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1.
Nature ; 610(7930): 182-189, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36131013

RESUMO

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.


Assuntos
Anticorpos , Especificidade de Anticorpos , Proteínas de Membrana , Proteólise , Ubiquitina-Proteína Ligases , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Neoplasias Colorretais/metabolismo , Ligantes , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Especificidade por Substrato , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo
2.
J Biol Chem ; 293(3): 906-919, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29191832

RESUMO

Anti-hinge antibodies (AHAs) are an autoantibody subclass that, following proteolytic cleavage, recognize cryptic epitopes exposed in the hinge regions of immunoglobulins (Igs) and do not bind to the intact Ig counterpart. AHAs have been postulated to exacerbate chronic inflammatory disorders such as inflammatory bowel disease and rheumatoid arthritis. On the other hand, AHAs may protect against invasive microbial pathogens and cancer. However, despite more than 50 years of study, the origin and specific B cell compartments that express AHAs remain elusive. Recent research on serum AHAs suggests that they arise during an active immune response, in contrast to previous proposals that they derive from the preexisting immune repertoire in the absence of antigenic stimuli. We report here the isolation and characterization of AHAs from memory B cells, although anti-hinge-reactive B cells were also detected in the naive B cell compartment. IgG AHAs cloned from a single human donor exhibited restricted specificity for protease-cleaved F(ab')2 fragments and did not bind the intact IgG counterpart. The cloned IgG-specific AHA-variable regions were mutated from germ line-derived sequences and displayed a high sequence variability, confirming that these AHAs underwent class-switch recombination and somatic hypermutation. Consistent with previous studies of serum AHAs, several of these clones recognized a linear, peptide-like epitope, but one clone was unique in recognizing a conformational epitope. All cloned AHAs could restore immune effector functions to proteolytically generated F(ab')2 fragments. Our results confirm that a diverse set of epitope-specific AHAs can be isolated from a single human donor.


Assuntos
Autoanticorpos/metabolismo , Linfócitos B/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Autoanticorpos/imunologia , Linfócitos B/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/metabolismo
3.
Cell Chem Biol ; 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-38056465

RESUMO

Selective and precise activation of signaling transduction cascades is key for cellular reprogramming and tissue regeneration. However, the development of small- or large-molecule agonists for many signaling pathways has remained elusive and is rate limiting to realize the full clinical potential of regenerative medicine. Focusing on the Wnt pathway, here we describe a series of disulfide-constrained peptides (DCPs) that promote Wnt signaling activity by modulating the cell surface levels of ZNRF3, an E3 ubiquitin ligase that controls the abundance of the Wnt receptor complex FZD/LRP at the plasma membrane. Mechanistically, monomeric DCPs induce ZNRF3 ubiquitination, leading to its cell surface clearance, ultimately resulting in FZD stabilization. Furthermore, we engineered multimeric DCPs that induce expansive growth of human intestinal organoids, revealing a dependence between valency and ZNRF3 clearance. Our work highlights a strategy for the development of potent, biologically active Wnt signaling pathway agonists via targeting of ZNRF3.

4.
Semin Cell Dev Biol ; 21(9): 866-73, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20709619

RESUMO

Cytokinesis is emerging as a control system defined by interacting biochemical and mechanical modules, which form a system of feedback loops. This integrated system accounts for the regulation and kinetics of cytokinesis furrowing and demonstrates that cytokinesis is a whole-cell process in which the global and equatorial cortices and cytoplasm are active players in the system. Though originally defined in Dictyostelium, features of the control system are recognizable in other organisms, suggesting a universal mechanism for cytokinesis regulation and contractility.


