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  • 1. Abramczyk, Olga
    et al.
    Zien, Piotr
    Zielinski, Rafał
    Pilecki, Marek
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Szyszka, Ryszard
    The protein kinase 60S is a free catalytic CK2alpha' subunit and forms an inactive complex with superoxide dismutase SOD12003In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 307, no 1, p. 31-40Article in journal (Refereed)
    Abstract [en]

    The 60S ribosomes from Saccharomyces cerevisiae contain a set of acidic P-proteins playing an important role in the ribosome function. Reversible phosphorylation of those proteins is a mechanism regulating translational activity of ribosomes. The key role in regulation of this process is played by specific, second messenger-independent protein kinases. The PK60S kinase was one of the enzymes phosphorylating P-proteins. The enzyme has been purified from yeast and characterised. Pure enzyme has properties similar to those reported for casein kinase type 2. Peptide mass fingerprinting (PMF) has identified the PK60S as a catalytic alpha(') subunit of casein kinase type 2 (CK2alpha(')). Protein kinase activity is inhibited by SOD1 and by highly specific CK2 inhibitor-4,5,6,7-tetrabromo-benzotriazole (TBBt). The possible mechanism of regulation of CK2alpha(') activity in stress conditions, by superoxide dismutase in regulation of 80S-ribosome activity, is discussed.

  • 2. Agüero, Fernán
    et al.
    Noé, Griselda
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Repetto, Yolanda
    Zaha, Arnaldo
    Cazzulo, Juan José
    Purification, cloning, and expression of the mitochondrial malate dehydrogenase (mMDH) from protoscolices of Echinococcus granulosus2004In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 137, no 2, p. 207-214Article in journal (Refereed)
    Abstract [en]

    Protoscolices of the parasitic helminth Echinococcus granulosus contain two malate dehydrogenases (EC 1.1.1.37), one cytosolic and one mitochondrial. The latter has been separated from the other isoform and purified to protein homogeneity. Sequencing of tryptic peptides by Edman degradation allowed the design of oligonucleotide primers for PCR, leading to the cloning and sequencing of a full length cDNA. The encoding gene is present as a single copy per haploid genome and codes for a protein with high sequence identity (56-58%) with the similar enzymes from mammals, Caenorhabditis elegans and yeast. Active recombinant mitochondrial malate dehydrogenase was expressed in Escherichia coli, as protein fusions with glutathione S-transferase or a poly-His tail. The purified recombinant enzymes had a kinetic behaviour similar to that of the native enzyme, being inhibited by excess of the substrate oxaloacetate and unaffected by excess L-malate. The results indicate that E. granulosus contains two typical eukaryotic malate dehydrogenases, with relative levels quite different from those present in mammalian tissues like heart, in good agreement with the predominantly fermentative metabolism of the protoscolices.

  • 3. Bardales, José R.
    et al.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Villamarí­n, J. Antonio
    CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis2007In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 461, no 1, p. 130-137Article in journal (Refereed)
    Abstract [en]

    Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta. Even in the absence of CK2, R(myt2) was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R(myt2) to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R(myt2) showed that mussel protein contains the signature sequence common to all PKA family members, within the "phosphate binding cassette" (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R(myt2), as a whole, and those from type II R subunits was 68-75%, but the global identity percentage with type I R subunits was only about 30%, so that R(myt2) can be classified as a type II R subunit.

  • 4.
    Bardales, José R.
    et al.
    Departamento de Bioquímica e Bioloxía Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Villamarín, J. Antonio
    Departamento de Bioquímica e Bioloxía Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain.
    Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis2008In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 275, no 18, p. 4479-4489Article in journal (Refereed)
    Abstract [en]

    Several isoforms of the cAMP-dependent protein kinase catalytic subunit (C-subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange chromatography, and identified by: (a) protein kinase activity; (b) antibody recognition; and (c) peptide mass fingerprinting. Some of the isozymes seemed to be tissue-specific, and all them were phosphorylated at serine and threonine residues and showed slight but significant differences in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa. The results from the MS analysis suggest that at least some of the mussel C-subunit isoforms arise as a result of alternative splicing events. Furthermore, several peptide sequences from mussel C-subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Calpha-isoform, and contained some structural motifs that are essential for catalytic function. On the other hand, no significant differences were observed in the kinetic parameters of C-subunit isoforms, determined by using synthetic peptides as substrate and inhibitor. However, the C-subunit isoforms separated from the mantle tissue differed in their ability to phosphorylate in vitro some proteins present in a mantle extract.

  • 5. Ben-Saadon, Ronen
    et al.
    Fajerman, Ifat
    Ziv, Tamar
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Schwartz, Alan L
    Ciechanover, Aaron
    The tumor suppressor protein p16(INK4a) and the human papillomavirus oncoprotein-58 E7 are naturally occurring lysine-less proteins that are degraded by the ubiquitin system: Direct evidence for ubiquitination at the N-terminal residue2004In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 40, p. 41414-41421Article in journal (Refereed)
    Abstract [en]

    Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16(INK4a). For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16(INK4a) is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes.

  • 6. Berger, Karin
    et al.
    Sivars, Ulf
    Sörhede Winzell, Maria
    Johansson, Peter
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Rippe, Catarina
    Erlanson-Albertsson, Charlotte
    Mitochondrial ATP synthase--a possible target protein in the regulation of energy metabolism in vitro and in vivo2002In: Nutritional neuroscience, ISSN 1028-415X, E-ISSN 1476-8305, Vol. 5, no 3, p. 201-210Article in journal (Refereed)
    Abstract [en]

    The increasing prevalence of obesity in the Western world has stimulated an intense search for mechanisms regulating food intake and energy balance. A number of appetite-regulating peptides have been identified, their receptors cloned and the intracellular events characterized. One possible energy-dissipating mechanism is the mitochondrial uncoupling of ATP-synthesis from respiratory chain oxidation through uncoupling proteins, whereby energy derived from food could be dissipated as heat, instead of stored as ATP. The exact role of the uncoupling proteins in energy balance is, however, uncertain. We show here that mitochondrial F1F0-ATP synthase itself is a target protein for an anorectic peptide, enterostatin, demonstrated both after affinity purification of rat brain membranes and through a direct physical interaction between enterostatin and purified F1-ATP synthase. In insulinoma cells (INS-1) enterostatin was found to target F1F0-ATP synthase, causing an inhibition of ATP production, an increased thermogenesis and increased oxygen consumption. The experiments suggest a role of mitochondrial F1F0-ATP synthase in the suppressed insulin secretion induced by enterostatin. It could be speculated that this targeting mechanism is involved in the decreased energy efficiency following enterostatin treatment in rat.

  • 7.
    Bergström, Joakim
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Murphy, Charles L.
    Weiss, Deborah T.
    Solomon, Alan
    Sletten, Knut
    Hellman, Ulf
    Ludwiginstitutet för Cancerforskning.
    Westermark, Per
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Two different types of amyloid deposits - apolipoprotein A-IV and transthyretin - in a patient with systemic amyloidosis2004In: Lab. Invest., Vol. 84, p. 981-988Article in journal (Refereed)
    Abstract [en]

    Certain forms of systemic amyloidosis have been associated with the pathologic deposition as fibrils of three different apolipoprotein-related proteins--apolipoprotein A-I, apolipoprotein A-II, and serum amyloid A. We have previously reported (Bergstrom et al, Biochem Biophys Res Commun 2001;285:903-908) that amyloid fibrils extracted from the heart of an elderly male with senile systemic amyloidosis contained, in addition to wild-type transthyretin-related molecules, an N-terminal fragment of yet a fourth apolipoprotein--apolipoprotein A-IV (apoA-IV). We now provide the results of our studies that have established the complete amino-acid sequence of this approximately 70-residue component and, additionally, have shown this protein to be the product of an unmutated apoA-IV gene. Notably, the apoA-IV and transthyretin fibrils were not codeposited but, rather, had anatomically distinct patterns of distribution within the heart and other organs, as evidenced immunohistochemically, by variation in the ultra structural morphology and by differences in the intensity of Congo red birefringence. These findings provide the first conclusive evidence that two separate forms of amyloid, each derived from a wild-type amyloidogenic precursor protein, were present in a patient with systemic amyloidosis.

  • 8.
    Bhaskaran, Nimesh
    et al.
    Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden.
    Lin, Kah Wai
    Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden.
    Gautier, Aude
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Woksepp, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Souchelnytskyi, Serhiy
    Karolinska Biomics Center, Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden.
    Comparative proteome profiling of MCF10A and 184A1 human breast epithelial cells emphasized involvement of CDK4 and cyclin D3 in cell proliferation2009In: Proteomics Clinical Applications, ISSN 1862-8354, Vol. 3, no 1, p. 68-77Article in journal (Refereed)
    Abstract [en]

    Acquiring high proliferation rate is crucial for carcinogenic transformation of cells. We reporthere proteome profiling of human breast epithelial cells with low (184A1) and high (MCF10A)proliferation rates.We identified 183 proteins in 184A1 and 318 proteins in MCF10A cells. Thesedatasets provide the most comprehensive proteome annotations of 184A1 and MCF10A cells.Proteins were taken for identification from 2-D gels in a systematic and unbiased way. Functionalclustering of the identified proteins showed similarities in distribution of proteins to the samefunctional domains, indicating similarities in proteomes of 184A1 and MCF10A cells. Amongobserved differences in protein expression, we validated correlation of expression of endogenouscyclin-dependent kinase 4 (CDK4), cyclin D3, cdc25B, and p38g with cell proliferation. Furthermore,down-regulation of CDK4 and cyclin D3 with specific siRNA inhibited cell proliferation,which emphasized the role of CDK4 and cyclin D3 in enhancement of cell proliferation rate ofhuman breast epithelial cells.

