Albert Lasker
Basic Medical Research Award

Award Presentation by Titia de Lange

The nineteenth century philosopher Schoppenhauer said: "Talent hits a target no one else can hit; Genius hits a target no one else can see."

The target hit by the two basic scientists we honor today concerns the question of how proteins achieve their active state in the cell. At the time of their discoveries, Ulrich Hartl and Arthur Horwich were the only ones to see that this problem was unresolved. The rest of us were blinded by the false tenet that this issue had long been settled.

The misconception about how proteins fold into their native structure originated from key observations made some thirty years earlier, by Christian Anfinsen at NIH. Anfinsen, who shared the 1972 Nobel Prize in Chemistry for his discovery, had taken a purified active enzyme and denatured it so that its long chain of amino acids would unfold. He then diluted out the denaturant and discovered that the protein could spontaneously regain its enzymatic activity. The conclusion was clear: the amino acid sequence of the enzyme's polypeptide chain is sufficient to specify its enzymatic activity and since the enzymatic activity depends on the three dimensional structure of the protein, it followed that a chain of amino acids can fold correctly without addition of other proteins or ATP, nature's energy currency.

Although Anfinsen was right in principle, in the practical world of the living cell, things are different. A newly made protein emerges like toothpaste out of the exit channel of the ribosome — the protein factory where the amino acids are stitched together — and then faces a major problem: some parts of its amino acid chain are greasy and tacky, whereas others are watery and slick. The sticky parts need to gum together at the inner core of the protein, whereas the watery bits are meant to face out.

There are many ways in which the sticky patches can be joined, but only one will generate the right overall structure needed for the protein to fulfill its particular purpose in the cell: to catalyze a chemical reaction, or break down what needs to be destroyed, or help the cell perceive signals from the outside.

Each of the thousands of different proteins at work in our cells has to solve the problem of finding its proper structure — a challenge made worse by the high density of proteins in the cell. The intracellular milieu is like a Tokyo subway car at peak hour, so the emerging protein also faces the risk of gumming on to other proteins in this crowd of diligent protein workers, ruining its own chance of fulfilling its promise and potentially wrecking others.

How does the cell make sure each protein finds its destined fold so that it can go about its business without bothering others? How does nature prevent the packed Tokyo subway car from turning into a mosh pit? Hartl and Horwich were the first to discover this existential problem faced by newly-made proteins and described the remarkable way in which it is solved.

These two mustached, wire-rim glassed, quiet researchers followed remarkably parallel paths. Both started out training as physicians. Hartl was studying medicine at the University of Heidelberg — an isolated, bucolic center of learning near the Black Forest in Germany — while Horwich's medical training started at equally idyllic and provincial Brown University. But while Horwich moved to Yale's School of Medicine and remains active as a pediatrician to this day, Hartl decided early on that it was better for him (and his patients), if he focused on bench work, setting up his own research program first at Memorial Sloan Kettering Cancer Center in New York and then at the Max Planck Institute near Munich. Both were initiated in the budding disciplines of biochemistry and cell biology during postdoctoral studies in Southern California and both were drawn to studying mitochondria — membrane-enveloped compartments where proteins work to generate energy for the cell.

For the narrative culminating into today's luncheon, the key issue about mitochondria is that its constituent proteins are made in the cytosol and then need to pass through the mitochondrial membrane to find their workstations. After they snake through the membrane as an extended chain, just like a new protein emerging from the ribosome, they have to fold to become active.

Horwich, driven by an interest in a life-threatening genetic disorder that causes ammonia built-up in the blood of newborns, studied the healthy version of the culprit gene — the mitochondrial enzyme ornithine transcarbamylase or OTC. He wanted to understand how OTC gets into the mitochondria and quickly discovered that he could use baker's yeast as a genetic tool to get information on the human enzyme. In a genetic screen, he found a strange yeast mutant that appeared to import OTC into the mitochondria, yet the enzyme failed to become active.

What was wrong? Perhaps the OTC did not really get into the mitochondria and was just hanging on to the outside? This is where Hartl came in. Hartl had become an expert in examining mitochondrial import and took a good look at the OTC enzyme in Horwich's mutant yeast. He had no doubt: this OTC was definitely inside the mitochondria, yet, it was not active.

The implication was OTC was unable to fold correctly in the mutant yeast strain. But according to the Anfinsen doctrine, folding was a spontaneous process so how could a yeast mutant fail at a process that was not supposed to require any assistance?

When Horwich identified the gene at fault in the mutant yeast, it turned out to be Hsp60, or heat shock protein 60: a highly conserved protein that was known to increase in abundance at high temperature. Hsp60 clearly assisted OTC in its attempt to become active. The era of the Anfinsen doctrine had ended. A new era had begun in which it was now understood that protein folding involved the help of other proteins, generally referred to as chaperones. But how did the Hsp60 chaperonin work?

Over the next decade, the work of Hartl and Horwich revealed the remarkable trick nature uses to ensure that their proteins can find their active state.

Remarkably, they found that the Hsp60 chaperonin system resembles an isolation chamber where a protein can be put in solitary confinement, somewhat like the American penal system but without the inhumane aspects and much more effective. Hsp60 forms a barrel that grabs the sticky patches of the unfolded protein, moves it into the barrel, and closes a lid on top of it. In isolation, the protein can now try out alternate conformations, giving it a chance to fold into its correct structure, without bumping into other proteins. The wall of the chamber is highly charged, acting as Teflon. The protein is kept in the chamber for about 10 seconds during which one imagines that it struggles and contorts, bouncing of the walls until the lid flips open. The escaping inmate may or may not have folded into a virtuous and productive denizen of the cell. Repeat offenders are quickly recaptured and put through another stint in the isolation cell. This may go on for 10 rounds but eventually, with high probability, a polypeptide will find its proper fold, probation is lifted, and the protein goes to work, behaving as a model citizen and making no inappropriate contact with other proteins. The mosh pit is avoided.

The concept that the cell contains proteinaceous chambers for specific tasks has been extended to another parts of the cellular penal system: execution of the death sentence, a verdict often imposed by cell's regulatory mechanisms, which also takes place in a barrel-shaped protein compartment, the proteasome.

The basic principles uncovered by Hartl and Horwich have found wide application throughout biology and medicine. Virtually all proteins require some form of a chaperone system during their maturation and all organisms employ the solitary confinement trick to whip a subset of their proteins into shape. Importantly, the work on chaperone-assisted protein folding has brought us insights into disorders such as Alzheimer's, Parkinson's, Lou Gehrig's disease in which clumps of entangled mis-folded proteins cause neurological symptoms.

Hartl and Horwich opened the door to these insights by letting us look into the cellular folding chamber and we thank them today for this monumental achievement.