RESUMO
The role of Nucleoplasmin (NP) as a H2A-H2B histone chaperone has been extensively characterized. To understand its putative interaction with other histone ligands, we have characterized its ability to bind H3-H4 and histone octamers. We find that the chaperone forms distinct complexes with histones, which differ in the number of molecules that build the assembly and in their spatial distribution. When complexed with H3-H4 tetramers or histone octamers, two NP pentamers form an ellipsoidal particle with the histones located at the center of the assembly, in stark contrast with the NP/H2A-H2B complex that contains up to five histone dimers bound to one chaperone pentamer. This particular assembly relies on the ability of H3-H4 to form tetramers either in solution or as part of the octamer, and it is not observed when a variant of H3 (H3C110E), unable to form stable tetramers, is used instead of the wild-type protein. Our data also suggest that the distal face of the chaperone is involved in the interaction with distinct types of histones, as supported by electron microscopy analysis of the different NP/histone complexes. The use of the same structural region to accommodate all type of histones could favor histone exchange and nucleosome dynamics.
Assuntos
Histonas/química , Nucleoplasminas/química , Sequência de Aminoácidos , Animais , Histonas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Dados de Sequência Molecular , Nucleoplasminas/metabolismo , Multimerização Proteica , Proteólise , Xenopus laevisRESUMO
MRE11-RAD50 is a key early response protein for processing DNA ends of broken chromosomes for repair, yet how RAD50 nucleotide dynamics regulate MRE11 nuclease activity is poorly understood. We report here that ATP binding and ATP hydrolysis cause a striking butterfly-like opening and closing of the RAD50 subunits, and each structural state has a dramatic functional effect on MRE11. RAD50-MRE11 has an extended conformation in solution when MRE11 is an active nuclease. However, ATP binding to RAD50 induces a closed conformation, and in this state MRE11 is an endonuclease. ATP hydrolysis opens the RAD50-MRE11 complex, and MRE11 maintains exonuclease activity. Thus, ATP hydrolysis is a molecular switch that converts MRE11 from an endonuclease to an exonuclease. We propose a testable model in which the open-closed transitions are used by RAD50-MRE11 to discriminate among DNA ends and drive the choice of recombination pathways.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Complexos Multienzimáticos/metabolismo , Pyrococcus furiosus/enzimologia , Recombinação Genética/fisiologia , Trifosfato de Adenosina/genética , Proteínas Arqueais/genética , DNA Arqueal/genética , DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Hidrólise , Complexos Multienzimáticos/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Pyrococcus furiosus/genéticaRESUMO
Recent reviews have focused on the structure and function of histone chaperones involved in different aspects of somatic cell chromatin metabolism. One of the most dramatic chromatin remodeling processes takes place immediately after fertilization and is mediated by egg histone storage chaperones. These include members of the nucleoplasmin (NPM2/NPM3), which are preferentially associated with histones H2A-H2B in the egg and the nuclear autoantigenic sperm protein (NASP) families. Interestingly, in addition to binding and providing storage to H3/H4 in the egg and in somatic cells, NASP has been shown to be a unique genuine chaperone for histone H1. This review revolves around the structural and functional roles of these two families of chaperones whose activity is modulated by their own post-translational modifications (PTMs), particularly phosphorylation. Beyond their important role in the remodeling of paternal chromatin in the early stages of embryogenesis, NPM and NASP members can interact with a plethora of proteins in addition to histones in somatic cells and play a critical role in processes of functional cell alteration, such as in cancer. Despite their common presence in the egg, these two histone chaperones appear to be evolutionarily unrelated. In contrast to members of the NPM family, which share a common monophyletic evolutionary origin, the different types of NASP appear to have evolved recurrently within different taxa.
