Tuning protein–protein interactions using cosolvents: specific effects of ionic and non-ionic additives on protein phase behavior

2016 ◽  
Vol 18 (15) ◽  
pp. 10270-10280 ◽  
Author(s):  
Jan Hansen ◽  
Florian Platten ◽  
Dana Wagner ◽  
Stefan U. Egelhaaf
Keyword(s):  
Phase Behavior ◽  
Protein Interactions ◽  
Phase Behaviour ◽  
Protein Protein Interactions ◽  
Specific Effects ◽  
The Stability

Cosolvents modulate not only the stability of proteins, but also protein–protein interactions and protein phase behaviour.

scholarly journals Crosstalk between β-catenin and WT1 signalling activity in acute myeloid leukemia

2021 ◽  
Author(s):  
Megan Payne ◽  
Olga Tsaponina ◽  
Gillian Caalim ◽  
Hayley Greenfield ◽  
Leanne Milton-Harris ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Myeloid Leukemia ◽  
Normal Development ◽  
Signal Transduction Pathway ◽  
Myeloid Cell ◽  
Wnt Signalling ◽  
Protein Protein Interactions ◽  
The Stability ◽  
First Time ◽  
Acute Myeloid

Wnt signalling is an evolutionary conserved signal transduction pathway heavily implicated in normal development and disease. The central mediator of this pathway, β-catenin, is frequently overexpressed, mislocalised and overactive in acute myeloid leukaemia (AML) where it mediates the establishment, maintenance and drug resistance of leukaemia stem cells. Critical to the stability, localisation and activity of β-catenin are the protein-protein interactions it forms, yet these are poorly defined in AML. We recently performed the first β-catenin interactome study in blood cells of any kind and identified a plethora of novel interacting partners. This study shows for the first time that β-catenin interacts with Wilms tumour protein (WT1), a protein frequently overexpressed and mutated in AML, in both myeloid cell lines and also primary AML samples. We demonstrate crosstalk between the signalling activity of these two proteins in myeloid cells, and show that modulation of either protein can affect expression of the other. Finally, we demonstrate that WT1 mutations frequently observed in AML can increase stabilise β-catenin and augment Wnt signalling output. This study has uncovered new context-dependent molecular interactions for β-catenin which could inform future therapeutic strategies to target this dysregulated molecule in AML.

scholarly journals Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid–Liquid Phase Separation

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4705
Author(s):  
Adiran Garaizar ◽  
Ignacio Sanchez-Burgos ◽  
Rosana Collepardo-Guevara ◽  
Jorge R. Espinosa
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Protein Interactions ◽  
Conformational Dynamics ◽  
Liquid Phase Separation ◽  
Phase Behaviour ◽  
Coarse Grained ◽  
Protein Protein Interactions ◽  
Intrinsically Disordered ◽  
Liquid Liquid Phase Separation

Proteins containing intrinsically disordered regions (IDRs) are ubiquitous within biomolecular condensates, which are liquid-like compartments within cells formed through liquid–liquid phase separation (LLPS). The sequence of amino acids of a protein encodes its phase behaviour, not only by establishing the patterning and chemical nature (e.g., hydrophobic, polar, charged) of the various binding sites that facilitate multivalent interactions, but also by dictating the protein conformational dynamics. Besides behaving as random coils, IDRs can exhibit a wide-range of structural behaviours, including conformational switching, where they transition between alternate conformational ensembles. Using Molecular Dynamics simulations of a minimal coarse-grained model for IDRs, we show that the role of protein conformation has a non-trivial effect in the liquid–liquid phase behaviour of IDRs. When an IDR transitions to a conformational ensemble enriched in disordered extended states, LLPS is enhanced. In contrast, IDRs that switch to ensembles that preferentially sample more compact and structured states show inhibited LLPS. This occurs because extended and disordered protein conformations facilitate LLPS-stabilising multivalent protein–protein interactions by reducing steric hindrance; thereby, such conformations maximize the molecular connectivity of the condensed liquid network. Extended protein configurations promote phase separation regardless of whether LLPS is driven by homotypic and/or heterotypic protein–protein interactions. This study sheds light on the link between the dynamic conformational plasticity of IDRs and their liquid–liquid phase behaviour.

