Bitter tastants can activate bitter taste receptors on constricted smooth muscle cells to inhibit L-type calcium channels and induce bronchodilation.
Bronchodilators are a standard medicine for treating airway obstructive diseases, and β2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than β2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca2+ signals, as revealed in cultured ASM cells, to activate large-conductance Ca2+-activated K+ channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca2+ events nor alter spontaneous local Ca2+ transients. Interestingly, they increase global intracellular [Ca2+]i, although to a much lower level than bronchoconstrictors. We show that these Ca2+ changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cβ [PLCβ]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca2+]i induced by bronchoconstrictors, and this lowering of the [Ca2+]i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca2+ channels (VDCCs), resulting in reversal in [Ca2+]i, and this inhibition can be prevented by pertussis toxin and G-protein βγ subunit inhibitors, but not by the blockers of PLCβ and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca2+ signaling pathways via Gβγ to increase [Ca2+]i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca2+]i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.
Bitter taste receptors (TAS2Rs), a G-protein-coupled receptor family long thought to be solely expressed in taste buds on the tongue, have recently been detected in airways. Bitter substances can activate TAS2Rs in airway smooth muscle to cause greater bronchodilation than β2 adrenergic receptor agonists, the most commonly used bronchodilators. However, the mechanisms underlying this bronchodilation remain elusive. Here we show that, in resting primary airway smooth muscle cells, bitter tastants activate a TAS2R-dependent signaling pathway that results in an increase in intracellular calcium levels, albeit to a level much lower than that produced by bronchoconstrictors. In bronchoconstricted cells, however, bitter tastants reverse the bronchoconstrictor-induced increase in calcium levels, which leads to the relaxation of smooth muscle cells. We find that this reversal is due to inhibition of L-type calcium channels. Our results suggest that under normal conditions, bitter tastants can activate TAS2Rs to modestly increase calcium levels, but that when smooth muscle cells are constricted, they can block L-type calcium channels to induce bronchodilation. We postulate that this novel mechanism could operate in other extraoral cells expressing TAS2Rs.
An age-associated isoform of ING1, ING1a, induces cell senescence by altering endocytosis, subsequently activating the retinoblastoma tumor suppressor.
The INhibitor of Growth (ING) proteins act as type II tumor suppressors and epigenetic regulators, being stoichiometric members of histone acetyltransferase and histone deacetylase complexes. Expression of the alternatively spliced ING1a tumor suppressor increases >10-fold during replicative senescence. ING1a overexpression inhibits growth; induces a large flattened cell morphology and the expression of senescence-associated β-galactosidase; increases Rb, p16, and cyclin D1 levels; and results in the accumulation of senescence-associated heterochromatic foci. Here we identify ING1a-regulated genes and find that ING1a induces the expression of a disproportionate number of genes whose products encode proteins involved in endocytosis. Intersectin 2 (ITSN2) is most affected by ING1a, being rapidly induced >25-fold. Overexpression of ITSN2 independently induces expression of the p16 and p57KIP2 cyclin-dependent kinase inhibitors, which act to block Rb inactivation, acting as downstream effectors of ING1a. ITSN2 is also induced in normally senescing cells, consistent with elevated levels of ING1a inducing ITSN2 as part of a normal senescence program. Inhibition of endocytosis or altering the stoichiometry of endosome components such as Rab family members similarly induces senescence. Knockdown of ITSN2 also blocks the ability of ING1a to induce a senescent phenotype, confirming that ITSN2 is a major transducer of ING1a-induced senescence signaling. These data identify a pathway by which ING1a induces senescence and indicate that altered endocytosis activates the Rb pathway, subsequently effecting a senescent phenotype.
Alternative splicing of several genes including the p16 and p53 tumor suppressors has been reported to increase during replicative senescence of normal diploid cells, but the biological functions of most alternative transcripts are unknown. We have found that a splicing product of the ING1 epigenetic regulator, ING1a, also increases during senescence; moreover, forced expression of ING1a at these levels in otherwise growth-competent cells can induce senescence. In this study we have determined that a major mechanism by which ING1a induces senescence is through inhibiting endocytosis; this subsequently activates the retinoblastoma (Rb) tumor suppressor pathway by increasing Rb levels and preventing its inactivation through multiple mechanisms. Our study also establishes a link between endocytosis and oxidative stress and suggests that multiple mechanisms that induce cellular senescence may do so by inhibiting normal endocytic processes, thereby affecting normal signal transduction pathways including those mitogenic pathways required for cell growth.
