Corticotropin-releasing factor overexpression gives rise to sex differences in Alzheimer's disease-related signaling.

Several neuropsychiatric and neurodegenerative disorders share stress as a risk factor and are more prevalent in women than in men. Corticotropin-releasing factor (CRF) orchestrates the stress response, and excessive CRF is thought to contribute to the pathophysiology of these diseases. We previously found that the CRF1 receptor (CRF1) is sex biased whereby coupling to its GTP-binding protein, Gs, is greater in females, whereas ß-arrestin-2 coupling is greater in males. This study used a phosphoproteomic approach in CRF-overexpressing (CRF-OE) mice to test the proof of principle that when CRF is in excess, sex-biased CRF1 coupling translates into divergent cell signaling that is expressed as different brain phosphoprotein profiles. Cortical phosphopeptides that distinguished female and male CRF-OE mice were overrepresented in unique pathways that were consistent with Gs-dependent signaling in females and ß-arrestin-2 signaling in males. Notably, phosphopeptides that were more abundant in female CRF-OE mice were overrepresented in an Alzheimer's disease (AD) pathway. Phosphoproteomic results were validated by demonstrating that CRF overexpression in females was associated with increased tau phosphorylation and, in a mouse model of AD pathology, phosphorylation of ß-secretase, the enzyme involved in the formation of amyloid ß. These females exhibited increased formation of amyloid ß plaques and cognitive impairments relative to males. Collectively, the findings are consistent with a mechanism whereby the excess CRF that characterizes stress-related diseases initiates distinct cellular processes in male and female brains, as a result of sex-biased CRF1 signaling. Promotion of AD-related signaling pathways through this mechanism may contribute to female vulnerability to AD.

S-Adenosylmethionine decarboxylase from the archaeon Methanococcus jannaschii: identification of a novel family of pyruvoyl enzymes.

Polyamines are present in high concentrations in archaea, yet little is known about their synthesis, except by extrapolation from bacterial and eucaryal systems. S-Adenosylmethionine (AdoMet) decarboxylase, a pyruvoyl group-containing enzyme that is required for spermidine biosynthesis, has been previously identified in eucarya and Escherichia coli. Despite spermidine concentrations in the Methanococcales that are several times higher than in E. coli, no AdoMet decarboxylase gene was recognized in the complete genome sequence of Methanococcus jannaschii. The gene encoding AdoMet decarboxylase in this archaeon is identified herein as a highly diverged homolog of the E. coli speD gene (less than 11% identity). The M. jannaschii enzyme has been expressed in E. coli and purified to homogeneity. Mass spectrometry showed that the enzyme is composed of two subunits of 61 and 63 residues that are derived from a common proenzyme; these proteins associate in an (alphabeta)(2) complex. The pyruvoyl-containing subunit is less than one-half the size of that in previously reported AdoMet decarboxylases, but the holoenzyme has enzymatic activity comparable to that of other AdoMet decarboxylases. The sequence of the M. jannaschii enzyme is a prototype of a class of AdoMet decarboxylases that includes homologs in other archaea and diverse bacteria. The broad phylogenetic distribution of this group suggests that the canonical SpeD-type decarboxylase was derived from an archaeal enzyme within the gamma proteobacterial lineage. Both SpeD-type and archaeal-type enzymes have diverged widely in sequence and size from analogous eucaryal enzymes.

Investigation of the substrate spectrum of the human mismatch-specific DNA N-glycosylase MED1 (MBD4): fundamental role of the catalytic domain.

The human DNA repair protein MED1 (also known as MBD4) was isolated as an interactor of the mismatch repair protein MLH1 in a yeast two-hybrid screening. MED1 has a tripartite structure with an N-terminal 5-methylcytosine binding domain (MBD), a central region, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. Indeed, MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil paired with guanine. The glycosylase activity of MED1 preferentially targets G:T mismatches in the context of CpG sites; this indicates that MED1 is involved in the repair of deaminated 5-methylcytosine. Interestingly, frameshift mutations of the MED1 gene have been reported in human colorectal, endometrial, and pancreatic cancers. For its putative role in maintaining genomic fidelity at CpG sites, it is important to characterize the biochemical properties and the substrate spectrum of MED1. Here we show that MED1 works under a wide range of temperature and pH, and has a limited optimum range of ionic strength. MED1 has a weak glycosylase activity on the mutagenic adduct 3,N(4)-ethenocytosine, a metabolite of vinyl chloride and ethyl carbamate. The differences in glycosylase activity on G:U and G:T substrates are not related to differences in substrate binding and likely result from intrinsic differences in the chemical step. Finally, the isolated catalytic domain of MED1 retains the preference for G:T and G:U substrates in the context of methylated or unmethylated CpG sites. This suggests that the catalytic domain is fundamental, and the 5-methylcytosine binding domain dispensable, in determining the substrate spectrum of MED1.

