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Introduction to Prion Biology: Part 1 Lecture Notes
Key words and termsProtein folding, prion, amyloid, protein-based inheritance, translation termination, neurodegenerative diseases, chaperones, stress response Lecture NotesThese notes follow Dr. Lindquist’s lecture, except that a few concepts are introduced in a different order than they were presented in the lecture, for pedagogical purposes.
Protein folding is as ancient as life itself All living organisms use proteins for structure, energy production, information processing, etc. Finished proteins have complicated three-dimensional structures that are necessary for their diverse activities. However, proteins are first produced as a linear string of amino acids, and they must then fold into the right conformations in the crowded environment of a cell (~300mg/ml). Diseases can ensue when proteins have problems reaching their functional conformations: sickle cell anemia, cystic fibrosis, Alzheimer’s etc.
Using simple model organisms to study protein misfolding Protein folding is a complex problem, but because this basic problem, and its solution, is shared among all organisms, we can use simple, tractable organisms like yeast to understand it. Yeast have been used by man for making bread, beer, and wine for thousands of years. Biologists often work with them because they are easy to manipulate experimentally. Yeast allow the use of many tricks and tools of molecular biology, including the genetic techniques to make site-specific mutations in their DNA, and the cell biological and biochemical techniques to study protein-protein interactions. For an introduction to yeast methods, see Fred Sherman’s guide, “Getting Started with Yeast,” at http://dbb.urmc.rochester.edu/labs/sherman_f/StartedYeast.html
The stress response Cells are always creating new proteins and ensuring that they fold into their correct shapes. Cells help proteins fold by producing helper proteins known as chaperones. These proteins specifically recognize parts of proteins that are not correctly folded, and shield them from other proteins until they can achieve their correct shape. Protein folding is a delicate process that can go haywire when cells are exposed to stressful conditions like high temperatures. At the first sign of such stress, cells ramp up their production of chaperones, which for this reason are also known as Heat Shock Proteins (HSPs).
The heat-shock response is protective against stressful events. For instance, pre-treating yeast with mild heat strongly protects them from a subsequent exposure to heat that would otherwise be lethal. The same is true for bacteria, plants, and animals – the stress response has been conserved since the dawn of life. Even humans mount a stress response under many conditions. In fact, our ability to produce a fever in response to illness is thought to be our body’s way of turning on our heat-shock response to protect our proteins while incapacitating those of the pathogen. Sometimes, however, chaperones may be too good at what they do. Cancer cells, for instance, have many abnormal changes that increase their protein misfolding, but are allowed to survive and continue propagating because they have also elevated their use of chaperones.
The most important chaperone for protecting against extreme stress is Hsp104. Most heat-shock proteins work by preventing misfolded proteins from associating with one another into highly stable protein aggregates. We discovered, however, that Hsp104 actually takes aggregates back apart. When cells are heat shocked, blobs of protein aggregates can be seen in cells examined by light microscopy. These aggregates eventually go away due to the action of Hsp104, but they persist in cells that do not have Hsp104. Hsp104 uses tremendous amounts of ATP in order to break up aggregated proteins.
Mammalian prions Mad cow disease (also known as Kuru and Creutzfeldt-Jacob Disease in humans; scrapie in sheep and goats; and chronic wasting disease in deer) is a bizarre disease of the nervous system that is:
Regardless of how they arise, prion diseases can be infectious. Stanley Prusiner discovered that there appears to be no nucleic acid in the infectious material (no genetic code with which to make new infectious particles). He discovered that instead, prions were composed of a misfolded form of one of our own proteins, called PrP. This misfolded form of the prion starts a chain reaction by converting the normal PrP molecules that it encounters into the same misfolded form. These new prions go on to convert the rest of the PrP.
The Yeast Prion [PSI+] Since protein misfolding is such a deeply rooted biological problem, we might expect that nature might have stumbled across ways to take advantage of it. Indeed, prions also exist in organisms such as yeast, and in these cases the prions appear not to be diseases.
Brian Cox, when studying why some yeast colonies of a particular strain were red and others were white, found that the white trait was dominant and segregated in a non-Mendelian fashion (i.e. when white yeast divided by meiosis all progeny were white rather than half white and half red as would have been expected). He named the white trait [PSI+] due to its unusual inheritance, with all capitals to indicate dominance and brackets to indicate its non-Mendelian character.
What distinguished the red and white cells were their efficiencies in one part of the process of decoding genetic messages. Specifically, the activity of the Sup35 protein, which is necessary for terminating protein synthesis at the end of mRNA messages, was decreased in the white cells. We now know that the altered translation fidelity caused by [PSI+] affects mRNA messages from throughout the genome. Thus, not only does it change the color of the cells, but also many other processes that affect the cells’ abilities to survive in diverse conditions.
