bluuebunny/crossref_metadata_2025 huggingface dataset:
Preclinical Trials of Amblyomin‐X: a Toxicity Study in Mice
Authors: Chudzinski‐Tavassi Ana Marisa , Will Sonia Elisabete , Sciani Juliana Mozer , Bosch Rosemary , Goldfeder Mauricio B. , Maria Durvanei A.
Published: November 2023

Introduction

Amblyomin‐X is a recombinant protein, which cause apoptosis of tumor cells, in a selective way, beside tumor regression, in in vivo tumor models. Due to the interesting effects, this protein is being developed as an antitumor drug, and toxicity studies in animals are required by regulatory agencies. Amblyomin‐X selectively induces apoptosis in tumor cells and was able to promote a reduction in tumor size in a melanoma model.

Aim

The aim of this study was to perform preclinical assays to evaluate the toxicity of Amblyomin‐X in mice after acute (200 mg/kg) and chronic (5 and 50 mg/kg per day) intravenous administration. During the experimental period, indications of toxicity, body weight variations and water and food consumption were evaluated. No mortality was observed following treatment with Amblyomin‐X.

Results

Amblyomin‐X is well tolerated by mice, both male and female, that were treated with a high concentration of the drug (200 times higher than the effective dose therapy) and therefore is not be expected to have any acute or long‐term adverse effects in humans. The therapeutic concentration of Amblyomin‐X, which is less than 5 mg/kg, causes tumor reduction in mouse models and is pharmacologically safe. In general, Amblyomin‐X did not cause clinical alterations, and treated animals exhibited no changes in hematological and biochemical parameters. The macroscopic and histologic analysis of organs demonstrated that the protein increased the white pulp of the spleen, caused vacuolization in hepatocytes and induced intestinal lesions, which can be considered toxic effects of Amblyomin‐X, at concentrations of 50 mg/kg to 200 mg/kg. However, these effects do not appear to alter the function of the organ or homeostasis.

Conclusion

After this study, we can conclude that the liver changes that were observed in animals treated with high doses of Amblyomin‐X are changes that can take place without jeopardizing the integrity of the organ, which is responsible for the most important metabolic processes in the body. Careful interpretation and extrapolation of the data obtained from these models are essential to reduce the current clinical costs of new anticancer therapeutic agents for humans. This study provides data regarding important safety parameters that enable the implementation of further experiments aimed at developing this molecule as an anti‐tumor drug.

Support or Funding Information

Support by FAPESP
Ataxin3 Deubiquitination Activity is Stimulated by Association with Specific Binding Partners
Authors: Rao Maya V. , Loll Patrick J.
Published: November 2023

Ataxin3 is a cysteine protease that carries out deubiquitination (DUB) and contributes to protein quality control in the cell. It specifically interacts with the molecular motor protein p97 to extract misfolded protein substrates from the endoplasmic reticulum to the cytosol, and thus plays a crucial role in endoplasmic ‐reticulum‐associated degradation. The extraction depends on the ubiquitination of the substrate, which helps recruit p97 and also tags the substrate for degradation. We believe this tag is trimmed or cleaved by ataxin3 to facilitate entry of the substrate into the extraction or degradation apparatus. Furthermore, ataxin3 also directly recognizes and binds both mono‐ubiquitin and poly‐ubiquitin chains. We propose that ataxin3 interacts with both p97 and the ubiquitinated substrate, forming an active complex enabling cleavage of the ubiquitin chain and handoff of the substrate to the degradation machinery. Using surface plasmon resonance, we have characterized the affinity of p97 for ataxin3, and assessed the effect of p97's nucleotide‐binding state on its association with ataxin3. We also propose that ataxin3's DUB activity is regulated by its interaction with these binding partners. By use of a fluorogenic enzyme assay, we have shown that ataxin3 activity is enhanced in the presence of ubiquitin and is further increased by p97. Such robust stimulation of DUB activity has not been described previously.
Expression of Herpud1 during osteoclast and osteoblast differentiation
Authors: Quiroga Clara , Da Silva Luan Americo , Mansilla Georthan , Oyarzún Ingrid , Bustamante Mario , Castro Pablo , Verdejo Hugo
Published: November 2023

Bone is a dynamic tissue characterized by its hardness, rigidity, and high power of regeneration and repair. Its integrity is the result of balance among synthesis mediated by osteoblasts and resorption mediated by osteoclast and the the cross‐talk between them is key to maintain tissue homeostasis.

Multiple extra and intracellular signals play an important role in differentiation and function of osteoblasts and osteoclast. Protein quality control (QC) mechanisms is a newly discovered actor in osteoblastogenesis, which requires the activation of proteins involved in endoplasmic reticulum (ER) stress response, particularly the PERK/EIF2AK3‐eIF2A‐ATF4 axis. In contrast, the resorptive phenotype of osteoclast requires activation of macroautophagy machinery.

To maintain cellular homeostasis and control the increase of proteotoxic aggregates into the ER, cells activate a program of response (Unfolded Protein Response or UPR) that include the reduction of general protein synthesis, an increase in chaperones synthesis and an increase in the ER‐associated degradation (ERAD).

Herpud1, an ER‐membrane integral protein, is highly expressed in response to ER‐stress and is involved in the regulation of ERAD protein complex. Recently we described that a reduction in Herpud1 levels promotes an increase in autophagic flux, showing that this protein negatively controls this mechanism.

