Stabilities and activities of the N- and C-domains of FKBP22 from a psychrotrophic bacterium overproduced in Escherichia coli

FKBP22 from a psychrotrophic bacterium Shewanella sp. SIB1, is a dimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) activity. According to homology modeling, it consists of an N-terminal domain, which is involved in dimerization of the protein, and a C-terminal catalytic domain. A long alpha3 helix spans these domains. An N-domain with the entire alpha3 helix (N-domain+) and a C-domain with the entire alpha3 helix (C-domain+) were overproduced in Escherichia coli in a His-tagged form, purified, and their biochemical properties were compared with those of the intact protein. C-domain+ was shown to be a monomer and enzymatically active. Its optimum temperature for activity (10 degrees C) was identical to that of the intact protein. Determination of the PPIase activity using peptide and protein substrates suggests that dimerization is required to make the protein fully active for the protein substrate or that the N-domain is involved in substrate-binding. The differential scanning calorimetry studies revealed two distinct heat absorption peaks at 32.5 degrees C and 46.6 degrees C for the intact protein, and single heat absorption peaks at 44.7 degrees C for N-domain+ and 35.6 degrees C for C-domain+. These results indicate that the thermal unfolding transitions of the intact protein at lower and higher temperatures represent those of C- and N-domains, respectively. Because the unfolding temperature of C-domain+ is much higher than its optimum temperature for activity, SIB1 FKBP22 may adapt to low temperatures by increasing a local flexibility around the active site. This study revealed the relationship between the stability and the activity of a psychrotrophic FKBP22.     

Cigarette smoke extract induces endothelial cell injury via JNK pathway

Cigarette smoking is the most crucial factor responsible for chronic obstructive pulmonary disease (COPD). The precise mechanisms of the development of the disease have, however, not been fully understood. Recently, impairment of pulmonary endothelial cells has been increasingly recognized as a critical pathophysiological process in COPD. To verify this hypothesis, we examined how cigarette smoke extract (CSE) damages human umbilical vein endothelial cells (HUVECs). CSE activated c-Jun N-terminal kinase (JNK), and treatment of HUVECs with SP600125, a specific inhibitor of the JNK pathway, significantly suppressed endothelial cell damage by CSE. In contrast, inhibition of the extracellular-regulated kinase or the p38 pathway did not affect the cytotoxicity of CSE. Furthermore, anti-oxidants superoxide dismutase and catalase reduced CSE-induced JNK phosphorylation and endothelial cell injury. These results indicate that CSE damages vascular endothelial cells through the JNK pathway activated, at least partially, by oxidative stress.     

Importance of a repetitive nine-residue sequence motif for intracellular stability and functional structure of a family I.3 lipase

PML5 is a functional derivative of a family I.3 lipase from Pseudomonas sp. MIS38 and contains five repeats of a nine-residue sequence motif. Two aspartate residues within the second and third repetitive sequences of PML5 were replaced by Ala. The secretion level, intracellular accumulation level, and stability of the resultant mutant protein were greatly reduced as compared to those of PML5. In addition, this mutant protein was inactive and did not bind Ca2+ ion. We propose that the repetitive sequences of PML5 form a beta-roll structure in the cells and thereby contribute to the intracellular stability and secretion efficiency of the protein.     

Conversion of diacetyl-C-(beta-D-glucopyranosyl)phloroglucinol to spiroketal compounds

Diacetyl-C-(beta-D-glucopyranosyl)phloroglucinol was converted by refluxing in water to spiro(benzofuran-[2H]furan) a new compound, along with spiro(benzofuran-[2H]pyran). The stereochemistry of the quaternary carbon of both spiro compounds had an S-configuration.     

Synthesis and enzyme-catalyzed hydrolysis of a radical-masked glycosylated spin-label reagent

N1-Acetoxy-2,2,6,6-tetramethylpiperidin-4-yl 2,3,4,6-tetra-O-benzyl-alpha- and -beta-D-glucopyranosides (3-alpha, beta) and N1-acetoxy-2,2,5,5-tetramethylpyrrolin-3-oyl 2,3,4,6-tetra-O-benzyl-alpha- and -beta-D-glucopyranosylamines (9-alpha, beta) were synthesized in good yield by Schmidt's glycosylation method. Their subsequent O-debenzylation was proceeded successfully to give the desired products 1-alpha, and 1-beta in good yield, and 2-alpha in a low yield, without 2-beta by only short-timed hydrogenolysis in the presence of palladium-on-carbon (Pd-C) in a CHCl3-MeOH solvent system that included concentrated HCl. Upon enzyme-catalyzed hydrolysis, only 2-alpha was hydrolyzed by the esterase, while both of 1-alpha and 1-beta were not hydrolyzed by any other enzyme such as lipase. These 2-alpha can likely be used as a new water-soluble radical-masked glycosylated spin-label reagent.     