Removal of confidentiality claims on biosafety data is necessary to adhere to standard scientific procedures of quality assurance, to increase transparency, to minimize impacts of conflicts of interests, and ultimately to improve public confidence in GMOs.
Structural analyses show that a viral protein and immunosuppressant drugs inhibit the phosphatase calcineurin by preventing substrate binding, and provide a model of a phosphatase engaged with its substrate.
Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca2+/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, “LxVP.” The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that “LxVP” sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent “LxVP”-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.
Transplantation medicine was revolutionized by the introduction of the immunosuppressant drugs cyclosporin A and FK506 that prevent rejection of transplanted organs by the recipient's immune system. These drugs work by inhibiting calcineurin, a conserved protein phosphatase. Calcineurin regulates the immune response by dephosphorylating and activating the members of the NFAT family of transcription factors, which in turn activate genes required for the antigen-dependent stimulation of T-cells. Despite its biological and clinical importance, we have only a limited understanding of how calcineurin and other protein phosphatases interact with their substrates and target specific phosphorylated residues for dephosphorylation. Here, we determined the structure of calcineurin in complex with A238L, a viral peptide inhibitor of its function. This study shows that the viral peptide inhibits calcineurin not by targeting its active site but rather by occupying two critical substrate-binding regions of calcineurin (distant from each other and from the active site), thereby preventing its interaction with protein substrates. These findings allow us to present the first computational model of calcineurin bound to a phospho-substrate at its active site. Furthermore, by elucidating the structural basis for one particular mode of substrate–calcineurin interaction, this study reveals that both this viral peptide and immunosuppressant drugs inhibit calcineurin by blocking substrate access to a single critical region of the enzyme.
Ecological selection on an adaptive allele causes a tightly linked hybrid incompatibility factor to rapidly hitchhike to high frequency in a population of the wildflower Mimulus guttatus.
Most species are superbly and intricately adapted to the environments in which they live. Adaptive evolution by natural selection is the primary force shaping biological diversity. Differences between closely related species in ecologically selected characters such as habitat preference, reproductive timing, courtship behavior, or pollinator attraction may prevent interbreeding in nature, causing reproductive isolation. But does ecological adaptation cause reproductive incompatibilities such as hybrid sterility or lethality? Although several genes causing hybrid incompatibilities have been identified, there is intense debate over whether the genes that contribute to ecological adaptations also cause hybrid incompatibilities. Thirty years ago, a genetic study of local adaptation to copper mine soils in the wildflower Mimulus guttatus identified a locus that appeared to cause copper tolerance and hybrid lethality in crosses to other populations. But do copper tolerance and hybrid lethality have the same molecular genetic basis? Here we show, using high-resolution genome mapping, that copper tolerance and hybrid lethality are not caused by the same gene but are in fact separately controlled by two tightly linked loci. We further show that selection on the copper tolerance locus indirectly caused the hybrid incompatibility allele to go to high frequency in the copper mine population because of hitchhiking. Our results provide a new twist on Darwin's original supposition that hybrid incompatibilities evolve as an incidental by-product of ordinary adaptation to the environment.
Adaptive evolution by natural selection is the primary force generating biological diversity. A critical question is whether the evolution of hybrid incompatibility, which is essential for the maintenance of species diversity, is caused by adaptive evolution. In this article, we investigate one of the most widely cited examples of ecological divergence driving the evolution of reproductive incompatibility, the strong association between hybrid lethality and copper tolerance in a copper mine population of the wildflower Mimulus guttatus. Hybrid lethality and tolerance of high levels of copper co-segregate as a single Mendelian locus. While copper tolerance and hybrid lethality are nearly universal in the mine population at Copperopolis, California, they are absent from adjacent off-mine populations, suggesting that reproductive isolation evolved rapidly as a pleiotropic by-product of recent adaptation to the mine environment. We find that copper tolerance and hybrid lethality are controlled by distinct loci, in tight genetic linkage. We also demonstrate that this genomic region has experienced strong recent selection and conclude that ecological selection for copper tolerance indirectly caused the neighboring hybrid lethality allele to hitchhike to high frequency. To our knowledge, this is the first case to demonstrate that reproductive isolation factors can evolve as an incidental by-product of adaptation to novel environments through genetic hitchhiking.
Andrew Dobson on the inestimable value, and beauty, of the artful field journal.
