scholarly journals Single-embryo phosphoproteomics reveals the importance of intrinsic disorder in cell cycle dynamics

2021 ◽  
Author(s):  
Juan Manuel Valverde ◽  
Geronimo Dubra ◽  
Henk van den Toorn ◽  
Guido van Mierlo ◽  
Michiel Vermeulen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Intrinsically Disordered Proteins ◽  
Protein Complexes ◽  
Disordered Proteins ◽  
Cell Cycles ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Egg Extracts ◽  
Cell Cycle Dynamics

Switch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

scholarly journals Intrinsically disordered proteins identified in the aggregate proteome serve as biomarkers of neurodegeneration

2021 ◽  
Author(s):  
Srinivas Ayyadevara ◽  
Akshatha Ganne ◽  
Meenakshisundaram Balasubramaniam ◽  
Robert J. Shmookler Reis
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Protein Complexes ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Physiological Processes ◽  
Protein Partners ◽  
And Function ◽  
Computational Analyses ◽  
Disordered Regions

AbstractA protein’s structure is determined by its amino acid sequence and post-translational modifications, and provides the basis for its physiological functions. Across all organisms, roughly a third of the proteome comprises proteins that contain highly unstructured or intrinsically disordered regions. Proteins comprising or containing extensive unstructured regions are referred to as intrinsically disordered proteins (IDPs). IDPs are believed to participate in complex physiological processes through refolding of IDP regions, dependent on their binding to a diverse array of potential protein partners. They thus play critical roles in the assembly and function of protein complexes. Recent advances in experimental and computational analyses predicted multiple interacting partners for the disordered regions of proteins, implying critical roles in signal transduction and regulation of biological processes. Numerous disordered proteins are sequestered into aggregates in neurodegenerative diseases such as Alzheimer’s disease (AD) where they are enriched even in serum, making them good candidates for serum biomarkers to enable early detection of AD.

scholarly journals Evolutionary Study of Disorder in Protein Sequences

Biomolecules ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1413
Author(s):  
Kristina Kastano ◽  
Gábor Erdős ◽  
Pablo Mier ◽  
Gregorio Alanis-Lobato ◽  
Vasilis J. Promponas ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Protein Complexes ◽  
Cell Types ◽  
Disordered Proteins ◽  
Protein Protein Interactions ◽  
Globular Structure ◽  
Large Variability ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

Intrinsically disordered proteins (IDPs) contain regions lacking intrinsic globular structure (intrinsically disordered regions, IDRs). IDPs are present across the tree of life, with great variability of IDR type and frequency even between closely related taxa. To investigate the function of IDRs, we evaluated and compared the distribution of disorder content in 10,695 reference proteomes, confirming its high variability and finding certain correlation along the Euteleostomi (bony vertebrates) lineage to number of cell types. We used the comparison of orthologs to study the function of disorder related to increase in cell types, observing that multiple interacting subunits of protein complexes might gain IDRs in evolution, thus stressing the function of IDRs in modulating protein-protein interactions, particularly in the cell nucleus. Interestingly, the conservation of local compositional biases of IDPs follows residue-type specific patterns, with E- and K-rich regions being evolutionarily stable and Q- and A-rich regions being more dynamic. We provide a framework for targeted evolutionary studies of the emergence of IDRs. We believe that, given the large variability of IDR distributions in different species, studies using this evolutionary perspective are required.

scholarly journals Combinatorial phosphorylation modulates the structure and function of the G protein gamma subunit in yeast

2020 ◽  
Author(s):  
Zahra Nassiri Toosi ◽  
Xinya Su ◽  
Shilpa Choudhury ◽  
Wei Li ◽  
Yui Tik Pang ◽  
...  
Keyword(s):  
G Protein ◽  
Intrinsically Disordered Proteins ◽  
Protein Complexes ◽  
Disordered Proteins ◽  
Post Translational Modification ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Gpcr Activation ◽  
Essential Components ◽  
And Function

AbstractProtein intrinsically disordered regions (IDRs) are often targets of combinatorial post-translational modifications (PTMs) that serve to regulate protein structure and/or function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits – essential components of heterotrimeric G protein complexes – are intrinsically disordered, highly phosphorylated governors of G protein signaling. Here, we demonstrate that the yeast Gγ Ste18 undergoes combinatorial, multi-site phosphorylation within its N-terminal IDR. Phosphorylation at S7 is responsive to GPCR activation and osmotic stress while phosphorylation at S3 is responsive to glucose stress and is a quantitative indicator of intracellular pH. Each site is phosphorylated by a distinct set of kinases and both are also interactive, such that phosphomimicry at one site affects phosphorylation on the other. Lastly, we show that phosphorylation produces subtle yet clear changes in IDR structure and that different combinations of phosphorylation modulate the activation rate and amplitude of the scaffolded MAPK Fus3. These data place Gγ subunits among the growing list of intrinsically disordered proteins that exploit combinatorial post-translational modification to govern signaling pathway output.

