RESUMO
Most antibody-drug conjugates (ADC) approved for the treatment of cancer contain protease-cleavable linkers. ADCs that traffic to lysosomes traverse highly acidic late endosomes, while ADCs that recycle to the plasma membrane traffic through mildly acidic sorting and recycling endosomes. Although endosomes have been proposed to process cleavable ADCs, the precise identity of the relevant compartments and their relative contributions to ADC processing remain undefined. Here we show that a METxMET biparatopic antibody internalizes into sorting endosomes, rapidly traffics to recycling endosomes, and slowly reaches late endosomes. In agreement with the current model of ADC trafficking, late endosomes are the primary processing site of MET, EGFR, and prolactin receptor ADCs. Interestingly, recycling endosomes contribute up to 35% processing of the MET and EGFR ADCs in different cancer cells, mediated by cathepsin-L, which localizes to this compartment. Taken together, our findings provide insight into the relationship between transendosomal trafficking and ADC processing and suggest that receptors that traffic through recycling endosomes might be suitable targets for cleavable ADCs.
Assuntos
Vacinas Anticâncer , Imunoconjugados , Humanos , Imunoconjugados/farmacologia , Anticorpos , Endossomos , Receptores ErbBRESUMO
Protein aggregation is linked to many chronic and devastating neurodegenerative human diseases and is strongly associated with aging. This work demonstrates that protein aggregation and oligomerization can be evaluated by a solid-state nanopore method at the single molecule level. A silicon nitride nanopore sensor was used to characterize both the amyloidogenic and native-state oligomerization of a model protein ß-lactoglobulin variant A (ßLGa). The findings from the nanopore measurements are validated against atomic force microscopy (AFM) and dynamic light scattering (DLS) data, comparing ßLGa aggregation from the same samples at various stages. By calibrating with linear and circular dsDNA, this study estimates the amyloid fibrils' length and diameter, the quantity of the ßLGa aggregates, and their distribution. The nanopore results align with the DLS and AFM data and offer additional insight at the level of individual protein molecular assemblies. As a further demonstration of the nanopore technique, ßLGa self-association and aggregation at pH 4.6 as a function of temperature were measured at high (2 M KCl) and low (0.1 M KCl) ionic strength. This research highlights the advantages and limitations of using solid-state nanopore methods for analyzing protein aggregation.
Assuntos
Nanoporos , Humanos , Agregados Proteicos , Envelhecimento , Difusão Dinâmica da Luz , LactoglobulinasRESUMO
Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Animais , Linfócitos T CD8-Positivos , Citocinas/uso terapêutico , Humanos , Linfócitos do Interstício Tumoral , Macaca fascicularis , Camundongos , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos , Microambiente Tumoral , ZircônioRESUMO
T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.
Assuntos
Anticorpos Biespecíficos , Complexo CD3 , Citocinas , Citocinas/metabolismo , Ativação Linfocitária , Distribuição TecidualRESUMO
Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%-5% of lung cancers), and acquired resistance limits response durability. An antibody-drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.