The modern evolutionary synthesis codified the idea that species exist as distinct entities because intrinsic reproductive barriers prevent them from merging together. Understanding the origin of species therefore requires understanding the evolution and genetics of reproductive barriers between species. In most cases, speciation is an accident that happens as different populations adapt to different environments and, incidentally, come to differ in ways that render them reproductively incompatible. As with other reproductive barriers, the evolution and genetics of interspecific hybrid sterility and lethality were once also thought to evolve as pleiotripic side effects of adaptation. Recent work on the molecular genetics of speciation has raised an altogether different possibility—the genes that cause hybrid sterility and lethality often come to differ between species not because of adaptation to the external ecological environment but because of internal evolutionary arms races between selfish genetic elements and the genes of the host genome. Arguably one of the best examples supporting a role of ecological adaptation comes from a population of yellow monkey flowers, Mimulus guttatus, in Copperopolis, California, which recently evolved tolerance to soil contaminants from copper mines and simultaneously, as an incidental by-product, hybrid lethality in crosses with some off-mine populations. However, in new work, Wright and colleagues show that hybrid lethality is not a pleiotropic consequence of copper tolerance. Rather, the genetic factor causing hybrid lethality is tightly linked to copper tolerance and spread to fixation in Copperopolis by genetic hitchhiking.
Gps1 provides a novel molecular polarity cue at the cell division site that guides Rho1- and Cdc42-dependent polarization during and after cytokinesis in budding yeast.
The spatiotemporal control of cell polarity is crucial for the development of multicellular organisms and for reliable polarity switches during cell cycle progression in unicellular systems. A tight control of cell polarity is especially important in haploid budding yeast, where the new polarity site (bud site) is established next to the cell division site after cell separation. How cells coordinate the temporal establishment of two adjacent polarity sites remains elusive. Here, we report that the bud neck associated protein Gps1 (GTPase-mediated polarity switch 1) establishes a novel polarity cue that concomitantly sustains Rho1-dependent polarization and inhibits premature Cdc42 activation at the site of cytokinesis. Failure of Gps1 regulation leads to daughter cell death due to rebudding inside the old bud site. Our findings provide unexpected insights into the temporal control of cytokinesis and describe the importance of a Gps1-dependent mechanism for highly accurate polarity switching between two closely connected locations.
In budding yeast, cell polarization (or the asymmetric distribution of subcellular components) ensures the targeted transport of proteins and membrane material to the sites of cell growth or cell division in late mitosis. Two conserved members of the Rho-GTPase family, Rho1 and Cdc42, are master regulators of cell polarity. While Rho1 has a well-established role in cytokinesis and cell separation, Cdc42 helps to establish the new polarity site from which the future daughter cell will grow after cytokinesis. Interestingly, despite the fact that Cdc42 is recruited to the site of cell division at the same time as Rho1, the new daughter cell never emerges from the site previously used for cytokinesis during the preceding cell cycle, and it remains elusive how cells coordinate the distinct functions of Rho1 and Cdc42 during cytokinesis. Here, we show that the novel protein Gps1 marks the cell division site, where it maintains Rho1-dependent polarity until cell separation is completed. We also demonstrate that Gps1 prevents activation of Cdc42 at the site of cell division during cytokinesis. We propose that Gps1 provides a novel polarity cue that guides the establishment of a new polarity site, away from the old site of cell division, where the new daughter cell then emerges.
Weaving the visual arts into a science curriculum can both help develop scientific imagination and engage non-scientists.
A crystal structure reveals an elegant mechanistic switch whereby helical bending and catalytic domain rotation allow self-activation of a histidine kinase during a bacterial stress response.
Two-component systems (TCSs) are important for the adaptation and survival of bacteria and fungi under stress conditions. A TCS is often composed of a membrane-bound sensor histidine kinase (SK) and a response regulator (RR), which are relayed through sequential phosphorylation steps. However, the mechanism for how an SK is switched on in response to environmental stimuli remains obscure. Here, we report the crystal structure of a complete cytoplasmic portion of an SK, VicK from Streptococcus mutans. The overall structure of VicK is a long-rod dimer that anchors four connected domains: HAMP, Per-ARNT-SIM (PAS), DHp, and catalytic and ATP binding domain (CA). The HAMP, a signal transducer, and the PAS domain, major sensor, adopt canonical folds with dyad symmetry. In contrast, the dimer of the DHp and CA domains is asymmetric because of different helical bends in the DHp domain and spatial positions of the CA domains. Moreover, a conserved proline, which is adjacent to the phosphoryl acceptor histidine, contributes to helical bending, which is essential for the autokinase and phosphatase activities. Together, the elegant architecture of VicK with a signal transducer and sensor domain suggests a model where DHp helical bending and a CA swing movement are likely coordinated for autokinase activation.
