A predictive model for prostate cancer incorporating PSA molecular forms and age.

The diagnostic specificity of prostate specific antigen (PSA) is limited. We aimed to characterize eight anti-PSA monoclonal antibodies (mAbs) to assess the prostate cancer (PCa) diagnostic utility of different PSA molecular forms, total (t) and free (f) PSA and PSA complexed to α1-antichymotrypsin (complexed PSA). MAbs were obtained by immunization with PSA and characterized by competition studies, ELISAs and immunoblotting. With them, we developed sensitive and specific ELISAs for these PSA molecular forms and measured them in 301 PCa patients and 764 patients with benign prostate hyperplasia, and analyzed their effectiveness to discriminate both groups using ROC curves. The free-to-total (FPR) and the complexed-to-total PSA (CPR) ratios significantly increased the diagnostic yield of tPSA. Moreover, based on model selection, we constructed a multivariable logistic regression model to predictive PCa that includes tPSA, fPSA, and age as predictors, which reached an optimism-corrected area under the ROC curve (AUC) of 0.86. Our model outperforms the predictive ability of tPSA (AUC 0.71), used in clinical practice. In conclusion, The FPR and CPR showed better diagnostic yield than tPSA. In addition, the PCa predictive model including age, fPSA and complexed PSA, outperformed tPSA detection efficacy. Our model may avoid unnecessary biopsies, preventing harmful side effects and reducing health expenses.

Kinetics of regulatory serine variants of tyrosine hydroxylase with cyclic AMP-dependent protein kinase and extracellular signal-regulated protein kinase 2.

Rat tyrosine hydroxylase is phosphorylated at four serine residues, at positions 8, 19, 31, and 40 in its amino terminal regulatory domain by multiple protein kinases. Cyclic AMP-dependent protein kinase phosphorylates S40, which results in alleviation of inhibition by dopamine. Extracellular signal-regulated protein kinase 2 phosphorylates S8 and S31. Site-directed serine-to-glutamate mutations were introduced into tyrosine hydroxylase to mimic prior phosphorylation of the regulatory serines; these proteins were used as substrates for cAMP-dependent kinase and extracellular signal-regulated kinase 2. The activity of cAMP-dependent kinase was unaffected by the substitution of serines 8, 19 or 31 with glutamate and the activity of extracellular signal-regulated kinase 2 was unaffected by substitution of serines 19 or 40 with glutamate. Cyclic AMP-dependent kinase was less active in phosphorylating S40 if dopamine was bound to tyrosine hydroxylase, but extracellular signal-regulated kinase 2 phosphorylation at S31 was unaffected by the presence of dopamine.

Mechanistic studies of mouse polyamine oxidase with N1,N12-bisethylspermine as a substrate.

In mammalian cells, the flavoprotein polyamine oxidase catalyzes a key step in the catabolism of polyamines, the oxidation of N1-acetylspermine and N1-acetylspermidine to spermidine and putrescine, respectively. The mechanism of the mouse enzyme has been studied with N1,N12-bisethylspermine (BESPM) as a substrate. At pH 10, the pH optimum, the limiting rate of reduction of the flavin in the absence of oxygen is comparable to the k(cat) value for turnover, establishing reduction as rate-limiting. Oxidation of the reduced enzyme is a simple second-order reaction. No intermediates are seen in the reductive or oxidative half-reactions. The k(cat) value decreases below a pK(a) of 9.0. The k(cat)/K(m) value for BESPM exhibits a bell-shaped pH profile, with pK(a) values of 9.8 and 10.8. These pK(a) values are assigned to the substrate nitrogens. The rate constant for the reaction of the reduced enzyme with oxygen is not affected by a pH between 7.5 and 10. Active site residue Tyr430 is conserved in the homologous protein monoamine oxidase. Mutation of this residue to phenylalanine results in a 6-fold decrease in the k(cat) value and the k(cat)/K(m) value for oxygen due to a comparable decrease in the rate constant for flavin reduction. This moderate change is not consistent with this residue forming a tyrosyl radical during catalysis.

Mutation of regulatory serines of rat tyrosine hydroxylase to glutamate: effects on enzyme stability and activity.

Tyrosine hydroxylase is phosphorylated at four serine residues in its amino-terminus by multiple kinases. Phosphorylation of serine 40 by cAMP-dependent protein kinase results in alleviation of dopamine inhibition [J. Biol. Chem. 267 (1992) 12639]. The other serines are at positions 8, 19, and 31. The effect of phosphorylation at these serines has been investigated using mutated forms of tyrosine hydroxylase containing glutamates at the positions of the serines. The S8E, S19E, and S31E tyrosine hydroxylase variants have similar steady-state kinetic parameters and similar binding affinity for catecholamines to wild-type enzyme. The S8E, S19E, S31E, and S40E variants differ in stability at elevated temperatures. The S40E variant is the least stable, while the others are all more stable than wild-type enzyme. The increased stability of S8E, S19E, and S31E tyrosine hydroxylases may be one of the physiological effects of phosphorylation. It may also have implications for the interpretation of activities of heterogeneous mixtures of tyrosine hydroxylase which have been phosphorylated.

