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 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.

NMR for the design of functional mimetics of protein-protein interactions: one key is in the building of bridges

1998 ◽  
Vol 76 (2-3) ◽  
pp. 177-188 ◽  
Author(s):  
Jianxing Song ◽  
Feng Ni
Keyword(s):  
Protein Interactions ◽  
Binding Sites ◽  
Experimental Approach ◽  
Structural Information ◽  
Peptide Ligands ◽  
Protein Protein Interactions ◽  
Binding Residues ◽  
Related Proteins ◽  
Binding Inhibitors

Using the design of bivalent and bridge-binding inhibitors of thrombin as an example, we review an NMR-based experimental approach for the design of functional mimetics of protein-protein interactions. The strategy includes: (i) identification of binding residues in peptide ligands by differential resonance perturbation, (ii) determination of protein-bound structures of peptide ligands by use of transferred NOEs, (iii) minimization of larger protein and peptide ligands on the basis of NMR structural information, and (iv) linkage of two weakly binding mimetics to produce an inhibitor with enhanced affinity and specificity. This approach can be especially effective for the design of potent and selective functional mimetics of protein-protein interactions because it is less likely that the surfaces of two related proteins or enzymes share two identical binding sites or regions.Key words: NMR, protein-protein interactions, functional mimetics, bridge-binding inhibitors, thrombin.

scholarly journals Comparative Proteomic Analysis of Paulownia fortunei Response to Phytoplasma Infection with Dimethyl Sulfate Treatment

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Zhen Wei ◽  
Zhe Wang ◽  
Xiaoyu Li ◽  
Zhenli Zhao ◽  
Minjie Deng ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Dimethyl Sulfate ◽  
Morphological Characteristics ◽  
Forest Tree ◽  
Protein Protein Interactions ◽  
Absolute Quantitation ◽  
Phytoplasma Infection ◽  
Paulownia Fortunei ◽  
Isobaric Tags ◽  
Related Proteins

Paulownia fortunei is a widely cultivated economic forest tree species that is susceptible to infection with phytoplasma, resulting in Paulownia witches’ broom (PaWB) disease. Diseased P. fortunei is characterized by stunted growth, witches’ broom, shortened internodes, and etiolated and smaller leaves. To understand the molecular mechanism of its pathogenesis, we applied isobaric tags for relative and absolute quantitation (iTRAQ) and liquid chromatography coupled with tandem mass spectrometry approaches to study changes in the proteomes of healthy P. fortunei, PaWB-infected P. fortunei, and PaWB-infected P. fortunei treated with 15 mg·L−1 or 75 mg·L−1 dimethyl sulfate. We identified 2969 proteins and 104 and 32 differentially abundant proteins that were phytoplasma infection responsive and dimethyl sulfate responsive, respectively. Based on our analysis of the different proteomes, 27 PaWB-related proteins were identified. The protein-protein interactions of these 27 proteins were analyzed and classified into four groups (photosynthesis-related, energy-related, ribosome-related, and individual proteins). These PaWB-related proteins may help in developing a deeper understanding of how PaWB affects the morphological characteristics of P. fortunei and further establish the mechanisms involved in the response of P. fortunei to phytoplasma.

Site-specific recombination by φC31 integrase and other large serine recombinases

2010 ◽  
Vol 38 (2) ◽  
pp. 388-394 ◽  
Author(s):  
Margaret C.M. Smith ◽  
William R.A. Brown ◽  
Andrew R. McEwan ◽  
Paul A. Rowley
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Serine Recombinases ◽  
Tyrosine Recombinases ◽  
Dna Binding Sites ◽  
Attachment Sites ◽  
Φc31 Integrase ◽  
Related Proteins ◽  
Control Protein

Most temperate phages encode an integrase for integration and excision of the prophage. Integrases belong either to the λ Int family of tyrosine recombinases or to a subgroup of the serine recombinases, the large serine recombinases. Integration by purified serine integrases occurs efficiently in vitro in the presence of their cognate (~50 bp) phage and host attachment sites, attP and attB respectively. Serine integrases require an accessory protein, Xis, to promote excision, a reaction in which the products of the integration reaction, attL and attR, recombine to regenerate attP and attB. Unlike other directional recombinases, serine integrases are not controlled by proteins occupying accessory DNA-binding sites. Instead, it is thought that different integrase conformations, induced by binding to the DNA substrates, control protein–protein interactions, which in turn determine whether recombination proceeds. The present review brings together the evidence for this model derived from the studies on φC31 integrase, Bxb1 integrase and other related proteins.

