What does a molecular thermometer look like? This seemed to be a simple question, not much different from the many science fair projects I had done in grade school and high school in Chicago. But rather than the simple solutions I’d present on triptych posterboards, the answer to this question has kept me fascinated for my entire career. The cell’s thermometer appears to be a network of stress-sensing transcription factors and specialized proteins—molecular chaperones—that function as the guardian of the proteome, sensing damage and keeping the cell’s proteins properly folded as they roll off the production line of ribosomes. Exciting as that was, none of us working on this question at the time could have predicted that this thermometer might also control the body’s fountain of youth and provide new ways of thinking about disease.

Proteins are fundamental building blocks for the cell; they are the predominant products of the genome that provide much of the shape and functionality of cells, tissues, and organisms. The proper synthesis, folding, assembly, translocation, and clearance of proteins is essential for the health of the cell and the organism. Proteins also provide the essential parts to replenish molecular machines for biosynthetic processes and ensure their efficient functioning in the adult cell, a process critical for longevity. At the root of the problem is a fundamental process: protein folding. When quality control—as overseen by heat shock proteins and molecular chaperones—slips, errors occur and persist. This interferes with molecular processes, which can lead to disease. When these events occur in neurons, the consequences can be devastating, leading to major classes of neurological disorders, like multiple sclerosis, Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease.

I first became intrigued by heat shock proteins while listening to a seminar by Matt Meselson, who was visiting the University of Chicago to receive an honorary doctorate in the late 1970s. At the time, I had just begun my second year as a graduate student in Murray Rabinowitz’s laboratory in the Department of Biochemistry. While the seminar piqued my interest in this newly discovered heat shock response, it was through my subsequent conversations with Susan Lindquist—who had completed her research with Meselson and had just recently arrived on campus as a postdoctoral fellow—that I really began to appreciate the complexities of this biological response to stress.

In between my efforts to complete my thesis research on the yeast mitochondrial genome and compete in intramural sports with our team, the Kimwipes, I made time to learn more from Lindquist about the heat shock response. Discovered in 1962 by Ferruccio Ritossa, the heat shock response was described as the temperature-induced change in the organization of the tightly packed Drosophila salivary gland chromosome, leading to the appearance of puffs.

Ritossa’s “heat shock” puffs, which by light microscopy looked like cotton balls compressed between sections of tightly packed chromosomes, were also induced by exposure to dinitrophenol, ethanol, and salicylate. However, it was the demonstration that these puffs were new sites of transcription that was most impressive—Ritossa could detect newly synthesized RNA within minutes of puffing. The significance of this was soon revealed by Lindquist and Steven Henikoff in the Meselson lab and Allan Spradling with Mary-Lou Pardue at MIT. These heat shock–induced messenger RNAs encoded a set of proteins, now widely known as the heat shock proteins. These are widely studied as Hsp90, Hsp70, and a family of proteins that guide conformation and folding, and prevent misfolding in cells of all species.

Using a clever trick of cellular biochemistry to enrich for heat shock messenger RNA, the laboratories of David Hogness at Stanford, Walter Gehring at University of Basel, Brian McCarthy at UCSF, Alfred Tissieres at the University of Geneva, and Meselson all cloned the first heat shock genes from Drosophila in the late 1970s and early 1980s.

By this time, I had received my PhD from Chicago and was a postdoc with Matt Meselson in the Harvard biolabs. During this period, I became intrigued with the possibility that heat shock genes might be expressed in other eukaryotes. The counter opinion at the time was that heat shock response was a fruit fly thing. While in hindsight this might seem an odd and naïve question, our knowledge in the early 1980s was very limited and there was no reason to assume that a temperature response in fruit flies should have anything to do with humans. Moreover, the concepts of heat shock proteins as molecular chaperones would not appear for nearly another decade. Nevertheless, armed with the newly developed method of Southern blotting, I decided to search for the heat shock genes in other eukaryotes. Being located in the Harvard biolabs where there was an impressive collection of organisms under investigation, it was just one day’s work to wander the long halls with an ice bucket and return with genomic DNA from a wide array of life’s creatures. After running my collection of DNA on an agarose gel and incubating the nitrocellulose transfer paper with my radiolabeled Hsp70 gene fragments, I searched for the DNA fragments that were complementary.