Direct evidence for the cooperative unfolding of cytochrome c in lipid membranes from H-(2)H exchange kinetics.

The interaction of cytochrome c (cyt c) with anionic lipid membranes is known to disrupt the tightly packed native structure of the protein. This process leads to a lipid-inserted denatured state, which retains a native-like alpha-helical structure but lacks any specific tertiary interactions. The structural and dynamic properties of cyt c bound to vesicles containing an anionic phospholipid (DOPS) were investigated by amide H-(2)H exchange using two-dimensional NMR spectroscopy and electrospray ionisation mass spectrometry. The H-(2)H exchange kinetics of the core amide protons in cyt c, which in the native protein undergo exchange via an uncorrelated EX2 mechanism, exchange in the lipid vesicles via a highly concerted global transition that exposes these protected amide groups to solvent. The lack of pH dependence and the observation of distinct populations of deuterated and protonated species by mass spectrometry confirms that exchange occurs via an EX1 mechanism with a common rate of 1(+/-0.5) h(-1), which reflects the rate of transition from the lipid-inserted state, H(l), to an unprotected conformation, D(i), associated with the lipid interface.

Biphasic kinetics of the human DNA repair protein MED1 (MBD4), a mismatch-specific DNA N-glycosylase.

The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.

Structural determinants of metal-induced conformational changes in HIV-1 integrase.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) undergoes a reversible metal-induced conformational change that activates the enzyme (Asante-Appiah, E., and Skalka, A. M. (1997) J. Biol. Chem. 272, 16196-16205). In this report, key structural features that mediate this conformational change have been identified by site-directed mutagenesis, limited proteolysis, and mass spectrometry studies. The results reveal two separable metal-induced effects. One depends on residues in the N-terminal domain (amino acids 1-50) and a C-terminal tail (amino acids 274-288) and is detected by increased resistance of the full-length protein to proteolytic digestion. This effect appears to depend on metal binding at an undefined location distinct from the known sites in the N-terminal and catalytic core domains. The second conformational change depends on metal binding at the active site in the catalytic core domain. Substitution of acidic residues Asp64 or Glu152 in the catalytic core D,D(35)E motif or truncation of the Src homology 3 (SH3)-like domain in the C-terminal region of the enzyme abolishes this metal-induced change. Comparison of tryptic digests of an HIV-1 IN derivative competent for metal-induced conformational change and a conformation-defective D64N derivative identified specific regions in HIV-1 IN that are affected by this second change. A region in the N terminus that spans Lys14, an extended loop and the adjacent region in the core domain (including lysines 136, 156, and 160 and Arg173), and residues at the C terminus beyond the SH3-like domain all become less accessible to proteolysis in the conformation-competent protein. In contrast, a region that encompasses Lys258 in the putative DNA binding groove of the SH3-like domain becomes more sensitive to proteolysis in the presence of Mn2+. The results are consistent with a model in which the binding of the metal ion by residues of the D,D(35)E motif elicits specific changes in all three domains of HIV-1 IN, inducing the restructuring of the enzyme for catalytic competence.

Phosphoglucose isomerase: a ketol isomerase with aldol C2-epimerase activity.

