Wei-Yuan Chou

Selective Oxidative Modification and Affinity Cleavage of Pigeon Liver Malic Enzyme by the Cu2+-Ascorbate System

Pigeon liver malic enzyme was rapidly inactivated by micromolar concentration of Fe2+ in the presence of ascorbate at neutral pH. The inactivated enzyme was subsequently cleaved by the Fe2+-ascorbate system at the chemical bond between Asp258 and Ile259 (Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., and Chang, G. G.(1994) Biochemistry, 33, 7931-7936), which was confirmed by site-specific mutagenesis (Wei, C. H., Chou, W. Y., and Chang, G. G.(1995) Biochemistry 34, 7949-7954). In the present study, at neutral pH, Cu2+ was found to be more reactive in the oxidative modification of malic enzyme and the enzyme was cleaved in a similar manner as Fe2+ did. At acidic pH, however, Fe2+ was found to be ineffective in oxidative modification of the enzyme. Nevertheless, Cu2+ still caused enzyme inactivation and cleaved the enzyme at Asp141-Gly142, Asp194-Pro195, or Asp464-Asp465. Mn2+ and L-malate synergistically protect the enzyme from Cu2+ inactivation at acidic pH. Cu2+ is also a competitive inhibitor versus Mn2+ in the malic enzyme-catalyzed reaction with Ki value 70.3 ± 5.8 μM. The above results indicated that, in addition to the previously determined Asp258 at neutral pH, Asp141, Asp194, and Asp464 are also the coordination sites for the metal binding of malic enzyme. We suggest that the mechanism of affinity modification and cleavage of malic enzyme by the Cu2+-ascorbate system proceed in the following sequence. First, Cu2+ binds with the enzyme at the Mn2+ binding site and reduces to Cu+ by ascorbate. Next, the local oxygen molecules are reduced by Cu+, thereby generating superoxide or other reactive free radicals. These radicals interact with the susceptible essential amino acid residues at the metal-binding site, ultimately causing enzyme inactivation. Finally, the modified enzyme is cleaved into several peptide fragments, allowing the identification of metal site of the enzyme. The pH-dependent different specificities of metal-catalyzed oxidation system may be generally applicable for other enzymes or proteins.

Structural Studies of the Pigeon Cytosolic Nadp+ -Dependent Malic Enzyme

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO2 in the presence of divalent cations (Mg2+, Mn2+). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD+ or NADP+, but not both. Structural studies of the human mitochondrial NAD+‐dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP+‐dependent malic enzyme, in a closed form, in a quaternary complex with NADP+, Mn2+, and oxalate. This represents the first structural information on an NADP+‐dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2′‐phosphate group of the NADP+ cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP+ selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.

Heat shock modulates prion protein expression in human NT-2 cells

The pathological hallmarks of Prion disease are cortical spongi-form changes and neuronal loss, which are induced by the accumulation of the scrapie-isoform prion protein (PrPSc). PrPSc is derived from a post-translational modification of the cellular form of prion protein (PrPC). Heat-shock proteins, a group of molecular chaperones, are involved in the degradation of denatured proteins and post-translational folding of newly synthesized polypeptides. In an attempt to examine any possible relationship between heat shock stress and an induction of prion protein (PrP), human NT-2 cells were treated with heat shock at 42°C for 30 min. After heat-shock treatment, both the level of mRNA and PrPC protein were analyzed at various time points by Northern and Western blot, respectively. There was a 1.5-to 2.5-fold increase in PrP mRNA levels 1 and 3 h following heat shock. In addition, a two-fold increase in protein level of PrP was found 3 h after heat-shock treatment. These results suggest that cellular stress induces the elevation of both PrP mRNA and protein synthesis. The up-regulation of prion-protein mRNA and protein, implies that PrP may play a role in cellular stress.

Effect of Metal Binding on the Structural Stability of Pigeon Liver Malic Enzyme

The cytosolic malic enzyme from the pigeon liver is sensitive to chemical denaturant urea. When monitored by protein intrinsic fluorescence or circular dichroism spectral changes, an unfolding of the enzyme in urea at 25 °C and pH 7.4 revealed a biphasic phenomenon with an intermediate state detected at 4–5m urea. The enzyme activity was activated by urea up to 1 m but was completely lost before the intermediate state was detected. This suggests that the active site region of the enzyme was more sensitive to chemical denaturant than other structural scaffolds. In the presence of 4 mm Mn2+, the urea denaturation pattern of malic enzyme changed to monophasic. Mn2+ helped the enzyme to resist phase I urea denaturation. The [urea]0.5 for the enzyme inactivation shifted from 2.2 to 3.8 m. Molecular weight determined by the analytical ultracentrifuge indicated that the tetrameric enzyme was dissociated to dimers in the early stage of phase I denaturation. In the intermediate state at 4–5 m urea, the enzyme showed polymerization. However, the polymer forms were dissociated to unfolded monomers at a urea concentration greater than 6 m. Mn2+retarded the polymerization of the malic enzyme. Three mutants of the enzyme with a defective metal ligand (E234Q, D235N, E234Q/D235N) were cloned and purified to homogeneity. These mutant malic enzymes showed a biphasic urea denaturation pattern in the absence or presence of Mn2+. These results indicate that the Mn2+ has dual roles in the malic enzyme. The metal ion not only plays a catalytic role in stabilization of the reaction intermediate, enol-pyruvate, but also stabilizes the overall tetrameric protein architecture.