Assuntos
Citocinese , Dictyostelium/citologia , Actinas/metabolismo , Animais , Divisão Celular , Membrana Celular/metabolismo , Dictyostelium/metabolismo , Retroalimentação Fisiológica , Miosinas/metabolismo
5.
Curr Biol ; 18(18): R860-2, 2008 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-18812080

RESUMO

Mechanosensation is emerging as a general principle of myosin motors. As demonstrated in a recent study, the single-headed myosin I molecule is an exquisite mechanosensor, able to sense strain over a very small force range.


Assuntos
Mecanorreceptores/fisiologia , Mecanotransdução Celular/fisiologia , Miosinas/fisiologia , Actinas/química , Actinas/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Cinética , Proteínas Motores Moleculares/fisiologia , Miosinas/química
6.
Curr Biol ; 16(19): 1962-7, 2006 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-17027494

RESUMO

Because cell-division failure is deleterious, promoting tumorigenesis in mammals, cells utilize numerous mechanisms to control their cell-cycle progression. Though cell division is considered a well-ordered sequence of biochemical events, cytokinesis, an inherently mechanical process, must also be mechanically controlled to ensure that two equivalent daughter cells are produced with high fidelity. Given that cells respond to their mechanical environment, we hypothesized that cells utilize mechanosensing and mechanical feedback to sense and correct shape asymmetries during cytokinesis. Because the mitotic spindle and myosin II are vital to cell division, we explored their roles in responding to shape perturbations during cell division. We demonstrate that the contractile proteins myosin II and cortexillin I redistribute in response to intrinsic and externally induced shape asymmetries. In early cytokinesis, mechanical load overrides spindle cues and slows cytokinesis progression while contractile proteins accumulate and correct shape asymmetries. In late cytokinesis, mechanical perturbation also directs contractile proteins but without apparently disrupting cytokinesis. Significantly, this response only occurs during anaphase through cytokinesis, does not require microtubules, and is independent of spindle orientation, but is dependent on myosin II. Our data provide evidence for a mechanosensory system that directs contractile proteins to regulate cell shape during mitosis.


Assuntos
Forma Celular , Dictyostelium/citologia , Mecanotransdução Celular/fisiologia , Proteínas dos Microfilamentos/fisiologia , Mitose/fisiologia , Miosina Tipo II/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Dictyostelium/metabolismo , Dictyostelium/ultraestrutura , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Miosina Tipo II/análise , Miosina Tipo II/metabolismo , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , Fuso Acromático/fisiologia , Fuso Acromático/ultraestrutura
7.
Dev Cell ; 32(5): 561-73, 2015 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-25684354

RESUMO

Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an "attacking" cell drills finger-like protrusions into the "receiving" cell to promote cell fusion. Here, we show that the receiving cell mounts a Myosin II (MyoII)-mediated mechanosensory response to its invasive fusion partner. MyoII acts as a mechanosensor, which directs its force-induced recruitment to the fusion site, and the mechanosensory response of MyoII is amplified by chemical signaling initiated by cell adhesion molecules. The accumulated MyoII, in turn, increases cortical tension and promotes fusion pore formation. We propose that the protrusive and resisting forces from fusion partners put the fusogenic synapse under high mechanical tension, which helps to overcome energy barriers for membrane apposition and drives cell membrane fusion.


Assuntos
Membrana Celular/metabolismo , Drosophila melanogaster/metabolismo , Mecanotransdução Celular , Fusão de Membrana/fisiologia , Miosina Tipo II/metabolismo , Animais , Apoptose , Western Blotting , Comunicação Celular , Proliferação de Células , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Técnicas Imunoenzimáticas , Imunoprecipitação , Bicamadas Lipídicas/metabolismo , Modelos Biológicos , Miosina Tipo II/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
8.
Methods Mol Biol ; 983: 367-82, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23494318

RESUMO

Micropipette aspiration (MPA) is a widely applied method for studying cortical tension and deformability. Based on simple hydrostatic principles, this assay allows the application of a specific magnitude of mechanical stress on cells. This powerful method has revealed insights about cell mechanics and mechanosensing, not only in Dictyostelium discoideum but also in other cell types. In this chapter, we present how to set up a micropipette aspiration system and the experimental procedures for determining cortical tension and mechanosensory responses.


