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1.
J Chem Inf Model ; 60(3): 1652-1665, 2020 03 23.
Article in English | MEDLINE | ID: mdl-32134653

ABSTRACT

The human sodium iodide symporter (hNIS) is a theranostic reporter gene which concentrates several clinically approved SPECT and PET radiotracers and plays an essential role for the synthesis of thyroid hormones as an iodide transporter in the thyroid gland. Development of hNIS mutants which could enhance translocation of the desired imaging ions is currently underway. Unfortunately, it is hindered by lack of understanding of the 3D organization of hNIS and its relation to anion transport. There are no known crystal structures of hNIS in any of its conformational states. Homology modeling can be very effective in such situations; however, the low sequence identity between hNIS and relevant secondary transporters with available experimental structures makes the choice of a template and the generation of 3D models nontrivial. Here, we report a combined application of homology modeling and molecular dynamics refining of the hNIS structure in its semioccluded state. The modeling was based on templates from the LeuT-fold protein family and was done with emphasis on the refinement of the substrate-ion binding pocket. The consensus model developed in this work is compared to available biophysical and biochemical experimental data for a number of different LeuT-fold proteins. Some functionally important residues contributing to the formation of putative binding sites and permeation pathways for the cotransported Na+ ions and I- substrate were identified. The model predictions were experimentally tested by generation of mutant versions of hNIS and measurement of relative (to WT hNIS) 125I- uptake of 35 hNIS variants.


Subject(s)
Symporters , Binding Sites , Humans , Iodides/metabolism , Symporters/metabolism , Thyroid Gland/metabolism
2.
Virology ; 454-455: 237-46, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24725950

ABSTRACT

The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Hemagglutinins, Viral/genetics , Hemagglutinins, Viral/immunology , Morbillivirus/genetics , Morbillivirus/immunology , Animals , Antibodies, Monoclonal/immunology , Humans , Male , Measles/immunology , Measles Vaccine/immunology , Neutralization Tests
4.
PLoS One ; 8(1): e52306, 2013.
Article in English | MEDLINE | ID: mdl-23300970

ABSTRACT

The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs.


Subject(s)
Antibodies, Monoclonal/immunology , Hemagglutinins, Viral/genetics , Measles Vaccine/immunology , Mutation , Adenoviridae/genetics , Animals , Antibodies, Neutralizing/immunology , CHO Cells , Chlorocebus aethiops , Cricetinae , Epitopes/genetics , Epitopes/immunology , Glycosylation , Hemagglutinins, Viral/immunology , Humans , Measles virus/genetics , Measles virus/immunology , Mice , Mice, Inbred BALB C , Mutagenesis, Site-Directed , Neutralization Tests , Plasmids , Protein Structure, Tertiary , Rabbits , Recombinant Proteins/immunology , Vero Cells
5.
Expert Rev Vaccines ; 9(11): 1275-302, 2010 Nov.
Article in English | MEDLINE | ID: mdl-21087107

ABSTRACT

Paramyxoviruses, measles virus (MV), mumps virus (MuV) and Newcastle disease virus (NDV), are well known for causing measles and mumps in humans and Newcastle disease in birds. These viruses have been tamed (attenuated) and successfully used as vaccines to immunize their hosts. Remarkably, pathogenic MuV and vaccine strains of MuV, MV and NDV efficiently infect and kill cancer cells and are consequently being investigated as novel cancer therapies (oncolytic virotherapy). Phase I/II clinical trials have shown promise but treatment efficacy needs to be enhanced. Technologies being developed to increase treatment efficacy include: virotherapy in combination with immunosuppressive drugs (cyclophosphamide); retargeting of viruses to specific tumor types or tumor vasculature; using infected cell carriers to protect and deliver the virus to tumors; and genetic manipulation of the virus to increase viral spread and/or express transgenes during viral replication. Transgenes have enabled noninvasive imaging or tracking of viral gene expression and enhancement of tumor destruction.


Subject(s)
Measles virus/growth & development , Mumps virus/growth & development , Neoplasms/therapy , Newcastle disease virus/growth & development , Oncolytic Virotherapy/methods , Clinical Trials as Topic , Humans , Measles virus/pathogenicity , Mumps virus/pathogenicity , Newcastle disease virus/pathogenicity , Treatment Outcome
6.
Proc Natl Acad Sci U S A ; 100(22): 12694-9, 2003 Oct 28.
Article in English | MEDLINE | ID: mdl-14557549

ABSTRACT

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation.


Subject(s)
Carrier Proteins/chemistry , Cysteine Endopeptidases/chemistry , DNA Repair , DNA-Binding Proteins/chemistry , Multienzyme Complexes/chemistry , Amino Acid Sequence , Binding Sites , Carrier Proteins/metabolism , Cysteine Endopeptidases/metabolism , DNA Repair Enzymes , DNA-Binding Proteins/metabolism , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Multienzyme Complexes/metabolism , Proteasome Endopeptidase Complex , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA-Binding Proteins
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