
Published: June 2021

Mutations at the active site of isocitrate dehydrogenase 1 gene (IDH1; R132H) occur at a high frequency (>70%) early in the oncogenesis of lower grade malignant gliomas, and result in a dramatic accumulation of the oncometabolite D‐2 hydroxyglutarate (D‐2HG), that effectively replaces the normal metabolite α‐ketoglutarate (α‐KG) in cell physiology. Surprisingly, IDH1 mutations bestow superior therapeutic responses to alkylating agents and better patient survival. D‐2HG, effectively competes with α‐KG and potently inhibits various dioxygenase reactions, including the TET1&2 DNA ‐demethylases and histone demethylases thereby, re‐shaping and reprogramming the epigenetic landscape, and consequent transcriptional silencing; the DNA repair protein O6‐lguanine DNA methyltransferase (MGMT), which confers drug resistance is one major target for such repression. As a novel and innovative strategy of turning the tide against GBMs by exploiting the mechanistic aspects of the oncometabolite, we hypothesized that D‐2HG and α‐KG derivatives that can replace the natural metabolite in epigenomic dioxygenase reactions will serve as potent anti‐glioma drugs either by themselves or in combination with the alkylating agents. To this end, we synthesized a 2,4‐dimethyleneglutaric acid (DMG), an α‐KG mimic with methylene groups inserted at the C2 and C4 positions. The hydrophobic DMG ester, by itself, was cytotoxic with IC50 values up to 300 μM against brain tumor cell lines, however, when combined at 100 μM with TMZ resulted in >20‐fold increased synergistic cell killing. DMG induced oxidative stress inhibited α‐KG dehydrogenase, MGMT and TET1&2. DMG induced oxidative stress further caused mitochondrial damage as confirmed with JC‐1 staining. Mitochondrial damage further affected its biosynthetic, bioenergetic and signaling functions as confirmed with respective assays. We also observed a marked reduction in ATP and NADH levels and rapid metabolic flux changes indicating an altered cellular metabolism. Metabolomic analysis of major Krebs cycle intermediates revealed significant decrease in their levels. We, further observed that, bioenergetic stress induced by DMG activated AMPK and inhibited mTOR signaling cascade. Sub‐acute toxicity on mice revealed the safety of DMG in mice and we observed significant regression in tumor growth in intracranial xenograft tumor models. Collectively, these data reveal that acute treatments of α‐KG analogs can alter the cellular metabolic and epigenetic makeup in a manner ascribed to D‐2HG, and open up much‐needed novel and exciting avenues of oncometabolite‐based therapies for brain tumors

Support or Funding Information

CPRIT grants RP130266 and RP170207 to KSS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Analysis of Darunavir Analogs Against Primary Resistance Mutations in HIV Protease
Authors: Lockbaum Gordon Joseph , Leidner Florian , Rusere Linah , Nalivaika Ellen , Ali Akbar , Kurt‐Yilmaz Nese , Schiffer Celia
Published: June 2021

Antiretroviral therapies (ARTs) involve combinations of small molecule inhibitors that suppress the viral load of Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Symptom (AIDS). ARTs, currently the only defense against HIV, target critical proteins necessary for viral replication, such as HIV‐1 protease. The most potent FDA approved protease inhibitor, darunavir (DRV), is still susceptible to resistance if the protease acquires mutations in and around the active site. One method to avoid resistance is to optimize the inhibitor to fit the consensus volume that the protease's natural substrates occupy. Using this strategy, DRV analogs with larger moieties at the P1’ position were designed and synthesized to leverage unexploited space and create additional protease‐inhibitor interactions, resulting in inhibitors that are more potent and less susceptible to resistance. To elucidate the molecular mechanisms of active site resistance mutations against these inhibitors, we used a multidisciplinary approach, including biochemical assays and x‐ray crystallography. Results suggest that modifications to the DRV scaffold result in inhibitors with potency comparable to DRV, but structural analysis highlights differences in intermolecular and intramolecular interactions, which will inform future drug design.