scholarly journals Protein–protein interactions in intracellular Ca2+-release channel function

1999 ◽  
Vol 337 (3) ◽  
pp. 345-361 ◽  
Author(s):  
John J. MACKRILL
Keyword(s):  
Protein Interactions ◽  
Ryanodine Receptors ◽  
Accessory Proteins ◽  
Protein Protein Interactions ◽  
Release Channel ◽  
Specific Effects ◽  
Complex Protein ◽  
A Cell ◽  
And Function ◽  
Opposing Effects

Release of Ca2+ ions from intracellular stores can occur via two classes of Ca2+-release channel (CRC) protein, the inositol 1,4,5-trisphosphate receptors (InsP3Rs) and the ryanodine receptors (RyRs). Multiple isoforms and subtypes of each CRC class display distinct but overlapping distributions within mammalian tissues. InsP3Rs and RyRs interact with a plethora of accessory proteins which modulate the activity of their intrinsic channels. Although many aspects of CRC structure and function have been reviewed in recent years, the properties of proteins with which they interact has not been comprehensively surveyed, despite extensive current research on the roles of these modulators. The aim of this article is to review the regulation of CRC activity by accessory proteins and, wherever possible, to outline the structural details of such interactions. The CRCs are large transmembrane proteins, with the bulk of their structure located cytoplasmically. Intra- and inter-complex protein–protein interactions between these cytoplasmic domains also regulate CRC function. Some accessory proteins modulate channel activity of all CRC subtypes characterized, whereas other have class- or even isoform-specific effects. Certain accessory proteins exert both direct and indirect forms of regulation on CRCs, occasionally with opposing effects. Others are themselves modulated by changes in Ca2+ concentration, thereby participating in feedback mechanisms acting on InsP3R and RyR activity. CRCs are therefore capable of integrating numerous signalling events within a cell by virtue of such protein–protein interactions. Consequently, the functional properties of InsP3Rs and RyRs within particular cells and subcellular domains are ‘customized ’ by the accessory proteins present.

scholarly journals The mechanistic GEMMs of oncogenic histones

2020 ◽  
Vol 29 (R2) ◽  
pp. R226-R235 ◽  
Author(s):  
Anders M Lindroth ◽  
Yoon Jung Park ◽  
Verónica Matía ◽  
Massimo Squatrito
Keyword(s):  
Protein Interactions ◽  
Drug Testing ◽  
Age Groups ◽  
Tumor Formation ◽  
Model Systems ◽  
Protein Protein Interactions ◽  
Efficient Treatment ◽  
Histone Modifiers ◽  
Second Tumors ◽  
The Stability

Abstract The last decade’s progress unraveling the mutational landscape of all age groups of cancer has uncovered mutations in histones as vital contributors of tumorigenesis. Here we review three new aspects of oncogenic histones: first, the identification of additional histone mutations potentially contributing to cancer formation; second, tumors expressing histone mutations to study the crosstalk of post-translational modifications, and; third, development of sophisticated biological model systems to reproduce tumorigenesis. At the outset, we recapitulate the firstly discovered histone mutations in pediatric and adolescent tumors of the brain and bone, which still remain the most pronounced histone alterations in cancer. We branch out to discuss the ramifications of histone mutations, including novel ones, that stem from altered protein-protein interactions of cognate histone modifiers as well as the stability of the nucleosome. We close by discussing animal models of oncogenic histones that reproduce tumor formation molecularly and morphologically and the prospect of utilizing them for drug testing, leading to efficient treatment and cure of deadly cancers with histone mutations.

