RESUMO
A workflow has been evaluated that utilizes a single tissue section to obtain spatially co-registered, molecular, and phenotypical information suitable for AI-enabled image analysis. Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was used to obtain molecular information followed by conventional histological staining and immunolabelling. The impact of varying DESI-MSI conditions (e.g., heated transfer line (HTL) temperature, scan rate, acquisition time) on the detection of small molecules and lipids as well as on tissue integrity crucial for integration into typical clinical pathology workflows was assessed in human kidney. Increasing the heated transfer line temperature from 150 to 450 °C resulted in a 1.8-fold enhancement in lipid signal at a scan rate of 10 scans/s, while preserving histological features. Moreover, increasing the acquisition speed to 30 scans/s yielded superior lipid signal when compared to 10 scans/s at 150 °C. Tissue morphology and protein epitopes remained intact allowing full histological assessment and further multiplex phenotyping by immunofluorescence (mIF) and immunohistochemistry (mIHC) of the same section. The successful integration of the workflow incorporating DESI-MSI, H&E, and immunolabelling on a single tissue section revealed an accumulation of ascorbic acid in regions of focal chronic inflammatory cell infiltrate within non-cancerous kidney tissue. Additionally, a strong positive correlation between PI 38:3 and proliferating cells was observed in clear cell renal cell carcinoma (ccRCC) showing the utility of this approach in uncovering molecular associations in disease pathology.
Assuntos
Carcinoma de Células Renais , Proliferação de Células , Neoplasias Renais , Imagem Multimodal , Espectrometria de Massas por Ionização por Electrospray , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/diagnóstico por imagem , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Neoplasias Renais/diagnóstico por imagem , Espectrometria de Massas por Ionização por Electrospray/métodos , Imagem Multimodal/métodos , Fenótipo , Rim/metabolismo , Rim/patologiaRESUMO
The enzyme 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (DNPH1) catalyzes the N-ribosidic bond cleavage of 5-hydroxymethyl-2'-deoxyuridine 5'-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2'-deoxynucleoside 5'-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2'-deoxyuridine 5'-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography-mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid-base catalysis is operational and that for maximum kcat/KM, two groups with an average pKa of 6.4 must be deprotonated, while two groups with an average pKa of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to kcat/KM. Solvent deuterium isotope effects on kcat/KM and kcat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.
Assuntos
Desoxiuridina , Hidrolases , Humanos , Hidrólise , Catálise , Solventes , Fosfatos , Cinética , Concentração de Íons de HidrogênioRESUMO
Nucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2'-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2'-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2'-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2'-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis (CtNDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5-9.5, with two very distinct pKa values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2'-fluoro-2'-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2'-difluoro-2'-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2'-deoxy)ribonucleoside selection, as CtNDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of CtNDT in biocatalysis.
RESUMO
The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of N1-(5-phospho-ß-D-ribosyl)-ATP (PRATP) to N1-(5-phospho-ß-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to N-(5'-phospho-D-ribosylformimino)-5-amino-1-(5â³-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV-VIS spectroscopy and LC-MS to show Acinetobacter baumannii putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall kcat increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on kcat and kcat/KM ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized N1-(5-phospho-ß-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel.
RESUMO
INTRODUCTION: Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. METHODS: Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC-MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. RESULTS: We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. CONCLUSION: Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.
Assuntos
Neoplasias Colorretais , Timidilato Sintase , Humanos , Timidilato Sintase/genética , Fluordesoxiuridilato/farmacologia , Fluordesoxiuridilato/uso terapêutico , Fluoruracila/uso terapêutico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Antimetabólitos , Neoplasias Colorretais/genética , DNARESUMO
The aim of this study was to identify oxysterols and any down-stream metabolites in placenta, umbilical cord blood plasma, maternal plasma and amniotic fluid to enhance our knowledge of the involvement of these molecules in pregnancy. We confirm the identification of 20S-hydroxycholesterol in human placenta, previously reported in a single publication, and propose a pathway from 22R-hydroxycholesterol to a C27 bile acid of probable structure 3ß,20R,22R-trihydroxycholest-5-en-(25R)26-oic acid. The pathway is evident not only in placenta, but pathway intermediates are also found in umbilical cord plasma, maternal plasma and amniotic fluid but not non-pregnant women.
Assuntos
Oxisteróis , Feminino , Humanos , Gravidez , Cromatografia Líquida , Espectrometria de Massas , Líquido Amniótico/metabolismo , Sangue Fetal/metabolismoRESUMO
Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography - tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient.
Assuntos
Oxisteróis , Colesterol , Cromatografia Líquida , Humanos , Espectrometria de Massas , EsteróisRESUMO
Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ5-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol. Using liquid chromatography - mass spectrometry (LC-MS) we have uncovered a pathway of bile acid biosynthesis in SLOS avoiding cholesterol starting with 7-DHC and proceeding through 7-oxo and 7ß-hydroxy intermediates. This pathway also occurs to a minor extent in healthy humans, but elevated levels of pathway intermediates could be responsible for some of the features SLOS. The pathway is also active in SLOS affected pregnancies as revealed by analysis of amniotic fluid. Importantly, intermediates in the pathway, 25-hydroxy-7-oxocholesterol, (25R)26-hydroxy-7-oxocholesterol, 3ß-hydroxy-7-oxocholest-5-en-(25R)26-oic acid and the analogous 7ß-hydroxysterols are modulators of the activity of Smoothened (Smo), an oncoprotein that mediates Hedgehog (Hh) signalling across membranes during embryogenesis and in the regeneration of postembryonic tissue. Computational docking of the 7-oxo and 7ß-hydroxy compounds to the extracellular cysteine rich domain of Smo reveals that they bind in the same groove as both 20S-hydroxycholesterol and cholesterol, known activators of the Hh pathway.