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- Weibrecht, Irene, 1983-
(författare)
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Visualizing Interacting Biomolecules In Situ
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Intra- and intercellular information is communicated by posttranslational modifications (PTMs) and protein-protein interactions, transducing information over cell membranes and to the nucleus. A cells capability to respond to stimuli by several highly complex and dynamic signaling networks provides the basis for rapid responses and is fundamental for the cellular collaborations required in a multicellular organism. Having received diverse stimuli, being positioned at various stages of the cell cycle or, for the case of cancer, containing altered genetic background, each cell in a population is slightly different from its neighbor. However, bulk analyses of interactions will only reveal an average, but not the true variation within a population. Thus studies of interacting endogenous biomolecules in situ are essential to acquire a comprehensive view of cellular functions and communication. In situ proximity ligation assay (in situ PLA) was developed to investigate individual endogenous protein-protein interactions in fixed cells and tissues and was later applied for detection for PTMs. Progression of signals in a pathway can branch out in different directions and induce expression of different target genes. Hence simultaneous measurement of protein activity and gene expression provides a tool to determine the balance and progression of these signaling events. To obtain this in situ PLA was combined with padlock probes, providing an assay that can interrogate both PTMs and mRNA expression at a single cell level. Thereby different nodes of the signaling pathway as well as drug effects on different types of molecules could be investigated simultaneously. In addition to regulation of gene expression, protein-DNA interactions present a mechanism to manage accessibility of the genomic DNA in an inheritable manner, providing the basis for lineage commitment, via e.g. histone PTMs. To enable analyses of protein-DNA interactions in situ we developed a method that utilizes the proximity dependence of PLA and the sequence selectivity of padlock probes. This thesis presents new methods providing researchers with a set of tools to address cellular functions and communication in complex microenvironments, to improve disease diagnostics and to contribute to hopefully finding cures.
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| 2. |
- Sander, Marie Rubin, 1988-
(författare)
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Analysis of PDGF receptor internalization and signaling using proximity ligation assays
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell signaling is mediated by signaling proteins that relay the signal in an intricate network of interactions before the signal is translated into a biological response. Short linear motifs (SLiMs) in intrinsically disordered regions of proteins serve as docking sites for protein interaction in all aspects of cell regulation including signal transduction. SLiM-mediated interactions are transient and low affinity and can be hijacked by virus. Only a small fraction of SLiMs have been described, but many more exist. Platelet derived growth factor receptor β (PDGFRβ) belongs to the family of receptor tyrosine kinases (RTKs) and controls cell growth, proliferation and migration. Dysregulation of PDGFRβ mediated signaling pathways is seen in many cancer types. To discover and characterize protein interactions, large scale high through-put methods are needed in concert with low through-put methods, that can characterize the interaction in a cellular context. The aim of this thesis has been to study protein-protein interactions in internalization and signaling of PDGFRβ and motif-mediated host-virus interactions through the use of in situ proximity ligation assay (PLA). Signaling via PDGFRβ is compartmentalized and depends on receptor internalization. In paper I we investigated the effects of dynamin inhibition for activation and signaling of PDGFRβ, and found that dynamin inhibition leads to impaired dimerization of the PDGFRβ. The results indicate that membrane localization of PDGFRβ is affected by dynamin. In paper II we developed a new method, Molboolean, for localized simultaneous detection of both free protein and protein in complex in cells. Molboolean is based on the principles from PLA with a fluorescent read out detectable with fluorescence microscopy. In paper III we mapped SLiM based host-virus interactions and explored their mechanisms. Using proteomic peptide phage display, we identified 1712 potential virus-host interactions by screening a library covering intrinsically disordered regions of the proteome for 229 RNA viruses. Clathrin mediated endocytosis was found to be a common target for viral hijacking, and viral binding of clathrin impaired PDGFRβ internalization. Some RTKs are proteolytically cleaved following ligand activation. In paper IV we characterized the Ca2+-dependent proteolytic cleavage of PDGFRβ. The cleavage resulted in two PDGFRβ fragments and was dependent on receptor internalization. Inhibition of the proteasome with bortezomib prevented internalization and cleavage and resulted in increased activation of PLCγ and STAT3. This thesis provides insight in the regulation of PDGFRβ signaling and internalization, and highlights contributions of both large-scale screenings and low through-put methods for studying protein-protein interactions.
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| 3. |
- Wu, Di
(författare)
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Proximity Ligation and Barcoding Assays : Tools for analysis of proteins and protein complexes
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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