  • 9. Bilyy, Rostyslav
    et al.
    Kit, Yuriy
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Stoika, Rostyslav
    AMID: new insights on its intracellular localization and expression at apoptosis2008In: Apoptosis (London), ISSN 1360-8185, E-ISSN 1573-675X, Vol. 13, no 5, p. 729-732Article in journal (Refereed)
    Abstract [en]

    AMID (apoptosis-inducing factor (AIF)-like mitochondrion-associated inducer of death) is a poorly studied member of the AIF family; despite the given name AMID, predicting its association with mitochondria, its real cellular localization, as well as its role and changes during apoptosis are currently unclear. By means of MALDI-TOF mass spectrometry, we have identified as AMID (accession number AAH38129, sequence coverage 31%) the protein isolated by Pisum sativum lectin-affinity chromatography from the plasma membrane fraction of apoptotic murine leukemia L1210 cells, lacking in the intact cells. The obtained results suggest its possible glycosylation that was further suggested by finding N-glycosylation sequon in the signal peptide of AMID protein (in silica), and by predicting transmembrane localization of its N-terminal part. Using monoclonal antibodies to AMID, we demonstrated an increased expression of AMID in human leukemia Jurkat T-cells after apoptosis induction. Immunocytochemical study suggested its association to the plasma membrane.

  • 10. Bilyy, Rostyslav
    et al.
    Kit, Yuriy
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Tryndyak, Volodymyr
    Kaminskyy, Vitaliy
    Stoika, Rostyslav
    In vivo expression and characteristics of novel alpha-D-mannose-rich glycoprotein markers of apoptotic cells2005In: Cell Biology International, ISSN 1065-6995, E-ISSN 1095-8355, Vol. 29, no 11, p. 920-928Article in journal (Refereed)
    Abstract [en]

    We recently established that an increased expression of alpha-D-mannose (Man)- and beta-D-galactose-rich plasma membrane glycoproteins (GPs) is characteristic for apoptotic cells in vitro [Bilyy, R.O., Stoika, R.S., 2003. Lectinocytochemical detection of apoptotic murine leukemia L1210 cells. Cytometry 56A, 89-95]. It was independent of cell line or apoptosis-inducing agent, and can therefore be considered as a selective marker for identification and isolation of apoptotic cells [Bilyy, R.O., Antonyuk, V.O., Stoika, R.S., 2004. Cytochemical study of role of alpha-D-mannose- and beta-D-galactose-containing glycoproteins in apoptosis. J. Mol. Histol. 35, 829-838]. The main goals of the present study were: (1) to determine whether an increased expression of specific GPs also takes place after apoptosis induction in vivo; and (2) to identify additional characteristics of the membrane GP markers of the apoptotic cells. To reach these goals, we studied the expression of alpha-Man-rich membrane GPs in murine leukemia L1210 cells inoculated into abdominal cavities of mice which were then subjected to the action of apoptosis inducer doxorubicin. Another experimental model used in the present work was splenocytes obtained from mice treated with dexamethasone. Lectin-affinity chromatography and PAGE electrophoresis, or PAGE electrophoresis and lectinoblot analysis were applied for isolation of plasma membrane GPs (34 kDa, and high M(W) of approximately 600 and 800 kDa) whose expressions were increased during apoptosis. Triton X-114 treatment of cell membrane samples showed that the apoptotic cell-specific GPs were localized in the peripheral and integral compartments of plasma membrane. Apoptosis in vitro and in vivo was accompanied by an increased expression of the same GP, identified by MALDI-TOF MS analysis as the microtubule-actin cross-linking factor 1. Other GPs, whose expressions were also increased at apoptosis, were similarly identified as G-protein beta-subunit like (Acc# BAA06185.1) and dystonin isoform beta.

  • 11. Björk, Petra
    et al.
    Baurén, Göran
    Jin, ShaoBo
    Tong, Yong-Guang
    Bürglin, Thomas R.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Wieslander, Lars
    A novel conserved RNA-binding domain protein, RBD-1, is essential for ribosome biogenesis2002In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 13, no 10, p. 3683-3695Article in journal (Refereed)
    Abstract [en]

    Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20-30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

  • 12. Brena, Beatriz M.
    et al.
    Díaz, Laura
    Sienra, Daniel
    Ferrari, Graciela
    Ferraz, Natalia
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Gonzalez-Sapienza, Gualberto
    Last, Jerold A.
    ITREOH building of regional capacity to monitor recreational water: development of a non-commercial microcystin ELISA and its impact on public health policy2006In: International journal of occupational and environmental health, ISSN 1077-3525, E-ISSN 2049-3967, Vol. 12, no 4, p. 377-385Article in journal (Refereed)
    Abstract [en]

    In 2001, a University of California, Davis-University of the Republic, Montevideo, partnership created a Fogarty ITREOH program to exploit the potential of ELISA to provide a low-cost environmental analysis attractive to economically distressed countries of temperate South America. This paper describes the development and validation of an ELISA method for the determination of Cyanobacteria microcystin toxins in algal blooms, which release hepatotoxic metabolites that can reach toxic levels in rivers, lakes, or coastal estuaries used for recreation or water supplies. The assay made possible the first systematic monitoring of water from the Rio de la Plata at Montevideo over two summers. The project has been integrated into a bi-national effort to monitor the Rio de la Plata.

  • 13. Cabrera, Gonzalo
    et al.
    Cabrejos, María Eugenia
    Morassutti, Alessandra Loureiro
    Cabezón, Carolina
    Orellana, Juana
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Zaha, Arnaldo
    Galanti, Norbel
    DNA damage, RAD9 and fertility/infertility of Echinococcus granulosus hydatid cysts2008In: Journal of Cellular Physiology, ISSN 0021-9541, E-ISSN 1097-4652, Vol. 216, no 2, p. 498-506Article in journal (Refereed)
    Abstract [en]

    Hydatidosis, caused by the larval stage of the platyhelminth parasite Echinococcus granulosus, affects human and animal health. Hydatid fertile cysts are formed in intermediate hosts (human and herbivores) producing protoscoleces, the infective form to canines, at their germinal layers. Infertile cysts are also formed, but they are unable to produce protoscoleces. The molecular mechanisms involved in hydatid cysts fertility/infertility are unknown. Nevertheless, previous work from our laboratory has suggested that apoptosis is involved in hydatid cyst infertility and death. On the other hand, fertile hydatid cysts can resist oxidative damage due to reactive oxygen and nitrogen species. On these foundations, we have postulated that when oxidative damage of DNA in the germinal layers exceeds the capability of DNA repair mechanisms, apoptosis is triggered and hydatid cysts infertility occurs. We describe a much higher percentage of nuclei with oxidative DNA damage in dead protoscoleces and in the germinal layer of infertile cysts than in fertile cysts, suggesting that DNA repair mechanisms are active in fertile cysts. rad9, a conserved gene, plays a key role in cell cycle checkpoint modulation and DNA repair. We found that RAD9 of E. granulosus (EgRAD9) is expressed at the mRNA and protein levels. As it was found in other eukaryotes, EgRAD9 is hyperphosphorylated in response to DNA damage. Our results suggest that molecules involved in DNA repair in the germinal layer of fertile hydatid cysts and in protoscoleces, such as EgRAD9, may allow preserving the fertility of hydatid cysts in the presence of ROS and RNS.

  • 14.
    Chen, Zhi Xiong
    et al.
    Ludwig Institute for Cancer Research Ltd., Karolinska Institutet, Nobels väg 3, SE-17177, Stockholm, Sweden.
    Wallis, Karin
    Ludwig Institute for Cancer Research Ltd., Karolinska Institutet, Nobels väg 3, SE-17177, Stockholm, Sweden.
    Fell, Stuart M
    Ludwig Institute for Cancer Research Ltd., Karolinska Institutet, Nobels väg 3, SE-17177, Stockholm, Sweden.
    Sobrado, Veronica R
    Ludwig Institute for Cancer Research Ltd., Karolinska Institutet, Nobels väg 3, SE-17177, Stockholm, Sweden.
    Hemmer, Marie C
    Ludwig Institute for Cancer Research Ltd., Karolinska Institutet, Nobels väg 3, SE-17177, Stockholm, Sweden.
    Ramsköld, Daniel
    Department of Cell and Molecular Biology, Karolinska Institutet, Nobels väg 3, SE-17177, Stockholm, Sweden.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Sandberg, Rickard
    Department of Cell and Molecular Biology, Karolinska Institutet, Nobels väg 3, SE-17177, Stockholm, Sweden.
    Kenchappa, Rajappa S
    Moffi tt Cancer Center, Neuro-Oncology Program, Tampa, Florida, USA.
    Martinson, Tommy
    Department of Clinical Genetics, Institute of Biomedicine, University of Gothenburg, Sahlgrenska University Hospital, Göteborg, Sweden.
    Johnsen, John I
    Department of Women’s and Children’s Health, Karolinska University Hospital, Stockholm.
    Kogner, Per
    Department of Women’s and Children’s Health, Karolinska University Hospital, Stockholm.
    Schlisio, Susanne
    Ludwig Institute for Cancer Research Ltd., Karolinska Institutet, Nobels väg 3, SE-17177, Stockholm, Sweden.
    RNA helicase A is a downstream mediator of KIF1Bβ tumor-suppressor function in neuroblastoma2014In: Cancer Discovery, ISSN 2159-8274, E-ISSN 2159-8290, Vol. 4, no 4, p. 434-451Article in journal (Refereed)
    Abstract [en]

