Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

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Enhancing c-MYC degradation via 20S proteasome activation induces in vivo anti-tumor efficacy

10.1101/2020.08.24.265470 ◽

2020 ◽

Cited By ~ 1

Author(s):

Evert Njomen ◽

Theresa A. Lansdell ◽

Allison Vanecek ◽

Vanessa Benham ◽

Matt P. Bernard ◽

...

Keyword(s):

Therapeutic Strategy ◽

Target Genes ◽

Intrinsically Disordered Proteins ◽

Therapeutic Potential ◽

Human Cancer ◽

Disordered Proteins ◽

20S Proteasome ◽

Intrinsically Disordered

SUMMARYEnhancing proteasome activity is a potential new therapeutic strategy to prevent the accumulation of aberrant high levels of protein that drive the pathogenesis of many diseases. Herein, we examine the use of small molecules to activate the 20S proteasome to reduce aberrant signaling by the undruggable oncoprotein c-MYC, to treat c-MYC driven oncogenesis. Overexpression of c-MYC is found in more than 50% of all human cancer but remains undruggable because of its highly dynamic intrinsically disordered 3-D conformation, which renders traditional therapeutic strategies largely ineffective. We demonstrate herein that small molecule activation of the 20S proteasome targets dysregulated intrinsically disordered proteins (IDPs), including c-MYC, and reduces cancer growth in vitro and in vivo models of multiple myeloma, and is even effective in bortezomib resistant cells and unresponsive patient samples. Genomic analysis of various cancer pathways showed that proteasome activation results in downregulation of many c-MYC target genes. Moreover, proteasome enhancement was well tolerated in mice and dogs. These data support the therapeutic potential of 20S proteasome activation in targeting IDP-driven proteotoxic disorders, including cancer, and demonstrate that this new therapeutic strategy is well tolerated in vivo.

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Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2000223117 ◽

2020 ◽

Vol 117 (21) ◽

pp. 11421-11431 ◽

Cited By ~ 21

Author(s):

Benjamin S. Schuster ◽

Gregory L. Dignon ◽

Wai Shing Tang ◽

Fleurie M. Kelley ◽

Aishwarya Kanchi Ranganath ◽

...

Keyword(s):

Phase Separation ◽

Intrinsically Disordered Proteins ◽

Lipid Membrane ◽

Coarse Grained ◽

Disordered Proteins ◽

Sequence Features ◽

Intrinsically Disordered ◽

Arginine Residues

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

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Interplay of Structural Disorder and Short Binding Elements in the Cellular Chaperone Function of Plant Dehydrin ERD14

Cells ◽

10.3390/cells9081856 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1856

Author(s):

Nikoletta Murvai ◽

Lajos Kalmar ◽

Bianka Szalaine Agoston ◽

Beata Szabo ◽

Agnes Tantos ◽

...

Keyword(s):

Heat Stress ◽

Intrinsically Disordered Proteins ◽

Stress Protein ◽

Structural Disorder ◽

Disordered Proteins ◽

Sequence Motifs ◽

E Coli ◽

Intrinsically Disordered

Details of the functional mechanisms of intrinsically disordered proteins (IDPs) in living cells is an area not frequently investigated. Here, we dissect the molecular mechanism of action of an IDP in cells by detailed structural analyses based on an in-cell nuclear magnetic resonance experiment. We show that the ID stress protein (IDSP) A. thaliana Early Response to Dehydration (ERD14) is capable of protecting E. coli cells under heat stress. The overexpression of ERD14 increases the viability of E. coli cells from 38.9% to 73.9% following heat stress (50 °C × 15 min). We also provide evidence that the protection is mainly achieved by protecting the proteome of the cells. In-cell NMR experiments performed in E. coli cells show that the protective activity is associated with a largely disordered structural state with conserved, short sequence motifs (K- and H-segments), which transiently sample helical conformations in vitro and engage in partner binding in vivo. Other regions of the protein, such as its S segment and its regions linking and flanking the binding motifs, remain unbound and disordered in the cell. Our data suggest that the cellular function of ERD14 is compatible with its residual structural disorder in vivo.

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Desiccation tolerance: an unusual window into stress biology

Molecular Biology of the Cell ◽

10.1091/mbc.e17-04-0257 ◽

2019 ◽

Vol 30 (6) ◽

pp. 737-741 ◽

Cited By ~ 5

Author(s):

Douglas Koshland ◽

Hugo Tapia

Keyword(s):

