Summary by Dr. Louise Pinet
Tongyao Wei, Heng Liu, Bizhu Chu, Pilar Blasco, Zheng Liu, Ruijun Tian, David Xiang Li, and Xuechen Li*
TL; DR: Phosphorylation of the chromatin-associated protein HMGA1a acidic tail favors intramolecular interaction with KR-rich regions, competing with binding of these regions to p53. Phosphorylation is therefore a conformational switch indirectly regulating protein-protein interactions.
HMGA1a (High-Mobility Group A 1a) is an intrinsically disordered chromatin factor involved in many nuclear functions mainly during development, but also in several types of cancer. Its C-terminal tail (residues 92-106) is acidic due to its high content in glutamic acids. Its function is unknown, since HMGA1a AT-hooks and flanking regions have been identified as responsible for DNA- and protein-binding. Here the group of X. Li studied the influence of phosphorylation of the acidic tail on conformation and protein-protein interactions.
The authors chemically synthesized homogeneous samples of di- (S101, S102) and tri- (S98, S101, S102) phosphorylated forms of HMGA1a, that are the predominant in the cell, as well as a mono-phosphorylated form (S102), using ligation. They showed that increasing HMGA1a phosphorylation level (and introducing phospho-mimetic glutamate mutations) decreases affinity for p53 compared to unphosphorylated WT protein, with no interaction at all detected by pull-down for the tri-phosphorylated sample. The phospho-mimetic mutant also shows reduced p53 inhibition in dual-luciferase assays compared to a charge-neutral triple-alanine mutant. The authors then used fragments to show that the HMGA1a-p53 interaction is mediated by KR-rich regions of HMGA1a, and therefore involves the acidic tail only indirectly. They showed by NMR (NOEs) that phosphorylation of the acidic tail increases intramolecular contacts in HMGA1a. More precisely, pull-down and ITC studies of HMGA1a fragments prove that the phosphorylated tail can interact intermolecularly with the KR-rich region in a stronger way than the non-phosphorylated form, efficiently competing with p53. The authors therefore assume that in the full-length phosphorylated HMGA1a, intramolecular long-range contacts between the phosphorylated acidic tail and KR-rich regions prevent binding of the latter to p53. Finally, synthetic protein-affinity purification mass spectrometry showed that several proteins with acidic regions also bind preferentially to unphosphorylated HMGA1a rather than to its tri-phosphorylated counterpart, suggesting a common regulation mechanism.
Xuechen Li is a Professor in the Department of Chemistry at the University of Hong Kong.
Link to Paper
Yi Lu, Tiantian Wu, Orit Gutman, Huasong Lu, Qiang Zhou, Yoav I. Henis* and Kunxin Luo*
TL; DR: Liquid-liquid phase separation of transcription factors can promote transcriptional activation.
YAP and TAZ are key transcriptional factors of the Hippo pathway regulating cell proliferation and apoptosis. Previously, YAP was found undergoing liquid-liquid phase separation (LLPS) in vivo following osmotic stress. In this paper, Lu and his coworker found the TAZ can form liquid-like biomolecular condensates in vitro and in vivo.
Most proteins undergoing LLPS are disordered proteins. TAZ is predicted to be a highly disordered protein based on IUPred’s prediction. TAZ appears to form phase condensate in different constructs under the regular environmental condition without osmotic stress in vivo and in vitro. Several TAZ domains are deleted or swapped with corresponding domains in YAP to identify which domain determines TAZ LLPS potential. CC domain and WW domain are found to be necessary for TAZ LLPS but are not sufficient for triggering the TAZ LLPS individually.
To reveal how LLPS contributes to TAZ functions, serum starvation, high cell density, and other treatments are applied to cell lines to trigger Hippo signaling. These treatments prohibit the formation of TAZ condensate. Fluorescence microscopy shows the formation of TAZ condensate in the nucleus can recruit and colocalize TEAD4, BRD4, and other transcriptional regulation components in the nuclear puncta. By monitoring the expression rate of the TAZ target gene, they also found TAZ demonstrates a higher transcriptional activity compared to YAP and non-LLPS TAZ mutants. These results suggest TAZ LLPS can be regulated by the Hippo pathway and can be essential for efficient transcription activation.
Yoav Henis is an Emeritus Professor in the Department of Neurobiology at Tel Aviv University.
Kunxin Luo is a Professor of Cell and Developmental Biology at University of California, Berkeley.
