Reversibility and Low Commitment to Forward Catalysis in the Conjugation of Lipid Alkenals by Glutathione Transferase A4-4.

High concentrations of electrophilic lipid alkenals formed during oxidative stress are implicated in cytotoxicity and disease. However, low concentrations of alkenals are required to induce antioxidative stress responses. An established clearance pathway for lipid alkenals includes conjugation to glutathione (GSH) via Michael addition, which is catalyzed mainly by glutathione transferase isoform A4 (GSTA4-4).

Key Challenges and Recommendations for Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol.

The influence of proline isomerization on potency and stability of anti-HIV antibody 10E8.

Monoclonal antibody (mAb) 10E8 recognizes a highly conserved epitope on HIV and is capable of neutralizing > 95% of circulating viral isolates making it one of the most promising Abs against HIV. Solution instability and biochemical heterogeneity of 10E8 has hampered its development for clinical use. We identify the source of 10E8 heterogeneity being linked to cis/trans isomerization at two prolines within the YPP motif in the CRD3 loop that exists as two predominant conformers that interconvert on a slow timescale.

Gas-Phase Hydrogen/Deuterium Exchange for Distinguishing Isomeric Carbohydrate Ions.

The structural diversity of carbohydrates presents a major challenge for glycobiology and the analysis of glycoconjugates. Mass spectrometry has become a primary tool for glycan analysis thanks to its speed and sensitivity, but the information content regarding the glycan structure of protonated glycoconjugates is hindered by the inability to differentiate linkage and stereoisomers. Here, we examine a variety of protonated carbohydrate structures by gas-phase hydrogen/deuterium exchange (HDX) to discover that the exchange rates are distinct for isomeric carbohydrates with even subtle structural differences.

Membrane Fluidity Modulates Thermal Stability and Ligand Binding of Cytochrome P4503A4 in Lipid Nanodiscs.

Cytochrome P4503A4 (CYP3A4) is a peripheral membrane protein that plays a major role in enzymatic detoxification of many drugs and toxins. CYP3A4 has an integral membrane N-terminal helix and a localized patch comprised of the G' and F' helix regions that are embedded in the membrane, but the effects of membrane composition on CYP3A4 function are unknown. Here, circular dichroism and differential scanning calorimetry were used to compare the stability of CYP3A4 in lipid bilayer nanodiscs with varying ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine to 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).

The Myeloablative Drug Busulfan Converts Cysteine to Dehydroalanine and Lanthionine in Redoxins.

The myeloablative agent busulfan (1,4-butanediol dimethanesulfonate) is an old drug that is used routinely to eliminate cancerous bone marrow prior to hematopoietic stem cell transplant. The myeloablative activity and systemic toxicity of busulfan have been ascribed to its ability to cross-link DNA. In contrast, here we demonstrate that incubation of busulfan with the thiol redox proteins glutaredoxin or thioredoxin at pH 7.

Efficient Syntheses of Vitamin K Chain-Shortened Acid Metabolites.

Vitamin K sequentially undergoes ω-oxidation followed by successive rounds of β-oxidation to ultimately produce two chain-shortened carboxylic acid metabolites, vitamin K acid 1 and vitamin K acid 2. Two facile syntheses of these acid metabolites are described, each starting from commercially available menadione-cyclopentadiene adduct . Vitamin K acid 1 was synthesized in five steps alkylation with a geranyl halide followed by subsequent oxidation reactions, while fully retaining the configuration of the side chain 2',3'-double bond.

Supporting data for characterization of the busulfan metabolite EdAG and the Glutaredoxins that it adducts.

This article describes data related to a research article titled "The Busulfan Metabolite EdAG Irreversibly Glutathionylates Glutaredoxins" [1]. EdAG is an electrophilic GSH analog formed in vivo from busulfan, which is used in hematopoietic stem cell transplants. EdAG glutathionylates Glutaredoxins (Grx's) but not glutathione transferase A1-1 (GSTA1-1) in vitro.

Comparison of epsilon- and delta-class glutathione S-transferases: the crystal structures of the glutathione S-transferases DmGSTE6 and DmGSTE7 from Drosophila melanogaster.

Cytosolic glutathione transferases (GSTs) comprise a large family of enzymes with canonical structures that diverge functionally and structurally among mammals, invertebrates and plants. Whereas mammalian GSTs have been characterized extensively with regard to their structure and function, invertebrate GSTs remain relatively unstudied. The invertebrate GSTs do, however, represent potentially important drug targets for infectious diseases and agricultural applications.

Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix.

Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown.

The busulfan metabolite EdAG irreversibly glutathionylates glutaredoxins.

The DNA alkylating agent busulfan is used to 'precondition' patients with leukemia, lymphomas and other hematological disorders prior to hematopoietic stem cell transplants. Busulfan is metabolized via conjugation with glutathione (GSH) followed by intramolecular rearrangement to the GSH analog γ-glutamyl-dehydroalanyl -glycine (EdAG). EdAG contains the electrophilic dehydroalanine, which is expected to react with protein nucleophiles, particularly proteins with GSH binding sites such as glutaredoxins (Grx's).

