scholarly journals Homologue Scanning Mutagenesis Reveals Cd66 Receptor Residues Required for Neisserial Opa Protein Binding

1999 ◽  
Vol 190 (3) ◽  
pp. 331-340 ◽  
Author(s):  
Martine P. Bos ◽  
Daniel Hogan ◽  
Robert J. Belland
Keyword(s):  
Epithelial Cells ◽  
Protein Binding ◽  
Protein Interactions ◽  
Human Neutrophils ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Binding Characteristics ◽  
Protein Receptor ◽  
Strain Ms11 ◽  
Differential Binding

The immunoglobulin-like family of CD66 antigens, present on human neutrophils and epithelial cells, are used as receptors for adhesins expressed by the pathogenic Neisseriae. N. gonorrhoeae strain MS11 can express 11 isoforms of these adhesins, called opacity-related (Opa) proteins. Each MS11 Opa protein recognizes a distinct spectrum of CD66 receptors. CD66–Opa binding is mediated by the NH2-terminal domain of the receptor and occurs through protein–protein interactions. In this report, we have investigated the molecular basis for the binding between the CD66 and Opa protein families by mapping amino acids in CD66 receptors that determine Opa protein binding. We performed homologue scanning mutagenesis between CD66e, which binds multiple Opa variants, and CD66b, which binds none, and tested both loss-of-function by CD66e and gain-of-function by CD66b in solution assays and in assays involving full-length receptors expressed by epithelial cells. We found that three residues in the CD66e N-domain are required for maximal Opa protein receptor activity. Opa proteins that recognize the same spectrum of native CD66 molecules showed differential binding of receptors with submaximal activity, indicating that the binding characteristics of these Opa proteins are actually slightly different. These data provide a first step toward resolving the structural requirements for Opa–CD66 interaction.

scholarly journals Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch

2016 ◽  
Vol 474 (1) ◽  
pp. 79-104 ◽  
Author(s):  
Dakshnamurthy Selvakumar ◽  
Marian J. Drescher ◽  
Nathan A. Deckard ◽  
Neeliyath A. Ramakrishnan ◽  
Barbara J. Morley ◽  
...  
Keyword(s):  
Hair Cell ◽  
Protein Interactions ◽  
Molecular Switch ◽  
Protein Protein Interactions ◽  
Inner Hair Cell ◽  
Binding Characteristics ◽  
Protein Receptor ◽  
Kd Values ◽  
Sensitive Factor ◽  
Related Proteins

Dopamine receptors regulate exocytosis via protein–protein interactions (PPIs) as well as via adenylyl cyclase transduction pathways. Evidence has been obtained for PPIs in inner ear hair cells coupling D1A to soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-related proteins snapin, otoferlin, N-ethylmaleimide-sensitive factor (NSF), and adaptor-related protein complex 2, mu 1 (AP2mu1), dependent on [Ca2+] and phosphorylation. Specifically, the carboxy terminus of dopamine D1A was found to directly bind t-SNARE-associated protein snapin in teleost and mammalian hair cell models by yeast two-hybrid (Y2H) and pull-down assays, and snapin directly interacts with hair cell calcium-sensor otoferlin. Surface plasmon resonance (SPR) analysis, competitive pull-downs, and co-immunoprecipitation indicated that these interactions were promoted by Ca2+ and occur together. D1A was also found to separately interact with NSF, but with an inverse dependence on Ca2+. Evidence was obtained, for the first time, that otoferlin domains C2A, C2B, C2D, and C2F interact with NSF and AP2mu1, whereas C2C or C2E do not bind to either protein, representing binding characteristics consistent with respective inclusion or omission in individual C2 domains of the tyrosine motif YXXΦ. In competitive pull-down assays, as predicted by KD values from SPR (+Ca2+), C2F pulled down primarily NSF as opposed to AP2mu1. Phosphorylation of AP2mu1 gave rise to a reversal: an increase in binding by C2F to phosphorylated AP2mu1 was accompanied by a decrease in binding to NSF, consistent with a molecular switch for otoferlin from membrane fusion (NSF) to endocytosis (AP2mu1). An increase in phosphorylated AP2mu1 at the base of the cochlear inner hair cell was the observed response elicited by a dopamine D1A agonist, as predicted.

scholarly journals Genetic and Molecular Characterization of the Caenorhabditis elegans Gene, mel-26, a Postmeiotic Negative Regulator of MEI-1, a Meiotic-Specific Spindle Component

Genetics ◽  
1998 ◽  
Vol 150 (1) ◽  
pp. 119-128
Author(s):  
M Rhys Dow ◽  
Paul E Mains
Keyword(s):  
Caenorhabditis Elegans ◽  
Protein Interactions ◽  
Negative Regulator ◽  
Meiotic Spindle ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Gain Of Function ◽  
Recessive Mutations ◽  
Female Germline

