24 January, 2024
The DNA of our cells contains all the instructions we need to develop, survive and reproduce. But the real workhorses of this process are the proteins, and for them to “fulfill their function,” as the citation states “they must adopt specific three-dimensional structures that are achieved in cells with the help of a family of proteins called chaperones.” The four laureates made the twin discoveries that threw open this vibrant field: Hartl and Arthur Horwich identified the first cellular pathway that regulates protein folding, by describing the role played by the Hsp60 chaperone, while Mori and Walter discovered the mechanism cells turn to when protein folding goes wrong, attempting their repair and, failing that, ensuring their timely destruction.
These insights into a biological process so essential to life have enormous biomedical implications, since the molecular machinery that controls both protein folding and the response to failures in this mechanism is at the origin of multiple diseases, from cancer to neurodegenerative disorders like Alzheimer’s, Parkinson’s and amyotrophic lateral sclerosis (ALS) or the aging process itself. Hence the committee, in its citation, hailed the four awardees for their “groundbreaking findings,” which revealed “how cells control protein biogenesis and degradation, central not only to physiology but also disease pathogenesis and therapy.”
“The two discoveries distinguished by the award are fundamental for the health of each cell and therefore of the organism itself. Some diseases, indeed, are due to the accumulation of proteins that have misfolded and then become toxic,” explains Oscar Marín, Professor of Neuroscience and Director of the MRC Centre for Neurodevelopmental Disorders at King’s College London (United Kingdom), and secretary of the award committee. “If proteins do not fold properly, this leads to the impairment and loss of cell function, as occurs, for example, in some degenerative diseases of the nervous system.”
“The findings of the four awardees are important for our understanding of fundamental biology, and also because they lead to new understanding on diseases and how better to cure them in the future,” adds Dario Alessi, committee member and Director of the MRC Protein Phosphorylation and Ubiquitylation Unit at Dundee University (United Kingdom). “There’s huge interest, in the field of neurodegeneration, to maintain proteins properly folded in cells, and also to boost the process of removing unfolded proteins, because that is bad for the cells. And for cancer, it’s thought that if you can inhibit the enzymes that cause folding in some cancers, this could boost the ability to remove the cancer cells that are growing very fast and are very dependent on this process.”
The ‘heretical’ discovery that disproved the conclusions of a Nobel laureate
In 1972, Christian Anfinsen won the Nobel Prize for a series of experiments that found that some small proteins folded spontaneously in a test tube. His work established the idea, later disproved by Hartl and Horwich, that all proteins folded spontaneously, even within the cell.
In the 1980s, Hartl and Horwich were separately studying how proteins get into the cell compartments known as mitochondria, which are enclosed within a membrane. Hartl had found that proteins must be in an unfolded state to pass through this membrane, and his result set Horwich thinking along new, unconventional lines: “Maybe proteins, inside cells at least, don’t spontaneously refold after they go through the mitochondrial membrane.”
As it turned out, he had various mutant yeast strains in his lab. Examining them with his team to see how the proteins entered mitochondria, he thought he spotted a case where the proteins crossed the membrane correctly but, once inside, showed no signs of activity. If this result was confirmed, it would mean that proteins were not folding spontaneously within these cells: there had to be something that was preventing it. “We were terrified by the result,” says Horwich now, “because we thought nobody in the world will ever believe this. It was just heretical to the principles of Anfinsen.”
By chance, a few months later, he received a phone call from Hartl’s lab. Still not apprised of the result of the “heretical” experiment, Hartl too had been interrogating the assumption of spontaneous protein folding in the cell, and wanted to test the process with Horwich’s mutant yeasts. This, for Horwich, was a providential call; the chance to get access to the sophisticated experimental techniques available in Hartl’s lab, which would permit a detailed reconstruction of the process of protein entry into the mitochondria.
He set off immediately from Yale to Munich hoping that Hartl would check his preliminary finding with the mutant yeast. On his return, he mailed Hartl the sample in which he had apparently seen the protein folding fail, and a few weeks later, Hartl came back to him with the good news that his result was confirmed.
