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Dive into the research topics where Tanja Mittag is active.

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Featured researches published by Tanja Mittag.


Cell | 2015

Phase Separation by Low Complexity Domains Promotes Stress Granule Assembly and Drives Pathological Fibrillization

Amandine Molliex; Jamshid Temirov; Jihun Lee; Maura Coughlin; Anderson P. Kanagaraj; Hong Joo Kim; Tanja Mittag; J. Paul Taylor

Stress granules are membrane-less organelles composed of RNA-binding proteins (RBPs) and RNA. Functional impairment of stress granules has been implicated in amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy-diseases that are characterized by fibrillar inclusions of RBPs. Genetic evidence suggests a link between persistent stress granules and the accumulation of pathological inclusions. Here, we demonstrate that the disease-related RBP hnRNPA1 undergoes liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by a low complexity sequence domain (LCD). While the LCD of hnRNPA1 is sufficient to mediate LLPS, the RNA recognition motifs contribute to LLPS in the presence of RNA, giving rise to several mechanisms for regulating assembly. Importantly, while not required for LLPS, fibrillization is enhanced in protein-rich droplets. We suggest that LCD-mediated LLPS contributes to the assembly of stress granules and their liquid properties and provides a mechanistic link between persistent stress granules and fibrillar protein pathology in disease.


Journal of Molecular Recognition | 2009

Protein dynamics and conformational disorder in molecular recognition

Tanja Mittag; Lewis E. Kay; Julie D. Forman-Kay

Recognition requires protein flexibility because it facilitates conformational rearrangements and induced‐fit mechanisms upon target binding. Intrinsic disorder is an extreme on the continuous spectrum of possible protein dynamics and its role in recognition may seem counterintuitive. However, conformational disorder is widely found in many eukaryotic regulatory proteins involved in processes such as signal transduction and transcription. Disordered protein regions may in fact confer advantages over folded proteins in binding. Rapidly interconverting and diverse conformers may create mean electrostatic fields instead of presenting discrete charges. The resultant “polyelectrostatic” interactions allow for the utilization of post‐translational modifications as a means to change the net charge and thereby modify the electrostatic interaction of a disordered region. Plasticity of disordered protein states enables steric advantages over folded proteins and allows for unique binding configurations. Disorder may also have evolutionary advantages, as it facilitates alternative splicing, domain shuffling and protein modularity. As proteins exist in a continuous spectrum of disorder, so do their complexes. Indeed, disordered regions in complexes may control the degree of motion between domains, mask binding sites, be targets of post‐translational modifications, permit overlapping binding motifs, and enable transient binding of different binding partners, making them excellent candidates for signal integrators and explaining their prevalence in eukaryotic signaling pathways. “Dynamic” complexes arise if more than two transient protein interfaces are involved in complex formation of two binding partners in a dynamic equilibrium. “Disordered” complexes, in contrast, do not involve significant ordering of interacting protein segments but rely exclusively on transient contacts. The nature of these interactions is not well understood yet but advancements in the structural characterization of disordered states will help us gain insights into their function and their implications for health and disease. Copyright


Structure | 2010

Structure/Function Implications in a Dynamic Complex of the Intrinsically Disordered Sic1 with the Cdc4 Subunit of an SCF Ubiquitin Ligase

Tanja Mittag; Joseph A. Marsh; Alexander Grishaev; Stephen Orlicky; Hong Lin; Frank Sicheri; Mike Tyers; Julie D. Forman-Kay

Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.


Proceedings of the National Academy of Sciences of the United States of America | 2007

Polyelectrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity

Tanja Mittag; Tony Pawson; Mike Tyers; Julie D. Forman-Kay; Hue Sun Chan

Regulation of biological processes often involves phosphorylation of intrinsically disordered protein regions, thereby modulating protein interactions. Initiation of DNA replication in yeast requires elimination of the cyclin-dependent kinase inhibitor Sic1 via the SCFCdc4 ubiquitin ligase. Intriguingly, the substrate adapter subunit Cdc4 binds to Sic1 only after phosphorylation of a minimum of any six of the nine cyclin-dependent kinase sites on Sic1. To investigate the physical basis of this ultrasensitive interaction, we consider a mean-field statistical mechanical model for the electrostatic interactions between a single receptor site and a conformationally disordered polyvalent ligand. The formulation treats phosphorylation sites as negative contributions to the total charge of the ligand and addresses its interplay with the strength of the favorable ligand–receptor contact. Our model predicts a threshold number of phosphorylation sites for receptor–ligand binding, suggesting that ultrasensitivity in the Sic1–Cdc4 system may be driven at least in part by cumulative electrostatic interactions. This hypothesis is supported by experimental affinities of Cdc4 for Sic1 fragments with different total charges. Thus, polyelectrostatic interactions may provide a simple yet powerful framework for understanding the modulation of protein interactions by multiple phosphorylation sites in disordered protein regions.


