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Protein Folding in the Cell
Summary: Arthur Horwich has been interested in understanding how chaperonin ring machines assist protein folding in the cell and, more recently, in discovering how a misfolded protein causes motor neuron degeneration resembling ALS (Lou Gehrig's 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 in the cellular environment, where the temperature is relatively high and where macromolecules are present at high concentration.
The common feature recognized by molecular chaperones is exposed hydrophobic surface, which is ultimately buried to the interior of a folded, native protein. Exposed hydrophobic 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. Molecular 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 focused for many years on one type of chaperone, called a chaperonin, a double-ring "machine" that has the remarkable ability to promote proper folding of an initially captured protein to its native state inside an encapsulated chamber.
GroEL, the bacterial chaperonin that we studied, assists proper folding through two principal actions. One action involves binding a nonnative protein in an open ring, via a hydrophobic lining of the ring that recognizes exposed hydrophobic surfaces in the substrate protein. Such binding can serve to "mask" hydrophobic surfaces, preventing them from aggregating, and can even reverse misfolding by effectively pulling the protein conformation apart, giving it a fresh chance for correct folding. The second assisting action involves releasing the protein, in the presence of ATP and a small "lid" structure, GroES, into a now GroES-encapsulated chamber where folding can proceed in solitary confinement in a chamber, without the chance of aggregation. The walls of the GroES-bound chamber are electrostatic, and our studies of the past few years suggest that this "no-stick" surface does not interfere with the folding process, allowing folding to occur as if at infinite dilution in a sea of buffer.
Our most recent studies, using electron microscopy in collaboration with Helen Saibil (Birkbeck College, University of London), have revealed the action of ATP in "activating" a ring to bind GroES. A series of movements of the seven subunits of a ring shows a trajectory in which, upon ATP binding, they first tilt over sideways, then elevate their hydrophobic binding surface while separating some of the contacts between the subunits. Through all of this movement, the hydrophobic regions remain pointed into the central cavity, enabling polypeptide to remain bound. But at the end of the trajectory, the downwardly protruding loops of GroES, itself composed of seven subunits, can now directly dock 1:1 with the GroEL subunits via their own hydrophobic surface, and in doing so, prevent the bound polypeptide from escaping the cavity. After GroES docking, a large and forceful elevation and a clockwise twisting motion release the polypeptide into the GroES-domed chamber, where folding proceeds.
The Neurodegenerative Disease ALS
Mutations in the abundant cytosolic free radical–scavenging enzyme superoxide dismutase 1 (SOD1) have been associated with ~2 percent of cases of amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease). Overexpression of such mutant forms of SOD1 in mice likewise leads to motor neuron dysfunction and death, associated with muscle weakness and ultimately paralysis. Studies have shown that, as with other neurodegenerative diseases, disease is not due to a loss of function (because, for example, many patients retain normal enzyme activity) but rather to a gain of function that is toxic.
We have engineered a transgenic mouse strain that overproduces a fusion protein of a completely misfolded mutant version of SOD1, G85R, which is unable to fold properly, with the yellow fluorescent protein (YFP). Unlike a wild-type SOD1-YFP transgenic mouse expressing the same amount of protein, these animals develop paralysis by 5–6 months of age; this paralysis is associated with the presence of YFP-fluorescent aggregates in their motor neuron cell bodies (appearing as early as 1–2 months of age). Such aggregation occurs despite the observed association of a substantial amount of the misfolded protein with the molecular chaperone Hsc70. At early times before clinical disease is apparent, we also observe altered morphology of mitochondria in cell bodies of the ALS animals, and neuromuscular junctions in their lower extremities, for example, are also abnormal. We are seeking to understand how the mutant protein interacts with components of these systems to produce toxic injury, and we are carrying out a variety of genetic experiments aimed at protecting the animals from development of disease.
Grants from the National Institutes of Health provided support for a portion of these projects.
As of May 30, 2012
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