I still remember the water dripping down my arm as I pulled out the developed X-ray film and saw bands of different sizes in each of the lanes of the gel. Hsp70 was everywhere and in all eukaryotic genomes, including humans’. The characterization of the human Hsp70 gene then became the focus of my new laboratory in the Department of Biochemistry, Molecular Biology, and Cell Biology at Northwestern University in Evanston, Ill. Around the same time, Jim Bardwell and Betty Craig at the University of Wisconsin at Madison had identified a gene in E. coli, dnaK, which was the bacterial equivalent of Hsp70, leading to the realization that heat shock genes were essentially identical from bacteria to humans and subsequently shown to be present in all five kingdoms.¹

With the cloning of human Hsp70 and subsequently other members of the human Hsp70 gene family, our efforts at Northwestern initially focused on learning how this gene was regulated in fruit flies. While looking for sequence homologies across species, we spent a lot of time looking at the regulatory regions of the human heat shock genes that would be bound by the heat shock transcription factor. Efforts mostly from Carl Wu at NIH, Bob Kingston at Harvard, John Lis at Cornell, and my lab led to the identification of a family of heat shock transcription factors that controlled the first step of the heat shock response. Was this the molecular thermometer that I had been searching for? These heat shock factors changed their shape when the temperature changed, binding to the heat shock gene promoters and initiating the expression of heat shock genes (see graphic below). Moreover, humans expressed three such heat shock factor genes—as opposed to one in yeast, Drosophila, and C. elegans—suggesting that these genes, with such exquisite regulation, might have other functions that we had yet to discover.

Additional insights on the molecular thermometer came from studies showing that the heat shock proteins were regulating the very transcription factor that initiated their production in the nucleus. The function of heat shock proteins as molecular chaperones to guide folding, to ensure alternate conformational states, and to recognize and suppress misfolding was becoming well established. Therefore, the cellular thermometer wasn’t just measuring temperature, it also monitored for the appearance of damaged proteins. Heat shock and other stressors would lead to a flux of misfolded and damaged proteins, shifting the chaperone equilibrium in the cell toward capturing these damaged proteins and releasing Hsf1 to self-associate and form trimers capable of binding to the heat shock elements in the genome. The chaperones, therefore, had yet another role: to repress the transcriptional activity of Hsf1, providing a feedback loop for the heat shock response when sufficient levels of heat shock proteins were available.

As my lab started exploring the normal functions of heat shock proteins in the cell, I began to feel somewhat constrained by the human tissue culture cell lines we had been using. While the cells allowed us to probe the identity and function of the relevant molecules, it was unclear how these changes related to a whole organism. In around 1999, we introduced C. elegans into our lab as a new model system to address a question on the regulation of the heat shock response. We drew inspiration from a 1994 paper by Max Perutz showing that a mutation in polyglutamine that caused the protein to aggregate was responsible for Huntington’s disease, a profound and devastating neurodegenerative disease. In short time, C. elegans became our system of choice for exploring new stress signals such as the expression of expanded polyglutamine proteins.² Could misfolded proteins, as prompted by the expression of polyglutamine, provide insights into the molecular thermometer?

Buoyed by these possibilities, I submitted the renewal for my long-standing NIH grant and was nominated for a Merit Award. This gift of 10-year support from the National Institute of General Medical Sciences (NIGMS) came at exactly the right time. In addition to developing new animal models for expression of polyglutamine expansion in neurons and muscle cells, we observed that the aggregation and associated toxicity of these proteins was proportional to their length and also to the age of the animal. This led us to ask whether suppressing the insulin signalling pathways that regulate lifespan might affect the aggregation of polyglutamine. The results showed a clear relationship,³ moreover subsequent studies revealed that Hsf1 was essential for lifespan via the insulin signalling pathway. These observations revealed that the heat shock response was not only important for survival of acute stress but equally for day-to-day events of protein toxicity that could affect the health of the cell’s proteins and lifespan.

By this point, an increasing amount of work in our lab had shifted toward the use of C. elegans, and this whole organismal thinking gave me leeway to probe beyond the concept of stress at a cellular level to begin to address how the animal senses and integrates external information at the molecular level. If stress affected aging, then perhaps these heat shock proteins that sensed and protected the cell from accumulated damage might help slow the effects of aging.