Structure | 2013

From Sequence and Forces to Structure, Function, and Evolution of Intrinsically Disordered Proteins

Julie D. Forman-Kay; Tanja Mittag

Intrinsically disordered proteins (IDPs), which lack persistent structure, are a challenge to structural biology due to the inapplicability of standard methods for characterization of folded proteins as well as their deviation from the dominant structure/function paradigm. Their widespread presence and involvement in biological function, however, has spurred the growing acceptance of the importance of IDPs and the development of new tools for studying their structure, dynamics, and function. The interplay of folded and disordered domains or regions for function and the existence of a continuum of protein states with respect to conformational energetics, motional timescales, and compactness are shaping a unified understanding of structure-dynamics-disorder/function relationships. In the 20(th) anniversary of Structure, we provide a historical perspective on the investigation of IDPs and summarize the sequence features and physical forces that underlie their unique structural, functional, and evolutionary properties.


Proceedings of the National Academy of Sciences of the United States of America | 2012

Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase

Xiaojing Tang; Stephen Orlicky; Tanja Mittag; Veronika Csizmok; Tony Pawson; Julie D. Forman-Kay; Frank Sicheri; Mike Tyers

The ubiquitin ligase SCFCdc4 (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. Composite dynamic interactions of low affinity sites may be a general mechanism to establish phosphorylation thresholds in biological responses.


The EMBO Journal | 2016

Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.

Melissa R. Marzahn; Suresh Marada; Jihun Lee; Amanda Nourse; Sophia Kenrick; Huaying Zhao; Gili Ben-Nissan; Regina Maria Kolaitis; Jennifer L. Peters; Stanley Pounds; Wesley J. Errington; Gilbert G. Privé; J. Paul Taylor; Michal Sharon; Peter Schuck; Stacey K. Ogden; Tanja Mittag

Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.


Trends in Cell Biology | 2018

Protein Phase Separation: A New Phase in Cell Biology

Steven Boeynaems; Simon Alberti; Nicolas L. Fawzi; Tanja Mittag; Magdalini Polymenidou; Frederic Rousseau; Joost Schymkowitz; James Shorter; Benjamin Wolozin; Ludo Van Den Bosch; Peter Tompa; Monika Fuxreiter

Cellular compartments and organelles organize biological matter. Most well-known organelles are separated by a membrane boundary from their surrounding milieu. There are also many so-called membraneless organelles and recent studies suggest that these organelles, which are supramolecular assemblies of proteins and RNA molecules, form via protein phase separation. Recent discoveries have shed light on the molecular properties, formation, regulation, and function of membraneless organelles. A combination of techniques from cell biology, biophysics, physical chemistry, structural biology, and bioinformatics are starting to help establish the molecular principles of an emerging field, thus paving the way for exciting discoveries, including novel therapeutic approaches for the treatment of age-related disorders.


Nature Communications | 2017

An allosteric conduit facilitates dynamic multisite substrate recognition by the SCF Cdc4 ubiquitin ligase

Veronika Csizmok; Stephen Orlicky; Jing Cheng; Jianhui Song; Alaji Bah; Neda Delgoshaie; Hong Lin; Tanja Mittag; Frank Sicheri; Hue Sun Chan; Mike Tyers; Julie D. Forman-Kay

The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.


Journal of Molecular Biology | 2016

Multiple Weak Linear Motifs Enhance Recruitment and Processivity in SPOP-Mediated Substrate Ubiquitination.

Wendy K. Pierce; Christy Rani R. Grace; Jihun Lee; Amanda Nourse; Melissa R. Marzahn; Edmond R. Watson; Anthony A. High; Junmin Peng; Brenda A. Schulman; Tanja Mittag

Primary sequence motifs, with millimolar affinities for binding partners, are abundant in disordered protein regions. In multivalent interactions, such weak linear motifs can cooperate to recruit binding partners via avidity effects. If linear motifs recruit modifying enzymes, optimal placement of weak motifs may regulate access to modification sites. Weak motifs may thus exert physiological relevance stronger than that suggested by their affinities, but molecular mechanisms of their function are still poorly understood. Herein, we use the N-terminal disordered region of the Hedgehog transcriptional regulator Gli3 (Gli3(1-90)) to determine the role of weak motifs encoded in its primary sequence for the recruitment of its ubiquitin ligase CRL3(SPOP) and the subsequent effect on ubiquitination efficiency. The substrate adaptor SPOP binds linear motifs through its MATH (meprin and TRAF homology) domain and forms higher-order oligomers through its oligomerization domains, rendering SPOP multivalent for its substrates. Gli3 has multiple weak SPOP binding motifs. We map three such motifs in Gli3(1-90), the weakest of which has a millimolar dissociation constant. Multivalency of ligase and substrate for each other facilitates enhanced ligase recruitment and stimulates Gli3(1-90) ubiquitination in in vitro ubiquitination assays. We speculate that the weak motifs enable processivity through avidity effects and by providing steric access to lysine residues that are otherwise not prioritized for polyubiquitination. Weak motifs may generally be employed in multivalent systems to act as gatekeepers regulating post-translational modification.

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Melissa R. Marzahn

St. Jude Children's Research Hospital

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Amanda Nourse

St. Jude Children's Research Hospital

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Jihun Lee

St. Jude Children's Research Hospital

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Mike Tyers

Université de Montréal

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Brenda A. Schulman

St. Jude Children's Research Hospital

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