Molecular mechanisms of canalization: Hsp90 and beyond

The Hsp90 chaperone machine facilitates the maturation of a diverse set of ‘client’ proteins. Many of these Hsp90 clients are essential nodes in signal transduction pathways and regulatory circuits, accounting for the important role Hsp90 plays in organismal development and responses to the environment. Recent findings suggest a broader impact of the chaperone on phenotype: fully functional Hsp90 canalizes wild-type phenotypes by suppressing underlying genetic and epigenetic variation. This variation can be expressed upon challenging the Hsp90 machinery by environmental stress, genetic or pharmaceutical targeting of Hsp90. The existence of Hsp90-buffered genetic and epigenetic variation together with plausible release mechanisms has wide-ranging implication for phenotype and possibly evolutionary processes. Here, we discuss the role of Hsp90 in canalization and organismal plasticity, and highlight important questions for future experimental inquiry.     

The Hsp90 capacitor, developmental remodeling, and evolution: the robustness of gene networks and the curious evolvability of metamorphosis

Genetic capacitors moderate expression of heritable variation and provide a novel mechanism for rapid evolution. The prototypic genetic capacitor, Hsp90, interfaces stress responses, developmental networks, trait thresholds and expression of wide-ranging morphological changes in Drosophila and other organisms. The Hsp90 capacitor hypothesis, that stress-sensitive storage and release of genetic variation through Hsp90 facilitates adaptive evolution in unpredictable environments, has been challenged by the belief that Hsp90-buffered variation is unconditionally deleterious. Here we review recent results supporting the Hsp90 capacitor hypothesis, highlighting the heritability, selectability, and potential evolvability of Hsp90-buffered traits. Despite a surprising bias toward morphological novelty and typically invariable quantitative traits, Hsp90-buffered changes are remarkably modular, and can be selected to high frequency independent of the expected negative side-effects or obvious correlated changes in other, unselected traits. Recent dissection of cryptic signal transduction variation involved in one Hsp90-buffered trait reveals potentially dozens of normally silent polymorphisms embedded in cell cycle, differentiation and growth control networks. Reduced function of Hsp90 substrates during environmental stress would destabilize robust developmental processes, relieve developmental constraints and plausibly enables genetic network remodeling by abundant cryptic alleles. We speculate that morphological transitions controlled by Hsp90 may fuel the incredible evolutionary lability of metazoan life-cycles.     

A naturally occurring variant of Hsp90 that is associated with decanalization

The heat shock protein Hsp90 has been the focus of many studies since it was suggested that it acts to mediate the buffering of phenotypic variation. Hsp90-mediated buffering may result in the accumulation of cryptic genetic variation that, when released either as a consequence of environmental or genetic stress, increases the evolvability of a population. Recent studies using laboratory-induced mutations of Hsp90 and/or chemical inhibition to disrupt Hsp90 function confirm that Hsp90 can buffer cryptic genetic variation. We have previously identified a naturally occurring variant in the charged linker region of the Hsp90 gene, and now examine whether this variant is associated with altered levels of trait variability. The variant is associated with the release of cryptic genetic variation for canalized morphological (bristle) traits, but not for uncanalized morphological (wing and bristle) traits, and the effect on canalized traits depends on culture temperature. This suggests that natural genetic variation in Hsp90 may mediate the evolution of canalized morphological traits even if it does not influence the expression of variation for uncanalized traits.     

