Guo Research

Welcome to the Guo lab at Thomas Jefferson University! Liquid-liquid phase separation drives the formation of membrane-less organelles such as stress granule. Aberrant protein phase transition leads to the formation of fibrils and aggregates implicated in various neurodegenerative diseases. We are interested in understanding the molecular mechanisms underlying these aberrant phase transitions and leveraging our understanding of phase transition to develop strategies with therapeutic potential to prevent and reverse these toxic events. We use a combination of biochemistry, molecular biology, cell biology, and biophysics techniques to tackle these problems.

It is now universally appreciated that accumulation of misfolded proteins, which can acquire alternative proteotoxic states, causes a series of deleterious molecular events resulting in numerous lethal neurodegenerative diseases. Numerous RNA binding proteins (RBPs) with Prion-like Domains (PrLDs) have been connected via pathology and genetics to the etiology of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic mislocalization and inclusion formation are common pathological features of these proteinopathies. The PrLD of these RBPs drives a phase transition into protein-rich reversible liquid droplets that share biophysical properties with RNP granules. “Aging” of these liquid droplets leads to an aberrant phase transition to a more solid-like irreversible fibrillar hydrogel state, which induces neurotoxicity by sequestering RNP cargo and impairing RNP granule function. Elucidating the mechanism of protein aberrant phase transition will be critical for understanding ALS pathogenesis. Furthermore, it is important to develop methods to rescue toxicity by reversing aggregation and aberrant phase transitions.

Aberrant phase transition of FUS and hnRNPA1 between dispersed solution, liquid droplet and hydrogel has been linked to impaired RNP granule function and ALS pathogenesis. Moreover, the function and localization of RNP granules can be tuned by the biophysical properties of these protein/RNA assemblies. However, the molecular mechanism of these transitions is not clear. We will develop biophysical methods especially single molecule FCS (Fluorescence Correlation Spectroscopy) and single molecule FRET methods to quantitatively monitor the molecular events during these transitions. This study will provide a detailed quantitative view of the aberrant phase transition of ALS disease proteins and shed a light on the mechanism of ALS pathogenesis.

We discovered that nuclear import receptor Kapβ2 can also function as chaperon and protein disaggregase to rapidly dissolves FUS fibrils and macroscopic hydrogels. Elevating Kapβ2 expression prevents recruitment of FUS into stress granules, restores nuclear localization, and rescues degeneration caused by disease-linked FUS. Thus, Kapβ2 could be important therapeutic target to restore RBP homeostasis in several fatal neurodegenerative disorders. However, for ALS-linked FUS variants that have mutations in the PY-NLS, the activity of Kapβ2 is impaired due to decreased binding affinity. Therefore, it is essential to define how Kapβ2 disaggregates FUS fibrils and to potentiate Kapβ2 activity against ALS-linked FUS variants.

RNA binding is essential for RBP function, cellular localization, cytotoxicity and incorporation into stress granules. Consistent with these in vivo results, in vitro studies show that RNA binding contributes to liquid droplet formation of FUS. We hypothesize that RNAs could be designed to delay and reverse aberrant phase transition and aggregation of FUS and rescue proteotoxicity by restoring normal stress granule function. Therefore, the third project in our lab focuses on developing such RNAs. We will study how combinations of these RNAs regulate FUS phase transition, which could give us insight on how cell regulates the biophysical properties of RNP granule by its RNA population. This study will provide insights for developing novel RNA therapeutics for ALS as well as other diseases that are characterized by accumulation of RBPs.