PubMed | Laboratory of Structural Biology, Federal University of Pará and Evandro Chagas Institute
Type: | Journal: Experimental parasitology | Year: 2016
Leishmaniasis are worldwide diseases that occur in 98 countries including Brazil, transmitted by the bite of female phlebotomines during blood feeding. In Brazil it is known that some species of sand flies as Lutzomyia longipalpis sensun latum (vector of Leishmania infantum chagasi), Lutzomyia flaviscutellata (vector of Leishmania (Leishmania) amazonensis) and Lutzomyia antunesi [suspected vector of Leishmania (Viannia) lindenbergi] are incriminated of transmitting the parasite Leishmania for the vertebrate host. The phlebotomine-parasite is mediated by the attachment of the promastigote lipophosphoglycan (LPG) to the midgut epithelium. However, another mechanism that is LPG-independent and mediated by N-acetyl-galactosamine (GalNAc) seems to occur in some species of phlebotomines that are classified as permissive. The aim of this study was to characterize the carbohydrate residues that, probably, play a role in parasite attachment to the midgut of phlebotomine from colony and field populations from the Brazilian Amazonian region. We observed the presence of GalNAc, mannose, galactose and GlcNAc in all phlebotomine species. A binding assay between L. (L.) amazonensis and L. i.chagasi to the midguts of different species of phlebotomines was performed. The attachment of both Leishmania and vector species suggests the presence of GalNAc on the midgut surfaces. Thus, these results suggested that GalNAc is a possible binding sites of Leishmania in sand flies from the Brazilian Amazonian region.
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.
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.
Kirby T.W.,Laboratory of Structural Biology |
Derose E.F.,Laboratory of Structural Biology |
Cavanaugh N.A.,Laboratory of Structural Biology |
Beard W.A.,Laboratory of Structural Biology |
And 4 more authors.
Nucleic Acids Research | Year: 2012
Binding of the catalytic divalent ion to the ternary DNA polymerase β/gapped DNA/dNTP complex is thought to represent the final step in the assembly of the catalytic complex and is consequently a critical determinant of replicative fidelity. We have analyzed the effects of Mg 2+ and Zn 2+ on the conformational activation process based on NMR measurements of [methyl- 13C]methionine DNA polymerase β. Unexpectedly, both divalent metals were able to produce a template base-dependent conformational activation of the polymerase/1-nt gapped DNA complex in the absence of a complementary incoming nucleotide, albeit with different temperature thresholds. This conformational activation is abolished by substituting Glu295 with lysine, thereby interrupting key hydrogen bonds necessary to stabilize the closed conformation. These and other results indicate that metal-binding can promote: translocation of the primer terminus base pair into the active site; expulsion of an unpaired pyrimidine, but not purine, base from the template-binding pocket; and motions of polymerase subdomains that close the active site. We also have performed pyrophosphorolysis studies that are consistent with predictions based on these results. These findings provide new insight into the relationships between conformational activation, enzyme activity and polymerase fidelity. © 2011 The Author(s).
Imai T.,Kyushu University |
Nakamura T.,Kyushu University |
Maeda T.,Kyushu University |
Nakayama K.,Kyushu University |
And 7 more authors.