The action of calf rennet and other proteolytic enzymes on κ-casein

1969 ◽  
Vol 36 (1) ◽  
pp. 11-20 ◽  
Author(s):  
R. C. Lawrence ◽  
L. K. Creamer
Keyword(s):  
Protein Interactions ◽  
Proteolytic Enzymes ◽  
Enzyme Concentration ◽  
Ion Concentration ◽  
Protein Protein Interactions ◽  
Low Concentrations ◽  
Lag Period ◽  
High Concentrations ◽  
The Stability ◽  
Calcium Ion Concentration

SummaryThe hydrolysis of κ-casein by a number of rennets and other proteolytic enzymes has been followed by measuring the increase in opacity due to the formation of insoluble aggregates of para-κ-caseins. The stability of these precipitates varied markedly, some being solubilized rapidly by the further action of the enzyme. The turbidity obtained with certain enzymes was dependent upon the calcium ion concentration, indicating that the para-κ-caseins produced were not identical for all enzymes.For high concentrations of calf rennet, the rate of aggregation was linear with respect to time. With low concentrations of enzyme, increase in turbidity was preceded by a lag period which was lengthened by decreasing the enzyme concentration or increasing the κ-casein concentration. This increase in lag is favoured by a high κ-casein/para-κ-casein ratio, suggesting that the aggregation of newly formed para-κ-casein is prevented by the unchanged κ-casein. In addition, small amounts of αs1- or β-caseins present in the κ-casein also markedly affected the aggregation of para-κ-casein, indicating that all 3 major casein components can inhibit the aggregation of para-κ-casein in the absence of calcium ions. In the light of these observations the possible role of protein-protein interactions in casein coagulation by calf rennet is discussed.

scholarly journals A knowledge–based scoring function to assess the stability of quaternary protein assemblies

2019 ◽  
Author(s):  
Abhilesh S. Dhawanjewar ◽  
Ankit Roy ◽  
M.S. Madhusudhan
Keyword(s):  
Protein Interactions ◽  
Binary Classification ◽  
Scoring Function ◽  
Protein Docking ◽  
Scoring Functions ◽  
Protein Protein Interactions ◽  
Residue Contact ◽  
Knowledge Based ◽  
Cellular Biochemistry ◽  
The Stability

AbstractMotivationElucidation of protein-protein interactions is a necessary step towards understanding the complete repertoire of cellular biochemistry. Given the enormity of the problem, the expenses and limitations of experimental methods, it is imperative that this problem is tackled computationally. In silico predictions of protein interactions entail sampling different conformations of the purported complex and then scoring these to assess for interaction viability. In this study we have devised a new scheme for scoring protein-protein interactions.ResultsOur method, PIZSA (Protein Interaction Z Score Assessment) is a binary classification scheme for identification of stable protein quaternary assemblies (binders/non-binders) based on statistical potentials. The scoring scheme incorporates residue-residue contact preference on the interface with per residue-pair atomic contributions and accounts for clashes. PIZSA can accurately discriminate between native and non-native structural conformations from protein docking experiments and outperform other recently published scoring functions, demonstrated through testing on a benchmark set and the CAPRI Score_set. Though not explicitly trained for this purpose, PIZSA potentials can identify spurious interactions that are artefacts of the crystallization process.AvailabilityPIZSA is implemented as awebserverat http://cospi.iiserpune.ac.in/pizsa/[email protected]

scholarly journals Nuclear membrane-tethered FRAP method for measuring protein complex off-rates in live cells

2021 ◽  
Author(s):  
Lindsey R. Pack ◽  
Leighton H. Daigh ◽  
Mingyu Chung ◽  
Tobias Meyer
Keyword(s):  
Protein Interactions ◽  
Nuclear Membrane ◽  
Protein Complex ◽  
Protein Complexes ◽  
Live Cells ◽  
Cdk Inhibitors ◽  
Cyclin Dependent Kinase ◽  
Protein Protein Interactions ◽  
The Stability

Abstract Understanding the stability or binding affinity of protein complex members is important for understanding their regulation and roles in cells. While there are many biochemical methods to measure protein-protein interactions in vitro, these methods often rely on the ability to robustly purify components individually. Moreover, few methods have been developed to study protein complexes within live cells. Binding parameters for cyclin-dependent kinase (CDK) complexes have been challenging to measure due to difficulty expressing and purifying CDKs separately from activating cyclins. Here, we develop a method to measure off-rates of protein complex components in live-cells. Our method relies on the stable tethering of CDK to the inner nuclear membrane (Figure 1), and the utilization of FRAP to measure the off-rate of soluble, fluorescently-tagged CDK binding proteins. We use this method to study dimeric CDK complexes, measuring the off-rates of cyclins or INK4 CDK inhibitor p16 from CDKs, and trimeric CDK complexes, measuring the off-rate of cyclins and CIP/KIP CDK inhibitors p21 and p27 when bound together.

