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Ramström, Margareta
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Publications (10 of 33) Show all publications
Andersson, S., Sundberg, M., Pristovsek, N., Ibrahim, A., Jonsson, P., Katona, B., . . . Asplund, A. (2017). Insufficient antibody validation challenges oestrogen receptor beta research. Nature Communications, 8, Article ID 15840.
Open this publication in new window or tab >>Insufficient antibody validation challenges oestrogen receptor beta research
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2017 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 15840Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-329670 (URN)10.1038/ncomms15840 (DOI)000403317200001 ()28643774 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2017-09-19 Created: 2017-09-19 Last updated: 2017-11-29Bibliographically approved
Jansson, R., Lau, C. H., Ishida, T., Ramström, M., Sandgren, M. & Hedhammar, M. (2016). Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris.. Biotechnology Journal, 11(5), 687-699
Open this publication in new window or tab >>Functionalized silk assembled from a recombinant spider silk fusion protein (Z-4RepCT) produced in the methylotrophic yeast Pichia pastoris.
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2016 (English)In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 11, no 5, p. 687-699Article in journal (Refereed) Published
Abstract [en]

Functional biological materials are a growing research area with potential applicability in medicine and biotechnology. Using genetic engineering, the possibility to introduce additional functions into spider silk-based materials has been realized. Recently, a recombinant spider silk fusion protein, Z-4RepCT, was produced intracellularly in Escherichia coli and could after purification self-assemble into silk-like fibers with ability to bind antibodies via the IgG-binding Z domain. In this study, the use of the methylotrophic yeast Pichia pastoris for production of Z-4RepCT has been investigated. Temperature, pH and production time were influencing the amount of soluble Z-4RepCT retrieved from the extracellular fraction. Purification of secreted Z-4RepCT resulted in a mixture of full-length and degraded silk proteins that failed to self-assemble into fibers. A position in the C-terminal domain of 4RepCT was identified as being subjected to proteolytic cleavage by proteases in the Pichia culture supernatant. Moreover, the C-terminal domain was subjected to glycosylation during production in P. pastoris. These observed alterations of the CT domain are suggested to contribute to the failure in fiber assembly. As alternative approach, Z-4RepCT retrieved from the intracellular fraction, which was less degraded, was used and shown to retain ability to assemble into silk-like fibers after enzymatic deglycosylation.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-287507 (URN)10.1002/biot.201500412 (DOI)000375078600011 ()26814048 (PubMedID)
Funder
VINNOVASwedish Research Council, VR-NT 2013-6104Knut and Alice Wallenberg FoundationSwedish Research Council Formas, 2013-883Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2016-04-25 Created: 2016-04-25 Last updated: 2017-11-30Bibliographically approved
Maksimov, V., Nakamura, M., Wildhaber, T., Nanni, P., Ramström, M., Bergquist, J. & Hennig, L. (2016). The H3 chaperone function of NASP is conserved in Arabidopsis.. The Plant Journal, 88(3), 425-436
Open this publication in new window or tab >>The H3 chaperone function of NASP is conserved in Arabidopsis.
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2016 (English)In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 88, no 3, p. 425-436Article in journal (Refereed) Published
Abstract [en]

Histones are abundant cellular proteins but, if not incorporated into chromatin, they are usually bound by histone chaperones. Here, we identify Arabidopsis NASP as a chaperone for histones H3.1 and H3.3. NASP interacts in vitro with monomeric H3.1 and H3.3 as well as with histone H3.1-H4 and H3.3-H4 dimers. However, NASP does not bind to monomeric H4. NASP shifts the equilibrium between histone dimers and tetramers towards tetramers but does not interact with tetramers in vitro. Arabidopsis NASP promotes [H3-H4]2 tetrasome formation, possibly by providing preassembled histone tetramers. However, NASP does not promote disassembly of in vitro preassembled tetrasomes. In contrast to its mammalian homolog, Arabidopsis NASP is a predominantly nuclear protein. In vivo, NASP binds mainly monomeric H3.1 and H3.3. Pulldown experiments indicated that NASP may also interact with the histone chaperone MSI1 and a HSC70 heat shock protein.