This was the start of a productive collaboration between Hartl and Horwich, which led them to discover that the culprit for the proteins’ failure to fold was another protein, Hsp60 (Hsp standing for heat shock protein), which was not present in the mutant yeast. They deduced from this that it was Hsp60 that was ensuring the proteins folded properly, i.e., acted as their chaperone, definitively ousting the previously accepted view that folding happened spontaneously, even inside the cell. Hartl and Horwich published these conclusions in Nature in 1989.
“The results of the first experiments were highly controversial for a couple of years,” Hartl recalls today. “There was a lot of skepticism, in particular from people who had been looking at the folding of smaller proteins that can fold spontaneously in the test tube.” But after successive experiments, he and Horwich finally convinced the scientific community that proteins indeed needed chaperones to fold themselves correctly in the far more adverse environment of the cell.”
“The conceptual paradigm shift introduced by Hartl and Horwich was that proteins were previously thought to be able to fold on their own. But what they described was that nature, through evolution, had required additional machinery – the chaperones – in the protein folding process in order for the the protein to become fully functional,” explains Óscar Millet, Principal Investigator in the Precision Medicine and Metabolism Lab at CIC bioGUNE in Bilbao (Spain).
The chaperone, in effect, acts by encapsulating the protein to isolate it from the environment. “The chaperone provides a vessel, what we now call a nano test tube,” says Hartl, which proteins can only enter one by one. This averts the risk of the unfolded protein aggregating with others and being unable to fold.
“The cell is a ruthless world, a place where there are great masses of proteins continually colliding with each other. The chaperones provide the proteins by various means with a supportive environment so they can fold themselves without unwanted interactions,” adds José María Valpuesta, head of the Department of Macromolecular Structures at the National Centre for Biotechnology in Madrid (Spain).
In an experiment that would prove critical in determining the essential role of chaperones, Hartl and Horwich took single-celled organisms like the bacteria Escherichia coli or yeast itself and knocked out the genes responsible for chaperone production. And what they observed was that these organisms experienced an aggregation of unfolded proteins that caused their death.
An alarm system for breakdowns in the protein folding machinery
When proteins misfold and cannot fulfill their function, cells have a mechanism in reserve to either repair or, failing that, destroy them. This is the unfolded protein response (UPR), a process discovered in simultaneous but separate studies by Kazutoshi Mori and Peter Walter.
When the cell’s capacity to fold proteins is exceeded by the quantity of proteins in the pool or when folding conditions are subject to stress (due, for example, to lack of oxygen or nutrients), proteins cannot fold themselves correctly and acquire toxicity through aggregation. The next step is to either repair or remove them, very much as you would deal with “a rubbish bin that needs emptying,” as Dario Alessi describes it. While this machinery is in motion, the cellular processes that make more proteins are placed on hold until such time as the cell can “reboot” and resume normal function.
Mori and Walter arrived at their conclusions independently, and though they have never signed a joint paper, published their findings in Cell in the same year, 1993. “We were competing to achieve the same goal,” Mori relates, “and that competition helped move the field dramatically forward.”
“Peter Walter and I identified the molecular mechanisms of the UPR, which allowed many researchers to work in this field,” says Mori. Specifically, both men identified an enzyme, named IRE1, that acts as a sensor of unfolded proteins and sends alarm signals to the cell nucleus to correct the misfolding and eliminate misfolded proteins.
Walter looks back on his own contribution. “We started out very simplistic, asking how cells communicate intracellularly, how cells figure out when they need to change their molecular composition. And in this case, it means making more chaperones according to need. But in order for the cell to make that decision, they have to know if protein folding is going to plan. That’s how we discovered the IRE1 sensors, which act like the canary in the coal mine.”
