Conserved properties of individual Ca2+-binding sites in calmodulin

Calmodulin (CaM) is a Ca²⁺-sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca²⁺ concentration into cellular signals by binding to a wide range of target proteins in a Ca²⁺-dependent manner. We do not fully understand how CaM performs its role as a high-fidelity signal transducer for more than 300 target proteins, but diversity among its four Ca²⁺-binding sites, called EF-hands, may contribute to CaM’s functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require CaM to decode Ca²⁺ signals using four specialized EF-hands, each with specific, conserved traits. In addition, we provide an extensive map of sites associated with target proteins and with human disease and correlate these with evolutionary sequence diversity. Our comprehensive evolutionary analysis provides a basis for understanding the sequence space associated with CaM function and should help guide future work on the relationship between structure, function, and disease.Eukaryotes use Ca²⁺ in numerous intracellular signaling pathways. Calmodulin (CaM) is a highly versatile Ca²⁺ signaling protein that is essential for at least dozens of cellular processes in eukaryotic cells. In humans it binds to more than 300 targets (1–3). Humans have three genes that encode identical CaM proteins, but mutations in just one of the three copies can cause disease (4–8), as can altered gene expression (9). Although CaM has been extensively studied, many details about its function are still poorly understood. The high evolutionary conservation along with the wide range of targets brings up the question of how a single Ca²⁺-binding protein displays both selectivity and flexibility in the context of its various signaling pathways.CaM binds Ca²⁺ at four, nonidentical sites that contain the structural motif called an EF-hand (10, 11), each of which contains an acidic Ca²⁺-coordinating loop, or “EF-loop” (Fig. 1A). The EF-loop spans 12 amino acids and provides at least six oxygen atoms for coordinating Ca²⁺ (12). The coordinating oxygen atoms are provided by the side chains at the first, third, fifth, and 12th positions of the EF-loop, and an oxygen from a main chain carbonyl group is provided at the seventh position (10). Water molecules participate in the Ca² coordination geometry (13). CaM functions as a sensor over a broad range of Ca²⁺ signals that vary in amplitude, duration, and location. Although biophysical and evolutionary sequence studies have resulted in a general understanding of the bulk properties of EF-hand–binding sites, the implications of differences in Ca²⁺ affinity among the four EF-hands deserves a thorough investigation.Open in a separate windowFig. 1.(A) Example of a Ca²⁺-bound EF-hand structure from PDBID 1CLL. A cartoon of an EF-hand peptide chain threads through a semitransparent representation of its molecular surface. The surface is the interface between molecular atoms and solvent rendered in PyMOL. Only atoms nearest the Ca²⁺ are shown and are depicted as spheres—green for Ca²⁺ and red for oxygens. A Ca²⁺-coordinating water is depicted as a semitransparent red sphere. Helices are gray, and the EF-loop is tan. (B) Maximum likelihood branch lengths of CaM and tubulin constrained to match the species tree in Torruella et al. (40). This tree covers much of eukaryotic diversity. Holozoa and Holomycota include animals and fungi, respectively, and their closely related protist lineages. SARPAE is described in the text. Both proteins are highly constrained, but whereas tubulin’s rate has been fairly consistent across eukaryotes, CaM underwent a dramatic speed-up in Ascomycete fungi, which include the model system S. cerevisiae.Previous reports showed that the large family of EF-hand proteins likely arose from a founder protein with a single EF-hand in the most recent common ancestor of all extant eukaryotes (11, 14–18). Different EF-hand–containing proteins bind Ca²⁺ with different affinities, suggesting that a protein with multiple EF-hands, such as CaM, may bind Ca²⁺ with a different affinity at each site (19–28). It has therefore been suggested that CaM’s four sites display different affinities and perhaps cooperativity (29, 30). We therefore hypothesized that CaM’s four, nonidentical loops may generate some of their functional flexibility by binding Ca²⁺ using different physical properties and explored whether such differences could be discerned in the evolutionary record.Evolutionary analyses can provide mechanistic insight into how CaM is used as a Ca²⁺ sensor across eukaryotes. Prior work showed that the protein sequence of CaM is evolving at a faster pace in fungal species (11, 31–33), reflecting the fact that although CaM is essential in Saccharomyces cerevisiae, the cells can survive with all four EF-hands ablated (34). However, previous evolutionary studies focused on a small subset of eukaryotes, either because few sequences were available at the time of publication or because the study was focused on a particular lineage. The vast expansion of taxonomic coverage in sequence databases, and the recent availability of new NMR and X-ray crystal structures of CaM, therefore demands a more comprehensive analysis. Unfortunately, CaM is a small, ancient, and highly conserved protein and therefore does not contain enough information to infer phylogenetic tree topologies. Kretsinger and Nakayama and coworkers (11, 16, 17, 35), for instance, found little correspondence between phylogenies inferred from protein, DNA, or intron–exon structure.To overcome this hurdle, we used a variety of techniques to explore sequence and structural conservation in CaM across eukaryotes. Our approach allows us to address several key questions: (i) How fast is CaM diverging in different phyla? (ii) How does the function of a site, or its association with disease, correlate with sequence conservation? (iii) What properties of the EF-hands are conserved over deep evolutionary time, and how might this correspond to functional plasticity?     

