One of the most intriguing recent developments in cell and molecular biology are the findings regarding the critical role of protein liquid-liquid phase separation (LLPS) in many biological and biochemical processes. LLPS is typically driven by weak multivalent protein-protein or protein-RNA interactions, leading to formation of highly dynamic liquid droplets. The transient nature of these interactions allows the cell to use this phenomenon to assemble and disassemble protein complexes in response to varying stimuli in order to affect metabolism. An important example of LLPS is formation of stress granules, cytoplasmic membraneless organelles (MLOs) which form in response to cellular stress, where proteins and RNAs interact, sequestering RNAs from translation machinery to conserve energy. When the stress inducing stimulus is removed, the stress granule dissociates and RNA translation resumes as normal. Many other biological processes may be regulated by MLOs, as research into this phenomenon is quite young.
While LLPS plays an important role in normal cell biology, there is also a darker side to this phenomenon. .Indeed, LLPS appears to be associated with the pathogenesis of some of the most devastating neurodegenerative disorders, including amyotrophic lateral sclerosis, Alzheimer's disease and frontotemporal dementia. Using biophysical, biochemical and cellular techniques, our lab is seeking to understand the mechanism of LLPS of proteins involved in neurodegenerative diseases and uncover the relationship between LLPS, protein aggregation, and the pathogenic process. The proteins of particular interest in this regard are tau and Tar-DNA Binding Protein of 43 kDa (TDP-43). Tau is a major player in Alzheimer's disease and several other disorders collectively known as tauopathies, whereas aggregation of TDP-43 is associated with amyotrophic lateral sclerosis and frontotemporal dementia. For more information regarding the link between protein LLPS and neurodegenerative diseases see our recent review article (Babinchak WM and Surewicz WK, J Mol Biol 432, 1910-1925 (2020).
Amyloid fibrils are highly ordered protein aggregates characterized by a fibrillar morphology, cross-beta sheet structure and ability to be stained by dyes such as Congo red. Accumulation of amyloid-like fibrils in the nervous system is a common feature of many neurodegenerative diseases such as Alzheimer’s disease (AD), prion diseases (or Transmissible Spongiform Encephalopathies, TSEs), Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD), even though the identity of aggregating protein is disease-specific. A growing number of observations indicate that these fibrillar aggregates can propagate between cells in a prion-like fashion, spreading disease pathology from one brain region to another.
Recent findings indicate that amyloid fibrils formed by the same protein can exist in a multitude of structurally distinct forms, and that these structurally distinct amyloid "strains" may be responsible for phenotypic variability of not only prion diseases but also other neurodegenerative disorders (e.g., sporadic and familial cases of AD, different forms of tauopathies). Our lab is using cryo-electron microscopy and other biophysical methods to determine high-resolution structures of amyloid fibrils associated with different forms of prion diseases and other neurodegenerative disorders and to gain insight into the relationship between amyloid structural polymorphism and disease phenotype.
The prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that include scrapie in sheep, mad cow disease in cattle, and kuru, Creutzfeldt-Jakob disease, GSS disease and fatal familial insomnia in humans. Pathogenesis in these unusual diseases is associated with a conformational rearrangement of the cellular prion protein (PrPC) to an abnormal "scrapie" conformer, PrPSc. While the benign PrPC conformer is monomeric and rich in alpha-helical structure, PrPSc is a protein aggregate (typically amyloid) characterized by high proportion of ß-structure and resistance to proteolytic digestion. Although molecular details of the pathogenic process in TSE diseases remain controversial, a large body of evidence supports the protein-only model, according to which PrPSc itself is the infectious prion pathogenm that self-perpetuates by a mechanism involving binding to PrPc and inducing a conversion of the latter protein to the PrPSc state. The notion that an infectious agent can be devoid of nucleic acids and propagate by a mechanism based on self-perpetuating changes in protein conformation constitutes a new paradigm in molecular biology and medicine.
Our group is interested in biophysical and biochemical aspects of prion protein folding/misfolding and the molecular basis of prion strains and TSE transmissibility barriers. The current focus of our research is on (i) Elucidating the molecular mechanisms and structural basis of PrP conformational conversion; (ii) Determining high-resolution structures of different prion strains; (iii) Understanding the role of non-proteinaceous cofactors in prion protein conversion to the infectious form; (iv) Understanding the molecular/structural basis of TSE transmissibility barriers. Methodologically, this research constitutes a combination of state-of-the-art methods of protein chemistry, structural biology (including cryo-electron microscopy) and studies using transgenic mice.