RESUMO
BACKGROUND: Leishmaniasis is a newly emerging infection in Thailand. Most of the previous human cases have presented with the clinical features of visceral leishmaniasis and were mainly found in southern Thailand. Here we report the first two patients from northern Thailand presenting with disseminated cutaneous leishmaniasis. OBJECTIVES: To determine the nature of the infection of leishmaniasis and to identify the species of parasite responsible. METHODS: Clinical investigations included the taking of biopsy samples and histology. Parasitological diagnosis was performed by establishment of Leishmania promastigote cultures, and identification was performed by DNA sequencing of four independent gene loci (ribosomal RNA internal transcribed spacer 1; large subunit of RNA polymerase II; heat shock protein 70; RPL23a intergenic sequence). RESULTS: Both patients were infected with HIV, and had multiple cutaneous lesions and accompanying visceral leishmaniasis. They had similar cutaneous manifestations characterized by chronic generalized fibrotic lesions, which were more prominent on traumatic areas. In both patients the parasite was identified as Leishmania martiniquensis. This is a recently described species that is distinct and only distantly related to the classical agents of cutaneous leishmaniasis in Asia (Leishmania major and Leishmania tropica) or of visceral leishmaniasis (Leishmania donovani and Leishmania infantum). Each patient responded well to therapy with intravenous amphotericin B followed by oral itraconazole. CONCLUSIONS: Leishmania martiniquensis is a cause of cutaneous leishmaniasis in Thailand.
Assuntos
Coinfecção/complicações , Otopatias/complicações , Infecções por HIV/complicações , Dermatoses da Mão/complicações , Leishmaniose Cutânea/complicações , Pele/patologia , Adulto , Doença Crônica , Coinfecção/patologia , Otopatias/patologia , Orelha Externa , Fibrose/parasitologia , Infecções por HIV/patologia , Dermatoses da Mão/patologia , Humanos , Leishmaniose Cutânea/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , TailândiaRESUMO
Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.
Assuntos
Carcinoma de Células Renais/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Renais/genética , Mutação , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Replicação do DNA , Heterogeneidade Genética , Histonas/metabolismo , Humanos , Neoplasias Renais/metabolismo , Instabilidade de Microssatélites , Nucleossomos/patologiaRESUMO
We performed a kinome-wide siRNA screen and identified 70 kinases altering cell migration in A549 lung cancer cells. In particular, ribosomal S6 kinase 1 (RSK1) silencing increased, whereas RSK2 and RSK4 downregulation inhibited cell motility. In a secondary collagen-based three-dimensional invasion screen, 38 of our hits cross-validated, including RSK1 and RSK4. In two further lung cancer cell lines, RSK1 but not RSK4 silencing showed identical modulation of cell motility. We therefore selected RSK1 for further investigation. Bioinformatic analysis followed by co-immunoprecipitation-based validation revealed that the actin regulators VASP and Mena interact with RSK1. Moreover, RSK1 phosphorylated VASP on T278, a site regulating its binding to actin. In addition, silencing of RSK1 enhanced the metastatic potential of these cells in vivo using a zebrafish model. Finally, we investigated the relevance of this finding in human lung cancer samples. In isogenically matched tissue, RSK1 was reduced in metastatic versus primary lung cancer lesions. Moreover, patients with RSK1-negative lung tumours showed increased number of metastases. Our results suggest that the findings of our high-throughput in vitro screen can reliably identify relevant clinical targets and as a proof of principle, RSK1 may provide a biomarker for metastasis in lung cancer patients.
