Overview
The central theme of our research is to explore the impact of protein conformational changes on diverse processes in cellular and organismal biology. We are exploiting our understanding of protein folding to gain insights into the basis of neurodegenerative diseases and spongiform encephalopathies, and to design therapeutic strategies. In addition to the role that misfolded proteins play in disease, we have also identified potentially important beneficial effects of self-perpetuating alternate protein conformations, including mechanisms of evolutionary change and long-term memory.

 

Research Summary
Prions and Protein-based Inheritance: Prions are proteins that can acquire self-perpetuating changes in structure that alter protein function and cell phenotype. They represent an epigenetic mechanism of inheritance because the altered phenotype is passed from generation to generation through the heritable changes in protein structure, with no underlying changes in nucleic acids. We have shown by cell biological, genetic and biochemical data that this protein-only mechanism controls the inheritance of the yeast prion [PSI+]. [PSI+] is propagated through a self-perpetuating change in the conformational state of the translation termination factor Sup35p, which forms an amyloid structure in its [PSI+] prion state. Most recently we have been examining how many other proteins (in S. cerevisiae and C. elegans) can produce heritable switches in conformation and function, and whether this mechanism can serve as a new, general method for manipulating phenotypes.

An extraordinary phenomenon of [PSI+] that we have discovered is its ability to mask a vast store of phenotypic variation. In more than 150 assays, conversion from [psi-] to [PSI+] provided the means to uncover a wide variety of hidden phenotypes and produce new, sometimes beneficial, heritable phenotypes in multiple genetic backgrounds. We suggest that the epigenetic and metastable nature of [PSI+] inheritance allows yeast cells to exploit pre-existing genetic variation to thrive in fluctuating environments without necessitating a change in genotype (see figure).  Further, the capacity of [PSI+] to convert previously neutral genetic variation to a non-neutral state may facilitate the evolution of new traits.

To further understand the mechanism of prion amyloid formation and transmission, we created a large number of cysteine-containing variants and employed fluorescent tags, crosslinking, and other methods to determine the nature of Sup35p's cooperatively folded amyloid core. We identified specific segments that form intermolecular contacts in a 'Head-to-Head', 'Tail-to-Tail' fashion, while a central region forms exclusively intramolecular contacts. This provided the first definition of the contacts that hold any amyloid fiber together. These structural insights allowed us to attack two central questions in prion biology, the mechanism of nucleation and the structural basis of prion strains. We established that the Head region acquires amyloidogenic interactions first and that these are sufficient to nucleate assembly. Prion strains, in both yeast and mammals, are distinct physical forms that produce different prion phenotypes in vivo. We found that variations in the length of the amyloid core and the nature of intermolecular interfaces were both characteristic of distinct strains of [PSI+] and sufficient to create them anew.

Protein Conformational Diseases: Some of the most devastating and intractable human diseases involve proteins that change their conformation. We have imported several of these proteins (the mammalian prion, PrP; huntingtin and alpha-synuclein) into yeast, providing a flexible system to study folding transitions and a high-throughput screening system to test therapeutic strategies. Hypotheses developed in yeast are now being tested in mammalian cells and transgenic mice.

In collaborative work with the labs of Jeffrey Macklis and Harvey Lodish, we have been investigating the normal function of mammalian PrP. PrP is normally expressed on the surface of bone marrow stem cells and neuronal precursors. In both systems lack of normal PrP expression, as seen in PrP knockout mice, seems to result in fewer mature cells. Misfolded PrP, on the other hand, can cause the devastating diseases commonly known as CJD in humans and “mad cow disease” in cattle. They are unique among diseases in having infectious, spontaneous and dominantly heritable forms. In the transmissible form, the misfolded conformation of PrP (PrPSc) induces the normal cellular protein (PrPC) to convert to the PrPSc conformation, but little is known about how PrPSc causes neurodegeneration. Our recent experiments suggest that the presence of PrP in the cytosol rather than in its normal cell surface location may be the cause of neurodegeneration in prion diseases.

Hsp104p and Stress Tolerance: Heat shock proteins (Hsps) are induced when organisms are exposed to high temperatures and other stresses. These stresses cause proteins to unfold and potentially to aggregate, creating a protein-folding crisis in the cell. Hsps are the chaperone proteins that help the cell cope with this crisis by binding to folding intermediates and rescuing them. Hsp104p has the unusual ability to disassemble protein aggregates formed from denatured proteins. At normal temperatures, the remodeling activities of Hsp104p control the inheritance of several protein-based genetic elements (prions), including [PSI+]. To understand how Hsp104p functions, we are employing a variety of biochemical and genetic approaches.

Hsp90p and Evolution: Another chaperone protein, Hsp90p, has very different properties from Hsp104p. It is specialized to chaperone a distinct class of unstable substrates including a wide variety of signal transducers. In both Drosophila melanogaster and Arabidopsis thaliana we have found that Hsp90p buffers the effects of a multitude of silent polymorphisms under normal conditions. When the Hsp90p buffering capacity is compromised by stress the effects of these polymorphisms are exposed. In D. melonogaster we found that with selection for the new traits they rapidly became independent of the buffering action of Hsp90p.

In studies of pathogenic fungi, we recently revealed an entirely new role for Hsp90p in promoting the emergence of new traits. Rather than buffering pre-existing mutations, Hsp90p potentiated the evolution of drug resistance by allowing new mutations to have immediate and beneficial consequences. It did so by chaperoning calcineurin, a key signal transducer for several stress responses, allowing it to function in the cellular response pathways that prevent external stressors such as antifungal drugs from becoming lethal. Thus, Hsp90p can act in very different ways to couple environmental contingency to the evolution and assimilation of new traits.

The strength and breadth of effects on the buffering and release of genetic variation by both Hsp90p and [PSI+] suggest they may have a large influence on evolutionary processes.

 

Selected Publications
Steele, A.D., Emsley, J.G., Özdinler, P.H., Lindquist, S. and Macklis, J.D. Prion protein (PrPc) positively regulates neural precursor proliferation during developmental and adult mammalian neurogenesis. Proc. Natl. Acad. Sci. USA 103(9): 3416-21. (2006)

Homann, O.R., Cai, H., Becker, J.M. and Lindquist, S.L. Harnessing Natural Diversity to Probe Metabolic Pathways. PLoS Genet. 1(6): e80. (2005).

Cowen, L.E. and Lindquist, S. Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309: 2185-89. (2005).

Krishnan, R., Lindquist, S. L. Structural insights into a yeast prion illuminate nucleation and strain diversity. Nature 435:765-72. (2005).

True, H., Berlin, I., Lindquist, S., Epigenetic regulation of translation reveals hidden genetic variation to produce complex traits. Nature 431:184-7. (2004).

Shorter, J., Lindquist, S., Hsp104 Catalyzes Formation and Elimination of Self-Replicating Sup35 Prion Conformers. Science 304:1793-97. (2004).

Si, K., Lindquist, S., Kandel, E.R., A Neuronal Isoform of the Aplysia CPEB Has Prion-Like Properties. Cell 115:879-91. (2003).

Outeiro, T.F., Lindquist, S., Yeast Cells Provide Insight into Alpha-Synuclein Biology and Pathobiology. Science 302:1772-75. (2003).

Ma, J., Wollman, R. and Lindquist, S., Neurotoxicity and Neurodegeneration When PrP Accumulates in the Cytosol. Science 298:1781-1785. (2002).

Queitsch, C., Sangster, T.A. and Lindquist, S., Hsp90 as a capacitor of phenotypic variation. Nature 417: 618-624. (2002).

Search PubMed for Lindquist Lab publications.

 

 

 

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