scholarly journals Effects of Pathological Mutations on the Prion-Like Polymerisation of MyD88

2018 ◽  
Author(s):  
Ailís O’Carroll ◽  
Brieuc Chauvin ◽  
James Brown ◽  
Ava Meagher ◽  
Joanne Coyle ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Full Length ◽  
Binary Response ◽  
Death Domain ◽  
Tir Domain ◽  
The Impact

AbstractA novel concept has emerged whereby the higher-order self-assembly of proteins provides a simple and robust mechanism for signal amplification. This appears to be a universal signalling mechanism within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns need to trigger a strong, binary response within cells. Previously, multiple structural studies have been limited to single domains, expressed and assembled at high protein concentrations. We therefore set out to develop new in vitro strategies to characterise the behaviour of full-length proteins at physiological levels. In this study we focus on the adaptor protein MyD88, which contains two domains with different self-assembly properties: a TIR domain that can polymerise similarly to the TIR domain of Mal, and a Death Domain that has been shown to oligomerise with helical symmetry in the Myddosome complex. To visualize the behaviour of full-length MyD88 without purification steps, we use single-molecule fluorescence coupled to eukaryotic cell-free protein expression. These experiments demonstrate that at low protein concentration, only full-length MyD88 forms prion-like polymers. We also demonstrate that the metastability of MyD88 polymerisation creates the perfect binary response required in innate signalling: the system is silenced at normal concentrations but upstream signalling creates a “seed” that triggers polymerisation and amplification of the response. These findings pushed us to re-interpret the role of polymerisation in MyD88-related diseases and we studied the impact of disease-associated point mutations L93P, R196C and L252P/L265P at the molecular level. We discovered that all mutations completely block the ability of MyD88 to polymerise. We also confirm that L252P, a gain-of-function mutation, allows the MyD88 mutant to form extremely stable oligomers, even when expressed at low nanomolar concentrations. Thus, our results are consistent with and greatly add to the findings on the Myddosomes digital ‘all-or-none’ responses and the behaviour of the oncogenic mutation of MyD88.

scholarly journals Large domain movements upon UvrD dimerization and helicase activation

2017 ◽  
Vol 114 (46) ◽  
pp. 12178-12183 ◽  
Author(s):  
Binh Nguyen ◽  
Yerdos Ordabayev ◽  
Joshua E. Sokoloski ◽  
Elizabeth Weiland ◽  
Timothy M. Lohman
Keyword(s):  
Escherichia Coli ◽  
Single Molecule ◽  
Self Assembly ◽  
Total Internal Reflection ◽  
Dna Helicase ◽  
Conformational State ◽  
Helicase Activity ◽  
Multiple Conformations ◽  
Dna Substrate

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.

scholarly journals Allosteric activation of preformed EGF receptor dimers by binding of a single ligand

2021 ◽  
Author(s):  
Endang Purba ◽  
Ei-ichiro Saita ◽  
Reetesh Akhouri ◽  
Lars-Göran Öfverstedt ◽  
Gunnar Wilken ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Egf Receptor ◽  
Electron Tomography ◽  
Growth Factor Receptor ◽  
Full Length ◽  
Cancer Therapies ◽  
Receptor Dimer

Abstract Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after activation. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilised by ligand binding. Consistently, optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

scholarly journals Topical Antibiotic Use Coselects for the Carriage of Mobile Genetic Elements Conferring Resistance to Unrelated Antimicrobials in Staphylococcus aureus

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Glen P. Carter ◽  
Mark B. Schultz ◽  
Sarah L. Baines ◽  
Anders Gonçalves da Silva ◽  
Helen Heffernan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
New Zealand ◽  
Single Molecule ◽  
Antibiotic Use ◽  
Multidrug Resistant ◽  
Topical Antibiotic ◽  
Content Type ◽  
Topical Antibiotics ◽  
The Impact

ABSTRACTTopical antibiotics, such as mupirocin and fusidic acid, are commonly used in the prevention and treatment of skin infections, particularly those caused by staphylococci. However, the widespread use of these agents is associated with increased resistance to these agents, potentially limiting their efficacy. Of particular concern is the observation that resistance to topical antibiotics is often associated with multidrug resistance, suggesting that topical antibiotics may play a role in the emergence of multidrug-resistant (MDR) strains. New Zealand (NZ) has some of the highest globally recorded rates of topical antibiotic usage and resistance. Using a combination of Pacific Biosciences single-molecule real-time (SMRT) whole-genome sequencing, Illumina short-read sequencing, and Bayesian phylogenomic modeling on 118 new multilocus sequence type 1 (ST1) communityStaphylococcus aureusisolates from New Zealand and 61 publically available international ST1 genome sequences, we demonstrate a strong correlation between the clinical introduction of topical antibiotics and the emergence of MDR ST1S. aureus. We also providein vitroexperimental evidence showing that exposure to topical antibiotics can lead to the rapid selection of MDRS. aureusisolates carrying plasmids that confer resistance to multiple unrelated antibiotics, from within a mixed population of competitor strains. These findings have important implications regarding the impact of the indiscriminate use of topical antibiotics.

