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Stockholm, Sweden

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PubMed | CNRS Institute of Organic and Analytical Chemistry, Janssen Pharmaceutical, Biomotif AB, Vlaams Institute for Biotechnology and Karolinska Institutet
Type: Journal Article | Journal: Journal of proteome research | Year: 2015

Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases.

Chingin K.,Karolinska Institutet | Astorga-Wells J.,Karolinska Institutet | Astorga-Wells J.,Biomotif AB | Pirmoradian Najafabadi M.,Karolinska Institutet | And 4 more authors.
Analytical Chemistry | Year: 2012

We introduce an online multiple-junction capillary isoelectric focusing fractionator (OMJ-CIEF) for separation of biological molecules in solution by pI. In OMJ-CIEF, the separation capillary is divided into seven equal sections joined with each other via tubular Nafion membrane insertions. Each junction is communicated with its own external electrolytic buffer which is used both to supply electrical contact and for solvent exchange. The performance of the fractionator was explored using protein and peptide samples covering broad pI range. Separation was achieved in ionic and ampholytic buffers, including ammonium formate, ammonium hydroxide, histidine, and arginine. By maintaining electric potential across upstream segments of the capillary after the focusing stage, selective release of downstream analyte fractions could be achieved. The selective release mode circumvents the problem of peak broadening during mobilization and enables convenient comprehensive sampling for orthogonal separation methods. Using single-component ampholyte buffers with well-defined pI cutoff values, controlled separation of protein mixture into basic and acidic fractions was demonstrated. The device is cheap and easy to fabricate in-house, simple in operation, and straightforward in interfacing to hyphened analytical platforms. OMJ-CIEF has a potential of becoming a practical add-on unit in a wide range of bioanalytical setups, in particular as a first-dimension separation in mass spectrometry based proteomics or as a preparative tool for analyte purification, fractionation, and preconcentration. © 2012 American Chemical Society.

Cherry A.L.,Karolinska Institutet | Finta C.,Karolinska Institutet | Karlstrom M.,Karolinska Institutet | Jin Q.,Karolinska Institutet | And 9 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics. © 2013 International Union of Crystallography.

Agency: European Commission | Branch: FP7 | Program: CP-IP | Phase: KBBE-2007-2-2-08 | Award Amount: 8.29M | Year: 2009

Dysregulation of lipid homeostasis is related to multiple major global healthcare problems today, including aging, diabetes and cardiovascular disease. It has already been shown that nutritional modulation of lipid homeostasis via direct supplementation, e.g., n-3 fatty acids, or via indirect mechanisms, e.g., dietary polyphenols, has beneficial effects on human health. There is growing evidence that ether phospholipids such as plasmalogens play a central role in mediating the beneficial effects, but the underlying mechanisms are not understood. ETHERPATHS will develop systems biology tools that will facilitate studies of dietary interventions aiming to modulate lipid homeostasis. Specifically, we will develop (1) models that enable studies of gut microbiota and its effect on host cell metabolism, (2) dynamic models of systemic lipid metabolism, and (3) pathway reconstruction methods to study tissue-specific effects of dietary interventions. All models will be optimized in the context of studies of dietary interventions and will be integrated into a sophisticated software platform. In silico strategies will be complemented by multiple experimental approaches, including (1) dietary interventions involving n-3 fatty acids and polyphenols, combined with tracer studies in vitro and in vivo (2) in vitro colon model (3) in vivo germ-free and conventional models of altered lipid metabolism, specifically of plasmalogen deficiency. ETHERPATHS includes academic and industrial partners with combined unique expertise in information technology, bioinformatics, metabolic and physiological modelling, systems engineering, biochemistry, microbiology, lipid metabolism, metabolomics, obesity and metabolic syndrome, and clinical nutrition. We expect the ETHERPATHS tools to be broadly applied in nutrition research, and anticipate that the novel findings generated within the project will be applied for development of new food products for better health.

