Charge segregation in the intrinsically disordered region governs VRN1 and DNA liquid-like phase separation robustness

In Wnt/β-catenin signaling, the β-catenin protein level is deliberately controlled by the assembly of the multiprotein β-catenin destruction complex composed of Axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β), casein kinase 1α (CK1α), and others. Here we provide compelling evidence that formation of the destruction complex is driven by protein liquid–liquid phase separation (LLPS) of Axin. An intrinsically disordered region in Axin plays an important role in driving its LLPS. Phase-separated Axin provides a scaffold for recruiting GSK3β, CK1α, and β-catenin. APC also undergoes LLPS in vitro and enhances the size and dynamics of Axin phase droplets. The LLPS-driven assembly of the destruction complex facilitates β-catenin phosphorylation by GSK3β and is critical for the regulation of β-catenin protein stability and thus Wnt/β-catenin signaling.

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Phase separation of Nur77 mediates celastrol-induced mitophagy by promoting the liquidity of p62/SQSTM1 condensates

Nature Communications ◽

10.1038/s41467-021-26295-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shuang-zhou Peng ◽

Xiao-hui Chen ◽

Si-jie Chen ◽

Jie Zhang ◽

Chuan-ying Wang ◽

...

Keyword(s):

Phase Separation ◽

Cellular Functions ◽

Intrinsically Disordered ◽

Mechanistic Insight ◽

Intrinsically Disordered Region ◽

Uba Domain ◽

Pb1 Domain ◽

Additional Interaction ◽

Insight Into ◽

Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation promotes the formation of membraneless condensates that mediate diverse cellular functions, including autophagy of misfolded proteins. However, how phase separation participates in autophagy of dysfunctional mitochondria (mitophagy) remains obscure. We previously discovered that nuclear receptor Nur77 (also called TR3, NGFI-B, or NR4A1) translocates from the nucleus to mitochondria to mediate celastrol-induced mitophagy through interaction with p62/SQSTM1. Here, we show that the ubiquitinated mitochondrial Nur77 forms membraneless condensates capable of sequestrating damaged mitochondria by interacting with the UBA domain of p62/SQSTM1. However, tethering clustered mitochondria to the autophagy machinery requires an additional interaction mediated by the N-terminal intrinsically disordered region (IDR) of Nur77 and the N-terminal PB1 domain of p62/SQSTM1, which confers Nur77-p62/SQSTM1 condensates with the magnitude and liquidity. Our results demonstrate how composite multivalent interaction between Nur77 and p62/SQSTM1 coordinates to sequester damaged mitochondria and to connect targeted cargo mitochondria for autophagy, providing mechanistic insight into mitophagy.

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The Ligand of Ate1 is intrinsically disordered and participates in nucleolar phase separation regulated by Jumonji Domain Containing 6

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2015887118 ◽

2020 ◽

Vol 118 (1) ◽

pp. e2015887118

Author(s):

Akshaya Arva ◽

Yasar Arfat T. Kasu ◽

Jennifer Duncan ◽

Mosleh A. Alkhatatbeh ◽

Christopher S. Brower

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Protein Degradation ◽

Unknown Function ◽

Low Complexity ◽

Bimolecular Fluorescence Complementation ◽

Hydroxylase Activity ◽

Intrinsically Disordered ◽

Intrinsically Disordered Region ◽

Liquid Liquid Phase Separation

The Ligand of Ate1 (Liat1) is a protein of unknown function that was originally discovered through its interaction with arginyl-tRNA protein transferase 1 (Ate1), a component of the Arg/N-degron pathway of protein degradation. Here, we characterized the functional domains of mouse Liat1 and found that its N-terminal half comprises an intrinsically disordered region (IDR) that facilitates its liquid–liquid phase separation (LLPS) in the nucleolus. Using bimolecular fluorescence complementation and immunocytochemistry, we found that Liat1 is targeted to the nucleolus by a low-complexity poly-K region within its IDR. We also found that the lysyl-hydroxylase activity of Jumonji Domain Containing 6 (Jmjd6) modifies Liat1, in a manner that requires the Liat1 poly-K region, and inhibits its nucleolar targeting and potential functions. In sum, this study reveals that Liat1 participates in nucleolar LLPS regulated by Jmjd6.

