Crystal structures of human muscle fructose-1,6-bisphosphatase: novel quaternary states, enhanced AMP affinity, and allosteric signal transmission pathway.

Fructose-1,6-bisphosphatase, a key enzyme in gluconeogenesis, is subject to metabolic regulation. The human muscle isozyme is significantly more sensitive towards the allosteric inhibitor, AMP, than the liver isoform. Here we report crystal structures and kinetic studies for wild-type human muscle Fru-1,6-Pase, the AMP-bound (1.6 Å), and product-bound complexes of the Q32R mutant, which was firstly introduced by an error in the cloning. Our high-resolution structure reveals for the first time that the higher sensitivity of the muscle isozyme towards AMP originates from an additional water-mediated, H-bonded network established between AMP and the binding pocket. Also present in our structures are a metaphosphate molecule, alternate conformations of Glu97 coordinating Mg(2+), and possible metal migration during catalysis. Although the individual subunit is similar to previously reported Fru-1,6-Pase structures, the tetrameric assembly of all these structures deviates from the canonical R- or T-states, representing novel tetrameric assemblies. Intriguingly, the concentration of AMP required for 50% inhibition of the Q32R mutant is increased 19-fold, and the cooperativity of both AMP and Mg(2+) is abolished or decreased. These structures demonstrate the Q32R mutation affects the conformations of both N-terminal residues and the dynamic loop 52-72. Also importantly, structural comparison indicates that this mutation in helix α2 is detrimental to the R-to-T conversion as evidenced by the absence of quaternary structural changes upon AMP binding, providing direct evidence for the critical role of helix α2 in the allosteric signal transduction.

Comparative proteomic analysis of colon cancer cells in response to oxaliplatin treatment.

Colon cancer is one of the most common malignancies in the world. Oxaliplatin, a third-generation platinum compound, is widely used in clinical chemotherapy of colon cancer. Although the mechanisms of the antitumor effect of Oxaliplatin have been investigated in recent years, the proteomic changes that are associated with the cellular response to this compound are poorly understood. In this study, we performed a comparative proteomic analysis to survey the global changes in protein expression levels after Oxaliplatin treatment in three colon cancer cell lines: HT29, SW620, and LoVo. Two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF mass spectrometry revealed 57, 48, and 53 differentially expressed proteins in the three cell lines (HT29, SW620 and LoVo, respectively) after Oxaliplatin treatment. Of these proteins, 21 overlapped among all three cell lines. These overlapping proteins participate in many cellular processes, such as apoptosis, signal transduction, transcription and translation, cell structural organization, and metabolism. Additionally, the expression levels of ezrin (EZRI), heat-shock protein beta-1 (HSPB1), translationally controlled tumor protein (TCTP), and cell division control protein 2 homolog (CDC2) were confirmed by immunoblotting. This is the first direct proteomic analysis of Oxaliplatin-treated colon cancer cells. Several interesting proteins that we found warrant further investigation owing to their potential significant functions in the antitumor effect of Oxaliplatin.

Ferritin heavy chain-mediated iron homeostasis and subsequent increased reactive oxygen species production are essential for epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) plays a critical role in tumor progression. To obtain a broad view of the molecules involved in EMT, we carried out a comparative proteomic analysis of transforming growth factor-beta1 (TGF-beta1)-induced EMT in AML-12 murine hepatocytes. A total of 36 proteins with significant alterations in abundance were identified. Among these proteins, ferritin heavy chain (FHC), a cellular iron storage protein, was characterized as a novel modulator in TGF-beta1-induced EMT. In response to TGF-beta1, there was a dramatic decrease in the FHC levels, which caused iron release from FHC and, therefore, increased the intracellular labile iron pool (LIP). Abolishing the increase in LIP blocked TGF-beta1-induced EMT. In addition, increased LIP levels promoted the production of reactive oxygen species (ROS), which in turn activated p38 mitogen-activated protein kinase. The elimination of ROS inhibited EMT, whereas H2O2 treatment rescued TGF-beta1-induced EMT in cells in which the LIP increase was abrogated. Overexpression of exogenous FHC attenuated the increases in LIP and ROS production, leading to a suppression of EMT. We also showed that TGF-beta1-mediated down-regulation of FHC occurs via 3' untranslated region-dependent repression of the translation of FHC mRNA. Moreover, we found that FHC down-regulation is an event that occurs between the early and highly invasive advanced stages in esophageal adenocarcinoma and that depletion of LIP or ROS suppresses the migration of tumor cells. Our data show that cellular iron homeostasis regulated by FHC plays a critical role in TGF-beta1-induced EMT.

