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Titus M.,Roswell Park Cancer Institute | Tomer K.B.,Laboratory of Structural Biology
Methods in Molecular Biology | Year: 2011

Prostate cancer that recurs after androgen deprivation therapy is the second leading cause of cancer-related death in North American men. Clinical and experimental evidences indicate that the development of recurrent prostate cancer is dependent on re-activation of the androgen receptor signaling pathway. Androgen is required for androgen receptor translocation to the nucleus, interaction with androgen response elements, expression of target genes, and prostate cancer cell proliferation. The intra-tissue and serum testosterone and dihydrotestosterone levels are important biomarkers to monitor androgen deprivation therapy efficacy in prostate cancer and recurrent prostate cancer. We have measured testosterone and dihydrotestosterone in procured recurrent prostate cancer specimens using liquid chromatography tandem mass spectrometry. The measured androgen levels are sufficient to activate androgen receptor and suggest that the recurrent prostate cancer microenvironment is capable of intracrine androgen biosynthesis. © 2011 Springer Science+Business Media, LLC. Source

Bereszczak J.Z.,University Utrecht | Bereszczak J.Z.,Netherlands Proteomics Center | Rose R.J.,University Utrecht | Rose R.J.,Netherlands Proteomics Center | And 8 more authors.
Journal of the American Chemical Society | Year: 2013

Infection of humans by hepatitis B virus (HBV) induces the copious production of antibodies directed against the capsid protein (Cp). A large variety of anticapsid antibodies have been identified that differ in their epitopes. These data, and the status of the capsid as a major clinical antigen, motivate studies to achieve a more detailed understanding of their interactions. In this study, we focused on the Fab fragments of two monoclonal antibodies, E1 and 3120. E1 has been shown to bind to the side of outward-protruding spikes whereas 3120 binds to the "floor" region of the capsid, between spikes. We used hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the effects on HBV capsids of binding these antibodies. Conventionally, capsids loaded with saturating amounts of Fabs would be too massive to be readily amenable to HDX-MS. However, by focusing on the Cp protein, we were able to acquire deuterium uptake profiles covering the entire 149-residue sequence and reveal, in localized detail, changes in H/D exchange rates accompanying antibody binding. We find increased protection of the known E1 and 3120 epitopes on the capsid upon binding and show that regions distant from the epitopes are also affected. In particular, the α2a helix (residues 24-34) and the mobile C-terminus (residues 141-149) become substantially less solvent-exposed. Our data indicate that even at substoichiometric antibody binding an overall increase in the rigidity of the capsid is elicited, as well as a general dampening of its breathing motions. © 2013 American Chemical Society. Source

Cajanek L.,Karolinska Institutet | Cajanek L.,University of Basel | Ganji R.S.,Masaryk University | Henriques-Oliveira C.,Karolinska Institutet | And 5 more authors.
Molecular and Cellular Biology | Year: 2013

Understanding the mechanisms that drive the differentiation of dopaminergic (DA) neurons is crucial for successful development of novel therapies for Parkinson's disease, in which DA neurons progressively degenerate. However, the mechanisms underlying the differentiation-promoting effects of Wnt5a on DA precursors are poorly understood. Here, we present the molecular and functional characterization of a signaling pathway downstream of Wnt5a, the Wnt/Dvl/Rac1 pathway. First, we characterize the interaction between Rac1 and Dvl and identify the N-terminal part of Dvl3 as necessary for Rac1 binding. Next, we show that Tiam1, a Rac1 guanosine exchange factor (GEF), is expressed in the ventral midbrain, interacts with Dvl, facilitates Dvl-Rac1 interaction, and is required for Dvl- or Wnt5a-induced activation of Rac1. Moreover, we show that Wnt5a promotes whereas casein kinase 1 (CK1), a negative regulator of the Wnt/Dvl/Rac1 pathway, abolishes the interactions between Dvl and Tiam1. Finally, using ventral midbrain neurosphere cultures, we demonstrate that the generation of DA neurons in culture is impaired after Tiam1 knockdown, indicating that Tiam1 is required for midbrain DA differentiation. In summary, our data identify Tiam1 as a novel regulator of DA neuron development and as a Dvl-associated and Rac1-specific GEF acting in the Wnt/ Dvl/Rac1 pathway. © 2013, American Society for Microbiology. Source

Sugiura N.,Aichi Medical University | Baba Y.,Laboratory of Structural Biology | Kawaguchi Y.,Laboratory of Structural Biology | Iwatani T.,Laboratory of Structural Biology | And 7 more authors.
Journal of Biological Chemistry | Year: 2010

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA β1-4 and GlcNAc α1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source

Sassa A.,Japan National Institute of Health Sciences | Sassa A.,Tokyo University of Pharmacy and Life Science | Sassa A.,Laboratory of Structural Biology | Suzuki T.,Japan National Institute of Health Sciences | And 16 more authors.
DNA Repair | Year: 2014

Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N2-deoxyguanine (BPDE-N2-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N2-dG. In the present study, we established human cell lines that express wild-type Pol κ (POLK+/-), F171A (POLK F171A/-) or lack expression of Pol κ (POLK-/-) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (-)- or (+)-trans-anti-BPDE-N2-dG in the supF gene. The frequencies of mutations were in the order of POLK-/->POLK+/->POLK F171A/- both in (-)- and (+)-trans-anti-BPDE-N2-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N2-dG by Pol κ and raise the possibility that the cognate substrates for Pol κ are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens. © 2014 Elsevier B.V. Source

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