Convergence of ion channel genome content in early animal evolution

Multicellularity has evolved multiple times, but animals are the only multicellular lineage with nervous systems. This fact implies that the origin of nervous systems was an unlikely event, yet recent comparisons among extant taxa suggest that animal nervous systems may have evolved multiple times independently. Here, we use ancestral gene content reconstruction to track the timing of gene family expansions for the major families of ion-channel proteins that drive nervous system function. We find that animals with nervous systems have broadly similar complements of ion-channel types but that these complements likely evolved independently. We also find that ion-channel gene family evolution has included large loss events, two of which were immediately followed by rounds of duplication. Ctenophores, cnidarians, and bilaterians underwent independent bouts of gene expansion in channel families involved in synaptic transmission and action potential shaping. We suggest that expansions of these family types may represent a genomic signature of expanding nervous system complexity. Ancestral nodes in which nervous systems are currently hypothesized to have originated did not experience large expansions, making it difficult to distinguish among competing hypotheses of nervous system origins and suggesting that the origin of nerves was not attended by an immediate burst of complexity. Rather, the evolution of nervous system complexity appears to resemble a slow fuse in stem animals followed by many independent bouts of gene gain and loss.Animal nervous systems are complex cellular networks that encode internal states and behavioral output. They achieve this complexity primarily in two ways. First, nervous systems encode information in a wiring scheme whose connections differ in strength and sign (excitatory or inhibitory). The strengths can often change in an activity-dependent fashion (1). Second, nervous systems have a dynamic neural code made up of all-or-none action potentials and subtler graded potentials (2). The shape, timing, and duration of evoked electrical potentials vary greatly among—and even within—neurons and can also be activity-dependent. These two types of complex signaling, respectively, among and within cells are the fundamental work of nervous systems (1), and they are made possible by the great variety of ion channel proteins expressed in neurons.Recent studies have found that most ion channels and proteins involved in the formation of synapses are ancient, having originated long before the advent of nervous systems or even of animal multicellularity (3–7). However, the nature of the first animals and of the cells from which nervous systems evolved are not well understood, although many theories exist (8–11), and little is known about the genomic events that facilitated the rise of complex nervous systems. New information about animal phylogeny has demanded a return to these old questions concerning the nature of the first animals and the evolutionary history of nervous systems (12–15).This new information concerns the placement of the ctenophores, or comb jellies. Recent studies place ctenophores as the sister group to all other metazoans, a surprising finding given that ctenophores are complex predators with fairly sophisticated nervous systems (15). In contrast, sponges, which traditionally were considered to be the sister group of the remaining animals (16), and placozoans do not have nervous systems (but see refs. 17 and 18). Recent genomic analyses have found that ctenophores are lacking many nervous system and muscle-associated genes, suggesting independent origins of these structures in ctenophores (15, 19, 20). Conversely, the genomic presence and expression of certain developmental genes involved in nervous system differentiation (13, 14) and genes expressed in the synapse (13, 21) indicate deep similarities between ctenophore nervous systems and others. These findings have revived the debate about whether animal nervous systems have one or more origins. Although it is clear that there has been some degree of homoplasy and/or secondary simplifications or losses, the nature and timing of these events remains debatable (refs. 14, 20, and 21 are excellent reviews on this subject).Many studies have addressed the origin of animal nervous systems by using comparative physiological, developmental, or morphological evidence (22–24). We used a different technique: ancestral gene content reconstruction. This approach has been used to explore the origin of multicellularity (25), the evolution of prokaryotic metabolism (26), and the expansion of G-protein–coupled receptors in animals (27). Gene duplication has long been known to be a major source of novelty and complexity (28), and many of the families we analyzed play few known roles outside of nervous systems. We therefore hypothesized that the elaboration of nervous systems coincided with an expansion of the ion-channel families that are expressed there. We used two methods (27, 29) to reconstruct the ancestral copy number for a variety of ion channel families and tracked the evolution of gene duplications across the animal and fungal tree. The evolution of some of these families have been studied by other groups (15, 27, 30–32), but here we combine current methods of ancestral genome content reconstruction with dense sampling of early branching species and gene families to search for patterns of gene duplication that might illuminate the early history of nervous systems.     

Hours worked and patient visits provided by dentists in Australia

Background

The aim was to examine the numbers of hours worked and patient visits provided by age and gender of dentists in Australia, and compare with previous estimates to describe trends.

Methods

Data were collected from a random sample (N = 2961) of Australian dentists. Private sector dentists working in clinical practise were included in the analysis.

Results

The response rate was 49% (N = 1345 dentists). Hours per year worked and number of patient visits per year were lower for dentists aged 65 years and older compared with younger dentists, and were higher for male compared with female dentists aged 35–45 to 55–64 years. Hours per year worked were lower in 2013–2014 than reported in 2009–2010, but the number of patient visits in 2013–2014 was similar to the previously reported estimate from 2009–2010.

Conclusions

Hours worked and visits provided were only lower among older dentists aged 65 years or more. Male dentists tend to work more hours per year and provide more patient visits per year than female dentists. Over the last decade, Australian dentists maintained a stable output of visits per year despite a trend towards fewer hours worked per year.     

Dental insurance,service use and health outcomes in Australia: a systematic review

Private health insurance plays a key role in financing dental care in Australia. Having private dental insurance has been associated with higher levels of access to dental care, visiting for a check‐up and receiving a favourable pattern of services. Associations with better oral health have also been reported. In the absence of any existing review, this paper aims to systematically review the relationship between dental insurance and dental service use and/or oral health outcomes in Australia. A systematic search of online databases and subsequent sifting resulted in 36 publications, 33 of which were cross sectional and three cohort analyses. Dental service outcomes were more commonly reported than oral health outcomes. There was considerable heterogeneity in the outcome measures reported, for both service use and health outcomes. Overall, the majority of the evidence was from cross sectional studies and few studies reported analyses adjusted for confounding factors. The consolidated evidence points towards a positive association between dental insurance and dental visiting. Dentally insured adults are likely to have more regular access to dental care and have a more favourable pattern of service use than the uninsured. However, evidence of associations between dental insurance and oral health are mixed.