Both used yeast cells to start their research, and it was in them they found the sensors. “They’re sort of like little test tubes in a living system,” says Walter. “Very simple to manipulate for genetics and biochemistry. And later on, it turned out that pretty much all the salient features that we discovered in yeast hold true in pretty much every cell of the human body.”
Biomedical potential against neurodegenerative diseases and cancer
The four laureates are convinced that their findings on the molecular machinery that regulates both protein folding and its failures can drive the development of new, effective treatments against multiple diseases, and may even contribute to understanding and acting on the aging process. “Parkinson’s disease, Alzheimer’s, Huntington’s, maybe amyotrophic lateral sclerosis, all have in common that, at a particular age, patients develop problems with their brain, with their nerve cells, because of the accumulation of misfolded protein aggregates. Generally, the likelihood that this will happen is much greater when you get old,” explains Hartl. The Max Planck Institute investigator believes for this reason that it may be possible to combat these conditions “by interfering with the production of the proteins that undergo aggregation.” In fact, he adds, significant experimental advances have been reported in the use of this therapeutic strategy against ALS and Huntington’s disease.
Horwich too is excited about the biomedical potential of his basic research: “The implications are that there are folding assistants inside cells that bind misfolded proteins, and in so doing, can prevent them from being toxic to the cell or preventing them from lacking any normal function.” From this perspective, the Yale professor believes that the therapeutic use of chaperones capable of recognizing these misfolded proteins “might be one way to resolve neurodegenerative conditions.”
Moreover, as misfolded protein aggregation apparently increases with age, Hartl sees some prospect that their findings could help slow down the aging process: “If you could enhance the cell’s own machinery that prevents the toxic aggregation of proteins, it would probably also be beneficial in terms of aging more generally.”
In fact, researcher Tomás Aragón from the CIMA center in Navarra (Spain), who wrote his doctoral thesis under the supervision of Peter Walter, explains that failures in protein folding mechanisms and the unfolded protein response are associated with human aging and most human neurodegenerative diseases: “The capacity of our body’s cells to develop these protein repair and quality control processes declines over time. Enhancing or depotentiating them may help us address aging and multiple pathologies.”
Mori, for his part, points to the fact that molecules with the ability to mitigate protein folding defects are beginning to be used against both ALS and certain liver disorders. “In the future, we hope to be able to treat various chronic conditions like neurodegenerative and liver diseases.”
Walter, finally, talks about how this therapeutic avenue could advance the fight against cancer. “Cancer cells, “ he explains, “have an intrinsic problem, in that they are genetically unstable. They make many unfolded proteins, proteins that cannot assemble correctly. And the unfolded protein response now provides an inappropriate set of protections to these cells, and that keeps them alive, despite the fact that normally this response would be programmed for them to kill themselves.” So it could be that “inhibiting that response would take away that inappropriate growth advantage and allow us to affect cancer cells very selectively in treating the disease.”
There is already in fact, explains Óscar Millet “a whole line of pharmacological treatment that attempts to emulate the effect of chaperones with what’s known as the drug chaperone or molecular chaperones, which would be chemical molecules that simply bind to the protein in the manner of a chaperone.”
A total of 155 nominations were received in this edition. The awardee researchers were nominated by Sebastián Bernales, CEO of Praxis Biotech (United States); Thomas Boehm, Director of the Max Planck Institute for Immunobiology and Epigenetics (Germany); Rikardo Bueno, Director General of the Basque Research & Technology Alliance (Spain); Andrew Dillin, Howard Hughes Medical Institute Investigator and Professor in the Departments of Molecular and Cell Biology and Immunology and Molecular Medicine and the Helen Wills Neuroscience Institute at the University of California, Berkeley (United States); James E. Rothman, Sterling Professor of Cell Biology and Professor of Chemistry at Yale University (United States); Martin Stratmann, Director of the Max-Planck-Institut für Eisenforschung (Max Planck Institute for Iron Research), Germany; and Alexander Varshavsky, Thomas Hunt Morgan Professor of Biology at the California Institute of Technology (Caltech), United States, and 2011 Frontiers of Knowledge Laureate in Biology and Biomedicine.