    UNLABELLED: Inherited KIF1B loss-of-function mutations in neuroblastomas and pheochromocytomas implicate the kinesin KIF1B as a 1p36.2 tumor suppressor. However, the mechanism of tumor suppression is unknown. We found that KIF1B isoform β (KIF1Bβ) interacts with RNA helicase A (DHX9), causing nuclear accumulation of DHX9, followed by subsequent induction of the proapoptotic XIAP-associated factor 1 (XAF1) and, consequently, apoptosis. Pheochromocytoma and neuroblastoma arise from neural crest progenitors that compete for growth factors such as nerve growth factor (NGF) during development. KIF1Bβ is required for developmental apoptosis induced by competition for NGF. We show that DHX9 is induced by and required for apoptosis stimulated by NGF deprivation. Moreover, neuroblastomas with chromosomal deletion of 1p36 exhibit loss of KIF1Bβ expression and impaired DHX9 nuclear localization, implicating the loss of DHX9 nuclear activity in neuroblastoma pathogenesis.

    SIGNIFICANCE: KIF1Bβ has neuroblastoma tumor-suppressor properties and promotes and requires nuclear-localized DHX9 for its apoptotic function by activating XAF1 expression. Loss of KIF1Bβ alters subcellular localization of DHX9 and diminishes NGF dependence of sympathetic neurons, leading to reduced culling of neural progenitors, and, therefore, might predispose to tumor formation.

  • 15. Conrotto, Paolo
    et al.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Lys Tag: an easy and robust chemical modification for improved de novo sequencing with a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer2008In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 22, no 12, p. 1823-33Article in journal (Refereed)
    Abstract [en]

    Mass spectrometry using a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) instrument is a widespread technique for various types of proteomic analysis. Along with an expanding interest in proteomics, there is a strong requirement for the identification of proteins with high confidence from biological samples. Peptide modification by a wide variety of post-translational modifications (PTMs), the existence of different protein isoforms and the presence of uncharacterized genomes of many species, make protein identification through peptide mass fingerprinting (PMF) often unachievable. Peptide de novo sequencing has been proven to be a useful approach to overcome these variables, and efficient derivatization processes are important tools to achieve this goal. In the present work we describe the methodology and experimental applications of a fast, efficient and cheap lysine derivatization. This chemical modification improves the signals from lysine-terminated peptides and can be efficiently used as a lysine-blocking agent in combination with other derivatization techniques. Most importantly, upon peptide fragmentation it generates a neat series of predominantly y-ions, allowing the determination of unambiguous amino acid sequences. Moreover, this chemical compound was used with target-eluted samples, enabling a second, alternative analysis of the same sample in the MALDI mass spectrometer.

  • 16.
    Conrotto, Paolo
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Sulfonation chemistry as a powerful tool for MALDI TOF/TOF de novo sequencing and post-translational modification analysis2005In: Journal of biomolecular techniques, ISSN 1524-0215, Vol. 16, no 4, p. 441-452Article in journal (Refereed)
    Abstract [en]

    Mass spectrometry using matrix-assisted laser desorption/ionization (MALDI) is a widespread technique for various types of proteomic analysis. In the identification of proteins using peptide mass fingerprinting, samples are enzymatically digested and resolved into a number of peptides, whose masses are determined and matched with a sequence data-base. However, the presence inside the cell of several splicing variants, protein isoforms, or fusion proteins gives rise to a complex picture, demanding more complete analysis. Moreover, the study of species with yet uncharacterized genomes or the investigation of post-translational modifications are not possible with classical mass fingerprinting, and require specific and accurate de novo sequencing. In the last several years, much effort has been made to improve the performance of peptide sequencing with MALDI. Here we present applications using a fast and robust chemical modification of peptides for improved de novo sequencing. Post-source decay of derivatized peptides generates at the same time peaks with high intensity and simple spectra, leading to a very easy and clear sequence determination.

  • 17. Cortez, Leonardo
    et al.
    Marino-Buslje, Cristina
    de Jiménez Bonino, Mirtha Biscoglio
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Interactions between alpha-conotoxin MI and the Torpedo marmorata receptor alpha-delta interface2007In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 355, no 1, p. 275-279Article in journal (Refereed)
    Abstract [en]

    The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. Moreover, we previously reported that alpha-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jimenez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791-795]. Herein, to identify T. marmorata receptor regions involved in alpha-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. alpha-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the alpha/delta interface. A proposal for receptor-toxin interaction is discussed based on experimental results and docking studies.

  • 18. Dalmasso, Marí­a C.
    et al.
    Echeverria, Pablo C.
    Zappia, Marí­a P.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Dubremetz, Jean François
    Angel, Sergio O.
    Toxoplasma gondii has two lineages of histones 2b (H2B) with different expression profiles2006In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 148, no 1, p. 103-107Article in journal (Refereed)
  • 19.
    Enqvist, Stina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sletten, Knut
    Stevens, Fred J.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Westermark, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Germ Line Origin and Somatic Mutations Determine the Target Tissues in Systemic AL-Amyloidosis2007In: PLoS ONE, ISSN 1932-6203, Vol. 2, no 10, p. e981-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Amyloid is insoluble aggregated proteins deposited in the extra cellular space. About 25 different proteins are known to form amyloid in vivo and are associated with severe diseases such as Alzheimeŕs disease, prion diseases and type-2 diabetes. Light chain (AL) -amyloidosis is unique among amyloid diseases in that the fibril protein, a monoclonal immunoglobulin light chain, varies between individuals and that no two AL-proteins with identical primary structures have been described to date. The variability in tissue distribution of amyloid deposits is considerably larger in systemic AL-amyloidosis than in any other form of amyloidosis. The reason for this variation is believed to be based on the differences in properties of the amyloidogenic immunoglobulin light chain. However, there is presently no known relationship between the structure of an AL-protein and tissue distribution. METHODOLOGY/PRINCIPAL FINDINGS: We compared the pattern of amyloid deposition in four individuals with amyloid protein derived from variable light chain gene O18-O8, the source of a high proportion of amyloidogenic light chains, and in whom all or most of the fibril protein had been determined by amino acid sequencing. In spite of great similarities between the structures of the proteins, there was a pronounced variability in deposition pattern. We also compared the tissue distribution in these four individuals with that of four other patients with AL-amyloid derived from the L2-L16 gene. Although the interindividual variations were pronounced, liver and kidney involvement was much more evident in the latter four. CONCLUSIONS/SIGNIFICANCE: We conclude that although the use of a specific gene influences the tissue distribution of amyloid, each light chain exhibits one or more determinants of organ-specificity, which originate from somatic mutations and post-translational modifications. Eventual identification of such determinants could lead to improved treatment of patients with AL amyloidosis.

  • 20.
    Feliziani, Constanza
    et al.
    Laboratorio de Microbiología e Inmunología, Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC - CONICET, Friuli 2434, (5000) Córdoba, Argentina.
    Merino, María C
    Laboratorio de Microbiología e Inmunología, Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC - CONICET, Friuli 2434, (5000) Córdoba, Argentina.
    Rivero, María R
    Laboratorio de Microbiología e Inmunología, Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC - CONICET, Friuli 2434, (5000) Córdoba, Argentina.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Pistoresi-Palencia, María C
    Departamento de Bioquímica Clínica, CIBICICONICET, Facultad de Ciencias Químicas, Haya de la Torre y Medina Allende, UNC, (5000) Córdoba, Argentina.
    Rópolo, Andrea S
    Laboratorio de Microbiología e Inmunología, Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC - CONICET, Friuli 2434, (5000) Córdoba, Argentina.
    Immunodominant proteins α-1 giardin and β-giardin are expressed in both assemblages A and B of Giardia lamblia2011In: BMC Microbiology, E-ISSN 1471-2180, Vol. 11, p. 233-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: To date, eight assemblages of Giardia lamblia have been described, but only assemblages A and B are known to infect humans. Despite the fact that the genomic, biological, and clinical differences found between these two assemblages has raised the possibility that they may be considered different species, there is relatively limited information on their phenotypic differences. In the present study, we developed monoclonal antibodies against alpha-1 and beta giardin, two immunodominant proteins produced during G. lamblia infection, and studied their expression and localization in WB (assemblage A) and GS trophozoites (assemblage B).

    RESULTS: The polyclonal antibodies generated against WB trophozoites, particularly those recognizing intracellular proteins as well as the proteins present at the plasma membrane (variable-specific surface proteins), showed cross-reactivity with intracellular proteins in GS trophozoites. The use of monoclonal antibodies against beta giardin indicated ventral disc localization, particularly at the periphery in WB trophozoites. Interestingly, although beta giardin was also restricted to the ventral disc in GS trophozoites, the pattern of localization clearly differed in this assemblage. On the other hand, monoclonal antibodies against alpha-1 giardin showed plasma membrane localization in both assemblages with the bare area of GS trophozoites also being distinguished. Moreover, the same localization at the plasma membrane was observed in Portland-1 (Assemblage A) and in P15 (Assemblage E) trophozoites.

