Electron transport lifetimes in InSb/Al1-xInxSb quantum well 2DEGs

We report magnetotransport measurements of InSb/Al1-xInxSb modulation doped quantum well (QW) structures and the extracted transport ( ) tt and quantum (tq) lifetime of carriers at low temperature (<2K.) We consider conventional transport lifetimes over a range of samples with different doping levels and carrier densities, and deduce different transport regimes dependent on QW state filling calculated from self-consistent Schrödinger–Poisson modelling. For samples where only the lowest QW subband is occupied at electron densities of 2.13 10 ´ 11 cm−2 and 2.54 10 ´ 11 cm−2 quantum lifetimes of tq » 0.107 ps, and tq » 0.103 ps are extracted from Shubnikov–de Haas oscillations below a magnetic field of 0.8 T. The extracted ratios of transport to quantum lifetimes, t t t q » 17 and t t t q » 20 are similar to values reported in other binary QW two-dimensional electron gas systems, but are inconsistent with predictions from transport modelling which assumes that remote ionized donors are the dominant scattering mechanism. We find the low t t t q ratio and the variation in transport mobility with carrier density cannot be explained by reasonable levels of background impurities or well width fluctuations. Thus, there is at least one additional scattering mechanism unaccounted for, most likely arising from structural defects

Smoothing a rugged protein folding landscape by sequence-based redesign

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as $\alpha_1$-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create $\textit{conserpin}$, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics.BTP is a Medical Research Council Career Development Fellow. AAN and JJH are supported by the Wellcome Trust (grant number WT 095195). SM acknowledges fellowship support from the Australian Research Council (FT100100960). NAB is an Australian Research Council Future Fellow (110100223). GIW is an Australian Research Council Discovery Outstanding Researcher Award Fellow (DP140100087). AMB is a National Health and Medical Research Senior Research Fellow (1022688). JCW is an NHMRC Senior Principal Research fellow and also acknowledges the support of an ARC Federation Fellowship. We thank the Australian Synchrotron for beam-time and technical assistance. This work was supported by the Multi-modal Australian ScienceS Imaging and Visualisation Environment (MASSIVE) (www.massive.org.au). We acknowledge the Monash Protein Production Unit and Monash Macromolecular Crystallization Facilit

Smoothing a rugged protein folding landscape by sequence-based redesign

The rugged folding landscapes of functional proteins puts them at risk of misfolding and aggregation. Serine protease inhibitors, or serpins, are paradigms for this delicate balance between function and misfolding. Serpins exist in a metastable state that undergoes a major conformational change in order to inhibit proteases. However, conformational labiality of the native serpin fold renders them susceptible to misfolding, which underlies misfolding diseases such as α1-antitrypsin deficiency. To investigate how serpins balance function and folding, we used consensus design to create conserpin, a synthetic serpin that folds reversibly, is functional, thermostable, and polymerization resistant. Characterization of its structure, folding and dynamics suggest that consensus design has remodeled the folding landscape to reconcile competing requirements for stability and function. This approach may offer general benefits for engineering functional proteins that have risky folding landscapes, including the removal of aggregation-prone intermediates, and modifying scaffolds for use as protein therapeutics

Peripheral Blood CD64 Levels Decrease in Crohn's Disease following Granulocyte and Monocyte Adsorptive Apheresis

Granulocyte and monocyte adsorptive apheresis (GMA) is reportedly useful as induction therapy for Crohn's disease (CD). However, the effects of GMA on CD64 have not been well characterized. We report here our assessment of CD64 expression on neutrophils before and after treatment with GMA in two patients with CD. The severity of CD was assessed with the CD activity index (CDAI). The duration of each GMA session was 60 min at a flow rate of 30 ml/min as per protocol. CD64 expression on neutrophils was measured by analyzing whole blood with a FACScan flow cytometer. In case 1, CD64 levels after each session of GMA tended to decrease compared to pretreatment levels, whereas in case 2, CD64 levels dropped significantly after treatment. The CDAI decreased after GMA in both cases 1 and 2. A significant correlation was noted between CDAI scores and CD64 levels in both cases. In conclusion, GMA reduced blood CD64 levels, which would be an important factor for the decrease of CDAI scores

A Major Role for Side-Chain Polyglutamine Hydrogen Bonding in Irreversible Ataxin-3 Aggregation