In function of these antecedents, we aim to assess the role of Herpud1 during the osteoblast and osteoclast differentiation. Pre‐osteoblast MC3T3 and pre‐osteoclast RAW264.7 cell lines were treated with different ER stressors and autophagy inducers in order to compare cellular responses and were maintained in differentiation medium for 3, 7 and 14 days. In case of MC3T3, cell differentiation was evaluated through increases in calcium deposits, alkaline phosphatase (ALP) activity and expression of gene markers of differentiation like RUNX2, OPN, OCN. On the other hand, differentiation of RAW264.7 was assessed by expression and activity of Tartrate‐resistant acid phosphate (TRAP), MMP9 and others. The levels of Herpud1 were determinated through western‐blot and real time PCR.

Our results show that Herpud1 levels increase in response to ER‐stressors. Although in initial stages of differentiation (12, 24 h) Herpud1 increases in both cellular lineages, long times of differentiation showed a divergent regulation in its expression. Herpud1 increases during osteoblastogenesis and decreases during osteoclastogenesis, suggesting that Herpud1 has a differential role in osteoblasto‐ and osteoclastogenesis, and could be a candidate to regulate the bone turnover.

Support or Funding Information

This work is supported by FONDECYT 11140470 (CQ), FONDECYT 1150359 (HV) and FONDAP 15130011 (PC, HV).
N‐end‐rule‐mediated Degradation of the Proteolytically Activated Form of PKC‐theta Kinase attenuates its Pro‐Apoptotic Function
Authors: Eldeeb Mohamed Ali , Esmaili Mansoore , Fahlman Richard
Published: November 2023

Cellular stresses and signalling that lead to the initiation of apoptotic pathways often result in the activation of caspases or calpains which in turn leads to the generation of proteolytically generated protein fragments with new or altered functions. Mounting number of studies reveal that the activity of these proteolytically activated protein fragments can be counteracted via their selective degradation by the N‐End Rule pathway. Here we investigate the proteolytically generated fragment of the PKC theta kinase, where we report the first study on the stability of this pro‐apoptotic protein fragment. We have determined that the pro‐apoptotic cleaved fragment of PKC‐theta is unstable in cells as its N‐terminal lysine targets it for proteasomal degradation via the N‐end rule pathway and this degradation is inhibited by mutating the destabilizing N‐Termini, knockdown of the UBR1 and UBR2 E3 ligases. Tellingly, we demonstrate that the metabolic stabilization of the cleaved fragment of PKC‐theta or inhibition of the N‐end rule augments the apoptosis‐inducing effect of staurosporine in Jurkat cells. Notably, we have demonstrated that the cleaved fragment of PKC theta, per se, can induce apoptotic cell death in Jurkat T‐cell leukemia. Our results expand the functional scope of N‐end rule pathway and support the notion that targeting N‐end rule machinery may have therapeutic implications.

Support or Funding Information

Mohamed Eldeeb is supported by Alberta‐Innovates Technology Futures (AITF) scholarship.
Bioinformatic and Functional Analysis of Venom from the Jewel Wasp Ampulex compressa
Authors: Arvidson Ryan , Kaiser Maayan , Pan SongQin , Libersat Frederic , Adams Michael E
Published: November 2023

The parasitoid jewel wasp Ampulex compressa exploits its host, the American cockroach Periplaneta americana, as a food source for its young. The host is subdued through direct envenomation into the central nervous system, inducing a days‐long “zombie‐like state” referred to as hypokinesia. Hypokinesia is a specific, venom‐induced behavioral state characterized by suppression of the escape response and reduced spontaneous walking, leaving other motor functions intact. This condition is reversible, provided that egg deposition is prevented. To facilitate elucidation of biochemical mechanisms underlying venom‐induced hypokinesia, we developed a comprehensive inventory of the venom by combining: 1) next‐generation RNA sequencing, transcriptomic analysis of venom gland components, and 2) proteomics of venom apparatus extracts and whole milked venom via mass spectroscopy‐based Multi‐Dimensional Protein Identification Technology. These analyses yielded a total of 201 proteins and peptides in the venom, 105 with Pfam domains, 146 similar to other proteins in the NCBI‐nr or Uniprot databases, and 45 novel proteins absent in global databases. A. compressa venom proteins include members of the M13 family of proteases (11), phospholipase A2 isoforms (10), along with hyaluronidase, chitinase, icarapin, venom acid phosphatase, serpins, and signaling peptides including tachykinin, corazonin, and myosuppressin. Differential expression analysis and proteomics of the two major tissues of the venom apparatus ‐ venom sac and venom gland ‐identified tissue‐specific expression of key venom components.

We show that A. compressa venom tachykinins (AcVTK) are able to activate the cockroach brain tachykinin receptor with high affinity (EC50 ~1 nM) in vitro. Injection of synthetic AcVTK into the subesophageal ganglion, an established target of the wasp venom, increases escape response threshold for one hour post‐treatment. These results indicate that tachykinin may participate in the behavioral changes resulting from envenomation of cockroach cerebral ganglia.

We isolated and characterized two novel, paralogous venom sac peptides found in milked venom. These peptides are alpha‐helical, amphipathic, and constitute a new family of venom toxins termed ampulexins. Injection of the most abundant venom peptide, ampulexin‐1, into the subesophageal ganglion of cockroaches resulted in a short‐term increase in escape threshold.

