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Chaperone Action in Protein Folding and Neurodegenerative Disease
Summary: Arthur Horwich is interested in understanding how chaperonin ring machines assist protein folding in the cell, and in how protein misfolding causes neurodegenerative disease.
The final step of information transfer from DNA to effector protein involves the proper folding of a newly translated polypeptide into its biologically active, native form. Although all of the information necessary to direct this process is contained in the sequence of amino acid–building blocks that are strung together to make a polypeptide chain, there is a class of specialized proteins known as molecular chaperones that provide assistance to the folding process in the intact cell, in particular preventing polypeptide chains from aggregating with each other during the folding process.
The common feature recognized by molecular chaperones is exposed hydrophobic surface, which is ultimately buried in the interior of the folded, native protein. These exposed surfaces allow nonnative proteins to stick to each other, causing multimolecular aggregation, a state that carries no biological activity and that also can be damaging to the cell, as in a number of neurodegenerative diseases. Chaperones prevent such aggregation from occurring by proffering their own hydrophobic binding sites, capturing nonnative proteins. Following release, a chaperoned protein has a fresh chance to fold properly.
We have been focusing on one type of chaperone, called a chaperonin, a double-ring "machine" that has the remarkable ability to promote folding of an initially captured protein to its native state inside an encapsulated chamber. More recently, we have also been focusing on how a cytosolic protein could misfold, even in a setting where molecular chaperones are present, to cause neurodegenerative disease.
We originally discovered chaperonin-mediated folding in mitochondria when we uncovered an action of the mitochondrial chaperonin, called heat-shock protein 60 (Hsp60), in support of the proper folding of newly imported proteins. Structurally related relatives of Hsp60 are present in the bacterial cytoplasm (GroEL) and chloroplast, and we observed more distant relatives in archaebacteria and the cytosol of higher organisms. We have directed our research toward understanding how these machines work, focusing on the bacterial machine, GroEL. Analogous to Hsp60, GroEL assists the folding of many newly translated proteins in the bacterial cytoplasm.
To dissect the mechanism by which GroEL assists folding, we have used a variety of techniques, including biochemical studies, x-ray crystallography (carried out with the late Paul Sigler), cryoEM studies (with Helen Saibil [Birkbeck College, London]), and NMR studies (with Kurt Wüthrich [Swiss Federal Institute of Technology, ETH, Zürich]). This has given us a working model of GroEL action, involving cooperation of a small cochaperonin "lid," GroES, and consumption of ATP (see Figure 1 for a schematic illustration of this model).
Two Steps of Chaperonin Assistance: Polypeptide Binding by an Open Ring, and Folding in an Encapsulated Chamber
Binding. GroEL is a back-to-back arrangement of two seven-membered rings, with a large central cavity at either end. This is the site of binding nonnative protein. The inside of this cavity is lined, at its apical end, with hydrophobic residues that are essential for binding nonnative proteins. Such binding in many cases involves interaction between the nonnative protein and several surrounding apical domains. It is apparently passive multivalent capture by this surface of GroEL that prevents a nonnative protein from aggregation.
Folding. Farther down the central cavity, at the equatorial waist of the GroEL cylinder, each subunit contains an ATP-binding pocket. Binding and hydrolysis of ATP in these sites drive the cycle of binding and release of polypeptide and GroES (see Figure 1). Our earlier studies showed that the binding of ATP and GroES to the same ring as polypeptide (i.e., in cis) releases the polypeptide into a now GroES-encapsulated cavity where folding commences. The polypeptide folds in solitary confinement—there is no other protein with which it can aggregate. Furthermore, the walls of the chamber are switched to hydrophilic character by rigid body movements of the apical domains that occur upon ATP/GroES binding, providing a "no stick" surface that allows the polypeptide to fold on its own but without the complication of multimolecular aggregation that would occur outside the cavity. Indeed, in a recent study we observed that when folding conditions in free solution were adjusted to prevent a substrate protein from aggregating, the rate of folding to native form was the same both outside and inside.
The Neurodegenerative Disease ALS
We have also begun to investigate a neurodegenerative condition, Lou Gehrig's Disease, also known as ALS (amyotrophic lateral sclerosis), in which progressive loss of function and death of motor neurons occur in association with cytosolic aggregates in these cells. We have focused on one form of this condition associated with inherited mutations affecting the cytosolic enzyme superoxide dismutase (SOD1). While initially it was thought that disease resulted from loss of the free radical–scavenging function of this abundant cytosolic enzyme, subsequent studies have supported a gain of function related to instability and misfolding of the enzyme. (For example, mice deleted of SOD1 do not develop ALS disease, whereas mice transgenic for mutant forms of SOD1 develop ALS, even when there is some residual enzyme activity.) We have further modeled the condition produced by mutant SOD1 by producing both Caenorhabditis elegans worms and mice that are transgenic for an entirely misfolded form of SOD1, called G85R, that produces ALS in humans and transgenic mice. To follow the fate of this protein in vivo, we have fused a yellow fluorescent protein (YFP) marker to its terminus. Both worms and mice that are transgenic for the mutant G85R SOD1-YFP develop locomotor paralysis, compared to normal behavior with wild-type SOD1-YFP counterparts. There is specific development of yellow fluorescent aggregates in neurons of the mutant animals (Figure 2). To our surprise, we observe in the spinal cord of affected mice that the misfolded protein, before it aggregates, is bound to a major molecular chaperone, called Hsc70. Why doesn't this chaperone prevent aggregation and disease? Ongoing genetic analyses in the worms and biochemical studies in the mice are under way to address this question.
Grants from the National Institutes of Health provided support for a portion of these projects.
Last updated March 16, 2009
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