Assuntos
Autoantígenos/metabolismo , Chaperonas de Histonas/metabolismo , Proteínas Nucleares/metabolismo , Nucleoplasminas/metabolismo , Animais , Autoantígenos/genética , Montagem e Desmontagem da Cromatina/genética , Evolução Molecular , Feminino , Chaperonas de Histonas/genética , Humanos , Proteínas Nucleares/genética , Nucleoplasminas/genética , Óvulo/crescimento & desenvolvimento , Óvulo/metabolismo , Vertebrados/genética , Vertebrados/metabolismoRESUMO
BACKGROUND: The optimal radiological follow-up of prostate lesions negative on magnetic resonance imaging (MRI)-targeted biopsy (MRI-TB) is yet to be optimised. OBJECTIVE: To present medium-term radiological and clinical follow-up of biopsy-negative lesions. DESIGN, SETTING, AND PARTICIPANTS: The records for men who underwent multiparametric MRI at the UCLH one-stop clinic for suspected prostate cancer between September 2017 and March 2020 were reviewed (n = 1199). Patients with Likert 4 or 5 lesions were considered (n = 495), and those with a subsequent negative MRI-TB comprised the final study population (n = 91). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Baseline and follow-up MRI and biopsy data (including prostate-specific antigen [PSA], prostate volume, radiological scores, and presence of any noncancerous pathology) were extracted from reports. The last follow-up date was the date of the last test or review in clinic. RESULTS AND LIMITATIONS: Median follow-up was 1.8 yr (656 d, interquartile range [IQR] 359-1008). At baseline, the median age was 65.4 yr (IQR 60.7-70.0), median PSA was 7.1 ng/ml (IQR 4.7-10.0), median prostate volume was 54 ml (IQR 39.5-75.0), and median PSA density (PSAD) was 0.13 ng/ml2 (IQR 0.09-0.18). Eighty-six men (95%) had Likert 4 lesions, while the remaining five (5%) had Likert 5 lesions. Only 21 men (23%) had a single lesion; most had at least two. Atrophy was the most prevalent pathology on MRI-TB, present in 64 men (74%), and followed by acute inflammation in 42 (46%), prostatic intraepithelial neoplasia in 33 (36%), chronic inflammation in 18 (20%), atypia in 13 (14%), and granulomatous inflammation in three (3%). Fifty-eight men had a second MRI study (median 376 d, IQR 361-412). At the second MRI, median PSAD decreased to 0.11 ng/ml2 (IQR 0.08-0.18). A Likert 4 or 5 score persisted only in five men (9%); 40 men (69%) were scored Likert 3, while the remaining 13 (22%) were scored Likert 2 (no lesion). Of 45 men with a Likert ≥3 score, most only had one lesion at the second MRI (28 men; 62%). Of six men with repeat MRI-TB during the study period, two were subsequently diagnosed with prostate cancer and both had persistent Likert 4 scores (at baseline and at least one follow-up MRI). CONCLUSIONS: Most biopsy-negative MRI lesions in the prostate resolve over time, but any persistent lesions should be closely monitored. PATIENT SUMMARY: Lesions in the prostate detected via magnetic resonance imaging (MRI) scans that are negative for cancer on biopsy usually resolve. Repeat MRI can indicate persistent lesions that might need a second biopsy.
Assuntos
Próstata , Neoplasias da Próstata , Masculino , Humanos , Idoso , Próstata/diagnóstico por imagem , Próstata/patologia , Antígeno Prostático Específico , Seguimentos , Biópsia/métodos , Imageamento por Ressonância Magnética/métodos , Neoplasias da Próstata/patologia , InflamaçãoRESUMO
Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. These pathogenic E. coli express a syringe-like protein machine, known as the type III secretion system (T3SS), used for the injection of virulence factors into the cytosol of the host epithelial cell. Breaching the epithelial plasma membrane requires formation of a translocation pore that contains the secreted protein EspD. Here we demonstrate that the N-terminal segment of EspD, encompassing residues 1-171, contains two amphipathic domains spanning residues 24-41 and 66-83, with the latter of these helices being critical for EspD function. Fluorescence and circular dichroism analysis revealed that, in solution, His6-EspD1â171 adopts a native disordered structure; however, on binding anionic small unilamellar vesicles composed of phosphatidylserine, His6-EspD1â171 undergoes a pH depended conformational change that increases the α-helix content of this protein approximately sevenfold. This change coincides with insertion of the region circumscribing Trp47 into the hydrophobic core of the lipid bilayer. On the HeLa cell plasma membrane, His6-EspD1â171 forms a homodimer that is postulated to promote EspD-EspD oligomerization and pore formation. Complementation of ΔespD null mutant bacteria with an espDΔ66-83 gene showed that this protein was secreted but non-functional.