Our findings show that the venom of A. compressa is a rich collection of both well‐known and novel, pharmacologically diverse compounds. Our study provides new and comprehensive information on A. compressa venom contents that inform hypotheses regarding its mode of action.
Characterization of retinal thickness measurements and their relationship to visual acuity in progressing cases of dry macular degeneration
Authors: Klein Brandon J. , Khadjavi Lily S.
Published: November 2023

Dry macular degeneration has been linked to thinning of the retinal tissue layers bound between the internal limiting membrane and retinal pigment epithelium (ILM‐RPE). Development of optical coherence tomography (OCT) technologies has introduced a quick, non‐invasive method of measuring the ILM‐RPE thickness in a clinical environment. In this study, we analyzed clinical data including OCT measurements and standard exam data to investigate the relationship between retinal characteristics and visual acuity in 46 dry macular degeneration patients, ranging in age from 47 to 94 years. Strong positive correlations were noted between visual acuity and quantitative, qualitative, and longitudinal OCT measurements. In particular, a multiple linear regression model using only OCT outputs as explanatory variables could predict the patient's visual acuity.

Support or Funding Information

Loyola Marymount University Summer Undergraduate Research Program 2015
Syntrophic metabolism of vitamins and amino acids in gut microbial community as revealed by in silico genomic analyses
Authors: Khoroshkin Matvei Sergeevich , Rodionov Dmitry
Published: November 2023

Metabolite exchange is an important process shaping the structure of microbial communities. However, very little is known about crossfeeding by specific nutrients between microbial community members. Among micronutrients, vitamins of B‐group are of high significance as they are precursors of nearly all metabolic coenzymes that are universally essential in all forms of life. Amino acids are building blocks of proteins and peptides, and also serve as precursors of many essential metabolites including nucleotides, cofactors, etc. Many members of microbial communities are not capable to synthesize some vitamins or amino acids (auxotrophs), while other members possess complete biosynthetic pathways for these nutrients (prototrophs). Therefore, vitamin and amino acid exchange between auxotrophs and prototrophs can be an important factor shaping microbial community structure.

We used the metabolic subsystem approach implemented in SEED genomic platform to study potential metabolite exchange between bacteria that inhabit human gastrointestinal tract. We reconstructed metabolic pathways for synthesis of 8 B‐vitamins and 20 amino acids in 430 human gut bacteria with genomes sequenced by Human Microbiome Project. We also analyzed genomic distributions of known uptake transporters for vitamins and amino acids. The inferred vitamin and amino acid proto‐/auxotrophic phenotypes and predicted transporters allowed us to classify the studied organisms with respect to their biosynthetic and transport capabilities. The studied bacteria showed high level of conservation of proto‐, auxotrophic phenotypes on the taxonomic level of species. Incomplete biosynthesis pathways for some vitamins, such as biotin, thiamine and pantothenate, suggest certain vitamin deficiencies can be alternatively supplemented by their metabolic precursors (e.g. dethiobiotin, thiazole, pantoate). Overall, vitamin auxotrophic phenotypes are much more abundant in the human gut microbiota, whereas the larger number of studied bacteria is capable of de novo synthesis of all 20 amino acids. Histidine and tryptophan have the most variable phenotypes within the Clostridiales lineage suggesting that crossfeeding by these amino acids is important for gut community. In summary, the obtained in silico reconstructions of vitamin and amino acid metabolic pathways contribute to our understanding of metabolic crossfeeding processes in human gut microbiome.

Support or Funding Information

This research was supported by the Russian Science Foundation (grant #14‐14‐00289) and National Institute of Health (GM108527).
Molecular design and photophysics of curcuminoid chromophores for application as photoacoustic contrast agents
Authors: Mithila Farha J , Bellinger‐Buckley Stephanie , Frenette Mattheiu , Hatamimoslehabadi Maryam , Yelleswarapu Chandra , Rochford Jonathan
Published: November 2023

Photoacoustic Imaging (PAI) is a new emerging technique that combines optical absorption and ultrasonic emission to enable high resolution imaging in biological media. Targeted PAI requires engineering of exogenous photoacoustic contrast agents that are capable of converting light energy to sound energy. However, there is currently a lack of optimal contrast agents available specifically designed for PAI applications. Quadrupolar dyes, such as the curcuminBF2 chromophore, have recently been shown to possess important characteristics necessary for molecular photoacoustic contrast agents (MPACs). This study demonstrates that selective functionalization at the terminal positions of the curcuminoid chromophore, one can tune their absorption spectra toward the ideal transparent biological window. With this in mind, novel curcumin chromophores have been synthesized by adding aryl and heterocyclic substituents at the terminal position of the β‐diketonate difluoroboron backbone to investigate changes in their photophysical and photoacoustic properties. The 5‐methyl‐2,2′‐bithiophene substituent shows significant promise as it shifts the absorption maximum toward the biological window, similar to the dimethylaminobenzene substituent, in contrast to the p‐butoxyphenyl substituted system. Emission and absorption properties have been investigated along with computational analysis and preliminary photoacoustic studies.
Characterization of Leadmium Green for Fluorescence Detection of Intracellular Cadmium
Authors: Malaiyandi Latha M. , Sharthiya Harsh , Dineley Kirk E.
Published: November 2023