Assuntos
Anticorpos Monoclonais/química , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Imunoconjugados/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Feminino , Humanos , Imunoconjugados/farmacocinética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macaca fascicularis , Masculino , Camundongos , Camundongos SCID , Mutação , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) blocking antibodies including cemiplimab have generated profound clinical activity across diverse cancer types. Tumorous PD-L1 expression, as assessed by immunohistochemistry (IHC), is an accepted predictive marker of response to therapy in some cancers. However, expression is often dynamic and heterogeneous, and therefore not reliably captured by IHC from tumor biopsies or archival samples. Thus, there is significant need for accurate whole-body quantification of PD-L1 levels. METHODS: We radiolabeled the novel human anti-PD-L1 antibody REGN3504 with zirconium-89 (89Zr) using the chelator p-SCN-Bn-Deferoxamine to enable non-invasive immuno-positron emission tomography (immuno-PET) of PD-L1 expression. PET imaging assessed the localization of 89Zr-REGN3504 to multiple human tumor xenografts. Mice genetically humanized for PD-1 and PD-L1 were used to assess the biodistribution of 89Zr-REGN3504 to normal tissues and the estimated human radiation dosimetry of 89Zr-REGN3504 was also determined. Pharmacokinetics of REGN3504 was assessed in monkeys. RESULTS: Clear localization of 89Zr-REGN3504 to human tumor xenografts was observed via PET imaging and ex vivo biodistribution studies demonstrated high (fourfold to sixfold) tumor:blood ratios. 89Zr-REGN3504 specifically localized to spleen and lymph nodes in the PD-1/PD-L1 humanized mice. 89Zr-REGN3504 immuno-PET accurately detected a significant reduction in splenic PD-L1 positive cells following systemic treatment with clodronate liposomes. Radiation dosimetry suggested absorbed doses would be within guidelines for other 89Zr radiolabeled, clinically used antibodies. Pharmacokinetics of REGN3504 was linear. CONCLUSION: This work supports the clinical translation of 89Zr-REGN3504 immuno-PET for the assessment of PD-L1 expression. Future clinical studies will aim to investigate the utility of 89Zr-REGN3504 immuno-PET for predicting and monitoring response to anti-PD-1 therapy.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígeno B7-H1/metabolismo , Neoplasias/diagnóstico por imagem , Radioisótopos/química , Zircônio/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Estudos de Casos e Controles , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Haplorrinos , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Neoplasias/imunologia , Tomografia por Emissão de Pósitrons , Distribuição TecidualRESUMO
Patients with hematologic cancers have improved outcomes after treatment with bispecific antibodies that bind to CD3 on T cells and that redirect T cells toward cancer cells. However, clinical benefit against solid tumors remains to be shown. We made a bispecific antibody that targets both the common prostate tumor-specific antigen PSMA and CD3 (PMSAxCD3) and provide evidence for tumor inhibition in several preclinical solid tumor models. Mice expressing the human extracellular regions of CD3 and PSMA were generated to examine antitumor efficacy in the presence of an intact immune system and PSMA expression in normal tissues. PSMAxCD3 accumulated in PSMA-expressing tissues and tumors as detected by immuno-PET imaging. Although PSMAxCD3 induced T-cell activation and showed antitumor efficacy in mice with low tumor burden, PSMAxCD3 lost efficacy against larger solid tumors, mirroring the difficulty of treating solid tumors in the clinic. Costimulatory receptors can enhance T-cell responses. We show here that costimulation can enhance the antitumor efficacy of PSMAxCD3. In particular, 4-1BB stimulation in combination with PSMAxCD3 enhanced T-cell activation and proliferation, boosted efficacy against larger tumors, and induced T-cell memory, leading to durable antitumor responses. The combination of CD3 bispecific antibodies and anti-4-1BB costimulation represents a therapeutic approach for the treatment of solid tumors.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais/farmacologia , Complexo CD3/imunologia , Glutamato Carboxipeptidase II/antagonistas & inibidores , Neoplasias da Próstata/tratamento farmacológico , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Biespecíficos/imunologia , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Complexo CD3/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glutamato Carboxipeptidase II/imunologia , Glutamato Carboxipeptidase II/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Tecidual , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Zircônio/farmacocinéticaRESUMO
