scholarly journals Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

2018 ◽  
Author(s):  
Sindhuja Sridharan ◽  
Nils Kurzawa ◽  
Thilo Werner ◽  
Ina Guenthner ◽  
Dominic Helm ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Nucleic Acid Binding ◽  
Macromolecular Assemblies ◽  
Intrinsically Disordered ◽  
Cellular Environment ◽  
Nucleic Acid Binding Proteins ◽  
High Concentrations ◽  
In Cells

Nucleotide triphosphates (NTPs) regulate numerous biochemical processes in cells as (co-)substrates, allosteric modulators, biosynthetic precursors, and signaling molecules. Apart from its roles as energy source fueling cellular biochemistry, adenosine triphosphate (ATP), the most abundant NTP in cells, has been reported to affect macromolecular assemblies, such as protein complexes and membrane-less organelles. Moreover, both ATP and guanosine triphosphate (GTP) have recently been shown to dissolve protein aggregates. However, system-wide studies to characterize NTP- interactions under conditions approximating the native cellular environment are lacking, which limits our perspective of the diverse physiological roles of NTPs. Here, we have mapped and quantified proteome-wide NTP-interactions by assessing thermal stability and solubility of proteins using mechanically disrupted cells. Our results reveal diverse biological roles of ATP depending on its concentration. We found that ATP specifically interacts with proteins that utilize it as substrate or allosteric modulator at doses lower than 500 μM, while it affects protein-protein interactions of protein complexes at mildly higher concentrations (between 1-2 mM). At high concentrations (> 2 mM), ATP modulates the solubility state of a quarter of the insoluble proteome, consisting of positively charged, intrinsically disordered, nucleic acid binding proteins, which are part of membrane-less organelles. The extent of solubilization depends on the localization of proteins to different membrane-less organelles. Furthermore, we uncover that ATP regulates protein-DNA interactions of the Barrier to autointegration factor (BANF1). Our data provides the first quantitative proteome-wide map of ATP affecting protein structure and protein complex stability and solubility, providing unique clues on its role in protein phase transitions.

scholarly journals The intracellular environment affects protein–protein interactions

2021 ◽  
Vol 118 (11) ◽  
pp. e2019918118
Author(s):  
Shannon L. Speer ◽  
Wenwen Zheng ◽  
Xin Jiang ◽  
I-Te Chu ◽  
Alex J. Guseman ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Complex ◽  
Hard Core ◽  
Protein Protein Interactions ◽  
Chemical Interactions ◽  
E Coli ◽  
Cellular Environment ◽  
High Concentrations ◽  
In Cells

Protein–protein interactions are essential for life but rarely thermodynamically quantified in living cells. In vitro efforts show that protein complex stability is modulated by high concentrations of cosolutes, including synthetic polymers, proteins, and cell lysates via a combination of hard-core repulsions and chemical interactions. We quantified the stability of a model protein complex, the A34F GB1 homodimer, in buffer, Escherichia coli cells and Xenopus laevis oocytes. The complex is more stable in cells than in buffer and more stable in oocytes than E. coli. Studies of several variants show that increasing the negative charge on the homodimer surface increases stability in cells. These data, taken together with the fact that oocytes are less crowded than E. coli cells, lead to the conclusion that chemical interactions are more important than hard-core repulsions under physiological conditions, a conclusion also gleaned from studies of protein stability in cells. Our studies have implications for understanding how promiscuous—and specific—interactions coherently evolve for a protein to properly function in the crowded cellular environment.

scholarly journals SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells

2016 ◽  
Vol 113 (37) ◽  
pp. 10322-10327 ◽  
Author(s):  
Matthew J. Smola ◽  
Thomas W. Christy ◽  
Kaoru Inoue ◽  
Cindo O. Nicholson ◽  
Matthew Friedersdorf ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Rna Structure ◽  
Noncoding Rna ◽  
Ex Vivo ◽  
Living Cells ◽  
Mammalian Development ◽  
Cellular Environment ◽  
Nucleotide Resolution ◽  
In Cells