Cadmium is a common industrial pollutant, but the study of cadmium cytotoxicity is difficult in part due to the absence of a probe that can detect cadmium in living cells without the confounds of contaminating metals. Leadmium Green (LG) is a commercially available, small molecule, fluorescent probe advertised as a detector of intracellular cadmium and lead. LG has been used to monitor cadmium sequestration in plant cells, heavy metal export in a protozoan, and lead absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for cadmium, lead, and other metals. Using a cell‐impermeant form, we evaluated the in vitro selectivity of LG using spectrofluorometry. Consistent with manufacturer's claims, LG responded robustly to cadmium (KD 603nM) and also lead (KD 8.65nM) in a concentration‐dependent manner, and furthermore proved insensitive to calcium, cobalt, manganese and nickel. However, LG also responded to zinc with a KD of 81.9nM. Using fluorescence microscopy, we evaluated the utility of LG in live mouse hippocampal HT22 cells. We demonstrated that LG detects ionophore‐mediated elevations of cadmium or zinc in a concentration‐dependent manner. In comparison to zinc changes reported by the widely used zinc fluorescent probe FluoZin‐3, LG yielded a relatively small dynamic range. In conclusion, LG may have utility as an intracellular cadmium probe, however data must be interpreted cautiously due to potential contribution of zinc to the signal.
Purification of Truncated Wild‐Type and Mutant Recombinant Lipoprotein Lipase and Investigating Their Ability to Regulate ABCA1
Authors: Madhwani Kimberly Rose , Medh Jheem D.
Published: November 2023

Background

Lipoprotein lipase (LPL) catalyzes the hydrolysis of triglycerides in extracellular lipoprotein particles. It is expressed by macrophages and secreted to the cell surface. The LPL molecule is comprised of a large amino‐terminal domain that contains its lipolytic activity, and a small carboxyl‐terminal domain that functions as an adapter between lipoprotein receptors/cell surface proteoglycans and apolipoproteins. From previous studies, we discovered that silencing the LPL gene in THP‐1 macrophages promotes the expression and function of an atheroprotective cholesterol transporter, ABCA1. Thus, the objective of this project is to synthesize recombinant LPLC, a truncated molecule lacking the catalytic domain; and LPLC‐WW, a mutant carboxyl‐terminal domain that is unable to bind lipoproteins. Our long‐term goal is to determine the molecular components of LPL that contribute to its ability to modulate ABCA1 expression.

Methods

Recombinant glutathione‐S‐transferase (GST) fusion proteins were prepared using pGEX‐2T plasmids. These plasmids, containing the genes for the WT and WW LPLC domain were obtained as a gift. pGEX‐2T LPLC‐WT and LPLC‐WW plasmids were transformed into competent E.coli cells and plated on LB+ampicillin agar plates. Viable colonies were picked and inoculated into 2‐ml cultures of 2X‐YTA medium, before being transferred to a large‐batch culture. The larger cultures were induced with 100 mM IPTG for 2–6 hours, pelleted and resuspended with 1X PBS. Resuspended pellets were sonicated at 37º for 10 minutes and solubilized in 20% Triton X‐100. Glutathione Sepharose 4B was used to immobilize the GST fusion proteins while the contaminants were washed away with 1X PBS. Thereafter, the fusion proteins were eluted from a small aliquot of beads by Elution Buffer containing 10 mM Glutathione. The elution of GST‐LPLC was confirmed by 15% SDS‐PAGE. Next, a thrombin mixture was added to the Glutathione Sepharose 4B‐bound fusion proteins, and incubated for 12–16 hours to cleave and separate the LPLC proteins from the immobilized GST. The presence of purified LPLC proteins was confirmed by 15% SDS‐PAGE and Coomassie Blue staining.

Results

Both LPLC‐WT and LPLC‐WW were obtained free of any contaminants including GST. We obtained 106 μg of LPLC‐WT and 116.7 μg of LPLC‐WW and are continuing to scale‐up the purification.

Conclusions

The pGEX‐2T plasmids are suitable tools for the preparation of GST fusion proteins. The use of thrombin and a glutathione‐sepharose column efficiently removed the GST component to obtain pure protein. The recombinant LPLC‐WT and LPLC‐WW will be valuable reagents for studies on the molecular components required for the modulation of ABCA1 expression.

Support or Funding Information

This work was supported by National Institutes of Health Award SC3GM095413.
Optical Tools to Assess Heart Development
Authors: Jenkins Michael W
Published: November 2023

For years, researchers have shown that altered cardiac function can lead to congenital heart defects (CHDs); yet many years later, we still have limited information on when, how and to what degree the developing cardiovascular system is most vulnerable to abnormal cardiac function. Studying the impact of abnormal function becomes even more urgent when we consider that altered function may be a contributing factor to a large percentage of CHDs regardless of whether the initial trigger is environmental or genetic. Most tools available to developmental cardiology researchers assay embryos after they have been sacrificed, which makes assessment of the impact of function extremely difficult. To assess the impact of hemodynamics on development, we need to monitor and reproducibly perturb blood flow, and then quantify the resultant effects on morphology. Our lab has developed optical pacing technology to increase regurgitant flow in early heart development along with optical coherence tomography technology to measure the perturbed hemodynamics and structural defects. Briefly, pacing‐induced regurgitation led to a series of CHDs including valve defects, ventricular septal defects, hypoplastic right ventricles and common atrioventricular canals. Utilizing optical tools to understand the influence of hemodynamics in development is an important step towards determining CHD mechanisms and ultimately developing earlier treatments.
Regulating Skeletal Progenitor Cells at the Interface Between Tendon and Bone
Authors: Merrill Amy Elizabeth , Roberts Ryan
Published: November 2023