Support or Funding Information

This work was supported in part by a grant from the National Institute of General Medical Sciences, National Institutes of Health (P01‐GM109767).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression and Purification of Hydrophobic PEP‐Inhibiting Peptides from Bovine αS1 Casein
Authors: Sheffield Logan E. , Whalen Kaylei D. , Goode Courtney A. , Acosta Francisco M. , Lenert Melissa E. , Clack Beatrice A.
Published: June 2021

According to previous studies, there is a prolyl endopeptidase‐inhibiting sequence (LNENLLRFFVAPFPEVFG) within a relatively hydrophobic region of αS1 casein (Juillerat‐Jeanneret et al., 2011). Recently, sixteen recombinant peptides were designed in assorted lengths, each containing the inhibitory sequence at varied positions within the αS1 casein sequence. The corresponding cDNA sequences were originally ligated into the pET15b vector, which provides an N‐terminal poly‐histidine tag for purification. The vector was in turn transformed into a BL21(DE3)pLysS E. coli expression host (Vishram et al., 2016). However, the system proved unsuccessful in adequate expression of the hydrophobic peptide. The intention of this study was to assemble a new pET28a expression system, kindly provided by Dr. X. Cheng (MD Anderson in Houston, TX) that utilizes a SUMO (small ubiquitin‐like modifier) fusion tag. The SUMO tag is known for enhancing expression levels of potentially toxic or insoluble recombinant peptides (Bommarius et al., 2010). The expressed peptides were purified via Ni‐NTA column chromatography, with subsequent UlpI cleavage. This was followed by reversed‐phase high performance liquid chromatography (RP‐HPLC) to further purify the peptides. Tricine‐SDS‐PAGE, a useful screening tool for hydrophobic peptides smaller than 30kDa, was used to analyze expression and purification. A comparison was made between SUMO‐fused expression and non‐fused expression of the hydrophobic casein peptides.

Support or Funding Information

Office of Research and Sponsored Projects, SFASU and Ed & Gwen Cole

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Covalent attachment of protein cargo to a computationally designed toroid scaffold
Authors: Polkinghorn Caley Ray , Hallinan Jazmine , Doyle Lindsey , Bradley Phil , Stoddard Barry , Kaiser Brett K.
Published: June 2021

Recent developments in computational design have expanded the accessible protein structures to those beyond what was selected for through the evolutionary process. Closed alpha‐solenoid tandem repeat structures (alpha‐toroids) are one class of rationally designed proteins. Four designed toroids, each containing a central pore and a varied number of repeats, were previously validated by x‐ray crystallography. We envision using toroids as stabilizing scaffolds upon which other proteins can be attached. The goal of this study is to develop an approach for attaching protein cargo to the toroid using the Spycatcher/Spytag peptide tagging system. Spycatcher (protein) and Spytag (peptide) are split protein partners derived from the fibronectin binding protein of Streptococcus pyogenes that form an irreversible, stable isopeptide bond. We demonstrated that 24‐repeat toroids containing four Spycatcher domains embedded within loop regions (“toroid‐SpyCatcher”) properly fold and assemble by size exclusion chromatography. We then showed that a Spytag‐GFP fusion protein binds efficiently and irreversibly to the toroid‐SpyCatcher using fluorescent binding assays. This proof of principle experiment demonstrates that this approach is feasible for attaching proteins to the toroid scaffold and could be expanded to attach cargo proteins with diverse activities.

Support or Funding Information

Murdock Charitable Trust

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational design and molecular mechanism in oligomerization of C‐terminal binding protein 2
Authors: Miyashita Yurina , Matsuo Naoya , Yamamoto Yuta , Nakano Shogo , Ito Sohei , Numoto Nobutaka , Ikura Teikichi , Ito Nobutoshi , Sekiya Motohiro , Shimano Hitoshi , Tokiwa Hiroaki
Published: June 2021

Introduction

C‐terminal binding protein 2 (CtBP2) is moonlighting proteins, which has multiple functions and plays a key role in transcriptional control and in the maintenance of cellular metabolism (1). CtBP2 consist of D‐isomer specific 2‐hydroxyacid dehydrogenase (D2‐HDH) domains and C‐terminal intrinsically unstructured region. Recent studies have shown that the recruitment of a corepressor complex by CtBP2 depends on NADH/NAD ratio. Molecular mechanism of oligomerization of CtBP2 may provide a basis for the formation of a transcriptional corepressor complex.

Methods

We attempted to construct artificial designed proteins on the basis of in silico screening using the INTMSAlign program (2). Overexpression in E. coli cells, and purification of artificial designed CtBP2 (ArCtBP2) were performed. The oligomeric state and the binding affinity to NADH of ArCtBP2 were compared to human CtBP2 (hCtBP2) by size exclusion chromatography and intrinsic Trp fluorescence. To evaluate structural information, X‐ray crystallographic analysis of ArCtBP2 was performed.