Antibodies labeled with positron-emitting isotopes have been used for tumor detection, predicting which patients may respond to tumor antigen-directed therapy, and assessing pharmacodynamic effects of drug interventions. Prolactin receptor (PRLR) is overexpressed in breast and prostate cancers and is a new target for cancer therapy. We evaluated REGN2878, an anti-PRLR monoclonal antibody, as an immunoPET reagent. REGN2878 was labeled with Zr-89 after conjugation with desferrioxamine B or labeled with I-131/I-124. In vitro determination of the half-maximal inhibitory concentration (IC50) of parental REGN2878, DFO-REGN2878, and iodinated REGN2878 was performed by examining the effect of the increasing amounts of these on uptake of trace-labeled I-131 REGN2878. REGN1932, a non-PRLR binding antibody, was used as a control. Imaging and biodistribution studies were performed in mice bearing tumor xenografts with various expression levels of PRLR, including MCF-7, transfected MCF-7/PRLR, PC3, and transfected PC3/PRLR and T4D7v11 cell lines. The specificity of uptake in tumors was evaluated by comparing Zr-89 REGN2878 and REGN1932, and in vivo competition compared Zr-89 REGN2878 uptake in tumor xenografts with and without prior injection of 2 mg of nonradioactive REGN2878. The competition binding assay of DFO-REGN2878 at ratios of 3.53-5.77 DFO per antibody showed IC50 values of 0.4917 and 0.7136 nM, respectively, compared to 0.3455 nM for parental REGN2878 and 0.3343 nM for I-124 REGN2878. Imaging and biodistribution studies showed excellent targeting of Zr-89 REGN2878 in PRLR-positive xenografts at delayed times of 189 h (presented as mean ± 1 SD, percent injected activity per mL (%IA/mL) 74.6 ± 33.8%IA/mL). In contrast, MCF-7/PRLR tumor xenografts showed a low uptake (7.0 ± 2.3%IA/mL) of control Zr-89 REGN1932 and a very low uptake and rapid clearance of I-124 REGN2878 (1.4 ± 0.6%IA/mL). Zr-89 REGN2878 has excellent antigen-specific targeting in various PRLR tumor xenograft models. We estimated, using image-based kinetic modeling, that PRLR antigen has a very rapid in vivo turnover half-life of â¼14 min from the cell membrane. Despite relatively modest estimated tumor PRLR expression numbers, PRLR-expressing cells have shown final retention of the Zr-89 REGN2878 antibody, with an uptake that appeared to be related to PRLR expression. This reagent has the potential to be used in clinical trials targeting PRLR.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Imunoconjugados/administração & dosagem , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/química , Imunoconjugados/imunologia , Imunoconjugados/farmacocinética , Camundongos , Camundongos Nus , Imagem Molecular/métodos , Neoplasias/patologia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/imunologia , Compostos Radiofarmacêuticos/farmacocinética , Receptores da Prolactina/imunologia , Receptores da Prolactina/metabolismo , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.
Assuntos
Antineoplásicos/farmacologia , Imunotoxinas/farmacologia , Maitansina/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Receptores ErbB/imunologia , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunotoxinas/química , Imunotoxinas/imunologia , Cinética , Masculino , Maitansina/síntese química , Maitansina/química , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed in a subset of breast cancers and may contribute to its pathogenesis. It is relatively overexpressed in approximately 25% of human breast tumors while expressed at low levels in some normal human tissues including the mammary gland. We developed an anti-PRLR antibody-drug conjugate (ADC), to target PRLR-positive breast cancer. REGN2878-DM1 is comprised of a fully human high-affinity function-blocking anti-PRLR IgG1 antibody (REGN2878) conjugated via a noncleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated REGN2878 and conjugated REGN2878-DM1 block PRL-mediated activation in vitro and are rapidly internalized into lysosomes. REGN2878-DM1 induces potent cell-cycle arrest and cytotoxicity in PRLR-expressing tumor cell lines. In vivo, REGN2878-DM1 demonstrated significant antigen-specific antitumor activity against breast cancer xenograft models. In addition, REGN2878-DM1 showed additive activity when combined with the antiestrogen agent fulvestrant. These results illustrate promising antitumor activity against PRLR-positive breast cancer xenografts and support the evaluation of anti-PRLR ADCs as potential therapeutic agents in breast cancer. Mol Cancer Ther; 16(7); 1299-311. ©2017 AACR.