Because we had earlier observed that many diseases associated with aggregation-prone proteins exhibit age-related phenotypes and that all protein misfolding diseases are associated with aging, it seemed logical to ask whether temperature-sensitive metastable proteins lost function during aging. We had observed earlier that polyglutamine expressed in different tissues of C. elegans caused temperature-sensitive proteins in the same tissue to completely misfold and aggregate. This result really turned our heads, for none of us had expected that a single aggregation-prone protein would have such global effects! Indeed, when the same temperature-sensitive proteins were followed during aging alone, we observed that they all misfolded and lost function early on in natural aging.⁴ When I saw these results, I was shocked. It suggested that the machines that keep proteins folded properly would have already started to fall apart as an organism reaches maturity. Returning to Hsf1, we learned that the heat shock response and other cytoprotective responses were compromised during aging. However, despite the rather profound consequences of these observations, we found that activation of Hsf1 in early development could suppress this collapse of protein-folding homeostasis (proteostasis). The implications are intriguing. While it is always possible that such observations do not extend to more complex metazoans, we are all made of proteins and it seems likely that the tales of worms may help us understand human aging.

But there was more to the story. Our recent work suggests there may be ways of modulating proteostasis that don’t involve interfering with Hsf1 directly. We decided to plunge into a classical genetic screen of C. elegans mutants that demonstrated protein misfolding in the muscle cells. While one might have expected to get many of the same genes already identified, we found something completely new. The mutation that affected misfolding in the muscle cell corresponded to a transcription factor that controls the production of the neurotransmitter GABA, responsible for mediating excitatory neurotransmitters and controlling muscle tone! Remarkably, any imbalance in the GABA pathway leading to overstimulation caused the postsynaptic muscle cell to think it was stressed and to start misfolding proteins.⁵ Suddenly, the molecular thermometer got more complicated with the realization that the activity of neurons could affect the thermometer of another cell.

If this mechanism also occurs in humans, we may be able to find the neuronal signature that controls protein misfolding in cells and activate the heat shock proteins in the skeletal muscle system to help restore function in muscular dystrophy and other motor neuron diseases. The findings also tease the possibility that there might be a way to control, stall, or regulate the misfolding associated with aging via a neurological mechanism. However, the story with neurons seems to be more nuanced. We observed that two neurons in C. elegans control the regulation of the heat shock response in all adult somatic cells. The role of active neuronal signalling and feedback control would seem to provide a basis by which cells and tissues activate a heat shock response according to need. This makes sense, as different tissues will have varied protein biosynthetic needs and environmental exposures. A “one-size-fits-all” approach to the heat shock response would not work.

With these exciting ideas that our research has led us to ponder, I and several of my colleagues (Jeff Kelly at The Scripps Research Institute and Andy Dillin at the Salk Institute) cofounded Proteostasis Therapeutics in Cambridge, Mass., to discover small molecule therapeutics that could correct the effects of misfolded proteins in disease. The approach is not without risk—the level of heat shock proteins are increased in many cancers—but the promise of the technology is certainly too exciting not to explore.

The heat shock response is a good story. It has humble beginnings of pure curiosity. Starting with a small band of “heat shockers,” the field has grown immensely and encompasses a multitude of disciplines and approaches fundamental to biology. In it are all of the elements of intrigue and surprise, with a remarkable cast of characters. Who would have predicted the chance observation of Ritossa’s that chromosomal puffs in Drosophila, induced by elevated temperature, would lead to discoveries across all of biology? Well beyond the satisfaction of these observations alone has been the recognition that many human diseases thought to be unrelated may share commonalities of defects in protein folding homeostasis and that the correction of these defects could have broad-reaching global effects on proteome stability and the health of the cell. All throughout this, I have been fortunate to have a wonderful family—my wife Joyce and children Emiko and Kenji and a fantastic set of colleagues and lab members. Perhaps the efforts to apply the discoveries of heat shock proteins successfully to maintaining health and curing disease is one way to thank them for their enthusiastic support.

Have a comment? Email us at mail@the-scientist.com

Richard Morimoto is the Bill and Gayle Cook Professor of Biology and director of the Rice Institute for Biomedical Research at Northwestern University, and a cofounder of the Proteostasis Therapeutics, Inc. in Cambridge, Mass.

In cancer biology, HSPs are the DAMP (or SAMP), danger or stress associated molecular patterns, necessary for causing an immunogenic stimulation for dendritic cells. Other HSP like calreticulin and perhaps calnexin are connected with immunogenicity of tumors or virus infected cells.

Thus it is not simply aging, it is the survival of the species to face invaders from both within and without

Though I believe that Morimoto is correct in his diagnosis, I think probably not in his idea of the cure by Heat shock Proteins Therapy. Heat Shock Proteins are no longer able to do their task optimally in the current scenario as human cells experience unprecedented stress in today?s times. For a long time, I have been studying factors affecting protein structure, protein folding, and misfolding, effect of stress on protein unfolding/misfolding, role of heat shock proteins in refolding misfolded proteins. I think, apoptosis is a consequence of the failure of cellular machinery to refold misfolded proteins and thus stopping all metabolic processes. That links so many ?apparently?un-related diseases - all due to protein misfolding. Do not forget that Heat Shock Proteins are proteins too and may become misfolded under certain conditions.

