Comparison of Protein N-Homocysteinylation in Rat Plasma under Elevated Homocysteine Using a Specific Chemical Labeling Method

Elevated blood concentrations of homocysteine have been well established as a risk factor for cardiovascular diseases and neuropsychiatric diseases, yet the etiologic relationship of homocysteine to these disorders remains poorly understood. Protein N-homocysteinylation has been hypothesized as a contributing factor; however, it has not been examined globally owing to the lack of suitable detection methods. We recently developed a selective chemical method to label N-homocysteinylated proteins with a biotin-aldehyde tag followed by Western blotting analysis, which was further optimized in this study. We then investigated the variation of protein N-homocysteinylation in plasma from rats on a vitamin B12 deficient diet. Elevated “total homocysteine” concentrations were determined in rats with a vitamin B12 deficient diet. Correspondingly, overall levels of plasma protein N-homocysteinylation displayed an increased trend, and furthermore, more pronounced and statistically significant changes (e.g., 1.8-fold, p-value: 0.03) were observed for some individual protein bands. Our results suggest that, as expected, a general metabolic correlation exists between “total homocysteine” and N-homocysteinylation, although other factors are involved in homocysteine/homocysteine thiolactone metabolism, such as the transsulfuration of homocysteine by cystathionine β-synthase or the hydrolysis of homocysteine thiolactone by paraoxonase 1 (PON1), may play more significant or direct roles in determining the level of N-homocysteinylation

Comparison of Protein N-Homocysteinylation in Rat Plasma under Elevated Homocysteine Using a Specific Chemical Labeling Method

Elevated blood concentrations of homocysteine have been well established as a risk factor for cardiovascular diseases and neuropsychiatric diseases, yet the etiologic relationship of homocysteine to these disorders remains poorly understood. Protein N-homocysteinylation has been hypothesized as a contributing factor; however, it has not been examined globally owing to the lack of suitable detection methods. We recently developed a selective chemical method to label N-homocysteinylated proteins with a biotin-aldehyde tag followed by Western blotting analysis, which was further optimized in this study. We then investigated the variation of protein N-homocysteinylation in plasma from rats on a vitamin B12 deficient diet. Elevated “total homocysteine” concentrations were determined in rats with a vitamin B12 deficient diet. Correspondingly, overall levels of plasma protein N-homocysteinylation displayed an increased trend, and furthermore, more pronounced and statistically significant changes (e.g., 1.8-fold, p-value: 0.03) were observed for some individual protein bands. Our results suggest that, as expected, a general metabolic correlation exists between “total homocysteine” and N-homocysteinylation, although other factors are involved in homocysteine/homocysteine thiolactone metabolism, such as the transsulfuration of homocysteine by cystathionine β-synthase or the hydrolysis of homocysteine thiolactone by paraoxonase 1 (PON1), may play more significant or direct roles in determining the level of N-homocysteinylation

Selective AKR1C3 Inhibitors Potentiate Chemotherapeutic Activity in Multiple Acute Myeloid Leukemia (AML) Cell Lines

We report the design, synthesis, and evaluation of potent and selective inhibitors of aldo-keto reductase 1C3 (AKR1C3), an important enzyme in the regulatory pathway controlling proliferation, differentiation, and apoptosis in myeloid cells. Combination treatment with the nontoxic AKR1C3 inhibitors and etoposide or daunorubicin in acute myeloid leukemia cell lines, elicits a potent adjuvant effect, potentiating the cytotoxicity of etoposide by up to 6.25-fold and the cytotoxicity of daunorubicin by >10-fold. The results validate AKR1C3 inhibition as a common adjuvant target across multiple AML subtypes. These compounds in coadministration with chemotherapeutics in clinical use enhance therapeutic index and may avail chemotherapy as a treatment option to the pediatric and geriatric population currently unable to tolerate the side effects of cancer drug regimens