Results

Size exclusion chromatography of ArCtBP2 shows 4 peaks for each corresponding oligomeric state, whereas that of hCtBP2 was observed as a single peak. The dissociation constants (Kd) value of NADH to ArCtBP2 was determined as 3.6 ± 0.9 μM by fluorescence spectroscopy. In addition, crystal structure of apo and NAD–bound form of ArCtBP2 at 2.0 and 1.7 Å resolution, respectively. Crystal structure of ArCtBP2 showed opened‐like structure in D2‐HDH domain compared to hCtBP2. These results suggested that the open–closed form of D2‐HDH domain is involved in the binding affinity for NADH and oligomeric state of CtBP2.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of New Molecular Tools for Antibody Detection and Purification
Authors: de Lourdes Borba Magalhaes MAria , da Silva Gustavo Felippe , Echeverri Lina Maria Salazar , Fernandes Leonardo Antonio , Batista Franciane
Published: June 2021

Primary antibody detection is crucial for biomarker detection in research and diagnosis. Molecular tools for antibody detection and purification currently available consist of secondary antibodies as well as bacterial proteins such as Proteins A and G. Disadvantages of the current methods include the fact that secondary antibody production is expensive and may require the use of animals. In the case of Proteins A and G, they either do not recognize all IgG isotypes or require acidic elution conditions for antibody purification, which may lead to antibody denaturation. We have produced chimeric proteins, using an IgG binding protein recently discovered (TRIM21) linked to detection proteins such as streptavidin, and alkaline phosphatase. TRIM21 is a cytosolic protein that binds all IgG isotypes from many species. Bacterial expression, purification and refolding of TRIM21‐streptavidin as well as TRIM21‐alkaline phosphatase were successful. TRIM21‐streptavidin chimeric protein was incubated with biotinylated HRP or alkaline phosphatase for immunoassays. ELISA and western blotting experiments demonstrated that the chimeric proteins efficiently detect monoclonal and polyclonal antibodies from a wide range of species, including human, mouse, rat, dog, horse and bovine. Binding experiments are currently underway to determine the binding constants for TRIM21 chimeric proteins and IgG from different species. Future experiments will be performed using imobilized TRIM21 for antibody purification.

Support or Funding Information

This work was supported by the “ State University of Santa Catarina” (UDESC), “Fundação de Amparo a Pesquisa e Inovação do Estado de Santa Catarina” (FAPESC) and CNPQ.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How to design artificial protein surpassing native enzyme function ~ Design and multidisciplinary analysis of artificial L‐threonine 3‐dehydrogenase ~
Authors: Nakano Shogo , Motoyama Tomoharu , Miyashita Yurina , Ishizuka Yuki , Matsuo Naoya , Tokiwa Hiroaki , Shinoda Suguru , Asano Yasuhisa , Ito Sohei
Published: June 2021

Introduction

Currently, we can easily obtain a large amount of protein sequence data through database, such as PubMed. The data are being used for protein engineering to reconstruct sequence of target protein, like full‐consensus protein design (FPD) and ancestral sequence reconstruction (ASR). However, redesign of highly functional protein is challenging tasks. In this study, we attempt to get a clue how to succeed in the redesign through protein sequence reconstruction of native l‐threonine 3‐dehydrogenase from Cupriavidus necator (CnTDH) (1).

Methods

Firstly, total 5000 of homologous sequences with CnTDH were obtained by submitting sequence of CnTDH to Blastp: the sequences were called “library”. Artificial TDH, FpdTDH‐N0, was designed by the FPD method using the library. Next, utilizing INTMSAlign (2, 3), the curated library was generated to maximize frequency of each consensus residues of artificial TDH. Two artificial TDHs were designed by FPD (FpdTDH‐N1) and ASR (AsrTDH) method, respectively, utilizing the curated library.

Results & Discussion

Specific activity was measured for the three artificial TDHs; and the activity could be confirmed for FpdTDH‐N1 and AsrTDH which designed using the curated library. FpdTDH‐N1 and AsrTDH share 69 and 65% of sequence identity with CnTDH, respectively. Enzyme properties of CnTDH, FpdTDH‐N1 and AsrTDH were estimated by the following multidisciplinary analysis. Thermal stability of both FpdTDH‐N1 and AsrTDH was better than that of CnTDH: Tm values of FpdTDH‐N1 and AsrTDH were higher about 10 and 5 °C than that of CnTDH. Enzyme kinetics parameters of FpdTDH‐N1 and AsrTDH (kcat/Km value) were comparable to CnTDH. Furthermore, dissociation constant (Kd) for AsrTDH and FpdTDH‐N1 toward NAD+ which measured by ITC were about 8‐ and 2‐fold lower than that of CnTDH. Structural comparison among CnTDH, FpdTDH‐N1 (2.9 Å resolution) and ArsTDH (2.2 Å resolution) indicated that there is no mutation around 7 Å of oxygen atom of catalytic Tyr. The mutation was assembled at NAD+ binding site and protein surface.