An individual could die when he has aged sufficiently due to such accumulated misfolded proteins in the cells of vital organs (heart, brain, liver, pancreas, kidneys, lungs, blood etc). Excessive stress in the modern times could be the cause of new diseases resulting in this way. Repeated stress causes proteins to unfold repeatedly and increase the chance of an imperfect refolding to happen which can lead to amyloid formations. Amyloid proteins (misfolded proteins with predominant beta structures) are found in many diseases like Beta amyloid (in Alzheimer), IAPP in Type II Diabetes, Alpha synnuclein (Parkinson?s disease), Huntingtin (Huntingtons Disease), Calcitonin ( Medullary carcinoma of Thyroid), Atrial Natriuretic factor in Cardiac arrhythmias, Serum Amyloid A in Rheumatoid arthritis. These are a really a small number of a large group of diseases.

Though normally protein structures are controlled via the integrity of the genes producing them, prions and amyloids have the capacity to infect normally folded proteins to make them misfolded too (into amyloids). This can create an overwhelming task for Heat Shock Proteins in which they can fail easily. Thus treating protein folding disorders will be a major challenge.
Diets and lifestyles which can reduce all kinds of protein stress can probably reduce diseases and ageing and delay death. I would say a good sleep, and some regular daily meditation would prevent production of stress hormones. Preventing mechanical stress to proteins could be achieved by avoiding physical injury on the body (for instance, it is known that head injuries are associated with Alzheimer). That is a good reason to ban corporal punishments in schools and elsewhere. I think that avoiding fermented foods containing microbial proteins would be helpful ? as many microbes (not all) such as yeast produce functional prions which they need for their normal growth. These prions could infect our proteins to misfold. For infecting a normal protein, all that a prion protein needs is physical proximity to a normal protein. Similarly all alcohol and some kinds of cheese can probably be risky too.

As is his forte, Rick has reminded us of the varied and important roles of molecular chaperones. However I would be remiss if I did not point out that the genes dnaK, dnaJ, groEL and groES, were all discovered in the 1970?s as E.coli genes involved in the replication (hence the name dnaK, dnaJ) and morphogenesis (groEL and groES) of bacteriophage, in particular, phage Lambda, T4 and T5. I wish Rick and fellow ?age? researchers god-speed ? for obvious reasons.

Rick's seminars are just as interesting and well constructed as this article. I wish more scientists could admit they are human beings.

I am a heat shocker who got into the field studying the expression of these genes in the various tissues of cold blooded vertebrates. After a sabbatical in John Subjeck's lab at Roswell Park Cancer Institute in Buffalo, my interests shifted to what at the time were very poorly understood members of the Hsp70 family, Hsp110 and its ER resident paralog,Grp170. Most of that work was done in collaboration with John's Lab. Currently I an investigating the role of the very large maternal supply of hsp110 in Drosophila oocytes.

All through the various phases of my career in the heat shock protein field, Rick has been generous in supplying antibodies,advice and encouragement. I am just a bit player in the study of proteostaisis but Rick has always treated me with the same respect as he would any other researcher in the field.

Rick's very nice article reminds me of how Balkanized the heat shock field was in the early days. Investigators were walled in by disciplinary boundaries and geographical ones as well. It was Rick and a handful of community-minded researchers who took time away from their scholarly work to serve as presidents of the Cell Stress Society International. Although he was too modest to include it, his efforts and those of his colleagues really stimulated cross-fertilization of ideas in this field on a global scale. To a large extent, this is what has led to the realization of the role and potential for stress responses in human health and disease.

I was attracted to the article by Morimoto's statement: ?All protein misfolding diseases are associated with aging.? He is fortunate that he became interested in the problem of protein misfolding from his grade school days. I am from an older generation, and first heard about the problem of protein folding from Buzz Baldwin, while he was an Assistant Professor at the Biochemistry Department, University of Wisconsin. I came to the same laboratory as a graduate student to the adjoining laboratory in 1957. Subsequently, I kept up a passive interest on the subject, but it has amazed me like so many other basic science findings that a little observation leads to very profound thesis on a fundamental principle and in life sciences relevant to human diseases. I wish this kind of an article is read by younger people first taking science courses.

This is a very interesting story, well written, very intriguing and full of fun!