The Hsp90 Capacitor,Developmental Remodeling,and Evolution: The Robustness of Gene Networks and the Curious Evolvability of Metamorphosis

ABSTRACT

Genetic capacitors moderate expression of heritable variation and provide a novel mechanism for rapid evolution. The prototypic genetic capacitor, Hsp90, interfaces stress responses, developmental networks, trait thresholds and expression of wide-ranging morphological changes in Drosophila and other organisms. The Hsp90 capacitor hypothesis, that stress-sensitive storage and release of genetic variation through Hsp90 facilitates adaptive evolution in unpredictable environments, has been challenged by the belief that Hsp90-buffered variation is unconditionally deleterious. Here we review recent results supporting the Hsp90 capacitor hypothesis, highlighting the heritability, selectability, and potential evolvability of Hsp90-buffered traits. Despite a surprising bias toward morphological novelty and typically invariable quantitative traits, Hsp90-buffered changes are remarkably modular, and can be selected to high frequency independent of the expected negative side-effects or obvious correlated changes in other, unselected traits. Recent dissection of cryptic signal transduction variation involved in one Hsp90-buffered trait reveals potentially dozens of normally silent polymorphisms embedded in cell cycle, differentiation and growth control networks. Reduced function of Hsp90 substrates during environmental stress would destabilize robust developmental processes, relieve developmental constraints and plausibly enables genetic network remodeling by abundant cryptic alleles. We speculate that morphological transitions controlled by Hsp90 may fuel the incredible evolutionary lability of metazoan life-cycles.     

Incapacitating the evolutionary capacitor: Hsp90 modulation of disease

The nature-nurture argument surrounding the mechanisms of disease causation cannot be resolved, as the roles of genes and environment are inextricably entwined. Environmental fluctuation is clearly a major modifier of phenotype, as well as a promoter of evolutionary change. Both types of variability can be mediated by the stress response pathway, with the Hsp90 chaperone family as key components. Hsp90 has been hailed as a capacitor for evolutionary change, because partial inhibition of its functions can uncover cryptic mutations, leading to unexpected phenotypes that, although generally deleterious, will under rare new environmental conditions provide improved survival to the carrier of that variant. There is, therefore, a strong environmentally elicited link between the capacity to reveal hidden variation as human disease phenotype and as novel morphological forms for evolutionary selection.     

Hsp90 canalizes developmental perturbation

Stochastic processes are intrinsic phenomena that perturb developmental processes. However, the canalization process restricts the magnitude of perturbation and hence the magnitude of morphological variation during development. Heat-shock protein 90 (Hsp90) chaperones are a class of proteins stabilizing a network of 'client' proteins that are involved in diverse signal transduction pathways affecting development. Here it is reported that a reduction of Hsp90 gene dose creates canalization perturbations that affect many aspects of Arabidopsis development and results in a plethora of morphological alterations. Hence, Hsp90 restricts stochastic phenomena by minimizing perturbations, thereby canalizing development. It is also shown that morphogenesis is determined by three mutually inter-related parameters: genotype, environment, and time. Hsp90 is involved in the interaction of these three parameters which ultimately affect developmental processes. The amount of phenotypic variation upon the reduction of Hsp90 function could be perceived as adaptive and could have an impact on the evolutionary process.     

Hsp90 inhibition and the expression of phenotypic variability in the rainforest species Drosophila birchii

Recently a heat shock protein (Hsp90) has been implicated as controlling the expression of cryptic genetic variation through buffering developmental processes. The release of variability in canalized characters following Hsp90 inhibition has been established in model species including Drosophila melanogaster and Arabidopsis thaliana , but has not yet been examined in species with limited distributions. To test if Hsp90 has a role in releasing phenotypic variation in rainforest Drosophila species, developing larvae from a large (> 1000 individuals) outbred population of Drosophila birchii were treated with the Hsp90 inhibitors geldanamycin and radicicol, and morphological traits, desiccation resistance, and life-history traits were measured. The means of all traits were influenced by inhibition. Although only the phenotypic variances of two canalized bristle traits were affected consistently, variability for two of the continuously varying traits (fecundity and development time) were also affected, albeit inconsistently. There was also no effect of Hsp90 inhibition on the developmental stability of the morphological traits as measured by fluctuating asymmetry. Hsp90 inhibition did not increase phenotypic variability in desiccation resistance, a trait previously shown to represent an evolutionary limit in this species. These results question the extent to which Hsp90 buffers variation for both quantitative and discrete traits, and highlight the need for further empirical studies to determine the involvement of Hsp90 in canalization and developmental stability. Nevertheless the results demonstrated increased variability in canalized traits, consistent with observations in model systems. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 457–465.     