Taken together, we succeeded in design artificial TDHs bearing high stability, activity and affinity toward NAD+ compared with native TDH, CnTDH. This result also inferred that curated library designed by maximizing frequency of amino acids for consensus residues of target protein is effective to design “super enzyme” surpassing native enzyme function.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Number 16K18688, 17KT0010, 17H06169 and JST by ERATO grant number JPMJER1102.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring Binding Determinants of (s)‐allantoin with Proteins via Docking and Molecular Modelling
Authors: Reeves Matthew E. , Allard Marco M. , Payne Marvin A.
Published: June 2021

(s)‐allantoin is one of the breakdown products of uric acid in many animals, including humans. It has been shown to be a product of the oxidation of uric acid. Allantoin concentration in plasma and urine has been correlated with the level of oxidative stress in the organism. The goal of this research is to study the binding modes of allantoin to natural binding partners for which allantoin is a product or substrate, and other unrelated proteins that bind with reasonable affinity. The proteins are first subjected to docking using Swiss‐Dock to identify binding clusters with optimized affinity. The most favorable are then subjected to ab initio quantum mechanical calculations on limited volumes around the binding sites using Gaussian. H‐bond networks and other interactions are predicted. The overall results have been used to design synthetic peptides with good predicted binding affinities. Besides adding to the understanding of allantoin binding to proteins, this method has applicability in designing detection methods for the presence of allantoin and related compounds.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of an Immersion Model for Tooth Decay Observed via Scanning Electron Microscopy, Atomic Force Microscopy and Flame Atomic Absorption Spectroscopy
Authors: Shipman Richard David , Quelle Benjamin G. , Grant Jennifer E. , Lee Eun Joo
Published: June 2021

A model for immersive tooth decay was developed to assess the loss of calcium from hydroxyapatite (calcium phosphate) discs. Hydroxyapatite discs are routinely used as a target model of a tooth. Similarly, immersion in an acidic solution is commonly used to emulate conditions within the human mouth; it is believed that a pH of 3.26 is a reasonable approximation of a biological pH experienced by the mouth upon eating acidic foods.

Discs were incubated the acidic solution for one or three months. Acid‐treated hydroxyapatite discs were removed from the solution and dried via vacuum centrifuge before being analyzed; the immersion solution was also retained. Scanning electron microscopy (SEM), atomic force microscopy (AFM) and flame atomic absorption spectroscopy (FAAS) were performed to document the extent of tooth damage. Samples were sputter‐coated with gold prior to SEM analysis, where surface morphology of treated hydroxyapatite discs upon acid damage was monitored. AFM was used to measure the average surface roughness of acid‐treated discs compared to control discs. Disc immersion solution was saved, and calcium dissolution was quantified using FAAS.

This research improves on known protocols for evaluating damage to hydroxyapatite discs treated in an acidic immersion assay. By providing a model for the development of caries in teeth, this protocol offers a system for studying the impact of biological and nanobiological materials on tooth decay. SEM surface morphology measurements provided a visual assessment of the erosion of the hydroxyapatite disc when immersed in an acidic solution with changes in the topographic determination of roughness quantified with AFM. FAAS analysis was used to document the release calcium from the hydroxyapatite discs.

We have implemented a protocol for analyzing damage caused to teeth as modeled in a hydroxyapatite disc assay, and set standards for analyzing the damage by three complementary techniques.

Support or Funding Information

This research was supported by University of Wisconsin‐Stout Student Research Grant to RDS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Polymeric Nanovehicles for siRNA Delivery in Cardiomyocytes
Authors: Lázaro‐Alfaro Anay , Lozano‐García Omar , García‐Rivas Gerardo
Published: June 2021

Cardiovascular diseases are the first cause of death worldwide. In these diseases, Ca2+ handling plays an essential role. Mitochondria, the main sources of energy in eukaryotic cells, are important Ca2+ regulators. However, under pathological conditions, mitochondrial energy production may be altered due to mitochondrial Ca2+ overload. Therefore, there is an increasing interest in developing cardiovascular therapies with mitochondrial molecular targets. Among these therapies, gene therapy has great importance due to its overwhelming effects. However, an effective in vivo cardiovascular gene transfection vehicle has not been developed. This project consists in the design and development of a biodegradable and biocompatible nanovehicle suitable for siRNA delivery to in vivo systems, with the objective of decreasing mitochondrial dysfunction in myocardial cells through the reduced expression of the Mitochondrial Calcium Uniporter (MCU), protein which transports Ca2+ from the cytosol into the mitochondria.

