An Interview with Manuel Varela and Ann Varela:  Susan Lindquist and Prions, Yeast, and Heat Shock Proteins

Nov 12, 2020 by

Michael F. Shaughnessy

1) Susan Lindquist—When was she born, where was she born, and what about her early formative years? 

Dr. Susan Lee Lindquist McKenzie is famous for her discoveries involving protein folding, molecular chaperones such as heat-shock proteins, prions in yeast microbes, and cancer. On June 5, 1949, Lindquist was born in Chicago, Illinois, into the home of Iver and Eleanor (Maggio) Lindquist, who were first-generation immigrants. Their aspirations for her future involved being a homemaker. Providentially, Lindquist would carve out her future by studying microbiology and biology in some of the country’s finest higher learning institutions.

Lindquist’s parents valued education, yet their daughter’s expectations were low, as was customary of the times. Lindquist herself admits to not having any career goals in her youth. Lindquist’s fifth-grade science teacher was her inspiration and influenced her way of thinking big and questioning style. Lindquist also learned techniques to cope with mild dyslexia and earned a scholarship to college. She earned her bachelor’s degree in microbiology from the University of Illinois at Urbana—Champaign in 1971.

2) Her Ph.D. was from Harvard—who did she study under, and what was her main field of interest?

Lindquist earned her Ph.D. in biology in 1976 from Harvard University. She worked in the laboratory of molecular biologist Matthew Stanley Meselson. There she studied the fruit fly, Drosophila melanogaster, and its heat-shock proteins (Hsps)—proteins synthesized more quickly in larger quantities following cellular exposure to sudden rises in temperatures.

After earning her Ph.D., Lindquist conducted her postdoctoral research in the laboratory of Hewson Swift at the University of Chicago. In 1978, she was employed in the U of C molecular genetics and cell biology department, where she remained until 2001. Lindquist flourished in this environment and became a Howard Hughes Medical Institute Investigator and full professor in 1988 and the Albert D. Lasker Professor of Medical Sciences in 1999.

3) First, prions—what are they—why are they important?

Prions are infectious agents that cause a constellation of slowly progressive degenerative diseases of the brain. Surprisingly, the prion particles consist entirely of protein. As causative agents of ailments, it was thought early on after their discovery by Stanley Prusiner that the prions were viruses. Viruses are known to harbor nucleic acid enclosed by a protein coat. Instead, it was found that these contagious prion agents lacked any form of nucleic acids. Therefore, the prions are named as such because they are proteinaceous infectious entities.

The prion diseases constellation has been renamed transmissible spongiform encephalopathies (TSE). One notorious prion disease is colloquially called “mad cow disease,” known as bovine spongiform encephalopathy (BSE). Due to neurological pathology, the cattle infected by the prions and exhibiting BSE appear to have gone mad. Other potentially susceptible mammals include elk, deer, mice, cats, minks, sheep, and humans.

The sheep version of the prion disease is called “scrapie.” In addition to neurological manifestations, the sheep have an uncontrollable desire to scrape themselves against objects like fences, poles, or trees until they bleed. The prion disease-causing agent is called prion protein, “PrPSC,” in which “SC” denotes scrapie, the pathogenic form of the PrP. The standard non-pathogenic form is called the cellular prion protein. It is designated as “PrPC,” where the “C” stands for cellular. The PrPC form functions to chaperone the folding of a protein into its standard shape. However, a primary function of the PrPSC is to bind the healthy PrPC version and convert it into another PrPSC type of protein. The PrPC harbors mainly alpha-helices. The PrPSC consists primarily of beta-strands. See Figure 36, which depicts a secondary structure format.

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Figure 36. Differences between secondary structures of the normal PrPC protein and scrapie prion PrPSC. (Labels have been translated to English.)

Humans can be susceptible to the prions either by infection, genetic inheritance, or sporadically. The human prion versions of the illness acquired by infection are called Creutzfeldt-Jakob disease (CJD) and variant CJD. Another transmissible human version is called kuru, which emerged in New Guinea. The kuru form of the prion disease is transmitted by a cannibalistic foodborne mode in which natives of New Guinea consume their dead relatives’ brains, who succumbed to the agent. The prions embedded in their victim’s brain tissue is transferred to the cannibals, who then can acquire the kuru. Carlton Gajdusek took the Nobel prize for discovering that kuru was infectious and for developing a technique for diagnosis.