The 18-kb Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation during female eutherian mammalian development. Global structural architecture, cell-induced conformational changes, and protein–RNA interactions within Xist are poorly understood. We used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to examine these features of Xist at single-nucleotide resolution both in living cells and ex vivo. The Xist RNA forms complex well-defined secondary structure domains and the cellular environment strongly modulates the RNA structure, via motifs spanning one-half of all Xist nucleotides. The Xist RNA structure modulates protein interactions in cells via multiple mechanisms. For example, repeat-containing elements adopt accessible and dynamic structures that function as landing pads for protein cofactors. Structured RNA motifs create interaction domains for specific proteins and also sequester other motifs, such that only a subset of potential binding sites forms stable interactions. This work creates a broad quantitative framework for understanding structure–function interrelationships for Xist and other lncRNAs in cells.

scholarly journals The disordered boundary of the cell: emerging properties of membrane-bound intrinsically disordered proteins

2019 ◽  
Vol 10 (1) ◽  
pp. 25-36 ◽  
Author(s):  
Irrem-Laareb Mohammad ◽  
Borja Mateos ◽  
Miquel Pons
Keyword(s):  
Lipid Bilayers ◽  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Intrinsically Disordered Protein ◽  
Disordered Proteins ◽  
Protein Protein Interactions ◽  
Intrinsically Disordered ◽  
High Concentrations ◽  
The Individual ◽  
Confined Environment

AbstractWe define the disordered boundary of the cell (DBC) as the system formed by membrane tethered intrinsically disordered protein regions, dynamically coupled to the underlying membrane.The emerging properties of the DBC makes it a global system of study, which cannot be understood from the individual properties of their components. Similarly, the properties of lipid bilayers cannot be understood from just the sum of the properties of individual lipid molecules.The highly anisotropic confined environment, restricting the position and orientation of interacting sites, is affecting the properties of individual disordered proteins. In fact, the collective effect caused by high concentrations of disordered proteins extend beyond the sum of individual effects.Examples of emerging properties of the DBC include enhanced protein-protein interactions, protein-driven phase separations, Z-compartmentalization, and protein modulated electrostatics.

scholarly journals InterPep2: global peptide–protein docking using interaction surface templates

2020 ◽  
Vol 36 (8) ◽  
pp. 2458-2465 ◽  
Author(s):  
Isak Johansson-Åkhe ◽  
Claudio Mirabello ◽  
Björn Wallner
Keyword(s):  
Protein Interactions ◽  
Protein Complexes ◽  
Structural Features ◽  
Protein Docking ◽  
Supplementary Information ◽  
Peptide Ligand ◽  
Protein Protein Interactions ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Improved Performance

Abstract Motivation Interactions between proteins and peptides or peptide-like intrinsically disordered regions are involved in many important biological processes, such as gene expression and cell life-cycle regulation. Experimentally determining the structure of such interactions is time-consuming and difficult because of the inherent flexibility of the peptide ligand. Although several prediction-methods exist, most are limited in performance or availability. Results InterPep2 is a freely available method for predicting the structure of peptide–protein interactions. Improved performance is obtained by using templates from both peptide–protein and regular protein–protein interactions, and by a random forest trained to predict the DockQ-score for a given template using sequence and structural features. When tested on 252 bound peptide–protein complexes from structures deposited after the complexes used in the construction of the training and templates sets of InterPep2, InterPep2-Refined correctly positioned 67 peptides within 4.0 Å LRMSD among top10, similar to another state-of-the-art template-based method which positioned 54 peptides correctly. However, InterPep2 displays a superior ability to evaluate the quality of its own predictions. On a previously established set of 27 non-redundant unbound-to-bound peptide–protein complexes, InterPep2 performs on-par with leading methods. The extended InterPep2-Refined protocol managed to correctly model 15 of these complexes within 4.0 Å LRMSD among top10, without using templates from homologs. In addition, combining the template-based predictions from InterPep2 with ab initio predictions from PIPER-FlexPepDock resulted in 22% more near-native predictions compared to the best single method (22 versus 18). Availability and implementation The program is available from: http://wallnerlab.org/InterPep2. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Development of Phosphodiesterase-Protein Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

2021 ◽  
Author(s):  
Nikhil Kumar Tulsian ◽  
Valerie Jia-En Sin ◽  
Ganesh Srinivasan Anand ◽  
Hwee-Ling Koh
Keyword(s):  
Protein Kinases ◽  
Protein Interactions ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Drug Targets ◽  
Cross Reactivity ◽  
Nucleotide Receptors ◽  
Macromolecular Assemblies ◽  
Fluorescence Polarization Assay ◽  
In Cells