Is Hsp90 a regulator of evolvability?

In a recent paper, Rutherford and Lindquist (1998. Nature 396:336-342) identified mutations in the Hsp90 protein that act to unmask hidden genetic variation with a variety of phenotypic effects. The Hsp90 protein has a number of properties that suggest a role in regulating the expression of genetic variation and therefore in adjusting the evolvability of the organism. In this paper we reflect upon the evolutionary feasibility of such mechanisms and suggest some possible ways of testing the adaptation-for-evolvability hypothesis in more detail. We conclude that Hsp90 holds promise as a molecular model system for the evolution of evolvability. J. Exp. Zool. ( Mol. Dev. Evol. ) 285:116-118, 1999.     

The evolution of fungal drug resistance: modulating the trajectory from genotype to phenotype

The emergence of drug resistance in pathogenic microorganisms provides an excellent example of microbial evolution that has had profound consequences for human health. The widespread use of antimicrobial agents in medicine and agriculture exerts strong selection for the evolution of drug resistance. Selection acts on the phenotypic consequences of resistance mutations, which are influenced by the genetic variation in particular genomes. Recent studies have revealed a mechanism by which the molecular chaperone heat shock protein 90 (Hsp90) can alter the relationship between genotype and phenotype in an environmentally contingent manner, thereby 'sculpting' the course of evolution. Harnessing Hsp90 holds great promise for treating life-threatening infectious diseases.     

Phenotypic diversity,population structure and stress protein-based capacitoring in populations of Xeropicta derbentina,a heat-tolerant land snail species

The shell colour of many pulmonate land snail species is highly diverse. Besides a genetic basis, environmentally triggered epigenetic mechanisms including stress proteins as evolutionary capacitors are thought to influence such phenotypic diversity. In this study, we investigated the relationship of stress protein (Hsp70) levels with temperature stress tolerance, population structure and phenotypic diversity within and among different populations of a xerophilic Mediterranean snail species (Xeropicta derbentina). Hsp70 levels varied considerably among populations, and were significantly associated with shell colour diversity: individuals in populations exhibiting low diversity expressed higher Hsp70 levels both constitutively and under heat stress than those of phenotypically diverse populations. In contrast, population structure (cytochrome c oxidase subunit I gene) did not correlate with phenotypic diversity. However, genetic parameters (both within and among population differences) were able to explain variation in Hsp70 induction at elevated but non-pathologic temperatures. Our observation that (1) population structure had a high explanatory potential for Hsp70 induction and that (2) Hsp70 levels, in turn, correlated with phenotypic diversity while (3) population structure and phenotypic diversity failed to correlate provides empirical evidence for Hsp70 to act as a mediator between genotypic variation and phenotype and thus for chaperone-driven evolutionary capacitance in natural populations.     

Selection Transforms the Landscape of Genetic Variation Interacting with Hsp90

The protein-folding chaperone Hsp90 has been proposed to buffer the phenotypic effects of mutations. The potential for Hsp90 and other putative buffers to increase robustness to mutation has had major impact on disease models, quantitative genetics, and evolutionary theory. But Hsp90 sometimes contradicts expectations for a buffer by potentiating rapid phenotypic changes that would otherwise not occur. Here, we quantify Hsp90’s ability to buffer or potentiate (i.e., diminish or enhance) the effects of genetic variation on single-cell morphological features in budding yeast. We corroborate reports that Hsp90 tends to buffer the effects of standing genetic variation in natural populations. However, we demonstrate that Hsp90 tends to have the opposite effect on genetic variation that has experienced reduced selection pressure. Specifically, Hsp90 tends to enhance, rather than diminish, the effects of spontaneous mutations and recombinations. This result implies that Hsp90 does not make phenotypes more robust to the effects of genetic perturbation. Instead, natural selection preferentially allows buffered alleles to persist and thereby creates the false impression that Hsp90 confers greater robustness.     

