Docking of protein molecular surfaces with evolutionary trace analysis

We have developed a new method to predict protein- protein complexes based on the shape complementarity of the molecular surfaces, along with sequence conservation obtained by evolutionary trace (ET) analysis. The docking is achieved by optimization of an object function that evaluates the degree of shape complementarity weighted by the conservation of the interacting residues. The optimization is carried out using a genetic algorithm in combination with Monte Carlo sampling. We applied this method to CAPRI targets and evaluated the performance systematically. Consequently, our method could achieve native-like predictions in several cases. In addition, we have analyzed the feasibility of the ET method for docking simulations, and found that the conservation information was useful only in a limited category of proteins (signal related proteins and enzymes).     

Light chain subunit of a poorly soluble human IgG2λ crystallizes in physiological pH environment both in cellulo and in vitro

Prominent inclusion bodies can develop in the endoplasmic reticulum (ER) when overexpressed antibodies possess intrinsically high condensation propensities. These observations suggest that antibodies deemed to show notable solubility problems may reveal such characteristics preemptively in the form of ER-associated inclusion bodies during antibody overexpression. To define the relationships between solubility problems and inclusion body phenotypes, we investigated the biosynthesis of a model human IgG2λ that shows severe opalescence in an acidic formulation buffer yet retains high solubility at physiological pH. Consistent with the pH-dependent solubility characteristics, the model antibody did not induce notable inclusion body in the physiological pH environment of the ER lumen. However, when individual subunit chains of the antibody were expressed separately, the light chain (LC) spontaneously induced notable crystal-like inclusion bodies in the ER. The LC crystallization event was readily reproducible in vitro by simply concentrating the purified LC protein at physiological pH. Two independent structural determinants for the LC crystallization were identified through rational mutagenesis approach by monitoring the effect of amino acid substitutions on intracellular LC crystallogenesis. The effect of mutations on crystallization was also recapitulated in vitro using purified LC proteins. Importantly, when introduced directly into the model antibody, a mutation that prevents the LC crystallization remediated the antibody's solubility problem without compromising the secretory output or antigen binding. These results illustrate that the ER can serve as a “physiological test tube” that not only reports secretory cargo's high condensation propensity at physiological pH, but also provides an orthogonal method that guides antibody engineering strategy.     

Age-Related Decrease of Meiotic Cohesins in Human Oocytes

Aneuploidy in fetal chromosomes is one of the causes of pregnancy loss and of congenital birth defects. It is known that the frequency of oocyte aneuploidy increases with the human maternal age. Recent data have highlighted the contribution of cohesin complexes in the correct segregation of meiotic chromosomes. In mammalian oocytes, cohesion is established during the fetal stages and meiosis-specific cohesin subunits are not replenished after birth, raising the possibility that the long meiotic arrest of oocytes facilitates a deterioration of cohesion that leads to age-related increases in aneuploidy. We here examined the cohesin levels in dictyate oocytes from different age groups of humans and mice by immunofluorescence analyses of ovarian sections. The meiosis-specific cohesin subunits, REC8 and SMC1B, were found to be decreased in women aged 40 and over compared with those aged around 20 years (P<0.01). Age-related decreases in meiotic cohesins were also evident in mice. Interestingly, SMC1A, the mitotic counterpart of SMC1B, was substantially detectable in human oocytes, but little expressed in mice. Further, the amount of mitotic cohesins of mice slightly increased with age. These results suggest that, mitotic and meiotic cohesins may operate in a coordinated way to maintain cohesions over a sustained period in humans and that age-related decreases in meiotic cohesin subunits impair sister chromatid cohesion leading to increased segregation errors.     

Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5′ located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.     