Water Loss ◽

Desiccation Tolerance ◽

Intrinsically Disordered Proteins ◽

Membrane Integrity ◽

Disordered Proteins ◽

In Vivo Studies ◽

Intrinsically Disordered ◽

As Stress

Climate change has accentuated the importance of understanding how organisms respond to stresses imposed by changes to their environment, like water availability. Unusual organisms, called anhydrobiotes, can survive loss of almost all intracellular water. Desiccation tolerance of anhydrobiotes provides an unusual window to study the stresses and stress response imposed by water loss. Because of the myriad of stresses that could be induced by water loss, desiccation tolerance seemed likely to require many established stress effectors. The sugar trehalose and hydrophilins (small intrinsically disordered proteins) had also been proposed as stress effectors against desiccation because they were found in nearly all anhydrobiotes, and could mitigate desiccation-induced damage to model proteins and membranes in vitro. Here, we summarize in vivo studies of desiccation tolerance in worms, yeast, and tardigrades. These studies demonstrate the remarkable potency of trehalose and a subset of hydrophilins as the major stress effectors of desiccation tolerance. They act, at least in part, by limiting in vivo protein aggregation and loss of membrane integrity. The apparent specialization of individual hydrophilins for desiccation tolerance suggests that other hydrophilins may have distinct roles in mitigating additional cellular stresses, thereby defining a potentially new functionally diverse set of stress effectors.

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A 20S proteasome receptor for degradation of intrinsically disordered proteins

10.1101/210898 ◽

2017 ◽

Cited By ~ 1

Author(s):

Assaf Biran ◽

Nadav Myers ◽

Julia Adler ◽

Karin Broennimann ◽

Nina Reuven ◽

...

Keyword(s):

Intrinsically Disordered Proteins ◽

Proteasomal Degradation ◽

Disordered Proteins ◽

20S Proteasome ◽

Intrinsically Disordered ◽

C Terminus ◽

Protein Interactome

AbstractDegradation of intrinsically disordered proteins (IDPs) by the 20S proteasome, unlike ubiquitin-dependent 26S proteasomal degradation, does not require proteasomal targeting by polyubiquitin. However, how these proteins are recognized by the proteasome was unknown. We report here on a mechanism of 20S proteasome targeting. Analysis of protein interactome datasets revealed that the proteasome subunit PSMA3 interacts with many IDPs. By employing in vivo and cell-free experiments we demonstrated that the PSMA3 C-terminus binds p21, c-Fos and p53, all IDPs and 20S proteasome substrates. A 69 amino-acids long fragment is autonomously functional in interacting with IDP substrates. Remarkably, this fragment in isolation blocks the degradation of a large number of IDPs in vitro and increases the half-life of proteins in vivo. We propose a model whereby the PSMA3 C-terminal region plays a role of substrate receptor in the process of proteasomal degradation of many IDPs.

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Interactions of nuclear transport factors and surface-conjugated FG nucleoporins: Insights and limitations

10.1101/435651 ◽

2018 ◽

Author(s):

Ryo Hayama ◽

Mirco Sorci ◽

John J. Keating ◽

Lee M. Hecht ◽

Joel L. Plawsky ◽

...

Keyword(s):

Protein Interactions ◽

Intrinsically Disordered Proteins ◽

Binding Mode ◽

Nucleocytoplasmic Transport ◽

Disordered Proteins ◽

Protein Protein Interactions ◽

Force Microscopy ◽

Intrinsically Disordered ◽

Mutant Variant

ABSTRACTProtein-protein interactions are central to biological processes and the methods to thoroughly characterize them are of great interest. In vitro methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions involved in nucleocytoplasmic transport as a model system, we examined the utility of three surface-based methods in characterizing rapid interactions involving intrinsically disordered proteins: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the existence of previously overlooked mass transport limitations was revealed. Additional experiments with a loss-of-interaction mutant variant demonstrated the existence of additional physical events and an uncharacterized binding mode. These results suggest the binding events that take place on the surface are more complex than initially assumed, prompting a need for re-interpretation of previous data.

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Converging on the function of intrinsically disordered nucleoporins in the nuclear pore complex

Biological Chemistry ◽

10.1515/bc.2010.092 ◽

2010 ◽

Vol 391 (7) ◽

Cited By ~ 30

Author(s):

Orit Peleg ◽

Roderick Y.H. Lim

Keyword(s):

Nuclear Pore Complex ◽

Intrinsically Disordered Proteins ◽

Molecular Transport ◽

Disordered Proteins ◽

Nuclear Pore ◽

Biological Mechanisms ◽

Intrinsically Disordered ◽

Polymeric Chains ◽

Insight Into

Abstract Several biological mechanisms involve proteins or proteinaceous components that are intrinsically disordered. A case in point pertains to the nuclear pore complex (NPC), which regulates molecular transport between the nucleus and the cytoplasm. NPC functionality is dependent on unfolded domains rich in Phe-Gly (FG) repeats (i.e., FG-domains) that collectively act to promote or hinder cargo translocation. To a large extent, our understanding of FG-domain behavior is limited to in vitro investigations given the difficulty to resolve them directly in the NPC. Nevertheless, recent findings indicate a collective convergence towards rationalizing FG-domain function. This review aims to glean further insight into this fascinating problem by taking an objective look at the boundary conditions and contextual details underpinning FG-domain behavior in the NPC. Here, we treat the FG-domains as being commensurate with polymeric chains to address ambiguities such as for instance, how FG-domains tethered to the central channel of the NPC would behave differently as compared with their free-floating counterparts in solution. By bringing such fundamental questions to the fore, this review seeks to illuminate the importance of how such parameters can hold influence over the structure-function relation of intrinsically disordered proteins in the NPC and beyond.