Biochemical and Biophysical Research Communications | Year: 2014
Proteinaceous RNase P (PRORP1) in Arabidopsis thaliana is an endoribonuclease that catalyzes hydrolysis to remove the 5′-leader sequence of precursor tRNAs (pre-tRNAs). PRORP1 is composed of pentatricopeptide repeat (PPR) motifs, a central linker region, and a metal nuclease domain, the NYN domain. The PPR motifs are single-stranded RNA-binding motifs that recognize bases in a modular fashion. To obtain insight into the mechanism by which the PPR motifs in PRORP1 recognize a target sequence in catalysis, N-terminal successive deletion mutants were overproduced in Escherichia coli, and the resulting proteins were characterized in terms of enzymatic activity using chloroplast pre-tRNAPhe as a substrate. Although Δ89, in which all PPR motifs are present, retained the pre-tRNA cleavage activity, Δ129 devoid of the first PPR motif (PPR1) had significantly reduced cleavage activity. Likewise, deletions of the second (PPR2) or third PPR (PPR3) motif abolished the cleavage activity, suggesting that PPR motifs play a crucial role in catalysis. A proposed recognition code for PPR motifs predicted that PPR2-PPR5 in PRORP1 recognize C, A/U, A, and U, respectively, whose sequence is in good agreement with C56-A57-A58-A59 in the TψC loop in pre-tRNA Phe. Mutational analyses of nucleotide residues in the TψC loop as well as nucleotide-specifying residues (NSRs) in PPR motifs further suggested that PPR2 and PPR3 in PRORP1 favorably recognize nucleotide bases C56 and A57 at the TψC loop in pre-tRNAPhe, respectively. This prediction and previous biochemical data were combined to construct a fitting model of tRNA onto PRORP1, showing that the mechanism by which PRORP1 recognizes pre-tRNAs appears to be distinct from that by bacterial RNase P. © 2014 Elsevier Inc. All rights reserved.
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.
Hamasaki M.,Kyushu University |
Hazeyama K.,Kyushu University |
Iwasaki F.,Laboratory of Structural Biology |
Ueda T.,Laboratory of Structural Biology |
And 6 more authors.
Journal of Biochemistry | Year: 2016
PhoPop5 and PhoRpp30 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologues of human ribonuclease P (RNase P) proteins hPop5 and Rpp30, respectively, fold into a heterotetramer [PhoRpp30-(PhoPop5)2-PhoRpp30], which plays a crucial role in the activation of RNase P RNA (PhopRNA). Here, we examined the functional implication of PhoPop5 and PhoRpp30 in the tetramer. Surface plasmon resonance (SPR) analysis revealed that the tetramer strongly interacts with an oligonucleotide including the nucleotide sequence of a stem-loop SL3 in PhopRNA. In contrast, PhoPop5 had markedly reduced affinity to SL3, whereas PhoRpp30 had little affinity to SL3. SPR studies of PhoPop5 mutants further revealed that the C-terminal helix (α4) in PhoPop5 functions as a molecular recognition element for SL3. Moreover, gel filtration indicated that PhoRpp30 exists as a monomer, whereas PhoPop5 is an oligomer in solution, suggesting that PhoRpp30 assists PhoPop5 in attaining a functionally active conformation by shielding hydrophobic surfaces of PhoPop5. These results, together with available data, allow us to generate a structural and mechanistic model for the PhopRNA activation by PhoPop5 and PhoRpp30, in which the two C-terminal helices (α4) of PhoPop5 in the tetramer whose formation is assisted by PhoRpp30 act as binding elements and bridge SL3 and SL16 in PhopRNA. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
Ueda T.,Laboratory of Structural Biology |
Yamaguchi H.,Kyushu University |
Miyanoshita M.,Laboratory of Structural Biology |
Nakashima T.,Laboratory of Structural Biology |
And 5 more authors.
Journal of Biochemistry | Year: 2014
Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5′ leader sequence of precursor tRNA (pre-tRNA). The secondary structure of RNase P RNA (PhopRNA) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 has several peripheral stem-loops. In order to investigate their functional role, we prepared six mutants, ΔP1, ΔP3, ΔP8, ΔP9, ΔP12 and ΔP15, in which the stem-loops including helices P1, P3, P8, P9, P12/12.1/12.2 and P15/16 in PhopRNA were individually deleted, respectively, and characterized them with respect to pre-tRNA cleavage activity in the presence of five proteins and also to the ability to form a complex with the proteins. The reconstituted particles containing ΔP3, ΔP8 or ΔP9 retained considerable levels of activity (35-65%), while those containing ΔP1, ΔP12 or ΔP15 had markedly reduced activity (13%). It was further found that the reconstituted particles comprising ΔP3 or ΔP15 lacked PhoPop5 and PhoRpp30, whereas those containing ΔP1, ΔP8, ΔP9 or ΔP12 bound to all five proteins. Since it is known that PhoPop5 functions in a complex with PhoRpp30, the present result suggests that the peripheral stem-loops containing P3 or P15/16 are involved in the structural formation of a catalytic site by interacting with the protein complex PhoPop5-PhoRpp30. © 2013 The Authors.