Mutational and structural-based analyses of the osmolyte effect on protein stability

It is known that several naturally occurring substances known as osmolytes increase the conformational stability of proteins. Bolen and co-worker proposed the osmophobic theory, which asserts the osmolyte effect occurs because of an unfavorable interaction of osmolytes mainly with the protein backbone, based on the results on the transfer Gibbs energy of amino acids (Deltag) [Bolen and Baskakov (2001) J. Mol. Biol. 310, 955-963]. In this paper, we report the effect of sarcosine on the conformational stability (DeltaG) of RNase Sa (96 residues and one disulfide bond) and four mutant proteins. The thermal denaturation curves for RNase Sa in sarcosine fitted a two-state model on nonlinear least-squares analysis. All the RNase Sa proteins were stabilized by sarcosine. For example, the increase in stability of the wild-type protein in 4 M sarcosine due to the osmolyte effect (Delta(o)DeltaG) is 3.2 kcal/mol. Mutational analysis of the osmolyte effect indicated that the changed Delta(o)DeltaG values upon mutation (Delta(m)Delta(o)DeltaG), as estimated from the Deltag values, are similar to the experimental values. Structural-based analysis of the osmolyte effect was also performed using model denatured structures: (a) a fully extended model (single chain) with no disulfide bond, (b) two-part, unfolded models (two chains) with a disulfide bond constructed through molecular dynamic (MD) simulation, and (c) a two-part, folded model (two chains). The two-part, unfolded models were expected to be more suitable as denatured structures. The Delta(o)DeltaG values calculated using the two-part, unfolded models were more consistent with experimental values than those calculated using the fully extended and two-part, folded models. This suggests that MD simulation is useful for testing denatured structures. These results indicate that the osmophobic theory can explain the osmolyte effect on protein stability.     

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.     

DNA polymerase iota-dependent translesion replication of uracil containing cyclobutane pyrimidine dimers

Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type S-phase cells reveals that only approximately 25% of mutations occur at thymine (T), whilst 75% are targeted to cytosine (C). The mutational spectra changes dramatically in XP-V cells, devoid of poleta, where approximately 45% of mutations occur at Ts and approximately 55% at Cs. At the present time, it is unclear whether the C-->T mutations actually represent true misincorporations opposite C, or perhaps occur as the result of the correct incorporation of adenine (A) opposite a C in a UV-photoproduct that had undergone deamination to uracil (U). In order to assess the role that human poliota might play, if any, in the replicative bypass of such UV-photoproducts, we have analyzed the efficiency and fidelity of pol iota-dependent bypass of a T-U cyclobutane pyrimidine dimer (CPD) in vitro. Interestingly, pol iota-dependent bypass of a T-U CPD occurs more efficiently than that of a corresponding T-T CPD. Guanine (G) was misincorporated opposite the 3'U of the T-U CPD only two-fold less frequently than the correct Watson-Crick base, A. While pol iota generally extended the G:3'U-CPD mispairs less efficiently than the correctly paired primer, pol iota-dependent extension was equal to, or greater than that observed with human pols eta and kappa and S. cerevisiae pol zeta under the same assay conditions. Thus, we hypothesize that the ability of pol iota to bypass T-U CPDs through the frequent misincorporation of G opposite the 3'U of the CPD, may provide a mechanism whereby human cells can decrease the mutagenic potential of these lesions.     

Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source

Biosurfactant production by Pseudomonas aeruginosa A41, a strain isolated from seawater in the gulf of Thailand, was examined when grown in defined medium containing 2% vegetable oil or fatty acid as a carbon source in the presence of vitamins, trace elements and 0.4% NH(4)NO(3), at pH 7 and 30 degrees C with 200 rpm-shaking for 7 days. The yield of biosurfactant steadily increased even after a stationary phase. Under such conditions the surface tension of the medium was lowered from 55-70 mN/m to 27.8-30 mN/m with every carbon source tested. However, types of carbon sources were found to affect biosurfactant yield. The yields of rhamnolipid biosurfactant were 6.58 g/L, 2.91 g/L and 2.93 g/L determined as rhamnose content when olive oil, palm oil and coconut oil, respectively, were used as a carbon source. Among them, biosurfactant obtained from palm oil was the best in lowering surface tension of the medium. Increase in biosurfactant activities in terms of oil displacement test and rhamnose content were observed to be higher with shorter chain fatty acids than that of the longer chains (C12>C14>C16). In addition, we found that C18:2, highly unsaturated fatty acid, showed higher oil displacement activity and rhamnose content than that of C18:1. The optimal oil displacement activity was found at pH 7-9 and in the presence of 0.5-3% NaCl. The oil displacement activity was stable to temperatures up to 100 degrees C for 15 h. Surface tension reduction activity was relatively stable at pH 2-12 and 0-5% of NaCl. Emusification activity tested with various types of hydrocarbons and vegetable oils showed similarity of up to 60% stability. The partially purified biosurfactant via TLC and silica gel column chromatography gave three main peaks on HPLC with mass spectra of 527, 272, and 661 m/z respectively, corresponding to sodium-monorhamnodecanoate, hydroxyhexadecanoic acid and an unknown compound, respectively.