Purification of tapioca leaf rhodanese

Tapioca (Manihot esculenta, kekabu variety) leaf rhodanese was purified 7.8 fold by column chromatography on Sephadex G-10, acetone fractionation a     

A study of the apolar interaction of the enzyme rhodanese with octyl substituted agarose gel

The accessibilities of sites on the surface of the enzyme rhodanese for binding to macromolecular apolarity have been measured for the two forms of the enzyme related to obligatory catalytic intermediates: the free enzyme, E and the sulfur substituted enzyme, ES. This study was done using a micromethod developed for this purpose which allows facile assessment of the apolar binding of proteins to commercially available beads of cross-linked agarose on which hydrophobic groups have been immobilized. The results indicate that the enzyme rhodanese can bind to macromolecular apolarity and that there is considerably more binding of the E form than the ES form. The fact that the binding is relatively slow implicates a protein conformational change in the rate limiting binding step. In fact, there is a large increase in the binding when the temperature is raised from 23° to 40° which correlates with previous results showing a conformational change in rhodanese over the same temperature range. These results in comparison with other solution studies and with x-ray studies are consistent with a model for rhodanese which has an apolar active site and a mechanism for catalysis that includes a conformational change.     

Multivariate factor analysis of sulfur oxidation and rhodanese activity in soils

The role of rhodanese as an intermediate catalyst in the oxidation of elemental S (S°) is not well understood. This study investigated the effect of 26 soil properties and steam sterilization in relation to S° oxidation and rhodanese activity in 33 soils (27 Oregon soils and six Chinese soils). S° oxidation potential was determined by incubating (7 d at 23 °C) soil amended with 500 mg S° kg^-1 soil and measuring the SO₄ released. Both total S° oxidation (TSO) and rhodanese activity varied widely among the 33 soils, ranging from 0 to 143 mg SO₄-S kg^-1 soil 7 d^-1 and 22 to 2109 nmoles SCN^- g^-1 soil h^-1 respectively. S° oxidation but not rhodanese activity had a significant positive correlation with soil pH. In sterile soils, chemical S° oxidation (CSO) averaged 3% of the total S° oxidation and apparent rhodanese activity averaged 11% of the total rhodanese activity. S° oxidation was not significantly correlated with rhodanese activity. However, development of stepwise regression models predicting S° oxidation revealed that rhodanese activity was an important explanatory variable in predicting biological S° oxidation (TSO minus CSO). Also, microbial biomass C was found to be an important parameter in models for both S° oxidation and rhodanese activity. Investigations of the effect of acidification during S° oxidation showed that biological S° oxidation was negatively correlated with S° oxidation-induced-pH-change for soils with pH > 6 but no such significant relationship was found on soils with pH> 6. This suggested that extreme acidity may inhibit S° oxidation but not rhodanese activity.     

The "rhodanese" fold and catalytic mechanism of 3-mercaptopyruvate sulfurtransferases: crystal structure of SseA from Escherichia coli

3-Mercaptopyruvate sulfurtransferases (MSTs) catalyze, in vitro, the transfer of a sulfur atom from substrate to cyanide, yielding pyruvate and thiocyanate as products. They display clear structural homology with the protein fold observed in the rhodanese sulfurtransferase family, composed of two structurally related domains. The role of MSTs in vivo, as well as their detailed molecular mechanisms of action have been little investigated. Here, we report the crystal structure of SseA, a MST from Escherichia coli, which is the first MST three-dimensional structure disclosed to date. SseA displays specific structural differences relative to eukaryotic and prokaryotic rhodaneses. In particular, conformational variation of the rhodanese active site loop, hosting the family invariant catalytic Cys residue, may support a new sulfur transfer mechanism involving Cys237 as the nucleophilic species and His66, Arg102 and Asp262 as residues assisting catalysis.     

Determination of rhodanese activity by tetrazolium reduction

A colorimetric method for the assay of rhodanese activity based on the continuous determination of the sulfite product is described. 5-Ethylphenazinium ethyl sulfate is used as the intermediate electron carrier between sulfite and nitroblue tetrazolium to produce the colored reduced species. The present method is more sensitive than the usual procedure based on the colorimetric determination of thiocyanate. Furthermore, the color developed by nitroblue tetrazolium reduction affords a straightforward means to locate rhodanese activity in polyacrylamide gels.     