Assuntos
Membrana Celular/metabolismo , Escherichia coli Êntero-Hemorrágica/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Proteínas de Escherichia coli/química , Deleção de Genes , Teste de Complementação Genética , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/química , Dados de Sequência Molecular , Fosfatidilserinas/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Alinhamento de Sequência , Análise EspectralRESUMO
Nucleoplasmin (NP) is a pentameric chaperone that regulates the condensation state of chromatin extracting specific basic proteins from sperm chromatin and depositing H2A-H2B histone dimers. It has been proposed that histones could bind to either the lateral or distal face of the pentameric structure. Here, we combine different biochemical and biophysical techniques to show that natural, hyperphosphorylated NP can bind five H2A-H2B dimers and that the amount of bound ligand depends on the overall charge (phosphorylation level) of the chaperone. Three-dimensional reconstruction of NP/H2A-H2B complex carried out by electron microscopy reveals that histones interact with the chaperone distal face. Limited proteolysis and mass spectrometry indicate that the interaction results in protection of the histone fold and most of the H2A and H2B C-terminal tails. This structural information can help to understand the function of NP as a histone chaperone.
Assuntos
Histonas/química , Nucleoplasminas/química , Animais , Dimerização , Espectrometria de Massas/métodos , Microscopia Eletrônica/métodos , Fosforilação , Dobramento de Proteína , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Xenopus laevis/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Large-scale genetic aberrations that underpin prostate cancer development and progression, such as copy-number alterations (CNAs), have been described but the consequences of specific changes in many identified loci is limited. Germline SNPs in the 3q26.31 locus are associated with aggressive prostate cancer, and is the location of NAALADL2, a gene overexpressed in aggressive disease. The closest gene to NAALADL2 is TBL1XR1, which is implicated in tumour development and progression. Using publicly-available cancer genomic data we report that NAALADL2 and TBL1XR1 gains/amplifications are more prevalent in aggressive sub-types of prostate cancer when compared to primary cohorts. In primary disease, gains/amplifications occurred in 15.99% (95% CI: 13.02-18.95) and 14.96% (95% CI: 12.08-17.84%) for NAALADL2 and TBL1XR1 respectively, increasing in frequency in higher Gleason grade and stage tumours. Gains/amplifications result in transcriptional changes and the development of a pro-proliferative and aggressive phenotype. These results support a pivotal role for copy-number gains in this genetic region.
Assuntos
Cromossomos Humanos Par 3/genética , Loci Gênicos , Variação Genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Estudos de Coortes , Variações do Número de Cópias de DNA/genética , Amplificação de Genes , Genoma Humano , Humanos , Masculino , Invasividade Neoplásica , Oncogenes , Fenótipo , Transcrição Gênica , Transcriptoma/genéticaRESUMO
Histone variants play important roles in regulation of chromatin structure and function. To understand the structural role played by histone variants H2A.Z and H3.3, both of which are implicated in transcription regulation, we conducted extensive biochemical and biophysical analysis on mononucleosomes reconstituted from either random-sequence DNA derived from native nucleosomes or a defined DNA nucleosome positioning sequence and recombinant human histones. Using established electrophoretic and sedimentation analysis methods, we compared the properties of nucleosomes containing canonical histones and histone variants H2A.Z and H3.3 (in isolation or in combination). We find only subtle differences in the compaction and stability of the particles. Interestingly, both H2A.Z and H3.3 affect nucleosome positioning, either creating new positions or altering the relative occupancy of the existing nucleosome position space. On the other hand, only H2A.Z-containing nucleosomes exhibit altered linker histone binding. These properties could be physiologically significant as nucleosome positions and linker histone binding partly determine factor binding accessibility.
Assuntos
Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Animais , Fenômenos Bioquímicos , Fenômenos Biofísicos , Galinhas , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Histonas/genética , Humanos , Concentração Osmolar , Ligação Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ouriços-do-Mar , UltracentrifugaçãoRESUMO
NASP has been described as a histone H1 chaperone in mammals. However, the molecular mechanisms involved have not yet been characterized. Here, we show that this protein is not only present in mammals but is widely distributed throughout eukaryotes both in its somatic and testicular forms. The secondary structure of the human somatic version consists mainly of clusters of alpha-helices and exists as a homodimer in solution. The protein binds nonspecifically to core histone H2A-H2B dimers and H3-H4 tetramers but only forms specific complexes with histone H1. The formation of the NASP-H1 complexes is mediated by the N- and C-terminal domains of histone H1 and does not involve the winged helix domain that is characteristic of linker histones. In vitro chromatin reconstitution experiments show that this protein facilitates the incorporation of linker histones onto nucleosome arrays and hence is a bona fide linker histone chaperone.