PubMed | Laboratory of Structural Biology and Kyushu University
Type: | Journal: Biochemical and biophysical research communications | Year: 2016
Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the processing of 5 leader sequences of precursor tRNAs (pre-tRNA). RNase P proteins PhoRpp21 and PhoRpp29 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologs of human nuclear RNase P proteins Rpp21 and Rpp29 respectively, fold into a heterodimeric structure and synergistically function in the activation of the specificity domain (S-domain) in RNase P RNA (PhopRNA). To elucidate the molecular basis for their cooperativity, we first analyzed binding ability to PhopRNA using a pull-down assay. The result showed that PhoRpp21 is able to bind to PhopRNA in the absence of PhoRpp29, whereas PhoRpp29 alone has reduced affinity to PhopRNA, suggesting that PhoRpp21 primarily functions as a binding element for PhopRNA in the PhoRpp21-PhoRpp29 complex. Mutational analyses suggested that although individual positively charged clusters contribute little to the PhopRNA binding, Lys53, Lys54, and Lys56at the N-terminal helix (2) in PhoRpp21 and 10C-terminal residues in PhoRpp29 are essential for PhopRNA activation. Moreover, deletion of a single stranded loop linking P11 and P12 helices in the PhopRNA S-domain impaired the PhoRpp21-PhoRpp29 complex binding to PhopRNA. Collectively, the present results suggest that PhoRpp21 binds the loop between P11 and P12 helices through overall positively charged clusters on the surface of the complex and serves as a scaffold for PhoRpp29 to optimize structural conformation of its N-terminal helix (2) in PhoRpp21, as well as C-terminal residues in PhoRpp29, for RNase P activity.
PubMed | Laboratory of Structural Biology and Kyushu University
Type: Journal Article | Journal: Journal of biochemistry | Year: 2016
PhoPop5 and PhoRpp30 in the hyperthermophilic archaeon Pyrococcus horikoshii, homologues of human ribonuclease P (RNase P) proteins hPop5 and Rpp30, respectively, fold into a heterotetramer [PhoRpp30-(PhoPop5)2-PhoRpp30], which plays a crucial role in the activation of RNase P RNA (PhopRNA). Here, we examined the functional implication of PhoPop5 and PhoRpp30 in the tetramer. Surface plasmon resonance (SPR) analysis revealed that the tetramer strongly interacts with an oligonucleotide including the nucleotide sequence of a stem-loop SL3 in PhopRNA. In contrast, PhoPop5 had markedly reduced affinity to SL3, whereas PhoRpp30 had little affinity to SL3. SPR studies of PhoPop5 mutants further revealed that the C-terminal helix (4) in PhoPop5 functions as a molecular recognition element for SL3. Moreover, gel filtration indicated that PhoRpp30 exists as a monomer, whereas PhoPop5 is an oligomer in solution, suggesting that PhoRpp30 assists PhoPop5 in attaining a functionally active conformation by shielding hydrophobic surfaces of PhoPop5. These results, together with available data, allow us to generate a structural and mechanistic model for the PhopRNA activation by PhoPop5 and PhoRpp30, in which the two C-terminal helices (4) of PhoPop5 in the tetramer whose formation is assisted by PhoRpp30 act as binding elements and bridge SL3 and SL16 in PhopRNA.