Two-component signal transduction systems (TCSs) are promising targets for new antimicrobial research because they help bacteria and fungi adapt and survive. One of the main components of TCSs is a sensor histidine kinase (SK), which relays extracellular signals to intracellular pathways. Despite intensive research, a full-length structure of an SK has yet to be solved. In this study, we report the first crystal structure of the complete cytoplasmic region of VicK, an important SK in the tooth decay pathogen S. mutans. VicK is composed of several domains (HAMP, PAS, DHp, and catalytic and ATP binding domain [CA]) in addition to a short transmembrane domain. We find that the dimeric VicK protein has an elegant rod-shaped structure with the domains linearly connected like beads on a string. The structure suggests that VicK kinase activates itself by helical bending of the DHp domain and coordinated swinging around of the catalytic CA domain to engage with the target histidine. Structure-based mutagenesis experiments also helped us to identify key residues that are required for VicK's opposing phosphatase activity. Our studies of the multi-modular VicK protein suggest a sequential kinase activation model that may involve helical bending of the DHp domain and repositioning of the CA domains.
During the development of the Drosophila nervous system, the developmentally regulated Hedgehog pathway, together with a series of temporal transcription factors, schedules the end of neurogenesis.
In Drosophila postembryonic neuroblasts, transition in gene expression programs of a cascade of transcription factors (also known as the temporal series) acts together with the asymmetric division machinery to generate diverse neurons with distinct identities and regulate the end of neuroblast proliferation. However, the underlying mechanism of how this “temporal series” acts during development remains unclear. Here, we show that Hh signaling in the postembryonic brain is temporally regulated; excess (earlier onset of) Hh signaling causes premature neuroblast cell cycle exit and under-proliferation, whereas loss of Hh signaling causes delayed cell cycle exit and excess proliferation. Moreover, the Hh pathway functions downstream of Castor but upstream of Grainyhead, two components of the temporal series, to schedule neuroblast cell cycle exit. Interestingly, hh is likely a target of Castor. Hence, Hh signaling provides a link between the temporal series and the asymmetric division machinery in scheduling the end of neurogenesis.
In almost all metazoans, neurons are produced by a group of neural stem cells/progenitors in a precise temporal manner, which is important for generating a functional nervous system. In Drosophila, this “timing” mechanism is mainly governed by the sequential switching of transcription factors in neural stem cells called neuroblasts, such that neuronal fate is associated with its birth order. These temporal factors also coordinate the termination of neuroblast division towards the end of neurogenesis. In this study, we show that Hedgehog (Hh) signaling also regulates the division rate of neuroblasts during their proliferative phase at larval stage, as well as the cessation of proliferation at early pupal stage. Excessive Hh signaling causes premature neuroblast cell cycle exit and early termination of neurogenesis, while loss of Hh signaling results in prolonged proliferation of neuroblasts beyond its physiological window. We also find that Hh signaling acts in concert with the temporal transcription factors, and is itself regulated by these factors. We hypothesize that this mode of interaction (temporal transcription factors with developmentally regulated signals like Hh) during neurogenesis could be widely conserved in other organisms.
Partial inhibition of adipose tissue lipolysis does not increase fat mass but improves glucose metabolism and insulin sensitivity through modulation of fatty acid turnover and induction of fat cell de novo lipogenesis.
When energy is needed, white adipose tissue (WAT) provides fatty acids (FAs) for use in peripheral tissues via stimulation of fat cell lipolysis. FAs have been postulated to play a critical role in the development of obesity-induced insulin resistance, a major risk factor for diabetes and cardiovascular disease. However, whether and how chronic inhibition of fat mobilization from WAT modulates insulin sensitivity remains elusive. Hormone-sensitive lipase (HSL) participates in the breakdown of WAT triacylglycerol into FAs. HSL haploinsufficiency and treatment with a HSL inhibitor resulted in improvement of insulin tolerance without impact on body weight, fat mass, and WAT inflammation in high-fat-diet–fed mice. In vivo palmitate turnover analysis revealed that blunted lipolytic capacity is associated with diminution in FA uptake and storage in peripheral tissues of obese HSL haploinsufficient mice. The reduction in FA turnover was accompanied by an improvement of glucose metabolism with a shift in respiratory quotient, increase of glucose uptake in WAT and skeletal muscle, and enhancement of de novo lipogenesis and insulin signalling in liver. In human adipocytes, HSL gene silencing led to improved insulin-stimulated glucose uptake, resulting in increased de novo lipogenesis and activation of cognate gene expression. In clinical studies, WAT lipolytic rate was positively and negatively correlated with indexes of insulin resistance and WAT de novo lipogenesis gene expression, respectively. In obese individuals, chronic inhibition of lipolysis resulted in induction of WAT de novo lipogenesis gene expression. Thus, reduction in WAT lipolysis reshapes FA fluxes without increase of fat mass and improves glucose metabolism through cell-autonomous induction of fat cell de novo lipogenesis, which contributes to improved insulin sensitivity.
