Association of the eukaryotic V1VO ATPase subunits a with d and d with A

AbstractOwing to the complex nature of V1VO ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V1 headpiece and the VO-domain of the yeast V1VO ATPase via subunit A and d as well as the VO subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200nM have been determined for the a–d and d–A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A3B3 hexamer with VO.Structured summaryMINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107

High Affinity Human Antibody Fragments to Dengue Virus Non-Structural Protein 3

Dengue virus is the most prevalent mosquito transmitted infectious disease in humans and is responsible for febrile disease such as dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Dengue non-structural protein 3 (NS3) is an essential, multifunctional, viral enzyme with two distinct domains; a protease domain required for processing of the viral polyprotein, and a helicase domain required for replication of the viral genome. In this study ten unique human antibody fragments (Fab) that specifically bind dengue NS3 were isolated from a diverse library of Fab clones using phage display technology. The binding site of one of these antibodies, Fab 3F8, has been precisely mapped to the third α-helix within subdomain III of the helicase domain (amino acids 526–531). The antibody inhibits the helicase activity of NS3 in biochemical assays and reduces DENV replication in human embryonic kidney cells. The antibody is a valuable tool for studying dengue replication mechanisms

Weak Glycolipid Binding of a Microdomain-Tracer Peptide Correlates with Aggregation and Slow Diffusion on Cell Membranes

10.1371/journal.pone.0051222PLoS ONE712

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

AbstractOwing to the complex nature of V1VO ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V1 headpiece and the VO-domain of the yeast V1VO ATPase via subunit A and d as well as the VO subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200nM have been determined for the a–d and d–A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A3B3 hexamer with VO.Structured summaryMINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107

Real-time determination of the activity of ATPase by use of a water-soluble polythiophene

This contribution introduces a fluorescence assay for real-time determination of the activity of p97/VCP, a 540-kDa homo-hexameric enzyme, belonging to the AAA-ATPase family. A fluorescent reporter “poly 1-(3-((4-methylthiophen-3-yl)oxy)propyl)quinuclidin-1-ium” (poly PTQ) is used to monitor the hydrolysis of ATP to ADP by p97/VCP. The proposed assay relies on the different strength of coordination of ATP and ADP to the polymer backbone. We used recovery of fluorescence intensity on addition of p97/VCP to a poly PTQ/ATP solution to determine the enzymatic activity. The kinetic data K m and V max were 0.30 mmol L−1 ATP and 0.134 nmol ATP min−1 μg−1 enzyme, respectively. The specificity of the assay was investigated by using an unhydrolyzable ATP analogue and sensitivity against p97 mutagenesis was further examined by detection of the activity of wild type and truncated p97/VCP. Our study demonstrates that determination of the real-time activity of p97/VCP is possible, because of the superior sensitivity and very fast optical response of poly PTQ

A novel function of AAA-ATPase p97/VCP in the regulation of cell motility

High level of the multifunctional AAA-ATPase p97/VCP is often correlated to the development of cancer; however, the underlying mechanism is not understood completely. Here, we report a novel function of p97/VCP in actin regulation and cell motility. We found that loss of p97/VCP promotes stabilization of F-actin, which cannot be reversed by actin-destabilizing agent, Cytochalasin D. Live-cell imaging demonstrated reduced actin dynamics in p97/VCP-knockdown cells, leading to compromised cell motility. We further examined the underlying mechanism and found elevated RhoA protein levels along with increased phosphorylation of its downstream effectors, ROCK, LIMK, and MLC upon the knockdown of p97/VCP. Since p97/VCP is indispensable in the ubiquitination-dependent protein degradation pathway, we investigated if the loss of p97/VCP hinders the protein degradation of RhoA. Knockdown of p97/VCP resulted in a higher amount of ubiquitinated RhoA, suggesting p97/VCP involvement in the proteasome-dependent protein degradation pathway. Finally, we found that p97/VCP interacts with FBXL19, a molecular chaperone known to guide ubiquitinated RhoA for proteasomal degradation. Reduction of p97/VCP may result in the accumulation of RhoA which, in turn, enhances cytoplasmic F-actin formation. In summary, our study uncovered a novel function of p97/VCP in actin regulation and cell motility via the Rho-ROCK dependent pathway which provides fundamental insights into how p97/VCP is involved in cancer development.Ministry of Education (MOE)Published versionThis project is supported by Academic Research Fund RG44/13 and RG44/16, Ministry of Education, Singapore