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

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Andrei Vovk ◽  
Chad Gu ◽  
Michael G Opferman ◽  
Larisa E Kapinos ◽  
Roderick YH Lim ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Intrinsically Disordered Proteins ◽  
Nucleocytoplasmic Transport ◽  
Transport Proteins ◽  
Disordered Proteins ◽  
Biophysical Model ◽  
Nuclear Pore ◽  
Intrinsically Disordered

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.

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

2020 ◽  
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.

scholarly journals Random coil chemical shifts for serine, threonine and tyrosine phosphorylation over a broad pH range

2019 ◽  
Vol 73 (12) ◽  
pp. 713-725 ◽  
Author(s):  
Ruth Hendus-Altenburger ◽  
Catarina B. Fernandes ◽  
Katrine Bugge ◽  
Micha B. A. Kunze ◽  
Wouter Boomsma ◽  
...  
Keyword(s):  
Chemical Shift ◽  
Secondary Structure ◽  
Intrinsically Disordered Proteins ◽  
Chemical Shifts ◽  
Random Coil ◽  
Disordered Proteins ◽  
Phosphoryl Group ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Secondary Chemical

Abstract Phosphorylation is one of the main regulators of cellular signaling typically occurring in flexible parts of folded proteins and in intrinsically disordered regions. It can have distinct effects on the chemical environment as well as on the structural properties near the modification site. Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins (IDPs) and the reliability of the analysis depends on an appropriate choice of random coil model. Random coil chemical shifts and sequence correction factors were previously determined for an Ac-QQXQQ-NH2-peptide series with X being any of the 20 common amino acids. However, a matching dataset on the phosphorylated states has so far only been incompletely determined or determined only at a single pH value. Here we extend the database by the addition of the random coil chemical shifts of the phosphorylated states of serine, threonine and tyrosine measured over a range of pH values covering the pKas of the phosphates and at several temperatures (www.bio.ku.dk/sbinlab/randomcoil). The combined results allow for accurate random coil chemical shift determination of phosphorylated regions at any pH and temperature, minimizing systematic biases of the secondary chemical shifts. Comparison of chemical shifts using random coil sets with and without inclusion of the phosphoryl group, revealed under/over estimations of helicity of up to 33%. The expanded set of random coil values will improve the reliability in detection and quantification of transient secondary structure in phosphorylation-modified IDPs.

scholarly journals Optimization of Molecular Dynamics Simulations of c-MYC1-88—An Intrinsically Disordered System

Life ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 109 ◽  
Author(s):  
Sandra S. Sullivan ◽  
Robert O.J. Weinzierl
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Proliferation And Apoptosis ◽  
Conventional Structure ◽  
Experimental Approaches ◽  
Dynamics Simulations

Many of the proteins involved in key cellular regulatory events contain extensive intrinsically disordered regions that are not readily amenable to conventional structure/function dissection. The oncoprotein c-MYC plays a key role in controlling cell proliferation and apoptosis and more than 70% of the primary sequence is disordered. Computational approaches that shed light on the range of secondary and tertiary structural conformations therefore provide the only realistic chance to study such proteins. Here, we describe the results of several tests of force fields and water models employed in molecular dynamics simulations for the N-terminal 88 amino acids of c-MYC. Comparisons of the simulation data with experimental secondary structure assignments obtained by NMR establish a particular implicit solvation approach as highly congruent. The results provide insights into the structural dynamics of c-MYC1-88, which will be useful for guiding future experimental approaches. The protocols for trajectory analysis described here will be applicable for the analysis of a variety of computational simulations of intrinsically disordered proteins.

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

2020 ◽  
Vol 117 (21) ◽  
pp. 11421-11431 ◽  
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.

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

Cells ◽  
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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