Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein

PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.     

The relationship between muscle deoxygenation and activation in different muscles of the quadriceps during cycle ramp exercise

The relationship between muscle deoxygenation and activation was examined in three different muscles of the quadriceps during cycling ramp exercise. Seven young male adults (24 ± 3 yr; mean ± SD) pedaled at 60 rpm to exhaustion, with a work rate (WR) increase of 20 W/min. Pulmonary oxygen uptake was measured breath-by-breath, while muscle deoxygenation (HHb) and activity were measured by time-resolved near-infrared spectroscopy (NIRS) and surface electromyography (EMG), respectively, at the vastus lateralis (VL), rectus femoris (RF), and vastus medialis (VM). Muscle deoxygenation was corrected for adipose tissue thickness and normalized to the amplitude of the HHb response, while EMG signals were integrated (iEMG) and normalized to the maximum iEMG determined from maximal voluntary contractions. Muscle deoxygenation and activation were then plotted as a percentage of maximal work rate (%WR(max)). The HHb response for all three muscle groups was fitted by a sigmoid function, which was determined as the best fitting model. The c/d parameter for the sigmoid fit (representing the %WR(max) at 50% of the total amplitude of the HHb response) was similar between VL (47 ± 12% WR(max)) and VM (43 ± 11% WR(max)), yet greater (P < 0.05) for RF (65 ± 13% WR(max)), demonstrating a "right shift" of the HHb response compared with VL and VM. The iEMG also showed that muscle activation of the RF muscle was lower (P < 0.05) compared with VL and VM throughout the majority of the ramp exercise, which may explain the different HHb response in RF. Therefore, these data suggest that the sigmoid function can be used to model the HHb response in different muscles of the quadriceps; however, simultaneous measures of muscle activation are also needed for the HHb response to be properly interpreted during cycle ramp exercise.     

Molecular identification of ghrelin receptor (GHS-R1a) and its functional role in the gastrointestinal tract of the guinea-pig

Ghrelin stimulates gastric motility in vivo in the guinea-pig through activation of growth hormone secretagogue receptor (GHS-R). In this study, we identified GHS-R1a in the guinea-pig, and examined its distribution and cellular function and compared them with those in the rat. Effects of ghrelin in different regions of gastrointestinal tract were also examined. GHS-R1a was identified in guinea-pig brain cDNA. Amino acid identities of guinea-pig GHS-R1a were 93% to horses and 85% to dogs. Expression levels of GHS-R1a mRNA were high in the pituitary and hypothalamus, moderate in the thalamus, cerebral cortex, pons, medulla oblongata and olfactory bulb, and low in the cerebellum and peripheral tissues including gastrointestinal tract. Comparison of GHS-R1a expression patterns showed that those in the brain were similar but the expression level in the gastrointestinal tract was higher in rats than in guinea-pigs. Guinea-pig GHS-R1a expressed in HEK 293 cells responded to rat ghrelin and GHS-R agonists. Rat ghrelin was ineffective in inducing mechanical changes in the stomach and colon but caused a slight contraction in the small intestine. 1,1-Dimethyl-4-phenylpiperazinium and electrical field stimulation (EFS) caused cholinergic contraction in the intestine, and these contractions were not affected by ghrelin. Ghrelin did not change spontaneous and EFS-evoked [³H]-efflux from [³H]-choline-loaded ileal strips. In summary, guinea-pig GHS-R1a was identified and its functions in isolated gastrointestinal strips were characterized. The distribution of GHS-R1a in peripheral tissues was different from that in rats, suggesting that the functional role of ghrelin in the guinea-pig is different from that in other animal species.     

Phylogeography of Japanese population of Phelotrupes auratus (Coleoptera, Geotrupidae) inferred from mitochondrial DNA sequences

A partial sequence of the mitochondrial cytochrome c oxidase subunit I (COI) gene (745 bp) was determined for 57 specimens of a geotrupid beetle (Phelotrupes auratus) from throughout the Japanese archipelago. Of the 57 beetles examined, 42 haplotypes were identified. Phylogenetic trees inferred using maximum parsimony, neighbor joining, and Bayesian inference methods were highly congruent. Reconstructed phylogenetic relationships indicated that P. auratus from the Japanese archipelago was separated into two distinct lineages: Group A, which consisted of 35 haplotypes from Honshu, Shikoku, and Hokkaido Islands, and Group B, which consisted of seven haplotypes from Kyushu and Yakushima Islands. In addition, two sublineages were also recognized within Group A: Subgroup A-1, which consisted of 11 haplotypes from eastern Honshu and Hokkaido, and Subgroup A-2, which consisted of 10 haplotypes from western Honshu and Shikoku. Average genetic distances within Group A were positively correlated to geographic distance between sampling localities. Phylogenetic relationships among haplotypes did not correspond to subspecies classification.     

Endothelial lipase is synthesized by hepatic and aorta endothelial cells and its expression is altered in apoE-deficient mice

Both LPL and HL are synthesized in parenchymal cells, are secreted, and bind to endothelial cells. To learn where endothelial lipase (EL) is synthesized in adult animals, the localization of EL in mouse and rat liver was studied by immunohistochemical analysis. Furthermore, to test whether EL could play a role in atherogenesis, the expression of EL in the aorta and liver of apolipoprotein E knockout (EKO) mice was determined. EL in both mouse and rat liver was colocalized with vascular endothelial cells but not with hepatocytes. In contrast, HL was present in both hepatocytes and endothelial cells. By in situ hybridization, EL mRNA was present only in endothelial cells in liver sections. EL was also present at low levels in aorta of normal mice. We fed EKO mice and wild-type mice a variety of diets and determined EL expression in liver and aorta. EKO mice showed significant expression of EL in aorta. EL expression was lower in the liver of EKO mice than in normal mice. Cholesterol feeding decreased EL in liver of both types of mice. In the aorta, EL was higher in EKO than in wild-type mice, and cholesterol feeding had no effect. Together, these data suggest that EL may be upregulated at the site of atherosclerotic lesions and thus could supply lipids to the area.     

