Hai-Meng Zhou

Role of proline, glycerol, and heparin as protein folding aids during refolding of rabbit muscle creatine kinase.

Aggregation of 3 M guanidine hydrochloride denatured creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) occurs after dilution into the refolding solution. Proline, glycerol and heparin sodium act as folding aids which can effectively inhibit aggregation of creatine kinase during refolding. Proline at 1 M concentration, glycerol at 10% concentration and heparin at 25 mg/ml not only completely prevented creatine kinase aggregation but also enabled the creatine kinase to return to its native state as well as to recover most of its native activity. The reactivity after the aggregation was completely blocked by the presence of each folding aid reached 65-80% of the native activity. Results of turbidity, activity, intrinsic fluorescence and 1-anilinonaphthalene-8-sulfonate binding fluorescence measurements suggested that the effect of heparin differs from that of proline and glycerol in its artificial chaperone-like behavior. Heparin may bind with creatine kinase both in the native state and during the refolding course. The results showed that this heparin-creatine kinase complex favorably restored the creatine kinase reactivity.

The conserved Ala37 in the ERF/AP2 domain is essential for binding with the DRE element and the GCC box

Four AP2/EREBP genes encoding putative ethylene‐responsive element binding factor (ERF)/AP2 domains were cloned from Brassica napus, and these genes could be induced by low temperature, ethylene, drought, high salinity, abscisic acid and jasmonate treatments. These four genes, named BnDREBIII‐1 to BnDREBIII‐4, were highly homologous and the 37th amino acid was the only difference among their ERF/AP2 domains. BnDREBIII‐1 was demonstrated to be able to bind to both dehydration‐responsive element and the GCC box and transactivate the expression of downstream genes, while BnDREBIII‐4 could bind neither. Further results suggested that Ala37 might play a crucial role in the DNA binding or the stability of the ERF/AP2 domain.

Effect of osmolytes as folding aids on creatine kinase refolding pathway.

The influence of osmolytes, including dimethysulfoxide, glycine, proline and sucrose, on the refolding and reactivation courses of guanidine-denatured creatine kinase was studied by fluorescence emission spectra, circular dichroism spectra, recovery of enzymatic activity and aggregation. The results showed that low concentrations of dimethysulfoxide (<20%), glycine (<0.5 M), proline (<1 M) and sucrose (<0.75 M) improved the refolding yields of creatine kinase, but high osmolyte concentrations decreased its recovery. Sucrose favored the secondary structural formation of creatine kinase. Proline and sucrose facilitated refolding of the protein to its original conformation, while dimethysulfoxide and proline accelerated the hydrophobic collapse of creatine kinase to a packed protein. During the aggregation of creatine kinase, dimethysulfoxide and sucrose inhibited aggregation of creatine kinase, as did proline, but glycine was unable to inhibit aggregation. These systematic observations further support the suggestion that osmolytes, including low concentrations of dimethysulfoxide, proline or sucrose, possibly play a chaperone role in the refolding of creatine kinase. The results also indicate that sucrose and free amino acids are not only energy substrates and organic components in vivo, but also help correct protein folding.

Tyrosinase inhibition by isophthalic acid: kinetics and computational simulation.

Using inhibition kinetics and computational simulation, we studied the reversible inhibition of tyrosinase by isophthalic acid (IPA). IPA inhibited tyrosinase in a complex manner with K(i)=17.8 ± 1.8mM. Measurements of intrinsic and ANS-binding fluorescence showed that IPA induced no changes in tertiary protein structure. For further insight, we predicted the 3D structure of tyrosinase and used a docking algorithm to simulate binding between tyrosinase and IPA. Simulation was successful (binding energies for Dock6.3: -25.19 kcal/mol and for AutoDock4.2: -4.28 kcal/mol), suggesting that IPA interacts with PRO175 or VAL190. This strategy of predicting tyrosinase inhibition based on hydroxyl group number and orientation may prove useful for the screening of potential tyrosinase inhibitors.

Effect of Two Conserved Amino Acid Residues on DREB1A Function

Transcription factors of the DREBP subgroup and the EREBP subgroup contain conserved DNA-binding domains called AP2/EREBP domains, which specifically bind to DRE cis-element and GCC-box, respectively. The 14th and 19th amino acid residues of AP2/EREBP domains are absolutely conserved in the transcription factors of the DREBP subgroup as well as in the EREBP subgroup. However, these two residues of transcription factors of the DREBP subgroup are different from those of the EREBP subgroup. To assess the functional significance of these two residues in binding to the target sequence, the Val (14th residue) and Glu (19th residue) of the AP2/EREBP domain of DREB1A (a transcription factor of the DREBP subgroup) were mutated individually or doubly to Ala and Asp, respectively. This made the 14th and 19th amino acid residues of mutant DREB1A identical to the corresponding residues of transcription factors of the EREBP subgroup. Yeast in vivo analysis showed that: 1) on a selective medium plate of SD/His–Ura–Trp– + 30 mM ∼ 60 mM 3-AT, the growth of yeast cells containing HIS and lacZ double reporter genes was normal in the transformation of the 19th singly mutated DREB1A, obviously inhibited in the transformation of the 14th singly mutated DREB1A, and seriously inhibited in the transformation of the 14th/19th doubly mutated DREB1A; 2) quantitative assay of β-galactosidase activity showed that the intensities of lacZ expression decreased in the transformations of the 14th singly mutated and 14th/19th doubly mutated types. The experimental results revealed that the 19th site mutation did not affect the binding of the DREB1A transcription factor to the DRE cis-element; the 14th site mutation obviously inhibited their binding; and the double mutation of the 14th/19th sites seriously inhibited their binding. This suggests that the conserved Val (14th) and Glu (19th) residues are crucial in the regulation of the binding activity of DREB1A to the DRE cis-element.