Assuntos
Autoantígenos/química , Autoantígenos/ultraestrutura , Montagem e Desmontagem da Cromatina , Cromatina/química , Cromatina/ultraestrutura , Histonas/química , Histonas/ultraestrutura , Proteínas Nucleares/química , Proteínas Nucleares/ultraestrutura , Animais , Sítios de Ligação , Simulação por Computador , Reagentes de Ligações Cruzadas/química , Dimerização , Células Eucarióticas , Humanos , Modelos Químicos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/ultraestrutura , Ligação ProteicaRESUMO
Although the details of the structural involvement of histone H1 in the organization of the nucleosome are quite well understood, the sequential events involved in the recognition of its binding site are not as well known. We have used a recombinant human histone H1 (H1.1) in which the N- and C-terminal domains (NTD/CTD) have been swapped and we have reconstituted it on to a 208-bp nucleosome. We have shown that the swapped version of the protein is still able to bind to nucleosomes through its structurally folded wing helix domain (WHD); however, analytical ultracentrifuge analysis demonstrates its ability to properly fold the chromatin fibre is impaired. Furthermore, FRAP analysis shows that the highly dynamic binding association of histone H1 with the chromatin fibre is altered, with a severely decreased half time of residence. All of this suggests that proper binding of histone H1 to chromatin is determined by the simultaneous and synergistic binding of its WHD-CTD to the nucleosome.
Assuntos
Cromatina/metabolismo , Histonas/metabolismo , Sítios de Ligação , Cromatina/ultraestrutura , Dicroísmo Circular , Células HeLa , Histonas/genética , Humanos , Nucleossomos/metabolismo , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
Assuntos
Ultracentrifugação/métodos , Ultracentrifugação/normas , Calibragem , Reprodutibilidade dos TestesRESUMO
UNLABELLED: Our goal in this investigation was to develop a method for iodinating annexin V that would be suitable for the in vivo detection of apoptosis. METHODS: Annexin V was iodinated with (125)I using 2 different techniques: direct iodination with IODO-BEADS, resulting in the iodination of tyrosine residues; and use of the Bolton-Hunter reagent, which binds to lysine. The active fraction of the labeled preparation was purified by affinity chromatography. We assessed thyroid accumulation of free iodide by comparing mice with blocked and unblocked thyroids. We tested the ability of iodinated annexin V to bind apoptotic cells in vitro using irradiated neuroblastoma cells and immobilized phosphatidylserine and in vivo using C3H mice subjected to whole-body irradiation. RESULTS: The efficiency of IODO-BEADS iodination was just below 30%; with the Bolton-Hunter protocol we were able to achieve 40% efficiency. When the IODO-BEADS-labeled preparation was injected into nude mice, activity accumulated rapidly in the thyroid. Two hours after injection, uptake in the thyroid region was clearly visible on a gamma-camera scan. This uptake was absent in mice that had had their thyroids blocked. We concluded that the IODO-BEADS method of labeling resulted in a protein that was rapidly deiodinated in vivo. By contrast, when annexin V was labeled using the Bolton-Hunter protocol, there was no evidence of activity accumulating in the thyroid. The Bolton-Hunter-labeled annexin V bound to apoptotic cells and immobilized phosphatidylserine in vitro. The active fraction of Bolton-Hunter-labeled annexin V was approximately 0.75. In C3H mice given 5-Gy whole-body irradiation, there was a significant induction of apoptosis in the spleen, as measured by the terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay, and a 4-fold increase in (125)I activity in the spleens relative to that of the control animals. CONCLUSION: Direct iodination of annexin V on tyrosine residues is a poor technique suffering from rapid deiodination in vivo. With Bolton-Hunter chemistry, one can produce a molecule that retains its label in vivo and binds to apoptotic cells in vitro and in vivo.