Effects of mutations in tyrosine hydroxylase associated with progressive dystonia on the activity and stability of the protein.

Tyrosine hydroxylase (TyrH) catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA), the rate-limiting step in the biosynthesis of dopamine. Four mutations in the TyrH gene have recently been described in cases of autosomal recessive DOPA-responsive dystonia (Swaans et al., Ann Hum Genet 2000;64:25-31). All four are predicted to result in changes in single amino acid residues in the catalytic domain of the protein: T245P, T283M, R306H, and T463M. To determine the effects of these mutations on the molecular properties of the enzyme, mutant proteins containing the individual single amino acid changes have been expressed in bacteria and purified. Only the T283M mutation results in a decrease in the enzyme k(cat) value, while the T245P enzyme has a slightly higher value than the wild-type enzyme. The only case in which a K(m) value for either tyrosine or tetrahydrobiopterin is perturbed is the T245P enzyme, for which the K(m) value for tyrosine has increased about 50%. In contrast to the minor effects of the mutations on enzyme activity, the stability is decreased significantly by the mutations. The R306H and T283M enzymes are the least stable, losing activity 30- and 50-fold more rapidly than the wild-type enzyme. The apparent T(m) value for unfolding was decreased by 3.9, 8.2, and 7.2 degrees for the T245P, R306H, and T463M enzymes, while the T283M enzyme was too unstable for measurement of a T(m) value. The results establish that the physiological effects of the mutations are primarily due to the decreased stability of the mutant proteins rather than decreases in their intrinsic activities.

Specificity of the MAP kinase ERK2 for phosphorylation of tyrosine hydroxylase.

Short-term regulation of catecholamine biosynthesis involves reversible phosphorylation of several serine residues in the N-terminal regulatory domain of tyrosine hydroxylase. The MAP kinases ERK1/2 have been identified as responsible for phosphorylation of Ser31. As an initial step in elucidating the effects of phosphorylation of Ser31 on the structure and activity of tyrosine hydroxylase, the kinetics of phosphorylation of the rat enzyme by recombinant rat ERK2 have been characterized. Complete phosphorylation results in incorporation of 2mol of phosphate into each subunit of tyrosine hydroxylase. The S8A and S31A enzymes only incorporate a single phosphate, while the S19A and S40A enzymes incorporate two. Phosphorylation of S8A tyrosine hydroxylase is nine times as rapid as phosphorylation of the S31A enzyme, consistent with a ninefold preference of ERK2 for Ser31 over Ser8.

The proportion of prostate-specific antigen (PSA) complexed to alpha(1)-antichymotrypsin improves the discrimination between prostate cancer and benign prostatic hyperplasia in men with a total PSA of 10 to 30 microg/L.

BACKGROUND: The aim of this study was to assess the diagnostic accuracy of the proportion of prostate-specific antigen (PSA) complexed to alpha(1)-antichymotrypsin (PSA-alpha(1)ACT:PSA ratio) in the differential diagnosis of prostate cancer (CaP) and benign prostatic hyperplasia (BPH) in men with total PSA of 10-30 microg/L. METHODS: We used our immunoassays (ELISAs) for total PSA and PSA-alpha(1)ACT complex to study 146 men. In 123, total PSA was between 10 and 20 microg/L; 66 of these had CaP and 57 BPH. In 23 men, total PSA was between 20 and 30 microg/L; 14 of these had CaP and 9 BPH. We calculated the area under the ROC curves (AUC) for total PSA, PSA-alpha(1)ACT complex, and PSA-alpha(1)ACT:PSA ratio, and determined the cutoff points that gave sensitivities approaching 100%. RESULTS: In the total PSA range between 10 and 20 microg/L, the AUC was significantly higher for the PSA-alpha(1)ACT:PSA ratio (0.850) than for total PSA (0.507) and PSA-alpha(1)ACT complex (0.710; P <0.0001). A cutoff ratio of 0.62 would have permitted diagnosis of all 66 patients with CaP (100% sensitivity) and avoided 19% of unnecessary biopsies (11 of 57 patients). In the total PSA range between 20 and 30 microg/L, the AUC for the PSA-alpha(1)ACT:PSA ratio (0.980; 95% confidence interval, 0.82-0.99) was greater than the AUC for total PSA (0.750; 95% confidence interval, 0.51-0.89; P = 0.042). In this range, a cutoff point of 0.64 would have permitted the correct diagnosis of all 14 patients with CaP and 6 of the 9 with BPH. CONCLUSIONS: The diagnostic accuracy of the PSA-alpha(1)ACT:PSA ratio persists at high total PSA concentrations, increasing the specificity of total PSA. Prospective studies with large numbers of patients are needed to assess whether the ratio of PSA-alpha(1)ACT to total PSA is a useful tool to avoid unnecessary prostatic biopsy in patients with a total PSA >10 microg/L.