Chaperone-like activity of peptidyl-prolyl cis-trans isomerase during creatine kinase refolding.

Porcine kidney 18 kD peptidyl‐prolyl cis‐trans isomerase (PPIase) belongs to the cyclophilin family that is inhibited by the immunosuppressive drug cyclosporin A. The chaperone activity of PPIase was studied using inactive, active, and alkylated PPIase during rabbit muscle creatine kinase (CK) refolding. The results showed that low concentration inactive or active PPIase was able to improve the refolding yields, while high concentration PPIase decreased the CK reactivation yields. Aggregation was inhibited by inactive or active PPIase, and completely suppressed at 32 or 80 times the CK concentration (2.7 μM). However, alkylated PPIase was not able to prevent CK aggregation. In addition, the ability of inactive PPIase to affect CK reactivation and prevent CK aggregation was weaker than that of active PPIase. These results indicate that PPIase interacted with the early folding intermediates of CK, thus preventing their aggregation in a concentration‐dependent manner. PPIase exhibited chaperone‐like activity during CK refolding. The results also suggest that the isomerase activity of PPIase was independent of the chaperone activity, and that the proper molar ratio was important for the chaperone activity of PPIase. The cysteine residues of PPIase may be a peptide binding site, and may be an essential group for the chaperone function.

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase.

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).

COMPARISON OF INACTIVATION AND CONFORMATIONAL CHANGES OF AMINOACYLASE DURING GUANIDINIUM CHLORIDE DENATURATION

The inactivation and unfolding of aminoacylase (EC 3.5.1.14) during denaturation by different concentrations of guanidinium chloride (GuHCl) have been compared. A marked decrease in enzyme activity is already evident at low GuHCl concentrations before significant unfolding of the enzyme molecule, as monitored by fluorescence, ultraviolet difference absorption and CD measurement. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of aminoacylase during denaturation by GuHCl. The inactivation rate constants of free enzyme and substrate-enzyme complex were determined by Tsous method. The inactivation reaction kinetics were found to be a monophasic first-order reaction. The kinetics of the unfolding were a bisphasic process consisting of two first-order reactions. At lower GuHCl concentration (< 1.0 M), the enzyme activity was stripped at a high rate whereas its conformation was only slightly affected. At 1.0 M GuHCl, the inactivation rate of the enzyme was much faster than the unfolding rate. At higher GuHCl concentrations (> 1.0 M), the inactivation rate was too fast to be measured by conventional dynamic methods, whereas the unfolding remained as a bisphasic process with the fast reaction accruing very fast and the slow reaction occurring at a measurable rate. The results suggest that active sites of aminoacylase containing Zn2+ ions are situated in a limited region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.

Promoter hypermethylation correlates with the Hsulf‐1 silencing in human breast and gastric cancer

The HSulf‐1 gene is an important factor that modulates the sulfation status of heparan sulfate proteoglycans (HSPGs) in the extracellular matrix, resulting in disturbance of HSPG‐related signal transduction pathways. Recently, HSulf‐1 has been reported to be down‐regulated in several human cancers. In this study, we first cloned and characterized the 5′ promoter region of the HSulf‐1 gene (around 400 bp) that contained high basal promoter activity. We also found that this functional promoter region was hypermethylated in a number of human cancer cell lines. Furthermore, we found that hypermethylation in this promoter region correlated with the down‐regulation of the HSulf‐1 expression in human breast and gastric cancer cell lines and tissue samples. These results suggest that the promoter hypermethylation may be one of the mechanisms of the HSulf‐1 gene silencing in human breast and gastric cancers. Finally, we demonstrated that the HSulf‐1 promoter was more frequently (p < 0.05) methylated in cell‐free DNA extracted from serum samples of human breast and gastric cancer patients than that of healthy people (76.2%, 55.0% and 19.0%, respectively), indicating that detection of the HSulf‐1 promoter methylation in serum samples may have clinical implications in early detection and diagnosis of human breast and gastric cancers.

The guanidine like effects of arginine on aminoacylase and salt-induced molten globule state.

Aminoacylase is a dimeric enzyme containing one Zn(2+) ion per subunit. The arginine (Arg)-induced unfolding of Holo-aminoacylase and Apo-aminoacylase has been studied by measurement of enzyme activity, fluorescence emission spectra and 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra. Besides being the most alkaline amino acid, the arginine molecule contains a positively charged guanidine group, similar to guanidine hydrochloride, and has been used in many refolding systems to suppress protein aggregation. Our results showed that arginine caused the inactivation and unfolding of aminoacylase, with no aggregation during denaturation. A comparison between the unfolding of aminoacylase in aqueous and HCl (pH 7.5) arginine solutions indicated that the guanidine group of arginine had protein-denaturing effects similar to those of guanidine hydrochloride, which might help us understand the mechanism by which arginine suppresses incorrect refolding. The results showed that arginine-denatured aminoacylase could be reactivated and refolded correctly, indicating that arginine is as good a denaturant as the guanidine or urea for study of protein unfolding and refolding. Both the intrinsic fluorescence and the ANS fluorescence spectra showed that the arginine-unfolded aminoacylase formed a molten globule state in the presence of KCl, suggesting that intermediates exist during aminoacylase refolding. The results for the Apo-aminoacylase followed were similar to those for the Holo-enzyme, suggesting that Holo- and Apo-aminoacylase might have a similar unfolding and refolding pathway.