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Proximity Ligation and Barcoding Assays: Tools for analysis of proteins and protein complexes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are fundamental structural, enzymatic and regulatory components of cells. Analysis of proteins, such as by measuring their concentrations, characterizing their modifications, and detecting their interactions, provides insights in how biological systems work physiologically or pathologically at the molecular level. To perform such analysis, molecular tools with good sensitivity, specificity, high multiplexing and throughput capacity are needed.

In this thesis, four different assays were developed and applied to detect and profile proteins and protein complexes in human body fluids, and in cells or tissues. These assays are based on targeting proteins or protein complexes by oligonucleotide-conjugated antibodies, and subsequent proximity dependent enzymatic reactions involving the attached DNA reporter sequences.

In paper I, a solid-phase proximity ligation assay (SP-PLA) was applied to detect synthetic and endogenous amyloid beta protofibrils. The SP-PLA provided better sensitivity and increased dynamic range than a traditional enzyme-linked immunosorbent assay (ELISA).

In paper II, in situ PLA was applied to investigate the correlation between MARK2-dependent phosphorylation of tau and Alzheimer’s disease. Greater numbers of MARK2-tau interactions and of phosphorylated tau proteins were observed in brain tissues from Alzheimer’s patients than in healthy controls.

In paper III, a multiplex SP-PLA was applied to identify protein biomarker candidates in amyotrophic lateral sclerosis (ALS) disease and in the analgesic mechanism of spinal cord stimulation (SCS). Among 47 proteins in human cerebrospinal fluid (CSF) samples, four were found at significantly lower concentrations (p-values < 0.001) in the samples from ALS patients compared to those from healthy controls (follistatin, IL-1α, IL-1β, and KLK5). No significant changes of the analyzed proteins were found in the CSF samples of neuropathic pain patients in   the stimulated vs. non-stimulated condition using SCS.

In paper IV, a new technology termed the proximity barcoding assay (PBA) was developed to profile individual protein complexes. The performance of PBA was demonstrated on artificially assembled streptavidin-biotin oligonucleotide complexes. PBA was also proven to be capable of profiling transcriptional pre-initiation complexes from nuclear extract of a hepatic cell line.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 43 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 980
Keyword [en]
proximity ligation assay, proximity barcoding assay, sensitive, in situ, multiplex, profiling, amyloid-beta, MARK, tau, CSF, biomarker, protein complexes, Alzheimer’s disease, amyotrophic lateral sclerosis
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-220070ISBN: 978-91-554-8901-4 (print)OAI: oai:DiVA.org:uu-220070DiVA: diva2:704655
Public defence
2014-04-25, B21, Biomedicinskt centrum, Husargatan 3, SE-75123, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2014-04-03 Created: 2014-03-10 Last updated: 2014-04-29
List of papers
1. Sensitive detection of A beta protofibrils by proximity ligation: relevance for Alzheimer's disease
Open this publication in new window or tab >>Sensitive detection of A beta protofibrils by proximity ligation: relevance for Alzheimer's disease
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2010 (English)In: BMC neuroscience (Online), ISSN 1471-2202, E-ISSN 1471-2202, Vol. 11, 124- p.Article in journal (Refereed) Published
Abstract [en]

Background: Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A beta peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of A beta protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity. Results: For specific detection of A beta protofibrils we have used a monoclonal antibody, mAb158, selective for A beta protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Ab aggregates in solid- phase reactions, allowing detection of just 0.1 pg/ml A beta protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Ab protofibril detection by up to 25- fold. The assay was used to measure soluble Ab aggregates in brain homogenates from mice transgenic for a human allele predisposing to A beta aggregation. Conclusions: The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of A beta aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-133826 (URN)10.1186/1471-2202-11-124 (DOI)000283243900001 ()20923550 (PubMedID)
Available from: 2010-11-19 Created: 2010-11-16 Last updated: 2017-12-12Bibliographically approved
2. 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
3. Analyses of CSF reveal decreased levels of four proteins in ALS patients, but no changes upon analgesia in patients with neuropathic pain
Open this publication in new window or tab >>Analyses of CSF reveal decreased levels of four proteins in ALS patients, but no changes upon analgesia in patients with neuropathic pain
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(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-220139 (URN)
Available from: 2014-03-12 Created: 2014-03-11 Last updated: 2014-04-29
4. Profiling individual protein complexes by proximity-dependent barcoding
Open this publication in new window or tab >>Profiling individual protein complexes by proximity-dependent barcoding
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-220093 (URN)
Available from: 2014-03-11 Created: 2014-03-10 Last updated: 2015-02-03

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