Equilibrium folding of porcine insulin precursor in the presence of redox buffer: implications for the common intermediates shared by its unfolding/ refolding processes.

We use the procedure established for 'disulfide stability analysis in redox system' to investigate the unfolding process of porcine insulin precursor (PIP). Six major unfolding intermediates have been captured, in which four contain two disulfides, two contain one disulfide. Based on the characterization and analysis of the intermediates an unfolding pathway has been proposed, by which the native PIP unfolded through in turn 2SS and 1SS intermediates into fully reduced form. Besides, the comparison of the intermediates captured in PIP unfolding process with those intermediates captured in its refolding process revealed that some intermediates captured during both unfolding/refolding processes of PIP have identical disulfide pairing pattern, from which we suggest that the unfolding/refolding processes of PIP share some common intermediates but flow in the opposite direction.

Expression and characterization of two secreted His6-tagged endo-beta-1,4-glucanases from the mollusc Ampullaria crossean in Pichia pastoris.

Two endo-beta-1,4-glucanase cDNAs, eg27I and eg27II, from the mollusc Ampullaria crossean were expressed in Pichia pastoris cells. The secreted His6-tagged proteins were purified in a single chromatography step. The purified recombinant EG27I and EG27II showed enzymatic activity on carboxylmethyl cellulose sodium salt at 15.31 U/mg and 12.40 U/mg, respectively. The optimum pH levels of the recombinant EG27I and EG27II were 5.5 and 5.5-6.0, respectively, and the optimum temperatures were 50 degrees C and 50 degrees C-55 degrees C, respectively. The pH stability study revealed that both EG27I and EG27II showed their highest stability at pH 8.0. Analysis of their thermostability indicated that both EG27I and EG27II were relatively stable up to 40 degrees C. Site-directed mutagenesis of Asp43 and Asp153 of both EG27I and EG27II showed that the two Asp residues are critical for the enzymatic activity.

Stain efficiency and MALDI-TOF MS compatibility of seven visible staining procedures.

Visible stain is still the most popular protein staining method used in proteomic approaches. However, most published data have been derived from comparisons between visible dyes and fluorescent dyes. In this work, we have focused on seven widely used visible staining procedures--Neuhoff CCB, blue silver, and five silver stains (LKB SN, He SN, Yan SN, Vorum SN, and Blum SN)--and studied their stain efficiencies and MALDI-TOF MS compatibilities on 1-D and 2-D PAGE. It was concluded that blue silver is slightly better in terms of stain efficiency than Neuhoff CCB, but it presented worse MS compatibility. Neuhoff CCB presented better MS compatibility and superior linearity but worse sensitivity than silver stains. Among the five silvering procedures, He SN showed the best MS compatibility and a reasonable staining efficiency; Yan SN lowered the chances of obtaining the protein identity by PMF but gave the best stain efficiency; Vorum SN gave a very clear background and a great contrast, while Blum SN was the worst in this respect. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.

Molecular cloning and characterization of two novel cellulase genes from the mollusc Ampullaria crossean.

Cellulase genes have been reported not only from fungi, bacteria and plant, but also from some invertebrate animals. Here, two cellulase (endo-beta-1,4-glucanase, EC 3.2.1.4) genes, eg27I and eg27II, were cloned from the freshwater snail Ampullaria crossean cDNA using degenerate primers. The nucleotide sequences of the two genes shared 94.5% identity. The open reading frames of both genes consisted of 588 bp, encoding 195 amino acids. Both EG27I and EG27II belong to the glycoside hydrolase family 45, and each lacks a carbohydrate-binding module. The presence of introns demonstrated a eukaryotic origin of the EG27 gene, and, in addition, successful cloning of EG27 cDNA supported endogenous production of EG27 cellulase by Ampullaria crossean. Investigation of the EG27 cDNA from A. crossean will provide further information on GHF45 cellulases.

In vitro insulin refolding: characterization of the intermediates and the putative folding pathway.