Assuntos
Neoplasias Pulmonares/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Animais , Sítios de Ligação , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência , Metástase Neoplásica , Transplante de Neoplasias , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Treonina/genética , Treonina/metabolismo , Transplante Heterólogo , Peixe-Zebra/embriologiaRESUMO
The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin, Radixin, Moesin) homology domain comprising 19 conserved hydrophobic regions. To determine the role of this domain in governing recruitment of Jak1, but not Jak3, to the gp130 subunit of the interleukin-6 family of cytokine receptors, the interaction of three Jak1/Jak3 chimeras with gp130 was investigated. Chimeras 1, 2 and 3 (Jak1 FERM regions 1-19, 1-18 and 1-8/Jak3, respectively) were all enzymically active. Chimeras 1 and 2 interacted with the cytoplasmic domain of gp130, although less efficiently than Jak1. Only chimera 2, however, restored gp130 signalling in Jak1-negative cells. The data are consistent with recruitment of Jak1 to gp130 through the Jak1 FERM domain, but also emphasise the likely requirement for precise Jak/receptor orientation to sustain function.
Assuntos
Antígenos CD/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Receptor gp130 de Citocina , Citoplasma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibrossarcoma , Humanos , Janus Quinase 1 , Janus Quinase 3 , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator de Transcrição STAT1 , Transdução de Sinais , Transativadores/metabolismo , Células Tumorais CultivadasRESUMO
CEACAM1 on leukocytic, endothelial, and epithelial cells functions in homophilic adhesion, tumor suppression, regulating cell adhesion and proliferation, and in heterophilic adhesion as a receptor for E-selectin and Neisseria meningiditis, Neisseria gonorrhoeae, Haemophilus influenzae, and murine coronaviruses. The 8 transmembrane isoforms of human CEACAM1 possess an extracellular N-terminal IgV domain, followed by variable numbers of IgC2 domains. To establish which key amino acids contribute specifically to CEACAM1 homophilic adhesion, exposed amino acids in the N-terminal domain of a soluble form of CEACAM1 were subjected to mutagenesis. Analyses of mutant proteins with conformationally dependent antibodies indicated that most mutations did not substantially affect the structural integrity of CEACAM1. Nevertheless, decreased adhesion was observed for the single mutants V39A or D40A (single-letter amino acid codes) in the CC' loop and for the triple mutants located in the GFCC'C" face of the N-terminal domain. Interestingly, whereas single mutations in R64 or D82 that are predicted to form a salt bridge between the base of the D and F beta strands close to the critical V39 and D40 residues also abolish adhesion, an amino acid swap (R64D and D82R), which maintains the salt bridge was without significant effect. These studies indicate that the CC' loop plays a crucial role in the homophilic adhesion of CEACAM1. They further predict that specific hydrophobic amino acid residues on the nonglycosylated GFCC'C" face of CEACAM1 N-terminal domain are not only involved in heterophilic interactions with Opa proteins and H influenzae, but are also critical for protein-protein interactions between 2 CEACAM1 molecules on opposing cells.
Assuntos
Antígenos CD/fisiologia , Antígenos de Diferenciação/fisiologia , Adesão Celular/fisiologia , Isoformas de Proteínas/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação/química , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Sítios de Ligação , Células CHO , Antígeno Carcinoembrionário/classificação , Moléculas de Adesão Celular , Cricetinae , Cricetulus , Epitopos/imunologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Especificidade de Órgãos , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Infective metacyclic promastigote forms of Leishmania mexicana are introduced by the bite of sandfly vectors into their human hosts where they transform into the amastigote form. The kinetics of this process was examined in vitro in response to different combinations of temperature (26 degrees C or 32 degrees C), pH (7.2 or 5.5), and exposure to human serum. Little transformation occurred at 26 degrees C/pH 7.2, intermediate levels at 26 degrees C/pH 5.5 and 32 degrees C/pH 7.2, and the greatest response at 32 degrees C/pH 5.5. Transformation was stimulated by exposure to normal human serum, but was markedly reduced when serum previously incubated at 56 degrees C for 1 h was used (complement heat-inactivated). This stimulatory effect was reproduced by exposure to a single purified component of human serum, C-reactive protein (CRP). Binding of CRP to the whole surface of L. mexicana metacyclic promastigotes, including the flagella, was demonstrated by an indirect fluorescent antibody test. The effect of purified CRP was dose dependent and occurred using normal serum concentrations. The stimulatory effect of whole serum was oblated by CRP depletion and restored by addition of purified CRP. The effects of cAMP analogues indicated that transformation could be mediated via an adenylate cyclase cascade.