National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-306195 (URN)10.1111/tpj.13263 (DOI)000389202800007 ()27402088 (PubMedID)
Available from: 2016-10-26 Created: 2016-10-26 Last updated: 2017-11-29Bibliographically approved
Mehdi, S., Derkacheva, M., Ramström, M., Kralemann, L., Bergquist, J. & Hennig, L. (2016). The WD40 Domain Protein MSI1 Functions in a Histone Deacetylase Complex to Fine-Tune Abscisic Acid Signaling. The Plant Cell, 28(1), 42-54
Open this publication in new window or tab >>The WD40 Domain Protein MSI1 Functions in a Histone Deacetylase Complex to Fine-Tune Abscisic Acid Signaling
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2016 (English)In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 28, no 1, p. 42-54Article in journal (Refereed) Published
Abstract [en]

MSI1 belongs to a family of histone binding WD40-repeat proteins. Arabidopsis thaliana contains five genes encoding MSI1-like proteins, but their functions in diverse chromatin-associated complexes are poorly understood. Here, we show that MSI1 is part of a histone deacetylase complex. We copurified HISTONE DEACETYLASE19 (HDA19) with MSI1 and transcriptional regulatory SIN3-like proteins and provide evidence that MSI1 and HDA19 associate into the same complex in vivo. These data suggest that MSI1, HDA19, and HISTONE DEACETYLATION COMPLEX1 protein form a core complex that can integrate various SIN3-like proteins. We found that reduction of MSI1 or HDA19 causes upregulation of abscisic acid (ABA) receptor genes and hypersensitivity of ABA-responsive genes. The MSI1-HDA19 complex fine-tunes ABA signaling by binding to the chromatin of ABA receptor genes and by maintaining low levels of acetylation of histone H3 at lysine 9, thereby affecting the expression levels of ABA receptor genes. Reduced MSI1 or HDA19 levels led to increased tolerance to salt stress corresponding to the increased ABA sensitivity of gene expression. Together, our results reveal the presence of an MSI1-HDA19 complex that fine-tunes ABA signaling in Arabidopsis.

National Category
Botany Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:uu:diva-279517 (URN)10.1105/tpc.15.00763 (DOI)000369814300008 ()26704384 (PubMedID)
Funder
Swedish Research Council, 2011-5010Swedish Research Council, 621-2011-4423Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg Foundation
Available from: 2016-03-02 Created: 2016-03-02 Last updated: 2017-11-30Bibliographically approved
Sundberg, M., Bergquist, J. & Ramström, M. (2015). High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry. Biochemistry and Biophysics Reports, 3, 68-75
Open this publication in new window or tab >>High-abundant protein depletion strategies applied on dog cerebrospinal fluid and evaluated by high-resolution mass spectrometry
2015 (English)In: Biochemistry and Biophysics Reports, ISSN 2405-5808, Vol. 3, p. 68-75Article in journal (Refereed) Published
Abstract [en]

As the number of fully sequenced animal genomes and the performance of advanced mass spectrometry-based proteomics techniques are continuously improving, there is now a great opportunity to increase the knowledge of various animal proteomes. This research area is further stimulated by a growing interest from veterinary medicine and the pharmaceutical industry. Cerebrospinal fluid (CSF) is a good source for better understanding of diseases related to the central nervous system, both in humans and other animals. In this study, four high-abundant protein depletion columns, developed for human or rat serum, were evaluated for dog CSF. For the analysis, a shotgun proteomics approach, based on nanoLC-LTQ Orbitrap MS/MS, was applied. All the selected approaches were shown to deplete dog CSF with different success. It was demonstrated that the columns significantly improved the coverage of the detected dog CSF proteome. An antibody-based column showed the best performance, in terms of efficiency, repeatability and the number of proteins detected in the sample. In total 983 proteins were detected. Of those, 801 proteins were stated as uncharacterized in the UniProt database. To the best of our knowledge, this is the so far largest number of proteins reported for dog CSF in one single study.