In periods of energy demand, mobilization of fat stores in mammals (i.e., adipose tissue lipolysis) is essential to provide energy in the form of fatty acids. In excess, however, fatty acids induce resistance to the action of insulin, which serves to regulate glucose metabolism in skeletal muscle and liver. Insulin resistance (or low insulin sensitivity) is believed to be a cornerstone of the complications of obesity such as type 2 diabetes and cardiovascular diseases. In this study, our clinical observation of natural variation in fat cell lipolysis in individuals reveals that a high lipolytic rate is associated with low insulin sensitivity. Furthermore, partial genetic and pharmacologic inhibition of hormone-sensitive lipase, one of the enzymes involved in the breakdown of white adipose tissue lipids, results in improvement of insulin sensitivity in mice without gain in body weight and fat mass. We undertake a series of mechanistic studies in mice and in human fat cells to show that blunted lipolytic capacity increases the synthesis of new fatty acids from glucose in fat cells, a pathway that has recently been shown by others to be a major determinant of whole body insulin sensitivity. In conclusion, partial inhibition of adipose tissue lipolysis is a plausible strategy in the treatment of obesity-related insulin resistance.
Identifying evolutionary and ecological phenomena directly from sequencing of microbial communities is surprisingly challenging, even for one as simple as a single species adapting and diversifying in the laboratory.
Few areas of science have benefited more from the expansion in sequencing capability than the study of microbial communities. Can sequence data, besides providing hypotheses of the functions the members possess, detect the evolutionary and ecological processes that are occurring? For example, can we determine if a species is adapting to one niche, or if it is diversifying into multiple specialists that inhabit distinct niches? Fortunately, adaptation of populations in the laboratory can serve as a model to test our ability to make such inferences about evolution and ecology from sequencing. Even adaptation to a single niche can give rise to complex temporal dynamics due to the transient presence of multiple competing lineages. If there are multiple niches, this complexity is augmented by segmentation of the population into multiple specialists that can each continue to evolve within their own niche. For a known example of parallel diversification that occurred in the laboratory, sequencing data gave surprisingly few obvious, unambiguous signs of the ecological complexity present. Whereas experimental systems are open to direct experimentation to test hypotheses of selection or ecological interaction, the difficulty in “seeing ecology” from sequencing for even such a simple system suggests translation to communities like the human microbiome will be quite challenging. This will require both improved empirical methods to enhance the depth and time resolution for the relevant polymorphisms and novel statistical approaches to rigorously examine time-series data for signs of various evolutionary and ecological phenomena within and between species.
A single-molecule force study shows that rapid dissociation of a high-affinity protein interaction can be triggered by site-specific remodelling of one protein partner, and that prevention of remodelling maintains avidity.
Colicins are protein antibiotics synthesised by Escherichia coli strains to target and kill related bacteria. To prevent host suicide, colicins are inactivated by binding to immunity proteins. Despite their high avidity (Kd≈fM, lifetime ≈4 days), immunity protein release is a pre-requisite of colicin intoxication, which occurs on a timescale of minutes. Here, by measuring the dynamic force spectrum of the dissociation of the DNase domain of colicin E9 (E9) and immunity protein 9 (Im9) complex using an atomic force microscope we show that application of low forces (<20 pN) increases the rate of complex dissociation 106-fold, to a timescale (lifetime ≈10 ms) compatible with intoxication. We term this catastrophic force-triggered increase in off-rate a trip bond. Using mutational analysis, we elucidate the mechanism of this switch in affinity. We show that the N-terminal region of E9, which has sparse contacts with the hydrophobic core, is linked to an allosteric activator region in E9 (residues 21–30) whose remodelling triggers immunity protein release. Diversion of the force transduction pathway by the introduction of appropriately positioned disulfide bridges yields a force resistant complex with a lifetime identical to that measured by ensemble techniques. A trip switch within E9 is ideal for its function as it allows bipartite complex affinity, whereby the stable colicin:immunity protein complex required for host protection can be readily converted to a kinetically unstable complex whose dissociation is necessary for cellular invasion and competitor death. More generally, the observation of two force phenotypes for the E9:Im9 complex demonstrates that force can re-sculpt the underlying energy landscape, providing new opportunities to modulate biological reactions in vivo; this rationalises the commonly observed discrepancy between off-rates measured by dynamic force spectroscopy and ensemble methods.