The surfaces of bones are adorned with projections that mark their integration into the musculoskeletal system. These structures, known as bone eminences, take on diverse shapes and sizes to form anchoring points for tendons and ligaments. Bone eminences represent the mineralized side of the tendon‐bone attachment unit, which is composed of a transitional connective tissue, graded from tendinous to osseous. As such, the eminence plays a critical role transmitting forces generated by muscle contraction and dissipating stress concentrations across the hard‐soft tissue interface. During development, the tendon‐bone attachment unit descends from a unique pool of Scx+/Sox9+ progenitors that form secondary to those of the primary cartilage anlage and later become segregated into Scx+ tenocytes or Sox9+ chondrocytes of the eminence (Bltiz et al., 2009, 2013; Sugimoto et al., 2013). While many open questions remain about the molecular regulators that demarcate this boundary across the Scx+/Sox9+ progenitor field, chick embryo studies suggest a role for Fibroblast Growth Factor (FGF) signaling (Brent et al., 2003, 2004, 2005). We have uncovered evidence that Fgfr2 regulates cell fate choice during development of tendon‐bone attachment units in the craniofacial complex of mice. We have found that loss of Fgfr2 increases the eminence superstructure by promoting chondrogenic over tenocytic cell fate in the attachment unit progenitors. These findings contribute to the understanding of tendon‐bone attachment unit development and reveal a possible mechanism to explain the constellation of clinical abnormalities of the tendon‐bone interface that are found in the skeletal birth defects caused by gain and loss of FGFR2 function.

Support or Funding Information

NIH, NIDCR #1R01DE025222 to A.E.M.

March of Dimes, Gene Discovery and Translational Research Grant #6‐FY15‐233 to A.E.M.

NIH, NIDCR #T90DE021982 to R.R.
Mutational Analysis of the Catalytic Mechanism of a Peroxidase Enzyme from the Diatom Thalassiosira pseudonana
Authors: Bordeaux Evelyn , Rampur Saveeta , Frato Katherine
Published: November 2023

Heme iron is integral to numerous biochemical processes including electron transfer, oxygen transport, and catalysis of oxidation‐reduction reactions. Given that iron is only sparingly soluble in water, enzymes that contain iron in marine organisms must play an important physiological role. We have identified a putative heme‐containing enzyme, Tp21683, in the published genome of the marine diatom Thalassiosira pseudonana. Tp21683 has a C‐terminal predicted b‐type heme peroxidase domain and an N‐terminal cysteine‐rich domain of unknown function. We hypothesize that the role of Tp21683 is to relieve oxidative stress, a biological process critical to survival of photosynthetic organisms including diatoms. Here, we report the recombinant expression of the C‐terminal peroxidase domain of Tp21683 in Escherichia coli and successful refolding and purification to greater than 80% purity. Preliminary data indicate that Tp21683 turns over hydrogen peroxide in the presence of the small molecule electron donors ABTS and guaiacol. To confirm that the peroxidase activity of Tp21683 proceeds by a mechanism similar to other b‐type heme peroxidases, we constructed two separate site‐directed mutants at key amino acids in the predicted active site of Tp21683. We hypothesize that replacing the distal histidine residue, His221, with alanine will result in severe deficiencies in hydrogen peroxide turnover rate because a key proton donor and acceptor has been removed from the active site. Mutation of a conserved tryptophan located near the proximal histidine, Trp365, with a phenylalanine residue should highlight whether Tp21683 forms a protein‐based cation radical or a porphyrin‐based cation radical during the first step of hydrogen peroxide turnover. If Tp21683 uses a protein‐based radical, like the model enzyme yeast cytochrome c peroxidase, the W365F mutation will lead to a dramatic decrease in turnover rate because the enzyme is unable to form a stable compound I. Tp21683 mutants H221A and W365F will be recombinantly expressed and purified similarly to the wild‐type enzyme and the kinetic parameters will be assessed in the presence of hydrogen peroxide and ABTS. Verifying the roles of histidine 221 and tryptophan 365 in Tp21683 will further support our assignment of this previously uncharacterized enzyme as a heme peroxidase.
Fluorescent Assay of Ubiquitin Conjugation
Authors: Jackson Cassidy , Berndsen Christopher
Published: November 2023

Ubiquitination of proteins is a post translational protein modification linked to protein degradation, cell cycle regulation, and DNA damage repair. Many of the functions of ubiquitin and ubiquitin like proteins are still unknown and the catalytic mechanisms of the conjugating enzymes are partially known. The assays of ubiquitin/ubiquitin‐like protein conjugating include observing changes in migration on gel electrophoresis or radioactive tags which are expensive and require special handling. We have optimized a fluorescence based assay that quantifies the cleavage of ATP to AMP by the E1 ubiquitin/ubiquitin‐like protein activating enzyme. This assay permits study of the kinetics of ubiquitin conjugation in a continuous or discontinuous manner and is highly sensitive. We have optimized the conditions for monitoring E2 dependent ubiquitin chain formation in order to determine the mechanism of E1 and E2 enzymes. This assay will help in the understanding of the mechanism of ubiquitin and ubiquitin like protein conjugation.

