A novel mechanism of V-type zinc inhibition of glutamate dehydrogenase results from disruption of subunit interactions necessary for efficient catalysis.

Bovine glutamate dehydrogenase is potently inhibited by zinc and the major impact is on V(max) suggesting a V-type effect on catalysis or product release. Zinc inhibition decreases as glutamate concentrations decrease suggesting a role for subunit interactions. With the monocarboxylic amino acid norvaline, which gives no evidence of subunit interactions, zinc does not inhibit. Zinc significantly decreases the size of the pre-steady state burst in the reaction but does not affect NADPH binding in the enzyme-NADPH-glutamate complex that governs the steady state turnover, again suggesting that zinc disrupts subunit interactions required for catalytic competence. While differential scanning calorimetry suggests zinc binds and induces a slightly conformationally more rigid state of the protein, limited proteolysis indicates that regions in the vicinity of the antennae regions and the trimer-trimer interface become more flexible. The structures of glutamate dehydrogenase bound with zinc and europium show that zinc binds between the three dimers of subunits in the hexamer, a region shown to bind novel inhibitors that block catalytic turnover, which is consistent with the above findings. In contrast, europium binds to the base of the antenna region and appears to abrogate the inhibitory effect of zinc. Structures of various states of the enzyme have shown that both regions are heavily involved in the conformational changes associated with catalytic turnover. These results suggest that the V-type inhibition produced with glutamate as the substrate results from disruption of subunit interactions necessary for efficient catalysis rather than by a direct effect on the active site conformation.

Histone deacetylase 7 associates with Runx2 and represses its activity during osteoblast maturation in a deacetylation-independent manner.

UNLABELLED: HDAC7 associates with Runx2 and represses Runx2 transcriptional activity in a deacetylase-independent manner. HDAC7 suppression accelerates osteoblast maturation. Thus, HDAC7 is a novel Runx2 co-repressor that regulates osteoblast differentiation. INTRODUCTION: Runx2 is a key regulator of gene expression in osteoblasts and can activate or repress transcription depending on interactions with various co-factors. Based on previous observations that several histone deacetylases (HDACs) repress Runx2 activity and that HDAC inhibitors accelerate osteoblast differentiation in vitro, we hypothesized that additional HDACs may also affect Runx2 activity. MATERIALS AND METHODS: A panel of HDACs was screened for repressors of Runx2 activity. Immunofluorescence, co-immunoprecipitation, GST-pulldowns, and chromatin immunoprecipitations were used to characterize the interactions between Runx2 and HDAC7. Expression of osteoblast markers was examined in a C2C12 cell osteoblast differentiation model in which HDAC7 levels were reduced by RNAi. RESULTS: Runx2 activity was repressed by HDAC7 but not by HDAC9, HDRP, HDAC10, or HDAC11. HDAC7 and Runx2 were found co-localized in nuclei and associated with Runx2-responsive promoter elements in osseous cells. A carboxy-terminal domain of Runx2 associated with multiple regions of HDAC7. Although direct interactions with Runx2 were confined to the carboxy terminus of HDAC7, this region was dispensable for repression. In contrast, the amino terminus of HDAC7 bound Runx2 indirectly and was necessary and sufficient for transcriptional repression. Treatment with HDAC inhibitors did not decrease inhibition by HDAC7, indicating that HDAC7 repressed Runx2 by deacetylation-independent mechanism(s). Suppression of HDAC7 expression in C2C12 multipotent cells by RNAi accelerated their BMP2-dependent osteoblast differentiation program. Consistent with this observation, BMP2 decreased nuclear localization of HDAC7. CONCLUSIONS: These results establish HDAC7 as a regulator of Runx2's transcriptional activity and suggest that HDAC7 may be an important regulator of the timing and/or rate of osteoblast maturation.

Organelle and translocatable forms of glyoxysomal malate dehydrogenase. The effect of the N-terminal presequence.

Many organelle enzymes coded for by nuclear genes have N-terminal sequences, which directs them into the organelle (precursor) and are removed upon import (mature). The experiments described below characterize the differences between the precursor and mature forms of watermelon glyoxysomal malate dehydrogenase. Using recombinant protein methods, the precursor (p-gMDH) and mature (gMDH) forms were purified to homogeneity using Ni2+-NTA affinity chromatography. Gel filtration and dynamic light scattering have shown both gMDH and p-gMDH to be dimers in solution with p-gMDH having a correspondingly higher molecular weight. p-gMDH also exhibited a smaller translational diffusion coefficient (D(t)) at temperatures between 4 and 32 degrees C resulting from the extra amino acids on the N-terminal. Differential scanning calorimetry described marked differences in the unfolding properties of the two proteins with p-gMDH showing additional temperature dependent transitions. In addition, some differences were found in the steady state kinetic constants and the pH dependence of the K(m) for oxaloacetate. Both the organelle-precursor and the mature form of this glyoxysomal enzyme were crystallized under identical conditions. The crystal structure of p-gMDH, the first structure of a cleavable and translocatable protein, was solved to a resolution of 2.55 A. GMDH is the first glyoxysomal MDH structure and was solved to a resolution of 2.50 A. A comparison of the two structures shows that there are few visible tertiary or quaternary structural differences between corresponding elements of p-gMDH, gMDH and other MDHs. Maps from both the mature and translocatable proteins lack significant electron density prior to G44. While no portion of the translocation sequences from either monomer in the biological dimer was visible, all of the other solution properties indicated measurable effects of the additional residues at the N-terminal.