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Lönn, Peter
Publications (8 of 8) Show all publications
de Oliveira, F. M., Mereiter, S., Lönn, P., Siart, B., Shen, Q., Heldin, J., . . . Kamali-Moghaddam, M. (2018). Detection of post-translational modifications using solid-phase proximity ligation assay. New Biotechnology, 45, 51-59
Open this publication in new window or tab >>Detection of post-translational modifications using solid-phase proximity ligation assay
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2018 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 45, p. 51-59Article in journal (Refereed) Published
Abstract [en]

Post-translational modifications (PTMs) regulate protein activities to help orchestrate and fine-tune cellular processes. Dysregulation of PTMs is often related with disorders and malignancies, and may serve as a precise biomarker of disease. Developing sensitive tools to measure and monitor low-abundant PTMs in tissue lysates or serum will be instrumental for opening up new PTM-based diagnostic avenues. Here, we investigate the use of solid-phase proximity ligation assay (SP-PLA) for detection of different PTMs. The assay depends on the recognition of the target protein molecule and its modification by three affinity binders. Using antibodies and lectins, we applied the method for detection of glycosylated CD44 and E-Cadherin, and phosphorylated p53 and EGFR. The assay was found to have superior dynamic range and limit of detection compared to standard ELISAs. In summary, we have established the use of SP-PLA as an appropriate method for sensitive detection of PTMs in lysates and sera, which may provide a basis for future PTM-based diagnostic and prognostic biomarkers

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-334520 (URN)10.1016/j.nbt.2017.10.005 (DOI)000441913800008 ()29101055 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 316929
Available from: 2017-11-23 Created: 2017-11-23 Last updated: 2018-10-05Bibliographically approved
Lönn, P. & Landegren, U. (2017). Close Encounters: Probing Proximal Proteins in Live or Fixed Cells. TIBS -Trends in Biochemical Sciences. Regular ed., 42(7), 504-515
Open this publication in new window or tab >>Close Encounters: Probing Proximal Proteins in Live or Fixed Cells
2017 (English)In: TIBS -Trends in Biochemical Sciences. Regular ed., ISSN 0968-0004, E-ISSN 1362-4326, Vol. 42, no 7, p. 504-515Article, review/survey (Refereed) Published
Abstract [en]

The well-oiled machinery of the cellular proteome operates via variable expression, modifications, and interactions of proteins, relaying genomic and transcriptomic information to coordinate cellular functions. In recent years, a number of techniques have emerged that serve to identify sets of proteins acting in close proximity in the course of orchestrating cellular activities. These proximi dependent assays, including BiFC, BioID, APEX, FRET, and isPLA, have opened up new avenues to examine protein interactions in live or fixed cells. We review herein the current status of proximity-dependentin situ techniques. We compare the advantages and limitations of the methods, underlining recent progress and the growing importance of these techniques in basic research, and we discuss their potential as tools for drug development and diagnostics.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-333831 (URN)10.1016/j.tibs.2017.05.003 (DOI)000403569300004 ()28566215 (PubMedID)
Available from: 2017-11-24 Created: 2017-11-24 Last updated: 2017-11-24Bibliographically approved
Lönn, P. & Dowdy, S. F. (2015). Cationic PTD/CPP-mediated macromolecular delivery: charging into the cell. Expert Opinion on Drug Delivery, 12(10), 1627-1636
Open this publication in new window or tab >>Cationic PTD/CPP-mediated macromolecular delivery: charging into the cell
2015 (English)In: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 12, no 10, p. 1627-1636Article, review/survey (Refereed) Published
Abstract [en]

