RESUMEN
The use of multi-attribute method (MAM) for identity and purity testing of biopharmaceuticals offers the ability to complement and replace multiple conventional analytical technologies with a single mass spectrometry (MS) method. Phase-appropriate method validation is one major consideration for the implementation of MAM in a current Good Manufacturing Practice (cGMP) environment. We developed a MAM workflow for therapeutic monoclonal antibodies (mAbs) with optimized sample preparation using lysyl endopeptidase (Lys-C) digestion. In this study, we evaluated the assay performances of this platform MAM workflow for identity, product quality attributes (PQAs) monitoring and new peak detection (NPD) for single and coformulated mAbs. An IgG4 mAb-1 and its coformulations were used as model molecules in this study. The assay performance evaluation demonstrated the full potential of the platform MAM approach for its intended use for characterization and quality control of single mAb-1 and mAb-1 in its coformulations. To the best of our knowledge, this is the first performance evaluation of MAM for mAb identity, PQA monitoring, and new peak detection (NPD) in a single assay, featuring 1) the first performance evaluation of MAM for PQA monitoring using Lys-C digestion with a high-resolution MS, 2) a new approach for mAb identity testing capable of distinguishing single mAb from coformulations using MAM, and 3) the performance evaluation of NPD for MAM with Lys-C digestion. The developed platform MAM workflow and the MAM performance evaluation paved the way for its GMP qualification and enabled clinical release of mAb-1 in GMP environment with MAM.
Asunto(s)
Anticuerpos Monoclonales , Productos Biológicos , Anticuerpos Monoclonales/química , Espectrometría de Masas/métodos , Control de Calidad , DigestiónRESUMEN
Linear ubiquitin chains are implicated in the regulation of the NF-κB pathway, immunity, and inflammation. They are synthesized by the LUBAC complex containing the catalytic subunit HOIL-1-interacting protein (HOIP) and are disassembled by the linear ubiquitin-specific deubiquitinase OTULIN. Little is known about the regulation of these opposing activities. Here we demonstrate that HOIP and OTULIN interact and act as a bimolecular editing pair for linear ubiquitin signals in vivo. The HOIP PUB domain binds to the PUB interacting motif (PIM) of OTULIN and the chaperone VCP/p97. Structural studies revealed the basis of high-affinity interaction with the OTULIN PIM. The conserved Tyr56 of OTULIN makes critical contacts with the HOIP PUB domain, and its phosphorylation negatively regulates this interaction. Functionally, HOIP binding to OTULIN is required for the recruitment of OTULIN to the TNF receptor complex and to counteract HOIP-dependent activation of the NF-κB pathway.
Asunto(s)
Endopeptidasas/química , FN-kappa B/metabolismo , Ubiquitina-Proteína Ligasas/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Endopeptidasas/metabolismo , Células HeLa , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Termodinámica , Ubiquitina-Proteína Ligasas/metabolismo , Proteína que Contiene ValosinaRESUMEN
Cullin-RING ubiquitin ligase complexes (CRLs) rely on a vast array of adaptor proteins to recognize their substrates. Pierce et al. and related papers from Zemla et al. and Wu et al. in Nature Communications show that Cand1 promotes exchange of adaptor proteins to regulate the CRL repertoire.
RESUMEN
Cullin 4 (Cul4)-based ubiquitin ligases emerged as critical regulators of DNA replication and repair. Over 50 Cul4-specific adaptors (DNA damage-binding 1 (Ddb1)-Cul4-associated factors; DCAFs) have been identified and are thought to assemble functionally distinct Cul4 complexes. Using a live-cell imaging-based RNAi screen, we analysed the function of DCAFs and Cul4-linked proteins, and identified specific subsets required for progression through G1 and S phase. We discovered C6orf167/Mms22-like protein (Mms22L) as a putative human orthologue of budding yeast Mms22, which, together with cullin Rtt101, regulates genome stability by promoting DNA replication through natural pause sites and damaged templates. Loss of Mms22L function in human cells results in S phase-dependent genomic instability characterised by spontaneous double-strand breaks and DNA damage checkpoint activation. Unlike yeast Mms22, human Mms22L does not stably bind to Cul4, but is degraded in a Cul4-dependent manner and upon replication stress. Mms22L physically and functionally interacts with the scaffold-like protein Nfkbil2 that co-purifies with histones, several chromatin remodelling and DNA replication/repair factors. Together, our results strongly suggest that the Mms22L-Nfkbil2 complex contributes to genome stability by regulating the chromatin state at stalled replication forks.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/antagonistas & inhibidores , Técnicas de Silenciamiento del Gen/métodos , Inestabilidad Genómica , Humanos , Tamizaje Masivo/métodos , Proteínas Nucleares/antagonistas & inhibidores , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Fluorescence time-lapse imaging has become a powerful tool to investigate complex dynamic processes such as cell division or intracellular trafficking. Automated microscopes generate time-resolved imaging data at high throughput, yet tools for quantification of large-scale movie data are largely missing. Here we present CellCognition, a computational framework to annotate complex cellular dynamics. We developed a machine-learning method that combines state-of-the-art classification with hidden Markov modeling for annotation of the progression through morphologically distinct biological states. Incorporation of time information into the annotation scheme was essential to suppress classification noise at state transitions and confusion between different functional states with similar morphology. We demonstrate generic applicability in different assays and perturbation conditions, including a candidate-based RNA interference screen for regulators of mitotic exit in human cells. CellCognition is published as open source software, enabling live-cell imaging-based screening with assays that directly score cellular dynamics.
