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Blokzijl, Andries
Alternative names
Publications (5 of 5) Show all publications
Grannas, K., Arngården, L., Lönn, P., Mazurkiewicz, M., Blokzij, A., Zieba Wicher, A. & Söderberg, O. (2015). Crosstalk between Hippo and TGF beta: Subcellular Localization of YAP/TAZ/Smad Complexes. Journal of Molecular Biology, 427(21), 3407-3415
Open this publication in new window or tab >>Crosstalk between Hippo and TGF beta: Subcellular Localization of YAP/TAZ/Smad Complexes
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2015 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 21, p. 3407-3415Article in journal (Refereed) Published
Abstract [en]

The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGF beta. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGF beta (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGF beta stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGF beta induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGF beta signaling pathways.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-268434 (URN)10.1016/j.jmb.2015.04.015 (DOI)000363823200006 ()25937570 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 278568Swedish Research Council
Available from: 2015-12-04 Created: 2015-12-04 Last updated: 2017-12-01Bibliographically approved
Blokzijl, A., Nong, R., Darmanis, S., Hertz, E., Landegren, U. & Kamali-Moghaddam, M. (2014). Protein biomarker validation via proximity ligation assays. Biochimica et Biophysica Acta - Proteins and Proteomics, 1844(5), 933-939
Open this publication in new window or tab >>Protein biomarker validation via proximity ligation assays
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2014 (English)In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1844, no 5, p. 933-939Article, review/survey (Refereed) Published
Abstract [en]

The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PIA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. (C) 2013 Elsevier B.V. All rights reserved.

Keywords
Biomarkers, Protein detection, Proximity ligation assay, Diagnostic
National Category
Biochemistry and Molecular Biology Biophysics
Identifiers
urn:nbn:se:uu:diva-228134 (URN)10.1016/j.bbapap.2013.07.016 (DOI)000335878200010 ()
Available from: 2014-07-04 Created: 2014-07-04 Last updated: 2017-12-05Bibliographically approved
Shaw, A., Lundin, V., Petrova, E., Fordos, F., Benson, E., Al-Amin, A., . . . Teixeira, A. I. (2014). Spatial control of membrane receptor function using ligand nanocalipers. Nature Methods, 11(8), 841-846
Open this publication in new window or tab >>Spatial control of membrane receptor function using ligand nanocalipers
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2014 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 11, no 8, p. 841-846Article in journal (Refereed) Published
Abstract [en]

The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor mediated signaling.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-231209 (URN)10.1038/NMETH.3025 (DOI)000340075600024 ()
Funder
Swedish Research Council, 2010-6296, 2010-5060The Karolinska Institutet's Research FoundationVINNOVA
Available from: 2014-09-09 Created: 2014-09-05 Last updated: 2018-11-23Bibliographically approved
Gu, G. J., Lund, H., Wu, D., Blokzijl, A., Classon, C., von Euler, G., . . . Kamali-Moghaddam, M. (2013). Role of Individual MARK Isoforms in Phosphorylation of Tau at Ser(262) in Alzheimer's Disease. Neuromolecular medicine, 15(3), 458-469
Open this publication in new window or tab >>Role of Individual MARK Isoforms in Phosphorylation of Tau at Ser(262) in Alzheimer's Disease
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2013 (English)In: Neuromolecular medicine, ISSN 1535-1084, E-ISSN 1559-1174, Vol. 15, no 3, p. 458-469Article in journal (Refereed) Published
Abstract [en]

The microtubule-affinity regulating kinase (MARK) family consists of four highly conserved members that have been implicated in phosphorylation of tau protein, causing formation of neurofibrillary tangles in Alzheimer's disease (AD). Understanding of roles by individual MARK isoform in phosphorylating tau has been limited due to lack of antibodies selective for each MARK isoform. In this study, we first applied the proximity ligation assay on cells to select antibodies specific for each MARK isoform. In cells, a CagA peptide specifically and significantly inhibited tau phosphorylation at Ser(262) mediated by MARK4 but not other MARK isoforms. We then used these antibodies to study expression levels of MARK isoforms and interactions between tau and individual MARK isoforms in postmortem human brains. We found a strong and significant elevation of MARK4 expression and MARK4-tau interactions in AD brains, correlating with the Braak stages of the disease. These results suggest the MARK4-tau interactions are of functional importance in the progression of AD and the results also identify MARK4 as a promising target for AD therapy.

Keywords
Alzheimer's disease, MARK, Phosphorylation, Proximity ligation, Tau
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-206553 (URN)10.1007/s12017-013-8232-3 (DOI)000322722300002 ()
Available from: 2013-09-02 Created: 2013-09-02 Last updated: 2017-12-06Bibliographically approved
Al-Amin, R. A., Johansson, L., Landegren, N., Löf, L., Abdurakhmanov, E., Blokzijl, A., . . . Landegren, U.Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in Situ.
Open this publication in new window or tab >>Target Engagement-Mediated Amplification for Monitoring Drug-Target Interactions in Situ
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

It is important to determine the localization of drugs or drug candidates at cellular and subcellular resolution in relevant clinical specimens. This is necessary to evaluate drug candidates from early stages of drug development to clinical evaluation of mutations potentially causing resistance to targeted therapy. We describe a technology where oligonucleotide-conjugated drug molecules are used to visualize and measure target engagement in situ via rolling-circle amplification (RCA) of circularized oligonucleotide probes (padlock probes). We established this target engagement-mediated amplification (TEMA) technique using kinase inhibitor precursor compounds, and we applied the assay to investigate target interactions by microscopy in pathology tissue sections and using flow cytometry for blood samples from patients, as well as in commercial arrays including almost half of all human proteins.  In the variant proxTEMAtechnique, in situ proximity ligation assays were performed by combining drug-DNA conjugates with antibody-DNA conjugates to specifically reveal drug binding to particular on- or off-targets in pathological tissues sections. In conclusion, the TEMA methods successfully visualize drug-target interaction by experimental and clinically approved kinase inhibitors in situ and with kinases among a large collection of arrayed proteins. 

National Category
Natural Sciences
Research subject
Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-374262 (URN)
Available from: 2019-01-18 Created: 2019-01-18 Last updated: 2019-01-21
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