Powered By Bing

Mittag Lab: Research

We are interested in understanding the biological function of

  • intrinsic protein disorder and
  • multivalency.

In particular, we study how these properties relate to

  • liquid-liquid phase separation (LLPS) and compartmentalization in membrane-less organelles,
  • sequence-conformation-function relationships of IDPs and
  • disease processes caused by modulation of multivalency.

Our studies seek to provide insight into normal biological function and pathological mechanisms in cancer and neurological diseases.

Liquid-liquid phase separation and membrane-less organelles

"Membrane-less organelles" are µm-sized, typically spherical structures in cells with a clear boundary from the surrounding cytoplasm or nucleoplasm, while not being enclosed by a membrane (Figure 1, left). Notable membrane-less organelles include the nucleolus, stress granules and nuclear speckles and respectively function in ribosome assembly, mRNA sequestration upon stress and RNA splicing.

P granules, nucleoli, stress granules and an increasing number of membrane-less organelles have been shown to have liquid droplet properties. They flow, fuse, and coalesce into larger spherical droplets. These are classic properties of liquids.

Increasing evidence supports the hypothesis that these droplets are formed through a process called liquid-liquid phase separation (LLPS). LLPS is well known and studied in polymer physics. We will use this knowledge to improve our understanding of the role of LLPS in the formation of membrane-less organelles.

Critical components of membrane-less organelles can undergo LLPS in vitro (Figure 1, middle). Intrinsically disordered regions (IDRs) in these proteins typically mediate this behavior. This can happen through IDRs with low sequence complexity, or through multivalent domain-motif interactions (Figure 1, right).

Membrane-less organelles

Figure 1: We study membrane-less organelles such as stress granules and nuclear speckles, we reconstitute components that can undergo LLPS in vitro, and we characterize the molecular interactions leading to LLPS.

We are interested in characterizing

  1. sequence features and molecular interactions mediating LLPS,
  2. protein-protein interactions responsible for the recruitment of components to membrane-less organelles,
  3. impact on enzymatic function in a concentrated liquid compartment,
  4. 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 membrane-less organelles we study. But it reaches further, because LLPS has now been recognized to play a role in transcriptional regulation, DNA damage, membrane receptor clustering and the selectivity filter in the nuclear pore complex.

hnRNPA1, stress granules and ALS and other neurodegenerative diseases

We have demonstrated that a low complexity domain (LCD) within the RNA-binding protein hnRNPA1, a component of stress granules, undergoes reversible LLPS into protein-rich droplets. hnRNPA1 molecules are dynamically incorporated into droplets in vitro, allowing their entry, diffusion within, and exit, reminiscent of the dynamic properties of stress granules in cells. While the LCD of hnRNPA1 is sufficient to mediate phase separation, its folded RNA binding domains (RBDs) also contribute to phase separation in the presence of RNA, giving rise to several mechanisms of assembly. We have proposed that the architecture of the RBDs, and the specific composition and sequence patterns of the LCD, may promote dynamic compartmentalization of hnRNPA1, other RNA binding proteins and RNAs into RNP granules (Figure 2).

The LCD is the site of disease-causing mutations that enhance the fibrillization of hnRNPA1. Importantly, although not required for phase separation, fibrillization is enhanced in protein-rich droplets, presumably because the high local concentrations of hnRNPA1 in the protein-rich droplets drive nucleation processes. We suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties and provide a mechanistic link between persistent stress granules and fibrillar protein pathology in disease. (Molliex et al., Cell 2015. Figure 2.)

hnRNPA1 fibrillization model

Figure 2: Model proposing the role of LLPS in the formation of RNP granules and pathological inclusions in ALS and FTD (modified from Molliex et al., Cell 2015).

SPOP self-association leads to its localization in liquid compartments

The speckle-type POZ protein (SPOP) is a tumor suppressor and a substrate adaptor of a ubiquitin ligase and frequently mutated in cancers. SPOP forms higher-order oligomers via the synergistic function of two independent dimerization domains. Such higher-order oligomers do not have a defined oligomeric state, but exist as a distribution of different sizes, rendering their structural and functional characterization challenging. The oligomers’ structural organization and the influence of their size distribution on function were unclear. We demonstrated that the formation of large higher-order SPOP oligomers is linked to the localization of SPOP to nuclear speckles, thus elucidating the molecular basis for large protein assemblies in a membrane-less organelle. SPOP oligomerization stimulates ubiquitination activity. Based on this observation, we proposed nuclear speckles serve as critical hotspots of SPOP-mediated substrate ubiquitination, assigning a new functionality to nuclear speckles and highlighting their link to cancer pathogenesis (Marzahn et al., EMBO J 2016, Figure 3).

Data model speckle ubiquitination

Figure 3: SPOP oligomerizes via two dimerization domains, the BTB domain (red) and the BACK domain (blue). Substrate binding motifs are bound through the MATH domain (green). The resulting isodesmic self-association (left top) leads to a range of oligomers with a fixed size distribution (right top). SPOP oligomers are recruited to nuclear speckles and other liquid compartments. They also have a higher ubiquitination efficiency towards substrates than SPOP dimers and monomers. We thus propose that nuclear speckles are hotspots of SPOP-mediated ubiquitination (Marzahn et al., EMBO J 2016).