Introduction: Macromolecular therapeutics, including enzymes, transcription factors, siRNAs, peptides and large synthetic molecules, can potentially be used to treat human diseases by targeting intracellular molecular pathways and modulating biological responses. However, large macromolecules have no ability to enter cells and require delivery vehicles. Protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs), are a diverse class of peptides that can deliver macromolecules into cells.Areas covered: In this review, we cover the uptake and usage of arginine-rich PTDs/CPPs (TAT-PTD, Penetratin/Antp and 8R). We review the endocytosis-mediated uptake of these peptides and highlight three important steps: i) cell association; ii) internalization and iii) endosomal escape. We also discuss the array of different cargos that have been delivered by cationic PTDs/CPPs as well as cellular processes and biological responses that have been modulated.Expert opinion: PTDs/CPPs have shown great potential to deliver otherwise undeliverable macromolecular therapeutics into cells for experimentation in cell culture and in animal disease models in vivo. Moreover, over 25 clinical trials have been performed predominantly using the TAT-PTD. However, more work is still needed. Endosomal escape and target-cell specificity remain two of the major future challenges.

Keywords
CPP, delivery, macromolecular therapeutics, PTD
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-282739 (URN)10.1517/17425247.2015.1046431 (DOI)000369337400001 ()25994800 (PubMedID)
Funder
Swedish Research Council
Available from: 2016-04-14 Created: 2016-04-06 Last updated: 2018-01-10Bibliographically approved
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
Mansouri, L., Sutton, L.-A., Ljungström, V., Bondza, S., Arngården, L., Bhoi, S., . . . Rosenquist Brandell, R. (2015). Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia. Journal of Experimental Medicine, 212(6), 833-843
Open this publication in new window or tab >>Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia
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2015 (English)In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 212, no 6, p. 833-843Article in journal (Refereed) Published
Abstract [en]

NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-279237 (URN)10.1084/jem.20142009 (DOI)000355569300001 ()25987724 (PubMedID)
Funder
Swedish National Infrastructure for Computing (SNIC), b2011080Swedish Cancer SocietySwedish Research CouncilNIH (National Institute of Health), CA81554; CA081554EU, European Research Council, 259796EU, FP7, Seventh Framework Programme, 306242
Available from: 2016-02-29 Created: 2016-02-29 Last updated: 2018-01-10Bibliographically approved
Dahl, M., Maturi, V., Lönn, P., Papoutsoglou, P., Zieba, A., Vanlandewijck, M., . . . Moustakas, A. (2014). Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling. PLoS ONE, 9(8), e103651
Open this publication in new window or tab >>Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 8, p. e103651-Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

Initiation, amplitude, duration and termination of transforming growth factor β (TGFβ) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling.

METHODS:

A robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.

RESULTS:

TGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.

CONCLUSION:

Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFβ signaling.

National Category
Clinical Medicine
Identifiers
urn:nbn:se:uu:diva-231920 (URN)10.1371/journal.pone.0103651 (DOI)000341302700014 ()25133494 (PubMedID)
Available from: 2014-09-11 Created: 2014-09-11 Last updated: 2018-10-23Bibliographically approved
Björkesten, J., Patil, S., Fredolini, C., Lönn, P. & Landegren, U.Multiplex digital enumeration of circular DNA molecules on solid supports.
Open this publication in new window or tab >>Multiplex digital enumeration of circular DNA molecules on solid supports
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Digital PCR is a detection method with unprecedented accuracy for DNA quantification, but with some limitations in the form of complexity of instrumentation and limited multiplexing. Here we present an isothermal platform for digital enumeration of DNA reaction products in multiplex via standard fluorescence microscopy, to overcome limitations of digital PCR. In this method, sets of small DNA circles, resulting from detection reactions, are captured on a streptavidin-coated surface and subjected to rolling circle amplification (RCA). We found that the addition of 15% polyethylene glycol 4000 to RCA on planar surfaces ensured uniform, easily counted signals, each of which represents an individual reaction product. The DNA circles were immobilized and detected with efficiencies of 50 and 100%, respectively, as determined by droplet digital PCR. We confirmed previous reports about the effect on RCA efficiency by sequence composition and size of the RCA templates at the level of individual amplified molecules, and we developed an efficient one-step de- and re-staining procedure for sequential multiplexing via toehold-triggered DNA strand displacement.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-396324 (URN)
Available from: 2019-11-03 Created: 2019-11-03 Last updated: 2019-11-03
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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