The CeHSP17 basal expression level in 4-d-old untreated control worms was set to one. Given the fact that sHSP synthesis can be highly induced by many environmental stimuli such as such as extreme temperature, oxidative stress, heavy metals, and toxins Courgeon et al. We therefore investigated the expression of CeHSP17 in worms treated with heavy metal ions. However, the CeHSP17 protein level showed moderate increase after exposure to 0.
Expression levels of the CeHSP17 protein in worms treated with 0, 0. The expression level of untreated control worms 0 mM was set to one. The CeHSP17 protein level for 0. Zinc exposures also resulted in an increase in CeHSP17 protein expression at all assayed concentrations, with significant increase about twofold more than that found in control worms after exposure to 0. Small heat shock proteins as a molecular chaperone are basically known by the rapid induction of their expression in response to stresses such as heat, metal, reactive oxygen species, etc.
Therefore, they are defined by their ability to bind to denatured proteins arising from such stress, and prevent their irreversible aggregation. This suggests that CeHSP17 is not only highly induced in response to sublethal heat shock treatments, but the increased expression also preconditions the worms against subsequent high temperature challenge that would rather be lethal to the organism development.
Several studies have reported HSP to correlate with acquired stress resistance termed hormesis, which is the ability of a moderate stress to protect the animal from a subsequent and lethal stress Lindquist and Craig, ; Jaattela and Wissing, ; Mailhos et al. Regarding CeHSP17 induction by metals, our data also show that high concentrations of cadmium and zinc, which is clearly cytotoxic in wild-type C. This presumably would reflect the cellular requirement of CeHSP17 protein to confer the natural host with protection against exogenously imposed environmental stress.
It also suggests that the CeHSP17 protein might be a useful biomarker for assessing cadmium and zinc, and should be assayed for other heavy metals. This may be explained based on the fact that cadmium and zinc are closely related metals, both placed in the same group 12 on the periodic table, and based on their size and electron configuration similarities, could bind to identical macromolecular structure via nitrogen, oxygen, and sulfur Brzoska and Moniuszko-Jakoniuk, The responses of CeHSP17 protein expression may be an early biomarker for toxicity monitoring and environmental risk assessment.
However, future studies would determine if other heavy metals induce CeHSP17 protein expression, the specific cell components or processes targeted by CeHSP17 protein during thermal and metal stress, respectively, as are their interacting partners and client proteins necessary for explaining the mechanism involved in such molecular function. National Center for Biotechnology Information , U. Journal List J Nematol v. Ezemaduka , 1 Yunbiao Wang , and Xiujun Li 1.
We would like to thank Prof Chang Zengyi of Peking University for the opportunity to conduct some of the experiments in his laboratory. Received Apr Materials and Methods Nematode strain, maintenance, and propagation: Cadmium and zinc treatment for CeHSP17 protein expression: Immunoblotting analysis and antibodies: Open in a separate window.
CeHSP17 abundance in young worms during recovery from heat shock: Discussion Small heat shock proteins as a molecular chaperone are basically known by the rapid induction of their expression in response to stresses such as heat, metal, reactive oxygen species, etc. Chaperone activity of cytosolic small heat shock proteins from wheat.
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The small heat shock proteins of the nematode Caenorhabditis elegans: Structure, regulation and biology. Progress in Molecular and Subcellular Biology. A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by hsp The Journal of Biological Chemistry. Characterization of alpha-crystallin-plasma membrane binding. Hydrogen peroxide H 2 O 2 induces actin and some heat-shock proteins in drosophila cells. Insights into zinc and cadmium biology in the nematode Caenorhabditis elegans.
Archives of Biochemistry and Biophysics. Ding L, Candido EP. Association of several small heat-shock proteins with reproductive tissues in the nematode Caenorhabditis elegans. The differentially expressed kd heat shock genes of Caenorhabditis elegans exhibit differential changes in chromatin structure during heat shock. DNA and Cell Biology. A small heat shock protein enables Escherichia coli to grow at a lethal temperature of 50 degrees c conceivably by maintaining cell envelope integrity.