The protein ataxin-3 consists of an N-terminal globular Josephin domain (JD) and an unstructured C-terminal region containing a stretch of consecutive glutamines that triggers the neurodegenerative disorder spinocerebellar ataxia type 3, when it is expanded beyond a critical threshold. The disease results from misfolding and aggregation, although the pathway and structure of the aggregation intermediates are not fully understood. In order to provide insight into the mechanism of the process, we monitored the aggregation of a normal (AT3Q24) ataxin-3, an expanded (AT3Q55) ataxin-3, and the JD in isolation. We observed that all of them aggregated, although the latter did so at a much slower rate. Furthermore, the expanded AT3Q55 displayed a substantially different behavior with respect to the two other variants in that at the latest stages of the process it was the only one that did the following: i) lost its reactivity towards an anti-oligomer antibody, ii) generated SDS-insoluble aggregates, iii) gave rise to bundles of elongated fibrils, and iv) displayed two additional bands at 1604 and 1656 cm−1 in FTIR spectroscopy. Although these were previously observed in other aggregated polyglutamine proteins, no one has assigned them unambiguously, yet. By H/D exchange experiments we show for the first time that they can be ascribed to glutamine side-chain hydrogen bonding, which is therefore the hallmark of irreversibly SDS-insoluble aggregated protein. FTIR spectra also showed that main-chain intermolecular hydrogen bonding preceded that of glutamine side-chains, which suggests that the former favors the latter by reorganizing backbone geometry

Real-Time Imaging and Quantification of Amyloid-β Peptide Aggregates by Novel Quantum-Dot Nanoprobes

Background: Protein aggregation plays a major role in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease. However, direct real-time imaging of protein aggregation, including oligomerization and fibrillization, has never been achieved. Here we demonstrate the preparation of fluorescent semiconductor nanocrystal (quantum dot; QD)-labeled amyloid-b peptide (QDAb) and its advanced applications. Methodology/Principal Findings: The QDAb construct retained Ab oligomer-forming ability, and the sizes of these oligomers could be estimated from the relative fluorescence intensities of the imaged spots. Both QDAb coaggregation with intact Ab42 and insertion into fibrils were detected by fluorescence microscopy. The coaggregation process was observed by real-time 3D imaging using slit-scanning confocal microscopy, which showed a typical sigmoid curve with 1.5 h in the lag-time and 12 h until saturation. Inhibition of coaggregation using an anti-Ab antibody can be observed as 3D images on a microscopic scale. Microglia ingested monomeric QDAb more significantly than oligomeric QDAb, and the ingested QDAb was mainly accumulated in the lysosome. Conclusions/Significance: These data demonstrate that QDAb is a novel nanoprobe for studying Ab oligomerization an

Association between colony-stimulating factor 1 receptor gene polymorphisms and asthma risk

Colony-stimulating factor 1 receptor (CSF1R) is expressed in monocytes/macrophages and dendritic cells. These cells play important roles in the innate immune response, which is regarded as an important aspect of asthma development. Genetic alterations in the CSF1R gene may contribute to the development of asthma. We investigated whether CSF1R gene polymorphisms were associated with the risk of asthma. Through direct DNA sequencing of the CSF1R gene, we identified 28 single nucleotide polymorphisms (SNPs) and genotyped them in 303 normal controls and 498 asthmatic patients. Expression of CSF1R protein and mRNA were measured on CD14-positive monocytes and neutrophils in peripheral blood of asthmatic patients using flow cytometry and real-time PCR. Among the 28 polymorphisms, two intronic polymorphism (+20511C>T and +22693T>C) were associated with the risk of asthma by logistic regression analysis. The frequencies of the minor allele at CSF1R +20511C>T and +22693T>C were higher in asthmatic subjects than in normal controls (4.6 vs. 7.7%, p = 0.001 in co-dominant and dominant models; 16.4 vs. 25.8%, p = 0.0006 in a recessive model). CSF1R mRNA levels in neutrophils of the asthmatic patients having the +22693CC allele were higher than in those having the +22693TT allele (p = 0.026). Asthmatic patients with the +22693CC allele also showed significantly higher CSF1R expression on CD14-positive monocytes and neutrophils than did those with the +22693TT allele (p = 0.045 and p = 0.044). The +20511C>T SNP had no association with CSF1R mRNA or protein expression. In conclusion, the minor allele at CSF1R +22693T>C may have a susceptibility effect in the development of asthma, via increased CSF1R protein and mRNA expression in inflammatory cells

Autocatalytic Activation of the Furin Zymogen Requires Removal of the Emerging Enzyme's N-Terminus from the Active Site

Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg(107) downward arrowAsp(108), but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg(75) downward arrowSer(76)) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S' sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg(89) downward arrowGlu(90) site, is required for the efficient activation of furin.Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases