Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most common cause of cystic fibrosis. The F508 residue is located on nucleotide-binding domain 1 (NBD1) in contact with the cytosolic extensions of the transmembrane helices, in particular intracellular loop 4 (ICL4). To investigate how absence of F508 at this interface impacts the CFTR protein, we carried out a mutagenesis scan of ICL4 by introducing second-site mutations at 11 positions in cis with F508del. Using an image-based fluorescence assay, we measured how each mutation affected membrane proximity and ion-channel function. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains (F1068, R1070W, and F1074, mimicking F1068, F508, and F1074 in WT CFTR). F508del-CFTR displayed a distorted aromatic stack, with F1068 displaced toward the space vacated by F508, while in F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR included F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. These studies highlight how both hydrophobic interactions and conformational flexibility might be important at the ICL4/NBD1 interface, suggesting possible structural underpinnings of F508del-induced dysfunction.     

Do the utrophin tandem calponin homology domains bind F-actin in a compact or extended conformation?

Tandem calponin-homology (CH) domains play an important role in the actin-binding function of many spectrin superfamily proteins. Crystal structures from several of these proteins have suggested a flexibility between these domains, and the manner in which these domains bind to F-actin has been the subject of some controversy. A recent paper has used electron microscopy and three-dimensional reconstruction to examine the complex of the utrophin tandem CH domain with F-actin. In contrast to our previously published study, a closed conformation of the two calponin-homology domains was suggested in the new work. We show here that the new results can be explained by incomplete binding of utrophin to actin, heterogeneity in the mode of binding, and angular disorder in F-actin. We conclude that helical averaging applied to disordered filaments is responsible for their results, and that approaches designed to separate out homogeneous subsets within such filamentous complexes offer many advantages.     

What genes make a tree a tree?

Woody growth is evolutionarily ancient, yet has been gained and lost multiple times in plant evolution and is readily enhanced or minimized in eudicot speciation. New molecular genetic and genomic studies in Populus and Arabidopsis that are defining the genes responsible for cambium function and woody growth suggest that the genes regulating woody growth are not unique to woody plants. Surprisingly, key genetic mechanisms originally characterized as regulating the meristematic cells of the shoot apical meristem are also expressed in the vascular cambium during woody growth. This has important implications for the development of Populus as a model species and illustrates why forest trees constitute a contrived group of plants that have more in common with herbaceous relatives than we foresters like to admit.     

Translation initiation factor IF3: two domains, five functions, one mechanism?

Initiation factor IF3 contains two domains separated by a flexible linker. While the isolated N-domain displayed neither affinity for ribosomes nor a detectable function, the isolated C-domain, added in amounts compensating for its reduced affinity for 30S subunits, performed all activities of intact IF3, namely: (i) dissociation of 70S ribosomes; (ii) shift of 30S-bound mRNA from 'stand-by' to 'P-decoding' site; (iii) dissociation of 30S-poly(U)-NacPhe-tRNA pseudo- initiation complexes; (iv) dissociation of fMet-tRNA from initiation complexes containing mRNA with the non-canonical initiation triplet AUU (AUUmRNA); (v) stimulation of mRNA translation regardless of its start codon and inhibition of AUUmRNA translation at high IF3C/ribosome ratios. These results indicate that while IF3 performs all its functions through a C-domain-30S interaction, the N-domain function is to provide additional binding energy so that its fluctuating interaction with the 30S subunit can modulate the thermodynamic stability of the 30S-IF3 complex and IF3 recycling. The localization of IF3C far away from the decoding site and anticodon stem-loop of P-site-bound tRNA indicates that the IF3 fidelity function does not entail its direct contact with these structures.     

Gender as a historical kind: a tale of two genders?

Is there anything that members of each binary category of gender have in common? Even many non-essentialists find the lack of unity within a gender worrying as it undermines the basis for a common political agenda for women. One promising proposal for achieving unity is by means of a shared historical lineage of cultural reproduction with past binary models of gender (e.g. Bach in Ethics 122:231–272, 2012). I demonstrate how such an account is likely to take on board different binary and also non-binary systems of gender. This implies that all individuals construed as members of the category, “women” are in fact not members of the same historical kind after all! I then consider different possible means of modifying the account but conclude negatively: the problem runs deeper than has been appreciated thus far.     

Homoplasy and homology: dichotomy or continuum?

