The intracellular environment affects protein-protein interactions.
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ABSTRACT:
SUBMITTER: Speer SL
PROVIDER: S-EPMC7980425 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Conformational dynamics is important for protein functions, many of which are performed in cells. How the intracellular environment may affect protein conformational dynamics is largely unknown. Here, loop conformational dynamics is studied for a model protein in Escherichia coli cells by using nuclear magnetic resonance (NMR) spectroscopy. The weak interactions between the protein and surrounding macromolecules in cells hinder the protein rotational diffusion, which extends the dynamic detection timescale up to microseconds by the NMR spin relaxation method. The loop picosecond to microsecond dynamics is confirmed by nanoparticle-assisted spin relaxation and residual dipolar coupling methods. The loop interactions with the intracellular environment are perturbed through point mutation of the loop sequence. For the sequence of the protein that interacts stronger with surrounding macromolecules, the loop becomes more rigid in cells. In contrast, the mutational effect on the loop dynamics in vitro is small. This study provides direct evidence that the intracellular environment can modify protein loop conformational dynamics through weak interactions.
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Project description:Protein-protein interactions are important in many essential biological functions, such as transcription, translation, and signal transduction. Much progress has been made in understanding protein-protein association in dilute solution via experimentation and simulation. Cells, however, contain various macromolecules, such as DNA, RNA, proteins, among many others, and a myriad of non-specific interactions (usually weak) are present between these cellular constituents. In this review article, we describe the important developments in recent years that have furthered our understanding and even allowed prediction of the consequences of macromolecular crowding on protein-protein interactions. We outline the development of our crowding theory that can predict the change in binding free energy due to crowding quantitatively for both repulsive and attractive protein-crowder interactions. One of the most important findings from our recent work is that weak attractive interactions between crowders and proteins can actually destabilize protein complex formation as opposed to the commonly assumed stabilizing effect predicted based on traditional crowding theories that only account for the entropic-excluded volume effects. We also discuss the implications of macromolecular crowding on the population of encounter versus specific native complex.
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Project description:Inhibition of protein-protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. ?-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed "proteomimetics", which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N-alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified.
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Project description:Peptide drug discovery has great potential, but the cell membrane is a major obstacle when the target is an intracellular protein-protein interaction (PPI). It is difficult to target PPIs with small molecules; indeed, there are no intervention tools that can target any intracellular PPI. In this study, we developed a platform that enables the introduction of peptides into cells via mRNA-based gene delivery. Peptide-length nucleic acids do not enable stable ribosome binding and exhibit little to no translation into protein. In this study, a construct was created in which the sequence encoding dihydrofolate reductase (DHFR) was placed in front of the sequence encoding the target peptide, together with a translation skipping sequence, as a sequence that meets the requirements of promoting ribosome binding and rapid decay of the translated protein. This enabled efficient translation from the mRNA encoding the target protein while preventing unnecessary protein residues. Using this construct, we showed that it can inhibit Drp1/Fis1 binding, one of the intracellular PPIs, which governs mitochondrial fission, an important aspect of mitochondrial dynamics. In addition, it was shown to inhibit pathological hyperfission, normalize mitochondrial dynamics and metabolism, and inhibit apoptosis of the mitochondrial pathway.
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Project description:Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.
| S-EPMC5755618 | biostudies-literature
Project description:The fitness effect of mutations can be influenced by their interactions with the environment, other mutations, or both. Previously, we constructed 32 ( = 2⁵) genotypes that comprise all possible combinations of the first five beneficial mutations to fix in a laboratory-evolved population of Escherichia coli. We found that (i) all five mutations were beneficial for the background on which they occurred; (ii) interactions between mutations drove a diminishing returns type epistasis, whereby epistasis became increasingly antagonistic as the expected fitness of a genotype increased; and (iii) the adaptive landscape revealed by the mutation combinations was smooth, having a single global fitness peak. Here we examine how the environment influences epistasis by determining the interactions between the same mutations in two alternative environments, selected from among 1,920 screened environments, that produced the largest increase or decrease in fitness of the most derived genotype. Some general features of the interactions were consistent: mutations tended to remain beneficial and the overall pattern of epistasis was of diminishing returns. Other features depended on the environment; in particular, several mutations were deleterious when added to specific genotypes, indicating the presence of antagonistic interactions that were absent in the original selection environment. Antagonism was not caused by consistent pleiotropic effects of individual mutations but rather by changing interactions between mutations. Our results demonstrate that understanding adaptation in changing environments will require consideration of the combined effect of epistasis and pleiotropy across environments.
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Project description:Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-?-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both ?- and ?-amino acid residues ("?/?-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "?-peptides". This report documents an extension of the ?/?-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated ?/?-peptides based on a "stapled" Bim BH3 ?-peptide, which contains a hydrocarbon cross-link to enhance ?-helix stability. We show that a stapled ?/?-peptide can structurally and functionally mimic the parent stapled ?-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the ?/?-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled ?-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.
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Project description: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.
| S-EPMC1219985 | biostudies-other
Project description:BackgroundBiological processes are usually distributed over various intracellular compartments. Proteins from diverse cellular compartments are often involved in similar signaling networks. However, the difference in the reaction rates between similar proteins among different compartments is usually quite high. We suggest that the estimation of frequency of intracompartmental as well as intercompartmental protein-protein interactions is an appropriate approach to predict the efficiency of a pathway.ResultsUsing data from the databases STRING, ANDSystem, IntAct and UniProt, a PPI frequency matrix of intra/inter-compartmental interactions efficiencies was constructed. This matrix included 15 human-specific cellular compartments. An approach for estimating pathway efficiency using the matrix of intra/inter-compartmental PPI frequency, based on analysis of reactions efficiencies distribution was suggested. An investigation of KEGG pathway efficiencies was conducted using the developed method. The clusterization and the ranking of KEGG pathways based on their efficiency were performed. "Amino acid metabolism" and "Genetic information processing" revealed the highest efficiencies among other functional classes of KEGG pathways. "Nervous system" and "Signaling molecules interaction" contained the most inefficient pathways. Statistically significant differences were found between efficiencies of KEGG and randomly-generated pathways. Based on these observations, the validity of this approach was discussed.ConclusionThe estimation of efficiency of signaling networks is a complicated task because of the need for the data on the kinetic reactions. However, the proposed method does not require such data and can be used for preliminary analysis of different protein networks.
| S-EPMC4303950 | biostudies-literature
Project description:Inhibition of protein-protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. ?-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed "proteomimetics", which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N-alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified.
| S-EPMC4506525 | biostudies-other