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (Protein Kinases, PK) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PK generates an expanded active site which enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PK, and aid in signal termination. PDEs are important drug targets and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targets PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay has been adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode, and disrupt protein-protein interactions between PDEs and cyclic nucleotide activating protein kinases in a second mode. We tested this approach with three different systems: cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG and dual-specificity RegA-RD complexes and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

scholarly journals Increasing the precision of orthology-based complex prediction through network alignment

2014 ◽  
Author(s):  
Roland Pache ◽  
Patrick Aloy
Keyword(s):  
Protein Interactions ◽  
In Silico ◽  
Large Scale ◽  
Current Knowledge ◽  
Protein Complexes ◽  
Functional Divergence ◽  
Network Alignment ◽  
Model Organisms ◽  
Macromolecular Assemblies ◽  
Complex Components

Macromolecular assemblies play an important role in almost all cellular processes. However, despite several large-scale studies, our current knowledge about protein complexes is still quite limited, thus advocating the use of in silico predictions to gather information on complex composition in model organisms. Since protein-protein interactions present certain constraints on the functional divergence of macromolecular assemblies during evolution, it is possible to predict complexes based on orthology data. Here, we show that incorporating interaction information through network alignment significantly increases the precision of orthology-based complex prediction. Moreover, we performed a large-scale in silico screen for protein complexes in human, yeast and fly, through the alignment of hundreds of known complexes to whole organism interactomes. Systematic comparison of the resulting network alignments to all complexes currently known in those species revealed many conserved complexes, as well as several novel complex components. In addition to validating our predictions using orthogonal data, we were able to assign specific functional roles to the predicted complexes. In several cases, the incorporation of interaction data through network alignment allowed to distinguish real complex components from other orthologous proteins. Our analyses indicate that current knowledge of yeast protein complexes exceeds that in other organisms and that predicting complexes in fly based on human and yeast data is complementary rather than redundant. Lastly, assessing the conservation of protein complexes of the human pathogen Mycoplasma pneumoniae, we discovered that its complexes repertoire is different from that of eukaryotes, suggesting new points of therapeutic intervention, whereas targeting the pathogen’s Restriction enzyme complex might lead to adverse effects due to its similarity to ATP-dependent metalloproteases in the human host.

scholarly journals Development of Phosphodiesterase–Protein-Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

2021 ◽  
Vol 22 (10) ◽  
pp. 5242
Author(s):  
Nikhil K. Tulsian ◽  
Valerie Jia-En Sin ◽  
Hwee-Ling Koh ◽  
Ganesh S. Anand
Keyword(s):  
Protein Interactions ◽  
Cyclic Nucleotides ◽  
Cyclic Nucleotide ◽  
Drug Targets ◽  
Cross Reactivity ◽  
Nucleotide Receptors ◽  
Protein Protein Interactions ◽  
Macromolecular Assemblies ◽  
Fluorescence Polarization Assay ◽  
In Cells

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE–PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE–PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems—cAMP-specific PDE8–PKAR, cGMP-specific PDE5–PKG, and dual-specificity RegA–RD complexes—and ranked inhibitors according to their inhibition potency. Targeting PDE–PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

scholarly journals The role of disorder in RNA binding affinity and specificity

Open Biology ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 200328
Author(s):  
Diana S. M. Ottoz ◽  
Luke E. Berchowitz
Keyword(s):  
Protein Interactions ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Multiple Partners ◽  
Cellular Environment ◽  
Rna Targets

Most RNA-binding modules are small and bind few nucleotides. RNA-binding proteins typically attain the physiological specificity and affinity for their RNA targets by combining several RNA-binding modules. Here, we review how disordered linkers connecting RNA-binding modules govern the specificity and affinity of RNA–protein interactions by regulating the effective concentration of these modules and their relative orientation. RNA-binding proteins also often contain extended intrinsically disordered regions that mediate protein–protein and RNA–protein interactions with multiple partners. We discuss how these regions can connect proteins and RNA resulting in heterogeneous higher-order assemblies such as membrane-less compartments and amyloid-like structures that have the characteristics of multi-modular entities. The assembled state generates additional RNA-binding specificity and affinity properties that contribute to further the function of RNA-binding proteins within the cellular environment.

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