    CONCLUSIONS: We found differences in localization of the beta giardin protein between assemblages A and B, but the same pattern of localization of alpha-1 giardin in strains from Assemblages A, B and E. These findings reinforce the need for more studies based on phenotypic characteristics in order to disclose how far one assemblage is from the other.

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  • 21. Fernanda Troncoso, M.
    et al.
    Cerdá Zolezzi, Paula
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Wolfenstein-Todel, Carlota
    A novel trypsin inhibitor from Peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis2003In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 411, no 1, p. 93-104Article in journal (Refereed)
    Abstract [en]

    A trypsin inhibitor (PDTI) was isolated from Peltophorum dubium seeds by affinity chromatography on a thyroglobulin-agarose or a trypsin-agarose column. In both cases, SDS-PAGE showed two bands of M(r) 20,000 and 22,000, which could not be resolved. Their amino-terminal sequences were identical and similar to that of Kunitz-type soybean trypsin inhibitor (SBTI). Mass spectrometry analysis of tryptic digests of both bands showed 16 coincident peaks, suggesting that they are closely related proteins. The K(i)s for trypsin and chymotrypsin inhibitory activity of PDTI were 1.6 x 10(-7) and 1.3 x 10(-5)M, respectively. Lectin-like activity of PDTI and SBTI, detected by hemagglutination of rabbit erythrocytes, was inhibited by sialic acid-containing compounds. PDTI and SBTI caused apoptosis of Nb2 rat lymphoma cells, demonstrated by decrease of viability, DNA hypodiploidy, DNA fragmentation, and caspase-3-like activity. They had no effect on normal mouse splenocytes or lymphocytes, whereas they caused apoptosis of concanavalin A-stimulated mouse lymphocytes.

  • 22. Flensburg, John
    et al.
    Tangen, Anders
    Prieto, Maria
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Wadensten, Henrik
    Chemically-assisted fragmentation combined with multi-dimensional liquid chromatography and matrix-assisted laser desorption/ionization post source decay, matrix-assisted laser desorption/ionization tandem time-of flight or matrix-assisted laser desorption/ionization tandem mass spectrometry for improved sequencing of tryptic peptides2005In: European journal of mass spectrometry, ISSN 1469-0667, E-ISSN 1751-6838, Vol. 11, no 2, p. 169-179Article in journal (Refereed)
    Abstract [en]

    Derivatization of tryptic peptides using an Ettan CAF matrix-assisted laser desorption/ionization (MALDI) sequencing kit in combination with MALDI-post source decay (PSD) is a fast, accurate and convenient way to obtain de novo or confirmative peptide sequencing data. CAF (chemically assisted fragmentation) is based on solid-phase derivatization using a new class of water stable sulfonation agents, which strongly improves PSD analysis and simplifies the interpretation of acquired spectra. The derivatization is performed on solid supports, ZipTip(microC18, limiting the maximum peptide amount to 5 microg. By performing the derivatization in solution enabled the labeling of tryptic peptides derived from 100 microg of protein. To increase the number of peptides that could be sequenced, derivatized peptides were purified using multidimensional liquid chromatography (MDLC) prior to MALDI sequencing. Following the first dimension strong cation exchange (SCX) chromatography step, modified peptides were separated using reversed-phase chromatography (RPC). During the SCX clean up step, positively charged peptides are retained on the column while properly CAF-derivatized peptides (uncharged) are not. A moderately complex tryptic digest, prepared from six different proteins of equimolar amounts, was CAF-derivatized and purified by MDLC. Fractions from the second dimension nano RPC step were automatically sampled and on-line dispensed to MALDI sample plates and analyzed using MALDI mass spectrometry fragmentation techniques. All proteins in the derivatized protein mixture digest were readily identified using MALDI-PSD or MALDI tandem mass spectrometry (MS/MS). More than 40 peptides were unambiguously sequenced, representing a seven-fold increase in the number of sequenced peptides in comparison to when the CAF-derivatized protein mix digest was analyzed directly (no MDLC-separation) using MALDI-PSD. In conclusion, MDLC purification of CAF-derivatized peptides significantly increases the success rate for de novo and confirmative sequencing using various MALDI fragmentation techniques. This new approach is not only applicable to single protein digests but also to more complex digests and could, thus, be an alternative to electrospray ionization MS/MS for peptide sequencing.

  • 23. Galindo, Mario
    et al.
    Varela, Nelson
    Espinoza, Ingrid
    Toro, Gabriela Cecilia
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Galanti, Norbel
    Chromatin from two classes of platyhelminthes display both protist H1 and higher eukaryote core histones2004In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 567, no 2-3, p. 225-229Article in journal (Refereed)
    Abstract [en]

    Histones from the parasitic platyhelminthes, Echinococcus granulosus and Fasciola hepatica, were systematically characterized. Core histones H2A, H2B, H3 and H4, which were identified on the basis of amino acid sequencing and mass spectrometry data, showed conserved electrophoretic patterns. Histones H1, identified on the basis of physicochemical properties, amino acid composition and amino acid sequencing, showed divergence, both in their number and electrophoretic mobilities, between the two species and among other organisms. According to these data, core histones but not H1 histones, would be stabilized during evolution at the level of platyhelminthes.

  • 24.
    Gemoll, T.
    et al.
    Univ Lubeck, Lubeck, Germany..
    Masche, A.
    Univ Lubeck, Lubeck, Germany..
    Roblick, U. J.
    Univ Lubeck, Lubeck, Germany..
    Bader, F.
    Univ Lubeck, Lubeck, Germany..
    Unger, A.
    Univ Lubeck, Lubeck, Germany..
    Becker, S.
    Karolinska Inst, Stockholm, Sweden..
    Moeslein, G.
    Univ Klinikum Witten Herdecke, Zentrum Hereditare Tumorerkrankungen, Wuppertal, Germany..
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Disciplinary Domain of Medicine and Pharmacy, research centers etc., Ludwig Institute for Cancer Research.
    Joernvall, H.
    Karolinska Inst, Stockholm, Sweden..
    Bruch, H-P
    Auer, G.
    Karolinska Inst, Stockholm, Sweden..
    Habermann, J.
    Univ Lubeck, Lubeck, Germany..
    Protein expression of CSTF2T and ACTB discern sporadic from FAP-associated colon carcinomas at various stages of carcinogenesis2018In: Oncology Research and Treatment, ISSN 2296-5270, E-ISSN 2296-5262, Vol. 41, p. 173-173Article in journal (Other academic)
  • 25.
    Gemoll, Timo
    et al.
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles va¨g 2, 17177 Stockholm, Sweden.
    Habermann, Jens K.
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles va¨g 2, 17177 Stockholm, Sweden.
    Lahmann, Johanna
    Department of Surgery, University of Lu¨beck, 23538 Lu¨beck, Germany.
    Szymczak, Silke
    Institute of Medical Biometry and Statistics, University of Lu¨beck, 23562 Lu¨beck, Germany.
    Lundgren, Caroline
    Department of Oncology, Radiumhemmet, Karolinska University Hospital, 17176 Stockholm, Sweden.
    Bündgen, Nana K.
    Department of Surgery, University of Lu¨beck, 23538 Lu¨beck, Germany.
    Jungbluth, Thomas
    Department of Surgery, University of Lu¨beck, 23538 Lu¨beck, Germany.
    Nordström, Britta
    Department of Oncology, Radiumhemmet, Karolinska University Hospital, 17176 Stockholm, Sweden.
    Becker, Susanne
    Karolinska Biomic Center, Karolinska Institutet, 17176 Stockholm, Sweden.
    Lomnytska, Marta I.
    Karolinska Biomic Center, Karolinska Institutet, 17176 Stockholm, Sweden.
    Bruch, Hans-Peter
    Department of Surgery, University of Lu¨beck, 23538 Lu¨beck, Germany.
    Ziegler, Andreas
    Institute of Medical Biometry and Statistics, University of Lu¨beck, 23562 Lu¨beck, Germany.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Auer, Gert
    Karolinska Biomic Center, Karolinska Institutet, 17176 Stockholm, Sweden.
    Roblick, Uwe J.
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles va¨g 2, 17177 Stockholm, Sweden.
    Jörnvall, Hans
    Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Scheeles va¨g 2, 17177 Stockholm, Sweden.
    Protein profiling of genomic instability in endometrial cancer2012In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 69, no 2, p. 325-333Article in journal (Refereed)
    Abstract [en]

    DNA aneuploidy has been identified as a prognostic factor in the majority of epithelial malignancies. We aimed at identifying ploidy-associated protein expression in endometrial cancer of different prognostic subgroups. Comparison of gel electrophoresis-based protein expression patterns between normal endometrium (n = 5), diploid (n = 7), and aneuploid (n = 7) endometrial carcinoma detected 121 ploidy-associated protein forms, 42 differentially expressed between normal endometrium and diploid endometrioid carcinomas, 37 between diploid and aneuploid endometrioid carcinomas, and 41 between diploid endometrioid and aneuploid uterine papillary serous cancer. Proteins were identified by mass spectrometry and evaluated by Ingenuity Pathway Analysis. Targets were confirmed by liquid chromatography/mass spectrometry. Mass spectrometry identified 41 distinct polypeptides and pathway analysis resulted in high-ranked networks with vimentin and Nf-kappa B as central nodes. These results identify ploidy-associated protein expression differences that overrule histopathology-associated expression differences and emphasize particular protein networks in genomic stability of endometrial cancer.