The genetic versions of the human TSE diseases include familial-CJD (f-CJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome, and fatal familial insomnia (FFI). The f-CJD, GSS, and FFI disorders can involve mutations in the PrP molecule that destabilize the protein structures. The destabilized prion molecule spontaneously converts to the pathological PrPSC configuration.

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Figure 37. PrPSC induces a conformational change in PrPC.

While the molecular basis of the disease pathology is far from understood, a general mechanism has emerged. See Figure 37. First, the genetically mutated prion agent produces the PrPSC, or transmission to the patient occurs. The PrPSC then aggregates abnormally into giant molecular complexes, converging on brain cells’ membrane surfaces. The normal PrPC that’s anchored to phosphatidylinositol glycan molecules on cell surfaces are converted to the pathological PrPSC shape. The neuron compensates by making more PrPC. They form long chains consisting of repeating units outside the cell. The repeats create an extracellular component called an anionic glycosaminoglycan. Then, the long chains break from the action of phagocytes and shearing forces, releasing smaller aggregates of PrPSC, which then migrate to other cells to start the process over again.

Sometimes the PrPSC molecules get taken up by brain cells and accumulate, reaching high intracellular concentrations. The buildup is problematic because when phagocytosis attempts to eliminate the accrued PrPSC aggregates, the process fails. Instead of PrPSC eradication, abnormal brain tissue vacuolation occurs. The vacuolation somehow produces the diseased brain’s spongy-like texture.

Lindquist’s involvement with prions concerned her discovery of their presence in yeast microbes. Her findings were important discoveries because yeast cell harboring prions served as a useful laboratory model system for close study of the prions’ effects in living cells. Her studies of prions in yeast organisms showed that normal cellular proteins were associated with host cells’ prion biology. In particular, Lindquist showed how the folding of protein structures of ordinary versus prion proteins had similarities in their biochemical mechanisms.

4) Heat shock proteins—what exactly are these, where are they located, and why are they important?

Heat shock proteins are found in all taxa of living organisms, from bacteria to humans. These specialized proteins are produced at a rapid rate in cells that are exposed to higher-than-normal temperatures. For a cell’s proteins to function correctly, they must be folded into specific three-dimensional shapes; otherwise, misshaped proteins cannot work. The life of the cell will be in peril.

As soon as a protein is made fresh off the translational machine, the new protein molecule arranges its linear primary sequence of amino acids into an adequately shaped 3-D configuration. Some of these 3-D shaped proteins assemble into larger quaternary complexes, forming functional molecules necessary for life. Appropriately folded proteins are an essential requirement for the living cell. Such proteins can function only when a specific molecular structure is assumed.

Presumably, the heat shock proteins function to protect cellular proteins from denaturing. When temperatures are elevated, the heat will cause proteins to unfold or misfold, causing denaturation. A denatured protein will cease to function correctly and could even be destroyed by a cell. The heat shock proteins can protect cellular proteins from denaturation after exposure to heat. Lastly, for proteins that become unfolded or heat-denatured, the heat shock factors will recover the unfolded proteins and restore them to their original shapes. Such re-shaped proteins can reacquire their cellular function, allowing the cell to continue living.

As the ribosome and its translational machinery make new proteins, the newly constructed proteins are initially unfolded. Neighboring molecules and the cell’s internal environment can influence the new proteins’ nascent nature to form misfolded structures. Thus, an improperly folded protein can malfunction or associate with inappropriate molecules to form abnormal complexes, producing deviant cellular behavior that can be detrimental to the cell.

Today, we understand that these heat shock proteins serve a general biological role called molecular chaperoning. Molecular chaperones are proteins that assist the proper folding of newly made proteins into their correct molecular shapes. Molecular chaperones positively influence a cell’s life expectancy by helping new proteins form their appropriate 3-D shapes so that they can function normally.

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Figure 38. The diagram depicts molecular and cellular actions if stress is introduced to the cell.