Assuntos
Anexina A5/metabolismo , Apoptose , Radioisótopos do Iodo , Marcação por Isótopo , Succinimidas , Animais , Humanos , Camundongos , Camundongos Endogâmicos C3HRESUMO
UNLABELLED: This trial was performed to determine the maximum tolerated whole-body radiation-absorbed dose of fractionated (131)I-cG250. METHODS: This was a phase 1 dose escalation trial. Dose escalation refers here to the escalation of average whole-body absorbed dose. Fifteen patients with measurable metastatic renal cancer were studied. For each treatment cycle, patients initially received a "scout" administration consisting of 5 mg of cG250 antibody labeled with 185 MBq (5 mCi) of (131)I. Whole-body and serum activity was measured for 1 wk, and a simple pharmacokinetic model was fitted to the measured data. The pharmacokinetic model was used to calculate the required activities, administered in a fractionated pattern with 2-3 d between fractions, projected to deliver the prescribed whole-body absorbed dose. The initial cohort of 3 patients was prescribed an average whole-body absorbed dose of 0.50 Gy. In subsequent cohorts this was increased in 0.25-Gy increments. The first fraction in each cycle was 1,110 MBq (30 mCi) of (131)I conjugated to 5 mg of antibody. Subsequent fractions consisted of variable activities depending on the patient-specific whole-body clearance rates and the times between fractions. Patients without evidence of disease progression were retreated after recovery from toxicity if there was no evidence of altered pharmacokinetics or serum human antichimeric antibody titers, for a total of no more than 3 treatments. RESULTS: For the initial treatment course, the pharmacokinetics of the scout dose accurately predicted the pharmacokinetics of fractionated (131)I-cG250 therapy. In 2 patients, altered clearance accurately predicted development of human antichimeric antibody. Targeting to known disease >or= 2 cm in diameter was noted in all patients. Dose-limiting toxicity was hematopoietic, and the maximum tolerated dose per cycle was 0.75 Gy. CONCLUSION: Measurements of whole-body and serum clearance of cG250 antibody can be used to accurately predict the clearance of subsequent administrations, thus enabling rational treatment planning. An additional practical benefit of real-time pharmacokinetic monitoring is that therapy can be altered dynamically to reduce toxic side effects. However, there was no evidence for fractionation-induced sparing of the hematopoietic system in this study.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Carcinoma de Células Renais/radioterapia , Carcinoma de Células Renais/secundário , Radioisótopos do Iodo/efeitos adversos , Neoplasias Renais/radioterapia , Dose Máxima Tolerável , Lesões por Radiação/etiologia , Adulto , Idoso , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/uso terapêutico , Carga Corporal (Radioterapia) , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/diagnóstico por imagem , Fracionamento da Dose de Radiação , Relação Dose-Resposta à Radiação , Feminino , Humanos , Radioisótopos do Iodo/uso terapêutico , Neoplasias Renais/sangue , Neoplasias Renais/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Lesões por Radiação/prevenção & controle , Radioimunoterapia/efeitos adversos , Radioimunoterapia/métodos , Radiometria/métodos , CintilografiaRESUMO
Antibody-PET imaging might be of value for the selection of radioimmunotherapy (RIT) candidates to confirm tumor targeting and to estimate radiation doses to tumor and normal tissues. One of the requirements to be set for such a scouting procedure is that the biodistributions of the diagnostic and therapeutic radioimmunoconjugates should be similar. In the present study we evaluated the potential of the positron emitters zirconium-89 ((89)Zr) and iodine-124 ((124)I) for this approach, as these radionuclides have a relatively long half-life that matches with the kinetics of MAbs in vivo (t(1/2) 3.27 and 4.18 days, respectively). After radiolabeling of the head and neck squamous cell carcinoma (HNSCC)-selective chimeric antibody (cMAb) U36, the biodistribution of two diagnostic (cMAb U36-N-sucDf-(89)Zr and cMAb U36-(124)I) and three therapeutic radioimmunoconjugates (cMAb U36-p-SCN-Bz-DOTA-(88)Y-with (88)Y being substitute for (90)Y, cMAb U36-(131)I, and cMAb U36-MAG3-(186)Re) was assessed in mice with HNSCC-xenografts, at 24, 48, and 72 hours after injection. Two patterns of biodistribution were observed, one pattern matching for (89)Zr- and (88)Y-labeled cMAb U36 and one pattern matching for (124)I-, (131)I-, and (186)Re-cMAb U36. The most remarkable differences between both patterns were observed for uptake in tumor and liver. Tumor uptake levels were 23.2 +/- 0.5 and 24.1 +/- 0.7%ID/g for the (89)Zr- and (88)Y-cMAb U36 and 16.0 +/- 0.8, 15.7 +/- 0.79 and 17.1 +/- 1.6%ID/g for (124)I-, (131)I-, and (186)Re-cMAb U36-conjugates, respectively, at 72 hours after injection. For liver these values were 6.9 +/- 0.8 ((89)Zr), 6.2 +/- 0.8 ((88)Y), 1.7 +/- 0.1 ((124)I), 1.6 +/- 0.1 ((131)I), and 2.3 +/- 0.1 ((186)Re), respectively. These preliminary data justify the further development of antibody-PET with (89)Zr-labeled MAbs for scouting of therapeutic doses of (90)Y-labeled MAbs. In such approach (124)I-labeled MAbs are most suitable for scouting of (131)I- and (186)Re-labeled MAbs.
