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1.
Lancet Haematol ; 9(11): e810-e821, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36328040

RESUMO

BACKGROUND: Anti-CD38 monoclonal antibodies have consistently shown increased efficacy when added to standard of care for patients with multiple myeloma. We aimed to assess the efficacy of isatuximab in addition to lenalidomide, bortezomib, and dexamethasone in patients with newly diagnosed transplantation-eligible multiple myeloma. METHODS: This open-label, multicentre, randomised, active-controlled, phase 3 trial was done at 67 academic and oncology practice centres in Germany. This study is ongoing and divided into two parts; herein, we report results from part 1. Eligible patients were aged 18-70 years; had a confirmed diagnosis of untreated multiple myeloma requiring systemic treatment and a WHO performance status of 0-2; and were eligible for induction therapy, high-dose melphalan and autologous haematopoietic stem-cell transplantation, and maintenance treatment. Patients were randomly assigned (1:1) to receive three 42-day cycles of induction therapy either with isatuximab plus lenalidomide, bortezomib, and dexamethasone (isatuximab group) or lenalidomide, bortezomib, and dexamethasone alone (control group) using a web-based system and permuted blocks. Patients in both groups received lenalidomide (25 mg orally on days 1-14 and 22-35), bortezomib (1·3 mg/m2 subcutaneously on days 1, 4, 8, 11, 22, 25, 29, and 32), and dexamethasone (20 mg orally on days 1-2, 4-5, 8-9, 11-12, 15, 22-23, 25-26, 29-30, and 32-33). Isatuximab was given as 10 mg/kg intravenously on days 1, 8, 15, 22, and 29 of cycle 1 and on days 1, 15, and 29 of cycles 2 and 3. The primary endpoint was minimal residual disease (MRD) negativity assessed by flow cytometry, in the intention-to-treat (ITT) population. This study is registered with ClinicalTrials.gov, NCT03617731. FINDINGS: Between Oct 23, 2018, and Sep 22, 2020, 660 patients were included in the ITT analysis (331 in the isatuximab group and 329 in the control group). 654 (99%) patients were White, two were African, one was Arabic, and three were Asian. 250 (38%) were women and 410 (62%) were men. The median age was 59 years (IQR 54-64). MRD negativity after induction therapy was reached in 166 (50%) patients in the isatuximab group versus 117 (36%) in the control group (OR 1·82 [95% CI 1·33-2·48]; p=0·00017). Median follow-up time from start to end of induction therapy was 125 days (IQR 125-131) versus 125 days (125-132). At least one grade 3 or 4 adverse event occurred in 208 (63%) of 330 patients versus 199 (61%) of 328 patients. Neutropenia of grade 3 or 4 occurred in 77 (23%) versus 23 (7%) patients and infections of grade 3 or 4 occurred in 40 (12%) versus 32 (10%) patients. Among 12 deaths during induction therapy, one death due to septic shock in the isatuximab group and four deaths (one cardiac decompensation, one hepatic and renal failure, one cardiac arrest, and one drug-induced enteritis) in the control group were considered treatment-related. INTERPRETATION: Addition of isatuximab to lenalidomide, bortezomib, and dexamethasone for induction therapy improved rates of MRD negativity with no new safety signals in patients with newly diagnosed transplantation-eligible multiple myeloma. FUNDING: Sanofi and Bristol Myers Squibb (Celgene).


Assuntos
Mieloma Múltiplo , Masculino , Humanos , Feminino , Pessoa de Meia-Idade , Lenalidomida/uso terapêutico , Bortezomib/efeitos adversos , Mieloma Múltiplo/terapia , Quimioterapia de Indução , Dexametasona , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos
3.
Nature ; 572(7768): 254-259, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31316209

RESUMO

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs)2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D-a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells6-9-are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo.


Assuntos
Evasão da Resposta Imune , Leucemia Mieloide Aguda/patologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/patologia , Evasão Tumoral , Animais , Antígenos CD34/metabolismo , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Ligantes , Masculino , Camundongos , Células-Tronco Neoplásicas/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Mol Genet Genomics ; 293(5): 1217-1229, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29882166