In Figure 38, the effects of heat stress on a cell are shown. Stress induces the heat shock factor, HSF-1, which can form molecular chaperones. Cellular stresses, such as heat, can cause proteins to misfold as they denature. The molecular chaperones can influence misfolded proteins to fold correctly. However, if the misfolding is excessive, the protein will be completely degraded by a complex proteasome or through a process called autophagy, which is a debris elimination mechanism in cells.

Several of these molecular chaperones, known at the time as heat shock factors, were discovered by Lindquist during the 1980s. She first studied the heat stress behavior in fruit flies, in Drosophila melanogaster, at Harvard. Lindquist measured mRNA levels in flies that were exposed to high temperatures. As a postdoctoral fellow under Hewson Swift at the University of Chicago, Lindquist examined the translation’s efficiency in the new RNA messages in the heat- flies. She found that the protein synthesis patterns were not affected detrimentally but were altered in their translational programs. As an independent investigator and faculty at the University of Chicago, Lindquist studied the regulation patterns of protein synthesis and the intracellular locations of the proteins that emerged after heat exposures in the fruit flies.

She then compared the protein expression patterns observed in fruit flies with those in the yeast microbe called Saccharomyces cerevisiae. Lindquist discovered specific differences in heat stress responses between the fruit fly and yeast. In yeast, she found that particular RNA molecules disappeared from the microbial cells. In contrast, other RNA messages were faithfully translated into proteins. This expression phenomenon was in stark contrast to the protein expression patterns she had observed in the fruit flies. The flies had a control mechanism at play to increase specific protein production while suppressing the expression of other proteins in flies exposed to heat. In due course, Lindquist would go on to make new vital discoveries in the yeast microbes.

5) We are all vaguely familiar with yeast—but what was Susan Lindquist’s specific interest in yeast?

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Figure 39. Saccharomyces cerevisiae — baker’s yeast.

In yeast cells, see Figure 39, Lindquist made other startling discoveries involving heat shock proteins. One of these proteins was called Hsp90. In addition to observing that the rise in Hsp90 occurred after heat stress and helped unstable proteins fold correctly, Lindquist further discovered novel functions. She showed that Hsp90 had roles in signal transducing and developmental biology. The Hsp90 molecule worked on a completely different set of unstable mutated proteins to permit their functions to be carried out. Thus, Lindquist discovered that the Hsp90 allowed the evolution of new traits conferred by the newly stable mutated proteins. The discovery was of great importance because a heat shock protein, a chaperone, made it possible for new evolutionary adaptations to occur in novel environments.

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Figure 40. Molecular protein structure of the ATPase domain in the Hsp90 chaperone, registered with 1uyi code.

Lindquist studied the molecular mechanism of Hsp90. She and colleagues examined the amino acid sequences of the Hsp90 and found homology to a critical enzyme called ATPase, see Figure 40. It was further discovered that all Hsp proteins studied thus far harbor the ATPase domain. These ATPases contain two nucleotide-binding domains, called NBDs. These new findings pointed to ATP’s hydrolysis as a biochemical mechanism for driving the protein folding abilities of molecular chaperones. Interestingly, the ATPase domain structure has been conserved in other proteins, such as histidine kinase and DNA topoisomerase II enzymes.

Lindquist made another new significant discovery involving yeast microbes. This new finding concerned another heat shock protein, a chaperone protein called Hsp104, involved in yeast stress tolerance from heat exposure. This particular discovery would shake the foundations of prion biology, a field for which Lindquist had not previously been a member. In Lindquist’s laboratory, Hsp104 untangled inactive protein aggregates, restoring their functions. While the protein function restoring the ability of a heat shock protein was not a novel result on its own, the kind of protein that was reactivated was novel. That was an incredible achievement. Lindquist discovered that Hsp104 disentangled tangled prions!

The work was met with fierce resistance by many investigators. However, Lindquist would spend the next decades providing concrete experimental evidence for yeast proteins’ prion-like behavior. The discovery had shed light on the prion hypothesis, which implied a protein-based mode of genetic inheritance.

One unclear observation involving yeast was an inherited genetic element called [PSI+] first described by Brian Cox in 1965. The [PSI+] trait was a colorful yeast phenotype that did not obey standard Mendelian genetics. Lindquist showed that the [PSI+] genetic element was a prion-like aggregation of a protein called Sup35. The Sup35 molecule is a sub-unit of a translation-release factor that makes ribosomes stop protein synthesis when reaching nonsense codons on mRNA.