The in vitro refolding process of the double-chain insulin was studied based on the investigation of in vitro single-chain insulin refolding. Six major folding intermediates, named P1A, P2B, P3A, P4B, P5B, and P6B, were captured during the folding process. The refolding experiments indicate that all of these intermediates are on-pathway. Based on these intermediates and the formation of hypothetic transients, we propose a two-stage folding pathway of insulin. (1) At the early stage of the folding process, the reduced A chain and B chain individually formed the intermediates: two A chain intermediates (P1A and P3A), and four B chain intermediates (P2B, P4B, P5B, and P6B). (2) In the subsequent folding process, transient I was formed from P3A through thiol/disulfide exchange reaction; then, transients II and III, each containing two native disulfides, were formed through the recognition and interaction of transient I with P4B or P6B and the thiol group's oxidation reaction mainly using GSSG as oxidative reagent; finally, transients II and III, through thiol/mixture disulfide exchange reaction, formed the third native disulfide of insulin to complete the folding.

Identification of candidate prostate cancer biomarkers in prostate needle biopsy specimens using proteomic analysis.

Although serum prostate specific antigen (PSA) is a well-established diagnostic tool for prostate cancer (PCa) detection, the definitive diagnosis of PCa is based on the information contained in prostate needle biopsy (PNBX) specimens. To define the proteomic features of PNBX specimens to identify candidate biomarkers for PCa, PNBX specimens from patients with PCa or benign prostatic hyperplasia (BPH) were subjected to comparative proteomic analysis. 2-DE revealed that 52 protein spots exhibited statistically significantly changes among PCa and BPH groups. Interesting spots were identified by MALDI-TOF-MS/MS. The 2 most notable groups of proteins identified included latent androgen receptor coregulators [FLNA(7-15) and FKBP4] and enzymes involved in mitochondrial fatty acid beta-oxidation (DCI and ECHS1). An imbalance in the expression of peroxiredoxin subtypes was noted in PCa specimens. Furthermore, different post-translationally modified isoforms of HSP27 and HSP70.1 were identified. Importantly, changes in FLNA(7-15), FKBP4, and PRDX4 expression were confirmed by immunoblot analyses. Our results suggest that a proteomics-based approach is useful for developing a more complete picture of the protein profile of PNBX specimen. The proteins identified by this approach may be useful molecular targets for PCa diagnostics and therapeutics.

Molecular and biochemical characterization of Ba-EGA, a cellulase secreted by Bacillus sp. AC-1 from Ampullaria crosseans.

A novel gene (Ba-ega) of Bacillus sp. AC-1, encoding an endoglucanase (Ba-EGA), was cloned and expressed in Escherichia coli. Ba-ega, containing a 1,980-bp open reading frame (ORF), encoded a protein of 659 amino acids and had a molecular mass of 74.87 kDa. Ba-EGA was a modular enzyme composed of a family-9 glycosyl hydrolase catalytic module (CM9) and a family-3 carbohydrate-binding module (CBM3). To investigate the functions of the CBM3 and CM9, a number of truncated derivatives of Ba-EGA were constructed, and all were active. The catalytic module (rCM9) alone was less stable at high temperature than the recombinant Ba-EGA (rBa-EGA). The temperature stability for the complex of rCM9 and rCBM3 was still lower than rBa-EGA, but higher than rCM9 alone. These observations indicated the existence of a non-covalent interaction between CM9 and CBM3 that might strengthen the stability of CM9. However, this interaction is not strong enough to mimic the protective effect of the CBM in the wild-type enzyme.

The sequence determinant causing different folding behaviors of insulin and insulin-like growth factor-1.

Although insulin and insulin-like growth factor-1 (IGF-1) belong to the insulin superfamily and share highly homologous sequences, similar tertiary structure, and a common ancestor molecule, amphioxus insulin-like peptide, they have different folding behaviors: IGF-1 folds into two thermodynamically stable tertiary structures (native and swap forms), while insulin folds into one unique stable structure. To further understand which part of the sequence determines their different folding behavior, based on previous reports from the laboratory, two peptide models, [B9A][1-4]porcine insulin precursor (PIP) and [B10E][1-4]PIP, were constructed. The plasmids encoding the peptides were transformed into yeast cells for expression of the peptides; the results showed that the former peptide was expressed as single component, while the latter was expressed as a mixture of two components (isomer 1 and isomer 2). The expression results together with studies of circular dichoism, disulfide rearrangement, and refolding lead us to deduce that isomer 1 corresponds to the swap form and the isomer 2 corresponds to the native form. We further demonstrate that the sequence 1-4 plus B9 of IGF-1 B-domain can make PIP fold into two structures, while sequence 1-5 of insulin B-chain can make IGF-1 fold into one unique structure. In other words, it is the IGF-1 B-domain sequence that 1-4 allows IGF-1 folding into two thermodynamically stable tertiary structures; this sequence plus its residue B9E can change PIP folding behavior from folding into one unique structure to two thermodynamically stable structures, like that of IGF-1.