From genetic evidence, Reed Wickner first proposed that [PSI+] (and another mysterious genetic element, [URE3]) might be similar to the agent that causes mad cow disease, or a prion. Specifically, these elements, in addition to showing non-Mendelian inheritance patterns, could be induced by increasing the protein concentration of their associated proteins, were reversible, and required the continuous presence of their determining protein (Sup35 and Ure2, respectively).
The role of Hsp104 in propagating yeast prions Yury Chernoff discovered that overexpression of Hsp104, the protein discussed above that is so important for breaking up aggregates of misfolded protein, caused [PSI+] to be lost from cells. This brought him to work with the Lindquist lab on the mechanism of prion propagation. It turned out that lowering Hsp104 levels also caused the loss of [PSI+], meaning that [PSI+] only propagates successfully with intermediate levels of Hsp104. We now know that Hsp104 is necessary for other yeast prions as well.
Why might a prion require Hsp104? The ability of prions to template other proteins into their own shape requires both proteins to associate tightly with one another, perhaps in an aggregated form. But in order for there to be an increase in the number of prion particles these protein complexes need also to be taken apart into smaller pieces. Hsp104 turned out to be using its disaggregating activity to divide up prion protein complexes after they were templated.
Domain architecture of Sup35 The Sup35 protein has three distinct regions. The N-terminal domain is responsible for the protein’s ability to self-associate (aggregate) under normal conditions. It is highly enriched for residues like glutamine, giving it a similar chemical character as many other aggregation-prone proteins like Huntingtin (the protein involved in Huntington’s Disease) that contain an expanded poly-glutamine tract. The middle domain is highly enriched for charged residues, which helps keep the protein soluble when in the non-prion conformation. These two regions together, called NM, allow the protein to exist stably in either a soluble state or an aggregated state. Finally, the C-terminal domain is the business end of the molecule. It performs the protein’s important translation termination function.
When Sup35 is fused to green fluorescent protein (GFP), one can visualize fluorescent dots in [PSI+] cells that are small and can be passed on to daughter cells. When Hsp104 is inhibited, these clusters, or foci, of protein, which are composed of aggregated Sup35, become larger and fewer in number because they are no longer being cut into smaller pieces by Hsp104, and eventually fail to be passed on to daughter cells.
Prion formation in vitro Prions within these foci appear to be comprised of a specific type of protein aggregate known as amyloid. Amyloid is a highly stable and highly ordered fibrillar protein aggregate. It is a one-dimensional polymer of polypeptides – new subunits are templated and added on only at the two ends of the fiber. Purified Sup35 readily forms amyloid fibers in a test tube. The functional C-terminal domain is not involved in amyloid formation, as visualized by comparing high magnification images of NM fibers (which are smooth in appearance) with those formed from full-length Sup35 (which contain extra material protruding from the sides of the fibers).
After soluble NM is diluted into physiological conditions, there is a stable “lag phase” during which amyloid formation does not occur. But eventually some of the proteins fold into the amyloid conformation and then rapidly template most of the remaining protein into fibers. If a small amount of preformed fibers are added to the protein solution at the beginning, the reaction skips the lag phase and amyloid formation proceeds immediately. This example of templating in a test tube provided a completely logical explanation for prion behavior in living cells. And it stood up to genetic tests: mutants that slow amyloid formation in vitro also inhibit prion formation. Amyloid was ultimately proven to constitute infectious prion material when amyloid fibers formed from purified Sup35 prion domain were introduced into yeast cells and the cells became [PSI+]. These experiments confirmed the prion hypothesis – that protein conformations can act as genetic elements.
The prion aggregation reaction thus can be divided into three steps: 1) Spontaneous nucleation - in which non-prion protein subunits first acquire an amyloid conformation. 2) Conformational templating - the recruitment of further soluble conformers to the ends of the amyloid fiber. 3) Division – the process of fiber fragmentation to yield additional ends capable of templating (assisted by Hsp104 and other chaperones in the cell).
Amyloid deposits are also commonly observed with prion diseases, indicating that here, too, prion propagation may be related to the unique properties of amyloid. Additionally, the prion domains of both Sup35 and PrP (the mammalian prion protein) contain a series of amino acid repeats. PrP repeat expansions are a familial mutation that predisposes these families to form prion diseases. Analogously, expanding the repeats in Sup35 also causes increased prion appearance.
The prion strain phenomenon In addition to red and white yeast colonies, [PSI+] can also cause colonies to exist in different shades of pink. Likewise, different variants of prion disease caused by PrP have different pathological features in the brain. In both cases, these stable variants, known as “prion strains”, are caused by different conformations of the self-propagating amyloids. These conformations differ in their rates of templating and division, and thereby also in the extent to which they inactivate the prion protein.
Why are prions toxic to mammals but not to yeast? Many proteins when they polymerize into amyloid also form a variety of non-fibrillar oligomeric species. These intermediates are now thought to be the principal toxic species associated with amyloid diseases. It may be the case that yeast, or otherwise the yeast prion proteins themselves, have found a way to go through this step much faster and consequently do not build up as many toxic protein conformers. |