The chaperonin assisted and unassisted refolding of rhodanese can be modulated by its N-terminal peptide

Thein vitro refolding of the monomeric, mitochondrial enzyme rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), which is assisted by theE. coli chaperonins, is modulated by the 23 amino acid peptide (VHQVLYRALVSTKWLAESVRAGK) corresponding to the amino terminal sequence (1–23) of rhodanese. In the absence of the peptide, a maximum recovery of active enzyme of about 65% is achieved after 90 min of initiation of the chaperonin assisted folding reaction. In contrast, this process is substantially inhibited in the presence of the peptide. The maximum recovery of active enzyme is peptide concentration-dependent. The peptide, however, does not prevent the interaction of rhodanese with the chaperonin 60 (cpn60), which leads to the formation of the cpn60-rhodanese complex. In addition, the peptide does not affect the rate of recovery of active enzyme, although it does affect the extent of recovery. Further, the unassisted refolding of rhodanese is also inhibited by the peptide. Thus, the peptide interferes with the folding of rhodanese in either the chaperonin assisted or the unassisted refolding of the enzyme. A 13 amino acid peptide (STKWLAESVRAGK) corresponding to the amino terminal sequence (11–23) of rhodanese does not show any significant effect on the chaperonin assisted or unassisted refolding of the enzyme. The results suggest that other sequences of rhodanese, in addition to the N-terminus, may be required for the binding of cpn60, in accord with a model in which cpn60 interacts with polypeptides through multiple binding sites.     

cDNA expression array analysis of gene expression in human hepatocarcinoma Hep3B cells induced by BNIPL-1

Bcl-2/adenovirus E1B 19 kDa interacting protein 2 like-1 （BNIPL-1） is a novel human protein identified in our laboratory, which can interact with Bcl-2 and Cdc42GAP and induce apoptosis via the BNIP-2 and Cdc42GAP homology （BCH） domain. In the present study, we established the Hep3B-Tet-on stable cell line in which expression of BNIPL-1 can be induced by doxycycline. The cell proliferation activity assay showed that the overexpression of BNIPL-1 suppresses Hep3B cell growth in vitro. The differential expression profiles of 588 known genes from BNIPL-1-transfected Hep3B-Tet-on and vector control cells were determined using the Atlas human cDNA expression array. Fifteen genes were differentially expressed between these two cell lines, among which seven genes were up-regulated and eight genes were down-regulated by BINPL-1. Furthermore, the differential expression result was confirmed by semiquantitative RT-PCR. Among these differentially expressed genes, p16^INK4, IL-12, TRAIL and the lymphotoxin β gene involved in growth suppression or cell apoptosis were up-regulated, and PTEN involved in cell proliferation was down-regulated by BNIPL-1. These results suggest that BNIPL-1 might inhibit cell growth though cell cycle arrest and/or apoptotic cell death pathway（s）.     

Apoptosis induction by gamma-tocotrienol in human hepatoma Hep3B cells

We evaluated the antitumor activity of tocotrienol (T3) on human hepatoma Hep3B cells. At first, we examined the effect of T3 on the proliferation of human hepatoma Hep3B cells and found that gamma-T3 inhibited cell proliferation at lower concentrations and shorter treatment times than alpha-T3. Then, we examined the effect of gamma-T3 apoptosis induction and found that gamma-T3 induced poly (ADP-ribose) polymerase (PARP) cleavage and stimulated a rise in caspase-3 activity. In addition, gamma-T3 stimulated a rise in caspase-8 and caspase-9 activities. We also found that gamma-T3-induced apoptotic cell death was accompanied by up-regulation of Bax and a rise in the fragments of Bid and caspase-8. These data indicate that gamma-T3 induced apoptosis in Hep3B cells and that caspase-8 and caspase-9 were involved in apoptosis induction. Moreover, these results suggest that Bax and Bid regulated apoptosis induction by gamma-T3.     

Biological properties of human melanoma cells in culture

Summary Three human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary. THis work was supported in part by USPHS Grant No. 5 T01 AI00332-06 from the National Institutes of Health, Contract E73-2001-N01-CP-3-3237 from the Virus Cancer Program of the National Cancer Institute, and USPHS Grant No. 0H00714-02 from the National Institute for Occupational Safety and Health.     