With 13C NMR, phosphoglucose isomerase (PGI; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) is shown to produce mannose 6-phosphate (M6P) slowly from a much more rapidly catalyzed equilibrium between glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). The identity of M6P and its formation from G6P plus F6P are confirmed by 1H NMR and by the ability of PGI to convert M6P to F6P plus G6P. The possibility of contaminating phosphomannose isomerase (PMI, D-mannose-6-phosphate ketol-isomerase, EC 5.3.1.8) is ruled out by finding no exchange of the C1 proton of G6P or of M6P, whereas exchange occurs with a mixture of PMI and PGI in 2H2O. The pro-R and pro-S protons of F6P become the anomeric protons of M6P and G6P through the actions of PMI and PGI, respectively. Both isomerases exchange the C2 proton of their substrate with the medium; hence, when PGI and PMI are added together to hexose phosphate solutions in 2H2O, both the substrate and anomeric protons are exchange rapidly with deuterons from the medium. The rates of C2-epimerization of G6P and M6P by PGI are shown to be proportional to enzyme concentration and inhibited by 5-phosphoarabinoate, a competitive inhibitor of the previously demonstrated isomerase and anomerase activities of PGI. These data show that the epimerization is enzymatically catalyzed and suggest the involvement of the same active site for all three activities. A primary kinetic isotope effect of 7.5 (H/2H) on the rate constant kcat of the M6P C2-epimerase activity was determined by using a coupled enzymatic assay. A model of the mechanism of PGI is offered, which relates C2-epimerase activity to the isomerase and anomerase activities by allowing the cis-enediol intermediate to rotate about the C2-C3 bond axis followed by protonation at C2 but not at C1 from the si face.

Enolpyruvate: chemical determination as a pyruvate kinase intermediate.

Despite many studies suggesting the role of enolpyruvate as a bound intermediate in the pyruvate kinase reaction, direct evidence for it has been lacking. By use of a combination of chemical trapping and isolation of a derivative, significant amounts of enzyme-bound enolpyruvate have now been demonstrated. The method distinguishes enolpyruvate. It is based on reaction of bromine with enolpyruvate in acid, derivatization of formed bromopyruvate with thionitrobenzoate, and resolution by reversed-phase HPLC of the thioether derivative. As little as 10 pmol of the thioether derivative could be quantitated reliably. With this method, the internal equilibria, including the E.ATP.enolpyruvate intermediate, have been determined. Enzyme-enolpyruvate concentration was shown to be pH-dependent. Phosphoenolpyruvate also reacts with bromine to form bromopyruvate. To quantitate enolpyruvate specifically in a background of phosphoenolpyruvate, advantage was taken of phosphoenolpyruvate's much greater stability in acid. When bromide/was added 10 min after the acid quench, ketonization of enolpyruvate was complete, and only phosphoenolpyruvate was measured. Enolpyruvate is thus determined by difference between the bromopyruvate measured with and without delayed bromine addition.

Structural characterization of the interactions between calmodulin and skeletal muscle myosin light chain kinase: effect of peptide (576-594)G binding on the Ca2+-binding domains.

Calcium-containing calmodulin (CaM) and its complex with a peptide corresponding to the calmodulin-binding domain of skeletal muscle myosin light chain kinase [skMLCK(576-594)G] have been studied by one- and two-dimensional 1H NMR techniques. Resonances arising from the antiparallel beta-sheet structures associated with the calcium-binding domains of CaM and their counterparts in the CaM-skMLCK(576-594)G complex have been assigned. The assignments were initiated by application of the main chain directed assignment strategy. It is found that, despite significant changes in chemical shifts of resonances arising from amino acid residues in this region upon binding of the peptide, the beta-sheets have virtually the same structure in the complex as in CaM. Hydrogen exchange rates of amide NH within the beta-sheet structures are significantly slowed upon binding of peptide. These data, in conjunction with the observed nuclear Overhauser effect (NOE) patterns and relative intensities and the downfield shifts of associated amide and alpha resonances upon binding of peptide, show that the peptide stabilizes the Ca2+-bound state of calmodulin. The observed pattern of NOEs within the beta-sheets and their structural similarity correspond closely to those predicted by the crystal structure. These findings imply that the apparent inconsistency of the crystal structure with recently reported low-angle X-ray scattering profiles of CaM may lie within the putative central helix bridging the globular domains.

NMR studies of a complex of deuterated calmodulin with melittin.

