Overexpression of OLE1 Enhances Cytoplasmic Membrane Stability and Confers Resistance to Cadmium in Saccharomyces cerevisiae.

The heavy metal cadmium is widely used and released into the environment, posing a severe threat to crops and humans. Saccharomyces cerevisiae is one of the most commonly used organisms in the investigation of environmental metal toxicity. We investigated cadmium stress and the adaptive mechanisms of yeast by screening a genome-wide essential gene overexpression library. A candidate gene, OLE1, encodes a delta-9 desaturase and was associated with high anti-cadmium-stress activity. The results demonstrated that the expression of OLE1 was positively correlated with cadmium stress tolerance and induction was independent of Mga2p and Spt23p (important regulatory factors for OLE1). Moreover, in response to cadmium stress, cellular levels of monounsaturated fatty acids were increased. The addition of exogenous unsaturated fatty acids simulated overexpression of OLE1, leading to cadmium resistance. Such regulation of OLE1 in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. IMPORTANCE: A S. cerevisiae gene encoding a delta-9 desaturase, OLE1, was associated with high anti-cadmium-stress activity. The data suggest that the regulation of OLE1 in the synthesis of unsaturated fatty acids may serve as a positive feedback mechanism to help yeast cells counter the lipid peroxidation and cytoplasmic membrane damage caused by cadmium. The discovery of OLE1 involvement in membrane stability may indicate a novel defense strategy against cadmium stress.

A novel HAC1-based dual-luciferase reporter vector for detecting endoplasmic reticulum stress and unfolded protein response in yeast Saccharomyces cerevisiae.

Unfolded protein response (UPR) is an important cellular phenomenon induced by over-accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen. ER stress and UPR are implicated in human diseases such as diabetes, atherosclerosis and neurodegenerative diseases. Current methods for measuring ER stress levels and UPR activation usually include cells lysis and other complicated procedures such as reverse transcription-PCR (RT-PCR). These methods typically have low sensitivity and are not suitable for live detection. In this study, we developed a dual-luciferase gene reporter system to monitor UPR activation in live cells of the yeast Saccharomyces cerevisiae by taking advantage of the HAC1 intron and its unconventional splicing-regulation mechanism. We showed that this reporter can be used to monitor UPR in live cells with high sensitivity.

Insulin research in China and the U.K.

Insulin has been extensively studied since it was discovered by Banting and Best in 1921. Early in 1934, Dorothy Crowfoot and John Desmond Bernal obtained the first X-ray diffraction photograph of an enzyme protein: pepsin. In 1935, they took another photograph of a protein hormone: insulin. The chemical structure of protein was unknown until the amino acid sequence of bovine insulin was solved by Fred Sanger and colleagues in 1955. In 1958, the chemical synthesis of bovine insulin started in China through a nationwide collaboration of three institutions: the Institute of Biochemistry in Shanghai, the Institute of Organic Chemistry in Shanghai and Beijing University. The total synthesis of bovine insulin in crystalline form was accomplished in 1965. The success of the synthesis of the first protein in vitro greatly encouraged young researchers in China. Not long afterwards, the project of structural analysis of insulin crystal was carried out in China through the collaboration of the Institute of Biophysics, the Institute of Physics and Beijing University, and succeeded in 1971. In Dorothy Hodgkin's laboratory in Oxford, X-ray diffraction studies of insulin crystals were resumed after about 30 years, and the structure of rhombohedral insulin crystal was solved in 1969. Through insulin research, the Institute of Biophysics in Beijing and the Institute of Biochemistry in Shanghai established scientific collaboration and personal friendship with Dorothy Hodgkin's laboratory in Oxford, and later Guy Dodson's laboratory in York and Tom Blundell's laboratory in London. In 1975, Dorothy Hodgkin wrote a short note, 'Chinese work on insulin' in Nature, anticipating closer scientific exchange between the East and the West. In 1982, a bilateral meeting between the Biochemical Societies in the U.K. and China was held in Oxford. Now, the second bilateral meeting held in Shanghai will further promote the collaboration between our two countries.

Insulin analogs with B24 or B25 phenylalanine replaced by biphenylalanine.

B24 and B25 phenylalanines (Phe) play important roles in insulin structure and function. Insulin analogs with B24 Phe or B25 Phe replaced by biphenylalanine (Bip) were prepared by enzymatic semisynthesis. The biological activities were determined by receptor binding assay and in vivo mouse convulsion assay. The results showed that B25 Bip insulin has 139% receptor binding activity and 50% in vivo biological activity, whereas B24 Bip insulin is inactive, when compared with native insulin, suggesting that B24 Phe is crucial for insulin activity. The structures in solution were studied by circular dichroism and fluoremetry, and our results suggested that the insulin analogs with low activities tend to be more tightly packed. The association properties were studied by size exclusion chromatography. The Bip-amide replacement of B24 Phe in deshexapeptide insulin or B25 Phe in despentapeptide insulin will cause the monomeric B24 Phe-amide deshexapeptide insulin or B25 Phe-amide despentapeptide insulin to associate and form dimers, whereas the mutations of B24 Phe in insulin will make insulin dimers dissociate into insulin monomers.