Production of age-synchronous mass cultures of Caenorhabditis elegans. Molecular chaperones in the cytosol: From nascent chain to folded protein. Some like it hot: The structure and function of small heat-shock proteins. Nature Structural and Molecular Biology. Investigative Ophthalmology and Visual Science. Lifespan extension of Drosophila melanogaster through hormesis by repeated mild heat stress. Jaattela M, Wissing D.
Emerging role of heat shock proteins in biology and medicine. Small heat shock proteins are molecular chaperones. Heat shock responses for understanding diseases of protein denaturation. Small heat shock proteins, IbpA and IbpB, are involved in resistances to heat and superoxide stresses in Escherichia coli. Laksanalamai P, Robb FT. Small heat shock proteins from extremophiles: Heat shock resistance conferred by expression of the human hsp27 gene in rodent cells.
The Journal of Cell Biology. An emerging model in biomedical and environmental toxicology.
Both the expanded and polydisperse engineered forms of Hsp In contrast, deletion of the N-terminal region significantly reduces substrate affinity, suggesting this buried sequence is likely critical for substrate binding. While all molecular chaperones bind proteins in non-native states, they differ in their mechanism of substrate recognition, as well as in the conformation and fate of the bound substrate Despite a critical role in conferring thermotolerance 18 , sHSP involvement in cellular protein refolding is less clear. One current model postulates that under conditions of extreme stress sHSP provide an energy-independent mechanism to buffer the increase in non-native proteins 71 , Thus, sHSP can be a major contributor to the chaperone capacity of a cell.
Cooperative cellular chaperone networks have been proposed to play a central role in protein folding and in stress response Initial characterization of sHSP chaperone activity relied primarily on observing the suppression of light scattering by aggregating proteins. In brief, substrate proteins are exposed to conditions that promote unfolding, subsequent aggregation and precipitation. Chaperone efficiency is defined empirically as the reduction in light scattering by the substrate in the presence of the chaperone.
These convenient and experimentally simple assays confirmed the broad specificity of sHSP and defined the basic characteristics of their interactions with substrate proteins However, determination of binding parameters and structural analysis of the interaction complex are hindered by the non-equilibrium nature of these aggregation-based assays. The experimental observable, change in light scattering, reflects both the kinetic competition between self-association of the substrate and binding to the chaperone Furthermore, these assays employ strongly denaturing conditions resulting in a conformationally heterogeneous ensemble of bound substrates that is not readily amenable to structural analysis.
These extreme conditions may also compromise the structural and functional integrity of the chaperone. Thus, such assays do not recapitulate the predominant interactions of chaperones within the cellular environment that must precede nucleation of aggregation. These studies took advantage of the thorough analysis of T4L folding and stability that yielded a library of thermodynamically destabilized, site-directed mutants with known crystal structures 75 , Complex formation between substrate and sHSP was initially detected by altered spectroscopic signatures of spin or fluorescence labels 74 , 77 , 78 attached at a unique, solvent-exposed cysteine in the substrate.
A conceptual framework emerged from studies of T4L binding to sHSP that also incorporates previously reported mechanistic elements 54 , 74 , It is grounded in experiments that identify the conformational states recognized by sHSP, define the nature of the chaperone structural changes that are required for or accompany recognition and binding, and determine binding affinity and stoichiometry. The resulting minimalist model is summarized by three coupled equilibria. The substrate folding equilibrium eq. This phenomenological description captures reported experimental characteristics of sHSP chaperone activity, but simplifies the underlying distribution of oligomeric states and their relative affinities to non native proteins.
A more complete description should allow for low affinity substrate binding by inactivated sHSP. Equations 1 and 2 are coupled by the binding of sHSP a to partially I or globally unfolded U states of the substrate eq. Under steady-state conditions, the binding reflects an energetic preference of the non-native substrate states to associate with chaperones versus refolding to the native state N. The model predicts mutations or age-related substrate modifications that shift the equilibrium of eq. Similarly, the coupled equations predict that changes in the equilibrium of eq.
