Investigating mechanisms underlying biomolecular phase separation and how they determine functional and pathological processes
Proteins that lack a fixed structure and constantly change binding sites play a critical role in biology. The tendency and ability of proteins to change shape dictates how cellular components cluster and how sequence and conformation drive function. These protein features often contribute to the development of disease. Our work is aimed at understanding these phenomena to help unlock pathological mechanisms of cancer and neurological disorders.
Our research focuses on understanding the molecular interactions underlying the formation of membrane-less organelles or so-called biomolecular condensates and how they give rise to function and disease. We have made critical contributions to the conceptual understanding that biomolecular condensates are formed through phase separation and that multivalent interactions in disordered proteins and through domain-motif interactions mediate this process.
Our current studies specifically focus on phase separation and compartmentalization in biomolecular condensates; sequence-conformation-function relationships of IDPs, and disease processes caused by dysregulation of phase separation and the modulation of multivalency. We employ a combination of biophysical, biochemical, and cell biological approaches in our research, as well as specialized tools like NMR spectroscopy, light scattering, fluorescence approaches, microscopy, proteomics, and analytical ultracentrifugation.
An increasing number of biomolecular condensates – including the nucleolus, stress granules, and nuclear speckles – have been shown to be formed via a phase separation. Importantly, dysregulation of phase separation can result in disease. Mutations can disrupt functionally important phase separation, and the result can be cancer. On the other hand, the prolonged assembly of stress granules can result in their maturation and protein aggregation, giving rise to the hallmarks of neurodegenerative diseases such as ALS.
Our research will improve our understanding of the formation and function of stress granules and nuclear speckles, the two major biomolecular condensates we study. But the impact of our research reaches further, as phase separation is now recognized to play a role in transcriptional regulation, the DNA damage response, membrane receptor clustering, and the selectivity filter of the nuclear pore complex.
Our laboratory has shown that a low-complexity domain (LCD) within the RNA-binding protein hnRNPA1 undergoes reversible phase separation into protein-rich droplets. While the LCD of hnRNPA1 is sufficient to mediate phase separation, its folded RNA-binding domains also contribute to phase separation in the presence of RNA, giving rise to several mechanisms of assembly.
Building upon this understanding, we are now exploring how the architecture of the RNA-binding domains – as well as the specific composition and sequence patterns of the LCD – may promote dynamic compartmentalization of hnRNPA1, other RNA-binding proteins, and RNAs into RNP granules. We are particularly interested in understanding how naturally occurring sequence features determine the phase behavior of IDRs. We are further investigating the mechanism of the generation of insoluble protein deposits in persistent stress granules in neurodegenerative disease.
The speckle-type POZ protein (SPOP) is a tumor suppressor that is frequently mutated in cancers. It functions as a subunit of a ubiquitin ligase and is important for maintaining appropriate protein levels of many proto-oncogenes. Tumor-associated SPOP mutations disrupt substrate binding and ubiquitination, which leads to increased levels of oncogenic substrates. The SPOP protein assembles with its substrates in biomolecular condensates in the nucleus, but the underlying mechanisms are poorly understood. Our laboratory is studying these mechanisms, including SPOP’s association with the death-domain-associated protein (DAXX) and the androgen receptor. We showed that the resulting SPOP/DAXX bodies are biomolecular condensates and that they are active ubiquitination hubs. Our findings reveal that cancer-associated SPOP mutations disrupt phase separation and therefore substrate ubiquitination, resulting in cancer pathogenesis.
Tanja Mittag, PhD
MS311, Room D1034F
St. Jude Children's Research Hospital