Synthesis and biological activity of seleno sunflower trypsin inhibitor analog.

Sunflower trypsin inhibitor (SFTI-1) is a cyclic peptide with 14 amino acid residues and one disulfide bond. Its synthetic acyclic analog (aSFTI-1) with N-terminal Gly and C-terminal Asp was still active. Here, we report the synthesis of seleno aSFTI-1 with the disulfide bond of aSFTI-1 replaced by diselenide bond. The formation of the diselenide bond from selenol was achieved in a single step without the aid of oxidizing agent. For comparison, aSFTI-1 itself and aSFTI-1 with its disulfide bond replaced by two serines ([Ser(3,11)] aSFTI-1) were also synthesized. The trypsin inhibitory constants of seleno aSFTI-1, aSFTI-1 and [Ser(3,11)] aSFTI-1 were determined as 6.50 x 10(-9), 1.96 x 10(-9) and 8.10 x 10(-6) respectively, indicating that the disulfide bond is essential for the structure and function of aSFTI-1, and seleno aSFTI-1 is still active, although its inhibitory constant is reduced to 30% in comparison with that of aSFTI-1.

B22 Glu des-B30 insulin: a novel monomeric insulin.

Studies on monomeric insulin with reduced self-association are important in the development of insulin pharmaceutical preparations with rapid hypoglycemic action on patients with diabetes. Here we report a novel monomeric insulin, B22 Glu des-B30 insulin, prepared from a single chain insulin precursor with B22 Arg mutated to Glu, which was expressed in Pichia pastoris and converted to B22 Glu des-B30 insulin by tryptic digestion. It still retains 50% of the in vivo biological activity of porcine insulin and does not form a dimer even at a concentration of 10 mg/ml, showing that B22 Glu plays a key role in reducing the self-association of the insulin molecule without greatly reducing its biological activity. This novel monomeric insulin might have potential applications in the clinic.

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris.

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Expression of monomeric insulin precursor in Pichia pastoris and its conversion into monomeric B27 Lys destripeptide insulin by tryptic hydrolysis.

Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from its precursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient tryptic transpeptidation. The monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium of P. pastoris by a combination of hydrophobic, size-exclusion, and ion-exchange chromatography. The purified MIP was converted, by tryptic hydrolysis, to B27 Lys DTrI, which was then purified by ion-exchange chromatography to homogeneity as assessed by native gel electrophoresis, HPLC, amino acid composition, and electrospray mass-spectrometric analysis. B27 Lys DTrI exhibited superior monomeric properties in size-exclusion chromatography. The yield of MIP was 200 mg per liter of culture, and the overall yield of purified B27 Lys DTrI from the crude MIP was 70%. The in vivo biological activity of B27 Lys DTrI as determined by the mouse convulsion assay was 21 U/mg, identical to that obtained by semisynthesis.

Synthesis and characteristics of an aspartame analogue, L-asparaginyl L-3-phenyllactic acid methyl ester.

An aspartame analogue, L-asparaginyl L-3-phenyllactic acid methyl ester was synthesized with aspartic acid replaced by asparagine and peptide bond replaced by ester bond. The aspartic acid of aspartame could be replaced by asparagine as reported in the literature. In this analogue, the hydrogen of amide group could still form a hydrogen bond with the oxygen of ester bond and the ester bond was isosteric with peptide bond. However, the product was not sweet, showing that the peptide bond could not be replaced by ester bond. The peptide C-N bond behaves as a double bond that is not free to rotate and the C, O, N and H atoms are in the same plane. The replacement of peptide bond by ester bond destroyed the unique conformation of peptide bond, resulting in the loss of sweet taste.

Monomeric B27 Lys destripeptide insulin: semisynthesis, characterization and biological activity.

In this paper, we report the semisynthesis of B27 Lys destripeptide insulin (B27 Lys DTrI), i.e. destetrapeptide insulin with an additional Lys residue at the C-terminus of B-chain. B27 Lys DTrI is also monomeric as shown by gel filtration. Its in vivo biological activity is 80% in comparison with that of native insulin. The addition of a Lys residue at the C-terminus of B-chain makes it possible to obtain monomeric B27 Lys DTrI from a precursor expressed in Saccharomyces cerevesiae by tryptic hydrolysis instead of the less efficient tryptic transpeptidation.

