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Proximity Ligation Assay for High Performance Protein Analysis in Medicine
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (Prof. Ulf Landegren)
2012 (English)Doctoral thesis, comprehensive summary (Other academic) [Artistic work]
Abstract [en]

High quality reagents are preconditions for high performance protein analyses. But despite progress in some techniques, e.g. mass spectrometry, there is still a lack of affinity-based detection techniques with enhanced precision, specificity, and sensitivity. Building on the concept of multiple affinity recognition reactions and signal amplification, a proximity ligation assay (PLA) was developed as a molecular tool for analyzing proteins and their post-translational modification and interactions. PLA enhanced the analysis of protein expression levels and post-translational modifications in western blotting (Paper I), which had elevated sensitivity and specificity, and an ability to investigate protein phosphorylation.

A general and straightforward method was established for the functionalization of affinity reagents through adding DNA strands to protein domains for protein analysis in medicine (Paper II). A method for protein domain-mediated conjugation was developed to simplify the use of recombinant affinity reagents, such as designed ankyrin repeat protein (DARPin), in DNA-mediated protein analyses.

Alzheimer’s disease (AD) is characterized by progressive cognitive decline and memory impairment, and amyloid-beta plaques and neurofibrillary tangles (NFT) in the brain are clinical hallmarks of the disease. In order to understand the mechanisms underlying the formation of NFT, in situ PLA was used to explore the role of microtubule affinity related kinase 2 (MARK2) in phosphorylating tau protein during the pathological progress of AD (Paper III). The analyses of roles of MARK proteins 1-4 in phosphorylating tau protein in cells and in post-mortem human brains were performed in Paper IV.

The focus of this thesis was the study of post-translational modifications and interactions of proteins in medicine. Procedures for high performance protein analysis in western blotting via proximity ligation were developed, and a functionalization method for recombinant affinity reagents in DNA-mediated protein analysis was established. These and other techniques were used to investigate the roles of tau-phosphorylating MARK family proteins in AD.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2012. , 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 801
Keyword [en]
protein, post-translational modification, interactions, protein domain, western blotting, MARK, tau, AD
National Category
Biological Sciences
Research subject
Biology with specialization in Molecular Biotechnology
Identifiers
URN: urn:nbn:se:uu:diva-179827ISBN: 978-91-554-8449-1 (print)OAI: oai:DiVA.org:uu-179827DiVA: diva2:546512
Public defence
2012-10-05, Rudbeck hall, Rudbeck Laboratory, Dag Hammarskjölds väg20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2012-09-14 Created: 2012-08-23 Last updated: 2013-01-22Bibliographically approved
List of papers
1. Western Blotting via Proximity Ligation for High Performance Protein Analysis
Open this publication in new window or tab >>Western Blotting via Proximity Ligation for High Performance Protein Analysis
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2011 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 10, no 11, O111.011031- p.Article in journal (Refereed) Published
Abstract [en]

Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor beta by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-162697 (URN)10.1074/mcp.O111.011031 (DOI)000296759400019 ()
Available from: 2011-12-07 Created: 2011-12-05 Last updated: 2017-12-08Bibliographically approved
2. Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation
Open this publication in new window or tab >>Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation
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2013 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 30, no 2, 144-152 p.Article in journal (Refereed) Published
Abstract [en]

While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-mediated affinity-based assays, such as immuno-PCR and proximity ligation assays (PLA), use oligonucleotides attached to affinity reagents as reporter molecules. Conjugation of oligonucleotides to affinity reagents generally employs chemistries that target primary amines or cysteines. Because of the random nature of these processes neither the number of oligonucleotides conjugated per molecule nor their sites of attachment can be accurately controlled for affinity reagents with several available amines and cysteines. Here, we present a straightforward and convenient approach to functionalize recombinant affinity reagents for PLA by expressing the reagents as fusion partners with SNAP protein tags. This allowed us to conjugate oligonucleotides in a site-specific fashion, yielding precisely one oligonucleotide per affinity reagent. We demonstrate this method using designed ankyrin repeat proteins (DARPins) recognizing the tumor antigen HER2 and we apply the conjugates in different assay formats. We also show that SNAP or CLIP tags expressed as fusion partners of transfected genes, allow oligonucleotide conjugations to be performed in fixed cells, with no need for specific affinity reagents. The approach is used to demonstrate induced interactions between the fusion proteins FKBP and FRB by allowing the in situ conjugated oligonucleotides to direct the production of templates for localized rolling circle amplification reactions.

Keyword
genome reduction, SAR11, Alphaproteobacteria, mismatch repair system, ocean surface waters, Candidatus Pelagibacter ubique, recombination, gene loss
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-175988 (URN)10.1016/j.nbt.2012.05.005 (DOI)000313786400007 ()22664266 (PubMedID)
Note

De 2 första författarna delar förstaförfattarskapet.

Available from: 2012-06-14 Created: 2012-06-14 Last updated: 2017-12-07Bibliographically approved
3. Elevated MARK2-Dependent Phosphorylation of Tau in Alzheimer's Disease
Open this publication in new window or tab >>Elevated MARK2-Dependent Phosphorylation of Tau in Alzheimer's Disease
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2013 (English)In: Journal of Alzheimer's Disease, ISSN 1387-2877, E-ISSN 1875-8908, Vol. 33, no 3, 699-713 p.Article in journal (Refereed) [Artistic work] Published
Abstract [en]

The appearance of neurofibrillary tangles (NFT), one of the major hallmarks of Alzheimer's disease (AD), is most likely caused by inappropriate phosphorylation and/or dephosphorylation of tau, eventually leading to the accumulation of NFTs. Enhanced phosphorylation of tau on Ser(262) is detected early in the course of the disease and may have a role in the formation of tangles. Several kinases such as microtubule-affinity regulating kinase (MARK), protein kinase A, calcium calmodulin kinase II, and checkpoint kinase 2 are known to phosphorylate tau on Ser(262) in vitro. In this study, we took advantage of the in situ proximity ligation assay to investigate the role of MARK2, one of the four MARK isoforms, in AD. We demonstrate that MARK2 interacts with tau and phosphorylates tau at Ser(262) in stably transfected NIH/3T3 cells expressing human recombinant tau. Staurosporine, a protein kinase inhibitor, significantly reduced the interaction between MARK2 and tau, and also phosphorylation of tau at Ser(262). Furthermore, we observed elevated interactions between MARK2 and tau in post-mortem human AD brains, compared to samples from non-demented elderly controls. Our results from transfected cells demonstrate a specific interaction between MARK2 and tau, as well as MARK2-dependent phosphorylation of tau at Ser(262). Furthermore, the elevated interactions between MARK2 and tau in AD brain sections suggests that MARK2 may play an important role in early phosphorylation of tau in AD, possibly qualifying as a therapeutic target for intervention to prevent disease progression.

Keyword
Alzheimer's disease, MARK, phosphorylation, proximity ligation assay, tau
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-179823 (URN)10.3233/JAD-2012-121357 (DOI)000313620500005 ()
Available from: 2012-08-23 Created: 2012-08-23 Last updated: 2017-12-07Bibliographically approved
4. Roles of individual MARK isoforms in pathological phosphorylation of tau in Alzheimer’s disease revealed by proximity ligation
Open this publication in new window or tab >>Roles of individual MARK isoforms in pathological phosphorylation of tau in Alzheimer’s disease revealed by proximity ligation
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2012 (English)Article in journal, News item (Other academic) [Artistic work] Submitted
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-179825 (URN)
Available from: 2012-08-23 Created: 2012-08-23 Last updated: 2013-01-22Bibliographically approved

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