  • 26.
    Grimsby, Susanne
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Jaensson, Hanna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Dubrovska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Lomnytska, Marta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Proteomics-based identification of proteins interacting with Smad3: SREBP-2 forms a complex with Smad3 and inhibits its transcriptional activity2004In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 577, no 1-2, p. 93-100Article in journal (Refereed)
    Abstract [en]

    Smad3 is an important component of transforming growth factor-beta (TGFbeta) intracellular signalling. To identify novel interacting proteins of Smad3, we performed pull-down assays with Smad3 constructs fused to glutathione-S-transferase. Proteins which formed complexes with these constructs were analyzed by two-dimensional gel electrophoresis, and were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. We identified 14 proteins interacting with the Smad3 construct lacking the N-terminal Mad homology domain 1 (MH1), and 12 proteins interacting with the construct lacking the C-terminal MH2 domain. Proteins involved in signalling processes, in metabolism regulation, novel proteins, and components of cytoskeleton form four groups of interacting proteins. Interactions of AGP7, sex-determining region Y protein, actin beta and sterol regulatory element binding protein-2 (SREBP-2) proteins with Smad3 constructs were confirmed by immunoblotting with specific antibodies. Interaction of Smad3 with SREBP-2 was also confirmed by co-immunoprecipitation of myc-Smad3 and Flag-SREBP-2 upon expression in mammalian cells. We found that SREBP-2 inhibited the transcriptional activity of Smad3 in luciferase reporter assays.

  • 27.
    Grönroos, Eva
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Ericsson, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Control of Smad7 stability by competition between acetylation and ubiquitination2002In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 10, no 3, p. 483-493Article in journal (Refereed)
    Abstract [en]

    Smad proteins regulate gene expression in response to TGFbeta signaling. Here we present evidence that Smad7 interacts with the transcriptional coactivator p300, resulting in acetylation of Smad7 on two lysine residues in its N terminus. Acetylation or mutation of these lysine residues stabilizes Smad7 and protects it from TGFbeta-induced degradation. Furthermore, we demonstrate that the acetylated residues in Smad7 also are targeted by ubiquitination and that acetylation of these lysine residues prevents subsequent ubiquitination. Specifically, acetylation of Smad7 protects it against ubiquitination and degradation mediated by the ubiquitin ligase Smurf1. Thus, our data suggest that competition between ubiquitination and acetylation of overlapping lysine residues constitutes a novel mechanism to regulate protein stability.

  • 28.
    Hassel, Sylke
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Eichner, Annegret
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Yakymovych, Mariya
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Knaus, Petra
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Proteins associated with type II bone morphogenetic protein receptor (BMPR-II) and identified by two-dimensional gel electrophoresis and mass spectrometry2004In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 4, no 5, p. 1346-1358Article in journal (Refereed)
    Abstract [en]

    Bone morphogenetic proteins (BMP) are polypeptide growth factors that regulate cell differentiation and proliferation. BMPs bind to type I and type II serine/threonine kinase receptors to initiate intracellular signalling. BMPR-II is the type II receptor, its mutations lead to hereditary pulmonary hypertension, and knockout of Bmpr-II results in early embryonic lethality. To identify novel interacting proteins and explore signalling pathways that can be initiated by BMPR-II, we performed glutathione-S-transferase (GST) pull-down assays with BMPR-II protein constructs fused to GST and extracts of mouse myoblast C2C12 cells. We generated three constructs which contain different parts of the cytoplasmic region of BMPR-II: full-length cytoplasmic part of BMPR-II, only the kinase domain, or only the C-terminal tail of BMPR-II. Proteins which formed complexes with these BMPR-II constructs were analyzed by two-dimensional gel electrophoresis (2-D GE), and specifically interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). We identified 33 interacting proteins; 11 proteins interacted with the C-terminal tail of BMPR-II, 4 with full-length BMPR-II, and 18 with a short form of the receptor with a deleted tail. Fourteen proteins have assigned functions in various signalling processes, suggesting links of BMP signalling to regulation of MAP kinase pathway, apoptosis, transcription, PKCss, and PKA. Five of the identified proteins are components of the cytoskeleton, and four are enzymes involved in metabolism, e.g., processing of estrogens or lipids. We confirmed interaction of PKC beta and CtBP with BMPR-II using immunodetection. We showed that the C-terminal tail of BMPR-II provides binding sites for a number of regulatory proteins that may initiate Smad-independent signalling.

  • 29.
    Hassel, Sylke
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Yakymovych, Mariya
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Rönnstrand, Lars
    Knaus, Petra
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor2006In: Journal of Cellular Physiology, ISSN 0021-9541, E-ISSN 1097-4652, Vol. 206, no 2, p. 457-467Article in journal (Refereed)
    Abstract [en]

    Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR-II) which has a serine/threonine kinase domain, and stem cell factor receptor (c-kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2-dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2-dependent differentiation of C2C12 cells. We found that BMPR-II was phosphorylated at Ser757 upon co-expression with and activation of c-kit. BMPR-II phosphorylation required intact kinase activity of BMPR-II. Abrogation of the c-kit/SCF-dependent phosphorylation of BMPR-II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c-kit and BMPR-II leads to phosphorylation of BMPR-II at Ser757, which modulates BMPR-II-dependent signaling.

  • 30.
    Hegazy, Usama M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Replacement surgery with unnatural amino acids in the lock-and-key joint of glutathione transferase subunits2006In: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 13, no 9, p. 929-936Article in journal (Refereed)
    Abstract [en]

    Proteins contain amino acid residues essential to structure and function. Ribosomal protein synthesis is typically limited to the 20 amino acids of the genetic code, but posttranslational chemical modifications can greatly expand the diversity of side chain functionalities. In this investigation, a natural aromatic residue in the lock-and-key joint at the subunit interface of the dimeric glutathione transferase P1-1 was replaced by an S-alkylcysteine residue to give a functional enzyme. Introduction of Cys in the key position inactivates the enzyme, but subsequent alkylation of this residue enhances the catalytic efficiency up to 27,000-fold. Combinatorial modification of Cys by a mixture of reagents facilitated identification of an n-butyl group as the most efficient activator. Alkylation also enhanced binding affinity for active-site ligands and stabilized the enzyme against chemical denaturation and thermal inactivation.

  • 31.
    Hegazy, Usama M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Tars, Kaspars
    Department of Cell and Molecular Biology, Biomedical Center, Uppsala University, Box 590, SE-751 24 Uppsala, Sweden.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Modulating Catalytic Activity by Unnatural Amino Acid Residues in a GSH-Binding Loop of GST P1-12008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, no 3, p. 811-826Article in journal (Refereed)
    Abstract [en]

    The loop following helix alpha2 in glutathione transferase P1-1 has two conserved residues, Cys48 and Tyr50, important for glutathione (GSH) binding and catalytic activity. Chemical modification of Cys48 thwarts the catalytic activity of the enzyme, and mutation of Tyr50 generally decreases the k(cat) value and the affinity for GSH in a differential manner. Cys48 and Tyr50 were targeted by site-specific mutations and chemical modifications in order to investigate how the alpha2 loop modulates GSH binding and catalysis. Mutation of Cys48 into Ala increased K(M)(GSH) 24-fold and decreased the binding energy of GSH by 1.5 kcal/mol. Furthermore, the protein stability against thermal inactivation and chemical denaturation decreased. The crystal structure of the Cys-free variant was determined, and its similarity to the wild-type structure suggests that the mutation of Cys48 increases the flexibility of the alpha2 loop rather than dislocating the GSH-interacting residues. On the other hand, replacement of Tyr50 with Cys, producing mutant Y50C, increased the Gibbs free energy of the catalyzed reaction by 4.8 kcal/mol, lowered the affinity for S-hexyl glutathione by 2.2 kcal/mol, and decreased the thermal stability. The targeted alkylation of Cys50 in Y50C increased the affinity for GSH and protein stability. Characterization of the most active alkylated variants, S-n-butyl-, S-n-pentyl-, and S-cyclobutylmethyl-Y50C, indicated that the affinity for GSH is restored by stabilizing the alpha2 loop through positioning of the key residue into the lock structure of the neighboring subunit. In addition, k(cat) can be further modulated by varying the structure of the key residue side chain, which impinges on the rate-limiting step of catalysis.