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Deep-learning enables proteome-scale identification of phase-separated protein candidates from immunofluorescence images

10.1101/636738 ◽

2019 ◽

Author(s):

Chunyu Yu ◽

Boyan Shen ◽

Qi Huang ◽

Minglei Shi ◽

Kaiqiang You ◽

...

Keyword(s):

Phase Separation ◽

Binary Classification ◽

Classification Problem ◽

Protein Recognition ◽

Spherical Droplet ◽

Intrinsically Disordered ◽

Intrinsically Disordered Region ◽

Human Protein Atlas ◽

Subsequent Phase ◽

Binary Classification Problem

AbstractIntrinsically disordered region (IDR) analysis has been widely used in the screening of phase-separated proteins. However, the precise sequences determining phase separation remain unclear. Furthermore, a large number of phase-separated proteins that exhibit relatively low IDR content remain uncharacterized. Phase-separated proteins appear as spherical droplet structures in immunofluorescence (IF) images, which renders them distinguishable from non-phase-separated proteins. Here, we transformed the problem of phase-separated protein recognition into a binary classification problem of image recognition. In addition, we established a method named IDeepPhase to identify IF images with spherical droplet structures based on convolutional neural networks. Using IDeepPhase on proteome-scale IF images from the Human Protein Atlas database, we generated a comprehensive list of phase-separated candidates which displayed spherical droplet structures in IF images, allowing nomination of proteins, antibodies and cell lines for subsequent phase separation study.

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JRAB/MICAL-L2 undergoes liquid–liquid phase separation to form tubular recycling endosomes

Communications Biology ◽

10.1038/s42003-021-02080-7 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Ayuko Sakane ◽

Taka-aki Yano ◽

Takayuki Uchihashi ◽

Kazuki Horikawa ◽

Yusuke Hara ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Closed Form ◽

Liquid Phase Separation ◽

Cellular Functions ◽

Recycling Endosomes ◽

Intrinsically Disordered ◽

Intrinsically Disordered Region ◽

Multiple Molecules ◽

Liquid Liquid Phase Separation

AbstractElongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid–liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.

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Association/dissociation Mechanisms of Intrinsically Disordered Region of Protein Beyond Conformational Selection and Induced Fit

10.26434/chemrxiv.9874379 ◽

2019 ◽

Author(s):

Duy Phuoc Tran ◽

Akio Kitao

Keyword(s):

Free Energy ◽

Transcriptional Activation ◽

Binding Free Energy ◽

Energy Structure ◽

Induced Fit ◽

Hydrogen Bond Formation ◽

Conformational Selection ◽

Intrinsically Disordered ◽

Intrinsically Disordered Region ◽

Dissociation Mechanisms

<p>We investigate association and dissociation mechanisms of a typical intrinsically disordered region (IDR), transcriptional activation subdomain of tumor repressor protein p53 (TAD-p53) with murine double-minute clone 2 protein (MDM2). Using the combination of cycles of association and dissociation parallel cascade molecular dynamics, multiple standard MD, and Markov state model, we are successful in obtaining the lowest free energy structure of MDM2/TAD-p53 complex as the structure very close to that in crystal without prior knowledge. This method also reproduces the experimentally measured standard binding free energy, and association and dissociation rate constants solely with the accumulated MD simulation cost of 11.675 μs, in spite of the fact that actual dissociation occurs in the order of a second. Although there exist a few complex intermediates with similar free energies, TAD-p53 first binds MDM2 as the second lowest free energy intermediate dominantly (> 90% in flux), taking a form similar to one of the intermediate structures in its monomeric state. The mechanism of this step has a feature of conformational selection. In the second step, dehydration of the interface, formation of π-π stackings of the side-chains, and main-chain relaxation/hydrogen bond formation to complete α-helix take place, showing features of induced fit. In addition, dehydration (dewetting) is a key process for the final relaxation around the complex interface. These results demonstrate a more fine-grained view of the IDR association/dissociation beyond classical views of protein conformational change upon binding.</p>

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Faculty Opinions recommendation of Multiple interactions of the intrinsically disordered region between the helicase and nuclease domains of the archaeal Hef protein.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718458952.793496527 ◽

2014 ◽

Author(s):

Vladimir Uversky

Keyword(s):

Intrinsically Disordered ◽

Intrinsically Disordered Region ◽

Multiple Interactions

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