Crystal structure of YnjE from Escherichia coli,a sulfurtransferase with three rhodanese domains

Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple‐domain rhodanese‐like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well‐characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N‐terminal inactive rhodanese‐like domain. Phylogenetic analysis reveals that YnjE triple‐domain homologs can be found in a variety of other γ‐proteobacteria, in addition, some single‐, tandem‐, four and even six‐domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3‐mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C‐terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.     

Intracellular glutathione regulates Andrographolide-induced cytotoxicity on hepatoma Hep3B cells

Abstract

Andrographolide (ANDRO), a diterpenoid lactone isolated from the traditional herbal plant Andrographis paniculata, was reported to induce apoptosis in hepatoma Hep3B cells in our previous study (Ji LL, Liu TY, Liu J, Chen Y, Wang ZT. Andrographolide inhibits human hepatoma-derived Hep3B cells growth through the activation of c-Jun N-terminal kinase. Planta Med 2007; 73: 1397–1401). The present investigation was carried out to observe whether cellular reduced glutathione (GSH) plays important roles in ANDRO-induced apoptosis. ANDRO initially increased intracellular GSH levels which then decreased later, while inhibition of cellular GSH synthesis by L-Buthionine-(S,R)-sulfoximine (BSO) augmented ANDRO-induced cytotoxicity and apoptosis in Hep3B cells. On the other hand, the thiol antioxidant dithiothreitol (DTT) rescued ANDRO-depleted cellular GSH, and abrogated ANDRO-induced cytotoxicity and apoptosis. Furthermore, BSO pretreatment augmented ANDRO-decreased expression of antioxidant protein thioredoxin 1 (Trx1), while DTT reversed this decrease. Further results showed that ANDRO increased the activity of the GSH-related antioxidant enzyme glutathione peroxidase (GPx) and the production of intracellular reactive oxygen species (ROS). Taken together, this study demonstrates that the intracellular redox system plays important roles in regulating the cytotoxicity of ANDRO on hepatoma Hep3B cells.     

The N-terminal rhodanese domain from Azotobacter vinelandii has a stable and folded structure independently of the C-terminal domain

Sulfurtransferase are enzymes involved in the formation, conversion and transport of compounds containing sulfane-sulfur atoms. Although the three-dimensional structure of the rhodanese from the nitrogen-fixing bacterium Azotobacter vinelandii is known, the role of its two domains in the protein conformational stability is still obscure. We have evaluated the susceptibility to proteolytic degradation of the two domains of the enzyme. The two domains show different resistance to the endoproteinases and, in particular, the N-terminal domain shows to be more stable to digestion during time than the C-terminal one. Cloning and overexpression of the N-terminal domain of the protein was performed to better understand its functional and structural role. The recombinant N-terminal domain of rhodanese A. vinelandii is soluble in water solution and the spectroscopic studies by circular dichroism and heteronuclear NMR spectroscopy indicate a stable fold of the protein with the expected alpha/beta topology. The results indicate that this N-terminal domain has already got all the elements necessary for an C-terminal domain independent folding. Its solution structure by NMR, actually under course, will be a valid contribution to understand the role of this domain in the folding process of the sulfurtransferase.     

ASPP2以p53非依赖形式促进自噬引起Hep3B细胞凋亡

p53凋亡刺激蛋白2（apoptosis stimulating protein 2 of p53, ASPP2）能特异性地与p53蛋白结合并增强其促凋亡的功能,进而发挥抗肿瘤作用. 本室前期研究发现,ASPP2可以通过p53-DRAM自噬途径诱导细胞凋亡. 在本研究中,利用ASPP2 腺病毒感染Hep3B细胞(p53缺陷型肝癌细胞系)并用甲基磺酸(MMS)处理后; Calcein AM/PI和M30染色检测细胞凋亡;GFP-LC3质粒转染细胞后检测自噬; 荧光定量PCR和免疫印迹检测自噬基因表达. 结果表明,ASPP2在p53缺陷的Hep3B细胞内可诱导发生凋亡;在MMS存在和缺失条件下, Adr-ASPP2均引起自噬体水平升高及自噬基因的表达增 加,且MMS协同Adr-ASPP2能使自噬水平增加; 进一步用VPS34 siRNA和DRAM siRNA抑 制自噬发现,细胞凋亡水平下降, 说明由Adr-ASPP2诱发经损伤相关自噬调节蛋白（ DRAM）介导的自噬参与了肝癌细胞系凋亡的发生. 综上结果表明,ASPP2可以通过非p53依赖的DRAM介导自噬,并促进肝癌细胞凋亡. 该研究可为肝癌的基因治疗提供新的思路.     