The interpretive and predictive power of this model was tested in series of studies. Experiments confirm that manipulation of equation 1 affects the level of bound substrate equation 3 as predicted. This is a remarkable result considering that these mutants have similar structures in the folded state with no local static unfolding, as demonstrated by x-ray crystallography Figure 4A. Binding occurs in two modes with different affinities and stoichiometries Figure 4C.
Expression of CeHSP17 Protein in Response to Heat Shock and Heavy Metal Ions
Because binding occurs under conditions that strongly favor the folded state, the threshold for stable binding reflects the free energy balance between association with the sHSP and refolding to the native state. Consequently, in a set of mutants of similar native-state structures, sHSP sense the reduction in the stability of the native state.
A Structural superimposition of cysteineless pseudo wild type T4 Lysozyme blue and destabilized mutants L99A green and L46A red demonstrating preservation of tertiary structure. B Chemical denaturant unfolding curves of L99A and L46A as monitored by intrinsic tryptophan fluorescence depicting the relative destabilization of the two substrate mutations. Left shift of L99A curve demonstrates higher affinity binding.
K D of both low and high affinity substrate binding modes are reported adjacent to features of the binding isotherm corresponding to each mode. The reported spectrum of bound conformations appears to be substrate-specific and ranges from unfolded to loosely collapsed or molten globular 82 — Heterogeneity is accentuated in aggregation-based assays where binding is induced by global substrate unfolding and determined by kinetics as discussed above.
The possibility that the substrate intermediate recognized by sHSP differs substantially from the stably bound conformation due to subsequent rearrangements further confounds comparison of these studies. To overcome some of these issues, Claxton et al. They monitored proximities of residue pairs that fingerprint the tertiary fold and secondary structures of T4L Figure 5A.
In contrast, a systematic study monitoring hydrogen deuterium exchange, report limited protection of malate dehydrogenase MDH bound to Hsp This suggests that either the bound conformation is partially folded or specific regions of unfolded MDH are protected in the chaperone complex. The lack of any significant changes in the Hsp Understanding the origin of the differences in bound-substrate structures is critical for refining a mechanistic model of sHSP chaperone activity.
B Model depicting structural aspects of the low and high affinity binding modes. In each case there is extensive substrate unfolding with loss of proximities in labeled pairs tracking both secondary and tertiary structure. Labeled substrates report a net orientation with C termini located in a more conformationally restrictive, solvent inaccessible environment and N termini experiencing greater conformational mobility and solvent accessibility. The hydrodynamic radius of the high affinity binding mode is comparable to chaperone in absence of substrate and this radius increases in conditions favoring low affinity binding.
Long stretches of residues in the N-terminal domain possess flexibility characteristics of unfolded proteins in a sterically unhindered environment. The asymmetric pattern of contact suggests a preferential interaction of the sHSP with hydrophobic residues, considered a universal motif in substrate recognition by chaperones.
Neither region is readily accessible in the native oligomer suggesting that substrate binding requires substantial structural rearrangement. Substrate access can be achieved either by expansion of the oligomer as observed for Hsp The notion of sHSP activation by dissociation was originally invoked to explain the increase in chaperone efficiency at higher temperatures 49 , 53 , 95 — Together, these observations suggest that the oligomer architecture serves as a switch that could be activated by stress signals.
By regulating access to the substrate binding regions, the oligomer, with its complex symmetry and order, controls the sHSP apparent affinity and capacity and protects from aberrant interactions with folded proteins. In the context of this model, equilibrium dissociation allows sHSP to sense the presence of non native proteins and dynamically respond to shifts in cellular folding equilibria.