SH3-mediated targeting of Wrch1/RhoU by multiple adaptor proteins

2013 ◽  
Vol 394 (3) ◽  
pp. 421-432 ◽  
Author(s):  
Sarah L. Risse ◽  
Belen Vaz ◽  
Matthew F. Burton ◽  
Pontus Aspenström ◽  
Roland P. Piekorz ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Adaptor Protein ◽  
Structural Difference ◽  
Adaptor Proteins ◽  
Sh3 Domain ◽  
High Avidity ◽  
Sh3 Domains ◽  
Pxxp Motif ◽  
The Impact

Abstract Wrch1/RhoU is an atypical member of the Rho family. A major structural difference is the extended N-terminus of Wrch1 (nWrch1) containing three putative SH3 domain-binding motifs whose specificities are unknown. To define the impact of this extended region on coupling Wrch1 to cellular signaling, we analyzed in this study nWrch1 interaction with Src homology 3 (SH3) domains of different adaptor proteins. Using sedimentation and isothermal titration calorimetric (ITC) measurements, we identified isolated SH3 domains of growth factor receptor-bound protein 2 (Grb2), noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1), c-Src, chicken tumor virus no. 10 (CT 10) regulator kinase 1 (Crk1), and p120 as low-affinity Wrch1-binding partners. Interestingly, under cell-based conditions, nWrch1 bound tightly to endogenous Grb2 and Nck, but not to Crk, c-Src, or p120. Consistent with this, a very tight nWrch1 interaction with full-length Grb2 and Nck1 was confirmed in vitro by ITC measurements indicating that high avidity of the adaptor proteins can compensate for the low affinity of their SH3 domains. Peptide analysis revealed that the central PxxP motif of nWrch1, which employs a minimal consensus sequence of eight amino acids with an essential arginine next to the PxxP motif, is responsible for these interactions. Thus, novel functional insights from this study suggest that multiple upstream signals may converge on Wrch1 directly through its SH3 domain-binding properties.

scholarly journals Specificity of the Protective Antibody Response to Apical Membrane Antigen 1

2001 ◽  
Vol 69 (5) ◽  
pp. 3286-3294 ◽  
Author(s):  
Anthony N. Hodder ◽  
Pauline E. Crewther ◽  
Robin F. Anders
Keyword(s):  
Apical Membrane ◽  
Affinity Purification ◽  
Point Mutations ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Potential Vaccine ◽  
Inhibitory Antibodies ◽  
Domain I ◽  
The Impact

ABSTRACT Apical membrane antigen 1 (AMA1) is considered one of the leading candidates for inclusion in a vaccine against blood stages ofPlasmodium falciparum. Although the ama1 gene is relatively conserved compared to those for some other potential vaccine components, numerous point mutations have resulted in amino acid substitutions at many sites in the polypeptide. The polymorphisms in AMA1 have been attributed to the diversifying selection pressure of the protective immune responses. It was therefore of interest to investigate the impact of sequence diversity in P. falciparum AMA1 on the ability of anti-AMA1 antibodies to inhibit the invasion of erythrocytes in vitro by P. falciparummerozoites. For these studies, we used antibodies to recombinantP. falciparum 3D7 AMA1 ectodomain, which was prepared for testing in early clinical trials. Antibodies were raised in rabbits to the antigen formulated in Montanide ISA720, and human antibodies to AMA1 were isolated by affinity purification from the plasma of adults living in regions of Papua New Guinea where malaria is endemic. Both rabbit and human anti-AMA1 antibodies were found to be strongly inhibitory to the invasion of erythrocytes by merozoites from both the homologous and two heterologous lines of P. falciparum. The inhibitory antibodies targeted both conserved and strain-specific epitopes within the ectodomain of AMA1; however, it appears that the majority of these antibodies reacted with strain-specific epitopes in domain I, the N-terminal disulfide-bonded domain, which is the most polymorphic region of AMA1.

scholarly journals 5’ modifications to CRISPR Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage

2020 ◽  
Author(s):  
Grace Mullally ◽  
Kara van Aelst ◽  
Mohsin M. Naqvi ◽  
Fiona M. Diffin ◽  
Tautvydas Karvelis ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Cleavage ◽  
Rna Aptamers ◽  
Loop Formation ◽  
Cleavage Activity ◽  
Dna Cleavage Activity ◽  
Rnp Complex ◽  
Target Binding ◽  
The Impact

