Disulfide-linked propeptides stabilize the structure of zymogen and mature pancreatic serine proteases.

Chymotrypsinogen and proelastase 2 are the only pancreatic proteases with propeptides that remain attached to the active enzyme via a disulfide bridge. It is likely, although not proven, that these propeptides are functionally important in the active enzymes, as well as in the zymogens. A mutant chymotrypsin was constructed to test this hypothesis, but it was demonstrated that the lack of the propeptide had no effect on the catalytic efficiency, substrate specificity, or folding of the protein [Venekei, I., et al. (1996) FEBS Lett. 379, 139-142]. In this paper, we investigate the role of the disulfide-linked propeptide in the conformational stability of chymotrypsin(ogen). We compare the stabilities of the wild-type and mutant proteins (lacking propeptide-enzyme interactions) in their zymogen (chymotrypsinogen) and active (chymotrypsin) forms. The mutants exhibited a substantially increased sensitivity to heat denaturation and guanidine hydrochloride unfolding, and a faster loss of activity at extremes of pH relative to those of their wild-type counterparts. From guanidine hydrochloride denaturation experiments, we determined that covalently linked propeptide provides about 24 kJ/mol of free energy of extra stabilization (DeltaDeltaG). In addition, the mutant chymotrypsinogen lacked the normal resistance to digestion by pepsin. This may also explain (besides keeping the zymogen inactive) the evolutionary conservation of the propeptide-enzyme interactions. Tryptophan fluorescence, circular dichroism, microcalorimetric, and activity measurements suggest that the propeptide of chymotrypsin restricts the relative mobility between the two domains of the molecule. In pancreatic serine proteases, such as trypsin, that lose the propeptide upon activation, this function appears to be accomplished via alternative interdomain contacts.     

A scalable life cycle inventory of an electrical automotive traction machine—Part I: design and composition

Purpose

A scalable life cycle inventory (LCI) model of a permanent magnet electrical machine, containing both design and production data, has been established. The purpose is to contribute with new and easy-to-use data for LCA of electric vehicles by providing a scalable mass estimation and manufacturing inventory for a typical electrical automotive traction machine. The aim of this article (part I of two publications) is to present the machine design, the model structure, and an evaluation of the models’ mass estimations.

Methods

Data for design and production of electrical machines has been compiled from books, scientific papers, benchmarking literature, expert interviews, various specifications, factory records, and a factory site visit. For the design part, one small and one large reference machine were constructed in a software tool, which linked the machines’ maximum ability to deliver torque to the mass of its electromagnetically active parts. Additional data for remaining parts was then gathered separately to make the design complete. The two datasets were combined into one model, which calculates the mass of all motor subparts from an input of maximum power and torque. The range of the model is 20–200 kW and 48–477 Nm. The validity of the model was evaluated through comparison with seven permanent magnet electrical traction machines from established brands.

Results and discussion

The LCI model was successfully implemented to calculate the mass content of 20 different materials in the motor. The models’ mass estimations deviate up to 21% from the examples of real motors, which still falls within expectations for a good result, considering a noticeable variability in design, even for the same machine type and similar requirements. The model results form a rough and reasonable median in comparison to the pattern created by all data points. Also, the reference motors were assessed for performance, showing that the electromagnetic efficiency reaches 96–97%.

Conclusions

The LCI model relies on thorough design data collection and fundamental electromagnetic theory. The selected design has a high efficiency, and the motor is suitable for electric propulsion of vehicles. Furthermore, the LCI model generates representative mass estimations when compared with recently published data for electrical traction machines. Hence, for permanent magnet-type machines, the LCI model may be used as a generic component estimation for LCA of electric vehicles, when specific data is lacking.

     

Molecular dynamics of DNA quadruplex molecules containing inosine, 6-thioguanine and 6-thiopurine

The ability of the four-stranded guanine (G)-DNA motif to incorporate nonstandard guanine analogue bases 6-oxopurine (inosine, I), 6-thioguanine (tG), and 6-thiopurine (tI) has been investigated using large-scale molecular dynamics simulations. The simulations suggest that a G-DNA stem can incorporate inosines without any marked effect on its structure and dynamics. The all-inosine quadruplex stem d(IIII)(4) shows identical dynamical properties as d(GGGG)(4) on the nanosecond time scale, with both molecular assemblies being stabilized by monovalent cations residing in the channel of the stem. However, simulations carried out in the absence of these cations show dramatic differences in the behavior of d(GGGG)(4) and d(IIII)(4). Whereas vacant d(GGGG)(4) shows large fluctuations but does not disintegrate, vacant d(IIII)(4) is completely disrupted within the first nanosecond. This is a consequence of the lack of the H-bonds involving the N2 amino group that is not present in inosine. This indicates that formation of the inosine quadruplex could involve entirely different intermediate structures than formation of the guanosine quadruplex, and early association of cations in this process appears to be inevitable. In the simulations, the incorporation of 6-thioguanine and 6-thiopurine sharply destabilizes four-stranded G-DNA structures, in close agreement with experimental data. The main reason is the size of the thiogroup leading to considerable steric conflicts and expelling the cations out of the channel of the quadruplex stem. The G-DNA stem can accommodate a single thioguanine base with minor perturbations. Incorporation of a thioguanine quartet layer is associated with a large destabilization of the G-DNA stem whereas the all-thioguanine quadruplex immediately collapses.     

