Hartl, Franz Ulrich

Franz-Ulrich Hartl

Max Planck Institute of Biochemistry

Horwich, Arthur

Arthur L. Horwich

Yale University School of Medicine

For discoveries concerning the cell’s protein-folding machinery, exemplified by cage-like structures that convert newly made proteins into their biologically active forms.

The 2011 Albert Lasker Basic Medical Research Award honors two scientists for their discoveries concerning the cell’s protein-folding machinery, exemplified by cage-like structures that convert newly made proteins into their biologically active forms. With this work, Franz-Ulrich Hartl (Max Planck Institute of Biochemistry, Martinsried) and Arthur L. Horwich (Yale University School of Medicine) toppled traditional notions of how proteins fold inside cells and established new principles that operate from microbes to humans. This previously unexplored realm holds enormous importance for basic biology and biomedicine.

Protein folding is a vital process, as it converts linear amino acid chains into the three-dimensional forms that bestow the molecules’ unique activities. Greasy regions of new proteins, however, can grab one another and create useless globs. As proteins take shape, they bury these hydrophobic parts and expose hydrophilic, or water-loving, areas. Horwich and Hartl discovered that a special apparatus encases an unfolded protein and spurs folding by harnessing the energy of ATP, the small molecule that drives reactions inside cells.

In the late 1950s and early 1960s, Christian Anfinsen (National Institutes of Health) showed that the amino acid sequence of a protein supplies the information it needs to assume its final form. He added chemicals that unfold — or denature — a small protein and then removed these agents. The protein regained enzymatic activity without assistance, thus establishing that a protein can do its own origami. The impact of Anfinsen’s discovery was huge. Scientists assumed that newly synthesized proteins in cells fold unaided and without energy input, as they can in the test tube.

However, larger, more complicated proteins than those studied by Anfinsen aren’t as self-sufficient and obliging. Furthermore, inside cells, protein concentrations are orders of magnitude higher than those that Anfinsen used, and as concentration rises, so does the risk of aggregation. Finally, new proteins in living creatures face a challenge that denatured, full-length proteins in a test tube circumvent: Because they gain a single amino acid at a time, portions of the growing chain can potentially stick to one another before the entire molecule is available to fold properly.

Unanticipated molecular caretaker

In the late 1980s, Hartl and Horwich were studying how proteins that are made in the cytoplasm enter mitochondria. Gottfried Schatz (University of Basel) and Walter Neupert (University of Munich) had shown that proteins are imported into mitochondria in a stretched-out state. Once inside, the amino acid sequences that targeted them to the mitochondria are removed, the proteins refold and, in many cases, assemble into multi-part structures before gaining enzymatic activity.

Horwich sought cellular machinery that participates in the mitochondrial-import process. He isolated yeast strains that fail to perform this essential task under certain conditions and then teamed up with Hartl (then at the University of Munich) to analyze the cells’ misbehavior. One of the mutants could transport proteins into mitochondria and clip them to the correct size — but the proteins lacked function, the team reported in 1989. This result suggested that the deviant yeast strain carries a damaged version of a component that normally facilitates protein folding or subsequent events, such as multi-molecular assembly, necessary for protein activity. The evidence strongly favored a block in folding, but assembly requires that a protein has achieved its proper form, so the experiments could not absolutely distinguish these possibilities.

The idea that some proteins need help to assemble had emerged from work on bacteria and chloroplasts. In 1972, Costa Georgopoulos (then at Stanford University) discovered that viral proteins fail to congregate into normal “head” structures within a strain of Escherichia coli that harbors particular genetic flaws. He subsequently tracked the defects to neighboring genes, groEL and groES. In 1980, R. John Ellis (University of Warwick) identified a chloroplast factor that physically interacts with newly made subunits of Rubisco, a key metabolic enzyme. He proposed that this Rubisco-binding protein promotes assembly of the enzyme. In 1988, these observations coalesced when Ellis and Georgopoulos sequenced the GroEL and Rubisco-binding protein genes and established their strong similarity. The researchers dubbed GroEL and the Rubisco-binding protein chaperonins, thus defining a subset of the chaperone family, whose members were thought to encourage macromolecular assembly by helping components avoid improper liaisons with themselves and others.

Genetic analysis revealed that Horwich and Hartl’s yeast mitochondrial protein was identical to the previously discovered heat shock protein 60 (Hsp60), whose production increases in response to heat — and that it resembles the chaperonins. Soon afterward, the researchers performed their dogma-defying experiment by testing whether folding per se — rather than assembly — depends on Hsp60. A protein that operates on its own relied on an Hsp60-based molecular machine to fold inside mitochondria, and ATP powered this reaction. Hartl and Horwich had thus demonstrated the existence of an ATP-driven ‘folding catalyst’ and revealed an unimagined piece of nature: Proteins imported into the mitochondria cannot refold spontaneously.

The mechanism takes shape

In the next advance, George Lorimer (Du Pont de Nemours & Co.) established that chaperonin activity could be studied using isolated components. He restored the enzymatic activity of denatured Rubisco in a test tube by adding to it purified GroEL and GroES in the presence of ATP. Horwich and Hartl set up a similar system to peer into the folding reaction. They showed, for instance, that GroEL (Hsp60’s bacterial counterpart) binds proteins in their relatively unstructured forms — and that addition of GroES and ATP provokes folding.

A tremendous amount of subsequent work, anchored by Hartl’s and Horwich’s ongoing independent investigations, provided key details about chaperonin action. For instance, the GroEL cavity, capped by the GroES lid, provides a cage where amino acid chains can fold, much as denatured proteins did in Anfinsen’s experiments, sequestered from unproductive interactions with other unfolded proteins. Furthermore, GroES cycles on and off GroEL — and GroEL sometimes ejects a protein that has not yet reached its active state. In that situation, the substrate protein rebinds GroEL for another try. These reports and others began to illuminate the sequence of events by which ATP orchestrates a cyclical reaction (see diagram) in which GroES and protein associate with alternating GroEL rings to enact folding.