A novel thermoacidophilic endoglucanase, Ba-EGA, from a new cellulose-degrading bacterium, Bacillus sp.AC-1.

A newly discovered bacterium, strain AC1, containing cellulase was isolated from the gastric juice of the mollusca, Ampullaria crosseans. Analysis of the 16S rDNA sequence and carbon sources revealed that the bacterium belonged to the genus Bacillus. A novel endoglucanase (Ba-EGA) was purified from culture supernatants of the bacterium growing in CMC-Na (low viscosity) induction medium. The cellulase was purified about 150-fold by ammonium sulfate fractionation, ion exchange, hydrophobic, and gel filtration chromatography, with a specific activity of 35.0 IU/mg. The molecular mass of the enzyme was 67 kDa. N-terminal amino acid sequencing revealed a sequence of SDYNYVEVLQKSILF, which had high homology with endoglucanases from the Bacillus and Clostridium species. The maximal activity of the enzyme with the substrate of CM-cellulose is at pH 4.5-6.5 and 70 degrees C, respectively. The studies on pH and temperature stability showed that the Ba-EGA is stable enough between pH 7.5 and 10.5 at 30 degrees C for 2 h, and more than 80% of the activity still remains when incubation was prolonged to 1 h at 50 degrees C. The activity of the enzyme was significantly inhibited by Fe(2+), Cu(2+) (5.0 mM of each), and sodium dodecyl sulfate (SDS) (0.5%) and obviously activated by Tween 20 and Triton X-100 (0.25% each). Binding studies revealed that the Ba-EGA had cellulose-binding domain.

Study on substrate specificity at subsites for severe acute respiratory syndrome coronavirus 3CL protease.

Autocleavage assay and peptide-based cleavage assay were used to study the substrate specificity of 3CL protease from the severe acute respiratory syndrome coronavirus. It was found that the recognition between the enzyme and its substrates involved many positions in the substrate, at least including residues from P4 to P2'. The deletion of either P4 or P2' residue in the substrate would decrease its cleavage efficiency dramatically. In contrast to the previous suggestion that only small residues in substrate could be accommodated to the S1' subsite, we have found that bulky residues such as Tyr and Trp were also acceptable. In addition, based on both peptide-based assay and autocleavage assay, Ile at the P1' position could not be hydrolyzed, but the mutant L27A could hydrolyze the Ile peptide fragment. It suggested that there was a stereo hindrance between the S1' subsite and the side chain of Ile in the substrate. All 20 amino acids except Pro could be the residue at the P2' position in the substrate, but the cleavage efficiencies were clearly different. The specificity information of the enzyme is helpful for potent anti-virus inhibitor design and useful for other coronavirus studies.

Purification and characterization of two endo-beta-1,4-glucanases from mollusca, Ampullaria crossean.

Two novel endo-beta-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50 degrees and 60 degrees; for EG45 it was 50 degrees. The analysis on the stability of these two endo-beta-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 degrees, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 degrees for 24 h. However, less than 10% residual activity of EG45 was detected at 50 degrees. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan (from oat spelt) and Sigmacell 101, and they were inactive to p-nitrophenyl-beta-D-cellobioside, salicin and starch.

A novel auto-cleavage assay for studying mutational effects on the active site of severe acute respiratory syndrome coronavirus 3C-like protease.

The 3C-like protease (3CLpro) of severe acute respiratory syndrome (SARS) has been proposed as an attractive target for drug design. His41 and Cys145 were essential for the active site as the principal catalytic residues. In this study, we mutated the two sites, expressed four resulting mutants in Escherichia coli and characterized. All mutants showed undetectable activity in trans-cleavage assay. In addition, we introduced a 31-mer peptide containing an auto-cleavage site to the N-terminal of the proteases and found the peptide could be cleaved efficiently by 3CLsc itself, but, among the four mutants, only the mutant Cys145-->Ser showed residual activity as detected by the auto-cleavage assay. The data supported the proposition unequivocally that SARS-CoV 3CLpro was a member of serine proteases involving His41 and Cys145 residues at the active site. The auto-cleavage assay also provided a sensitive and reliable compensation to the traditional trans-cleavage assay.

The role of propeptide in the refolding of human group IB phospholipase A2.

