Divergence in Prion Inducing Ability of Paralogous Actin Asssociated Proteins Pubblico
Ali, Moiez (2012)
Abstract
Divergence in Prion Inducing Ability of Paralogous Actin Associated Proteins
Prions are infectious, amyloid-like protein aggregates that transmit neurodegenerative diseases in mammals and heritable traits in yeast. It is important to study the formation of prions to better understand the progression of prion diseases and related neural inclusive diseases including Alzheimer, Parkinson, and Huntington. Although the precise mechanism of initial prion formation remains unclear, de novo formation of a yeast prion is induced by transient overproduction of the prion-forming protein and is efficient only if other Q/N-rich protein aggregates are present in the same cell. Recently, it has been shown that overexpression of Q/N-rich protein Lsb2 (Las seventeen binding protein 2) promotes conversion of translation termination factor Sup35 into its prion form, [PSI+]. In contrast, Lsb1, a paralogous protein which shares 64% amino acid identity with Lsb2 does not promote [PSI+] formation. Here, we show that structural and sequence differences between the Lsb proteins may be responsible for facilitating Lsb2 prion inducing ability. However, we provide evidence that suggests that Lsb1 protein may regulate the prion inducing ability of Lsb2 through a direct association. In addition, we demonstrate that the protein levels and stability of the Lsb proteins depends on the presence of Las17, an actin polymerization factor, and are regulated by ubiquitination. Loss of either LSB1 or LSB2 results in destabilization of a weak [PSI+] variant under mild short-term heat shock. Together, this data provides evidence to show that the Lsb proteins may be involved in the formation and segregation of protein aggregates, indicating a possible biological significance of the Lsb proteins. Also, our findings elucidate the role of actin cytoskeleton machinery and ubiquitin proteolysis in regulating prion induction.
Divergence in Prion Inducing Ability of Paralogous Actin
Associated Proteins
By
Moiez Ali
Advisor: Keith D. Wilkinson, Ph.D.
An abstract of
A thesis submitted to the Faculty of the
James T. Laney School of Graduate Studies of Emory University
in partial fulfillment of the requirements for the degree of
Master of Science
Biology
2012
Table of Contents
Table of Contents
Chapter 1 General Introduction...1
Prions...1
Lifecycle of Prion: Formation...2
Lifecycle of Prion: Prion induction by heterologous
Proteins...4
Lifecycle of Prion: Loss or "curing" of a prion...6
Involvement of the Actin Cytoskeleton...7
Scope of this thesis...8
Chapter 2 Promotion of de novo induction of [PSI+] by Lsb2 and Lsb1...10
Introduction...10
Results...10
Lsb2, but not Lsb1, induces the formation of [PSI+] via Sup35...10
Discussion...12
Chapter 3 The Lsb proteins are both ubiquitinated...14
Introduction...14
Results...14
Lsb1 is ubiquitinated in the cell...14
Lsb1 is ubiquitinated by Rsp5 E3 ligase...15
Lsb2 and Lsb1 protein half-life is influenced by alterations in the
proteasome...15
Discussion...16
Chapter 4 Analysis of protein binding partners of Lsb2 and Lsb1...18
Introduction...18
Results...18
Lsb2 and Lsb1 bind Ub, Las17 and
Sup35...18
Lsb2 and Lsb1 interact in vivo...19
Lsb2 and Lsb1 interact in vitro...19
Lsb2 and Lsb1 are associated with cortical actin patches...20
Discussion...20
Chapter 5 Influence of Las17 on the protein dynamics and stability of the Lsb protein...25
Introduction...25
Results...25
Presence of Las17 influences the Lsb protein
stability...25
Inability to bind actin cytoskeleton further compromises the Lsb
protein stability...26
Mutation of major site of ubiquitination of Lsb1 and Lsb2 restores
protein levels...26
Discussion...27
Chapter 6 The Lsb proteins are processed within yeast...32
Introduction...32
Results...32
Lsb1 protein is processed at its C-terminal at
adjacent tyrosine residues Y187, Y188...32
Lsb2 protein is processed at its N-terminal...33
Lsb1 C-terminal processing may prevent de novo
[PSI+] induction...34
Discussion...35
Chapter 7 Effects of Lsb1 protein on the prion induction process via Lsb2...38
Introduction...38
Results...38
Absence of LSB1 enhances prion induction process via Lsb2...38
Discussion...39
Chapter 8 Role of the Lsb proteins in the maintenance of [PSI+] prion...41
Introduction...41
Results...41
Lsb2 Is a Stress-Inducible Protein Influencing
[PSI+] maintenance...41
Lsb1 protein influences [PSI+]
maintenance...42
Discussion...43
Chapter 9 Discussion and Future Directions...46
Interactions of Lsb1 and Lsb2 in Prion
Formation...46
Effects of Lsb proteins on Prion Maintenance...48
Processing of Lsb proteins...49
Summary...50
Chapter 10 Materials and Methods...52
Chapter 11 References...59
Table of Figures
Graph 1 Process of Ubiquitination and UPS-dependent degradation of
proteins...9
Figure 1 Homologous Proteins Lsb2 and Lsb1 Differ in Prion-Inducing
Ability...13
Figure 2 Ubiquitination and Degradation of the Lsb
Proteins...17
Figure 3 Protein binding partners of the Lsb proteins...22-24
Figure 4 Lsb protein stability is influenced by LAS17,
ubiquitination and association with actin patches...29-31
Figure 5 Analysis of Lsb protein processing...36-37
Figure 6 Efficiency of prion inducing ability of Lsb2 is influenced
by absence of LSB1...40
Figure 7 Lsb Levels and Effects during Stress...44-45
Figure 8 Model for Segregation and Retrograde Transport of Protein
Aggregates...51
Figure 9 Model for the Stress-Dependent Induction of Sup35
Prions...51
About this Master's Thesis
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