Research Interests
Biogenesis, Variability and Physiological Role of Prions
My research focuses on self-propagating infectious protein conformations, known as prions. The prion hypothesis postulates that some proteins can acquire stable unconventional conformations and pass these conformations on to newly synthesized protein molecules. Prions got into the spotlight as the causative agents of transmissible spongiform encephalopathies (TSEs), such as mad cow disease in cattle, and Creutzfeld-Jacob disease in humans. Noteworthy, prion phenomenon is not limited to the TSE-causing prion, PrP. The yeast non- Mendelian traits [PSI+] and [URE3] were shown to be prion forms of the Sup35 and Ure2 proteins, respectively, thus providing excellent model systems for the investigation of self-perpetuating protein conformations in genetically tractable unicellular eukaryotes.
Figure 1. Experimental system.
In [psi-] cells, Sup35 is in a functional conformation, but in [PSI+] cells most Sup35 takes on an alternative self-propagating protein conformation. This leads to the depletion of the functional Sup35p, that is a translational termination factor eRF3, and, consequently, to the increased level of suppression of nonsense mutations. Thus, the presence of [PSI+] can be scored using an easily detectable suppressor phenotype (Fig.1). My long-term research goal is to answer two fundamental questions: 1) how do prions appear, and what determines which of several possible variants of a prion will form in a particular prion appearance incident; 2) are prions rare abnormalities, or are they ubiquitous and manifest another level of regulation in the cell.
Figure 2. The "seeding" model.
In the course of my pre-doctoral and post-doctoral work in the laboratories of S.G. Inge-Vechtomov and S. W. Liebman I have established that the excess of Sup35 induces the de novo formation of [PSI+]. Indeed, it is presumed that prions arise either through a spontaneous misfolding of a protein molecule into the prion shape or by the interaction of two or more non-prion molecules leading to the formation of prion seeds, and either event is more likely when protein is overproduced. Later we established that the process of the de novo formation of [PSI+] is efficient only in the presence of one of several of heterologous prions (called [PIN+]s for [PSI+] inducibility). Furthermore, the de novo formation of other known and candidate prions appeared to be greatly facilitated by heterologous prions, suggesting that the underlying mechanism is universal in the world of self-propagating protein conformations. To explain this result, a 'seeding' model of prion formation was proposed (Fig.2). According to this model, a pre-existing prion can facilitate the formation of [PSI+] by providing a nidus for attachment and conformational conversion of Sup35 molecules. Now, we are utilizing the [PSI+]/[PIN+] two-prion system to directly test the feasibility of this model, to get an insight into the mechanism of the seeding process and to determine whether the compositional similarity of [PSI+] and [PIN+] is essential for this process (Fig.3).
Figure 3. Preliminary data supporting the "seeding" model for [PSI+] induction.
While searching for a heterologous prion element required for the de novo formation of [PSI+], I performed an in vivo screen for prion elements in yeast. Two known prions and nine candidate prions were identified. Strikingly, they all bear similar Gln/Asn-rich domains. This observation will be a starting point for me in addressing the question, how abundant are prions and can prionization be crucial for protein functioning. Indeed, prions could be beneficial because they increase variability at the physiological level: an alternative functional state is established and maintained semi-permanently without altering the genetic material encoded by nucleic acid. While the candidates identified in the screen will be used for designing novel experimental systems for prion studies and determining the role of prionization for functioning of a particular protein or protein family, minimal requirements for prion formation will be determined using a protein- design approach.