肝癌细胞系Hep3B的泛素化蛋白质组学

泛素化修饰是细胞内最重要的翻译后修饰形式之一,对细胞内蛋白质的稳定、降解、定位以及生物活性的调节起到重要作用。但因其在细胞内丰度低、降解周期短等特点而很难被检测。本研究中,制备的泛素结合结构域蛋白(Ubiquitin-binding domains,UBDs)用于富集肝癌细胞系Hep3B中的泛素化蛋白,并通过液相色谱-串联质谱联用的方法对富集的泛素化蛋白进行鉴定。实验共鉴定到1 900个潜在的泛素化蛋白和158个泛素化位点,这些被鉴定到的泛素化位点分属于102个蛋白。生物信息学分析发现泛素化蛋白显著富集的相关通路与肿瘤的发生发展密切相关,此结果暗示肿瘤细胞内泛素化-蛋白酶体的失调与肿瘤细胞的信号传导及细胞外基质的变化等具有较高的关联性。     

Sulfhydryl modification ofE. coli cpn60 leads to loss of its ability to support refolding of rhodanese but not to form a binary complex

Differential chemical modification ofE. coli chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents. For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4-dipyridyl disulfide (4-PDS). However, no sulfhydryl groups were modified when the even larger reagents 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) or 2-(4-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS), were employed, unless the chaperonin was unfolded. The cpn60 that had been covalently modified with NEM or IAANS, was not able to support the chaperonin-assisted refolding of the mitochondrial enzyme rhodanese, which also requires cpn10 and ATP hydrolysis. However, both modified forms of cpn60 were able to form binary complexes with rhodanese, as demonstrated by their ability to arrest the spontaneous refolding of the enzyme. That is, chemical modification with these sulfhydryl-directed reagents produced a species that was not prevented from interaction with partially folded rhodanese, but that was prevented from supporting a subsequent step(s) during the chaperonin-assisted refolding process.     

Susceptibility of Hep3B cells in different phases of cell cycle to tBid

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC.     

Heat shock induction of heme oxygenase mRNA in human Hep 3B hepatoma cells

Heat shock treatment of human Hep 3B hepatoma cells led to the induction of mRNA for microsomal heme oxygenase. The maximum induction of heme oxygenase mRNA (5----7-fold) was observed with treatment of cells at 43.5 degrees C, for 60 min. The heat-mediated induction of heme oxygenase mRNA was blocked by simultaneous treatment of cells with actinomycin D or cycloheximide. In contrast to Hep 3B cells, cells of another human hepatoma line, Hep G2, showed little induction of heme oxygenase mRNA by heat treatment. These findings suggest that heat shock treatment induces heme oxygenase mRNA in certain human hepatoma cells, but not in others.     

Gypenoside induces apoptosis in human Hep3B and HA22T tumour cells

The effect of gypenoside, an active component of the Chinese herb Gynostemma pentaphyllum (Thumb) Makino, on human hepatoma cell lines (Hep3B and HA22T) was investigated. Results demonstrated that gypenoside inhibited the proliferation or viability of the Hep3B and HA22T cells in a dose-dependent manner. The Hep3B and HA22T cells treated with gypenoside for 2 days were less DNA stainable and formed a sub-G1 peak. The treated cells increased cell numbers in the A0 region as well as shifting the ordinary S phase to the final S phase (D1 region), and induced a ladder pattern of fragmented DNA of about 200 base pairs. These data suggest that the cell death of the hepatoma cell lines Hep3B and HA22T induced by gypenoside was via apoptosis, and this was confirmed by morphological studies.     

N-containing compounds from Hymenocallis littoralis and their cytotoxicity against Hep3B cells

A chemical study focusing on the N-containing constituents of Hymenocallis littoralis was carried out, leading to the isolation of two new alkaloidal flavonoids (1–2) and five known alkaloids (3-7). Structures of the isolated compounds were established mainly by spectroscopic techniques, including NMR spectroscopy and mass spectrometry as well as the CD experiment. These compounds were subjected to the in vitro cytotoxicity assays using Hep3B cells, and among them only the known pancratistatine (3) and lycorine (5) could significantly inhibit the Hep3B cell proliferation.