Quantitative analysis of protease recognition by inhibitors in plasma using microscale thermophoresis

Dau, Therese; Edeleva, Evgeniia; Seidel, Susanne A. I.; Stockley, Robert A.; Braun, Dieter; Jenne, Dieter E.

doi:10.1038/srep35413

Cited by 6 publications

(5 citation statements)

References 27 publications

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“…PBS-T buffer 25 was also studied and produced repeatable binding curves that could be fitted. The initial fluorescence of the various capillaries was within the tolerance threshold (Fig.…”

Section: Resultsmentioning

confidence: 99%

PD-1/PD-L1 binding studies using microscale thermophoresis

Magnez

Thiroux

Taront

et al. 2017

Sci Rep

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The characterization of protein interactions has become essential in many fields of life science, especially drug discovery. Microscale thermophoresis (MST) is a powerful new method for the quantitative analysis of protein-protein interactions (PPIs) with low sample consumption. In addition, one of the major advantages of this technique is that no tedious purification step is necessary to access the protein of interest. Here, we describe a protocol using MST to determine the binding affinity of the PD-1/PD-L1 couple, which is involved in tumour escape processes, without purification of the target protein from cell lysates. The method requires the overexpression of fluorescent proteins in CHO-K1 cells and describes the optimal conditions for determining the dissociation constant. The protocol has a variety of potential applications in studying the interactions of these proteins with small molecules and demonstrates that MST is a valuable method for studying the PD-1/PD-L1 pathway.

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“…PBS-T buffer 25 was also studied and produced repeatable binding curves that could be fitted. The initial fluorescence of the various capillaries was within the tolerance threshold (Fig.…”

Section: Resultsmentioning

confidence: 99%

PD-1/PD-L1 binding studies using microscale thermophoresis

Magnez

Thiroux

Taront

et al. 2017

Sci Rep

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“…In contrast, the microscale thermophoresis method applied here disclosed a clear difference between the two variants. This method accounts for all interactions of AAT with components of the plasma matrix, which can modify the strength of interactions between AAT and NE, as we recently showed for the AAT-Z variant (36). Under these conditions, the dissociation constant between the enzymatically inactive NE and AAT was found to be lower for M1(V213) than for M1(A213), indicating that components of the plasma matrix interfered more strongly with the former allele.…”

Section: Discussionmentioning

confidence: 98%

Common coding variant in SERPINA1 increases the risk for large artery stroke

Malik

Dau

Gonik

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Large artery atherosclerotic stroke (LAS) shows substantial heritability not explained by previous genome-wide association studies. Here, we explore the role of coding variation in LAS by analyzing variants on the HumanExome BeadChip in a total of 3,127 cases and 9,778 controls from Europe, Australia, and South Asia. We report on a nonsynonymous single-nucleotide variant in serpin family A member 1 (SERPINA1) encoding alpha-1 antitrypsin [AAT; p.V213A; P = 5.99E-9, odds ratio (OR) = 1.22] and confirm histone deacetylase 9 (HDAC9) as a major risk gene for LAS with an association in the 3′-UTR (rs2023938; P = 7.76E-7, OR = 1.28). Using quantitative microscale thermophoresis, we show that M1 (A213) exhibits an almost twofold lower dissociation constant with its primary target human neutrophil elastase (NE) in lipoprotein-containing plasma, but not in lipid-free plasma. Hydrogen/deuterium exchange combined with mass spectrometry further revealed a significant difference in the global flexibility of the two variants. The observed stronger interaction with lipoproteins in plasma and reduced global flexibility of the Val-213 variant most likely improve its local availability and reduce the extent of proteolytic inactivation by other proteases in atherosclerotic plaques. Our results indicate that the interplay between AAT, NE, and lipoprotein particles is modulated by the gate region around position 213 in AAT, far away from the unaltered reactive center loop (357-360). Collectively, our findings point to a functionally relevant balance between lipoproteins, proteases, and AAT in atherosclerosis.genetics | ischemic stroke | large artery stroke | antitrypsin | variation S troke is the leading cause of long-term disability and the second most common cause of death worldwide (1, 2). About a quarter of ischemic stroke cases are caused by large artery atherosclerotic stroke (LAS) (3, 4). Atherosclerosis is a chronic inflammatory condition that involves a number of well-characterized steps. Initial stages include the deposition of lipids in vascular endothelial cells, whereas more advanced stages are characterized by fibrotic changes with formation of a fibrotic cap and, eventually, plaque rupture (5). LAS exhibits the highest heritability of all stroke subtypes, with estimates ranging from 40.3 to 66.6% (6, 7). This fact is reflected by recent genome-wide association studies that found common variants for LAS at multiple genomic loci (8-10). The lead SNPs from these regions all reside within intergenic (4, 7, 11) or SignificanceCommon single-amino acid variations of proteins are traditionally regarded as functionally neutral polymorphisms because these substitutions are mostly located outside functionally relevant surfaces. In this study, we present an example of a functionally relevant coding sequence variation, which, as we show here, confers risk for large artery atherosclerotic stroke. The singleresidue variation M1(A213V) in serpin family A member 1 (SERPINA1) [encoding alpha-1 antitrypsin (AAT)] is situated outside the...

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“…MST and fluorescence correlation spectroscopy (FCS) techniques were used to measure binding constant where the dissociation constants that determined by MST and F K d = 236 nM and K d = 282 nM, respectively. Different applications [43][44][45][46][47][48][49][50] of MST technique in the characterization of protein-protein interactions are summarized in Table 1.…”

Section: Protein-protein Interactionsmentioning

confidence: 99%

Thermophoresis for characterizing biomolecular interaction

Asmari

Ratih

Alhazmi

et al. 2018

Methods

101

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The study of biomolecular interactions is crucial to get more insight into the biological system. The interactions of protein-protein, protein-nucleic acids, protein-sugars, nucleic acid-nucleic acids and protein-small molecules are supporting therapeutics and technological developments. Recently, the development in a large number of analytical techniques for characterizing biomolecular interactions reflect the promising research investments in this field. In this review, microscale thermophoresis technology (MST) is presented as an analytical technique for characterizing biomolecular interactions. Recent years have seen much progress and several applications established. MST is a powerful technique in quantitation of binding events based on the movement of molecules in microscopic temperature gradient. Simplicity, free solutions analysis, low sample volume, short analysis time, and immobilization free are the MST advantages over other competitive techniques. A wide range of studies in biomolecular interactions have been successfully carried out using MST, which tend to the versatility of the technique to use in screening binding events in order to save time, cost and obtained high data quality.

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Quantitative analysis of protease recognition by inhibitors in plasma using microscale thermophoresis

Cited by 6 publications

References 27 publications

PD-1/PD-L1 binding studies using microscale thermophoresis

PD-1/PD-L1 binding studies using microscale thermophoresis

Common coding variant in SERPINA1 increases the risk for large artery stroke

Thermophoresis for characterizing biomolecular interaction

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