Assuntos
Proteína C-Reativa/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Adenilil Ciclases/metabolismo , Animais , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Glicoesfingolipídeos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Leishmania mexicana/metabolismo , Ligantes , Microscopia de Fluorescência , TemperaturaRESUMO
Proteins involved in DNA repair, or its coordination with DNA replication and mitosis through cell cycle checkpoints, are vital in the concerted cellular response to DNA damage that maintains the integrity of the genome. The "BRCT" domain (BRCA1 carboxy terminal) was noted as a putative protein-protein interaction motif in the breast cancer suppressor gene, BRCA1, and subsequently identified in over 50 proteins involved in DNA repair, recombination, or cell cycle control. The heterodimer of the DNA repair proteins, XRCC1 and DNA ligase III, was the first example of a functional interaction via BRCT modules. The only three-dimensional crystal structure of a BRCT domain was solved for this region of XRCC1. Key amino acid residues mediating the interaction with DNA ligase III were identified here by targeted mutagenesis of the XRCC1 BRCT domain. The consequences of these mutations on protein folding were assessed. A structural model of the DNA ligase III BRCT domain was constructed and similarly tested by mutation of corresponding residues required for the interaction with XRCC1. These data identify the XRCC1-DNA ligase III heterodimer interface and provide the first demonstration of the surface contacts coordinating a functional BRCT-BRCT protein interaction.
Assuntos
Proteína BRCA1/metabolismo , DNA Ligases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Substituição de Aminoácidos/genética , Proteína BRCA1/química , Proteína BRCA1/genética , Dicroísmo Circular , DNA Ligase Dependente de ATP , DNA Ligases/química , Proteínas de Ligação a DNA/química , Dimerização , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Ligação Proteica/genética , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Termodinâmica , Triptofano/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Proteínas de XenopusRESUMO
The BRCA1 C-terminal region contains a duplicated globular domain termed BRCT that is found within many DNA damage repair and cell cycle checkpoint proteins. The unique diversity of this domain superfamily allows BRCT modules to interact forming homo/hetero BRCT multimers, BRCT-non-BRCT interactions, and interactions with DNA strand breaks. The sequence and functional diversity of the BRCT superfamily suggests that BRCT domains are evolutionarily convenient interaction modules.
Assuntos
Proteína BRCA1/química , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Sequência de Aminoácidos , Proteína BRCA1/fisiologia , Proteínas de Bactérias/química , Biopolímeros , Neoplasias da Mama/genética , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Proteínas de Ciclo Celular/química , Dano ao DNA , DNA Ligases/química , DNA Nucleotidilexotransferase/química , DNA Nucleotidilexotransferase/fisiologia , Reparo do DNA , Proteínas de Ligação a DNA/química , Evolução Molecular , Feminino , Proteínas Fúngicas/química , Genes BRCA1 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Síndromes Neoplásicas Hereditárias/genética , Proteínas Nucleares , Poli(ADP-Ribose) Polimerases/química , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
The integrins are cell membrane receptors composed of alpha and beta subunits which orchestrate adhesive events in all tissues of the body (Hynes, R.O., 1992. Integrins: versatility, modulation, and signalling in cell adhesion. Cell 69, 11-25; and Hynes, R.O., 1999. Cell adhesion: old and new questions. Trends Cell Biol. 9, M33-37). At present 18 alpha subunits and 8 beta subunits have been identified which are loosely organised into families. There are three inherited autosomal recessive diseases in man which involve germline mutations in genes coding for integrins. Leukocyte adhesion deficiency-1 (LAD-1) is the result of mutations in the beta2 subunit of the CD11/CD18 integrins, LFA-1, Mac-1, p150,95 and alphadbeta2. The bleeding disorder Glanzmann thrombasthenia is caused by mutations in either the alpha or beta subunit of the platelet integrin, alphaIIbbeta3. Thirdly, it is now recognised than one of the variants of the usually lethal skin blistering disorder, epidermolysis bullosa (JEB-PA), is caused by mutation in either the alpha or beta subunit of the epithelial hemidesmosome integrin, alpha6beta4. Many of the mutations cause defective alphabeta heterodimer formation. The majority of the beta subunit mutations are in the conserved N-terminal region known as the betaI domain. It is suggested that this region participates in alphabeta heterodimer formation.
