scholarly journals Structural model of PORCN illuminates disease-associated variants and drug binding sites

2021 ◽  
Author(s):  
Jia Yu ◽  
Pei Ju Liao ◽  
Weijun Xu ◽  
Julie R. Jones ◽  
David B. Everman ◽  
...  
Keyword(s):  
Structural Model ◽  
Catalytic Mechanism ◽  
Drug Binding ◽  
Post Translational Modification ◽  
Catalytic Core ◽  
Uncertain Significance ◽  
Membrane Bound ◽  
Drug Binding Sites ◽  
Inherited Mutations ◽  
Pharmacologic Inhibition

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Based on homology to mammalian MBOAT proteins we developed and validated a structural model of PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model enhances our mechanistic understanding of PORCN substrate recognition and catalysis as well as the inhibition of its enzymatic activity and can facilitate the development of improved inhibitors and the understanding of disease relevant PORCN mutants.

scholarly journals Structural model of PORCN illuminates disease-associated variants and drug binding sites

2021 ◽  
Author(s):  
Jia Yu ◽  
Pei-Ju Liao ◽  
Weijun Xu ◽  
Julie R Jones ◽  
David B. Everman ◽  
...  
Keyword(s):  
Structural Model ◽  
Catalytic Mechanism ◽  
Drug Binding ◽  
Post Translational Modification ◽  
Catalytic Core ◽  
Uncertain Significance ◽  
Membrane Bound ◽  
Drug Binding Sites ◽  
Inherited Mutations ◽  
Pharmacologic Inhibition

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Here, based on homology to mammalian MBOAT proteins we develop and validate a molecular structural model of PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model provides mechanistic insights into PORCN substrate recognition and catalysis as well as the inhibition of its enzymatic activity and can facilitate the development of improved inhibitors and the understanding of disease relevant PORCN mutants.

scholarly journals First person – Jia Yu and Pei-Ju Liao

2021 ◽  
Vol 134 (24) ◽  
Keyword(s):  
Protein Interactions ◽  
Structural Model ◽  
Early Career ◽  
Drug Binding ◽  
First Person ◽  
Protein Protein Interactions ◽  
Early Career Researchers ◽  
Complex Simulation ◽  
Drug Binding Sites ◽  
Selection Of

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Jia Yu and Pei-Ju Liao are co-first authors on ‘ Structural model of human PORCN illuminates disease-associated variants and drug-binding sites’, published in JCS. Jia is a senior postdoc in the lab of David Virshup at Duke-NUS Medical School, Singapore, investigating Wnt secretion and signalling; in particular, how Wnt trafficking and secretion is regulated by two integral membrane proteins, porcupine and WLS. Pei-Ju is a research assistant in the same lab, investigating protein–protein interactions in the systems biology of signalling pathways using protein structure modelling and protein complex simulation.

Interaction Of Tritiated-Quinine With Human Platelets In The Presence Of A Quinine-Dependent Antibody

1981 ◽  
Author(s):  
D J Christie ◽  
R H Aster
Keyword(s):  
Binding Sites ◽  
Room Temperature ◽  
Hydrogen Exchange ◽  
Human Platelets ◽  
Drug Binding ◽  
Drug Induced ◽  
Catalytic Hydrogen ◽  
Normal Human ◽  
Membrane Bound ◽  
Drug Binding Sites

In drug-induced immune thrombocytopenia, it has not been established with certainty whether drug binds first to the platelet membrane or to antibody to promote platelet-antibody binding. The association of the drug, quinine, and a quinine dependent antibody (obtained from a patient with drug-induced thrombocytopenia) with human platelets was studied using a tritiated derivative of quinine prepared by catalytic hydrogen exchange. Tritiated quinine binding to platelets was measured in the presence of the antibody or normal human serum (NHS). Platelets were stirred with the appropriate reagents at room temperature for 20 minutes and washed. Bound tritiated quinine was measured by scintillation techniques.Two types of drug binding sites, antibody-dependent and antibody-independent, were demonstrated on platelets. In the presence of NHS, fewer than 20,000 molecules of drug became tightly bound to the membrane. In the presence of excess antibody, approximately 200,000 molecules of quinine became membrane-bound. Antibody did not bind to platelets in the absence of drug. These findings appear to provide the first demonstration of antibody-dependent binding of quinine to platelets and suggest that interaction of drug and antibody in the soluble phase precedes the binding of either constituent to the platelets (“innocent bystander mechanism”). The use of radioactive drugs appears to provide a useful tool for the investigation of the molecular basis of drug- induced immune disorders.