Support or Funding Information

US‐Israel Bi‐national Science Foundation 2013261
Probing the Role of a Catalytic Residue in a Lysine Deacetylase
Authors: Nichols Kyara A , Toro Tasha B , Watt Terry J
Published: November 2023

Acetylation of lysine is a common post‐translational protein modification. Lysine deacetylases (KDACs) reverse this modification. However, little information is known concerning the role of several conserved active site residues in KDACs. Studies have shown that if the reversal of this modification does not take place correctly, it can lead to many human diseases, including cancers. Recent studies also suggest that an active site residue that is found as either a tyrosine or histidine in human KDACs is important for enzymatic activity; however, how this active site residue functions is not entirely understood. Wild‐type KDAC7 contains a histidine at this position and is generally inactive with known KDAC substrates, whereas wild‐type KDAC8 contains a tyrosine residue and has considerable activity. To begin to understand how this residue affects catalytic activity of the enzyme, we created variants of KDAC7 and KDAC8 containing selected amino acids in this position of the active site, then expressed and purified the variants using both insect and E. coli cells. The resulting proteins were evaluated using several substrates to determine how each mutation affected the activity and substrate specificity of the enzymes. The KDAC variants tested demonstrated distinct specificity profiles for those substrates that have been tested thus far. Circular dichroism spectroscopy was used to analyze the secondary structure of the variants and revealed that reductions of enzyme activity were not due to major structural perturbations. We are currently determining kinetic parameters for the KDAC variants with multiple peptide substrates to determine whether the active site residue affects catalysis, substrate binding, or both. This approach will ultimately further our understanding of the mechanism by which KDACs function to deacetylate their target substrates.
Structural and Functional Substrate Binding in an Iterative Non‐ribosomal Peptide Synthetase Independent Siderophore (NIS) Enzyme from Streptomyces coelicolor
Authors: Hoffmann Katherine M
Published: November 2023

Prokaryotes scavenge iron using small molecule chelators called siderophores, made through a novel chemistry of peptide bond formation outside of a ribosome. One type of siderophore synthesis uses Non‐ribosomal peptide siderophore Independent Synthesis enzymes (NIS synthetases) to form amide bonds (peptide bonds) between metabolic intermediates in NIS siderophore biosynthesis pathways. Recently, primary structure analysis, correlated with known activity, has identified four subtypes (A, A′, B and C), only two of which have been structurally described. While subtype is based on the primary substrate specificity, an additional remarkable kinetic behavior exists in a sub‐category of enzymes whereby multiple iterative bonds may be catalyzed on the same substrate. Prior work in the field has identified a novel protein fold (including a novel ATP binding motif,) but several sub‐types and iterative proteins are yet to be structurally or biochemically described. The iterative activity is correlated with broad substrate specificity, but the binding affinity has never been quantified. In fact, iterative enzyme kinetic parameters have been elusive, as the first published biochemical characterization found the reporters limited the speed of the assays.

We are structurally and functionally characterizing a representative iterative NIS enzyme, DesD, from Streptomyces coelicolor, using the methodologies of X‐ray crystallography and isothermal titration calorimetry (ITC). Our results suggest a firm order of binding and significantly more substrate specificity than previously thought. Additionally, the innovative use of calorimetry to measure kinetic parameters has allowed a reporter‐free assay that may be broadly applicable to other processive enzymes. Finally, we are structurally characterizing the apo, partial complex, and full complex of DesD bound to it's high‐specificity substrates, to fully map binding interactions. We hope future work will include the design and synthesis of inhibitors, as well as site‐directed mutagenesis, to probe mechanism of the novel chemistry.
The Structural Basis of Cyclooxygenase‐2 Inhibition by Fenamates
Authors: Xu Shu , Banerjee Surajit , Kudalkar Shalley N , Perry Evan T , Ghebreselasie Kebreab , Marnett Lawrence J
Published: November 2023

Two cyclooxygenase (COX) isoforms, COX‐1 and COX‐2, catalyze the first two steps of prostaglandin biosynthesis from arachidonic acid (AA) and are the pharmacological targets of non‐steroidal anti‐inflammatory drugs (NSAIDs). Mefenamic acid is one of several NSAIDs that have shown selective inhibition of 2‐arachidonylglycerol (2‐AG) oxygenation by COX‐2 (Prusakiewicz J.J. et al, Biochemistry 2009). While the X‐ray crystal structures of most classes of NSAIDs bound to COX have been solved, the binding mode of fenamates is still unknown. Here we report the structural determination of murine COX‐2 structures complexed with several fenamates and biochemical investigation of the role of individual residues in their inhibition. The crystal structures for the first time revealed that the fenamates are bound in the cyclooxygenase channel in an inverted binding mode similar to those of propionic acid derivatives such as diclofenac and lumiracoxib. The side chains of Tyr‐385 and Ser‐530 form critical H‐bonds with the carboxylic group of the fenamates. The aniline ring substituents of mefenamic acid, tolfenamic acid and flufenamic acid exhibit a preference for the space beneath Leu‐531 as opposed to the side pocket off the active site channel. In contrast, when there is an additional chloride substituent, as in the case of meclofenamic acid, this extra substituent orients toward the side pocket. These observations provide a structural basis for the fact that meclofenamic acid is a slow, tight‐binding inhibitor of COX‐2 with a low IC50 (31 nM) whereas mefenamic acid, flufenamic acid, niflumic acid, and tolfenamic acid are weak, rapidly reversible inhibitors with IC50's greater than 10 μM for AA oxygenation. Strikingly, when 2‐AG is used as the substrate, these weak, rapidly reversible inhibitors become potent inhibitors with IC50's ranging from 20 to 50 nM. In contrast, meclofenamic acid exhibits the same potency for both substrates. Our results provide the first atomic level detail of the interaction between fenamates and COX‐2, structural insights of slow, irreversible and rapid reversible fenamates, and a biochemical and structural foundation for future design and development of substrate‐selective inhibitors of COX‐2.