Keyword
Cerebrospinal fluid, Dog, High-abundant protein depletion, Shotgun proteomics, Orbitrap, Mass spectrometry
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-259611 (URN)10.1016/j.bbrep.2015.07.013 (DOI)
Available from: 2015-08-10 Created: 2015-08-10 Last updated: 2015-10-01Bibliographically approved
Kiselova, N., Dierker, T., Spillmann, D. & Ramström, M. (2014). An automated mass spectrometry-based screening method for analysis of sulfated glycosaminoglycans. Biochemical and Biophysical Research Communications - BBRC, 450(1), 598-603
Open this publication in new window or tab >>An automated mass spectrometry-based screening method for analysis of sulfated glycosaminoglycans
2014 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 450, no 1, p. 598-603Article in journal (Refereed) Published
Abstract [en]

Glycosaminoglycans (GAGs) are linear polysaccharides, consisting of repeated disaccharide units, attached to core proteins in all multicellular organisms. Chondroitin sulfate (CS) and dermatan sulfate (DS) constitute a subgroup of sulfated GAGs for which the degree of sulfation varies between species and tissues. One major goal in GAG characterization is to correlate structure to function. A common approach is to exhaustively degrade the GAG chains and thereafter determine the amount of component disaccharide units. In large-scale studies, there is a need for high-throughput screening methods since existing methods are either very time- or samples consuming. Here, we present a new strategy applying MALDI-TOF MS in positive ion mode for semi-qualitative and quantitative analysis of CS/DS derived disaccharide units. Only a few picomoles of sample are required per analysis and 10 samples can be analyzed in 25 min, which makes this approach an attractive alternative to many established assay methods. The total CS/DS concentration in 19 samples derived from Caenorhabditis elegans and mammalian tissues and cells was determined. The obtained results were well in accordance with concentrations determined by a standard liquid chromatography-based method, demonstrating the applicability of the method for samples from various biological matrices containing CS/DS of different sulfation degrees.

Keyword
Mass spectrometry, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry, Glycosaminoglycan, Chondroitin sulfate, Dermatan sulfate, Bioanalytical methods
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-231045 (URN)10.1016/j.bbrc.2014.06.011 (DOI)000343641000098 ()
Available from: 2014-09-03 Created: 2014-09-03 Last updated: 2017-12-05Bibliographically approved
Sandh, G., Ramström, M. & Stensjö, K. (2014). Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments. BMC Genomics, 15, Article ID 1064.
Open this publication in new window or tab >>Analysis of the early heterocyst Cys-proteome in the multicellular cyanobacterium Nostoc punctiforme reveals novel insights into the division of labor within diazotrophic filaments
2014 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 15, article id 1064Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

In the filamentous cyanobacterium Nostoc punctiforme ATCC 29133, removal of combined nitrogen induces the differentiation of heterocysts, a cell-type specialized in N2 fixation. The differentiation involves genomic, structural and metabolic adaptations. In cyanobacteria, changes in the availability of carbon and nitrogen have also been linked to redox regulated posttranslational modifications of protein bound thiol groups. We have here employed a thiol targeting strategy to relatively quantify the putative redox proteome in heterocysts as compared to N2-fixing filaments, 24 hours after combined nitrogen depletion. The aim of the study was to expand the coverage of the cell-type specific proteome and metabolic landscape of heterocysts.