Assuntos
Epidermólise Bolhosa/genética , Integrinas/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Trombastenia/genética , Animais , Antígenos CD/genética , Antígenos de Superfície/genética , Antígenos CD11/genética , Antígenos CD18/genética , Mapeamento Cromossômico , Epidermólise Bolhosa/terapia , Humanos , Integrina alfa6 , Integrina alfa6beta4 , Integrina beta3 , Integrina beta4 , Síndrome da Aderência Leucocítica Deficitária/terapia , Mutação de Sentido Incorreto , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/genética , Trombastenia/terapiaRESUMO
p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion. It is thought to disassemble SNARE complexes formed during the process of membrane fusion. Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution. Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible. A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis. Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/ultraestrutura , Microscopia Crioeletrônica , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Arqueais , Sítios de Ligação , Proteínas de Transporte/química , Cristalografia por Raios X , Proteínas Fúngicas/química , Fusão de Membrana , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Sensíveis a N-Etilmaleimida , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Maleabilidade , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Alinhamento de Sequência , Proteína com ValosinaRESUMO
Carcinoembryonic antigen (CEA) is highly expressed by most tumours of gastrointestinal origin, but its use as a target for tumour therapy is complicated by the high levels of soluble CEA that are found circulating in the blood of cancer patients. A monoclonal antibody PR1A3 has been prepared, which binds preferentially to cell-surface rather than soluble CEA, this cell selectivity should make PR1A3 an ideal candidate for antibody-targeted tumour therapy. PR1A3 has been humanised and shown to retain its cell-surface specificity and affinity. Stable expression of the humanised antibody from chinese hamster ovary (CHO) cells has been achieved after transfection and amplification. Since PR1A3 binds preferentially to cell-associated CEA, a cell-free enzyme-linked immunosorbent assay (ELISA) has been developed to allow characterisation and routine assay of the antibody. This assay was developed using a recombinant chimeric protein constructed by cloning the domain of CEA that is bound by PR1A3 (the B3 domain) into a hybrid gene containing the Fc portion of IgG and three domains of biliary glycoprotein. Stable expression of this hybrid protein has been achieved from CHO cells. In ELISA both humanised and murine PR1A3 bound strongly to this antigen but only at a minimal level to soluble CEA. Two binding sites for the antibody were found on the gastric carcinoma cell line MKN45, one of higher affinity (1 nM) and the other at lower affinity (60 nM). Similar affinities were found for both murine and humanised antibodies. The data presented make it unlikely that the differential binding to cell-surface as distinct from soluble CEA can be accounted for by low affinity of PR1A3 for CEA, and provides further support for the hypothesis that some conformational change takes place on CEA release from cells and that it is this change that blocks PR1A3 binding to its epitope.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais Murinos , Afinidade de Anticorpos , Células CHO , Células COS/metabolismo , Antígeno Carcinoembrionário/metabolismo , Clonagem Molecular , Cricetinae , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismoRESUMO
The human XPG endonuclease cuts on the 3' side of a DNA lesion during nucleotide excision repair. Mutations in XPG can lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of structure-specific nucleases and exonucleases. To begin defining its catalytic mechanism, we changed highly conserved residues and determined the effects on the endonuclease activity of isolated XPG, its function in open complex formation and dual incision reconstituted with purified proteins, and its ability to restore cellular resistance to UV light. The substitution A792V present in two XP complementation group G (XP-G) individuals reduced but did not abolish endonuclease activity, explaining their mild clinical phenotype. Isolated XPG proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In the reconstituted repair system, alanine substitutions at these positions permitted open complex formation but were inactive for 3' cleavage, whereas D77E and E791D proteins retained considerable activity. The function of each mutant protein in the reconstituted system was mirrored by its ability to restore UV resistance to XP-G cell lines. Hydrodynamic measurements indicated that XPG exists as a monomer in high salt conditions, but immunoprecipitation of intact and truncated XPG proteins showed that XPG polypeptides can interact with each other, suggesting dimerization as an element of XPG function. The mutation results define critical residues in the catalytic center of XPG and strongly suggest that key features of the strand cleavage mechanism and active site structure are shared by members of the nuclease family.