scholarly journals Conservation and Variability of Synaptonemal Complex Proteins in Phylogenesis of Eukaryotes

2014 ◽  
Vol 2014 ◽  
pp. 1-16 ◽  
Author(s):  
Tatiana M. Grishaeva ◽  
Yuri F. Bogdanov
Keyword(s):  
Synaptonemal Complex ◽  
Protein Interactions ◽  
Phylogenetic Trees ◽  
Flowering Plants ◽  
Model Organisms ◽  
Protein Protein Interactions ◽  
Lateral Element ◽  
Origin And Evolution ◽  
Eukaryotic Proteomes ◽  
Related Proteins

The problems of the origin and evolution of meiosis include the enigmatic variability of the synaptonemal complexes (SCs) which, being morphology similar, consist of different proteins in different eukaryotic phyla. Using bioinformatics methods, we monitored all available eukaryotic proteomes to find proteins similar to known SC proteins of model organisms. We found proteins similar to SC lateral element (LE) proteins and possessing the HORMA domain in the majority of the eukaryotic taxa and assume them the most ancient among all SC proteins. Vertebrate LE proteins SYCP2, SYCP3, and SC65 proved to have related proteins in many invertebrate taxa. Proteins of SC central space are most evolutionarily variable. It means that different protein-protein interactions can exist to connect LEs. Proteins similar to the known SC proteins were not found in Euglenophyta, Chrysophyta, Charophyta, Xanthophyta, Dinoflagellata, and primitive Coelomata. We conclude that different proteins whose common feature is the presence of domains with a certain conformation are involved in the formation of the SC in different eukaryotic phyla. This permits a targeted search for orthologs of the SC proteins using phylogenetic trees. Here we consider example of phylogenetic trees for protozoans, fungi, algae, mosses, and flowering plants.

scholarly journals A conformational switch driven by phosphorylation regulates the activity of the evolutionarily conserved SNARE Ykt6

2021 ◽  
Vol 118 (12) ◽  
pp. e2016730118
Author(s):  
Kaitlyn McGrath ◽  
Shivani Agarwal ◽  
Marco Tonelli ◽  
Mykola Dergai ◽  
Anthony L. Gaeta ◽  
...  
Keyword(s):  
Conformational Change ◽  
Protein Interactions ◽  
Multidisciplinary Approach ◽  
Open Form ◽  
Conformational Switch ◽  
Protein Receptor ◽  
Evolutionarily Conserved ◽  
Sensitive Factor ◽  
Membrane Bound ◽  
Open Membrane

Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson’s disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.

The interaction of IQGAPs with calmodulin-like proteins

2011 ◽  
Vol 39 (2) ◽  
pp. 694-699 ◽  
Author(s):  
Sevvel Pathmanathan ◽  
Elaine Hamilton ◽  
Erwan Atcheson ◽  
David J. Timson
Keyword(s):  
Protein Interactions ◽  
Light Chain ◽  
Protein Protein Interactions ◽  
Iq Motif ◽  
Calcium Dependent ◽  
Essential Light Chain ◽  
Cellular Processes ◽  
Wide Range ◽  
Related Proteins ◽  
Do So

Since their identification over 15 years ago, the IQGAP (IQ-motif-containing GTPase-activating protein) family of proteins have been implicated in a wide range of cellular processes, including cytoskeletal reorganization, cell–cell adhesion, cytokinesis and apoptosis. These processes rely on protein–protein interactions, and understanding these (and how they influence one another) is critical in determining how the IQGAPs function. A key group of interactions is with calmodulin and the structurally related proteins myosin essential light chain and S100B. These interactions occur primarily through a series of IQ motifs, which are α-helical segments of the protein located towards the middle of the primary sequence. The three human IQGAP isoforms (IQGAP1, IQGAP2 and IQGAP3) all have four IQ motifs. However, these have different affinities for calmodulin, myosin light chain and S100B. Whereas all four IQ motifs of IQGAP1 interact with calmodulin in the presence of calcium, only the last two do so in the absence of calcium. IQ1 (the first IQ motif) interacts with the myosin essential light chain Mlc1sa and the first two undergo a calcium-dependent interaction with S100B. The significance of the interaction between Mlc1sa and IQGAP1 in mammals is unknown. However, a similar interaction involving the Saccharomyces cerevisiae IQGAP-like protein Iqg1p is involved in cytokinesis, leading to speculation that there may be a similar role in mammals.