RESULTS:

Here we report the first cell-type specific proteome of newly formed heterocysts, compared to N2-fixing filaments, using the cysteine-specific selective ICAT methodology. The data set defined a good quantitative accuracy of the ICAT reagent in complex protein samples. The relative abundance levels of 511 proteins were determined and 74% showed a cell-type specific differential abundance. The majority of the identified proteins have not previously been quantified at the cell-type specific level. We have in addition analyzed the cell-type specific differential abundance of a large section of proteins quantified in both newly formed and steady-state diazotrophic cultures in N. punctiforme. The results describe a wide distribution of members of the putative redox regulated Cys-proteome in the central metabolism of both vegetative cells and heterocysts of N. punctiforme.

CONCLUSIONS:

The data set broadens our understanding of heterocysts and describes novel proteins involved in heterocyst physiology, including signaling and regulatory proteins as well as a large number of proteins with unknown function. Significant differences in cell-type specific abundance levels were present in the cell-type specific proteomes of newly formed diazotrophic filaments as compared to steady-state cultures. Therefore we conclude that by using our approach we are able to analyze a synchronized fraction of newly formed heterocysts, which enabled a better detection of proteins involved in the heterocyst specific physiology.

National Category
Microbiology Botany Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-238129 (URN)10.1186/1471-2164-15-1064 (DOI)000349257400001 ()25476978 (PubMedID)
Available from: 2014-12-09 Created: 2014-12-09 Last updated: 2017-12-05Bibliographically approved
Eriksson, O., Ramström, M., Hörnaeus, K., Bergquist, J., Mokhtari, D. & Siegbahn, A. (2014). The Eph Tyrosine Kinase Receptors EphB2 and EphA2 Are Novel Proteolytic Substrates of Tissue Factor/Coagulation Factor VIIa. Journal of Biological Chemistry, 289(47)
Open this publication in new window or tab >>The Eph Tyrosine Kinase Receptors EphB2 and EphA2 Are Novel Proteolytic Substrates of Tissue Factor/Coagulation Factor VIIa
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2014 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 47Article in journal (Refereed) Published
Abstract [en]

Tissue factor (TF) binds the serine protease factor VIIa (FVIIa) to form a proteolytically active complex that can trigger coagulation or activate cell signaling. Here we addressed the involvement of tyrosine kinase receptors (RTKs) in TF/FVIIa signaling by antibody array analysis and subsequently found that EphB2 and EphA2 of the Eph RTK family were cleaved in their ectodomains by TF/FVIIa. We used N-terminal Edman sequencing and LC-MS/MS analysis to characterize the cleaved Eph isoforms and identified a key arginine residue at the cleavage site, in agreement with the tryptic serine protease activity of FVIIa. Protease-activated receptor 2 (PAR2) signaling and downstream coagulation activity was non-essential in this context, in further support of a direct cleavage by TF/FVIIa. EphB2 was cleaved by FVIIa concentrations in the subnanomolar range in a number of TF expressing cell types, indicating that the active cellular pool of TF was involved. FVIIa caused potentiation of cell repulsion by the EphB2 ligand ephrin-B1, demonstrating a novel proteolytical event to control Eph-mediated cell segregation. These results define Eph RTKs as novel proteolytical targets of TF/FVIIa and provide new insights into how TF/FVIIa regulates cellular functions independently of PAR2.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-240089 (URN)10.1074/jbc.M114.599332 (DOI)000345335000001 ()25281742 (PubMedID)
Available from: 2015-01-05 Created: 2015-01-05 Last updated: 2017-12-05Bibliographically approved
Kannicht, C., Ramström, M., Kohla, G., Tiemeyer, M., Casademunt, E., Walter, O. & Sandberg, H. (2013). Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII.. Thrombosis Research, 131(1), 78-88
Open this publication in new window or tab >>Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII.
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2013 (English)In: Thrombosis Research, ISSN 0049-3848, E-ISSN 1879-2472, Vol. 131, no 1, p. 78-88Article in journal (Refereed) Published
Abstract [en]