Many proteins interact with other proteins as part of their function. One method of modulating the activity of protein complexes is to break them apart. Some complexes, however, are extremely kinetically stable and it is unclear how these can dissociate on a biologically relevant timescale. In this study we address this question using protein complexes between colicin E9 (a bacterial toxin) and its immunity protein Im9. These highly avid complexes (with a lifetime of days) must be broken apart for colicin to be activated. By using single-molecule force methods we show that pulling on one end of colicin E9 drastically destabilises the complex so that it dissociates a million-fold faster than its intrinsic rate. We then show that preventing this destabilisation (by the insertion of cross-links that pin the N-terminus of E9 in place) yields a kinetically stable complex. It has previously been postulated that force can destabilise a protein complex by partially unfolding one or more binding partners. Our work provides new experimental evidence that shows this is the case and provides a mechanism for this phenomenon, which we term a trip bond. For the E9:Im9 complex, trip bond behaviour allows a stable complex to be rapidly dissociated by application of a surprisingly small force.
The divergence of Escherichia coli bacteria into metabolically distinct ecotypes has a similar genetic basis and similar evolutionary dynamics across independently evolved populations.
The causes and mechanisms of evolutionary diversification are central issues in biology. Geographic isolation is the traditional explanation for diversification, but recent theoretical and empirical studies have shown that frequency-dependent selection can drive diversification without isolation and that adaptive diversification occurring in sympatry may be an important source of biological diversity. However, there are no empirical examples in which sympatric lineage splits have been understood at the genetic level, and it is unknown how predictable this process is—that is, whether similar ecological settings lead to parallel evolutionary dynamics of diversification. We documented the genetic basis and the evolutionary dynamics of adaptive diversification in three replicate evolution experiments, in which competition for two carbon sources caused initially isogenic populations of the bacterium Escherichia coli to diversify into two coexisting ecotypes representing different physiological adaptations in the central carbohydrate metabolism. Whole-genome sequencing of clones of each ecotype from different populations revealed many parallel and some unique genetic changes underlying the derived phenotypes, including changes to the same genes and sometimes to the same nucleotide. Timelines of allele frequencies extracted from the frozen “fossil” record of the three evolving populations suggest parallel evolutionary dynamics driven at least in part by a co-evolutionary process in which mutations causing one type of physiology changed the ecological environment, allowing the invasion of mutations causing an alternate physiology. This process closely corresponds to the evolutionary dynamics seen in mathematical models of adaptive diversification due to frequency-dependent ecological interactions. The parallel genetic changes underlying similar phenotypes in independently evolved lineages provide empirical evidence of adaptive diversification as a predictable evolutionary process.
The causes and mechanisms of evolutionary diversification are central issues in biology. There is well-established theory that predicts that adaptive diversification can arise because of ecological interactions between individuals, such as competition or predation, but there are no empirical examples in which this process has been observed at the genetic level. We documented the genetic basis of adaptive diversification resulting from competition for resources in populations of the bacterium Escherichia coli. The populations diversified into two coexisting ecotypes representing different physiological adaptations. We found that similar but independently evolved phenotypes often shared mutations in the same gene and, in four cases, shared identical mutations at the same nucleotide position. Timelines of allele frequencies extracted from the frozen “fossil record” of three evolving populations showed parallel evolutionary dynamics, suggesting that mutations causing one type of physiology changed the ecological environment and allowed invasion of mutations causing an alternate physiology. The results provide empirical evidence of adaptive diversification as a predictable evolutionary process.
Characterization of bilaterian head patterning genes in a cnidarian reveals a key role for six3/6 in aboral domain development and provides new insight into the evolutionary origin of head development.
