Probe diffusion in aqueous poly(vinyl alcohol) solutions studied by fluorescence correlation spectroscopy

We report fluorescence correlation spectroscopy measurements of the translational diffusion coefficient of various probe particles in dilute and semidilute aqueous poly(vinyl alcohol) solutions. The range of sizes of the particles (fluorescent molecules, proteins, and polymers) was chosen to explore various length scales of the polymer solutions as defined by the polymer-polymer correlation length. For particles larger than the correlation length, we find that the diffusion coefficient, D, decreases exponentially with the polymer concentration. This can be explained by an exponential increase in the solution viscosity, consistent with the Stokes-Einstein equation. For probes on the order of the correlation length, the decrease of the diffusion coefficient cannot be accounted for by the Stokes-Einstein equation, but can be fit by a stretched exponential, D approximately exp(-alphacn), where we find n = 0.73-0.84 and alpha is related to the probe size. These results are in accord with a diffusion model of Langevin and Rondelez (Polymer 1978, 19, 1875), where these values of n indicate a good solvent quality.     

High CO2 Levels Cause Skeletal Muscle Atrophy via AMP-activated Kinase (AMPK), FoxO3a Protein,and Muscle-specific Ring Finger Protein 1 (MuRF1)

Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO₂ had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO₂ had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO₂ induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1^−/− mice exposed to high CO₂ did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO₂, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO₂ induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO₂ activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.     

Shape matters: Lifecycle of cooperative patches promotes cooperation in bulky populations

Natural cooperative systems take many forms, ranging from one‐dimensional cyanobacteria arrays to fractal‐like biofilms. We use in silico experimental systems to study a previously overlooked factor in the evolution of cooperation, physical shape of the population. We compare the emergence and maintenance of cooperation in populations of digital organisms that inhabit bulky (100 × 100 cells) or slender (4 × 2500) toroidal grids. Although more isolated subpopulations of secretors in a slender population could be expected to favor cooperation, we find the opposite: secretion evolves to higher levels in bulky populations. We identify the mechanistic explanation for the shape effect by analyzing the lifecycle and dynamics of cooperator patches, from their emergence and growth, to invasion by noncooperators and extinction. Because they are constrained by the population shape, the cooperator patches expand less in slender than in bulky populations, leading to fewer cooperators, less public good secretion, and generally lower cooperation. The patch dynamics and mechanisms of shape effect are robust across several digital cooperation systems and independent of the underlying basis for cooperation (public good secretion or a cooperation game). Our results urge for a greater consideration of population shape in the study of the evolution of cooperation across experimental and modeling systems.     

A phylogeographical perspective on the ex situ conservation of Aylacostoma (Thiaridae,Gastropoda) from the High Paraná River (Argentina–Paraguay)

Aylacostoma Spix, 1827, contains species that are the subject of focused conservation efforts under the auspices of the ‘Aylacostoma Project’, the only ex situ conservation programme for freshwater gastropods in South America. Two species from the High Paraná River (Argentina–Paraguay) are included in this programme (Aylacostoma chloroticum Hylton Scott, 1954 and Aylacostoma brunneum Vogler & Peso, 2014), as their habitats have disappeared as a consequence of the filling of the Yacyretá Reservoir in the 1990s. At present, A. chloroticum is restricted to only one known wild population in a small and fragile habitat, and wild populations of A. brunneum are presumed to have gone extinct. We used partial sequences of the cytochrome oxidase subunit I gene to provide the first phylogeographical perspective on these species from a limited dataset containing representatives of several wild populations that are successfully being bred in captivity. We found low genetic diversity and two haplotypes in A. chloroticum, and absence of variation with one haplotype in A. brunneum. The reservoir's entry zone was identified to be of great interest for conservation, and is where we suggest re‐introductions and translocations should be targeted, to preserve the future evolutionary potential of the extant diversity. © 2015 The Linnean Society of London     

Proteasomal degradation of preemptive quality control (pQC) substrates is mediated by an AIRAPL–p97 complex

The initial folding of secreted proteins occurs in the ER lumen, which contains specific chaperones and where posttranslational modifications may occur. Therefore lack of translocation, regardless of entry route or protein identity, is a highly toxic event, as the newly synthesized polypeptide is misfolded and can promiscuously interact with cytosolic factors. Mislocalized proteins bearing a signal sequence that did not successfully translocate through the translocon complex are subjected to a preemptive quality control (pQC) pathway and are degraded by the ubiquitin-proteasome system (UPS). In contrast to UPS-mediated, ER-associated degradation, few components involved in pQC have been identified. Here we demonstrate that on specific translocation inhibition, a p97–AIRAPL complex directly binds and regulates the efficient processing of polyubiquitinated pQC substrates by the UPS. We also demonstrate p97’s role in pQC processing of preproinsulin in cases of naturally occurring mutations within the signal sequence of insulin.     

Synthetic peptides mimicking the binding site of human acetylcholinesterase for its inhibitor fasciculin 2

Molecules capable of mimicking protein binding and/or functional sites present useful tools for a range of biomedical applications, including the inhibition of protein–ligand interactions. Such mimics of protein binding sites can currently be generated through structure‐based design and chemical synthesis. Computational protein design could be further used to optimize protein binding site mimetics through rationally designed mutations that improve intermolecular interactions or peptide stability. Here, as a model for the study, we chose an interaction between human acetylcholinesterase (hAChE) and its inhibitor fasciculin‐2 (Fas) because the structure and function of this complex is well understood. Structure‐based design of mimics of the hAChE binding site for Fas yielded a peptide that binds to Fas at micromolar concentrations. Replacement of hAChE residues known to be essential for its interaction with Fas with alanine, in this peptide, resulted in almost complete loss of binding to Fas. Computational optimization of the hAChE mimetic peptide yielded a variant with slightly improved affinity to Fas, indicating that more rounds of computational optimization will be required to obtain peptide variants with greatly improved affinity for Fas. CD spectra in the absence and presence of Fas point to conformational changes in the peptide upon binding to Fas. Furthermore, binding of the optimized hAChE mimetic peptide to Fas could be inhibited by hAChE, providing evidence for a hAChE‐specific peptide–Fas interaction. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

Background

F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin_N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin.

Results

We present the crystal structure of human F-spondin FS domain at 1.95Å resolution. The structure reveals a Ca²⁺-binding C2 domain variant with an 8-stranded antiparallel β-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain.

Conclusion

The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca²⁺- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.