Illustration of a protein-folding machine

As these biochemical studies proceeded, Horwich and the late Paul Sigler, a Yale colleague, generated pictures at atomic resolution to elucidate details of the chaperonin reaction mechanism. They first discerned the structure of GroEL on its own, using X-ray crystallography.

Previous electron microscopic analysis had shown that the protein looks like two 7-fold symmetric donuts atop each other, and Horwich and Sigler found that oily amino acids point toward GroEL’s cavity at the donut’s opening. This observation suggested how GroEL selectively grabs unfolded proteins: through their hydrophobic portions. The researchers tested this prediction and others by probing whether specific amino acid alterations perturb key aspects of GroEL’s function. Changing hydrophobic elements to hydrophilic ones at particular spots inside the donut’s opening disrupted protein binding — and the amino acids that grasp protein also grip GroES, suggesting that protein and GroES compete for the same sites on GroEL. These results confirmed the team’s idea about how non-native proteins affix to the central channel and suggested how GroES attachment forces protein release into the cavity.

Three years later, Horwich and Sigler captured an X-ray image of the step after GroES caps the GroEL hole. The scientists then compared their snapshot of the combined molecules to pictures of the separate components. Upon binding to GroES and ADP, part of the GroEL donut enlarges and twists dramatically, thus ripping the unfolded protein from its connections inside GroEL and freeing it within the cavity. The contortion radically changes the internal environment — from greasy to watery — thus encouraging the unfolded protein to expose its hydrophilic regions and bury its hydrophobic ones. Additional analysis revealed how ATP binding and hydrolysis drives the folding reaction.

As the GroEL/Hsp60 story developed, a puzzle emerged. Hugh Pelham (Medical Research Council, Cambridge) had proposed that a different heat shock protein, Hsp70, also binds to hydrophobic surfaces to limit inappropriate associations among partially denatured proteins. Hartl wondered why cells contain two systems that serve similar purposes.

In 1992, Hartl established the concept of a relay in which each chaperone plays a distinct role. He demonstrated that the E. coli version of Hsp70 prevents premature folding of the growing amino acid chain; it then transfers the complete protein to GroEL, which promotes folding. He and Horwich had previously discovered that cage-like chaperonins distinct from those in mitochondria, chloroplasts, and bacteria also exist in the eukaryotic cytoplasm, and Hartl showed that similar sequential pathways operate there as well.

Certain medical conditions underscore the significance of these findings. When proteins aggregate, illnesses such as Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis can arise, and adjusting chaperone activity might provide therapeutic benefit. In addition, a particular Hsp60 mutation has been associated with hereditary spastic paraplegia, an illness in which the legs weaken and stiffen.

Hartl and Horwich unveiled a hitherto unknown process that enables proteins to reach their biological potential. Across the tree of life, the folding machines isolate young proteins and create a transformative moment. Then the devices send forth the mature molecules to join the hustle and bustle that makes cells what they are.

by Evelyn Strauss

Key publications of Franz-Ulrich Hartl

Cheng, M.Y., Hartl, F.U., Martin, J., Pollock, R.A., Kalousek, F., Neupert, W., Hallberg, E.M., Hallberg, R.L. and Horwich, A.L. (1989). Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature. 337, 620-625.

Ostermann, J., Horwich, A.L., Neupert, W., and Hartl, F.U. (1989). Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature. 341, 125-130.

Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer, M.K., and Hartl, F.U. (1992). Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature. 356, 683-689.

Langer, T., Pfeifer, G., Martin, J., Baumeister, W., and Hartl, F.U. (1992). Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity. EMBO J. 11, 4757-4765.

Frydman, J., Nimmesgern, E., Ohtsuka, K., and Hartl, F.U. (1994). Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones. Nature. 370, 111-117.

Hartl, F.U., Bracher, A., and Hayer-Hartl, M. (2011). Molecular chaperones in protein folding and proteostasis. Nature. 475, 324-332.

Key publications of Arthur Horwich

Cheng, M.Y., Hartl, F.U., Martin, J., Pollock, R.A., Kalousek, F., Neupert, W., Hallberg, E.M., Hallberg, R.L., and Horwich, A.L. (1989). Mitochondrial heat-shock protein hsp60 is essential for assembly of proteins imported into yeast mitochondria. Nature. 337, 620-625.

Ostermann, J., Horwich, A.L., Neupert, W., and Hartl, F.U. (1989). Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature. 341, 125-130.

Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D.C., Joachimiak, A., Horwich, A.L., and Sigler, P.B. (1994). The crystal structure of the bacterial chaperonin GroEL at 2.8 A. Nature. 371, 578-86.

Fenton, W.A., Kashi, Y., Furtak, K., and Horwich, A.L. (1994). Residues in chaperonin GroEL required for polypeptide binding and release. Nature. 371, 614-619.

Rye, H.S., Burston, S.G., Fenton, W.A., Beechem, J.M., Xu, Z., Sigler, P.B., and Horwich, A.L. (1997). Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL. Nature. 388, 792-798.

Horwich, A.L. and Fenton, W.A. (2009). Chaperonin-mediated protein folding: using a central cavity to kinetically assist polypeptide chain folding. Q. Rev. Biophys. 42, 83-116.

Award presentation by Titia de Lange

The 19th-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 30 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, and 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.

Acceptance remarks

Interview with Franz-Ulrich Hartl and Arthur L. Horwich

Video Credit: Susan Hadary