scholarly journals Localization, Dynamics, and Protein Interactions Reveal Distinct Roles for ER and Golgi SNAREs

1998 ◽  
Vol 141 (7) ◽  
pp. 1489-1502 ◽  
Author(s):  
Jesse C. Hay ◽  
Judith Klumperman ◽  
Viola Oorschot ◽  
Martin Steegmaier ◽  
Christin S. Kuo ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Protein Interactions ◽  
The Other ◽  
Snare Proteins ◽  
Attachment Protein ◽  
Golgi Transport ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Coated Membranes ◽  
Factor Attachment Protein Receptor

ER-to-Golgi transport, and perhaps intraGolgi transport involves a set of interacting soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins including syntaxin 5, GOS-28, membrin, rsec22b, and rbet1. By immunoelectron microscopy we find that rsec22b and rbet1 are enriched in COPII-coated vesicles that bud from the ER and presumably fuse with nearby vesicular tubular clusters (VTCs). However, all of the SNAREs were found on both COPII- and COPI-coated membranes, indicating that similar SNARE machinery directs both vesicle pathways. rsec22b and rbet1 do not appear beyond the first Golgi cisterna, whereas syntaxin 5 and membrin penetrate deeply into the Golgi stacks. Temperature shifts reveal that membrin, rsec22b, rbet1, and syntaxin 5 are present together on membranes that rapidly recycle between peripheral and Golgi-centric locations. GOS-28, on the other hand, maintains a fixed localization in the Golgi. By immunoprecipitation analysis, syntaxin 5 exists in at least two major subcomplexes: one containing syntaxin 5 (34-kD isoform) and GOS-28, and another containing syntaxin 5 (41- and 34-kD isoforms), membrin, rsec22b, and rbet1. Both subcomplexes appear to involve direct interactions of each SNARE with syntaxin 5. Our results indicate a central role for complexes among rbet1, rsec22b, membrin, and syntaxin 5 (34 and 41 kD) at two membrane fusion interfaces: the fusion of ER-derived vesicles with VTCs, and the assembly of VTCs to form cis-Golgi elements. The 34-kD syntaxin 5 isoform, membrin, and GOS-28 may function in intraGolgi transport.

scholarly journals Downregulation of MYO1C mediated by cepharanthine inhibits autophagosome-lysosome fusion through blockade of the F-actin network

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Yanhao Zhang ◽  
Xiuxing Jiang ◽  
Qin Deng ◽  
Ziyi Gao ◽  
Xiangyu Tang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Protein Interactions ◽  
Human Breast Cancer ◽  
Ultrastructural Changes ◽  
Human Breast ◽  
Protein Protein Interactions ◽  
Transmission Electron ◽  
Promising Treatment ◽  
Related Proteins ◽  
First Time

Abstract Background MYO1C, an actin-based motor protein, is involved in the late stages of autophagosome maturation and fusion with the lysosome. The molecular mechanism by which MYO1C regulates autophagosome-lysosome fusion remains largely unclear. Methods Western blotting was used to determine the expression of autophagy-related proteins. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes. An immunoprecipitation assay was utilized to detect protein-protein interactions. Immunofluorescence analysis was used to detect autophagosome-lysosome fusion and colocalization of autophagy-related molecules. An overexpression plasmid or siRNA against MYO1C were sequentially introduced into human breast cancer MDA-MB-231 cells. Results We show here that cepharanthine (CEP), a novel autophagy inhibitor, inhibited autophagy/mitophagy through blockage of autophagosome-lysosome fusion in human breast cancer cells. Mechanistically, we found for the first time that MYO1C was downregulated by CEP treatment. Furthermore, the interaction/colocalization of MYO1C and F-actin with either LC3 or LAMP1 was inhibited by CEP treatment. Knockdown of MYO1C further decreased the interaction/colocalization of MYO1C and F-actin with either LC3 or LAMP1 inhibited by CEP treatment, leading to blockade of autophagosome-lysosome fusion. In contrast, overexpression of MYO1C significantly restored the interaction/colocalization of MYO1C and F-actin with either LC3 or LAMP1 inhibited by CEP treatment. Conclusion These findings highlight a key role of MYO1C in the regulation of autophagosome-lysosome fusion through F-actin remodeling. Our findings also suggest that CEP could potentially be further developed as a novel autophagy/mitophagy inhibitor, and a combination of CEP with classic chemotherapeutic drugs could become a promising treatment for breast cancer.

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