RESUMO

Recurrent oncogenic fusion genes play a critical role in the development of various cancers and diseases and provide, in some cases, excellent therapeutic targets. To date, analysis tools that can identify and compare recurrent fusion genes across multiple samples have not been available to researchers. To address this deficiency, we developed Co-occurrence Fusion (Co-fuse), a new and easy to use software tool that enables biologists to merge RNA-seq information, allowing them to identify recurrent fusion genes, without the need for exhaustive data processing. Notably, Co-fuse is based on pattern mining and statistical analysis which enables the identification of hidden patterns of recurrent fusion genes. In this report, we show that Co-fuse can be used to identify 2 distinct groups within a set of 49 leukemic cell lines based on their recurrent fusion genes: a multiple myeloma (MM) samples-enriched cluster and an acute myeloid leukemia (AML) samples-enriched cluster. Our experimental results further demonstrate that Co-fuse can identify known driver fusion genes (e.g., IGH-MYC, IGH-WHSC1) in MM, when compared to AML samples, indicating the potential of Co-fuse to aid the discovery of yet unknown driver fusion genes through cohort comparisons. Additionally, using a 272 primary glioma sample RNA-seq dataset, Co-fuse was able to validate recurrent fusion genes, further demonstrating the power of this analysis tool to identify recurrent fusion genes. Taken together, Co-fuse is a powerful new analysis tool that can be readily applied to large RNA-seq datasets, and may lead to the discovery of new disease subgroups and potentially new driver genes, for which, targeted therapies could be developed. The Co-fuse R source code is publicly available at https://github.com/sakrapee/co-fuse .


Assuntos
Genômica , Leucemia Mieloide Aguda/genética , Proteínas de Fusão Oncogênica/genética , Software , Biologia Computacional , Bases de Dados Genéticas , Humanos , Leucemia Mieloide Aguda/patologia , Proteínas de Fusão Oncogênica/isolamento & purificação , Análise de Sequência de RNA
6.
J Cell Sci ; 129(2): 277-89, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26644181

RESUMO

Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling.


Assuntos
Efrina-A5/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 12/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Células COS , Caspase 3/metabolismo , Membrana Celular/metabolismo , Chlorocebus aethiops , Citoesqueleto/metabolismo , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Transporte Proteico , Receptor EphA3
7.
Blood ; 123(8): 1218-28, 2014 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-24363400

RESUMO

Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in eliminating differentiated chronic myeloid leukemia (CML) cells, recent evidence suggests that leukemic stem and progenitor cells (LSPCs) persist long term, which may be partly attributable to cytokine-mediated resistance. We evaluated the expression of the interleukin 3 (IL-3) receptor α subunit (CD123), an established marker of acute myeloid leukemia stem cells, on CML LSPCs and the potential of targeting those cells with the humanized anti-CD123 monoclonal antibody CSL362. Compared with normal donors, CD123 expression was higher in CD34(+)/CD38(-) cells of both chronic phase and blast crisis CML patients, with levels increasing upon disease progression. CSL362 effectively targeted CML LSPCs by selective antibody-dependent cell-mediated cytotoxicity (ADCC)-facilitated lysis of CD123(+) cells and reduced leukemic engraftment in mice. Importantly, not only were healthy donor allogeneic natural killer (NK) cells able to mount an effective CSL362-mediated ADCC response, but so were CML patients' autologous NK cells. In addition, CSL362 also neutralized IL-3-mediated rescue of TKI-induced cell death. Notably, combination of TKI- and CSL362-induced ADCC caused even greater reduction of CML progenitors and further augmented their preferential elimination over normal hematopoietic stem and progenitor cells. Thus, our data support the further evaluation of CSL362 therapy in CML.


Assuntos
Anticorpos Monoclonais/imunologia , Subunidade alfa de Receptor de Interleucina-3/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Receptores de Interleucina-3/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Adulto , Idoso , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Antígenos CD34/metabolismo , Linhagem Celular , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Leucemia Eritroblástica Aguda/imunologia , Leucemia Eritroblástica Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Células-Tronco/imunologia , Células-Tronco/metabolismo , Células-Tronco/patologia
8.
Haematologica ; 98(6): 896-900, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23065516

RESUMO

Dasatinib is effective in most chronic phase chronic myeloid leukemia patients both in first-line therapy and following imatinib failure. While imatinib uptake into CD34(+) cells is low compared to mononuclear cells, few data evaluate how well dasatinib targets primitive CML cells. This study compares intracellular concentration of dasatinib and Bcr-Abl kinase inhibition in CML-CD34(+) progenitors and mononuclear cells induced by dasatinib. The intracellular concentrations of dasatinib were similar between CML-CD34(+) and mononuclear cells (P=0.8). Similarly, there was no significant difference in the degree of dasatinib-mediated Bcr-Abl kinase inhibition. ABCB1 (MDR1) and ABCG2 inhibitors neither increased dasatinib intracellular concentration nor enhanced dasatinib-mediated Bcr-Abl kinase inhibition. In contrast to nilotinib, we show that dasatinib is not an ABCB1 inhibitor. Thus, dasatinib targets CML-CD34(+) progenitors as effectively as it targets mononuclear cells. ABCB1 and ABCG2 efflux pumps do not appear to influence the intracellular dasatinib concentration in CML-CD34(+) progenitors.


