20 research outputs found
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
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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 (R)2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor
Discovery of Undefined Protein Cross-Linking Chemistry: A Comprehensive Methodology Utilizing <sup>18</sup>O‑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 workflowî—¸XChem-Finderî—¸that
involves four stages: (1) detection of cross-linked peptides via <sup>18</sup>O-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 Δ<sup>4</sup>-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)Âbutanoic 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 Δ<sup>4</sup>-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)Âbutanoic 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 Δ<sup>4</sup>-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)Âbutanoic 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