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Therapeutic Interventions of Cancers Using Intrinsically Disordered Proteins as Drug Targets: c-Myc as Model System

Cancer Informatics ◽

10.1177/1176935117699408 ◽

2017 ◽

Vol 16 ◽

pp. 117693511769940 ◽

Cited By ~ 31

Author(s):

Deepak Kumar ◽

Nitin Sharma ◽

Rajanish Giri

Keyword(s):

Drug Targets ◽

Intrinsically Disordered Proteins ◽

Hot Spot ◽

Intrinsic Disorder ◽

Therapeutic Interventions ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

In Vivo Experiments

The concept of protein intrinsic disorder has taken the driving seat to understand regulatory proteins in general. Reports suggest that in mammals nearly 75% of signalling proteins contain long disordered regions with greater than 30 amino acid residues. Therefore, intrinsically disordered proteins (IDPs) have been implicated in several human diseases and should be considered as potential novel drug targets. Moreover, intrinsic disorder provides a huge multifunctional capability to hub proteins such as c-Myc and p53. c-Myc is the hot spot for understanding and developing therapeutics against cancers and cancer stem cells. Our past understanding is mainly based on in vitro and in vivo experiments conducted using c-Myc as whole protein. Using the reductionist approach, c-Myc oncoprotein has been divided into structured and disordered domains. A wealth of data is available dealing with the structured perspectives of c-Myc, but understanding c-Myc in terms of disordered domains has just begun. Disorderness provides enormous flexibility to proteins in general for binding to numerous partners. Here, we have reviewed the current progress on understanding c-Myc using the emerging concept of IDPs.

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Multifunctionality of F-rich nucleoporins

Biochemical Society Transactions ◽

10.1042/bst20200357 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2603-2614

Author(s):

Nike Heinß ◽

Mikhail Sushkin ◽

Miao Yu ◽

Edward A. Lemke

Keyword(s):

High Speed ◽

Intrinsically Disordered Proteins ◽

Nucleocytoplasmic Transport ◽

Disordered Proteins ◽

Nuclear Pore ◽

Permeability Barrier ◽

Biological Processes ◽

Macromolecular Assembly ◽

Intrinsically Disordered ◽

The Family

Nucleoporins (Nups) represent a range of proteins most known for composing the macromolecular assembly of the nuclear pore complex (NPC). Among them, the family of intrinsically disordered proteins (IDPs) phenylalanine-glycine (FG) rich Nups, form the permeability barrier and coordinate the high-speed nucleocytoplasmic transport in a selective way. Those FG-Nups have been demonstrated to participate in various biological processes besides nucleocytoplasmic transport. The high number of accessible hydrophobic motifs of FG-Nups potentially gives rise to this multifunctionality, enabling them to form unique microenvironments. In this review, we discuss the multifunctionality of disordered and F-rich Nups and the diversity of their localizations, emphasizing the important roles of those Nups in various regulatory and metabolic processes.

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Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins

eLife ◽

10.7554/elife.52896 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 16

Author(s):

Chih-Yung S Lee ◽

Andrea Putnam ◽

Tu Lu ◽

ShuaiXin He ◽

John Paul T Ouyang ◽

...

Keyword(s):

Cell Fate ◽

Intrinsically Disordered Proteins ◽

Disordered Proteins ◽

Germline Development ◽

Independent Manner ◽

P Granules ◽

Rna Granules ◽

Intrinsically Disordered

RNA granules are protein/RNA condensates. How specific mRNAs are recruited to cytoplasmic RNA granules is not known. Here, we characterize the transcriptome and assembly of P granules, RNA granules in the C. elegans germ plasm. We find that P granules recruit mRNAs by condensation with the disordered protein MEG-3. MEG-3 traps mRNAs into non-dynamic condensates in vitro and binds to ~500 mRNAs in vivo in a sequence-independent manner that favors embryonic mRNAs with low ribosome coverage. Translational stress causes additional mRNAs to localize to P granules and translational activation correlates with P granule exit for two mRNAs coding for germ cell fate regulators. Localization to P granules is not required for translational repression but is required to enrich mRNAs in the germ lineage for robust germline development. Our observations reveal similarities between P granules and stress granules and identify intrinsically-disordered proteins as drivers of RNA condensation during P granule assembly.

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