Novel semi-synthetic glycopeptide antibiotics active against methicillin-resistant staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE): doubly-modified water-soluble derivatives of chloroorienticin B

A series of N-alkylated and aminomethylated derivatives of chloroorienticin B, a vancomycin-related glycopeptide antibiotic, were synthesized. Doubly-modified derivatives having both hydrophobic and hydrophilic substituents exhibited potent antibacterial activity against MRSA and VRE along with considerable water-solubility.     

Influence of Aerobic Training on the Reduced Vasoconstriction to Angiotensin II in Rats Exposed to Intrauterine Growth Restriction: Possible Role of Oxidative Stress and AT2 Receptor of Angiotensin II

Intrauterine growth restriction (IUGR) is associated with impaired vascular function, which contributes to the increased incidence of chronic disease. The aim of this study was to investigate whether aerobic training improves AngII-induced vasoconstriction in IUGR rats. Moreover, we assess the role of superoxide dismutase (SOD) isoforms and NADPH oxidase-derived superoxide anions in this improvement. Female Wistar rats were randomly divided into two groups on day 1 of pregnancy. A control group was fed standard chow ad libitum, and a restricted group was fed 50% of the ad libitum intake throughout gestation. At 8 weeks of age, male offspring from both groups were randomly assigned to 4 experimental groups: sedentary control (SC), trained control (TC), sedentary restricted (SRT), and trained restricted (TRT). The training protocol was performed on a treadmill and consisted of a continuous 60-min session 5 days/week for 10 weeks. Following aerobic training, concentration–response curves to AngII were obtained in endothelium-intact aortic rings. Protein expression of SOD isoforms, AngII receptors and the NADPH oxidase component p47^phox was assessed by Western blot analysis. The dihydroethidium was used to evaluate the in situ superoxide levels under basal conditions or in the presence of apocynin, losartan or PD 123,319. Our results indicate that aerobic training can prevent IUGR-associated increases in AngII-dependent vasoconstriction and can restore basal superoxide levels in the aortic rings of TRT rats. Moreover, we observed that aerobic training normalized the increased p47^phox protein expression and increased MnSOD and AT₂ receptor protein expression in thoracic aortas of SRT rats. In summary, aerobic training can result in an upregulation of antioxidant defense by improved of MnSOD expression and attenuation of NADPH oxidase component p47^phox. These effects are accompanied by increased expression of AT₂ receptor, which provide positive effects against Ang II–induced superoxide generation, resulting in attenuation of AngII-induced vasoconstriction.     

Thymine DNA Glycosylase Is a CRL4Cdt2 Substrate

The E3 ubiquitin ligase CRL4^Cdt2 targets proteins for destruction in S phase and after DNA damage by coupling ubiquitylation to DNA-bound proliferating cell nuclear antigen (PCNA). Coupling to PCNA involves a PCNA-interacting peptide (PIP) degron motif in the substrate that recruits CRL4^Cdt2 while binding to PCNA. In vertebrates, CRL4^Cdt2 promotes degradation of proteins whose presence in S phase is deleterious, including Cdt1, Set8, and p21. Here, we show that CRL4^Cdt2 targets thymine DNA glycosylase (TDG), a base excision repair enzyme that is involved in DNA demethylation. TDG contains a conserved and nearly perfect match to the PIP degron consensus. TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and PIP degron-dependent manner during DNA repair in Xenopus egg extract. The protein can also be destroyed during DNA replication in this system. During Xenopus development, TDG first accumulates during gastrulation, and its expression is down-regulated by CRL4^Cdt2. Our results expand the group of vertebrate CRL4^Cdt2 substrates to include a bona fide DNA repair enzyme.     

Crystal Structure of a Symmetric Football-Shaped GroEL:GroES2-ATP14 Complex Determined at 3.8 Å Reveals Rearrangement between Two GroEL Rings

The chaperonin GroEL is an essential chaperone that assists in protein folding with the aid of GroES and ATP. GroEL forms a double-ring structure, and both rings can bind GroES in the presence of ATP. Recent progress on the GroEL mechanism has revealed the importance of a symmetric 1:2 GroEL:GroES₂ complex (the “football”-shaped complex) as a critical intermediate during the functional GroEL cycle. We determined the crystal structure of the football GroEL:GroES₂-ATP₁₄ complex from Escherichia coli at 3.8 Å, using a GroEL mutant that is extremely defective in ATP hydrolysis. The overall structure of the football complex resembled the GroES-bound GroEL ring of the asymmetric 1:1 GroEL:GroES complex (the “bullet” complex). However, the two GroES-bound GroEL rings form a modified interface by an ~ 7° rotation about the 7-fold axis. As a result, the inter-ring contacts between the two GroEL rings in the football complex differed from those in the bullet complex. The differences provide a structural basis for the apparently impaired inter-ring negative cooperativity observed in several biochemical analyses.