Link to Paper
Conformational Expansion of Tau in Condensates Promotes Irreversible Aggregation
Jitao Wen, Liu Hong, Georg Krainer, Qiong-Qiong Yao, Tuomas P. J. Knowles, Si Wu,* and Sarah Perrett*
TL;DR: Tau proteins experience an expansion in LLPS which seems to be key for promoting aggregation.
Tau proteins are receiving a lot of attention because their aberrant aggregation is associated with highly debilitating diseases, like Alzheimer’s disease. Therefore, understanding the transition from the physiological state to the malignant one is a key step towards the pursuit of a treatment, or ideally a cure.
Wen and colleagues tried to describe the conditions at which such transition takes place. It is shown that, like many other proteins with intrinsically disordered regions (IDRs), also Tau in solution can generate liquid-liquid phase separation (LLPS), forming droplets where Tau is highly concentrated (also called biomolecular condensates). The underlying motivation of the study was to understand the role of LLPS in the irreversible aggregation of Tau, providing molecular insights on the structural process. To do so, the authors first mapped the phase space where Tau phase separates along the dimensions of salt concentration in solution, and the concentration of PEG (which has the role of a molecular crowder). Then, they prepared Tau variants with two fluorophores in specific sites for single molecule Forster resonance energy transfer (sm-FRET): in this way, they could measure that Tau experiences a conformational expansion when in the condensed phase compared to the dilute one. With other single labelled Tau variants, the authors also showed the propensity of Tau to form nanoscale condensates whose sizes are concentration dependent. But the most critical observation the authors made is that the formation of irreversible aggregates happens only if the proteins experience liquid-liquid phase separation as an intermediate step, showing that this is critical for the initiation of the self-aggregation of Tau. The information learned from the sm-FRET experiments allows the authors to conclude that the chain expansion measured inside the condensates has the effect of exposing the microtubule-binding regions, facilitating the intermolecular interaction and therefore the aberrant aggregation.
Sarah Perret is a Professor in the National Laboratory of Biomacromolecules in the Institute of Biophysics at the Chinese Academy of Sciences, Beijing. Si Wu is a scientist in the laboratory of Prof. Sarah Perret. Link to Paper
Structural insights into α-synuclein monomer–fibril interactions
Pratibha Kumari, Dhiman Ghosh, Agathe Vanas, Yanick Fleischmann, Thomas Wiegand, Gunnar Jeschke, Roland Riek* and Cédric Eichmann*
TL;DR: Conformational changes associated with monomeric α-synuclein binding it’s fibrillar counterpart reveals the molecular basis of secondary nucleation.
Aberrant aggregation of a-Synuclein, an IDP, into amyloid fibrils in neuronal cells engenders neurodegenerative diseases such as Parkinson’s disease. Mechanistic insights into the aggregation process of soluble a-Synuclein is crucial for designing therapeutic interventions. Of particular relevance in aggregation kinetics of a-Synuclein is the secondary nucleation mechanism where the surface of the fibril catalyses formation of new seeds for amyloid fibrils.
Kumari et al., in this communication using a combination of NMR and EPR spectroscopy probe the interaction of a-Synuclein with fibrils and elucidate the molecular underpinnings of secondary nucleation. Using NMR spectroscopy with labelled a-Synuclein and unlabelled fibrils, and by changing solution conditions (salt concentration) and the constructs (charge mutations and domain deletion constructs) they show that N-terminal of a-Synuclein monomer interacts with exposed C-terminal of the fibrillar a-Synuclein with millimolar affinity predominantly via transient electrostatic interactions. Using PRE and DEER measurements they further probe the conformational changes in fibril bound a-Synuclein monomers; they observe fibril bound a-Synuclein monomers to adopt a more extended conformer. This conformational transition in a-Synuclein upon fibril binding exposes the NAC region which is implicated for fibril formation. They hypothesize that partially ordered orientation of fibril bound a-Synuclein monomers, resulting from interaction of N-terminal of the a-Synuclein monomers with the C-terminal of fibrillar a-Synuclein, results in a high local concentration of a-Synuclein on fibril surface having a conformation and orientation that are conducive for formation of amyloids seeds. Their results uncover possible molecular basis of secondary nucleation in a-Synuclein aggregation.
Roland Riek is a Professor in the Laboratory of Physical Chemistry in the Department of Chemistry and Applied Biosciences at the ETH Zurich.
Cédric Eichmann is a senior scientist in the Department of Biological Regulation at the Weizmann Institute of Science.
Link to Paper