Biology and Biomedicine committee and evaluation support panel
The committee in this category was chaired by Angelika Schnieke, Emerita of Excellence in the School of Life Sciences at the Technical University of Munich (Germany). The secretary was Óscar Marín, Professor of Neuroscience and Director of the MRC Centre for Neurodevelopmental Disorders at King’s College London (United Kingdom). Remaining members were Dario Alessi, Director of the MRC Protein Phosphorylation and Ubiquitylation Unit at Dundee University (United Kingdom); Lélia Delamarre, Director and Distinguished Scientist in the Department of Cancer Immunology at Genentech (United States); Robin Lovell-Badge, Principal Group Leader and Head of the Laboratory of Stem Cell Biology and Developmental Genetics at the Francis Crick Institute (United Kingdom); Ursula Ravens, Guest Scientist in the Institute of Experimental Cardiovascular Medicine of the University of Freiburg (Germany); Ali Shilatifard, Robert Francis Furchgott Professor of Biochemistry and Pediatrics at Northwestern University Feinberg School of Medicine (United States); and Bruce Whitelaw, Director of the Roslin Institute and Professor of Animal Biotechnology in the Royal (Dick) School of Veterinary Studies (RDSVS) of the University of Edinburgh (United Kingdom).
The evaluation support panel was coordinated by José M. Mato,General Director of CIC bioGUNE and CIC biomaGUNE, and formed by Edurne Berra, CIC BioGUNE Associate Principal Investigator in the Hypoxia Lab; Arkaitz Carracedo, CIC bioGUNE Principal Investigator in the Cancer Lab; Abelardo Margolles Barros, Deputy Coordinator of the LIFE Global Area and Research Professor at the Dairy Research Institute of Asturias (IPLA, CSIC); Óscar Millet, CIC bioGUNE Principal Investigator in the Precision Medicine and Metabolism Lab; Jordi Pérez-Tur, Coordinator of the LIFE Global Area and Scientific Researcher at the Institute of Biomedicine of Valencia (IBV, CSIC); Liset M. de la Prida, Research Professor at the Cajal Institute (IC, CSIC); James D. Sutherland, CIC BioGUNE Associate Principal Investigator in the Developmental Biology Lab; and Isabel Varela Nieto, Research Professor at the Sols-Morreale Biomedical Research Institute (IIBM, CSIC-UAM).
About the BBVA Foundation Frontiers of Knowledge Awards
The BBVA Foundation centers its activity on the promotion of world-class scientific research and cultural creation, and the recognition of talent.
The BBVA Foundation Frontiers of Knowledge Awards, funded with 400,000 euros in each of their eight categories, recognize and reward contributions of singular impact in physics and chemistry, mathematics, biology and biomedicine, technology, environmental sciences (climate change, ecology and conservation biology), economics, social sciences, the humanities and music, privileging those that significantly enlarge the stock of knowledge in a discipline, open up new fields, or build bridges between disciplinary areas. The goal of the awards, established in 2008, is to celebrate and promote the value of knowledge as a public good without frontiers, the best instrument to take on the great global challenges of our time and expand the worldviews of each individual. Their eight categories address the knowledge map of the 21st century, from basic knowledge to fields devoted to understanding and interrelating the natural environment by way of closely connected domains such as biology and medicine or economics, information technologies, social sciences and the humanities, and the universal art of music.
The BBVA Foundation has been aided in the evaluation of nominees for the Frontiers Award in Climate Change by the Spanish National Research Council (CSIC), the country’s premier public research organization. CSIC appoints evaluation support panels made up of leading experts in the corresponding knowledge area, who are charged with undertaking an initial assessment of the candidates proposed by numerous institutions across the world, and drawing up a reasoned shortlist for the consideration of the award committees. CSIC is also responsible for designating each committee’s chair across the eight prize categories and participates in the selection of remaining members, helping to ensure objectivity in the recognition of innovation and scientific excellence.