In this work, the results of the first stage of this project will be presented. Two potential nanovehicles for siRNA delivery were designed, optimized and characterized with respect to size, surface charge and siRNA loading. The optimal size for both nanovehicles was ~100 nm, with surface charge close to neutrality through PEGylation, and a preliminary siRNA loading of 60 %. In addition, the nanovehicles have no cytotoxic effects up to 1000 μg/mL. Besides, the release profile and protection of siRNA is comparable to reported values using polymeric vehicles. These results show the possibility of developing an adequate siRNA delivery system with the potential of in vivo delivery to treat cardiovascular diseases through gene therapy.

Support or Funding Information

ALA is supported by CONACyT MSc scholarship

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards independent cellular release of a multi‐biomolecule system using gold nanorods: Simultaneous up and down regulation of cellular pathways with light control
Authors: Grossman Shannon , Morgan Erin , Reich Norbert
Published: June 2021

The control of cellular pathways using light has enormous potential to improve our understanding of biological processes for basic science and biomedical applications. No such technology is currently available to simultaneously up and down regulate multiple inputs. We have developed a robust gold nanorod platform that allows the controlled delivery of regulatory proteins and nucleic acids, relying on benign near infrared light exposure. Gold nanorods (GNR) are used because they absorb in the NIR region, they can release their cargo through breaking gold thiol bonds, and are capable of endosomal release. GNR that have peak absorbances at ~750 nm (GNR750) and ~1000 nm (GNR1000 ) were synthesized and functionalized with double stranded DNA via a gold‐thiol bond. GNR were irradiated with 800 nm pulsed light for 15 seconds, then separate samples were irradiated with 1150 nm pulsed light for 15 seconds. DNA release was quantified using TECAN fluorescence intensity assay and compared against controls. GNR750 showed ~50% release as expected when irradiated with 800 nm pulsed laser, and negligible release when irradiated with 1150 nm pulsed laser. GNR1000 showed the same release patterns after adjusting laser settings to lower energy/pulse at 730 nm. These results show that we have the ability to precisely release each cargo specifically at a single wavelength and not release it at another. We are currently working on using this platform to induce an “OFF‐ON‐OFF” sequence of red fluorescent protein expression in a mutant reporter cell line through the delivery of siRNA and Cre Recombinase protein. This proof of concept will move the project towards our long term goal of providing a novel method to control cellular events, thereby enabling fundamental interrogation in ways not presently achievable.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differentially expressed proteins are caused by increased chemokine ligand 2 in mice hippocampi after alcohol treatment
Authors: Cook Rachel , Lawrence James , Bray Jennifer
Published: June 2021

When alcohol enters the central nervous system (CNS) it disrupts the physiological balance, producing several negative effects on the nervous system, specifically learning and memory. It has been shown that the neuroimmune protein Chemokine (CC motif) Ligand 2 (CCL2), perturbs the negative effects alcohol has on the hippocampus, the region of the brain associated with learning and memory. CCL2 transgenic mice that overexpress this protein have been used to study the phenotypical and behavioral effects of alcohol use. Although the signaling cascade initiated by CCL2 is currently unknown, identification of proteins responsible for these effects is possible through the combination of SDS‐PAGE and high‐resolution mass spectrometry. Using these techniques, a molecular fingerprint was produced by identification of differentially expressed proteins located in hippocampus of CCL2‐transgenic mice when compared to wild type mice.