scholarly journals Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis

2009 ◽  
Vol 390 (2) ◽  
pp. 137-144 ◽  
Author(s):  
Yingmiao Liu ◽  
Chien-Tsun Kuan ◽  
Jing Mi ◽  
Xiuwu Zhang ◽  
Bryan M. Clary ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Rna Aptamers ◽  
Normal Brain ◽  
Post Translational Modification ◽  
Expression And Purification ◽  
Post Translational Modifications ◽  
Protein Expression And Purification ◽  
Epidermal Growth ◽  
Membrane Bound

Abstract Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant (Kd) of 33×10-9 m, and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.

scholarly journals Photocontrollable PROTAC molecules: Structure and mechanism of action

2021 ◽  
Vol 71 (3) ◽  
pp. 161-176
Author(s):  
Mladen Koravović ◽  
Gordana Tasić ◽  
Milena Rmandić ◽  
Bojan Marković
Keyword(s):  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
Binding Sites ◽  
E3 Ubiquitin Ligase ◽  
Drug Binding ◽  
Post Translational Modification ◽  
Protein Activity ◽  
Target Drug ◽  
Traditional Drug

Traditional drug discovery strategies are usually focused on occupancy of binding sites that directly affect functions of proteins. Hence, proteins that lack such binding sites are generally considered pharmacologically intractable. Modulators of protein activity, especially inhibitors, must be applied in appropriate dosage regimens that often lead to high systemic drug exposures in order to maintain sufficient protein inhibition in vivo. Consequently, there is a risk of undesirable off-target drug binding and side effects. Recently, PROteolysis TArgeting Chimera (PROTAC) technology has emerged as a new pharmacological modality that exploits PROTAC molecules for induced protein degradation. PROTAC molecule is a heterobifunctional structure consisting of a ligand that binds a protein of interest (POI), a ligand for recruiting an E3 ubiquitin ligase (an enzyme involved in the POI ubiquitination) and a linker that connects these two. After POI-PROTAC-E3 ubiquitin ligase ternary complex formation, the POI undergoes ubiquitination (an enzymatic post-translational modification in which ubiquitin is attached to the POI) and degradation. By merging the principles of photopharmacology and PROTAC technology, photocontrollable PROTACs for spatiotemporal control of induced protein degradation have recently emerged. The main advantage of photocontrollable over conventional PROTACs is the possible prevention of off-target toxicity thanks to local photoactivation.

scholarly journals Neutrophil azurophilic granule glycoproteins are distinctively decorated by atypical pauci- and phosphomannose glycans

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Karli R. Reiding ◽  
Yu-Hsien Lin ◽  
Floris P. J. van Alphen ◽  
Alexander B. Meijer ◽  
Albert J. R. Heck
Keyword(s):  
Immune System ◽  
Autoimmune Disease ◽  
Secreted Proteins ◽  
Receptor Interaction ◽  
Post Translational Modification ◽  
Site Specific ◽  
Healthy Donors ◽  
Asymmetric Hybrid ◽  
Membrane Bound ◽  
High Mannose

AbstractWhile neutrophils are critical first-responders of the immune system, they also cause tissue damage and act in a variety of autoimmune diseases. Many neutrophil proteins are N-glycosylated, a post-translational modification that may affect, among others, enzymatic activity, receptor interaction, and protein backbone accessibility. So far, a handful neutrophil proteins were reported to be decorated with atypical small glycans (paucimannose and smaller) and phosphomannosylated glycans. To elucidate the occurrence of these atypical glycoforms across the neutrophil proteome, we performed LC-MS/MS-based (glyco)proteomics of pooled neutrophils from healthy donors, obtaining site-specific N-glycan characterisation of >200 glycoproteins. We found that glycoproteins that are typically membrane-bound to be mostly decorated with high-mannose/complex N-glycans, while secreted proteins mainly harboured complex N-glycans. In contrast, proteins inferred to originate from azurophilic granules carried distinct and abundant paucimannosylation, asymmetric/hybrid glycans, and glycan phosphomannosylation. As these same proteins are often autoantigenic, uncovering their atypical glycosylation characteristics is an important step towards understanding autoimmune disease and improving treatment.

scholarly journals Systematic exploration of multiple drug binding sites

2017 ◽  
Vol 9 (1) ◽  
Author(s):  
Mónika Bálint ◽  
Norbert Jeszenői ◽  
István Horváth ◽  
David van der Spoel ◽  
Csaba Hetényi
Keyword(s):  
Binding Sites ◽  
Drug Binding ◽  
Multiple Drug ◽  
Systematic Exploration ◽  
Drug Binding Sites
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