Assuntos
Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/química , Endonucleases/genética , Teste de Complementação Genética , Células HeLa , Humanos , Dados de Sequência Molecular , Mutagênese , Proteínas Nucleares , Homologia de Sequência de Aminoácidos , Fatores de TranscriçãoRESUMO
The BRCT domain (BRCA1 C-terminus), first identified in the breast cancer suppressor protein BRCA1, is an evolutionarily conserved protein-protein interaction region of approximately 95 amino acids found in a large number of proteins involved in DNA repair, recombination and cell cycle control. Here we describe the first three-dimensional structure and fold of a BRCT domain determined by X-ray crystallography at 3.2 A resolution. The structure has been obtained from the C-terminal region of the human DNA repair protein XRCC1, and comprises a four-stranded parallel beta-sheet surrounded by three alpha-helices, which form an autonomously folded domain. The compact XRCC1 structure explains the observed sequence homology between different BRCT motifs and provides a framework for modelling other BRCT domains. Furthermore, the established structure of an XRCC1 BRCT homodimer suggests potential protein-protein interaction sites for the complementary BRCT domain in DNA ligase III, since these two domains form a stable heterodimeric complex. Based on the XRCC1 BRCT structure, we have constructed a model for the C-terminal BRCT domain of BRCA1, which frequently is mutated in familial breast and ovarian cancer. The model allows insights into the effects of such mutations on the fold of the BRCT domain.
Assuntos
Proteína BRCA1/genética , Proteínas de Ligação a DNA/química , Sequência de Aminoácidos , Sequência Conservada/genética , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
An analysis has been performed on the first example of a non-proline cis- peptide bond found within a complementarity determining region (CDR) of an antibody. The bond is located in CDR 3 of the heavy chain (H3) and makes substantial interactions to a peptide from a breast tumour-associated antigen. The antibody-peptide complex is compared, both in H3 length (six residues) and peptide conformation, to a number of other such complexes in the Brookhaven Data Bank (PDB). There is only one other H3 loop of the same length. Analysis of loop searches of the PDB, taken over the H3 framework of SM3, suggest that there is a limited repertoire of conformations for loops of length 6 compared to loops of length 5 and 7. It is argued that the cis-peptide bond is present because of the limited number of loop conformations of length 6, plus, the requirement of the H3 loop to contact the bound peptide. Modelling suggests that an all-trans-peptide loop conformation can replace the H3 loop and this raises the question of whether there is a trans- to cis-peptide bond isomerization upon peptide binding.