Modulation of glucocorticoid receptor-interacting protein 1 (GRIP1) transactivation and co-activation activities through its C-terminal repression and self-association domains.

Glucocorticoid receptor‐interacting protein 1 (GRIP1), a p160 family nuclear receptor co‐activator, possesses at least two autonomous activation domains (AD1 and AD2) in the C‐terminal region. AD1 activity appears to be mediated by CBP/p300, whereas AD2 activity is apparently mediated through co‐activator‐associated arginine methyltransferase 1 (CARM1). The mechanisms responsible for regulating the activities of AD1 and AD2 are not well understood. We provide evidence that the GRIP1 C‐terminal region may be involved in regulating its own transactivation and nuclear receptor co‐activation activities through primary self‐association and a repression domain. We also compared the effects of the GRIP1 C terminus with those of other factors that functionally interact with the GRIP1 C terminus, such as CARM1. Based on our results, we propose a regulatory mechanism involving conformational changes to GRIP1 mediated through its intramolecular and intermolecular interactions, and through modulation of the effects of co‐repressors on its repression domains. These are the first results to indicate that the structural components of GRIP1, especially those of the C terminus, might functionally modulate its putative transactivation activities and nuclear receptor co‐activator functions.

Human Spot 14 protein is a p53-dependent transcriptional coactivator via the recruitment of thyroid receptor and Zac1.

Spot 14 is an acidic homodimeric protein with no sequence similarity to other mammalian gene products. Its biochemical function remains unclear. Recent studies have shown that the human Spot 14 locus is in the chromosomal region 11q13 and is frequently amplified in breast cancers, suggesting that it plays a role in the gene regulation involved in cell growth during tumorigenesis. Our previous work has demonstrated that human Spot 14 protein physically and functionally interacts with thyroid receptor in the regulation of malic enzyme gene expression. In this study, we investigated the subcellular distribution of human Spot 14 protein using enhanced green fluorescence protein and infer that its localization might be affected by thyroid hormone. Our results also demonstrate that the potential transactivation activity of human Spot 14 protein is regulated by its C-terminal region. Human Spot 14 protein is involved both in the regulation of malic enzyme promoter activity, and in the regulation of p53-dependent transactivation. It does not interact directly with p53, whereas it is able to directly interact with thyroid receptor and Zac1 (zinc finger protein which regulates apoptosis and cell cycle arrest 1) using glutathione-S-transferase pull-down assay. Hence, human Spot 14 protein might regulate the p53 target gene, p21(WAF1/Cip1), via its direct interaction with the thyroid receptor or other p53 coactivators, such as Zac1. These findings provide a molecular rationale for the role of human Spot 14 protein in the p53-dependent transcriptional activation of specific genes via diverse pathways in cells.

Creutzfeldt-Jakob disease: heat shock protein 70 mRNA levels in mononuclear blood cells and clinical study

Abstract Prion diseases such as Creutzfeldt-Jakob disease (CJD) are associated in most cases with the accumulation of an unusual isoform of prion protein (PrPSC). PrPSC is derieved from the abnormal folding of the cellular isoform of prion protein (PrPC). On the other hand, heat shock protein is known to ensure proper protein assembly and folding and to facilitate proteolytic digestion of abnormal or denatured proteins. Many studies have therefore hypothesized that heat shock protein is linked to prion disease. We examined the relationship between heat shock protein HSP70 and prion disease in CJD patients. HSP70 mRNA levels in mononuclear blood cells (MBCs) were compared in 14 CJD patients (10 confirmed by histo-pathological study), 12 vascular dementia (VD) patients, 16 patients with Parkinsons disease and dementia (PD) and 14 nondemented control subjects. The possible correlation between HSP70 mRNA expression levels and clinical findings was also evaluated. HSP70 mRNA expression levels in MBCs were measured by northern blotting. HSP70 mRNA levels in MBCs from patients with CJD were significantly higher than those from patients with VD or PD and in nondemented controls. Age at symptom onset, dementia severity, disease duration and neuroimaging grade of CJD patients were not correlated with relative HSP70 mRNA levels. No significant relationship between HSP70 mRNA levels and aging was found. These results suggest that measurement of HSP70 mRNA in MBCs might provide an auxiliary tool for the diagnosis of CJD.