Assuntos
Antígenos CD34/metabolismo , Antineoplásicos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Células Cultivadas , Dasatinibe , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Especificidade por Substrato/efeitos dos fármacos
9.
Crit Rev Oncog ; 17(2): 211-32, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22471709

RESUMO

Intense research over the past 15 years has demonstrated Eph receptors and their cell surface ephrin ligands to be one of the most prevalent and complex cell-cell communication systems; this system guides cell positioning and orchestrates tissue patterning in multicellular organisms by coordinating synchronised cell-cell adhesion or segregation of interacting cells. The expression of many Eph and ephrin family members together with their embryonic patterning functions often re-emerge during oncogenesis and have generated considerable interest as targets for anticancer therapies. The first generation of monoclonal antibodies, kinase inhibitors, and vaccines suggests considerable promise in preclinical and early clinical development, but there is little doubt that successful clinical use will rely on a comprehensive understanding of the complex and sometimes puzzling activities of Eph receptors during tumor progression.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Receptor EphA1/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Progressão da Doença , Humanos , Metástase Neoplásica , Neoplasias/patologia
10.
Semin Cell Dev Biol ; 23(1): 43-50, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22261642

RESUMO

Polymeric receptor-ligand complexes between interacting Eph and ephrin-expressing cells are regarded as dynamic intercellular signalling scaffolds that control cell-to-cell contact: the resulting Eph-ephrin signalling clusters function as positional cues that facilitate cell navigation and tissue patterning during normal and oncogenic development. The considerable complexity of this task, coordinating a multitude of cell movements and cellular interactions, is achieved by accurate translation of spatial information from Eph and ephrin expression gradients into fine-tuned changes in cell-cell adhesion and position. Here we review emerging evidence suggesting that the required combinatorial diversity is not only achieved by the large number of possible Eph-ephrin interactions and selective use of Eph forward and ephrin reverse signals, but in particular through the composition and signal capacity of Eph-ephrin clusters, which is adjusted dynamically to reflect overall Eph and ephrin surface densities on interacting cells. Fine-tuning is provided through multi-layered cluster assembly, where homo- and heterotypic Eph and ephrin interactions define the composition - whilst intracellular signalling feedbacks determine the size and lifetime - of signalling clusters.


Assuntos
Microdomínios da Membrana/metabolismo , Transporte Proteico , Receptores da Família Eph/metabolismo , Animais , Efrinas/metabolismo , Humanos , Modelos Moleculares , Neoplasias/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Transdução de Sinais
11.
Cell Mol Life Sci ; 69(11): 1813-42, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22204021

RESUMO

Numerous studies attest to essential roles for Eph receptors and their ephrin ligands in controlling cell positioning and tissue patterning during normal and oncogenic development. These studies suggest multiple, sometimes contradictory, functions of Eph-ephrin signalling, which under different conditions can promote either spreading and cell-cell adhesion or cytoskeletal collapse, cell rounding, de-adhesion and cell-cell segregation. A principle determinant of the balance between these two opposing responses is the degree of receptor/ligand clustering and activation. This equilibrium is likely altered in cancers and modulated by somatic mutations of key Eph family members that have emerged as candidate cancer markers in recent profiling studies. In addition, cross-talk amongst Ephs and with other signalling pathways significantly modulates cell-cell adhesion, both between and within Eph- and ephrin-expressing cell populations. This review summarises our current understanding of how Eph receptors control cell adhesion and morphology, and presents examples demonstrating the importance of these events in normal development and cancer.


Assuntos
Adesão Celular , Neoplasias/metabolismo , Receptores da Família Eph/fisiologia , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Linhagem Celular Tumoral , Separação Celular , Efrinas/metabolismo , Humanos , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica/patologia , Neoplasias/patologia , Neovascularização Patológica , Receptores da Família Eph/metabolismo , Transdução de Sinais
12.
J Cell Biol ; 195(6): 1033-45, 2011 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-22144690

RESUMO

Eph receptors interact with ephrin ligands on adjacent cells to facilitate tissue patterning during normal and oncogenic development, in which unscheduled expression and somatic mutations contribute to tumor progression. EphA and B subtypes preferentially bind A- and B-type ephrins, respectively, resulting in receptor complexes that propagate via homotypic Eph-Eph interactions. We now show that EphA and B receptors cocluster, such that specific ligation of one receptor promotes recruitment and cross-activation of the other. Remarkably, coexpression of a kinase-inactive mutant EphA3 with wild-type EphB2 can cause either cross-activation or cross-inhibition, depending on relative expression. Our findings indicate that cellular responses to ephrin contact are determined by the EphA/EphB receptor profile on a given cell rather than the individual Eph subclass. Importantly, they imply that in tumor cells coexpressing different Ephs, functional mutations in one subtype may cause phenotypes that are a result of altered signaling from heterotypic rather from homotypic Eph clusters.