Assuntos
Imunoconjugados/farmacocinética , Radioisótopos do Iodo , Radioisótopos , Zircônio , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Linhagem Celular Tumoral , Feminino , Glicoproteínas/imunologia , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Receptores de Hialuronatos/imunologia , Imunoconjugados/química , Marcação por Isótopo , Camundongos , Camundongos Nus , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinética , Rênio , Fatores de Tempo , Distribuição Tecidual , Tomografia Computadorizada de Emissão , Ensaios Antitumorais Modelo de Xenoenxerto , Radioisótopos de ÍtrioRESUMO
UNLABELLED: The aim of this study was to develop a clinically applicable noninvasive method to quantify changes in androgen receptor (AR) levels based on (18)F-16beta-fluoro-5alpha-dihydrotestosterone ((18)F-FDHT) PET in prostate cancer patients undergoing therapy. METHODS: Thirteen patients underwent dynamic (18)F-FDHT PET over a selected tumor. Concurrent venous blood samples were acquired for blood metabolite analysis. A second cohort of 25 patients injected with (18)F-FDHT underwent dynamic PET of the heart. These data were used to generate a population-based input function, essential for pharmacokinetic modeling. Linear compartmental pharmacokinetic models of increasing complexity were tested on the tumor tissue data. Four suitable models were applied and compared using the Bayesian information criterion (BIC). Model 1 consisted of an instantaneously equilibrating space, followed by a unidirectional trap. Models 2a and 2b contained a reversible space between the instantaneously equilibrating space and the trap, into which metabolites were excluded (2a) or allowed (2b). Model 3 built on model 2b with the addition of a second reversible space preceding the unidirectional trap and from which metabolites were excluded. RESULTS: The half-life of the (18)F-FDHT in blood was between 6 and 7 min. As a consequence, the uptake of (18)F-FDHT in prostate cancer lesions reached a plateau within 20 min as the blood-borne activity was consumed. Radiolabeled metabolites were shown not to bind to ARs in in vitro studies with CWR22 cells. Model 1 produced reasonable and robust fits for all datasets and was judged best by the BIC for 16 of 26 tumor scans. Models 2a, 2b, and 3 were judged best in 7, 2, and 1 cases, respectively. CONCLUSION: Our study explores the clinical potential of using (18)F-FDHT PET to estimate free AR concentration. This process involved the estimation of a net uptake parameter such as the k(trap) of model 1 that could serve as a surrogate measure of AR expression in metastatic prostate cancer. Our initial studies suggest that a simple body mass-normalized standardized uptake value correlates reasonably well to model-based k(trap) estimates, which we surmise may be proportional to AR expression. Validation studies to test this hypothesis are underway.
Assuntos
Di-Hidrotestosterona/análogos & derivados , Radioisótopos de Flúor , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/diagnóstico por imagem , Idoso , Estudos de Coortes , Di-Hidrotestosterona/farmacocinética , Radioisótopos de Flúor/farmacocinética , Coração/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Estudos Prospectivos , Neoplasias da Próstata/sangue , Neoplasias da Próstata/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Receptores Androgênicos/metabolismoRESUMO
The genomes of myonecrotic Clostridium perfringens isolates contain genes encoding a large and fascinating array of highly modular glycoside hydrolase enzymes. Although the catalytic activities of many of these enzymes are somewhat predictable based on their amino acid sequences, the functions of their abundant ancillary modules are not and remain poorly studied. Here, we present the structural and functional analysis of a new family of ancillary carbohydrate-binding modules (CBMs), CBM51, which was previously annotated in data bases as the novel putative CBM domain. The high resolution crystal structures of two CBM51 members, GH95CBM51 and GH98CBM51, from a putative family 95 alpha-fucosidase and from a family 98 blood group A/B antigen-specific endo-beta-galactosidase, respectively, showed them to have highly similar beta-sandwich folds. However, GH95CBM51 was shown by glycan microarray screening, isothermal titration calorimetry, and x-ray crystallography to bind galactose residues, whereas the same analyses of GH98CBM51 revealed specificity for the blood group A/B antigens through non-conserved interactions. Overall, this work identifies a new family of CBMs with many members having apparent specificity for eukaryotic glycans, in keeping with the glycan-rich environment C. perfringens would experience in its host. However, a wider bioinformatic analysis of this CBM family also indicated a large number of members in non-pathogenic environmental bacteria, suggesting a role in the recognition of environmental glycans.