Assuntos
Dictyostelium/citologia , Mecanotransdução Celular , Análise de Célula Única/métodos , Fenômenos Biomecânicos , Calibragem , Técnicas de Cultura , Elasticidade , Microscopia de Fluorescência , Modelos Biológicos , Estresse Mecânico
9.
Mol Biol Cell ; 23(8): 1510-23, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22379107

RESUMO

The mitotic spindle is generally considered the initiator of furrow ingression. However, recent studies suggest that furrows can form without spindles, particularly during asymmetric cell division. In Dictyostelium, the mechanoenzyme myosin II and the actin cross-linker cortexillin I form a mechanosensor that responds to mechanical stress, which could account for spindle-independent contractile protein recruitment. Here we show that the regulatory and contractility network composed of myosin II, cortexillin I, IQGAP2, kinesin-6 (kif12), and inner centromeric protein (INCENP) is a mechanical stress-responsive system. Myosin II and cortexillin I form the core mechanosensor, and mechanotransduction is mediated by IQGAP2 to kif12 and INCENP. In addition, IQGAP2 is antagonized by IQGAP1 to modulate the mechanoresponsiveness of the system, suggesting a possible mechanism for discriminating between mechanical and biochemical inputs. Furthermore, IQGAP2 is important for maintaining spindle morphology and kif12 and myosin II cleavage furrow recruitment. Cortexillin II is not directly involved in myosin II mechanosensitive accumulation, but without cortexillin I, cortexillin II's role in membrane-cortex attachment is revealed. Finally, the mitotic spindle is dispensable for the system. Overall, this mechanosensory system is structured like a control system characterized by mechanochemical feedback loops that regulate myosin II localization at sites of mechanical stress and the cleavage furrow.


Assuntos
Divisão Celular , Dictyostelium/citologia , Dictyostelium/metabolismo , Cinesinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miosina Tipo II/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Mecanotransdução Celular , Proteínas de Protozoários/genética , Fuso Acromático/metabolismo , Fuso Acromático/fisiologia , Estresse Fisiológico
10.
Curr Biol ; 20(21): 1881-9, 2010 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-20951045

RESUMO

BACKGROUND: During cytokinesis, regulatory signals are presumed to emanate from the mitotic spindle. However, what these signals are and how they lead to the spatiotemporal changes in the cortex structure, mechanics, and regional contractility are not well understood in any system. RESULTS: To investigate pathways that link the microtubule network to the cortical changes that promote cytokinesis, we used chemical genetics in Dictyostelium to identify genetic suppressors of nocodazole, a microtubule depolymerizer. We identified 14-3-3 and found that it is enriched in the cortex, helps maintain steady-state microtubule length, contributes to normal cortical tension, modulates actin wave formation, and controls the symmetry and kinetics of cleavage furrow contractility during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac small GTPase (RacE), associates with myosin II heavy chain, and is needed to promote myosin II bipolar thick filament remodeling. CONCLUSIONS: 14-3-3 connects microtubules, Rac, and myosin II to control several aspects of cortical dynamics, mechanics, and cytokinesis cell shape change. Furthermore, 14-3-3 interacts directly with myosin II heavy chain to promote bipolar thick filament remodeling and distribution. Overall, 14-3-3 appears to integrate several critical cytoskeletal elements that drive two important processes-cytokinesis cell shape change and cell mechanics.


Assuntos
Proteínas 14-3-3/fisiologia , Citocinese/fisiologia , Dictyostelium/citologia , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Nocodazol/farmacologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Citocinese/genética , Dictyostelium/genética , Dictyostelium/ultraestrutura , Regulação para Baixo , Biblioteca Gênica , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Miosina Tipo II/fisiologia , Nocodazol/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/fisiologia
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