Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 3 de 3
Filter
Add more filters










Database
Language
Publication year range
1.
Methods Enzymol ; 586: 57-83, 2017.
Article in English | MEDLINE | ID: mdl-28137577

ABSTRACT

Protein folding is an intricate and precise process in living cells. Most exported proteins evade cytoplasmic folding, become targeted to the membrane, and then trafficked into/across membranes. Their targeting and translocation-competent states are nonnatively folded. However, once they reach the appropriate cellular compartment, they can fold to their native states. The nonnative states of preproteins remain structurally poorly characterized since increased disorder, protein sizes, aggregation propensity, and the observation timescale are often limiting factors for typical structural approaches such as X-ray crystallography and NMR. Here, we present an alternative approach for the in vitro analysis of nonfolded translocation-competent protein states and their comparison with their native states. We make use of hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS), a method based on differentiated isotope exchange rates in structured vs unstructured protein states/regions, and highly dynamic vs more rigid regions. We present a complete structural characterization pipeline, starting from the preparation of the polypeptides to data analysis and interpretation. Proteolysis and mass spectrometric conditions for the analysis of the labeled proteins are discussed, followed by the analysis and interpretation of HDX-MS data. We highlight the suitability of HDX-MS for identifying short structured regions within otherwise highly flexible protein states, as illustrated by an exported protein example, experimentally tested in our lab. Finally, we discuss statistical analysis in comparative HDX-MS. The protocol is applicable to any protein and protein size, exhibiting slow or fast loss of translocation competence. It could be easily adapted to more complex assemblies, such as the interaction of chaperones with nonnative protein states.


Subject(s)
Deuterium Exchange Measurement , Escherichia coli Proteins/chemistry , Mass Spectrometry , Proteome/chemistry , Amino Acid Sequence , Escherichia coli/metabolism , Escherichia coli Proteins/isolation & purification , Escherichia coli Proteins/metabolism , Protein Processing, Post-Translational , Protein Transport , Proteolysis , Proteome/isolation & purification , Proteome/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
2.
J Immunol Methods ; 375(1-2): 182-8, 2012 Jan 31.
Article in English | MEDLINE | ID: mdl-22301270

ABSTRACT

Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7-104%). The working range is 0.15-34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269-299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes.


Subject(s)
Collectins/blood , Enzyme-Linked Immunosorbent Assay/methods , Animals , Antibodies, Monoclonal/chemistry , CHO Cells , Cells, Cultured , Cricetinae , Humans , Reproducibility of Results , Sensitivity and Specificity
3.
Biochem Soc Trans ; 30(2): 177-83, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12023847

ABSTRACT

The ability to degrade the extracellular matrix by controlled proteolysis is an important property of malignant cancer cells, which enables them to invade the surrounding tissue and to gain access to the circulation by intravasation. One proteolytic system thought to be involved in these processes is urokinase-mediated plasminogen activation. Expression of a glycolipid-anchored receptor for urokinase-type plasminogen activator (uPA) targets this system to the cell surface. This receptor (uPAR) is composed of three homologous modules belonging to the Ly-6/uPAR/alpha-neurotoxin protein domain family. Integrity of the three-domain structure of uPAR is required for maintenance of its sub-nanomolar affinity for uPA, but the functional epitope for this interaction is primarily located in uPAR domain I. Using affinity maturation by combinatorial chemistry, we have recently identified a potent 9-mer peptide antagonist of the uPA-uPAR interaction having a high affinity for uPAR (K(d)< 1 nM). Photoaffinity labelling suggests that this peptide interacts with a composite binding site in uPAR involving both domains I and III. When tested in a chicken chorioallantoic membrane assay that was developed to quantify intravasation of human cells, this antagonist was able to reduce the intravasation of HEp-3 cancer cells by approx. 60%.


Subject(s)
Neoplasms/drug therapy , Neoplasms/metabolism , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Urokinase-Type Plasminogen Activator/chemistry , Urokinase-Type Plasminogen Activator/metabolism , Amino Acid Sequence , Animals , Humans , In Vitro Techniques , Models, Molecular , Molecular Sequence Data , Molecular Structure , Neoplasm Invasiveness , Oligopeptides/chemistry , Oligopeptides/pharmacology , Receptors, Cell Surface/drug effects , Receptors, Urokinase Plasminogen Activator , Urokinase-Type Plasminogen Activator/drug effects
SELECTION OF CITATIONS
SEARCH DETAIL
...