Hsp104 binds to Sup35 as it is freshly made, producing a partially folded transition state, which then aggregates to form the [PSI+] element. Thus, as part of the [PSI+] complex, the Sup35 is effectively sequestered from the translational machinery. Accordingly, the sequestered Sup35 cannot terminate translation. Therefore, the translational machinery reads through the termination codon of mRNA to produce a longer protein than is usual.

However, without [PSI+] complex formation, Sup35 reverts to its normal functional state, terminating translation at the stop codon on mRNA. To precisely regulate the Sup35 incorporation into the [PSI+] element, the Hsp104 concentration must be present in precise amounts—too little or too much would mess up the [PSI+] complex formation.

As the years went by, Lindquist would discover additional prion-like behaving proteins, showing how prion-like aggregations regulated their activities. Lindquist and her laboratory students and scientists would find dozens of prions and molecular chaperones from yeast cells.

6) We must mention that Lindquist served as a mentor to many female scientists. Professor Lindquist went out of her way to provide a warm welcome to up and coming colleagues. Can you name a few?

Dr. Susan Lindquist mentored generations of female students and postdoctoral fellows throughout her scientific career. Lindquist was a tremendously encouraging advocate for female colleagues, many of whom looked up to her as an influential and positive role model. Many of these fledgling scientists went on to become quite prominent independent investigators. For instance, Dr. Bonnie L. Bassler, a noted molecular biologist interested in bacterial quorum sensing at Princeton University, attributed much of her success to Lindquist’s mentorship. Dr. Bassler would become Squibb Professor, an endowed post, and chair of the molecular biology department at Princeton.

Another mentee, Dr. Dianne K. Newman, an endowed Binder and Amgen professor of biology and geobiology at Caltech, was motivated by Lindquist’s example of a stellar scientific investigator. Lindquist deeply inspired Newman. Likewise, Dr. Brit D’Arbeloff recalled that Lindquist’s career advice influenced her and her husband to pursue their investigations at the Whitehead Institute.

Lindquist’s publications were inspiring to young molecular biologists. Neurobiologist Dr. Cori Bargmann remembered reading Lindquist’s creatively brilliant scientific 1998 paper on Hsp90 functioning like a capacitor for directing molecular evolution as one of her favorite articles. The report would influence many to study misfolded proteins and their relationships to human neurodegenerative illnesses.

Many of Lindquist’s students and mentees were quite appreciative of her efforts to train them in the scientific method. Lindquist was awarded the Vanderbilt Prize for Women’s Excellence in Science and Mentorship 2014. It is an utmostly fitting tribute to a fine scientist.

She was honored in other ways. She was bestowed the President’s National Medal of Science in 2009, awarded by President Barack Obama. She was awarded the Dickson Prize in Medicine in 2003, the Otto-Warburg Prize in 2008, the Genetics Society of America Medal in 2008, the FASEB Excellence in Science Award in 2009, the Max Delbrück Medal in 2010, the Mendel Medal in 2010, the E.B. Wilson Medal in 2012, the Vallee Visiting Professorship in 2015, and the Albany Prize in 2016.

Lindquist was also elected as a member of the prestigious National Academy of Sciences, the National Academy of Medicine, the American Philosophical Society, the American Academy of Arts and Sciences, and the British Royal Society.

7) She was the Director of the Whitehead Institute—from 2001 to 2004—What exactly is the Whitehead Institute, see Figure 41, what gets researched there? And what did she investigate there?

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Figure 41. The Whitehead Institute for Biomedical Research (left building) in Cambridge, Massachusetts.

From 2001 until 2004, Lindquist became a biology department professor at the Massachusetts Institute of Technology (MIT). While there, she served as the Director of the MIT-affiliated Whitehead Institute for Biomedical Research until her death. She guided over 100 postdoctoral fellows at the Whitehead Institute, graduate students, and undergraduates to fruitful research careers.

In her laboratory, Lindquist examined the transcriptional response to heat stress and investigated induced proteins: molecular chaperones. She was captivated because, although all eukaryotic cells placed at an extreme temperature perish, those same cells survived if first exposed to an intermediate temperature. Lindquist’s laboratory showed a genetic program that responds to this, and other stresses are activated in all cells. Thus, she embarked on her career studying heat-shock factors whose functions made the difference between life and death after stressor exposures.