Abstract We have previously described the gene mei-1, which encodes an essential component of the Caenorhabditis elegans meiotic spindle. When ectopically expressed after the completion of meiosis, mei-1 protein disrupts the function of the mitotic cleavage spindles. In this article, we describe the cloning and the further genetic characterization of mel-26, a postmeiotic negative regulator of mei-1. mel-26 was originally identified by a gain-of-function mutation. We have reverted this mutation to a loss-of-function allele, which has recessive phenotypes identical to the dominant defects of its gain-of-function parent. Both the dominant and recessive mutations of mel-26 result in mei-1 protein ectopically localized in mitotic spindles and centrosomes, leading to small and misoriented cleavage spindles. The loss-of-function mutation was used to clone mel-26 by transformation rescue. As suggested by genetic results indicating that mel-26 is required only maternally, mel-26 mRNA was expressed predominantly in the female germline. The gene encodes a protein that includes the BTB motif, which is thought to play a role in protein-protein interactions.

scholarly journals ArabidopsisImmunophilin-like TWD1 Functionally Interacts with Vacuolar ABC Transporters

2004 ◽  
Vol 15 (7) ◽  
pp. 3393-3405 ◽  
Author(s):  
Markus Geisler ◽  
Marjolaine Girin ◽  
Sabine Brandt ◽  
Vincent Vincenzetti ◽  
Sonia Plaza ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Abc Transporters ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Binding Motifs ◽  
Calmodulin Binding ◽  
C Terminus ◽  
Domain Mapping ◽  
Tetratricopeptide Repeat Domain

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-β-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.

scholarly journals Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor

2018 ◽  
Vol 115 (12) ◽  
pp. 3036-3041 ◽  
Author(s):  
Yinglong Miao ◽  
J. Andrew McCammon
Keyword(s):  
Protein Binding ◽  
G Protein ◽  
Protein Interactions ◽  
Intracellular Signaling ◽  
Md Simulations ◽  
Binding Pocket ◽  
Signaling Proteins ◽  
Protein Protein Interactions ◽  
Intracellular Loops ◽  
G Protein Coupled

Protein–protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein–protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR–nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein–protein interactions.

scholarly journals Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

2016 ◽  
Vol 27 (5) ◽  
pp. 744-756 ◽  
Author(s):  
Yuki Niwa ◽  
Takehiro Suzuki ◽  
Naoshi Dohmae ◽  
Siro Simizu
Keyword(s):  
Mass Spectrometry ◽  
Enzymatic Activity ◽  
Wnt Signaling ◽  
Protein Interactions ◽  
Secreted Protein ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Rare Type ◽  
Synthesis Activity ◽  
Canonical Wnt

R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein–protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W153 and W156. Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W153 and W156 by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W156 but not W153 based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W156 and found that DPY19L3-mediated C-mannosylation of Rspo1 at W156 is required for its secretion.

scholarly journals Virus–Host Protein–Protein Interactions between Human Papillomavirus 16 E6 A1 and D2/D3 Sub-Lineages: Variances and Similarities

2020 ◽  
Vol 21 (21) ◽  
pp. 7980
Author(s):  
Guillem Dayer ◽  
Mehran L. Masoom ◽  
Melissa Togtema ◽  
Ingeborg Zehbe
Keyword(s):  
Human Papillomavirus ◽  
Asian American ◽  
Protein Interactions ◽  
Host Protein ◽  
Cellular Proteins ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Human Papillomavirus 16 ◽  
Novel Approach ◽  
Causative Agents

High-risk strains of human papillomavirus are causative agents for cervical and other mucosal cancers, with type 16 being the most frequent. Compared to the European Prototype (EP; A1), the Asian-American (AA; D2/D3) sub-lineage seems to have increased abilities to promote carcinogenesis. Here, we studied protein–protein interactions (PPIs) between host proteins and sub-lineages of the key transforming E6 protein. We transduced human keratinocyte with EP or AA E6 genes and co-immunoprecipitated E6 proteins along with interacting cellular proteins to detect virus–host binding partners. AAE6 and EPE6 may have unique PPIs with host cellular proteins, conferring gain or loss of function and resulting in varied abilities to promote carcinogenesis. Using liquid chromatography-mass spectrometry and stringent interactor selection criteria based on the number of peptides, we identified 25 candidates: 6 unique to AAE6 and EPE6, along with 13 E6 targets common to both. A novel approach based on pathway selection discovered 171 target proteins: 90 unique AAE6 and 61 unique EPE6 along with 20 common E6 targets. Interpretations were made using databases, such as UniProt, BioGRID, and Reactome. Detected E6 targets were differentially implicated in important hallmarks of cancer: deregulating Notch signaling, energetics and hypoxia, DNA replication and repair, and immune response.

