We prospectively evaluated the bone changes associated with proteasome inhibition using single agent bortezomib in relapsed or refractory myeloma patients. Ten patients received bortezomib 1.3 mg/m 2 per days 1, 4, 8 and 11 for three 21-day cycles, and 6 patients received 1 mg/m 2 per day with the same schedule. Bone architecture and metabolism changes were assessed by bone markers, micro-CT, bone histomorphometry, tetracycline labeling and serum parathormone levels. Bone parameter variations were compared by response to treatment. Microarchitectural changes were observed in all evaluable responsive patients. Bone alkaline phosphatase changes were associated with disease response (≥PR vs. others P=0.03 cycle 1, day 11) serum parathormone levels were also significantly increased (P=0.04 on days 11, 21, 33) in responding individuals.This study demonstrates that the myeloma control produced by proteasome inhibition is associated with bone changes and to a discrete pattern of hormonal variation. (Clinicaltrials.gov identifier: NCT00569868)

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Atypical protein kinase C zeta (PKCzeta) plays an important role in cell proliferation and survival. PKCzeta and its truncated form containing only the kinase domain, CATzeta, have been reported to be activated by the phosphorylation of threonine 410 in the activation loop. We expressed both the full length PKCzeta and CATzeta in a baculovirus/insect cell over-expression system and purified the proteins for biochemical characterization. Ion exchange chromatography of CATzeta revealed three species with different levels of phosphorylation at Thr-410 and allowed the isolation of the CATzeta protein devoid of phosphorylation at Thr-410. All three species of CATzeta were active and their activity was not correlated with phosphorylation at Thr-410, indicating that the kinase activity of CATzeta did not depend solely on activation loop phosphorylation. Tyrosine phosphorylation was detected in all three species of CATzeta and the full length PKCzeta. Homology structural modeling of PKCzeta revealed a conserved, predicted-to-be phosphorylated tyrosine residue, Tyr-428, in the close proximity of the RD motif of the catalytic loop and of Thr-410 in the activation loop. The structural analysis indicated that phospho-Tyr-428 would interact with two key, positively-charged residues to form a triad conformation similar to that formed by phospho-Thr-410. Based on these observations, it is possible that the Thr-410 phosphorylation-independent kinase activity of CATzeta is regulated by the phosphorylation of Tyr-428. This alternative mode of PKCzeta activation is supported by the observed stimulation of PKCzeta kinase activity upon phosphorylation at the equivalent site by Abl, and may be involved in resistance to drug-induced apoptosis.

Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3 1 21, with unit-cell parameters a = 70.2, c = 111.6 Å , and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme.

A procedure for the purification of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.341 solubilized from rat liver microsomes is reported. This enzyme has a specific activity of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. This represents a 4100 fold purification over the activity in microsomes, and a specific activity that is approximately 20-fold greater than the highest previously reported value. The enzyme is judged to be homogeneous on the basis of sodium dodecyl sulfate/polyacrylamide disc gel electrophoresis, polyacrylamide disc gel electrophoresis, and immunoanalysis. Data are also presented that indicate the separation of enzymatically active and inactive species of 3-hydroxy-3-methylglutaryl-coenzyme A reductase on affinity chromatography on a coenzyme A column.3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating); EC 1.1.1.34] catalyzes the reduction of HMG-CoA to mevalonic acid, the rate-limiting step of cholesterol biosynthesis in liver (1-3). Therefore, a number of researchers have focused their attention on the regulation of this enzyme. However, to study the modulation of HMG-CoA reductase under various physiological states a method is required to quantitate the amount of enzyme present. This can be achieved by either direct isolation of the enzyme by a reproducible purification procedure or by immunoprecipitation using monospecific antiserum to the enzyme.A number of procedures have been reported for the solubilization and partial purification of HMG-CoA reductase from the microsomal membrane (4-8). In addition, workers in three laboratories have reported the preparation of enzyme that yields only one band on immunodiffusion or sodium dodecyl sulfate (NaDodSO4) disc gel electrophoresis (9-11). However, the enzyme activities of these preparations were very low (10-516 nmol of mevalonate formed per min/mg of protein).In a previous study (12) we succeeded in purifying yeast HMG-CoA reductase to homogeneity. This enzyme had a specific activity of approximately 10,000 nmol of mevalonate formed per min/mg of protein. In this paper we report the purification of HMG-CoA reductase from rat liver by a combination of standard protein fractionation steps and coenzyme A affinity chromatography. This preparation also has a specific activity of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. This value is approximately 20-fold greater than the best value previously reported.As a part of this study we also show that enzymatically active and inactive species of HMG-CoA reductase are separated by affinity chromatography. This separation suggests the possibility that cholesterol synthesis may be regulated in vvo by the interconversion of these species.MATERIALS AND METHODS Materials. Chemicals were obtained from the following sources: 3-hydroxy-3-methyl[3-'4C]glutaric acid, New England Nuclear; coenzyme A, thioester-linked agarose-hexane-coenzyme A, and dithiothrei...