Pradzinska M.,University of Gdansk | Behrendt I.,University of Gdansk | Astorga-Wells J.,Karolinska Institutet | Astorga-Wells J.,Biomotif AB | And 5 more authors.
Amino Acids | Year: 2016

Human cystatin C (hCC) is a small cysteine protease inhibitor whose oligomerization by propagated domain swapping is linked to certain neurological disorders. One of the ways to prevent hCC dimerization and fibrillogenesis is to enable its interaction with a proper antibody. Herein, the sites of interaction of hCC with dimer-preventing mouse monoclonal anti-hCC antibodies Cyst28 are studied and compared with the binding sites found for mAb Cyst10 that has almost no effect on hCC dimerization. In addition, hCC epitopes in complexes with native polyclonal antibodies extracted from human serum were studied. The results obtained with hydrogen–deuterium exchange mass spectrometry (HDX MS) were compared with the previous findings made using the excision/extraction MS approach. The main results from the two complementary MS-based approaches are found to be in agreement with each other, with some differences being attributed to the specificity of each method. The findings of the current studies may be important for future design of hCC dimerization inhibitors. © 2016 The Author(s)

Pirmoradian M.,Karolinska Institutet | Pirmoradian M.,Biomotif AB | Zhang B.,Karolinska Institutet | Chingin K.,Karolinska Institutet | And 3 more authors.
Analytical Chemistry | Year: 2014

Recently, we introduced an online multijunction capillary isoelectric focusing (OMJ-CIEF) fractionator to fractionate proteins and peptides in electrospray-friendly solution. In this follow-up study, the original configuration of the fractionator was modified to improve the resolving power and reproducibility of separation. The major improvements include stabilization of the electrical current through the device using a voltage divider and stepwise elution of peptide zones in conjunction with the repeated refocusing of remaining peptides. Also, a novel algorithm was developed to calculate more accurately the pI values of peptides identified from experimental data. The standard deviation of calculated pI values for unmodified peptides from the theoretically predicted pI values was on average 0.21 pH units, which is more accurate than in standard-resolution gel-based methods. In order to characterize the analytical performance of the improved device, it was applied for the pI fractionation of yeast proteome digest into 18 fractions, with the collected fractions being analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry. Approximately 37% of 20047 identified peptides were detected in only one fraction and 27% - in two fractions. On average, every peptide was found in 2.4 fractions. These results strongly indicate the suitability of the improved device as a first dimension of separation in multidimensional shotgun proteomics analysis, with a potential for fully automated workflow. © 2014 American Chemical Society.

Pirmoradian M.,Karolinska Institutet | Pirmoradian M.,Biomotif AB | Budamgunta H.,Karolinska Institutet | Chingin K.,Karolinska Institutet | And 4 more authors.
Molecular and Cellular Proteomics | Year: 2013

Multiparameter optimization of an LC-MS/MS shotgun proteomics experiment was performed without any hardware or software modification of the commercial instrument. Under the optimized experimental conditions, with a 50-cm-long separation column and a 4-h LC-MS run (including a 3-h optimized gradient), 4,825 protein groups and 37,550 peptides were identified in a single run and 5,354 protein groups and 56,390 peptides in a triplicate analysis of the A375 human cell line, for approximately 50% coverage of the expressed proteome. The major steps enabling such performance included optimization of the cell lysis and protein extraction, digestion of even insoluble cell debris, tailoring the LC gradient profile, and choosing the optimal dynamic exclusion window in datadependent MS/MS, as well as the optimal m/z scan window. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Zhang B.,Karolinska Institutet | Pirmoradian M.,Karolinska Institutet | Pirmoradian M.,Biomotif AB | Chernobrovkin A.,Karolinska Institutet | Zubarev R.A.,Karolinska Institutet
Molecular and Cellular Proteomics | Year: 2014

Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of "cloning" chimeric tandem spectra for cofragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted cofragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average nine peptide-spectrum matches per second on a Q-Exactive workbench, whereas the actual MS/MS acquisition rate was close to seven spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant "one MS/MS spectrum - one peptide" paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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