scholarly journals Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (2) ◽  
pp. 1069-1077 ◽  
Author(s):  
J Sidorova ◽  
L Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Specific Complex ◽  
Dependent Cell ◽  
The Stability

SWI4 and SWI6 play a crucial role in START-specific transcription in Saccharomyces cerevisiae. SWI4 and SWI6 form a specific complex on the SCB (SWI4/6-dependent cell cycle box) sequences which have been found in the promoters of HO and G1 cyclin genes. Overproduction of SWI4 eliminates the SWI6 dependency of HO transcription in vivo and results in a new SWI6-independent, SCB-specific complex in vitro, which is heterogeneous and reacts with SWI4 antibodies. The C terminus of SWI4 is not required for SWI6-independent binding of SWI4 to SCB sequences, but it is necessary and sufficient for association with SWI6. Both SWI4 and SWI6 contain two copies of a 33-amino-acid TPLH repeat, which has been implicated in protein-protein interactions in other proteins. These repeats are not required for the SWI4-SWI6 association. Alanine substitutions in both TPLH repeats of SWI6 reduce its activity but do not affect the stability of the protein or its association with SWI4. However, these mutations reduce the ability of the SWI4/6 complex to bind DNA. Deletion of the lucine zipper motif in SWI6 also allows SWI4/6 complex formation, but it eliminates the DNA-binding ability of the SWI4/6 complex. This indicates that the integrity of two different regions of SWI6 is required for DNA binding by the SWI4/6 complex. From these data, we propose that the sequence-specific DNA-binding domain resides in SWI4 but that SWI6 controls the accessibility of this domain in the SWI4/6 complex.

scholarly journals Liquid network connectivity regulates the stability and composition of biomolecular condensates with many components

2020 ◽  
Vol 117 (24) ◽  
pp. 13238-13247 ◽  
Author(s):  
Jorge R. Espinosa ◽  
Jerelle A. Joseph ◽  
Ignacio Sanchez-Burgos ◽  
Adiran Garaizar ◽  
Daan Frenkel ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Critical Points ◽  
Network Connectivity ◽  
Coarse Grained ◽  
Physical Parameters ◽  
Protein Protein Interactions ◽  
Coarse Grained Model ◽  
Bond Network ◽  
The Stability ◽  
Liquid Liquid Phase Separation

One of the key mechanisms used by cells to control the spatiotemporal organization of their many components is the formation and dissolution of biomolecular condensates through liquid–liquid phase separation (LLPS). Using a minimal coarse-grained model that allows us to simulate thousands of interacting multivalent proteins, we investigate the physical parameters dictating the stability and composition of multicomponent biomolecular condensates. We demonstrate that the molecular connectivity of the condensed-liquid network—i.e., the number of weak attractive protein–protein interactions per unit of volume—determines the stability (e.g., in temperature, pH, salt concentration) of multicomponent condensates, where stability is positively correlated with connectivity. While the connectivity of scaffolds (biomolecules essential for LLPS) dominates the phase landscape, introduction of clients (species recruited via scaffold–client interactions) fine-tunes it by transforming the scaffold–scaffold bond network. Whereas low-valency clients that compete for scaffold–scaffold binding sites decrease connectivity and stability, those that bind to alternate scaffold sites not required for LLPS or that have higher-than-scaffold valencies form additional scaffold–client–scaffold bridges increasing stability. Proteins that establish more connections (via increased valencies, promiscuous binding, and topologies that enable multivalent interactions) support the stability of and are enriched within multicomponent condensates. Importantly, proteins that increase the connectivity of multicomponent condensates have higher critical points as pure systems or, if pure LLPS is unfeasible, as binary scaffold–client mixtures. Hence, critical points of accessible systems (i.e., with just a few components) might serve as a unified thermodynamic parameter to predict the composition of multicomponent condensates.

Sign in / Sign up

Export Citation Format

Share Document