Completely deuterated calmodulin ([2H]CaM) has been prepared by expressing the chicken gene for CaM in Escherichia coli grown in 2H2O on a deuterated medium. The structural and dynamic properties of a 1:1 CaM/melittin (Mel) complex have been investigated by proton NMR. The spectrum of bound Mel is obtained directly from the spectrum of the [2H]CaM X Mel complex and is found to resemble strongly the spectrum of the helical species in methanol rather than that of the random coil species in water. The spectrum of bound CaM is obtained indirectly from the difference spectrum between [1H]CaM X Mel and [2H]CaM X Mel. Many changes are observed between free and bound CaM and they are distributed in both halves of the molecule, indicating that the binding of Mel affects the structure in both parts of the molecule. The rates of exchange of the amide protons of [2H]CaM with 2H2O were compared to those of [2H]CaM X Mel. The results showed that most, but not all, of the protons exchanged more slowly in the complex; after 40 hr, the residual peaks number 7 in CaM and greater than 20 in the complex. Again, changes in rates in CaM due to binding of Mel occurred in both halves of the molecule. The relative rates of amide proton exchange in CaM and its complex with Mel prove to be a sensitive criterion of differences in conformational stability and/or structure.

Mathematical analysis of isotope labeling in the citric acid cycle with applications to 13C NMR studies in perfused rat hearts.

Rat hearts have been perfused in vitro with 5 mM glucose and either 5 mM acetate or 1 mM pyruvate to achieve steady state conditions, followed by replacement of the acetate with 90% enriched [2-13C]acetate or pyruvate with 90% enriched [3-13C]pyruvate. The hearts were frozen different times after addition of 13C-substrate and neutralized perchloric acid extracts from three pooled hearts per time point were used to obtain high resolution proton-decoupled 13C NMR spectra at 90.55 MHz. The 13C fractional enrichment of individual carbons of different metabolites was calculated from the area of the resolved resonances after correction for nuclear Overhauser enhancement and saturation effects. A mathematical flux model of the citric acid cycle and ancillary transamination reactions was constructed with the FACSIMILE program, and used to solve unknown flux parameters with constant pool sizes by nonlinear least squares analysis of the approximately 200 simultaneous differential equations required to describe the reactions. With [2-13C] acetate as substrate, resonances and line splittings due to 13C-13C spin coupling of the C-2, C-3, and C-4 carbons of glutamate were well resolved. The half-times to reach maximum 13C enrichment were 2.6 min for glutamate C-4 and 8 min for glutamate C-2 and C-3. From these data, a well determined citric acid cycle flux of 8.3 mumol/g dry weight X min was calculated for an observed oxygen consumption of 31 mumol/g dry weight X min. With [3-13C]pyruvate as substrate, resonances of aspartate C-2 and C-3 and of alanine C-3 were well resolved in addition to those of glutamate C-2, C-3, and C-4. Nonlinear least squares fitting of these data to the model gave nonrandomly distributed residuals for the 13C fractional enrichments of glutamate C-4, suggesting an incomplete model, but a well determined cycle flux of 11.9 mumol/g dry weight X min for an oxygen uptake of 35 mumol/g dry weight X min. Our studies demonstrate the practicality of 13C NMR, used in conjunction with mathematical modeling, for the measurement of metabolic flux parameters in living systems.

Intracellular pH mediates action of insulin on glycolysis in frog skeletal muscle.

In a glucose-free bicarbonate Ringer (5% CO2 in N2), insulin increased intracellular pH (pHi), as determined by [14C]dimethadione, by 0.12 +/- 0.02 and stimulated glycolysis, as monitored by anaerobic lactate production, by 42.9 +/- 3.5% in paired frog sartorius muscles. The effect of insulin on glycolysis was shown to vary approximately linearly with log [Na+]0, being converted in 0.12 mM Na+ Ringer to a 51.5 +/- 8.4% inhibition of glycolysis. As the Na+ free-energy gradient was varied by decreasing [Na+]0 from 104 to 6.8 mM, the changes in glycolytic flux produced by insulin consistently paralleled the changes in pHi produced by the hormone. The relationship between the change in pHi and percent change in glycolytic flux was the same regardless of whether the effects were produced by insulin or by changing CO2. When glycolysis was either stimulated or inhibited, intracellular levels of fructose 6-phosphate varied inversely with glycolytic flux. This indicates that the effect on glycolysis of either insulin or changes in CO2 is due to a change in the activity of phosphofructokinase. The results support the model that the acute effect of insulin on glycolysis is mediated by a change in pHi, consequent to activation by insulin of Na:H exchange at the plasma membrane.