Human group IB phospholipase A2 (IB-PLA2) and its zymogen (proIB-PLA2) were purified from E.coli. Refolding was carried out by diluting the denatured forms of both IB-PLA2 and proIB-PLA2 with renaturation buffer in which the disulfide bonds were completely reduced. The refolding yield of proIB-PLA2 was increased by about 50% over that of the mature enzyme. The refolding of IB-PLA2 usually produced aggregates under normal conditions, as determined by light scattering. In addition, the unfolding experiments showed that the mature enzyme was more stable than the proenzyme toward denaturants in the presence of DTT. Results suggested that the N-terminal sequence rather than its conformation of human proIB-PLA2 played an important role in the refolding process.

pH-dependent stability of EGX, a multi-functional cellulase from mollusca, Ampullaria crossean.

The cellulase activity and stability of EGX, a multi-functional cellulase previously purified from the mollusca Ampullaria crossean, was systematically studied under different pH. The pH induced con-formation and stability change of EGX have been investigated by using the intrinsic fluorescence, ANS fluorescence and CD spectrum. It has been found that the conformation and activity of this cellulase were strongly dependent on the pH. EGX was stable for both the enzyme activity and the conformation from pH 5.6 to pH 7.4. As shown by intrinsic and ANS fluorescence, no red shift of emission maximum occurred and a negligible intensity change was observed at pH 5.6-7.4. The activity of EGX remained about 80% in pH 5.6-7.4 and obviously decreased out of side the pH range. Urea-induced changes in EGX at pH 5.4 and pH 8.0 were measured by intrinsic fluorescence and CD spectrum. At pH 5.4, a significantly red shift of emission maximum occurred when the concentration of urea was 5 M compared to the concentration was 3 M at pH 8.0. The alpha-helix at pH 5.4 was 40.51% in the absence of urea and 31.04% in the presence of 4 M urea. At pH 8.0 the alpha-helix was 7.23% in the presence of 4 M urea. The data indicated that EGX was much susceptible to urea-induced unfolding at pH 8.0 and much stable at pH 5.4. The greater pH dependent stability of EGX may allow the enzyme to adequately catalyze the hydrolysis of cellulosic materials under natural or industrial extreme conditions.

Monoclonal antibodies assisting refolding of firefly luciferase.

The reactivation efficiency in the refolding of denatured luciferase in the presence and the absence of monoclonal antibodies (mAbs) has been studied. Luciferase could be partially reactivated when the protein was denatured in high concentrations of guanidium chloride (GdmCl; >4.5 M) and the refolding was carried out in very low protein concentrations. The refolding yield was, however, significantly lower when it was performed on luciferase that had been denatured with lower concentrations of GdmCl. The efficiency of refolding decreases when the formation of aggregates increases. Three of the five luciferase mAbs tested (4G3, N2E3, S2G10) dramatically increased the yield of reactivation and simultaneously eliminated the formation of aggregates. It is proposed that these mAbs assisted the refolding of luciferase by binding to the exposed hydrophobic surface of the refolding intermediate, thus preventing it from aggregating. The epitopes interacting with these refolding-assisting mAbs are all located in the A-subdomain of the N-terminal region of luciferase. These results have also shed light on the structural features of the intermediate and its interface involved in protein aggregate formation, contributing to the understanding of the protein folding mechanism.

Proteomic analysis of differential protein expression in a human hepatoma revertant cell line by using an improved two-dimensional electrophoresis procedure combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry.

The proteomic profiles of a human hepatoma revertant, CL1, and its original cell line, SMMC7721, were compared by using an improved two-dimensional electrophoresis (2-DE) procedure, with multi-IPGstrips gels (length

[A method for delineation of domains in proteins based on refolding free energy--application to continuous and discontinuous domains].

Domain is a protein architecture in a subunit. It might be defined as a basic unit for structure, function, folding, evolution and design. Different combinations of domains lead to the formation of various tertiary structures with various functions for proteins. The delineation of domains for a protein is important both conceptually and practically, which remains up to date a challenging and unsolved problem. Based on the above definition, a method was previously proposed based on refolding free energy to define continuous domains in proteins. By constructing a residue-residue contact matrix, using correspondence analysis, and then selecting optimal partition function of a protein according to refolding free energy and some empirical scoring functions, a new computer program, PDOM, was developed, which was applicable to both continuous and discontinuous domains. When compared with the manual partition results reported by crystallographers, PDOM has achieved an accuracy of 76% on a test data set including 55 protein structures frequently used. The differences in 13 proteins between PDOM, literature as well as SCOP have been discussed extensively.