Homology is the presence of the same feature in two organisms whose most recent common ancestor also possessed the feature. I discuss the bases on which we can tell that two features being compared share sufficient elements of sameness to allow them to be treated as homologous and therefore to be legitimately compared with one another in a way that informs comparative, evolutionary, and phylogenetic analysis. To do so, I discuss the relationship(s) between homology and homoplasy to conclude that we are dealing neither with a dichotomy between homoplasy as parallelism/convergence and homology as common descent nor with a dichotomy of homoplasy as the interrupted presence of the character in a lineage and homology as the continuous presence of the character. Rather, we are dealing with common descent with varying degrees of modification. Homoplasy and homology are not dichotomies but the extremes of a continuum, reflecting deep or more recent shared ancestry based on shared cellular mechanisms and processes and shared genes and gene pathways and networks. The same genes can be used to initiate the development of homoplastic and homologous structures. Consequently, structures may be lost but their developmental bases retained, providing the potential for homoplasy. It should not be surprising that similar features persist when a feature is present in the nearest common ancestor (homology). Neither should it be surprising to find that different environments or selective pressures can trigger the reappearance of similar features in organisms that do not share a recent common ancestor (homoplasy).     

How many possums make a cow?

The importance of possums as competitors with livestock for pasture is sometimes used as justification for possum control. Unfortunately a confusion of wet and dry weight values of pasture and daily consumption by possums appears to have resulted in a significant overestimate of the economic costs of possums as pasture pests. A correctly estimated daily dry matter intake of pasture by possums is 0.0144 kg pasture dry matter per possum per day. For a possum density of 1 ha^-1, this amounts to a reduction of about 1% of a stock unit ha^-1 year^-1. At higher possum densities, which often occur on farms adjacent to forest or scrub, losses would be correspondingly higher. A more direct way to measure possum impact on pasture production would be to use exclosures.     

Cellulose: how many cellulose synthases to make a plant?

Many questions remain about the biosynthesis of cellulose, the major plant cell wall component, not least of which is why plants have so many genes for the cellulose synthase catalytic subunit. Perhaps multiple isoforms of cellulose synthase are needed in the same cell for the formation of functional dimeric complexes.     

Pesticide resistance: can we make it a renewable resource?

Negative cross-resistance (NCR) occurs when a mutant allele confers (i) resistance to one toxic chemical and (ii) hyper-susceptibility to another. Sequential deployment of NCR toxins is useful for insect control in few situations (Pittendrigh et al., 2000). Using Monte Carlo simulations, we investigated the concurrent use of a pair of NCR toxins to control a hypothetical insect pest population. When the toxins killed more heterozygotes than homozygotes, the resistance allele became either extremely common or rare depending on starting allelic frequency. If the NCR toxins did not kill the two homozygous groups equally, then the toxin with lesser toxicity eventually played a greater role in the control of the pest population. Based on our results, we present an approach for the systematic development of an NCR toxin after the commercial release of the first toxin. First, large-scale screens are performed to find chemicals that kill the resistant homozygous insects, but not the susceptible ones. Chemicals that preferentially kill resistant insects are then tested for toxicity to the heterozygotes. Those highly toxic to both homo- and heterozygotes are given the highest priority for development. This screen can be adapted to identify compounds useful in controlling antibiotic-, herbicide- or fungicide-resistant organisms.     

Immunological exhaustion: How to make a disparate concept operational?

In this essay, we show that 3 distinct approaches to immunological exhaustion coexist and that they only partially overlap, generating potential misunderstandings. Exploring cases ranging from viral infections to cancer, we propose that it is crucial, for experimental and therapeutic purposes, to clarify these approaches and their interconnections so as to make the concept of exhaustion genuinely operational.     

Patterns of gene expression: homology or homocracy?

Numerous papers over the years have stated that the original meaning of the term homology is historical and morphological and denotes organs/structures in two or more species derived from the same structure in their latest common ancestor. However, several more recent papers have extended the use of the term to cover organs/structures which are organised through the expression of homologous genes. This usage has created an ambiguity about the meaning of the term, and we propose to remove this by proposing a new term, homocracy, for organs/structures which are organised through the expression of identical patterning genes. We want to emphasise that the terms homologous and homocratic are not mutually exclusive. Many homologous structures are in all probability homocratic, whereas only a small number of homocratic structures are homologous.     

Neuronal PDZ domains: a promising new molecular target for inhaled anesthetics?

Despite widespread use of volatile general anesthetics in millions of patients each year, the mechanisms by which they exert multiple effects on the behavior of central neurons are poorly understood. PDZ [postsynaptic density 95 (PSD-95), discs large (Dlg), and zonula occludens-1 (ZO-1)] domains are ubiquitous protein interaction modules that participate in neuronal signaling. Recent studies have indicated that clinically relevant concentrations of inhaled anesthetics dose-dependently and specifically inhibit the PDZ domain-mediated protein interactions among multiprotein signaling complexes. These inhibitory effects are immediate, potent, reversible, and occur at a hydrophobic peptidebinding groove on the surface of the PDZ domain. Thus, the PDZ domain might be a new molecular target for inhalational anesthetics.