INTRODUCTION: Host cell lines used for recombinant protein expression differ in their ability to perform post-translational modifications (PTMs). The currently available recombinant human FVIII (rhFVIII) products are produced in mammalian, non-human cell lines. For rhFVIII, glycosylation and sulfation are vital for functionality and von Willebrand factor (VWF)-binding affinity. Here we present the characterisation of the PTMs of a novel, human cell line-derived recombinant human FVIII (human-cl rhFVIII). rhFVIII expression in a human cell line avoids expression of undesirable mammalian glycoforms like Galα1-3Galβ1-GlcNAc-R (α-Gal) and N-glycolylneuraminic acid (Neu5Gc), which constitute epitopes antigenic to humans. MATERIALS AND METHODS: We describe sulfation analysis, glycan profiling and characterisation using liquid chromatography-mass spectrometry and high performance anion exchange chromatography with pulsed amperometric detection. RESULTS AND CONCLUSIONS: Human-cl rhFVIII is confirmed to be sulfated and glycosylated comparable to human plasma-derived FVIII. Most importantly, human-cl rhFVIII is devoid of the antigenic Neu5Gc or α-Gal epitopes observed in Chinese Hamster Ovary- and Baby Hamster Kidney-derived rFVIII products. Both the avoidance of non-human glycan structures and the achievement of complete sulfation are proposed to lower the intrinsic immunogenicity of human-cl rhFVIII compared with current rFVIII products.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-187919 (URN)10.1016/j.thromres.2012.09.011 (DOI)23058466 (PubMedID)
Available from: 2012-12-12 Created: 2012-12-12 Last updated: 2017-12-07
Ramström, M. & Sandberg, H. (2011). Characterization of γ-carboxylated tryptic peptides by collision-induced dissociation and electron transfer dissociation mass spectrometry.. European journal of mass spectrometry, 17(5), 497-506
Open this publication in new window or tab >>Characterization of γ-carboxylated tryptic peptides by collision-induced dissociation and electron transfer dissociation mass spectrometry.
2011 (English)In: European journal of mass spectrometry, ISSN 1469-0667, E-ISSN 1751-6838, Vol. 17, no 5, p. 497-506Article in journal (Refereed) Published
Abstract [en]

Vitamin K-dependent carboxylation of glutamic acid (Glu) residues into γ-carboxyglutamic acid (Gla) is a post-translational modification essential for normal protein activity of, for example, proteins involved in the blood coagulation system. These proteins may contain as many as 12 sites for γ-carboxylation within a protein sequence of 45 amino acid residues. In the biopharmaceutical industry, powerful analytical techniques are required for identification and localization of modified sites. We here present comparatively easy and rapid methods for studies of Gla-containing proteins using recent technology. The performances of two mass spectrometric fragmentation techniques, collision-induced dissociation (CID) and electron transfer dissociation (ETD), were evaluated with respect to γ-carboxylated peptides, applying on-line LC-ion trap MS. ETD MS has so far not been reported for Gla-containing peptides and the applicability of CID for heavily γ-carboxylated proteins has not been evaluated. The anticoagulant protein, protein C, containing nine Gla-sites, was chosen as a model protein. After tryptic digestion, three peptides containing Gla-residues were detected by MS; a 1.2 kDa fragment containing two Gla-residues, a 4.5 kDa peptide containing seven residues and also the 5.6 kDa tryptic peptides containing all nine Gla-residues. Regarding the shortest peptide, both CID and ETD provided extensive peptide sequencing. For the larger peptides, fragmentation by CID resulted in loss of the 44 Da CO(2)-group, while little additional fragmentation of the peptide chain was observed. In contrast, ETD resulted in comprehensive fragmentation of the peptide backbone. The study demonstrates that the combination of both techniques would be beneficial and complementary for investigation of γ-carboxylated proteins and peptides.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-172150 (URN)10.1255/ejms.1149 (DOI)22173536 (PubMedID)
Available from: 2012-04-02 Created: 2012-04-02 Last updated: 2017-12-07
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