Changes in receptor location affect the ability of oxytocin to stimulate proliferative growth in prostate epithelial cells

2019 ◽  
Vol 31 (6) ◽  
pp. 1166 ◽  
Author(s):  
M. L. Gould ◽  
H. D. Nicholson
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cells ◽  
Cell Membrane ◽  
Lipid Rafts ◽  
Protein Interactions ◽  
Cell Signalling ◽  
Signalling Pathways ◽  
Protein Protein Interactions ◽  
Normal Prostate ◽  
Prostate Epithelial Cells

In normal prostate cells, cell membrane receptors are located within signalling microdomains called caveolae. During cancer progression, caveolae are lost and sequestered receptors move out onto lipid rafts. The aim of this study was to investigate whether a change in the localisation of receptors out of caveolae and onto the cell membrane increased cell proliferation invitro, and to determine whether this is related to changes in the cell signalling pathways. Normal human prostate epithelial cells (PrEC) and androgen-independent (PC3) cancer cells were cultured with 10nM dihydrotestosterone (DHT). The effects of oxytocin (OT) and gonadal steroids on proliferation were assessed using the MTS assay. Androgen receptor (AR) and oxytocin receptor (OTR) expression was identified by immunofluorescence and quantified by western blot. OTR and lipid raft staining was determined using Pearson’s correlation coefficient. Protein–protein interactions were detected and the cell signalling pathways identified. Treatment with OT did not affect the proliferation of PrEC. In PC3 cells, OT or androgen alone increased cell proliferation, but together had no effect. In normal cells, OTR localised to the membrane and AR localised to the nucleus, whereas in malignant cells both OTR and AR were identified in the cell membrane. Colocalisation of OTR and AR increased following treatment with androgens. Significantly fewer OTR/AR protein–protein interactions were seen in PrEC. With OT treatment, several cell signalling pathways were activated. Movement of OTR out of caveolae onto lipid rafts is accompanied by activation of alternative signal transduction pathways involved in stimulating increased cell proliferation.

scholarly journals A High-Throughput Solid-Phase Microplate Protein-Binding Assay to Investigate Interactions between Myofilament Proteins

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Brandon J. Biesiadecki ◽  
J.-P. Jin
Keyword(s):  
Protein Binding ◽  
Protein Interactions ◽  
Protein Modification ◽  
Binding Assay ◽  
Solid Phase ◽  
Regulatory Protein ◽  
Protein Isoforms ◽  
Protein Protein Interactions ◽  
Protein Binding Assay ◽  
Phase Protein

To understand the structure-function relationship of muscle-regulatory-protein isoforms, mutations, and posttranslational modifications, it is necessary to probe functional effects at the level of the protein-protein interaction. Traditional methodologies assessing such protein-protein interactions are laborious and require significant amounts of purified protein, while many current methodologies require costly and specialized equipment or modification of the proteins, which may affect their interaction. To address these issues, we developed a novel method of microplate-based solid-phase protein-binding assay over the recent years. This method assesses specific protein-protein interactions at physiological conditions, utilizes relatively small amounts of protein, is free of protein modification, and does not require specialized instrumentation. Here we present detailed methodology for the solid-phase protein-binding assay with examples that we have successfully applied to quantify interactions of myofilament-regulatory proteins. We further provide considerations for optimization of the assay conditions and its broader application in studies of other protein-protein interactions.

Evolutionary selection for protein aggregation

2012 ◽  
Vol 40 (5) ◽  
pp. 1032-1037 ◽  
Author(s):  
Natalia Sanchez de Groot ◽  
Marc Torrent ◽  
Anna Villar-Piqué ◽  
Benjamin Lang ◽  
Salvador Ventura ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Protein Interactions ◽  
Current Knowledge ◽  
Protein Aggregates ◽  
Cellular Systems ◽  
Aggregation Process ◽  
Loss Of Function ◽  
Protein Protein Interactions ◽  
Wide Range ◽  
Range Of Functions

Protein aggregation is being found to be associated with an increasing number of human diseases. Aggregation can lead to a loss of function (lack of active protein) or to a toxic gain of function (cytotoxicity associated with protein aggregates). Although potentially harmful, protein sequences predisposed to aggregation seem to be ubiquitous in all kingdoms of life, which suggests an evolutionary advantage to having such segments in polypeptide sequences. In fact, aggregation-prone segments are essential for protein folding and for mediating certain protein–protein interactions. Moreover, cells use protein aggregates for a wide range of functions. Against this background, life has adapted to tolerate the presence of potentially dangerous aggregation-prone sequences by constraining and counteracting the aggregation process. In the present review, we summarize the current knowledge of the advantages associated with aggregation-prone stretches in proteomes and the strategies that cellular systems have developed to control the aggregation process.

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