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 the process of liquid-liquid phase separation (LLPS) and that multivalent interactions in disordered proteins and through domain-motif interactions mediate this process.
Our current studies specifically focus on liquid-liquid phase separation (LLPS) 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 process called liquid-liquid phase separation (LLPS). LLPS is mediated via multivalent interactions, and these can either be encoded in intrinsically disordered regions or occur via multivalent domain-motif interactions. Specifically, we are working to characterize the sequence features and molecular interactions mediating LLPS; the protein-protein interactions responsible for the recruitment of components to membrane-less organelles; the impact on enzymatic function in a concentrated liquid compartment; and the disease mechanisms related to LLPS, such as neurodegeneration and cancer.
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 LLPS 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 LLPS 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 ocurring 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. Tumor-associated SPOP mutations disrupt substrate binding and ubiquitination, which leads to increased levels of oncogenic substrates. Despite this understanding, how SPOP assembles with its substrates and becomes recruited to nuclear bodies remain poorly understood. Our laboratory is studying these mechanisms, including SPOP’s association with the death-domain-associated protein (DAXX) and the androgen receptor. We characterized these SPOP/DAXX bodies as biomolecular condensates and are studying whether they are active ubiquitination hubs. Our findings reveal that cancer-associated SPOP mutations disrupt phase separation, which normally concentrates the components required for substrate ubiquitination, resulting in loss of function.
After receiving her PhD from Johann Wolfgang Goethe University, Dr. Tanja Mittag trained as a postdoctoral fellow with Julie Forman-Kay at the Hospital for Sick Children in Toronto where she revealed how a highly dynamic complex with several interconverting interfaces can encode a cell cycle switch. She joined the Department of Structural Biology at St. Jude in 2010 and has focused her work on elucidating the role of liquid-liquid phase separation for functional compartmentalization in cells. She is the recipient of the prestigious Michael and Kate Bárány Award from the Biophysical Society for her “rigorous and foundational contributions to the field of macromolecular condensates and their biological relevance”.
Our research team is a colorful mix of biophysicists, biochemists and cell biologists with a diverse range of interests and expertise
Tanja Mittag, PhD
MS311, Room D1034F
St. Jude Children Research Hospital