  • 32.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Microanalysis of proteins and peptides2002In: Ukr Biokhim Zh, ISSN 0201-8470, Vol. 74, no 1, p. 128-132Article in journal (Refereed)
    Abstract [en]

    -

  • 33.
    Hellman, Ulf
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Bhikhabhai, Rama
    Easy amino acid sequencing of sulfonated peptides using post-source decay on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer equipped with a variable voltage reflector2002In: Rapid Communications in Mass Spectrometry, ISSN 0951-4198, E-ISSN 1097-0231, Vol. 16, no 19, p. 1851-1859Article in journal (Refereed)
    Abstract [en]

    Tryptic peptides were labeled with sulfonic acid groups at the N-termini using an improved chemistry. The derivatization was performed in common aqueous buffers on peptides adsorbed onto a ZipTip trade mark C(18), thus allowing simultaneous desalting/concentration of the sample. When only Arg-terminating peptides were considered, the procedure from adsorption onto the ZipTip until analysis by MALDI-PSD took about 10 min and several samples could be worked on in parallel. The resulting improved post-source decay (PSD) fragmentation produced spectra containing only y-ions. PSD amino acid sequencing of underivatized and derivatized synthetic peptides was compared. From the sequence information obtained from derivatized peptides isolated by ion selection from tryptic in-gel digests, a protein was correctly identified which was difficult to analyze from an unclear peptide mass fingerprint analysis. The method was also applied to the identification and localization of phosphorylated Ser and Tyr residues in native and synthetic peptides.

  • 34.
    Hörnsten, Lena
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Su, Chao
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Osbourn, Anne E.
    Hellman, Ulf
    Ludwiginstitutet för Cancerforskning.
    Oliw, Ernst H
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Cloning of the manganese lipoxygenase gene reveals homology with the lipoxygenase gene family2002In: Eur. J. Biochem, Vol. 269, p. 2690-7Article in journal (Refereed)
    Abstract [en]

    Manganese lipoxygenase was isolated to homogeneity from the take-all fungus, Gaeumannomyces graminis. The C-terminal amino acids and several internal peptides were sequenced, and the information was used to obtain a cDNA probe by RT/PCR. Screening of a genomic library of G. graminis yielded a full-length clone of the Mn-Lipoxygenase gene. cDNA analysis showed that the gene spanned 2.6 kb and contained one intron (133 bp). Northern blot analyses indicated two transcripts (2.7 and 3.1 kb). The deduced amino-acid sequence of the Mn-Lipoxygenase precursor (618 amino acids, 67.7 kDa) could be aligned with mammalian and plant lipoxygenases with 23-28% identity over 350-400 amino-acid residues of the catalytic domains. Lipoxygenases have one water molecule and five amino acids as Fe ligands. These are two histidine residues in the highly conserved 30 amino-acid sequence WLLAK-X15-H-X4-H-X3-E of alpha helix 9, one histidine and usually an asparaine residue in the sequence H-X3-N-X-G of alpha helix 18, and the carboxyl oxygen of the C-terminal isoleucine (or valine) residue. The homologous sequence of alpha helix 9 of Mn-Lipoxygenase [WLLAK-X14-H(294)-X3-H(297)-X3-E] contained two single-amino-acid gaps, but otherwise His294 and His297 aligned with the two His residues, which coordinate iron. Mn-Lipoxygenase [H(478)-X3-N(482)-X-G] could be aligned with the two metal ligands of alpha helix 18, and the C-terminal residue was Val618. We conclude that Mn-Lipoxygenase belongs to the lipoxygenase gene family and that its unique biochemical properties might be related to structural differences in the metal centre and alpha helix 9 of lipoxygenases rather than to the metal ligands.

  • 35.
    Ivarsson, Ylva
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Norrgård, Malena A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Engineering the enantioselectivity of glutathione transferase by combined active-site mutations and chemical modifications2007In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1770, no 9, p. 1374-1381Article in journal (Refereed)
    Abstract [en]

    Based on the crystal structure of human glutathione transferase M1-1, cysteine residues were introduced in the substrate-binding site of a Cys-free mutant of the enzyme, which were subsequently alkylated with 1-iodoalkanes. By different combinations of site-specific mutations and chemical modifications of the enzyme the enantioselectivity in the conjugation of glutathione with the epoxide-containing substrates 1-phenylpropylene oxide and styrene-7,8-oxide were enhanced up to 9- and 10-fold. The results also demonstrate that the enantioselectivity can be diminished, or even reversed, by suitable modifications, which can be valuable under some conditions. The redesign of the active-site structure for enhanced or diminished enantioselectivities have divergent requirements for different epoxides, calling for a combinatorial approach involving alternative mutations and chemical modifications to optimize the enantioselectivity for a targeted substrate. This approach outlines a general method of great potential for fine-tuning substrate specificity and tailoring stereoselectivity of recombinant enzymes.

  • 36.
    Iwahana, Hiroyuki
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Yakymovych, Ihor
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Dubrovska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Glycoproteome profiling of transforming growth factor-beta (TGF beta) signaling: Nonglycosylated cell death-inducing DFF-like effector A inhibits TGF beta 1-dependent apoptosis2006In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 6, no 23, p. 6168-6180Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor-beta (TGFbeta) is a potent regulator of cell growth, differentiation, and apoptosis. TGFbeta binds to specific serine/threonine kinase receptors, which leads to activation of Smad-dependent and Smad-independent signaling pathways. O-Glycosylation is a dynamic PTM which has been observed in many regulatory proteins, but has not been studied in the context of TGFbeta signaling. To explore the effect of TGFbeta1 on protein O-glycosylation in human breast epithelial cells, we performed analyses of proteins which were affinity purified with Helix pomatia agglutinin (HPA). HPA lectin allowed enrichment of proteins containing GalNAc and GlcNAc linked to serine and threonine residues. Using 2-DE and MALDI-TOF-MS, we identified 21 HPA-precipitated proteins, which were affected by treatment of cells with TGFbeta1. Among these proteins, regulators of cell survival, apoptosis, trafficking, and RNA processing were identified. We found that TGFbeta1 inhibited the appearance of cell death-inducing DFF-like effector A (CIDE-A) in 2-D gels with HPA-precipitated proteins. CIDE-A is a cell death activator which promotes DNA fragmentation. We observed that TGFbeta1 did not affect expression of CIDE-A, but inhibited its glycosylation. We found that deglycosylation of CIDE-A correlated with enhanced nuclear export of the protein, and that high level of nonglycosylated CIDE-A inhibited TGFbeta1-dependent cell death. Thus, inhibition of the glycosylation of CIDE-A may be a mechanism to protect cells from apoptosis.

  • 37. Jones, Iwan
    et al.
    Lindberg, Christina
    Jakobsson, Staffan
    Hellqvist, Anna
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Borg, Bertil
    Olsson, Per-Erik
    Molecular Cloning and Characterization of Spiggin: An androgen-regulated extraorganismal adhesive with structural similarities to von Willebrand Factor-related proteins2001In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 276, no 21, p. 17857-17863Article in journal (Refereed)
    Abstract [en]

    One of the most definitive examples of a vertebrate extraorganismal structural protein can be found in three-spined sticklebacks (Gasterosteus aculeatus). In the breeding male the kidney hypertrophies and synthesizes an adhesive protein called "spiggin," which is secreted into the urinary bladder from where it is employed as a structural thread for nest building. This paper describes the first molecular characterization of spiggin and demonstrates that this adhesive is a protein complex assembled from a potential of three distinct subunits (alpha, beta, and gamma). These subunits arise by alternative splicing, and 11-ketoandrogens induce their expression in stickleback kidneys. Analysis of the predicted amino acid sequence of each subunit reveals a modular organization whose structural elements display a similarity to the multimerization domains found within von Willebrand Factor-related proteins. These results implicate that spiggin utilizes a conserved multimerization mechanism for the formation of a viscous agglutinate from its constituent subunits in the urinary bladders of male sticklebacks. This novel extraorganismal structural protein is therefore ideally suited to its function as an adhesive thread.

  • 38. Kahata, Kaoru
    et al.
    Hayashi, Makoto
    Asaka, Masahiro
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kitagawa, Hirochika
    Yanagisawa, Jun
    Kato, Shigeaki
    Imamura, Takeshi
    Miyazono, Kohei
    Regulation of transforming growth factor-beta and bone morphogenetic protein signalling by transcriptional coactivator GCN52004In: Genes to Cells, ISSN 1356-9597, E-ISSN 1365-2443, Vol. 9, no 2, p. 143-151Article in journal (Refereed)
    Abstract [en]

    Smad proteins are intracellular signalling mediators of transforming growth factor-beta (TGF-beta) superfamily. In the nucleus, activated Smad complexes regulate transcriptional responses of the target genes in cooperation with transcriptional coactivators and corepressors. To identify new components of transcriptional complexes containing Smad proteins, we purified DNA-binding proteins from human breast cancer MCF-7 cell nuclear extract using a Smad-binding DNA element as bait, and identified a coactivator GCN5 as a direct partner of activated Smad complexes. GCN5 is structurally similar to PCAF, which was previously identified as a coactivator for receptor-regulated Smads (R-Smads) for TGF-beta signalling pathways. GCN5 functions like PCAF, in that it binds to TGF-beta-specific R-Smads, and enhances transcriptional activity induced by TGF-beta. In addition, GCN5, but not PCAF, interacts with R-Smads for bone morphogenetic protein (BMP) signalling pathways, and enhances BMP-induced transcriptional activity, suggesting that GCN5 and PCAF have distinct physiological functions in vivo. Moreover, silencing of the GCN5 gene by RNA interference results in repression of transcriptional activities induced by TGF-beta. In conclusion we identified GCN5 as a Smad-binding transcriptional coactivator which positively regulates both TGF-beta and BMP signalling pathways.