Tumor-associated SPOP mutations disrupt substrate binding and ubiquitination, leading to increased expression of oncogenic substrates, but the mechanisms by which SPOP assembles with its substrates and gets recruited to nuclear bodies remain poorly understood. We have recently investigated the mechanisms underlying colocalization of SPOP and its substrates, including the death-domain-associated protein (DAXX) and the androgen receptor (AR). When SPOP and DAXX were co-expressed, they both re-localized into nuclear bodies (termed SPOP/DAXX bodies) distinct from the PML bodies where DAXX usually resides and the nuclear speckles where SPOP generally localizes. We characterized these SPOP/DAXX bodies as liquid membraneless organelles, and the re-localization to these membrane-less bodies was disrupted by prostate cancer–associated SPOP mutations. 

The phase separation of SPOP and DAXX is mediated by weak multivalent interactions between DAXX and SPOP, which are facilitated by multiple SPOP-binding motifs in DAXX and oligomerization of SPOP. Mutations in SPOP or DAXX that disrupt phase separation, also prevent the co-localization of SPOP and DAXX, and reduce ubiquitination of DAXX.  Our evidence points to SPOP/DAXX bodies as active ubiquitination hubs, recruiting CUL3 to facilitate ubiquitination of DAXX. Similarly, other SPOP substrates harbor multiple SPOP-binding motifs, suggesting that SPOP may recruit them via phase separation. Consistent with this hypothesis, AR forms liquid droplet-like assemblies with SPOP, indicating a phase separation similar to DAXX.

These findings reveal that cancer-associated SPOP mutations disrupt the liquid-liquid phase separation that normally concentrates the components required for substrate ubiquitination, resulting in loss of function.

What can salad dressing tell us about cancer? Think oil and vinegar.

SPOP figure

Figure 4: SPOP and its substrate DAXX phase separate together, which leads to their colocalization in cells. SPOP cancer mutants are defective for phase separation, colocalization and ubiquitination activity. (Bouchard, Otero et al., Mol Cell 2018).

Sequence determinants of unperturbed global dimensions in proteins undergoing multi-site phosphorylation

Multi-site serine/threonine phosphorylation in proteins plays important roles in the regulation of transcription, translation, signaling from the cell surface to the nucleus, and protein/protein interactions. Phosphorylation typically occurs in intrinsically disordered regions (IDRs) of proteins, and multi-site phosphorylation has the potential to dramatically alter the global dimensions and local conformations of these IDRs. The degree of phosphorylation, and even the order in which individual sites become phosphorylated, may result in vastly different conformations and affect downstream signals. However, our recent work using SAXS, NMR and computational simulations (with Rohit Pappu) on an IDR of Ash1, a yeast transcription factor involved in mating-type switching, surprisingly showed that Ash1 has expanded coil-like features and that its global dimensions are insensitive to the degree of phosphorylation. We attributed this behavior to the patterning of proline and charged residues along the sequence (Figure 5), but other sequence features likely contribute to the behavior.

The expansion accompanying an FCR increase must be counteracted by a compensatory collapse. We have shown evidence that attractive and repulsive interactions screen each other, resulting in unperturbed global dimensions. The nature of the individual interactions, and how the contributing residues must be patterned along the sequence to allow Flory-type screening, are largely unknown, and we aim to uncover them.

The insensitivity to phosphorylation could be generally beneficial in proteins undergoing multi-site phosphorylation because it would largely maintain the conformational landscape of the IDR through multiple phosphorylation events, guarantee accessibility of phosphorylation motifs to readers, writers and erasers (i.e. binding partners, kinases and phosphatases). It would allow integration of multiple signaling pathways because it would uncouple the order of phosphorylation events from the downstream response.

Ash1 TOC

Figure 5: The global dimensions of non-phosphorylated and phosphorylated Ash1 are highly similar due to the patterning of proline and charged residues along the sequence (Martin et al., JACS 2016).

References

  1. A. Molliex, J. Temirov, J. Lee, M. Coughlin, A.P. Kanagaraj, H.J. Kim, T. Mittag*, J.P. Taylor*. (2015) Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization. Cell. 163(1): 123-33. PMID: 26406374
  2. M.R. Marzahn, S. Marada, J. Lee, A. Nourse, S. Kenrick, H. Zhao, G. Ben-Nissan, R.M. Kolaitis, J.L. Peters, S. Pounds, W.J. Errington, G.G. Prive, J.P. Taylor, M. Sharon, P. Schuck, S.K. Ogden*, T. Mittag*. (2016) Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles. EMBO J. 35(12): 1254-75. PMID: 27220849 Cover article
  3. W.K. Pierce, C.R. Grace, J. Lee, A. Nourse, M.R. Marzahn, E.R. Watson, A.A. High, J. Peng, B.A. Schulman, T. Mittag. (2016) Multiple weak linear motifs enhance recruitment and processivity in SPOP-mediated substrate ubiquitination. J. Mol. Biol. 428(6): 1256-71. PMID: 26475525
  4. J.J. Bouchard, J.H. Otero, D.C. Scott, E.M. Szulc, E.W. Martin, N. Sabri, D. Granata, M.R. Marzahn, K. Lindorff-Larsen, X. Salvatella, B.A. Schulman, T. Mittag. Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments. Mol Cell. Epub (2018).
  5. E.W. Martin, A. Holehouse, C.R. Grace, A. Hughes, R.V. Pappu*, T. Mittag*. (2016) Sequence determinants of the conformational properties of an intrinsically disordered protein prior to and upon multisite phosphorylation. J. Am. Chem. Soc. 138(47): 15323-15335. PMID: 27807972

*Co-corresponding authors