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/administração & dosagem , Receptores da Prolactina/imunologia , Animais , Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Imunoconjugados/imunologia , Camundongos , Receptores da Prolactina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Most in vitro investigations of alpha-Synuclein (alphaSyn) aggregation and amyloidogenesis use agitation in the presence of air and/or Teflon to accelerate kinetics. The effect of the agitation is implicitly or explicitly attributed to mass transfer or fibril fragmentation. This paper evaluates these hypotheses by agitating alphaSyn under typical amyloidogenic conditions with controlled numbers of balls made of polytetrafluoroethylene (PTFE), polymethylmethacrylate (PMMA), and borosilicate glass with no headspace. Amyloid was assayed using thioflavin T fluorescence and atomic force microscopy. The observed kinetics were proportional to the PTFE surface area; the effects of PMMA and glass balls were negligible by comparison. No amyloid was observed to form in the absence of mixing balls. Agitation with only air also showed accelerated kinetics but different aggregate morphology. The results indicate that the mechanism active in agitation experiments is dominated by reactions at the hydrophobic-water interface. Of the mass transfer, fragmentation, and hydrophobic interface hypotheses, only the last is capable of explaining the data. Condition and sequence determinants of amyloidogenic propensity that have thus far been reported must be reinterpreted as being reflective of partitioning to hydrophobic-water interfaces. Comparable hydrophobic interfaces are not found in vivo.
Assuntos
Amiloide/biossíntese , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Técnicas In Vitro , Microscopia de Força Atômica , Polímeros/metabolismoRESUMO
We combine atomic-force-microscopy particle-size-distribution measurements with earlier measurements on 1-anilino-8-naphthalene sulfonate, thioflavin T, and dynamic light scattering to develop a quantitative kinetic model for the aggregation of beta-lactoglobulin into amyloid. We directly compare our simulations to the population distributions provided by dynamic light scattering and atomic force microscopy. We combine species in the simulation according to structural type for comparison with fluorescence fingerprint results. The kinetic model of amyloidogenesis leads to an aggregation free-energy landscape. We define the roles of and propose a classification scheme for different oligomeric species based on their location in the aggregation free-energy landscape. We relate the different types of oligomers to the amyloid cascade hypothesis and the toxic oligomer hypothesis for amyloid-related diseases. We discuss existing kinetic mechanisms in terms of the different types of oligomers. We provide a possible resolution to the toxic oligomer-amyloid coincidence.
Assuntos
Amiloide/química , Amiloide/ultraestrutura , Naftalenossulfonato de Anilina/química , Animais , Bovinos , Simulação por Computador , Cinética , Lactoglobulinas/química , Luz , Microscopia de Força Atômica , Modelos Químicos , Estrutura Quaternária de Proteína , Reprodutibilidade dos Testes , Espalhamento de Radiação , Termodinâmica , Fatores de TempoRESUMO
We use time-dependent fluorescence Stokes shift (TDFSS) information to study the fluctuation rates of the lipocalin, beta-lactoglobulin A in the vicinity of an encapsulated coumarin 153 molecule. The system has three unique dielectric environments in which the fluorophore binds. We develop a method to decompose the static and dynamic contributions to the spectral heterogeneity. This method is applied to temperature-dependent steady-state fluorescence spectra providing us with site-specific information about thermodynamic transitions in beta-lactoglobulin. We confirm previously reported transitions and discuss the presence of an unreported transition of the central calyx at 18 degrees C. Our method also resolves the contributions to the TDFSS from the coumarin 153 centrally located in the calyx of beta-lactoglobulin despite overlapping signals from solvent exposed dyes. Our experiments show dynamics ranging from 3-12,00 ps. The analysis shows a decrease in the encapsulated dye's heterogeneity during the relaxation, which is taken as evidence of the breakdown of linear response.