Facilitation between extensor carpi radialis and pronator teres in humans: A study using a post-stimulus time histogram method

Group I muscle afferents modulate the excitability of motor neurons through excitatory and inhibitory spinal reflexes. Spinal reflex relationships between various muscle pairs are well described in experimental animals but not in the human upper limb, which exhibits a fine control of movement. In the present study, spinal reflexes between the extensor carpi radialis (ECR) and pronator teres (PT) muscles were examined in healthy human subjects using a post-stimulus time histogram method. Electrical stimulation of low-threshold afferents of ECR nerves increased the motor neuron excitability in 31 of 76 PT motor units (MUs) in all eight subjects tested, while stimulation of low-threshold afferents of PT nerves increased the motor neuron excitability in 36 of 102 ECR MUs in all 10 subjects. The estimated central synaptic delay was almost equivalent to that of homonymous facilitation. Mechanical stimulation (MS) of ECR facilitated 16 of 30 PT MUs in all five subjects tested, while MS of PT facilitated 17 of 30 ECR MUs in all six subjects. These results suggest excitatory reflex (facilitation) between PT and ECR. Group I afferents should mediate the facilitation through a monosynaptic path.     

The progression of comorbidity in IL-18 transgenic chronic obstructive pulmonary disease mice model

Patients with severe COPD are known to have comorbidities such as emaciation, cor pulmonale and right heart failure, muscle weakness, hyperlipemia, diabetes mellitus, osteoporosis, muscle atrophy, arterial sclerosis, hypertension, and depression. Therefore, treatment for COPD needs to focus on these comorbidities as well as the lungs. We previously reported a new mouse model of COPD utilizing the human surfactant protein C promoter SP-C to drive the expression of mature mouse IL-18 cDNA; constitutive IL-18 overproduction in the lungs of transgenic (Tg) mice induces severe emphysematous change, dilatation of the right ventricle, and mild pulmonary hypertension with aging. In the present study, we evaluated the progression of comorbidity in our COPD model. In female Tg mice, significant weight loss was observed at 16 weeks and beyond, when compared with control wild-type (WT) mice. This weight loss was suppressed in IL-13-deficient (knockout; KO) Tg mice. Muscle weight and bone mineral density were significantly decreased in aged Tg mice relative to control WT and IL-13 KO Tg mice. The aged Tg mice also showed impaired glucose tolerance. IL-18 and IL-13 may play important roles in the pathogenesis of comorbidity in COPD patients.     

Genetic architecture of variation in sex-comb tooth number in Drosophila simulans

The sex comb on the forelegs of Drosophila males is a secondary sexual trait, and the number of teeth on these combs varies greatly within and between species. To understand the relationship between the intra- and interspecific variation, we performed quantitative trait locus (QTL) analyses of the intraspecific variation in sex-comb tooth number. We used five mapping populations derived from two inbred Drosophila simulans strains that were divergent in the number of sex-comb teeth. Although no QTLs were detected on the X chromosome, we identified four QTLs on the second chromosome and three QTLs on the third chromosome. While identification and estimated effects of the second-chromosome QTLs depend on genetic backgrounds, significant and consistent effects of the two third-chromosome QTLs were found in two genetic backgrounds. There were significant epistatic interactions between a second-chromosome QTL and a third-chromosome QTL, as well as between two second-chromosome QTLs. The third-chromosome QTLs are concordant with the locations of the QTLs responsible for the previously observed differences in sex-comb tooth number between D. simulans and D. mauritiana.     

A hyperthermophilic protein acquires function at the cost of stability

Active-site residues are not often optimized for conformational stability (activity-stability trade-offs) in proteins from organisms that grow at moderate temperature. It is unknown if the activity-stability trade-offs can be applied to proteins from hyperthermophiles. Because enzymatic activity usually increases at higher temperature and hyperthermophilic proteins need high conformational stability, they might not sacrifice the stability for their activity. This study attempts to clarify the contribution of active-site residues to the conformational stability of a hyperthermophilic protein. We therefore examined the thermodynamic stability and enzymatic activity of wild-type and active-site mutant proteins (D7N, E8A, E8Q, D105A, and D135A) of ribonuclease HII from Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced denaturation was measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation were highly reversible in these proteins. All the mutations of these active-site residues, except that of Glu8 to Gln, reduced the enzymatic activity dramatically but increased the protein stability by 7.0 to 11.1 kJ mol(-1) at 50 degrees C. The mutation of Glu8 to Gln did not seriously affect the enzymatic activity and increased the stability only by 2.5 kJ mol(-1) at 50 degrees C. These results indicate that hyperthermophilic proteins also exhibit the activity-stability trade-offs. Therefore, the architectural mechanism for hyperthermophilic proteins is equivalent to that for proteins at normal temperature.