Asunto(s)
Células , Biología Computacional/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagen Molecular/métodos , Fenotipo , Programas Informáticos , Inteligencia Artificial , Automatización , Forma de la Célula , Supervivencia Celular , Células/citología , Simulación por Computador , Fluorescencia , Células HeLa , Humanos , Cinética , Cadenas de Markov , Mitosis , Factores de TiempoRESUMEN
Cul3 (Cullin3)-based E3 ubiquitin ligases recently emerged as critical regulators of mitosis. In this study, we identify two mammalian BTB (Bric-a-brac-Tramtrack-Broad complex)-Kelch proteins, KLHL21 and KLHL22, that interact with Cul3 and are required for efficient chromosome alignment. Interestingly, KLHL21 but not KLHL22 is necessary for cytokinesis and regulates translocation of the chromosomal passenger complex (CPC) from chromosomes to the spindle midzone in anaphase, similar to the previously described BTB-Kelch proteins KLHL9 and KLHL13. KLHL21 directly binds to aurora B and mediates ubiquitination of aurora B in vitro. In contrast to KLHL9 and KLHL13, KLHL21 localizes to midzone microtubules in anaphase and recruits aurora B and Cul3 to this region. Together, our results suggest that different Cul3 adaptors nonredundantly regulate aurora B during mitosis, possibly by ubiquitinating different pools of aurora B at distinct subcellular localizations.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anafase , Segregación Cromosómica , Proteínas Cullin/metabolismo , Citocinesis , Microtúbulos/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Anafase/genética , Aurora Quinasa B , Aurora Quinasas , Proteínas Cullin/genética , Citocinesis/genética , Células HeLa , Humanos , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Transfección , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Faithful cell-cycle progression is tightly controlled by the ubiquitin-proteasome system. Here we identify a human Cullin 3-based E3 ligase (Cul3) which is essential for mitotic division. In a complex with the substrate-specific adaptors KLHL9 and KLHL13, Cul3 is required for correct chromosome alignment in metaphase, proper midzone and midbody formation, and completion of cytokinesis. This Cul3-based E3 ligase removes components of the chromosomal passenger complex from mitotic chromosomes and allows their accumulation on the central spindle during anaphase. Aurora B directly binds to the substrate-recognition domain of KLHL9 and KLHL13 in vitro, and coimmunoprecipitates with the Cul3 complex during mitosis. Moreover, Aurora B is ubiquitylated in a Cul3-dependent manner in vivo, and by reconstituted Cul3/KLHL9/KLHL13 ligase in vitro. We thus propose that the Cul3/KLHL9/KLHL13 E3 ligase controls the dynamic behavior of Aurora B on mitotic chromosomes, and thereby coordinates faithful mitotic progression and completion of cytokinesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromosomas Humanos , Proteínas Cullin/metabolismo , Citocinesis , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Aurora Quinasa B , Aurora Quinasas , Proteínas de Ciclo Celular/genética , Proteínas Cullin/genética , Células HeLa , Humanos , Proteínas Serina-Treonina Quinasas/genética , Huso Acromático , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
We have previously reported that the DsbA signal sequence promotes efficient, cotranslational translocation of the cytoplasmic protein thioredoxin-1 via the bacterial signal recognition particle (SRP) pathway. However, two commonly used signal sequences, those of PhoA and MalE, which promote export by a posttranslational mechanism, do not export thioredoxin. We proposed that this difference in efficiency of export was due to the rapid folding of thioredoxin in the cytoplasm; cotranslational export by the DsbA signal sequence avoids the problem of cytoplasmic folding (C. F. Schierle, M. Berkmen, D. Huber, C. Kumamoto, D. Boyd, and J. Beckwith, J. Bacteriol. 185:5706-5713, 2003). Here, we use thioredoxin as a reporter to distinguish SRP-dependent from non-SRP-dependent cleavable signal sequences. We screened signal sequences exhibiting a range of hydrophobicity values based on a method that estimates hydrophobicity. Successive iterations of screening and refining the method defined a threshold hydrophobicity required for SRP recognition. While all of the SRP-dependent signal sequences identified were above this threshold, there were also a few signal sequences above the threshold that did not utilize the SRP pathway. These results suggest that a simple measure of the hydrophobicity of a signal sequence is an important but not a sufficient indicator for SRP recognition. In addition, by fusing a number of both classes of signal sequences to DsbA, we found that DsbA utilizes an SRP-dependent signal sequence to achieve efficient export to the periplasm. Our results suggest that those proteins found to be exported by SRP-dependent signal sequences may require this mode of export because of their tendency to fold rapidly in the cytoplasm.