The origin of the bilaterian head is a fundamental question for the evolution of animal body plans. The head of bilaterians develops at the anterior end of their primary body axis and is the site where the brain is located. Cnidarians, the sister group to bilaterians, lack brain-like structures and it is not clear whether the oral, the aboral, or none of the ends of the cnidarian primary body axis corresponds to the anterior domain of bilaterians. In order to understand the evolutionary origin of head development, we analysed the function of conserved genetic regulators of bilaterian anterior development in the sea anemone Nematostella vectensis. We show that orthologs of the bilaterian anterior developmental genes six3/6, foxQ2, and irx have dynamic expression patterns in the aboral region of Nematostella. Functional analyses reveal that NvSix3/6 acts upstream of NvFoxQ2a as a key regulator of the development of a broad aboral territory in Nematostella. NvSix3/6 initiates an autoregulatory feedback loop involving positive and negative regulators of FGF signalling, which subsequently results in the downregulation of NvSix3/6 and NvFoxQ2a in a small domain at the aboral pole, from which the apical organ develops. We show that signalling by NvFGFa1 is specifically required for the development of the apical organ, whereas NvSix3/6 has an earlier and broader function in the specification of the aboral territory. Our functional and gene expression data suggest that the head-forming region of bilaterians is derived from the aboral domain of the cnidarian-bilaterian ancestor.
The evolutionary origin of head development is a fundamental question for understanding the evolution of animal body plans. Bilaterally symmetrical animals (Bilaterians) have an anterior-posterior (head-to-tail) axis, whose anterior end is usually characterized by a nervous system centralization, the brain. This region is often associated with a distinct structure, the head, and its development is regulated by a set of conserved transcription factors and signalling molecules. Bilaterians evolved from an ancestor shared with cnidarians (corals, sea anemones, jellyfish), but brain-like structures are absent in cnidarians, although they have an obvious oral-aboral axis. Cnidarian larvae move with the aboral pole forward, but as adult polyps this pole is anchored to the ground, while the oral end is used for feeding. It is unclear whether one of the termini of cnidarians corresponds to the bilaterian head-forming region. We show here that in the sea anemone Nematostella vectensis genes regulating bilaterian head development are expressed at the larval aboral pole and that a key anterior developmental gene, six3/6, controls the development of the aboral pole. These findings support the hypothesis that the anterior, head-forming, region of bilaterians and the aboral region of cnidarians derived from the same domain of their last common ancestor and are therefore homologues.
A subunit of the chloroplast pyruvate dehydrogenase complex, which serves as a metabolic enzyme, also has a dual function as an RNA-binding protein and influences mRNA translation.
Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals and/or a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these dual functional proteins. We previously described the RNA binding activity of a 63 kDa chloroplast protein from Chlamydomonas reinhardtii, which has been implicated in expression of the psbA mRNA, encoding the D1 protein of photosystem II. Here, we identify this factor as dihydrolipoamide acetyltransferase (DLA2), a subunit of the chloroplast pyruvate dehydrogenase complex (cpPDC), which is known to provide acetyl-CoA for fatty acid synthesis. Analyses of RNAi lines revealed that DLA2 is involved in the synthesis of both D1 and acetyl-CoA. Gel filtration analyses demonstrated an RNP complex containing DLA2 and the chloroplast psbA mRNA specifically in cells metabolizing acetate. An intrinsic RNA binding activity of DLA2 was confirmed by in vitro RNA binding assays. Results of fluorescence microscopy and subcellular fractionation experiments support a role of DLA2 in acetate-dependent localization of the psbA mRNA to a translation zone within the chloroplast. Reciprocally, the activity of the cpPDC was specifically affected by binding of psbA mRNA. Beyond that, in silico analysis and in vitro RNA binding studies using recombinant proteins support the possibility that RNA binding is an ancient feature of dihydrolipoamide acetyltransferases. Our results suggest a regulatory function of DLA2 in response to growth on reduced carbon energy sources. This raises the intriguing possibility that this regulation functions to coordinate the synthesis of lipids and proteins for the biogenesis of photosynthetic membranes.
Metabolic control of gene expression coordinates the levels of specific gene products to meet cellular demand for their activities. This control can be exerted by metabolites acting as regulatory signals on a class of metabolic enzymes with dual functions as regulators of gene expression. However, little is known about how metabolic signals affect the balance between enzymatic and regulatory roles of these proteins. Here, we report an example of a protein with dual functions in gene expression and carbon metabolism. The chloroplast pyruvate dehydrogenase complex is well-known to produce activated di-carbon precursors for fatty acid, which is required for lipid synthesis. Our results show that a subunit of this enzyme forms ribonucleoprotein particles and influences chloroplast mRNA translation. Conversely, RNA binding affects pyruvate dehydrogenase (metabolic) activity. These findings offer insight into how intracellular metabolic signaling and gene expression are reciprocally regulated during membrane biogenesis. In addition, our results suggest that these dual roles of the protein might exist in evolutionary distant organisms ranging from cyanobacteria to humans.