Caffeine induces tumor cytotoxicity via the regulation of alternative splicing in subsets of cancer-associated genes

Caffeine causes a diverse range of pharmacological effects that are time- and concentration-dependent and reversible. The detailed mechanisms of caffeine in tumor suppression via tumor suppressor protein p53 remain unclear. The isoforms of p53 are physiological proteins that are expressed in normal cells and generated via alternative promoters, splicing sites and/or translational initiation sites. In this study, we investigated how caffeine modulated cell cycle arrest and apoptosis via the expression of various alternatively spliced p53 isoforms. Caffeine reduced p53α expression and induced the expression of p53β, which contains an alternatively spliced p53 C-terminus. In HeLa cells, the expression levels of many serine/arginine-rich splicing factors, including serine/arginine-rich splicing factors 2 and 3, were altered by caffeine. Serine/arginine-rich splicing factor 3 was a promising candidate for the serine/arginine-rich splicing factors responsible for the alternative splicing of p53 in response to caffeine treatment. In addition to p53-dependent functions, multiple target genes of serine/arginine-rich splicing factor 3 suggest that caffeine can regulate epithelial-mesenchymal-transition and hypoxic conditions to inhibit the survival of tumor cells. In summary, our data provide a new pathway of caffeine-modulated tumor suppression via the alternative splicing of the target genes of serine/arginine-rich splicing factor 3.

Engineering of a stable mutant malic enzyme by introducing an extra ion-pair to the protein

A double mutant (R9E/M17K) of pigeon liver malic enzyme with glutamate and lysine replaced for arginine and methionine at positions 9 and 17, respectively, was found to be much more stable in urea and thermal denaturation, but was enzymatically less active than the wild‐type enzyme (WT). Unfolding of the enzyme by urea produced a large red shifting of the protein fluorescence maximum from 320 to 360 nm, which was completely reversible upon dilution. Analysis of the denaturation curves monitored by enzyme activity lost suggested that a putative intermediate was involved in the denaturation process. The half unfolding urea concentration, measured by fluorescence spectral changes, increased from 2.24 M for WT to 3.13 M for R9E/M17K. The melting temperature increased by approximately 10°C for R9E/M17K compared with that for WT. Kinetic analysis of the thermal inactivation at 58°C also conformed to a three‐state model with the rate constant for the intermediate state of R9E/M17K (k2 = 0.03 min‐1) being much smaller than the WT value (k2= 2.39 min‐1). Results obtained from single mutants indicated that the decreasing enzyme activity of R9E/M17K was exclusively due to R9 mutation, which increased the KmMn and KmMal by at least one order of magnitude compared with WT. Consequently, a decrease occurred in the specificity constant [kcat/(KmMnKmNADPKmMal)] for the R9 mutants at least four orders of magnitude smaller than the WT. M17K has similar properties to the WT, while R9E is more labile than the WT enzyme. The above results indicate that the extra stability gained by the double mutant possibly occurs through the introduction of an extra ion‐pair between E9 and K17, which freezes the double mutant in the putative intermediate state. Examination of the N‐terminal amino acid sequence of pigeon liver malic enzyme reveals that position 15 is also a lysine residue. Since the R9E mutant, which has an extra Glu9‐Lys15 ion‐pair, is less stable than the WT, we conclude that the contribution to malic enzyme stability is specific for the Glu9‐Lys17 ion‐pair. Proteins 31:61–73, 1998.

Distinct interactions of αA-crystallin with homologous substrate proteins, δ-crystallin and argininosuccinate lyase, under thermal stress

δ-Crystallin is a taxon-specific eye lens protein that was recruited from argininosuccinate lyase (ASL) through gene sharing. ASL is a metabolic enzyme that catalyzes the reversible conversion of argininosuccinate into arginine and fumarate and shares about 70% sequence identity and similar overall topology with δ-crystallin. ASL has a lower thermal stability than δ-crystallin. In this study, we show that the small heat shock protein, αA-crystallin, functions as a molecular chaperone, and enhanced thermal stability of both δ-crystallin and ASL. The stoichiometry for efficient protection of the two substrate proteins by αA-crystallin was determined by slowly increasing the temperature. N- or C-terminal truncated mutants of δ-crystallin co-incubated with αA-crystallin showed higher thermal stability than wild-type enzyme, and the stoichiometry for efficient protection was the same. Thermal unfolding of δ-crystallin or ASL in the presence of αA-crystallin followed a similar three-state model, as determined by circular dichroism analyses. A stable intermediate which retained about 30% α-helical structure was observed. Protection from thermal denaturation by αA-crystallin was by interaction with partly unfolded ASL or δ-crystallin to form high molecular weight heteroligomers, as judged by size-exclusive chromatography and SDS-PAGE analyses. Aggregate formation of ASL was significantly reduced in the presence of αA-crystallin. The extent of protection of ASL and δ-crystallin at different ratios of αA-crystallin were described by hyperbolic and sigmoidal curves, respectively. These results suggest the preferential recognition of partly unfolded ASL by αA-crystallin. In contrast, unstable δ-crystallin might trigger a cooperative interaction by higher stoichiometries of αA-crystallin leading to fuller protection. The different interactions of αA-crystallin with the two homologous but functionally different substrate proteins show its behavior as a chaperone is variable.