RESUMO
Improving potencies through concomitant blockage of multiple epitopes and avid binding by fusing multiple (different) monovalent Nanobody building blocks via linker sequences into one multivalent polypeptide chain is an elegant alternative to affinity maturation. We explored a large and random formatting library of bivalent (combinations of two identical) and biparatopic (combinations of two different) Nanobodies for functional blockade of Pseudomonas aeruginosa PcrV. PcrV is an essential part of the P. aeruginosa type III secretion system (T3SS), and its oligomeric nature allows for multiple complex binding and blocking options. The library screening yielded a large number of promising biparatopic lead candidates, revealing significant (and non-trivial) preferences in terms of Nanobody building block and epitope bin combinations and orientations. Excellent potencies were confirmed upon further characterization in two different P. aeruginosa T3SS-mediated cytotoxicity assays. Three biparatopic Nanobodies were evaluated in a lethal mouse P. aeruginosa challenge pneumonia model, conferring 100% survival upon prophylactic administration and reducing lung P. aeruginosa burden by up to 2 logs. At very low doses, they protected the mice from P. aeruginosa infection-related changes in lung histology, myeloperoxidase production, and lung weight. Importantly, the most potent Nanobody still conferred protection after therapeutic administration up to 24 h post-infection. The concept of screening such formatting libraries for potency improvement is applicable to other targets and biological therapeutic platforms.
Assuntos
Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Técnicas de Química Combinatória/métodos , Ensaios de Triagem em Larga Escala/métodos , Proteínas Citotóxicas Formadoras de Poros/imunologia , Anticorpos de Domínio Único/imunologia , Potência de Vacina , Animais , Morte Celular , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Feminino , Humanos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Pneumonia/imunologia , Pneumonia/microbiologia , Pneumonia/patologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologiaRESUMO
The precise mechanism of chromosome condensation and decondensation remains a mystery, despite progress over the last 20 years aimed at identifying components essential to the mitotic compaction of the genome. In this study, we analyse the localization and role of the CAP-D2 non-SMC condensin subunit and its effect on the stability of the condensin complex. We demonstrate that a condensin complex exists in Drosophila embryos, containing CAP-D2, the anticipated SMC2 and SMC4 proteins, the CAP-H/Barren and CAP-G (non-SMC) subunits. We show that CAP-D2 is a nuclear protein throughout interphase, increasing in level during S phase, present on chromosome axes in mitosis, and still present on chromosomes as they start to decondense late in mitosis. We analysed the consequences of CAP-D2 loss after dsRNA-mediated interference, and discovered that the protein is essential for chromosome arm and centromere resolution. The loss of CAP-D2 after RNAi has additional downstream consequences on the stability of CAP-H, the localization of DNA topoisomerase II and other condensin subunits, and chromosome segregation. Finally, we discovered that even after interfering with two components important for chromosome architecture (DNA topoisomerase II and condensin), chromosomes were still able to compact, paving the way for the identification of further components or activities required for this essential process.
Assuntos
Adenosina Trifosfatases/metabolismo , Centrômero/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Linhagem Celular , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Complexos Multiproteicos/genética , Interferência de RNARESUMO
Chromatin states have to be faithfully duplicated during DNA replication to maintain cell identity. It is unclear whether or how ATP-dependent chromatin-remodelling factors are involved in this process. Here we provide evidence that the Williams syndrome transcription factor (WSTF) is targeted to replication foci through direct interaction with the DNA clamp PCNA, an important coordinator of DNA and chromatin replication. WSTF, in turn, recruits imitation switch (ISWI)-type nucleosome-remodelling factor SNF2H to replication sites. These findings reveal a novel recruitment mechanism for ATP-dependent chromatin-remodelling factors that is fundamentally different from the previously documented targeting by sequence-specific transcriptional regulators. RNA-interference-mediated depletion of WSTF or SNF2H causes a compaction of newly replicated chromatin and increases the amount of heterochromatin markers, including HP1beta. This increase in the amount of HP1beta protein is mediated by progression through S phase and is not the result of an increase in HP1beta mRNA levels. We propose that the WSTF-ISWI complex has a role in the maintenance of chromatin structures during DNA replication.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Replicação do DNA/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Transcrição/metabolismo , Síndrome de Williams/genética , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Humanos Par 7/genética , Marcadores Genéticos/genética , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Interferência de RNA , Fatores de Transcrição/genética , Síndrome de Williams/metabolismoRESUMO
Mammalian topoisomerase IIalpha (topo IIalpha) plays a vital role in the removal of topological complexities left on DNA during S phase. Here, we developed a new assay to selectively identify sites of catalytic activity of topo IIalpha with subcellular resolution. We show that topo IIalpha activity concentrates at replicating heterochromatin in late S in a replication-dependent manner and at centric heterochromatin during G2 and M phases. Inhibitor studies indicate that this cell cycle-dependent concentration over heterochromatin is sensitive to chromatin structure. We further show that catalytically active topo IIalpha concentrates along the longitudinal axis of mitotic chromosomes. Finally, we found that catalytically inert forms of the enzyme localize predominantly to splicing speckles in a dynamic manner and that this pool is differentially sensitive to changes in the activities of topo IIalpha itself and RNA polymerase II. Together, our data implicate several previously unsuspected activities in the partitioning of the enzyme between sites of activity and putative depots.