On the stability and layered organization of protein-DNA condensates

Chromatin Regulators ◽

Atomistic Models ◽

The Stability ◽

Experimental Findings ◽

Component Phase

Multi-component phase separation is emerging as a key mechanism for the formation of biological condensates that play essential roles in signal sensing and transcriptional regulation. The molecular factors that dictate these condensates' stability and spatial organization are not fully understood, and it remains challenging to predict their microstructures. Using a near-atomistic, chemically accurate force field, we studied the phase behavior of chromatin regulators that are crucial for heterochromatin organization and their interactions with DNA. Our computed phase diagrams recapitulated previous experimental findings on different proteins. They revealed a strong dependence of condensate stability on the protein-DNA mixing ratio as a result of balancing protein-protein interactions and charge neutralization. Notably, a layered organization was observed in condensates formed by mixing HP1, histone H1, and DNA. This layered organization may be of biological relevance as it enables cooperative DNA packaging between the two chromatin regulators: histone H1 softens the DNA to facilitate the compaction induced by HP1 droplets. Our study supports near atomistic models as a valuable tool for characterizing the structure and stability of biological condensates.

Spatial organization enhances versatility and specificity in cyclic di-GMP signaling

Biological Chemistry ◽

10.1515/hsz-2020-0202 ◽

2020 ◽

Vol 401 (12) ◽

pp. 1323-1334

Author(s):

Sandra Kunz ◽

Peter L. Graumann

Keyword(s):

Protein Interactions ◽

Cell Cycle Progression ◽

Spatial Organization ◽

Caulobacter Crescentus ◽

Second Messenger ◽

Spatial Cues ◽

Signaling Specificity ◽

Regulatory Circuits ◽

Second Messenger Signaling

AbstractThe second messenger cyclic di-GMP regulates a variety of processes in bacteria, many of which are centered around the decision whether to adopt a sessile or a motile life style. Regulatory circuits include pathogenicity, biofilm formation, and motility in a wide variety of bacteria, and play a key role in cell cycle progression in Caulobacter crescentus. Interestingly, multiple, seemingly independent c-di-GMP pathways have been found in several species, where deletions of individual c-di-GMP synthetases (DGCs) or hydrolases (PDEs) have resulted in distinct phenotypes that would not be expected based on a freely diffusible second messenger. Several recent studies have shown that individual signaling nodes exist, and additionally, that protein/protein interactions between DGCs, PDEs and c-di-GMP receptors play an important role in signaling specificity. Additionally, subcellular clustering has been shown to be employed by bacteria to likely generate local signaling of second messenger, and/or to increase signaling specificity. This review highlights recent findings that reveal how bacteria employ spatial cues to increase the versatility of second messenger signaling.

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

10.1101/2021.11.06.467095 ◽

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 ◽

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.

The mechanistic GEMMs of oncogenic histones

Human Molecular Genetics ◽

10.1093/hmg/ddaa143 ◽

2020 ◽

Vol 29 (R2) ◽

pp. R226-R235 ◽

Cited By ~ 1

Author(s):

Anders M Lindroth ◽

Yoon Jung Park ◽

Verónica Matía ◽

Massimo Squatrito

Keyword(s):

Protein Interactions ◽

Drug Testing ◽

Age Groups ◽

Tumor Formation ◽

Model Systems ◽

Efficient Treatment ◽

Histone Modifiers ◽

Second Tumors ◽

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.

A Conditional Role of U2AF in Splicing of Introns with Unconventional Polypyrimidine Tracts

Molecular and Cellular Biology ◽

10.1128/mcb.00627-07 ◽

2007 ◽

Vol 27 (20) ◽

pp. 7334-7344 ◽

Cited By ~ 16

Author(s):

Vinod Sridharan ◽

Ravinder Singh

Keyword(s):

Protein Interactions ◽

Splice Site ◽

Large Subunit ◽

Strong Dependence ◽

Rna Recognition Motif ◽

Branch Points ◽

Site Identification

ABSTRACT Recognition of polypyrimidine (Py) tracts typically present between the branch point and the 3′ splice site by the large subunit of the essential splicing factor U2AF is a key early step in pre-mRNA splicing. Diverse intronic sequence arrangements exist, however, including 3′ splice sites lacking recognizable Py tracts, which raises the question of how general the requirement for U2AF is for various intron architectures. Our analysis of fission yeast introns in vivo has unexpectedly revealed that whereas introns lacking Py tracts altogether remain dependent on both subunits of U2AF, introns with long Py tracts, unconventionally positioned upstream of branch points, are unaffected by U2AF inactivation. Nevertheless, mutation of these Py tracts causes strong dependence on the large subunit U2AF59. We also find that Py tract diversity influences the requirement for the conserved C-terminal domain of U2AF59 (RNA recognition motif 3), which has been implicated in protein-protein interactions with other splicing factors. Together, these results suggest that in addition to Py tract binding by U2AF, supplementary mechanisms of U2AF recruitment and 3′ splice site identification exist to accommodate diverse intron architectures, which have gone unappreciated in biochemical studies of model pre-mRNAs.

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

Journal of Dairy Research ◽

10.1017/s0022029900012486 ◽

1969 ◽

Vol 36 (1) ◽

pp. 11-20 ◽

Cited By ~ 27

Author(s):

R. C. Lawrence ◽

L. K. Creamer

Keyword(s):

Protein Interactions ◽

Proteolytic Enzymes ◽

Enzyme Concentration ◽

Ion Concentration ◽

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.

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

10.1101/562520 ◽

2019 ◽

Cited By ~ 3

Author(s):

Abhilesh S. Dhawanjewar ◽

Ankit Roy ◽

M.S. Madhusudhan

Keyword(s):

Protein Interactions ◽

Binary Classification ◽

Scoring Function ◽

Protein Docking ◽

Scoring Functions ◽

Residue Contact ◽

Knowledge Based ◽

Cellular Biochemistry ◽

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]

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

10.21203/rs.3.pex-1479/v1 ◽

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 ◽

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.

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

Physical Chemistry Chemical Physics ◽

10.1039/c5cp07285a ◽

2016 ◽

Vol 18 (15) ◽

pp. 10270-10280 ◽

Cited By ~ 17

Author(s):

Jan Hansen ◽

Florian Platten ◽

Dana Wagner ◽

Stefan U. Egelhaaf

Keyword(s):

Phase Behavior ◽

Protein Interactions ◽

Phase Behaviour ◽

Specific Effects ◽

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

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1069 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1069-1077 ◽

Cited By ~ 57

Author(s):

J Sidorova ◽

L Breeden

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Protein Interactions ◽

C Terminus ◽

Specific Complex ◽

Dependent Cell ◽

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1917569117 ◽

2020 ◽

Vol 117 (24) ◽

pp. 13238-13247 ◽

Cited By ~ 12

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 ◽

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.