Generation of Novel Yeast Prions through Domain Substitutions of Saccharomyces cerevisiae Sup35p Público

Qi, Xiaojie (2007)

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The yeast prion [PSI+], formed by the component protein Sup35p in Saccharomyces cerevisiae, has been employed as a model to study prion formation and aggregation. The studies on yeast prions have revealed the mechanism of prion formation and propagation, which provides important clues for studying mammalian prions and other protein aggregates that are involved in protein misfolding diseases. It was recently suggested that the prion forming domain of Sup35p can be further divided into the Asn/Gln-rich domain (NQ) and the oligopeptide repeats domain (NR), which are responsible for protein aggregation and prion propagation, respectively.

In this study, aggregation-prone peptide sequences were selected as the candidates that could potentially replace the NQ domain and generate novel prions. The substitution of a fragment from alpha-synuclein for the NQ domain of Sup35p generated prion-like phenotypes. The substitution of a fragment of islet amyloid polypeptide (IAPP) for the NQ domain of Sup35p generated a novel prion. The novel prion formed by IAPP-substituted Sup35p exhibited similar characteristics as prion state of the wild-type protein. In addition, the substitution of a bacterial Asn/Gln-rich oligopeptide repeat for the NR domain of Sup35p generated prion-like phenotypes. These results demonstrated that novel prions could arise from domain substitution with Sup35p, and that the aggregation units were not restricted to Asn/Gln-rich proteins. As the characteristics of the prions were shown to depend on the properties of the aggregation units, yeast prions can potentially be used as model systems to study the aggregation of selected protein sequences. These models can be useful in the identification of factors that are critical for protein aggregation, such as amino acid composition, growth condition, and the presence of small molecules. Moreover, these models may provide a tool to screen for drugs that can inhibit or counteract protein aggregation.

Table of Contents

Chapter 1. Introduction

Chapter 2. Selection of aggregation-prone protein candidates and their substitutions for the NQ domain of Sup35p (1) Introduction (2) Experimental procedures (3) Results

Selection of peptides and their substitutions for the NQ domain Viability of sup35 mutant strains Phenotypes of sup35 mutant strains

(4) Discussion

Chapter 3. The substitution of alpha-synuclein fragment for the NQ domain of Sup35p generated prion-like phenotypes (1) Introduction (2) Experimental procedures (3) Results

Cells containing the syn1-sup35 mutant gene showed two prion-like phenotypes The mutations A76E and A76R stabilized the non-prion state of Syn1-sup35p Wild type SUP35 restored the red phenotype when reintroduced into [SYN1+] The effect of molecular chaperones on [SYN1+] Overexpression of Syn1-M-GFP caused diffuse fluorescence Syn1 prions caused the aggregation of full-length alpha-synuclein Syn1-substituted Sup35NM protein formed amyloid fibers in vitro

(4) Discussion

Chapter 4. Substitution of islet amyloid polypeptide (IAPP) fragment for the NQ domain generated a novel prion. (1) Introduction (2) Experimental procedures (3) Results

The expression of IAPP-GFP-Sup35 fusion protein at different levels and its effect on the characteristics of IAPP-GFP-Sup35 fusion protein IAPP-substitute GFP-Sup35 fusion protein was able to form prion Genetic analysis of [I-PSI+] prion: cytoduction, mating, and tetrad analysis The effect of Hsp104p on [I-PSI+] prion Overexpression of IAPP-M-GFP induced the formation of [I-PSI+] prion IAPP-substituted Sup35NM protein formed amyloid fibers in vitro The kinetics of the aggregate formation by IAPP-substituted Sup35NM protein Introduction of S20G into IAPP and its effect on prion formation

(4) Discussion

Chapter 5. Substitution of a bacterial protein fragment for the NR domain generated prion-like phenotypes (1) Introduction (2) Experimental Procedures (3) Results

Selection of an NR-like bacterial oligopeptide repeats fragment The substitution of Bac6 repeats generated prion-like phenotypes The effect of Hsp104 chaperone on [BAC6+] prion Bac6-NM protein aggregated in a nucleation-dependent pattern

(4) Discussion

Chapter 6. Summary


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