Assuntos
Metabolismo dos Carboidratos , Clostridium perfringens/enzimologia , Glicosídeo Hidrolases/química , Sequência de Aminoácidos , Sítios de Ligação , Antígenos de Grupos Sanguíneos/química , Cálcio/metabolismo , Cristalografia por Raios X , Galactose/metabolismo , Variação Genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de ProteínaRESUMO
The import of PTS1 proteins into the glycosome or peroxisome requires binding of a PTS1-laden PEX5 receptor to the membrane-associated protein PEX14 to facilitate translocation of PTS1 proteins into the lumen of these organelles. Quaternary structure analysis of protozoan parasite Leishmania donovani PEX14 (LdPEX14) revealed that this protein forms a homomeric complex with a size > 670 kDa. Moreover, deletion mapping indicated that disruption of LdPEX14 oligomerization correlated with the elimination of the hydrophobic region and coiled-coil motif present in LdPEX14. Analysis of the LdPEX5-LdPEX14 interaction by isothermal titration calorimetry revealed a molar binding stoichiometry of 1:4 (LdPEX5: LdPEX14) and an in-solution dissociation constant (K(d)) of approximately 74 nm. Calorimetry, circular dichroism, intrinsic fluorescence, and analytical ultracentrifugation experiments showed that binding of LdPEX5 resulted in a dramatic conformational change in the LdPEX14 oligomeric complex that involved the reorganization of the hydrophobic segment in LdPEX14. Finally, limited tryptic proteolysis assays established that in the presence of LdPEX5, LdPEX14 became more susceptible to proteolytic degradation consistent with this protein interaction triggering a significant conformational change in the recombinant and native LdPEX14 structures. These structural changes provide essential clues to how LdPEX14 functions in the translocation of folded proteins across the glycosomal membrane.
Assuntos
Leishmania donovani/química , Leishmania donovani/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Calorimetria , Dicroísmo Circular , Leishmania donovani/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , TermodinâmicaRESUMO
Anthracycline antibiotics are an important group of antitumor drugs widely used in cancer chemotherapy. However, despite the increasing interest in these chemotherapeutic agents, their mechanism of action is not yet completely understood. Here, we review what is currently known about the molecular mechanisms involved with special emphasis on the interaction of these drugs with chromatin and its constitutive components: DNA and histones. The evidence suggests that one very important component of the activity of these drugs is the result of these manifold interactions that lead to a chromatin unfolding and aggregation. This chromatin structural disruption is likely to interfere with the metabolic processes of DNA (replication and transcription) and it may play an important role in the apoptosis undergone by the cells upon treatment with these drugs.
Assuntos
Antraciclinas/farmacologia , Antineoplásicos/farmacologia , Cromatina/química , Animais , Apoptose , Cromatina/metabolismo , DNA/química , DNA/metabolismo , Histonas/metabolismo , Humanos , Modelos Biológicos , Neoplasias/tratamento farmacológico , Ligação Proteica , Conformação Proteica , Dobramento de ProteínaRESUMO
We have previously characterized the interaction of nucleoplasmin with core histones and studied the possible involvement of this chaperone molecule in transcription. Here we study the interaction of nucleoplasmin with chromatin. We show that highly phosphorylated Xenopus laevis egg nucleoplasmin can unfold sperm and somatic chromatin in a way that involves the removal of chromosomal proteins from linker DNA regions without a stable interaction with the nucleosome. The complexes between egg nucleoplasmin and both somatic and sperm-specific linker proteins have been hydrodynamically characterized using sedimentation equilibrium in the analytical ultracentrifuge. The results are discussed within the context of the possible implication of nucleoplasmin in processes such as transcription and replication licensing which take place after egg fertilization at the onset of development.