  • 39.
    Kanamoto, Takashi
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Functional proteomics of transforming growth factor-beta1-stimulated Mv1Lu epithelial cells: Rad51 as a target of TGFbeta1-dependent regulation of DNA repair2002In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 21, no 5, p. 1219-1230Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor-beta (TGFbeta) conveys regulatory signals through multiple intracellular pathways, subsequently affecting various cellular functions. To identify new targets for TGFbeta, we studied the changes in the proteome of Mv1Lu lung epithelial cells in response to TGFbeta1 treatment. Thirty-eight non-abundant protein spots, affected by TGFbeta1, were selected, and proteins were identified by peptide mass-fingerprinting (PMF). Among them, proteins involved in regulation of immune response, apoptosis, regulation of TGFbeta signalling, metabolism and DNA repair were identified. Twenty-eight of the 38 proteins are new targets for TGFbeta1, thus suggesting novel ways of integration of TGFbeta signalling in intracellular regulatory processes. We show that TGFbeta1-dependent decrease in expression of one of the new targets, Rad51, correlates with a decrease in DNA repair efficiency. This was evaluated by formation of nuclear Rad51-containing DNA repair complexes in response to DNA damage, by single cell gel electrophoresis and by cell survival assay. The TGFbeta1-dependent inhibition of DNA repair was reversed by ectopic overexpression of Rad51. Therefore, TGFbeta can promote DNA instability through down-regulation of Rad51 and inhibition of DNA repair.

  • 40.
    Klint, Peter
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Hellman, Ulf
    Ludwiginstitutet för Cancerforskning.
    Wernstedt, Christer
    Ludwiginstitutet för Cancerforskning.
    Aman, Pierre
    Ron, David
    Claesson-Welsh, Lena
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Translocated in liposarcoma (TLS) is a substrate for fibroblast growth factor receptor-1.2004In: Cell Signal, ISSN 0898-6568, Vol. 16, no 4, p. 515-20Article in journal (Other scientific)
    Abstract [en]

    Binding of fibroblast growth factor (FGF) to the high affinity receptor-1 (FGFR-1) leads to activation of its endogenous tyrosine kinase activity. A number of substrates for the FGFR-1 kinase have been identified. Among those, FGF receptor-substrate-2 (FRS-2) was identified by virtue of its interaction with p13suc, a yeast protein involved in cell cycle regulation. We have used immobilized p13suc to identify a new substrate for FGRF-1, which is identical to "translocated in liposarcoma" (TLS). TLS is a RNA/DNA-binding protein which occurs in fusion products with different transcription factors in a variety of solid tumours. We show that TLS is tyrosine phosphorylated in intact cells by a number of different growth factors, indicating a role in growth regulation.

  • 41. Kubinski, Konrad
    et al.
    Zielinski, Rafal
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Mazur, Elzbieta
    Szyszka, Ryszard
    Yeast Elf1 factor is phosphorylated and interacts with protein kinase CK22006In: Journal of Biochemistry and Molecular Biology, ISSN 1225-8687, E-ISSN 0219-1024, Vol. 39, no 3, p. 311-318Article in journal (Refereed)
    Abstract [en]

    One of the biggest group of proteins influenced by protein kinase CK2 is formed by factors engaged in gene expression. Here we have reported recently identified yeast transcription elongation factor Elf1 as a new substrate for both monomeric and tetrameric forms of CK2. Elf1 serves as a substrate for both the recombinant CK2alpha' (K(m) 0.38 microM) and holoenzyme (K(m) 0.13 microM). By MALDI-MS we identified the two serine residues at positions 95 and 117 as the most probable in vitro phosphorylation sites. Coimmunoprecypitation experiments show that Elf1 interacts with catalytic (alpha and alpha') as well as regulatory (beta and beta') subunits of CK2. These data may help to elucidate the role of protein kinase CK2 and Elf1 in the regulation of transcription elongation.

  • 42.
    Lampropoulou, Evgenia
    et al.
    Univ Patras, Dept Pharm, Lab Mol Pharmacol, GR-26504 Patras, Greece..
    Logoviti, Ioanna
    Univ Patras, Dept Pharm, Lab Mol Pharmacol, GR-26504 Patras, Greece..
    Koutsioumpa, Marina
    Univ Patras, Dept Pharm, Lab Mol Pharmacol, GR-26504 Patras, Greece.;Univ Calif Los Angeles, David Geffen Sch Med, Vatche & Tamar Manoukian Div Digest Dis, Ctr Syst Biomed, Los Angeles, CA 90095 USA..
    Hatziapostolou, Maria
    Nottingham Trent Univ, Sch Sci & Technol, Dept Biosci, Nottingham NG11 8NS, England..
    Polytarchou, Christos
    Nottingham Trent Univ, Sch Sci & Technol, Dept Biosci, Nottingham NG11 8NS, England..
    Skandalis, Spyros S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Univ Patras, Dept Chem, Lab Biochem, GR-26504 Patras, Greece.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fousteris, Manolis
    Univ Patras, Dept Pharm, Lab Med Chem, GR-26504 Patras, Greece..
    Nikolaropoulos, Sotirios
    Univ Patras, Dept Pharm, Lab Med Chem, GR-26504 Patras, Greece..
    Choleva, Efrosini
    Univ Patras, Dept Pharm, Lab Mol Pharmacol, GR-26504 Patras, Greece..
    Lamprou, Margarita
    Univ Patras, Dept Pharm, Lab Mol Pharmacol, GR-26504 Patras, Greece..
    Skoura, Angeliki
    Univ Patras, Comp Engn & Informat Dept, Patras, Greece..
    Megalooikonomou, Vasileios
    Univ Patras, Comp Engn & Informat Dept, Patras, Greece..
    Papadimitriou, Evangelia
    Univ Patras, Dept Pharm, Lab Mol Pharmacol, GR-26504 Patras, Greece..
    Cyclin-dependent kinase 5 mediates pleiotrophin-induced endothelial cell migration2018In: Scientific Reports, E-ISSN 2045-2322, Vol. 8, article id 5893Article in journal (Refereed)
    Abstract [en]

    Pleiotrophin (PTN) stimulates endothelial cell migration through binding to receptor protein tyrosine phosphatase beta/zeta (RPTP beta/zeta) and alpha(nu)beta(3) integrin. Screening for proteins that interact with RPTP beta/zeta and potentially regulate PTN signaling, through mass spectrometry analysis, identified cyclindependent kinase 5 (CDK5) activator p35 among the proteins displaying high sequence coverage. Interaction of p35 with the serine/threonine kinase CDK5 leads to CDK5 activation, known to be implicated in cell migration. Protein immunoprecipitation and proximity ligation assays verified p35-RPTP beta/zeta interaction and revealed the molecular association of CDK5 and RPTP beta/zeta. In endothelial cells, PTN activates CDK5 in an RPTP beta/zeta- and phosphoinositide 3-kinase (PI3K)-dependent manner. On the other hand, c-Src, alpha(nu)beta(3) and ERK1/2 do not mediate the PTN-induced CDK5 activation. Pharmacological and genetic inhibition of CDK5 abolished PTN-induced endothelial cell migration, suggesting that CDK5 mediates PTN stimulatory effect. A new pyrrolo[2,3-alpha] carbazole derivative previously identified as a CDK1 inhibitor, was found to suppress CDK5 activity and eliminate PTN stimulatory effect on cell migration, warranting its further evaluation as a new CDK5 inhibitor. Collectively, our data reveal that CDK5 is activated by PTN, in an RPTP beta/zeta-dependent manner, regulates PTN-induced cell migration and is an attractive target for the inhibition of PTN pro-angiogenic properties.

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  • 43.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Wardega, Piotr
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Heldin, Carl-Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Alix Facilitates the Interaction between c-Cbl and Platelet-derived Growth Factor beta-Receptor and Thereby Modulates Receptor Down-regulation2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 51, p. 39152-39158Article in journal (Refereed)
    Abstract [en]

    Alix (ALG-2-interacting protein X) is an adaptor protein involved in down-regulation and sorting of cell surface receptors through the endosomal compartments toward the lysosome. In this study, we show that Alix interacts with the C-terminal region of the platelet-derived growth factor (PDGF) β-receptor (PDGFRβ) and becomes transiently tyrosine-phosphorylated in response to PDGF-BB stimulation. Increased expression levels of Alix resulted in a reduced rate of PDGFRβ removal from the cell surface following receptor activation, and this was associated with decreased receptor degradation. Furthermore, Alix was found to co-immunoprecipitate with the ubiquitin ligase c-Cbl, and elevated Alix levels increased the interaction between c-Cbl and PDGFRβ. Interestingly, Alix interacted constitutively with both c-Cbl and PDGFRβ. Moreover, c-Cbl was found to be hyperphosphorylated in cells engineered to overexpress Alix compared with control cells. The increased c-Cbl phosphorylation correlated with enhanced proteasomal degradation of c-Cbl, which in turn correlated with a decreased ubiquitination of PDGFRβ. Our data suggest that Alix inhibits down-regulation of PDGFRβ by modulating the interaction between c-Cbl and the receptor, thereby affecting the ubiquitination of the receptor.

  • 44.
    Lennartsson, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Engström, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Rönnstrand, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk2003In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 288, no 1, p. 110-118Article in journal (Refereed)
    Abstract [en]

    We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.

