| 1. |
- Al-Amin, Rasel A., Researcher, 1983-, et al.
(author)
-
Monitoring drug–target interactions through target engagement-mediated amplification on arrays and in situ
- 2022
-
In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:22, s. e129-e129
-
Journal article (peer-reviewed)abstract
- Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug–target interactions at spatial resolution in protein arrays, cells and in tissues.
|
|
| 2. |
|
|
| 3. |
|
|
| 4. |
- Hammond, Maria, 1984-
(author)
-
DNA-Mediated Detection and Profiling of Protein Complexes
- 2013
-
Doctoral thesis (other academic/artistic)abstract
- Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules.Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).
|
|
| 5. |
|
|
| 6. |
- Landegren, Ulf, et al.
(author)
-
Cancer diagnostics based on plasma protein biomarkers : hard times but great expectations
- 2021
-
In: Molecular Oncology. - : John Wiley & Sons. - 1574-7891 .- 1878-0261. ; 15:6, s. 1715-1726
-
Research review (peer-reviewed)abstract
- Cancer diagnostics based on the detection of protein biomarkers in blood has promising potential for early detection and continuous monitoring of disease. However, the currently available protein biomarkers and assay formats largely fail to live up to expectations, mainly due to insufficient diagnostic specificity. Here, we discuss what kinds of plasma proteins might prove useful as biomarkers of malignant processes in specific organs. We consider the need to search for biomarkers deep down in the lowest reaches of the proteome, below current detection levels. In this regard, we comment on the poor molecular detection sensitivity of current protein assays compared to nucleic acid detection reactions, and we discuss requirements for achieving detection of vanishingly small amounts of proteins, to ensure detection of early stages of malignant growth through liquid biopsy.
|
|
| 7. |
- Mattisson, Jonas, et al.
(author)
-
Leukocytes with chromosome Y loss have reduced abundance of the cell surface immunoprotein CD99
- 2021
-
In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
-
Journal article (peer-reviewed)abstract
- Mosaic loss of chromosome Y (LOY) in immune cells is a male-specific mutation associated with increased risk for morbidity and mortality. The CD99 gene, positioned in the pseudoautosomal regions of chromosomes X and Y, encodes a cell surface protein essential for several key properties of leukocytes and immune system functions. Here we used CITE-seq for simultaneous quantification of CD99 derived mRNA and cell surface CD99 protein abundance in relation to LOY in single cells. The abundance of CD99 molecules was lower on the surfaces of LOY cells compared with cells without this aneuploidy in all six types of leukocytes studied, while the abundance of CD proteins encoded by genes located on autosomal chromosomes were independent from LOY. These results connect LOY in single cells with immune related cellular properties at the protein level, providing mechanistic insight regarding disease vulnerability in men affected with mosaic chromosome Y loss in blood leukocytes.
|
|
| 8. |
- Natarajan, Karthick, et al.
(author)
-
Single-cell multimodal analysis in a case with reduced penetrance of Progranulin-Frontotemporal Dementia
- 2021
-
In: Acta neuropathologica communications. - : BioMed Central (BMC). - 2051-5960. ; 9:1
-
Journal article (peer-reviewed)abstract
- We identified an autosomal dominant progranulin mutation carrier without symptoms of dementia in her lifetime (Reduced Penetrance Mutation Carrier, RedPenMC). This resistance to develop expected pathology presents a unique opportunity to interrogate neurodegenerative mechanisms. We performed multimodal single-nuclei analyses of post-mortem frontal cortex from RedPenMC, including transcriptomics and global levels of chromatin marks. RedPenMC had an increased ratio of GRN-expressing microglia, higher levels of activating histone mark H3k4me3 in microglia and lower levels of the repressive chromatin marks H3k9me1 and H3k9me3 in the frontal cortex than her affected mutation carrier son and evidence of higher protein levels of progranulin in both plasma and brain homogenates. Although the study is limited to one case, the results support that restoring brain progranulin levels may be sufficient to escape neurodegeneration and FTD. In addition to previously identified modifier genes, it is possible that epigenetic marks may contribute to the increased progranulin expression in cases of reduced penetrance. These findings may stimulate similar follow-up studies and new therapeutic approaches.
|
|
| 9. |
|
|