A key aim in exploiting CRISPR-Cas is the engineering of gRNA to introduce additional functionalities, ranging from small nucleotide changes that increase efficiency of on-target binding to the inclusion of large functional RNA aptamers and ribonucleoproteins (RNPs. Interactions between gRNA and Cas9 are crucial for RNP complex assembly but several distinct regions of the gRNA are amenable to modification. Using a library of modified gRNAs, we used in vitro ensemble and single-molecule assays to assess the impact of RNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that R-loop formation and DNA cleavage activity are essentially unaffected by gRNA modifications of the Upper Stem, first Hairpin and 3’ end. In contrast, 5’ additions of only two or three nucleotides reduced R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such gRNA modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5’ end supported formation of a stable ~9 bp R-loop that could not activate DNA cleavage. These observations will assist in successful gRNA design.

scholarly journals Plasmodium falciparum K13 mutations in Africa and Asia present varying degrees of artemisinin resistance and an elevated fitness cost in African parasites

2021 ◽  
Author(s):  
Barbara H. Stokes ◽  
Kelly Rubiano ◽  
Satish K. Dhingra ◽  
Sachel Mok ◽  
Judith Straimer ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Southeast Asia ◽  
Survival Rates ◽  
Point Mutations ◽  
Artemisinin Resistance ◽  
Parasite Clearance ◽  
Definitive Evidence ◽  
The Impact

AbstractThe emergence of artemisinin (ART) resistance in Plasmodium falciparum parasites has led to increasing rates of treatment failure with first-line ART-based combination therapies (ACTs) in Southeast Asia. In this region, select mutations in K13 can result in delayed parasite clearance rates in vivo and enhanced survival in the ring-stage survival assay (RSA) in vitro. Our genotyping of 3,299 P. falciparum isolates across 11 sub-Saharan countries reveals the continuing dominance of wild-type K13 and confirms the emergence of a K13 R561H variant in Rwanda. Using gene editing, we provide definitive evidence that this mutation, along with M579I and C580Y, can confer variable degrees of in vitro ART resistance in African P. falciparum strains. C580Y and M579I were both associated with substantial fitness costs in African parasites, which may counter-select against their dissemination in high-transmission settings. We also report the impact of multiple K13 mutations, including the predominant variant C580Y, on RSA survival rates and fitness in multiple Southeast Asian strains. No change in ART susceptibility was observed upon editing point mutations in ferrodoxin or mdr2, earlier associated with ART resistance in Southeast Asia. These data point to the lack of an evident biological barrier to mutant K13 mediating ART resistance in Africa, while identifying their detrimental impact on parasite growth.

scholarly journals A Yersinia ruckeri TIR Domain-Containing Protein (STIR-2) Mediates Immune Evasion by Targeting the MyD88 Adaptor

2019 ◽  
Vol 20 (18) ◽  
pp. 4409 ◽  
Author(s):  
Tao Liu ◽  
Wen-Yan Wei ◽  
Kai-Yu Wang ◽  
Er-Long Wang ◽  
Qian Yang
Keyword(s):  
Immune Evasion ◽  
Adaptor Protein ◽  
Innate Immune ◽  
Luciferase Reporter ◽  
Yersinia Ruckeri ◽  
Reporter System ◽  
Tir Domain ◽  
Novel Strategy

TIR domain-containing proteins are essential for bacterial pathogens to subvert host defenses. This study describes a fish pathogen, Yersinia ruckeri SC09 strain, with a novel TIR domain-containing protein (STIR-2) that affects Toll-like receptor (TLR) function. STIR-2 was identified in Y. ruckeri by bioinformatics analysis. The toxic effects of this gene on fish were determined by in vivo challenge experiments in knockout mutants and complement mutants of the stir-2 gene. In vitro, STIR-2 downregulated the expression and secretion of IL-6, IL-1β, and TNF-α. Furthermore, the results of NF-κB-dependent luciferase reporter system, co-immunoprecipitation, GST pull-down assays, and yeast two-hybrid assay indicated that STIR-2 inhibited the TLR signaling pathway by interacting with myeloid differentiation factor 88 (MyD88). In addition, STIR-2 promoted the intracellular survival of pathogenic Yersinia ruckeri SC09 strain by binding to the TIR adaptor protein MyD88 and inhibiting the pre-inflammatory signal of immune cells. These results showed that STIR-2 increased virulence in Y. ruckeri and suppressed the innate immune response by inhibiting TLR and MyD88-mediated signaling, serving as a novel strategy for innate immune evasion.

scholarly journals 1P195 Single molecule observation of interaction between EGF receptor and an adaptor protein Grb2 reconstituted in vitro

2005 ◽  
Vol 45 (supplement) ◽  
pp. S80
Author(s):  
M. Morimatsu ◽  
K. Ohta ◽  
T. Yanagida ◽  
Y. Sako
Keyword(s):  
Single Molecule ◽  
Egf Receptor ◽  
Adaptor Protein

scholarly journals Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yann Gambin ◽  
Nicholas Ariotti ◽  
Kerrie-Ann McMahon ◽  
Michele Bastiani ◽  
Emma Sierecki ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Building Blocks ◽  
Scaffolding Protein ◽  
Cell Extracts ◽  
Single Molecule Analysis ◽  
Exquisite Specificity

In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.

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