Assuntos
Anticorpos/química , Região Variável de Imunoglobulina/química , Peptídeos/química , Cristalografia por Raios X , Bases de Dados Factuais , Modelos Moleculares , Prolina/química , Conformação ProteicaRESUMO
The anti-breast tumour antibody SM3 has a high selectivity in reacting specifically with carcinoma-associated mucin. SM3 recognises the core repeating motif (Pro-Asp-Thr-Arg-Pro) of aberrantly glycosylated epithelial mucin MUC1, and has potential as a therapeutic and diagnostic tool. Here we report the crystal structure of the Fab fragment of SM3 in complex with a 13-residue MUC1 peptide antigen (Thr1P-Ser2P-Ala3P-Pro4P-Asp5P-Thr6P -Arg7P-Pro8P-Ala9P-Pro10P-Gly11P- Ser12P-Thr13P). The SM3-MUC1 peptide structure was solved by molecular replacement, and the current model is refined at 1.95 A resolution with an R-factor of 21.3% and R-free 28.3%. The MUC1 peptide is bound both by non-polar interactions and hydrogen bonds in an elongated groove in the antibody-combining site through interactions with Complimentarity Determining Regions (CDRs), three of the light chain (L1, L2, L3) and two of the heavy chain (H1 and H3). The conformation of the peptide is mainly extended with no discernable standard secondary structure. There is a single non-proline cis-peptide bond in H3 (Val95H-Gly96H-Gln97H-Phe98H-Ala101H-Ty r102H) between Gly96H and Gln97H, which appears to play a role in SM3-peptide antigen interactions, and represents the first such example within an antibody hypervariable loop. The SM3-MUC1 peptide structure has implications for rational therapeutic and diagnostic antibody engineering.
Assuntos
Anticorpos Antineoplásicos/química , Neoplasias da Mama/imunologia , Epitopos/química , Fragmentos Fab das Imunoglobulinas/química , Mucinas/química , Peptídeos/química , Sequência de Aminoácidos , Reações Antígeno-Anticorpo , Modelos MolecularesRESUMO
ICP4 of herpes simplex virus (HSV) is essential for productive infection due to its central role in the regulation of HSV transcription. This study identified a region of ICP4 that is not required for viral growth in culture or at the periphery of experimentally inoculated mice but is critical for productive growth in the trigeminal ganglia. This region of ICP4 encompasses amino acids 184 to 198 and contains 13 nearly contiguous serine residues that are highly conserved among the alphaherpesviruses. A mutant in which this region is deleted (DeltaSER) was able to grow on the corneas of mice and be transported back to the trigeminal ganglia. DeltaSER did not grow in the trigeminal ganglia but did express low levels of several immediate-early (ICP4 and ICP27) and early (thymidine kinase [tk] and UL42) genes. It expressed very low levels of the late gC gene and did not appear to replicate DNA. This pattern of gene expression was similar to that observed for a tk mutant, dlsptk. Both DeltaSER and dlsptk expressed higher levels of the latency-associated transcript (LAT) per genome earlier in infected ganglia than did the wild-type virus, KOS. However, infected ganglia from all three viruses accumulated the same level of LAT per genome at 30 days postinfection (during latency). The data suggest that the polyserine tract of ICP4 provides an activity that is required for lytic infection in ganglia to progress to viral DNA synthesis and full lytic gene expression. In the absence of this activity, higher levels of LAT per genome accumulate earlier in infection than with wild-type virus.
Assuntos
Herpesvirus Humano 1/genética , Proteínas Imediatamente Precoces/fisiologia , Peptídeos/fisiologia , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Replicação do DNA , Expressão Gênica , Herpesvirus Humano 1/crescimento & desenvolvimento , Humanos , Proteínas Imediatamente Precoces/genética , Camundongos , Dados de Sequência Molecular , Peptídeos/genética , Fenótipo , Gânglio Trigeminal/virologia , Células Vero , Latência ViralRESUMO
PR1A3 antibody binds specifically to the tumour-associated cell-surface antigen, carcinoembryonic antigen. Crystals of the Fab fragment of the PR1A3 antibody were obtained by vapour diffusion against mother liquor containing Tris-HC1 buffer, pH 8.6, magnesium chloride and polyethylene glycol 4000 as precipitating agent. Crystals belong to the monoclinic space group P2(1) with cell dimensions a = 42.2, b = 216.7, c = 45.9 A and beta = 95.6 degrees. Two Fab fragments are proesent in the asymmetric unit. Diffracted intensities up to 2.9 A resolution have been measured from frozen crystals.