Discovery of Undefined Protein Cross-Linking Chemistry: A Comprehensive Methodology Utilizing ¹⁸O‑Labeling and Mass Spectrometry

Characterization of protein cross-linking, particularly without prior knowledge of the chemical nature and site of cross-linking, poses a significant challenge, because of their intrinsic structural complexity and the lack of a comprehensive analytical approach. Toward this end, we have developed a generally applicable workflowXChem-Finderthat involves four stages: (1) detection of cross-linked peptides via ¹⁸O-labeling at C-termini; (2) determination of the putative partial sequences of each cross-linked peptide pair using a fragment ion mass database search against known protein sequences coupled with a de novo sequence tag search; (3) extension to full sequences based on protease specificity, the unique combination of mass, and other constraints; and (4) deduction of cross-linking chemistry and site. The mass difference between the sum of two putative full-length peptides and the cross-linked peptide provides the formulas (elemental composition analysis) for the functional groups involved in each cross-linking. Combined with sequence restraint from MS/MS data, plausible cross-linking chemistry and site were inferred, and ultimately confirmed, by matching with all data. Applying our approach to a stressed IgG2 antibody, 10 cross-linked peptides were discovered and found to be connected via thioethers originating from disulfides at locations that had not been previously recognized. Furthermore, once the cross-link chemistry was revealed, a targeted cross-link search yielded 4 additional cross-linked peptides that all contain the C-terminus of the light chain

Discovery of (<i>R</i>)‑2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor

Type 5 17β-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ⁴-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (<i>R</i>)-2-(6-methoxynaphthalen-2-yl)­butanoic acid (in which the methyl group of <i>R</i>-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. <i>R</i>-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer <i>R</i>-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease

Discovery of (<i>R</i>)‑2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor

Type 5 17β-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ⁴-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (<i>R</i>)-2-(6-methoxynaphthalen-2-yl)­butanoic acid (in which the methyl group of <i>R</i>-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. <i>R</i>-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer <i>R</i>-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease

Discovery of (<i>R</i>)‑2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor

Type 5 17β-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ⁴-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (<i>R</i>)-2-(6-methoxynaphthalen-2-yl)­butanoic acid (in which the methyl group of <i>R</i>-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. <i>R</i>-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer <i>R</i>-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease

Potential Metabolic Activation of a Representative C4-Alkylated Polycyclic Aromatic Hydrocarbon Retene (1-Methyl-7-isopropyl-phenanthrene) Associated with the Deepwater Horizon Oil Spill in Human Hepatoma (HepG2) Cells

Exposure to petrogenic polycyclic aromatic hydrocarbons (PPAHs) in the food chain is the major human health hazard associated with the Deepwater Horizon oil spill. C4-Phenanthrenes are representative PPAHs present in the crude oil and could contaminate the seafood. We describe the metabolism of a C4-phenanthrene regioisomer retene (1-methyl-7-isopropyl-phenanthrene) in human HepG2 cells as a model for metabolism in human hepatocytes. Retene because of its sites of alkylation cannot be metabolized to a diol-epoxide. The structures of the metabolites were identified by HPLC-UV-fluorescence detection and LC–MS/MS. O-Monosulfonated-retene-catechols were discovered as signature metabolites of the ortho-quinone pathway of PAH activation catalyzed by aldo-keto reductases. We also found evidence for the formation of bis-ortho-quinones where the two dicarbonyl groups were present on different rings of retene. The identification of O-monosulfonated-retene-catechol and O-bismethyl-O-monoglucuronosyl-retene-bis-catechol supports metabolic activation of retene by P450 and aldo-keto reductase isozymes followed by metabolic detoxification of the ortho-quinone through interception of redox cycling by catechol-O-methyltransferase, uridine 5′-diphospho-glucuronosyltransferase, and sulfotransferase isozymes. We propose that catechol conjugates could be used as biomarkers of human exposure to retene resulting from oil spills