How do cell-surface receptors transmit signals into cells? This study resolves how signal relay occurs through the HAMP domains of bacterial chemoreceptors by causing them to switch between two conformational states.
HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between “on” and “off” conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses.
A central question in biological signal transduction is how cell-surface receptors transmit signals from the outside world across cell membranes and into the cells themselves. In bacteria and lower eukaryotes such receptors are composed of individual modules responsible for specific functions (e.g., sensing, relay, or output). HAMP domains act as the signal relay modules in many receptors, physically bridging input and output components and transferring signals between them. Through a combination of crystallographic, biophysical, spectroscopic, and functional studies we are able to associate two structurally defined HAMP conformational states with functional “on” and “off” signals in bacterial chemoreceptors, and thereby resolve the mechanism by which HAMPs can relay information. The two states differ in both their structure and dynamics and appear to enforce their properties on downstream output modules. Chemoreceptors allow bacteria to track chemical gradients with exquisite sensitivity and dynamic range; we further show that the response to chemoattractant depends critically on specific HAMP residues close to the membrane. Finally, based on the switching mechanism, we design and generate an inverse signaling HAMP domain that provides a new tool to engineer bacterial responses and may be especially advantageous in remediation efforts for directing bacteria towards chemicals that are normally repellants.
Brain-derived neurotrophic factor signaling is defective in Angelman syndrome and can be rescued by disruption of Arc/PSD95 binding.
Angelman syndrome (AS) is a neurodevelopment disorder characterized by severe cognitive impairment and a high rate of autism. AS is caused by disrupted neuronal expression of the maternally inherited Ube3A ubiquitin protein ligase, required for the proteasomal degradation of proteins implicated in synaptic plasticity, such as the activity-regulated cytoskeletal-associated protein (Arc/Arg3.1). Mice deficient in maternal Ube3A express elevated levels of Arc in response to synaptic activity, which coincides with severely impaired long-term potentiation (LTP) in the hippocampus and deficits in learning behaviors. In this study, we sought to test whether elevated levels of Arc interfere with brain-derived neurotrophic factor (BDNF) TrkB receptor signaling, which is known to be essential for both the induction and maintenance of LTP. We report that TrkB signaling in the AS mouse is defective, and show that reduction of Arc expression to control levels rescues the signaling deficits. Moreover, the association of the postsynaptic density protein PSD-95 with TrkB is critical for intact BDNF signaling, and elevated levels of Arc were found to impede PSD-95/TrkB association. In Ube3A deficient mice, the BDNF-induced recruitment of PSD-95, as well as PLCγ and Grb2-associated binder 1 (Gab1) with TrkB receptors was attenuated, resulting in reduced activation of PLCγ-α-calcium/calmodulin-dependent protein kinase II (CaMKII) and PI3K-Akt, but leaving the extracellular signal-regulated kinase (Erk) pathway intact. A bridged cyclic peptide (CN2097), shown by nuclear magnetic resonance (NMR) studies to uniquely bind the PDZ1 domain of PSD-95 with high affinity, decreased the interaction of Arc with PSD-95 to restore BDNF-induced TrkB/PSD-95 complex formation, signaling, and facilitate the induction of LTP in AS mice. We propose that the failure of TrkB receptor signaling at synapses in AS is directly linked to elevated levels of Arc associated with PSD-95 and PSD-95 PDZ-ligands may represent a promising approach to reverse cognitive dysfunction.
Angelman syndrome (AS) is a debilitating neurological disorder caused by a dysfunctional Ube3A gene. Most children with AS exhibit developmental delay, movement disorders, speech impairment, and often autistic features. The Ube3A enzyme normally regulates the degradation of the synaptic protein Arc, and in its absence the resulting elevated levels of Arc weaken synaptic contacts, making it difficult to generate long-term potentiation (LTP) and to process and store memory. In this study, we show that increased levels of Arc disrupt brain-derived neurotrophic factor (BDNF) signaling through the TrkB receptor (which is important for both the induction and maintenance of LTP). We find that the association of the postsynaptic density protein PSD-95 with TrkB is critical for intact BDNF signaling, and that the high levels of Arc in AS interfere with BDNF-induced recruitment of postsynaptic density protein-95 (PSD-95) and other effectors to TrkB. By disrupting the interaction between Arc and PSD-95 with the novel cyclic peptidomimetic compound CN2097, we were able to restore BDNF signaling and improve the induction of LTP in a mouse model of AS. We propose that the disruption of TrkB receptor signaling at synapses contributes to the cognitive dysfunction that occurs in Angelman syndrome.