Support or Funding Information

The work was supported by NIH CA089450 (to L. J. M.).
Cell polarity kinase MST4 cooperates with cAMP‐dependent kinase to orchestrate histamine‐stimulated acid secretion in gastric parietal cells
Authors: JIANG HAO , Wang Wenwen , Zhang Yin , Yao William W. , Ding Xia , Liu Xing , Yao Xuebiao
Published: November 2023

The digestive function of the stomach depends on acidification of the gastric lumen. Acid secretion into the lumen is triggered by activation of PKA cascade, which ultimately results in the insertion of gastric H, K‐ATPases into the apical plasma membranes of parietal cells. A coupling protein is ezrin whose phosphorylation at Ser66 by PKA is required for parietal cell activation. However, little is known regarding the molecular mechanism(s) by which this signaling pathway operates in gastric acid secretion. Here we show PKA cooperates with MST4 to orchestrate histamine‐elicited acid secretion by phosphorylating ezrin at Ser66 and Thr567, respectively. Histamine stimulation activates PKA which phosphorylates MST4 at Thr178 and then promotes MST4 kinase activity. Interestingly, activated MST4 then phosphorylates ezrin pre‐phosphorylated by PKA. Importantly, MST4 is important for acid secretion in parietal cells because either suppression of MST4 or overexpression of non‐phosphorylatable MST4 prevents the apical membrane reorganization and proton pump translocation elicited by histamine stimulation. In addition, overexpressing MST4 phosphorylation‐deficient ezrin results in an inhibition of gastric acid secretion. Taken together, these results define a novel molecular mechanism linking PKA‐MST4‐ezrin signaling cascade to polarized epithelial secretion in gastric parietal cells.

Support or Funding Information

This work was supported by Chinese 973 Project 2013CB911203; Ministry of Education 20113402130010; Anhui Project 08040102005; Chinese Natural Science Foundation Grants 31430054, 31271518, 31301105, 90508002 and 90913016; National Institutes of Health Grants CA164133 and DK56292; China Postdoctoral Science Foundation Grant 2014M560517; and Central University Grants WK2340000032 and WK2340000021.
Transcriptome Profiling of Burkholderia thailandensis Reveals that MftR is a Master Regulator
Authors: Gupta Ashish , Wang Guannan , Dassanayake Maheshi , Grove Anne
Published: November 2023

The Burkholderia thailandensis‐encoded major facilitator transport regulator (MftR) is a multiple antibiotic resistance regulator (MarR) homolog. mftR is divergently oriented to a gene encoding major facilitator transport protein (MftP), which is a major facilitator superfamily efflux pump. The mftR‐mftP locus is conserved in B. cenocepacia, which is a serious pathogen of patients with cystic fibrosis. Biofilm formation by this bacterium makes it less sensitive to antibiotics, making infection by B. cenocepacia difficult to treat. To combat the bacterial infection, the host produces reactive oxygen species (ROS), and one enzyme involved in this process is xanthine oxidase, which also produces urate. The invading pathogen may therefore detect urate and respond by upregulating the expression of virulence genes. MftR responds to urate by upregulation of mftR and mftP. To see the global effect of urate on B. thailandensis and the effect of MftR, we carried out transcriptome sequencing (RNA‐seq) analyses of wild type (WT) and mftRΔ cells grown in absence and presence of urate. The analysis of RNA‐seq data shows that ~330 genes are differentially expressed (>2‐fold) in the presence of urate and that MftR regulates ~310 genes. In mftRΔ and in WT grown in presence of urate, some genes are either up‐ or downregulated compared to WT, consistent with MftR controlling expression. For example, both mftRΔ and urate upregulate genes encoding enzymes responsible for the synthesis of 2‐heptyl‐4‐quinolone (HHQ). This signaling quinolone participates in cell‐cell communication. Genes encoding type III secretion system are repressed in mftRΔ and with urate. Some genes are differentially regulated by urate and not by MftR, suggesting the existence of additional factors that respond to urate; urate upregulates the whole flagellar gene cluster in the mftRΔ strain. Flagella play a vital role in biofilm formation and host invasion. Taken together, our data suggest that the urate‐responsive MftR is a master regulator that controls expression of genes associated with host colonization later in infection.

Support or Funding Information

NIH grant (1R15GM107825) to Anne Grove
Biochemical characterization of the substrate specificity of two unique members of the mammalian protein arginine methyltransferase family, PRMT7 and PRMT9
Authors: Hadjikyriacou Andrea , Feng You , Yang Yanzhong , Bedford Mark T. , Clarke Steven G.
Published: November 2023