RESUMO
Conformational clusters and consensus sequences for protein loops have been derived by computational analysis of their structures in a non-redundant set of 233 proteins with less than 25% sequence homology (X-ray resolution better than 2.5 A). Loops have been classified into five types (alpha-alpha, beta-beta links, beta-beta hairpins, alpha-beta and beta-alpha) according to the secondary structures they embrace. Four variables have been used to describe the loop geometry, three angles and one distance between the secondary structure elements embracing the loop. Ramachandran angles (phi, psi) are used to define the loop conformations within each brace geometry. All loops from the non-redundant set have been clustered by means of these geometric features. A total of 56 classes (9 alpha-alpha, 11 beta-beta links, 14 beta-beta hairpins, 13 alpha-beta and 9 beta-alpha) were identified with consensus Ramachandran angles in the loops. These classes were divided into subclasses based on the brace geometry. This clustering procedure captures most of the clusters analysed by predominantly visual inspection methods and finds other clusters that have hitherto not been described. Consensus sequence patterns were identified for the subclasses. An extensive characterisation of loop conformations has therefore been achieved and the computational approach is readily open to the incorporation of information from newly determined structures. These clusters should also enhance model building by comparison studies.
Assuntos
Estrutura Secundária de Proteína , Proteínas/química , Cristalografia por Raios X , Modelos Moleculares , Peptídeos/química , Conformação ProteicaRESUMO
SM3 antibody binds to a tumour-associated epitope on polymorphic epithelial mucin (PEM). Crystals of the Fab fragment of SM3 in complex with a peptide antigen were obtained by vapour diffusion against mother liquor containing acetate buffer, pH 6.5, cadmium chloride and polyethylene glycol (PEG) 4000 as precipitating agent. Crystals belong to the monoclinic space group P2(1) with cell dimensions a = 42.2, b = 83.9, c = 64.5 A and beta = 93.4 degrees. One Fab-antigen complex is present in the asymmetric unit. Diffracted intensities up to 1.95 A resolution have been measured from a frozen crystal using synchrotron radiation.
RESUMO
Expression of polysialic acid (PSA) involves its specific attachment to the neural cell adhesion molecule (NCAM). Here we identify the amino acid residues within NCAM that are polysialylated and structural domains of the NCAM polypeptide that are required for addition of PSA in cells. Chicken NCAM cDNAs containing amino acid mutations, domain deletions, and domain substitutions were expressed in the F11 rat/mouse hybrid cell line, which can produce polysialylated NCAM. Polysialylation of the chicken NCAM was evaluated by immunopurification and electrophoresis. Mutation of all three potential N-glycosylation sites within the fifth immunoglobulin domain (Ig5) abrogated polysialylation. Analysis of paired mutations revealed that Asn-459 is heavily polysialylated, Asn-430 has a lower level of substitution, and Asn-404 receives little or no PSA. Analysis of domain deletions established that the intracellular domain, Ig domains 1-3, and the COOH-terminal fibronectin-type III (FNIII) repeat are not required for polysialylation, but that deletion of either the adjacent Ig4 or FNIII-type domain prevented addition of PSA. Accordingly, a minimal polypeptide for polysialylation was found to contain Ig domains 4 and 5, the adjacent FNIII repeat, plus a membrane attachment. These results suggest that although all PSA is located within Ig5, regions outside Ig5 also play a role in PSA addition to NCAM. Furthermore, molecular modeling indicates spatial proximity of Asn-430 and Asn-459 and a tight-locking arrangement between Ig4, Ig5, and FNIII#1 that would be consistent with their formation of a spatially discrete enzyme recognition site for polysialylation.