Size and structure characterization of ethylhydroxyethyl cellulose by the combination of field-flow fractionation with other techniques. Investigation of ultralarge components

Ethylhydroxyethyl cellulose (EHEC) of three different viscosity classes (EHEC I, II, and III) was analyzed by programmed cross-flow asymmetrical flow field-flow fractionation coupled to multiangle light scattering and refractive index detectors to determine their size and molar mass distribution. Two size populations were detected in the two lower viscosity classes, EHEC I and II, one high molar mass and one ultrahigh molar mass (UHM). The two covered molar masses from 10(4) up to 10(9) g X mol(-1). The highest viscosity class EHEC III was less size-dispersed covering molar masses from 5 x 10(5) to 5 x 10(7) g.mol(-1). Filtering of the EHEC II solution removed small amounts of compact UHM material. Enzyme treatments were performed on EHEC II to further characterize it. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and anion ion-exchange chromatography coupled to pulsed amperometric detection showed that the UHM component contained EHEC.     

Probing the effect of point mutations at protein-protein interfaces with free energy calculations

We have studied the effect of point mutations of the primary binding residue (P1) at the protein-protein interface in complexes of chymotrypsin and elastase with the third domain of the turkey ovomucoid inhibitor and in trypsin with the bovine pancreatic trypsin inhibitor, using molecular dynamics simulations combined with the linear interaction energy (LIE) approach. A total of 56 mutants have been constructed and docked into their host proteins. The free energy of binding could be reliably calculated for 52 of these mutants that could unambiguously be fitted into the binding sites. We find that the predicted binding free energies are in very good agreement with experimental data with mean unsigned errors between 0.50 and 1.03 kcal/mol. It is also evident that the standard LIE model used to study small drug-like ligand binding to proteins is not suitable for protein-protein interactions. Three different LIE models were therefore tested for each of the series of protein-protein complexes included, and the best models for each system turn out to be very similar. The difference in parameterization between small drug-like compounds and protein point mutations is attributed to the preorganization of the binding surface. Our results clearly demonstrate the potential of free energy calculations for probing the effect of point mutations at protein-protein interfaces and for exploring the principles of specificity of hot spots at the interface.     

Direct Angiotensin II Type 2 Receptor Stimulation Ameliorates Insulin Resistance in Type 2 Diabetes Mice with PPARγ Activation

Objectives

The role of angiotensin II type 2 (AT₂) receptor stimulation in the pathogenesis of insulin resistance is still unclear. Therefore we examined the possibility that direct AT₂ receptor stimulation by compound 21 (C21) might contribute to possible insulin-sensitizing/anti-diabetic effects in type 2 diabetes (T2DM) with PPARγ activation, mainly focusing on adipose tissue.

Methods

T2DM mice, KK-Ay, were subjected to intraperitoneal injection of C21 and/or a PPARγ antagonist, GW9662 in drinking water for 2 weeks. Insulin resistance was evaluated by oral glucose tolerance test, insulin tolerance test, and uptake of 2-[³H] deoxy-D-glucose in white adipose tissue. Morphological changes of adipose tissues as well as adipocyte differentiation and inflammatory response were examined.

Results

Treatment with C21 ameliorated insulin resistance in KK-Ay mice without influencing blood pressure, at least partially through effects on the PPARγ pathway. C21 treatment increased serum adiponectin concentration and decreased TNF-α concentration; however, these effects were attenuated by PPARγ blockade by co-treatment with GW9662. Moreover, we observed that administration of C21 enhanced adipocyte differentiation and PPARγ DNA-binding activity, with a decrease in inflammation in white adipose tissue, whereas these effects of C21 were attenuated by co-treatment with GW9662. We also observed that administration of C21 restored β cell damage in diabetic pancreatic tissue.

Conclusion

The present study demonstrated that direct AT₂ receptor stimulation by C21 accompanied with PPARγ activation ameliorated insulin resistance in T2DM mice, at least partially due to improvement of adipocyte dysfunction and protection of pancreatic β cells.     

The molecular basis for Duchenne versus Becker muscular dystrophy: Correlation of severity with type of deletion

About 60% of both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) is due to deletions of the dystrophin gene. For cases with a deletion mutation, the "reading frame" hypothesis predicts that BMD patients produce a semifunctional, internally deleted dystrophin protein, whereas DMD patients produce a severely truncated protein that would be unstable. To test the validity of this theory, we analyzed 258 independent deletions at the DMD/BMD locus. The correlation between phenotype and type of deletion mutation is in agreement with the "reading frame" theory in 92% of cases and is of diagnostic and prognostic significance. The distribution and frequency of deletions spanning the entire locus suggests that many "in-frame" deletions of the dystrophin gene are not detected because the individuals bearing them are either asymptomatic or exhibit non-DMD/non-BMD clinical features.     

The Crystal Structure of a Trypsin-like Mutant Chymotrypsin: The Role of Position 226 in the Activity and Specificity of S189D Chymotrypsin

The crystal structure of the S189D+A226G rat chymotrypsin-B mutant has been determined at 2.2 angstroms resolution. This mutant is the most trypsin-like mutant so far in the line of chymotrypsin-to-trypsin conversions, aiming for a more complete understanding of the structural basis of substrate specificity in pancreatic serine proteases. A226G caused significant rearrangements relative to S189D chymotrypsin, allowing an internal conformation of Asp189 which is close to that in trypsin. Serious distortions remain, however, in the activation domain, including zymogen-like features. The pH-profile of activity suggests that the conformation of the S1-site of the mutant is influenced also by the P1 residue of the substrate.