Cell Biology, Neuroscience
Dr. Horwich is also Sterling Professor of Genetics and Pediatrics at Yale School of Medicine.
Protein Folding in the Cell
The final step in information flow from DNA to mature protein involves the proper folding of the newly synthesized polypeptide into its biologically active form. Although the blueprint for a protein’s three-dimensional shape is encoded in its amino acid sequence, a group of proteins called molecular chaperones often provides assistance during the folding process.
Art Horwich has spent much of his career investigating a ring-shaped chaperone called a chaperonin. Using a variety of methods, Horwich and his colleagues have captured snapshots of this protein-folding machine in action. By combining these static images with biochemical tests, the scientists were able to piece together the details of how the cylindrical chaperonin does its job. First, it binds an unfolded protein in a ring via a “greasy” surface, which eventually becomes buried in the folded state. This binding step prevents protein misfolding and aggregation. Second, a co-chaperonin “lid” caps the open end of the ring, encapsulating the unfolded protein, and allows it to fold in solitary confinement, without the possibility of aggregation. After 10 seconds, the lid pops off and the properly folded protein emerges.
Recently, Horwich’s team turned their attention to amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), a disease often caused by mutations in an enzyme called superoxide dismutase 1 (SOD1). Unable to fold correctly, the mutant protein forms toxic aggregates that disable motor neurons that extend down the body’s limbs and operate the muscles. Horwich and his colleagues have created a transgenic mouse model for ALS and are using it to understand how mutant SOD1 causes neuronal damage and malfunction. They’ve observed that fast-firing motor neurons are most susceptible to cell death. In parallel studies, they have overproduced in neurons of the ALS mice a chaperone, heat-shock protein 110 (Hsp110), that has recently been recognized to function as a rate-limiting component in a disaggregase chaperone complex in the mammalian cytosol. Overproduction of Hsp110 in motor neurons extended the median lifespan of the ALS mice by 30%, and the longest-lived mice lived twice as long.
Grants from the National Institutes of Health supported a portion of this work.
In 1987, as a postdoctoral researcher in the lab of Leon Rosenberg at Yale University, Art Horwich was studying an inherited disorder called OTC deficiency, in which a metabolic enzyme fails and toxic levels of ammonia build up in the blood. Babies born with OTC deficiency appear normal for a few days, then lapse into irreversible coma. Horwich, Rosenberg, and co-workers pinpointed the affected gene, making it possible to diagnose the disease with a DNA test, and Horwich went on to study the biogenesis of the OTC enzyme in his own lab at Yale. It was known that a precursor of the enzyme is produced in a cell’s cytoplasm, then imported across two membranes to the innermost space of a cellular organelle called the mitochondrion. During import, proteins were known to squeeze through narrow passageways in the mitochondrial membranes in an unfolded state.
When Horwich set up genetic screens in yeast to figure out how the import process occurs, he stumbled upon a mutant strain in which proteins entered the mitochondria normally, but then misfolded and aggregated. A Nobel Prize had been awarded to biochemist Christian Anfinsen for demonstrating that the blueprint for a protein’s three-dimensional shape is encoded in its amino acid sequence, and people assumed that newly transported mitochondrial proteins spontaneously refolded on the other side of the membranes. But Horwich and his student Ming Cheng had begun to wonder whether some sort of cellular machine might help with the refolding. Their mutant yeast strain supported that unconventional idea.
The yeast mutation affected a gene that encodes a mitochondrial protein dubbed heat-shock protein 60, or Hsp60. Hsp60 is a large, ring-shaped assembly called a chaperonin, which they showed is required for the proper folding of many proteins under normal conditions. Without Hsp60, OTC and other mitochondrial proteins were imported into mitochondria but could not assume functional shapes. More generally, all cells were shown to contain these cylindrical folding machines that assist in the folding of many newly synthesized proteins.
Horwich has studied chaperonin action in protein folding ever since that unexpected finding. The machine assists in two ways. First, the cylinder’s open ring binds an unfolded protein’s “greasy” surface, which eventually becomes buried in the folded state. Such binding to the chaperonin prevents misfolding and aggregation. Second, a lid structure called a co-chaperonin is added to the open ring, sequestering the unfolded protein from other cellular components and allowing folding to proceed undisturbed. Then, the lid pops off and the properly folded protein emerges.
Most of Horwich’s mechanism studies have focused on the bacterial chaperonin called GroEL. His genetic studies proceeded to biochemical studies, then to crystallographic studies, to electron micrograph studies, to structure-function studies, and finally to NMR studies. The result is a dynamic view of the working cycle of this machine.
While Horwich’s work on chaperonins progressed, other researchers showed that many neurodegenerative conditions result from protein misfolding. Recently, Horwich began investigating how a misfolded protein contributes to Lou Gehrig’s disease (amyotrophic lateral sclerosis, ALS), in which loss of motor neurons leads to paralysis. He discovered that the misfolded protein is bound by molecular chaperones, and that it nevertheless forms aggregates inside motor neurons. Horwich and his colleagues are testing whether increasing the supply of chaperones could slow the progression of disease. They have also learned that fast-firing motor neurons, innervating fast-twitch muscle, are most susceptible and die first.