Hsp90 modulates CAG repeat instability in human cells

The Hsp90 molecular chaperone has been implicated as a contributor to evolution in several organisms by revealing cryptic variation that can yield dramatic phenotypes when the chaperone is diverted from its normal functions by environmental stress. In addition, as a cancer drug target, Hsp90 inhibition has been documented to sensitize cells to DNA-damaging agents, suggesting a function for Hsp90 in DNA repair. Here we explore the potential role of Hsp90 in modulating the stability of nucleotide repeats, which in a number of species, including humans, exert subtle and quantitative consequences for protein function, morphological and behavioral traits, and disease. We report that impairment of Hsp90 in human cells induces contractions of CAG repeat tracks by tenfold. Inhibition of the recombinase Rad51, a downstream target of Hsp90, induces a comparable increase in repeat instability, suggesting that Hsp90-enabled homologous recombination normally functions to stabilize CAG repeat tracts. By contrast, Hsp90 inhibition does not increase the rate of gene-inactivating point mutations. The capacity of Hsp90 to modulate repeat-tract lengths suggests that the chaperone, in addition to exposing cryptic variation, might facilitate the expression of new phenotypes through induction of novel genetic variation.     

Stress,genomes, and evolution

Evolutionary change, whether in populations of organisms or malignant tumor cells, is contingent on the availability of inherited variation for natural selection to act upon. It is becoming clear that the Hsp90 chaperone, which normally functions to buffer client proteins against the effects of genetic variation, plays a central role in this process. Severe environmental stress can overwhelm the chaperone's buffering capacity, causing previously cryptic genetic variation to be expressed. Recent studies now indicate that in addition to exposing existing variation, Hsp90 can induce novel epigenetic and genetic changes. We discuss key findings that suggest a rich set of pathways by which Hsp90 can mediate the influences of the environment on the genome.     

Cdc37 goes beyond Hsp90 and kinases

Cdc37 is a relatively poorly conserved and yet essential molecular chaperone. It has long been thought to function primarily as an accessory factor for Hsp90, notably directing Hsp90 to kinases as substrates. More recent discoveries challenge this simplistic view. Cdc37 client proteins other than kinases have now been found, and Cdc37 displays a variety of Hsp90-independent activities both in vitro and in vivo. It can function as a molecular chaperone by itself, interact with other Hsp90 cochaperones in the absence of Hsp90, and even support yeast growth and protein folding without its Hsp90-binding domain. Thus, for many substrates, there may be many alternative chaperone pathways involving Cdc37, Hsp90, or both.     

Hsp90 and the quantitative variation of wing shape in Drosophila melanogaster

The molecular chaperone protein Hsp90 has been widely discussed as a candidate gene for developmental buffering. We used the methods of geometric morphometrics to analyze its effects on the variation among individuals and fluctuating asymmetry of wing shape in Drosophila melanogaster. Three different experimental approaches were used to reduce Hsp90 activity. In the first experiment, developing larvae were reared in food containing a specific inhibitor of Hsp90, geldanamycin, but neither individual variation nor fluctuating asymmetry was altered. Two further experiments generated lines of genetically identical flies carrying mutations of Hsp83, the gene encoding the Hsp90 protein, in heterozygous condition in nine different genetic backgrounds. The first of these, introducing entire chromosomes carrying either of two Hsp83 mutations, did not increase shape variation or asymmetry over a wild-type control in any of the nine genetic backgrounds. In contrast, the third experiment, in which one of these Hsp83 alleles was introgressed into the wild-type background that served as the control, induced an increase in both individual variation and fluctuating asymmetry within each of the nine genetic backgrounds. No effect of Hsp90 on the difference among lines was detected, pro,iding no evidence for cryptic genetic variation of wing shape. Overall, these results suggest that Hsp90 contributes to, but is not controlling, the buffering of phenotypic variation in wing shape.     