Lindquist and her laboratory at Whitehead pioneered new studies devoted to folding newly formed proteins in the cell. She had discovered a variety of heat shock factors, called Hsp, with each one given a number attesting to their molecular weights. For instance, she found that the chaperones, such as Hsp90, enhance and buffer potentially detrimental outcomes from genetic variation. Thus, the heat shock chaperone factors drove evolutionary processes. These evolution-based systems ranged from cellular transformation during malignant cell tumorigenesis to the emergence of microbial resistance factors against antimicrobial agents.

Lindquist’s scientific contributions also definitively established the cellular and molecular bases for protein-based avenues of inheritance, to transfer new traits to subsequent generations of organisms, a controversial hypothesis. Lindquist also implied that the molecular chaperones and the prions each confer unique but potential mechanisms for Lamarckian-like modes of inheritance.

The molecular processes involved in folding proteins can occasionally go awry. The consequence of aberrant folding, called misfolding, has been invoked to explain neurological disorders such as Alzheimer’s disease, cystic fibrosis, Parkinson’s disease, and Huntington’s disease chorea. Protein misfolding can also play roles in illnesses of cancer. Specific proteins that have malformed their molecular structures, such as the prions, actively seek and attack the brain’s neurons to confer transmissible spongiform encephalopathies. Consequently, these abnormalities produce diseases Creutzfeldt-Jakob in humans and scrapie in sheep and mad cow disease in bovines.

8) Although she never received a Nobel, she seemed to enjoy her research, the collegiality and cordial relationship Lindquist had with colleagues—What was she like as a person?

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Figure 42. Susan Lindquist.

A distinctive and characteristic attribute that has been noted about Lindquist was her mentorship. Her mentees reported that Lindquist took great care to teach younger people to write and communicate scientific findings. Her colleagues recognized Lindquist as a talented and gifted scientific communicator. She possessed a rare ability to explain molecular biology unambiguously.

Lindquist, Figure 42, was remembered as a bit absentminded when it came to everyday things. For example, in 2006, there was a meeting about Protein Folding in Vermont. Lindquist had accidentally left her computer at home. Since her colleagues were already on their way to the conference and could not bring her laptop, an alternate plan to deliver the forgotten laptop involved a helicopter. Luckily, the computer arrived just in time, and the conference-goers watched in astonishment as Lindquist retrieved her computer.

The positive influence of Dr. Susan Lindquist was powerful. She was recalled fondly by Dr. Rita Colwell, who became the first female Director of the prestigious National Science Foundation. Dr. Colwell would write that Lindquist will be long remembered for her significant mentorship towards younger female scientists. Her commitment to advancing education in the STEM fields was unparalleled. Further, Colwell stated, Dr. Lindquist would be commemorated for many scientific contributions for the ultimate betterment of humanity.

9) Sadly, she passed at age 67—and again, sadly, from cancer—what had she spent a lot of her time studying? 

Before her death, Lindquist studied cancer. On October 27, 2016, she died in Boston, Massachusetts, from ovarian cancer at 67 years of age.

Intriguingly, her work with the heat shock proteins had a direct relationship to studies of cancer. During tumor cell formation, the protective effects of the heat shock proteins are subverted. Thus, compromising molecular chaperones’ functions can facilitate carcinogenesis by converting benign tumors to malignant ones.

Conversely, molecular chaperones, such as Hsp90, present in tumorous tissues, permit mutated proteins to maintain function. The refolding of mutant proteins can allow cancers to regulate imbalanced signaling that is induced by oncogenic proteins. Investigators are actively pursuing studies on cancer cell function by using Hsp90 inhibitors.

These modulatory agents, as they become available, are applicable in cancer chemotherapy. Thus, improving the Hsp90 chaperone inhibitors’ pharmacological activities is an active field of anti-cancer therapy. New work that combines conventional anti-cancer agents with Hsp90 inhibitors is a promising avenue. Maybe these chaperones can guide cancer cells to become normal cells.

Learn more about Dr. Lindquist’s work in her own words. Some of her lectures and interviews are listed below.

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