Assuntos
Cumarínicos/química , Lactoglobulinas/química , Animais , Bovinos , Fluorescência , Conformação ProteicaRESUMO
We have investigated the aggregation and amyloid fibril formation of bovine beta-lactoglobulin variant A, with a focus on the early stages of aggregation. We used noncovalent labeling with thioflavin T and 1-anilino-8-naphthalenesulfonate to follow the conformational changes occurring in beta-lactoglobulin during aggregation using time resolved luminescence. 1-Anilino-8-naphthalenesulfonate monitored the involvement of the hydrophobic core/calyx of beta-lactoglobulin in the aggregation process. Thioflavin T luminescence monitored the formation of amyloid. The luminescence lifetime distributions of both probes showed changes that could be attributed to conformational changes occurring during and following aggregation. To correlate the luminescence measurements with the degree of aggregation and the morphology of the aggregates, we also measured dynamic light scattering and atomic force microscopy images. We evaluated the relative stability of the intermediates with an assay that is sensitive to aggregation reversibility. Our results suggest that initial aggregation during the first 5 days occurred with partial disruption of the characteristic calyx in beta-lactoglobulin. As the globular aggregates grew from days 5 to 16, the calyx was completely disrupted and the globular aggregates became more stable. After this second phase of aggregation, conversion into a fibrillar form occurred, marking the growth phase, and still more changes in the luminescence signals were observed. Based on these observations, we propose a three-step process by which monomer is converted first into weakly associated aggregates, which rearrange into stable aggregates, which eventually convert into protofibrils that elongate in the growth phase.
Assuntos
Amiloide/metabolismo , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Amiloide/química , Amiloide/ultraestrutura , Naftalenossulfonato de Anilina/metabolismo , Animais , Benzotiazóis , Sítios de Ligação , Bovinos , Fluorescência , Interações Hidrofóbicas e Hidrofílicas , Lactoglobulinas/ultraestrutura , Luz , Microscopia de Força Atômica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Espalhamento de Radiação , Tiazóis/metabolismoRESUMO
We introduce a new approach to global data fitting based on a regularization condition that invokes continuity in the global data coordinate. Stabilization of the data fitting procedure comes from probabilistic constraint of the global solution to physically reasonable behavior rather than to specific models of the system behavior. This method is applicable to the fitting of many types of spectroscopic data including dynamic light scattering, time-correlated single-photon counting (TCSPC), and circular dichroism. We compare our method to traditional approaches to fitting an inverse Laplace transform by examining the evolution of multiple lifetime components in synthetic TCSPC data. The global regularizer recovers features in the data that are not apparent from traditional fitting. We show how our approach allows one to start from an essentially model-free fit and progress to a specific model by moving from probabilistic to deterministic constraints in both Laplace transformed and nontransformed coordinates.
Assuntos
Algoritmos , Medições Luminescentes/métodos , Modelos Estatísticos , Fótons , Simulação por Computador , Entropia , Modelos Biológicos , Análise Espectral , Fatores de TempoRESUMO
The interpretation of single-molecule measurements is greatly complicated by the presence of multiple fluorescent labels. However, many molecular systems of interest consist of multiple interacting components. We investigate this issue using multiply labeled dextran polymers that we intentionally photobleach to the background on a single-molecule basis. Hidden Markov models allow for unsupervised analysis of the data to determine the number of fluorescent subunits involved in the fluorescence intermittency of the 6-carboxy-tetramethylrhodamine labels by counting the discrete steps in fluorescence intensity. The Bayes information criterion allows us to distinguish between hidden Markov models that differ by the number of states, that is, the number of fluorescent molecules. We determine information-theoretical limits and show via Monte Carlo simulations that the hidden Markov model analysis approaches these theoretical limits. This technique has resolving power of one fluorescing unit up to as many as 30 fluorescent dyes with the appropriate choice of dye and adequate detection capability. We discuss the general utility of this method for determining aggregation-state distributions as could appear in many biologically important systems and its adaptability to general photometric experiments.