Development of pyrazolone and isoxazol-5-one cambinol analogues as sirtuin inhibitors.

Sirtuins are a family of NAD(+)-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC50 ≈ 50 μM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein.

Human UGT1A4 and UGT1A3 conjugate 25-hydroxyvitamin D3: metabolite structure, kinetics, inducibility, and interindividual variability.

25-Hydroxyvitamin D3 (25OHD3) is used as a clinical biomarker for assessment of vitamin D status. Blood levels of 25OHD3 represent a balance between its formation rate and clearance by several oxidative and conjugative processes. In the present study, the identity of human uridine 5'-diphosphoglucuronyltransferases (UGTs) capable of catalyzing the 25OHD3 glucuronidation reaction was investigated.

Reaction dynamics of ATP hydrolysis catalyzed by P-glycoprotein.

P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug-drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible.

Circular Permutation of the Trp-cage: Fold Rescue upon Addition of a Hydrophobic Staple.

The Trp-cage, at 20 residues in length, is generally acknowledged as the smallest fully protein-like folding motif. Linking the termini by a two-residue unit and excising one residue affords circularly permuted sequences that adopt the same structure. This represents the first successful circular permutation of any fold of less than 50-residue length.

The urine microRNA profile may help monitor post-transplant renal graft function.

Noninvasive, cost-effective biomarkers that allow accurate monitoring of graft function are needed in kidney transplantation. Since microRNAs (miRNAs) have emerged as promising disease biomarkers, we sought to establish an miRNA signature in urinary cell pellets comparing kidney transplant patients diagnosed with chronic allograft dysfunction (CAD) with interstitial fibrosis and tubular atrophy and those recipients with normal graft function. Overall, we evaluated 191 samples from 125 deceased donor primary kidney transplant recipients in the discovery, initial validation, and the longitudinal validation studies for noninvasive monitoring of graft function.

13C structuring shifts for the analysis of model β-hairpins and β-sheets in proteins: diagnostic shifts appear only at the cross-strand H-bonded residues.

The present studies have shown that (13)C=O, (13)C(α) and (13)C(β) of H-bonded strand residues in β-hairpins provide additional probes for quantitating the extent of folding in β-hairpins and other β-sheet models. Large differences in the structuring shifts (CSDs) of these (13)C sites in H-bonded versus non-H-bonded sites are observed: the differences between H-bonded and non-H-bonded sites are greater than 1.2 ppm for all three (13)C probes.

Mutational effects on the folding dynamics of a minimized hairpin.

The fold stabilities and folding dynamics of a series of mutants of a model hairpin, KTW-NPATGK-WTE (HP7), are reported. The parent system and the corresponding DPATGK loop species display submicrosecond folding time constants. The mutational studies revealed that ultrafast folding requires both some prestructuring of the loop and a favorable interaction between the chain termini in the transition state.

MiRNAs in kidney transplantation: potential role as new biomarkers.

MiRNAs (miRNAs) are a class of small endogenous, noncoding RNAs with important roles in regulating gene expression known to play a role in many cellular functions including cellular differentiation, cell proliferation, cell development and functional regulation of the immune system, among others. As such, miRNAs are emerging not only as potential biomarkers but also as potential therapeutic targets. Here, we review the currently published work on miRNAs and renal transplantation as it pertains to ischemia-reperfusion injury, acute kidney injury, delayed graft function, calcineurin inhibitor toxicity, acute rejection, chronic allograft dysfunction and kidney fibrosis.

MicroRNAs as biomarkers in solid organ transplantation.

Important progress has been made in improving short-term outcomes in solid organ transplantation. However, long-term outcomes have not improved during the last decades. There is a critical need for biomarkers of donor quality, early diagnosis of graft injury and treatment response.

Stereoselective formation and metabolism of 4-hydroxy-retinoic Acid enantiomers by cytochrome p450 enzymes.

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously.

Identification of biomarkers to assess organ quality and predict posttransplantation outcomes.

Unlabelled: The increased disparity between organ supply and need has led to the use of extended criteria donors and donation after cardiac death donors with other comorbidities.

Methods: We have examined the preimplantation transcriptome of 112 kidney transplant recipient samples from 100 deceased-donor kidneys by microarray profiling. Subject groups were segregated based on estimated glomerular filtration rate (eGFR) at 1 month after transplantation: the GFR-high group (n=74) included patients with eGFR 45 mL/min per 1.

Crystal and NMR structures of a Trp-cage mini-protein benchmark for computational fold prediction.

To provide high-resolution X-ray crystallographic structures of a peptide with the Trp-cage fold, we prepared a cyclized version of this motif. Cyclized Trp-cage is remarkably stable and afforded two crystal forms suitable for X-ray diffraction. The resulting higher resolution crystal structures validate the prior NMR models and provide explanations for experimental observations that could not be rationalized by NMR structural data, including the structural basis for the increase in fold stability associated with motif cyclization and the manner in which a polar serine side chain is accommodated in the hydrophobic interior.