| 1. |
- Andersson, Sandra, et al.
(författare)
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Insufficient antibody validation challenges oestrogen receptor beta research
- 2017
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The discovery of oestrogen receptor beta (ER beta/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ER alpha (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ER beta antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ER beta in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ER beta protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.
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| 2. |
- Blokzijl, Andries, et al.
(författare)
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Single Chain Antibodies as Tools to Study transforming growth factor--Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens
- 2016
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 15:6, s. 1848-1856
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Tidskriftsartikel (refereegranskat)abstract
- The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF- signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF- signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF- signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA. Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.
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| 3. |
- Flowers, Sarah A., et al.
(författare)
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Lubricin binds cartilage proteins, cartilage oligomeric matrix protein, fibronectin and collagen II at the cartilage surface
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Lubricin, a heavily O-glycosylated protein, is essential for boundary lubrication of articular cartilage. Strong surface adherence of lubricin is required given the extreme force it must withstand. Disulfide bound complexes of lubricin and cartilage oligomeric matrix protein (COMP) have recently been identified in arthritic synovial fluid suggesting they may be lost from the cartilage surface in osteoarthritis and inflammatory arthritis. This investigation was undertaken to localise COMP-lubricin complexes within cartilage and investigate if other cartilage proteins are involved in anchoring lubricin to the joint. Immunohistochemical analysis of human cartilage biopsies showed lubricin and COMP co-localise to the cartilage surface. COMP knockout mice, however, presented with a lubricin layer on the articular cartilage leading to the further investigation of additional lubricin binding mechanisms. Proximity ligation assays (PLA) on human cartilage biopsies was used to localise additional lubricin binding partners and demonstrated that lubricin bound COMP, but also fibronectin and collagen II on the cartilage surface. Fibronectin and collagen II binding to lubricin was confirmed and characterised by solid phase binding assays with recombinant lubricin fragments. Overall, COMP, fibronectin and collagen II bind lubricin, exposed on the articular cartilage surface suggesting they may be involved in maintaining essential boundary lubrication.
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| 4. |
- Grannas, Karin, et al.
(författare)
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Crosstalk between Hippo and TGF beta : Subcellular Localization of YAP/TAZ/Smad Complexes
- 2015
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:21, s. 3407-3415
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Padhan, Narendra, et al.
(författare)
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Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation
- 2017
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.
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| 6. |
- Raja, Erna, et al.
(författare)
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The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling
- 2016
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 7:2, s. 1120-1143
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Tidskriftsartikel (refereegranskat)abstract
- The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease.
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| 7. |
- Zieba, Agata, et al.
(författare)
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In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation
- 2018
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Ingår i: Modern Pathology. - : Nature Publishing Group. - 0893-3952 .- 1530-0285. ; 31:2, s. 253-263
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are important tools in anatomical pathology and research, but the quality of in situ protein detection by immunohistochemistry greatly depends on the choice of antibodies and the abundance of the targeted proteins. Many antibodies used in scientific research do not meet requirements for specificity and sensitivity. Accordingly, methods that improve antibody performance and produce quantitative data can greatly advance both scientific investigations and clinical diagnostics based on protein expression and in situ localization. We demonstrate here protocols for antibody labeling that allow specific protein detection in tissues via bright-field in situ proximity ligation assays, where each protein molecule must be recognized by two antibodies. We further demonstrate that single polyclonal antibodies or purified serum preparations can be used for these dual recognition assays. The requirement for protein recognition by pairs of antibody conjugates can significantly improve specificity of protein detection over single-binder assays.
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| 8. |
- Zieba, Agata, et al.
(författare)
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The Human Endometrium-Specific Proteome Defined by Transcriptomics and Antibody-Based Profiling
- 2015
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Ingår i: Omics. - : MARY ANN LIEBERT, INC. - 1536-2310 .- 1557-8100. ; 19:11, s. 659-668
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Tidskriftsartikel (refereegranskat)abstract
- The human uterus includes the complex endometrial mucosa, the endometrium that undergoes dynamic, hormone-dependent alterations throughout the life of fertile females. Here we have combined a genome-wide transcriptomics analysis with immunohistochemistry-based protein profiling to analyze gene expression patterns in the normal endometrium. Human endometrial tissues from five women were used for deep sequencing (RNA-Seq). The mRNA and protein expression data from the endometrium were compared to 31 (RNA) and 44 (protein) other normal tissue types, to identify genes with elevated expression in the endometrium and to localize the expression of corresponding proteins at a cellular resolution. Based on the expression levels of transcripts, we could classify all putative human protein coding genes into categories defined by expression patterns and found altogether 101 genes that showed an elevated pattern of expression in the endometrium, with only four genes showing more than five-fold higher expression levels in the endometrium compared to other tissues. In conclusion, our analysis based on transcriptomics and antibody-based protein profiling reports here comprehensive lists of genes with elevated expression levels in the endometrium, providing important starting points for a better molecular understanding of human reproductive biology and disease.
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