  • 45. Lexander, Helena
    et al.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Palmberg, Carina
    Auer, Gert
    Hellström, Magnus
    Franzén, Bo
    Jörnvall, Hans
    Egevad, Lars
    Evaluation of two sample preparation methods for prostate proteome analysis2006In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 6, no 13, p. 3918-3925Article in journal (Refereed)
    Abstract [en]

    For laboratory techniques that require well-preserved proteins, such as 2-DE, fresh tissue must be harvested and processed as fast as possible to avoid proteolytic degradation. We describe a modified method for harvesting tissue from radical prostatectomy specimens for proteome analysis and compare it with the standard technique. Cells were scraped from cut surfaces of 11 prostate specimens. A fraction of the material was smeared on a glass slide and Giemsa stained for morphological control. The sample was collected in a medium with protease inhibitors, and the protein material was prepared for 2-DE. Filtering and Percoll centrifugation were omitted. Sample locations were noted on a specimen map. From the same area, a tissue block was harvested for comparison. The block was processed with the conventional technique including mechanical disintegration, filtering and Percoll centrifugation. Quality measures of 2-DE were similar with both methods. With the scrape sampling technique, control smears showed abundant epithelial cells and a cleaner background and processing was faster than with tissue block sampling. For proteomic analysis, the scrape sample technique has several advantages over the tissue block method.

  • 46. Lexander, Helena
    et al.
    Palmberg, Carina
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Auer, Gert
    Hellström, Magnus
    Franzén, Bo
    Jörnvall, Hans
    Egevad, Lars
    Correlation of protein expression, Gleason score and DNA ploidy in prostate cancer2006In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 6, no 15, p. 4370-4380Article in journal (Refereed)
    Abstract [en]

    The prognosis of prostate cancer correlates with tumor differentiation. Gleason score and DNA ploidy are two prognostic factors that correlate with prognosis. We analyzed differences in protein expression in prostate cancer of high and low aggressiveness according to these measures. From 35 prostatectomy specimens, 29 cancer samples and 10 benign samples were harvested by scraping cells from cut surfaces. DNA ploidy was assessed by image cytometry. Protein preparations from cell suspensions were examined by 2-DE. Protein spots that differed quantitatively between sample groups were identified by MS fingerprinting of tryptic fragments and MS/MS sequence analysis. We found 39 protein spots with expression levels that were raised or lowered in correlation with Gleason score and/or DNA ploidy pattern (31 overexpressed in high-malignant cancer, 8 underexpressed). Of these, 30 were identified by MS. Among overexpressed proteins were heat-shock, structural and membrane proteins and enzymes involved in gene silencing, protein synthesis/degradation, mitochondrial protein import (metaxin 2), detoxification (GST-pi) and energy metabolism. Stroma-associated proteins were generally underexpressed. The protein expression of prostate cancer correlates with tumor differentiation. Potential prognostic markers may be found among proteins that are differentially expressed and the clinical value of these should be validated.

  • 47. Liu, Xiao Li
    et al.
    Kilpeläinen, Pekka
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Sun, Yi
    Wartiovaara, Jorma
    Morgunova, Ekaterina
    Pikkarainen, Timo
    Yan, Kunimasa
    Jonsson, Anders P.
    Tryggvason, Karl
    Characterization of the interactions of the nephrin intracellular domain2005In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 272, no 1, p. 228-243Article in journal (Refereed)
    Abstract [en]

    Nephrin is a signalling cell-cell adhesion protein of the Ig superfamily and the first identified component of the slit diaphragm that forms the critical and ultimate part of the glomerular ultrafiltration barrier. The extracellular domains of the nephrin molecules form a network of homophilic and heterophilic interactions building the structural scaffold of the slit diaphragm between the podocyte foot processes. The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phosphorylated, and mediates signalling from the slit diaphragm into the podocytes. CD2AP, podocin, Fyn kinase, and phosphoinositide 3-kinase are reported intracellular interacting partners of nephrin, although the biological roles of these interactions are unclarified. To characterize the structural properties and protein-protein interactions of the nephrin intracellular domain, we produced a series of recombinant nephrin proteins. These were able to bind all previously identified ligands, although the interaction with CD2AP appeared to be of extremely low stoichiometry. Fyn phosphorylated nephrin proteins efficiently in vitro. This phosphorylation was required for the binding of phosphoinositide 3-kinase, and significantly enhanced binding of Fyn itself. A protein of 190 kDa was found to associate with the immobilized glutathione S-transferase-nephrin. Peptide mass fingerprinting and amino acid sequencing identified this protein as IQGAP1, an effector protein of small GTPases Rac1 and Cdc42 and a putative regulator of cell-cell adherens junctions. IQGAP1 is expressed in podocytes at significant levels, and could be found at the immediate vicinity of the slit diaphragm. However, further studies are needed to confirm the biological significance of this interaction and its occurrence in vivo.

  • 48.
    Lomnytska, Marta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Dubrovska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Volodko, Natalya
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Increased expression of cSHMT, Tbx3 and utrophin in plasma of ovarian and breast cancer patients2006In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 118, no 2, p. 412-421Article in journal (Refereed)
    Abstract [en]

    Plasma samples of ovarian and breast cancer patients were used to search for markers of cancer using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. Truncated forms of cytosolic serine hydroxymethyl transferase (cSHMT), T-box transcription factor 3 (Tbx3) and utrophin were aberrantly expressed in samples from cancer patients as compared to samples from noncancerous cases. Aberrant expression of proteins was validated by immunoblotting of plasma samples with specific antibodies to cSHMT, Tbx3 and utrophin. A cohort of 79 breast and 39 ovarian cancer patients and 31 individuals with noncancerous conditions was studied. We observed increased expression of truncated cSHMT, Tbx3 and utrophin in plasma samples obtained from patients at early stages of disease. Our data suggest that cSHMT, Tbx3 and utrophin can be used as components of multiparameter monitoring of ovarian and breast cancer (supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html).

  • 49.
    Lomnytska, Marta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Lukiyanchuk, Vasyl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Souchelnytskyi, Serhiy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Transforming growth factor-beta1-regulated proteins in human endothelial cells identified by two-dimensional gel electrophoresis and mass spectrometry2004In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 4, no 4, p. 995-1006Article in journal (Refereed)
    Abstract [en]

    Transforming growth factor-beta (TGFbeta) is a potent regulator of angiogenesis affecting proliferation, differentiation and migration of endothelial cells. The effect of TGFbeta on endothelial cells depends on the origin of the cells and on the experimental conditions. Global analysis of TGFbeta signalling is expected to unveil mechanisms of this variability and identify novel targets of the growth factor. Here, we report proteome profiling of human microvascular endothelial cells obtained from dermis, which were treated with TGFbeta1 and compared to nontreated cells. We identified 54 proteins affected by TGFbeta1 using two-dimensional gel electrophoresis and peptide mass fingerprinting. Thirteen of the identified proteins are involved in various signalling processes. Seven proteins are involved in cytoskeleton rearrangements and six are involved in regulation of metabolism. Ten proteins were identical to predicted hypothetical proteins with no assigned functions. In agreement with the effect of TGFbeta1 on components of the cytoskeleton, TGFbeta1 induces actin cytoskeleton rearrangements. TGFbeta1 also affected expression of E2F6, p57Kip2, G(q)alpha, hnRNP A1 and myosin light chain proteins as shown by immunoblotting. Down-regulation of the transcriptional repressor E2F6 by TGFbeta1 correlated with a weak growth-inhibitory activity of TGFbeta1 on HMVEC-d cells. Twenty-five of the identified proteins have not previously been described as being regulated by TGFbeta1, providing new insights into TGFbeta1 signalling in endothelial cells.

  • 50. Lorenzo, Carmen
    et al.
    Salinas, Gustavo
    Brugnini, Andreina
    Wernstedt, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Hellman, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    González-Sapienza, Gualberto
    Echinococcus granulosus antigen 5 is closely related to proteases of the trypsin family2003In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 369, no Pt 1, p. 191-198Article in journal (Refereed)
    Abstract [en]

    Antigen 5 (Ag5) is a dominant secreted component of the larval stage of Echinococcus granulosus, and is highly immunogenic in human infections. Although the diagnostic value of Ag5 has been thoroughly evaluated, there has been little progress in its molecular characterization and the understanding of its biological role. In the present study, the Ag5 gene was cloned by reverse transcription-PCR on the basis of the amino acid sequences of tryptic fragments. The nucleotide sequence indicates that Ag5 is synthesized as a single polypeptide chain that is afterwards processed into single disulphide-bridged 22 and 38 kDa subunits. Whereas the 22 kDa component contains a highly conserved glycosaminoglycan-binding motif that may help to confine Ag5 in the host tissue surrounding the parasite, the 38 kDa subunit is closely related to serine proteases of the trypsin family. The sequences in the vicinity of the active-site histidine, aspartic acid and serine residues, and critical cysteine residues involved in disulphide formation, are well conserved, but the catalytic serine residue is replaced by threonine. Since there are no significant chemical differences between the O gamma atoms of these residues, we performed a series of enzymic assays to find out whether Ag5 is a catalytic molecule. Neither proteolytic activity nor binding to protease inhibitors could be detected using the native purified antigen. Thus it may be possible that Ag5 possesses a highly specific physiological substrate or, more likely, that trypsin-like folding has been recruited to fulfil novel functions.

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