A fast mutagenesis procedure to recover soluble and functional scFvs containing amber stop codons from synthetic and semisynthetic antibody libraries

8 p.-4 fig.The selection and production of scFvs from phage display synthetic antibody libraries are frequently delayed by the presence of amber (TAG) stop codons within the sequences corresponding to the variable CDRs. This is due to the use of randomised oligonucleotides for library design and amber mutations for joining the scFv to the phage protein pIII. The screening of such libraries may lead to the selection of scFvs containing stop codons. Then, multiple site-directed mutagenesis is required for their removal or, alternatively, the proteins must be expressed as scFv-pIII fusions, which are not suitable for many functional assays. We describe here an alternative procedure to express soluble scFvs, despite the presence of TAG stop codons, in the currently used Escherichia coli suppressor strain TG1. It is based on a simple mutagenesis protocol that replaces the amber codon between the scFv and the pIII gene by a different stop codon (TAA), functional in E. coli TG1. The expression of soluble scFvs in the suppressor strain TG1 permits their fully functional characterization including the determination of affinity constants, which are critical for selecting the right scFvs for further studies.This project was partially supported by an EU grant COOP-CT-2004-512691 and the grant B102003-01481 from the Ministry of Science and Technology (MCYT) of Spain.Peer reviewe

ATP Binding to p97/VCP D1 Domain Regulates Selective Recruitment of Adaptors to Its Proximal N-Domain

<div><p>p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.</p> </div

Conformational changes upon ATP binding to p97/VCP and p97-N-D1 observed by differential scanning fluorimetry.

<p>Hexamers of either full length p97/VCP (A) or p97-N-D1 fragment (B) were subjected to differential scanning fluorimetry in the absence or presence of 1 mM ATP. In the absence of ATP, the melting transition of full length p97/VCP hexamers was a two step process with the major component occurring at 60°C. Minor shifts were detected in the presence of ATP (A). p97-N-D1 hexamers had a melting temperature of 50°C in the absence of ATP, which was significantly shifted by 14°C to 64°C in the presence of ATP (B). Concentration-dependent thermostabilizations were observed by measurements of melting temperatures of full length p97/VCP (C) or p97-N-D1 fragment (D) at the indicated concentrations of ATP.</p

Binding affinities of the interactions between p97/VCP and its adaptor proteins.

<p>p97/VCP was immobilized on a CM5 sensorchip surface, using amine coupling procedure, and a concentration series of p47 (two fold dilutions from 1.92 μM) or UN (two fold dilutions from 1.88 μM) were injected over the immobilized surface at a flow rate of 30 μl/min, at 25°C, using PBS as the sample and running buffer. Sensorgrams and the corresponding interaction maps for the p97/VCP-UN interaction without ATP (A), with ATP during UN association and dissociation (B) and for p97/VCP-p47 interactions when p47 is immobilized (C) and when p97/VCP is immobilized (D) are shown. Interaction Map Analysis shows that the interaction between UN and p97/VCP is complex, with a major component with ∼5 μM affinity, and a second component with ∼400 nM affinity. When ATP is added, the affinity of the second component increases to 100 nM. For the interaction between p97/VCP and p47, only one kinetic component was found, and the interaction could be fitted to a Langmuir 1∶1 interaction model, with ∼31.3 nM affinity when p47 is immobilized and p97 injected across (C) or ∼5 μM affinity when p97 is immobilized and p47 injected across (D).</p