Over a time course of ten days mice of varying ages consumed 1g/kg of 20% ethanol every day. After the alcohol treatment, the left‐hemisphere hippocampus was extracted for proteomic analysis and the right‐hemisphere hippocampus was used for quantification of CCL2 concentration. This ensured identical biological samples for cross‐comparison between CCL2 expression and the effects this had on the proteome of the hippocampus. ELISA was used to quantify CCL2 concentration. Proteins from the left hemisphere were extracted and subsequently resolved using large format SDS‐PAGE analysis. Differentially expressed proteins were identified by Quadropole‐Time of Flight tandem mass spectrometry. Identification of proteins was done using a combination of MassHunter Qualitative, ProFinder, and NCBI databases.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Candidate urine biomarker discovery from only five pairs of samples before and after tumor resection in glioma patients
Authors: Wu Jianqiang , Zhang Jun , Zhao Yuanli , Gao Youhe
Published: June 2021

Without control of the homeostatic mechanisms, urine accumulates systemic changes in the body to be an excellent biomarker source. But urine is affected by many factors other than the disease. Even though many candidate biomarkers are discovered in animal models, it may still require large number of clinical samples for successful candidate biomarkers discovery. Self‐control study may effectively reduce individual differences among patients. Gliomas are the most common primary malignant brain tumors with a very poor prognosis. Early diagnosis of gliomas and monitoring of tumor recurrence is crucial to improve the outcome of glioma patients. However, whether it is possible to find several promising biomarkers in relatively small samples of glioma patients remained unknown. In this study, urine samples of five glioma patients were collected at the time of tumor diagnosis and after total surgical removal of the brain tumor. A comparative proteomic analysis of urine samples before and after tumor resection were performed using liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS). Using label‐free proteome quantification, a total of 1422 urinary proteins were identified. It was found that 30 urine proteins changed significantly after tumor resection and 12 differential proteins had been reported to be associated with gliomas in previous studies, such as CEACAM1, ANXA7, CRYAB, pIgR, cathepsin D, beta‐glucuronidase, acid ceramidase and CD276 antigen. After functional annotation of these differential proteins, it was found that regulation of tissue remodeling, autophagy, negative regulation of gene expression, positive regulation of natural killer cell mediated cytotoxicity and angiogenesis were significantly enriched. In this study, candidate urinary biomarkers were identified in glioma patients that have the potential to detect the disease and monitor tumor recurrence after treatment. Our results suggested that self‐control before and after tumor resection is an effective way for finding differential proteins related to the tumor even in small number of urine samples. Our findings will provide clues for future biomarker studies involving brain cancers.

Support or Funding Information

This work was supported by the National Key Research and Development Program of China (2016YFC1306300), Beijing Natural Science Foundation (7173264, 7172076), and funds from Beijing Normal University (11100704, 10300‐310421102).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cerebrospinal fluid proteomics for identification of potential biomarkers to monitor pharmacological therapeutic efficacy in dopamine dictated disease states of Parkinson's disease and schizophrenia
Authors: HARIPRASAD GURURAO , Gupta Ashish Kumar , Pokhriyal Ruchika Kumar , Khan Mohd Imran , Kumar Domada Ratna , Goyal Vinay , Chadda Rakesh Kumar , Ramachandran Rashmi
Published: June 2021

Parkinson's disease and schizophrenia are disease end points of dopaminergic deficit and hyperactivity, respectively in the mid brain. Accordingly, current pharmacological interventions aim to restore normal dopamine levels, overshooting of which results in adverse effects of psychosis in Parkinson's disease and extra‐pyramidal symptoms in schizophrenia. Currently, there are no available laboratory tests to guide treatment decisions or help predict drug toxicity. Isobaric tag for relative and absolute quantification (iTRAQ) based proteomic experiments coupled with mass spectrometric analysis has been carried out using cerebro‐spinal fluid from drug naïve patients of Parkinson's disease (Unified Parkinson Disease Rating Scale), patients with urological diseases that served as neurological controls, and schizophrenia with hallucinations (International Statistical Classification of Diseases‐10). The qualitative and quantitative protein expression profiling across the three phenotypes helped to identify proteins that varied across the dopaminergic clinical spectrum. While ectonucleotide pyrophosphatase, somatomedin A and apolipoprotein D were directly proportional to mid‐brain dopamine concentrations; IgG Fc‐binding protein, complement C3, apolipoprotein C‐III, alpha‐2‐macroglobulin, complement 1, somatomedin A, and melanoma cell adhesion molecule were inversely proportional to mid‐brain dopamine concentrations. Validation included detailed pathway analysis that helped to establish the interactions of these proteins in dopamine release and signaling pathways, and also provided plausible rational for their regulation in Parkinson's disease and schizophrenia. Theses proteins either individually or as a panel are an ideal platform to develop biomarkers to assist clinicians to monitor pharmacological interventions in dopamine dictated disease states of Parkinson's disease and schizophrenia thereby increasing therapeutic efficacy.

Support or Funding Information

This research was supported by Department of Science and Technology, Government of India (SB/SO/BB‐0122/2013).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.