Assuntos
Polimerização , Receptores da Família Eph/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Glioma/enzimologia , Células HEK293 , Humanos , Masculino , Neoplasias da Próstata/enzimologia , Receptores da Família Eph/agonistas , Receptores da Família Eph/química , Transdução de Sinais
13.
J Cell Biol ; 191(6): 1189-203, 2010 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-21135139

RESUMO

Eph receptors orchestrate cell positioning during normal and oncogenic development. Their function is spatially and temporally controlled by protein tyrosine phosphatases (PTPs), but the underlying mechanisms are unclear and the identity of most regulatory PTPs are unknown. We demonstrate here that PTP1B governs signaling and biological activity of EphA3. Changes in PTP1B expression significantly affect duration and amplitude of EphA3 phosphorylation and biological function, whereas confocal fluorescence lifetime imaging microscopy (FLIM) reveals direct interactions between PTP1B and EphA3 before ligand-stimulated receptor internalization and, subsequently, on endosomes. Moreover, overexpression of wild-type (w/t) PTP1B and the [D-A] substrate-trapping mutant decelerate ephrin-induced EphA3 trafficking in a dose-dependent manner, which reveals its role in controlling EphA3 cell surface concentration. Furthermore, we provide evidence that in areas of Eph/ephrin-mediated cell-cell contacts, the EphA3-PTP1B interaction can occur directly at the plasma membrane. Our studies for the first time provide molecular, mechanistic, and functional insights into the role of PTP1B controlling Eph/ephrin-facilitated cellular interactions.


Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Receptor EphA1/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Microscopia Confocal
14.
Blood ; 112(3): 721-32, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18385452

RESUMO

Signaling by Eph receptors and cell-surface ephrin ligands modulates adhesive cell properties and thereby coordinates cell movement and positioning in normal and oncogenic development. While cell contact-dependent Eph activation frequently leads to cell-cell repulsion, also the diametrically opposite response, cell-cell adhesion, is a probable outcome. However, the molecular principles regulating such disparate functions have remained controversial. We have examined cell-biologic mechanisms underlying this switch by analyzing ephrin-A5-induced cell-morphologic changes of EphA3-positive LK63 pre-B acute lymphoblastic leukemia cells. Their exposure to ephrin-A5 surfaces leads to a rapid conversion from a suspended/nonpolarized to an adherent/polarized cell type, a transition that relies on EphA3 functions operating in the absence of Eph-kinase signaling. Cell morphology change and adhesion of LK63 cells are effectively attenuated by endogenous protein tyrosine phosphatase (PTP) activity, whereby PTP inhibition and productive EphA3-phosphotyrosine signaling reverse the phenotype to nonadherent cells with a condensed cytoskeleton. Our findings suggest that Eph-associated PTP activities not only control receptor phosphorylation levels, but as a result switch the response to ephrin contact from repulsion to adhesion, which may play a role in the pathology of hematopoietic tumors.


Assuntos
Adesão Celular , Efrinas/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Proteínas Tirosina Fosfatases/metabolismo , Receptores da Família Eph/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Polaridade Celular , Forma Celular , Efrina-A5/fisiologia , Humanos , Fosforilação , Receptor EphA3/fisiologia , Transdução de Sinais
15.
Cell ; 123(2): 291-304, 2005 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-16239146

RESUMO

The Eph family of receptor tyrosine kinases and their ephrin ligands are mediators of cell-cell communication. Cleavage of ephrin-A2 by the ADAM10 membrane metalloprotease enables contact repulsion between Eph- and ephrin-expressing cells. How ADAM10 interacts with ephrins in a regulated manner to cleave only Eph bound ephrin molecules remains unclear. The structure of ADAM10 disintegrin and cysteine-rich domains and the functional studies presented here define an essential substrate-recognition module for functional interaction of ADAM10 with the ephrin-A5/EphA3 complex. While ADAM10 constitutively associates with EphA3, the formation of a functional EphA3/ephrin-A5 complex creates a new molecular recognition motif for the ADAM10 cysteine-rich domain that positions the proteinase domain for effective ephrin-A5 cleavage. Surprisingly, the cleavage occurs in trans, with ADAM10 and its substrate being on the membranes of opposing cells. Our data suggest a simple mechanism for regulating ADAM10-mediated ephrin proteolysis, which ensures that only Eph bound ephrins are recognized and cleaved.


Assuntos
Proteínas ADAM/metabolismo , Efrina-A2/metabolismo , Efrina-A3/metabolismo , Efrina-A5/metabolismo , Proteínas de Membrana/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAM10 , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Sequência Conservada , Cristalografia por Raios X , Cisteína/química , Dissulfetos/química , Efrina-A3/química , Efrina-A5/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hidrólise , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Filogenia , Testes de Precipitina , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor EphA3/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
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