Modulation of the equilibrium appears to be central for the physiological roles of mammalian sHSP 99 — Hsp27 phosphorylation at three serines leads to complete dissociation into a small multimer — , presumably a dimer. The role of equilibrium dissociation in regulating sHSP affinity was systematically tested by correlation of Hsp27 oligomeric state to T4L binding Perturbation of Hsp27 oligomer equilibrium by phosphorylation mimicking mutations that alter the relative stability of the two major oligomeric states profoundly affects the affinity and level of T4L binding.
The mutations substantially increase T4L binding affinity and activate the high capacity mode Figure 6C. Conversely, mutations of Hsp27 or its phosphorylation mimic that reverse the dissociation, i. This phosphomimic shifts the oligomerization equilibrium in a manner favoring the complete disassembly of higher order oligomers observed in WT Hsp27 to smaller species.
B The equation represents the dissociation of Hsp27 from a large oligomer L to a multimer M where p is an integer that accounts for the difference in the number of subunits between the two oligomeric states. Below, this equilibrium is depicted graphically in colors corresponding to the predominant SEC peaks in A as well as the binding isotherms in C. C Binding is detected as quenching of a fluorescently labeled T4 lysozyme substrate. Phosphorylation induced dissociation of HspD3 drastically increases the affinity of Hsp27 for substrate compared to WT Hsp Combined with panel A , this data reinforces a model of chaperone activation and regulation through phosphorylation induced changes in oligomeric state.
There is evidence that the activation of some sHSP may occur without dissociation Such a mechanism may be particularly relevant for monodisperse and symmetric sHSP where there is no definitive evidence of equilibrium dissociation. Evidence exists suggesting mechanistic decoupling between subunit exchange and the chaperone activity of yeast Hsp26 , In the absence of directly measuring binding affinities, it is impossible to unequivocally evaluate the contribution of native oligomer binding. The extent to which sHSP from lower organisms utilize this proposed mechanism is yet to be fully explored.
This review presents an integrated structural and functional model of sHSP. Its central elements link sHSP affinity to oligomer structural dynamics and the free energy of substrate unfolding. Given the substantial binding capacity of sHSP, dynamic regulation of their affinity is critical for cellular response to stress and aging.
On the other hand, a static high affinity may promote substrate unfolding based on the coupled thermodynamic model Scheme 1. The buffering capacity of sHSP will be titrated out clogging the chaperone network and the degradation machinery.
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Despite remarkable advances in understanding the mechanism of sHSP chaperone activity, much remains to be discovered regarding their physiological and biochemical functions. The eleven human sHSP appear to have important roles in signaling pathways and we are just beginning to frame their contributions to health and disease. Emerging animal models, ex-vivo tools and cell culture systems promise to bridge the gap between the test tube and the organism to understand the contribution of sHSP to physiological and pathological states. The authors thank Derek P. Koteiche for discussion and critical reading of the manuscript.
National Center for Biotechnology Information , U. Author manuscript; available in PMC May Godar , and Phoebe L. The publisher's final edited version of this article is available at Biochemistry. See other articles in PMC that cite the published article. Abstract Small heat shock proteins sHSP are a remarkably diverse group of molecular chaperones possessing a degree of structural plasticity unparalleled in other protein superfamilies.
Open in a separate window. Small heat shock protein architecture A Schematic representation of Hsp Structural framework sHSP assemble into remarkably versatile oligomers with a level of divergence unparalleled in other protein superfamilies. CryoEM structural information for Hsp CryoEM structure and pseudoatomic model of Hsp Mechanism of sHSP chaperone activity While all molecular chaperones bind proteins in non-native states, they differ in their mechanism of substrate recognition, as well as in the conformation and fate of the bound substrate Destabilized model substrate for Hsp binding analysis A Structural superimposition of cysteineless pseudo wild type T4 Lysozyme blue and destabilized mutants L99A green and L46A red demonstrating preservation of tertiary structure.
The structure of bound substrate The reported spectrum of bound conformations appears to be substrate-specific and ranges from unfolded to loosely collapsed or molten globular 82 — Role of oligomeric assembly in recognition and binding: Implications of the model for understanding the role of sHSP in health and disease This review presents an integrated structural and functional model of sHSP.
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