Isosteric replacement of A3 Val of porcine insulin by allo-Thr.

A3 Val is important for insulin activity. It is invariant in insulins from different species studied thus far. Based on the three dimensional structure of insulin, it was thought to be involved in receptor binding. Its replacement by Leu resulted in remarkable lowering of insulin activity, indicating the crucial requirement of the side chain geometry at this position. When A3 Val was replaced by Thr, which is hydrophilic but isosteric with Val, substantial insulin activity was retained. Therefore, the isosteric requirement for receptor binding at this site is more stringent than the hydrophobic requirement. Here we report the replacement of A3 Val of porcine insulin by the unnatural allo-Thr. The in vivo biological activity of A3 allo-Thr insulin is similar to that of A3 Thr insulin or native insulin, but its receptor binding activity is 7.6% instead of 50% for A3 Thr insulin, indicating that at the A3 position the hydrophilic OH group of Thr could be more tolerated in receptor binding than the OH group of allo-Thr. The retention of insulin activity by substituting A3 Val with the unnatural isosteric allo-Thr demonstrates again the importance of isosteric interaction in the binding of insulin with its receptor.

Crystallization and preliminary crystallographic investigation of a low-pH native insulin monomer with flexible behaviour.

Insulin naturally aggregates as dimers and hexamers, whose structures have been extensively analysed by X-ray crystallography. Structural determination of the physiologically relevant insulin monomer, however, is an unusual challenge owing to the difficulty in finding solution conditions in which the concentration of insulin is high enough for crystallization yet the molecule remains monomeric. By utilizing solution conditions known to inhibit insulin assembly, namely 20% acetic acid, crystals of insulin in the monomeric state have been obtained. The crystals are strongly diffracting and a data set extending to 1.6 A has recently been collected. The crystals nominally belong to the space group I422, with unit-cell parameters a = b = 57.80, c = 54.61 A, giving rise to one molecule in the asymmetric unit. Preliminary electron-density maps show that whilst most of the insulin monomer is well ordered and similar in conformation to other insulin structures, parts of the B-chain C-terminus main chain adopt more than one conformation.

Secretory Expression of Human Insulin in Methylotrophic Yeast Pichia pastoris.

The porcine insulin precursor (PIP) gene and its derivative form sp-PIP gene, which had a nona-peptide (called spacer peptide, sp) added at the 5' terminus of PIP gene, were inserted into the plasmid pPIC9 of Pichia pastoris to obtain secretory plasmid pPIC9/PIP and pPIC9/sp-PIP, respectively. P.pastoris GS115 was transformed by pPIC9/PIP or pPIC9/sp-PIP and the high-copy strains, P39(-sp) and S51(+sp), were selected by dot-blotting. The expression levels of PIP and sp-PIP were 10 mg/L and 40 mg/L in 1 L shake flask, respectively, indicating that the spacer peptide could increase the expression. The expression level of PIP (sp-PIP) in P.pastoris was higher than that of PIP in S.cerevisiae and K.lactis reported in this laboratory. The expression level of sp-PIP was 250 mg/L in 10 L fermentor. Recombinant human insulin was obtained by means of transpeptidation of PIP or sp-PIP. The receptor binding capacity is identical with that of porcine insulin. In vivo biological activity of the recombinant human insulin is 27 IU/mg.

The Transferrins from Amphioxus and the Molecular Evolution of Transferrin.

An amphioxus transferring was isolated and purified from the Xiamen amphioxus. Its physio-chemical properties are the same as those of Qingdao amphioxus transferrin. It is a glyco-protein with a molecular weight of 26 kD that is about a quarter of that of the transferrin from vertebrates. Its partial amino acid sequence and its C-terminal fragment were determined. By comparison with the sequence of human serum transferrin, it has been found that the sequence of human serum transferrin can be divided into 4 fragments with about equal size as the sequences of the amphioxus transferrin. There is quite a high homology between the sequences of amphioxus transferrin and each of the 4 fragments as well as between the four fragments. The results demonstrate that the current transferrin may be evolved from an ancestor transferrin with one quarter of the molecular weight as a result of twice gene duplication. Amphioxus transferrin may be the representative from of such ancestor transferrin.

Secretory Expression of Porcine Insulin Precursor in Kluyveromyces lactis and Its Conversion into Human Insulin.

Porcine insulin precursor (PIP) was cloned to vectors derived from plasmid pKD1 and expressed in Kluyveromyces lactis. The secretory expression level of PIP was 20 to 30 mg per liter of the culture medium. Human insulin obtained from PIP through tryptic transpeptidation was characterized. Its amino acid composition, crystalline shape and biological activity are identical with those of native insulin.