Protein arginine methylation is a widespread and important posttranslational modification in eukaryotic cells, shown to be involved in the activation or repression of transcription, modification of the splicing machinery, signaling, and DNA repair. Mammalian protein arginine methyltransferases include a family of nine sequence‐related enzymes that transfer one or two methyl groups onto the terminal guanidino groups on arginine residues, producing monomethylarginine only (MMA, type III), symmetric dimethylarginine (SDMA) and MMA (Type II), or asymmetric dimethylarginine (ADMA) and MMA (Type I). While PRMT1, 2, 3, 4, 6, and 8 have been characterized as type I enzymes, and PRMT5 as a type II enzyme, the role and activity types of the two final members of this family of enzymes, PRMT7 and PRMT9, had been unclear due to conflicting results in the literature, and the substrates for these enzymes had been elusive. Both PRMT7 and PRMT9 are distinct members of the family with two methyltransferase or methyltransferase‐like domains and containing acidic residues in otherwise well‐conserved substrate double E binding motif, features not seen in the other PRMT enzymes. Recent work in our laboratory confirmed PRMT7 as the only type III MMA‐forming enzyme in the group, with a unusual low temperature optimum for activity, and a heretofore not seen preference for a basic stretch of residues in an R‐X‐R sequence for methylation. Mutations of the acidic residues in the substrate‐binding motif results in a loss of the specific R‐X‐R activity and the appearance of a G‐R‐G specificity typical of many of the other PRMTs. The physiological substrate of PRMT7 has yet to be confirmed, although histone H2B is an effective in vitro substrate. PRMT9, on the other hand, had no reported activity, until immunoprecipitation from HeLa cells showed it pulled down two splicing factors, SF3B2 and SF3B4, in a complex. Amino acid analysis showed that PRMT9 methylates SF3B2 to produce both MMA and SDMA, thus making it the second type II enzyme in mammals. PRMT9 knockdown results in modulation of alternative splicing events. This enzyme appears to be relatively specific for the SF3B2 protein; a peptide containing the methylatable arginine residue was not found to be a substrate, and typical substrates of other PRMTs are not recognized by PRMT9. We found that the position of the methylated arginine residue in SF3B2 is important, and the acidic residues in the substrate‐binding motif also play an important role in substrate recognition. Thus, PRMT7 and PRMT9 represent unique members of the mammalian PRMT family.

Support or Funding Information

NIH grants GM026020 to S.G.C., DK062248 to M.T.B. and GM007185, a Ruth L. Kirschstein National Research Service Award to A.H.
A Combination Course and Lab‐Based Approach To Teaching Research Skills to Undergraduates
Authors: Parente Amy Diegelman , Brown Ronald C. , Danowitz Amy M. , Jones Clinton D. , Taylor Chris E.
Published: November 2023

Undergraduate research is an important capstone experience that provides students with the conceptual and technical aptitude for graduate or industrial research. However, this experience is often compressed into a single term in a course‐based undergraduate research experience (CURE) or run by individual faculty members for select students on an ad hoc basis as an undergraduate research experience (URE). One alternative to these two possibilities is to use a team‐taught course during students’ third year to help them develop fundamental skills that they use to prepare and present a research proposal. This proposal serves as a vehicle for improving students’ ability to assimilate information from the primary research literature and for them to practice evaluating the safety and research ethics aspects of projects. Students also use their proposals as research plans for their remaining year and a half of college. Data gathered indicate that this course was effective and that it could be a useful addition to other departments’ curricula, particularly at teaching‐focused institutions.
Creating an outcomes‐based biochemistry major from ASBMB and ACS major guidelines that reflects MCAT needs
Authors: Bell Jessica K , Dwyer Tammy J , Loer Curtis M , Lowery Mary Sue , Provost Joseph J
Published: November 2023

Biochemistry and molecular biology curricula serve students pursuing the major, provide breadth to non‐biochemistry majors (chemistry, biology, biophysics, behavioral neuroscience, engineering) and a foundational knowledge of the topic to pre‐professional students in the biomedical sciences. To meet the needs of this diverse population, the University of San Diego redesigned the biochemistry major. Central to the redesign was retaining degree certification by ACS and meeting the criteria for ASBMB accreditation. ASBMB core concepts and learning objectives, as well as ACS guidelines for introductory, foundational and in‐depth coverage of the five areas of chemistry were mapped onto the curriculum of the major. Because many biochemistry majors and students taking these courses are interested in health professions, we also took into consideration core concept coverage against the MCAT requirements. From this analysis, gaps in foundational concepts or areas with only introductory concept coverage were identified and addressed by the development of new courses; a second semester biochemistry lecture and molecular biology techniques laboratory course. A research methods course was implemented in the 4th semester that introduces students to primary literature, ethical conduct in research and scholarship, and professional development. With this foundation, students complete a 100 h research project with faculty or via an internship as part of their 400 h experiential learning requirement. Communication skills (written and oral competency) were integrated into the 8 h biochemistry laboratory to showcase students’ course integrated research projects. To reflect the broad interests of students, flexibility for in‐depth concept coverage in an upper division elective was expanded to include biology (Microbiology, Immunology, Adv. Molecular Biology), chemistry (Biophysical Chemistry, Adv. Synthesis, Adv. Physical Methods), and physics (Biophysics) electives. As part of the major's redesign, MCAT 2015 topics in biochemistry were considered during development of the 2‐semester biochemistry course as pre‐professional students typically will complete only one semester of biochemistry lecture. Structure/function/regulation, enzyme kinetics/thermodynamics and catabolic pathways related to glucose were maintained in the first semester. In conjunction with other coursework, this concept coverage provides adequate preparation for MCAT 2015. For students who take only a select number of courses (i.e. non‐biochemistry majors), web‐hosted materials supplement this course work to provide preparatory information for standardized exams. Together, these curricular changes provide our students with the technical and transferrable skills to be successful in their chosen career path.