Constantly updated knowledge of Hsp90

Although protein folding, in principle is a spontaneous process which depends only upon the amino-acid sequence, the assistance of molecular chaperones is required for many proteins to achieve their final conformation in vivo. While Hsp90 is one of the major molecular chaperones, it has long been the most mysterious among them. Recent advances in our knowledge regarding Hsp90 structure and function, owing to both detailed biochemical and genetic characterizations of Hsp90 co-chaperones, as well as eminent structural studies have established Hsp90 as an ATPase-dependent chaperone, and have provided a paradigm of the Hsp90 chaperone cycle, which is sequentially tuned and coordinated by a variety of co-chaperones. Here we summarize the current knowledge regarding the structure and essential activities of Hsp90, which certainly promises a deeper understanding of the functions of Hsp90 in vivo.     

Probing molecular mechanisms of the Hsp90 chaperone: biophysical modeling identifies key regulators of functional dynamics

Deciphering functional mechanisms of the Hsp90 chaperone machinery is an important objective in cancer biology aiming to facilitate discovery of targeted anti-cancer therapies. Despite significant advances in understanding structure and function of molecular chaperones, organizing molecular principles that control the relationship between conformational diversity and functional mechanisms of the Hsp90 activity lack a sufficient quantitative characterization. We combined molecular dynamics simulations, principal component analysis, the energy landscape model and structure-functional analysis of Hsp90 regulatory interactions to systematically investigate functional dynamics of the molecular chaperone. This approach has identified a network of conserved regions common to the Hsp90 chaperones that could play a universal role in coordinating functional dynamics, principal collective motions and allosteric signaling of Hsp90. We have found that these functional motifs may be utilized by the molecular chaperone machinery to act collectively as central regulators of Hsp90 dynamics and activity, including the inter-domain communications, control of ATP hydrolysis, and protein client binding. These findings have provided support to a long-standing assertion that allosteric regulation and catalysis may have emerged via common evolutionary routes. The interaction networks regulating functional motions of Hsp90 may be determined by the inherent structural architecture of the molecular chaperone. At the same time, the thermodynamics-based "conformational selection" of functional states is likely to be activated based on the nature of the binding partner. This mechanistic model of Hsp90 dynamics and function is consistent with the notion that allosteric networks orchestrating cooperative protein motions can be formed by evolutionary conserved and sparsely connected residue clusters. Hence, allosteric signaling through a small network of distantly connected residue clusters may be a rather general functional requirement encoded across molecular chaperones. The obtained insights may be useful in guiding discovery of allosteric Hsp90 inhibitors targeting protein interfaces with co-chaperones and protein binding clients.     

NATURAL VARIATION IN THE EXPRESSION OF THE HEAT-SHOCK PROTEIN HSP70 IN A POPULATION OF DROSOPHILA MELANOGASTER AND ITS CORRELATION WITH TOLERANCE OF ECOLOGICALLY RELEVANT THERMAL STRESS

Although Hsp70, the principal inducible heat-shock protein of Drosophila melanogaster, has received intense scrutiny in laboratory strains, its variation within natural populations and the consequences of such variation for thermotolerance are unknown. We have characterized variation in first-instar larvae of 20 isofemale lines isolated from a single natural population of D. melanogaster, in which larvae are prone to thermal stress in nature. Hsp70 expression varied more than twofold among lines after induction by exposure to 36°C for one hour, with an estimated proportion of the variation due to genetic differences of 0.24 ± 0.08. Thermotolerance with and without a Hsp70-inducing pretreatment, survival at 25°C, and developmental time also varied significantly. As expected, expression of Hsp70 correlated positively with larval thermotolerance. By contrast, lines in which larval survival was high in the absence of heat stress showed lower than average Hsp70 expression and lower than average inducible thermotolerance. This conditional performance suggests an evolutionary trade-off between thermotolerance and the ability to produce higher concentrations of Hsp70, and survival in a benign environment.