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1.
Nat Commun ; 12(1): 4230, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244494

RESUMO

Extracellular matrix protein-1 (ECM1) promotes tumorigenesis in multiple organs but the mechanisms associated to ECM1 isoform subtypes have yet to be clarified. We report in this study that the secretory ECM1a isoform induces tumorigenesis through the GPR motif binding to integrin αXß2 and the activation of AKT/FAK/Rho/cytoskeleton signaling. The ATP binding cassette subfamily G member 1 (ABCG1) transduces the ECM1a-integrin αXß2 interactive signaling to facilitate the phosphorylation of AKT/FAK/Rho/cytoskeletal molecules and to confer cancer cell cisplatin resistance through up-regulation of the CD326-mediated cell stemness. On the contrary, the non-secretory ECM1b isoform binds myosin and blocks its phosphorylation, impairing cytoskeleton-mediated signaling and tumorigenesis. Moreover, ECM1a induces the expression of the heterogeneous nuclear ribonucleoprotein L like (hnRNPLL) protein to favor the alternative mRNA splicing generating ECM1a. ECM1a, αXß2, ABCG1 and hnRNPLL higher expression associates with poor survival, while ECM1b higher expression associates with good survival. These results highlight ECM1a, integrin αXß2, hnRNPLL and ABCG1 as potential targets for treating cancers associated with ECM1-activated signaling.


Assuntos
Processamento Alternativo , Carcinoma Epitelial do Ovário/genética , Proteínas da Matriz Extracelular/metabolismo , Recidiva Local de Neoplasia/epidemiologia , Neoplasias Ovarianas/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Carcinoma Epitelial do Ovário/mortalidade , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/terapia , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas da Matriz Extracelular/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/metabolismo , Estimativa de Kaplan-Meier , Camundongos , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Ovário/patologia , Ovário/cirurgia , Fosforilação/genética , Prognóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA-Seq , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto
2.
BJOG ; 128(8): 1282-1291, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33539617

RESUMO

OBJECTIVE: To study genetic variants and their function within genes coding for complement receptors in pre-eclampsia. DESIGN: A case-control study. SETTING: Pre-eclampsia is a common vascular disease of pregnancy. The clearance of placenta-derived material is one of the functions of the complement system in pregnancy. POPULATION: We genotyped 500 women with pre-eclamptic pregnancies and 190 pregnant women without pre-eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre-eclamptic pregnancies and 1905 controls from the national FINRISK cohort. METHODS: The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS-1 cells. MAIN OUTCOME MEASURES: Allele frequencies were compared between pre-eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays. RESULTS: The most significantly pre-eclampsia-linked CR3 variant M441K (P = 4.27E-4, OR = 1.401, 95% CI = 1.167-1.682) displayed a trend of increased adhesion to iC3b (P = 0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b. CONCLUSIONS: Results suggest that changes in complement-facilitated phagocytosis are associated with pre-eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro- and anti-inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre-eclampsia pathogenesis. TWEETABLE ABSTRACT: Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre-eclampsia.


Assuntos
Antígeno CD11b/genética , Integrina alfaXbeta2/genética , Antígeno de Macrófago 1/genética , Pré-Eclâmpsia/genética , Pré-Eclâmpsia/imunologia , Antígenos CD18/metabolismo , Citocinas/biossíntese , Feminino , Genótipo , Humanos , Integrina alfaXbeta2/metabolismo , Antígeno de Macrófago 1/metabolismo , Mutação , Fagocitose , Gravidez
3.
Mol Cells ; 43(12): 1023-1034, 2020 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-33372665

RESUMO

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMß2 and αXß2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective ß2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α'- chain (α'NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α'NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.


Assuntos
Complemento C3b/metabolismo , Integrina alfaXbeta2/química , Integrina alfaXbeta2/metabolismo , Sequência de Aminoácidos , Humanos , Ligação Proteica , Estrutura Terciária de Proteína
4.
Front Immunol ; 11: 2010, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922405

RESUMO

Dendritic cells (DCs) possess intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. In turn, HIV-1 has evolved strategies to evade innate immune sensing by DCs resulting in suboptimal maturation and poor antiviral immune responses. We previously showed that complement-opsonized HIV-1 (HIV-C) was able to efficiently infect various DC subsets significantly higher than non-opsonized HIV-1 (HIV) and therefore also mediate a higher antiviral immunity. Thus, complement coating of HIV-1 might play a role with respect to viral control occurring early during infection via modulation of DCs. To determine in detail which complement receptors (CRs) expressed on DCs was responsible for infection and superior pro-inflammatory and antiviral effects, we generated stable deletion mutants for the α-chains of CR3, CD11b, and CR4, CD11c using CRISPR/Cas9 in THP1-derived DCs. We found that CD11c deletion resulted in impaired DC infection as well as antiviral and pro-inflammatory immunity upon exposure to complement-coated HIV-1. In contrast, sole expression of CD11b on DCs shifted the cells to an anti-inflammatory, regulatory DC type. We here illustrated that CR4 comprised of CD11c and CD18 is the major player with respect to DC infection associated with a potent early pro-inflammatory immune response. A more detailed characterization of CR3 and CR4 functions using our powerful tool might open novel avenues for early therapeutic intervention during HIV-1 infection.


Assuntos
Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Integrina alfaXbeta2/metabolismo , Antígeno de Macrófago 1/metabolismo , Antígeno CD11b/genética , Antígeno CD11c/genética , Antígenos CD18/genética , Sistemas CRISPR-Cas , Proteínas do Sistema Complemento/metabolismo , Humanos , Imunidade , Integrina alfaXbeta2/genética , Antígeno de Macrófago 1/genética , Deleção de Sequência/genética , Transdução de Sinais , Células THP-1
5.
Biochim Biophys Acta Proteins Proteom ; 1867(6): 548-555, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30902766

RESUMO

CD23 is involved in a myriad of immune reactions. It is not only a receptor for IgE, but also functions in the regulation of IgE synthesis, isotype switching in B cells, and induction of the inflammatory response. These effector functions of CD23 arise through its interaction with another leukocyte-specific cell surface receptor - the ß2 integrin subfamily. It has been shown that CD23 is also capable of interacting with the ß3 and ß5 integrin ß-subunit of integrins via a basic RKC motif in a metal cation-independent fashion. In this study the interaction was probed for whether or not the RKC motif governs the interaction between CD23 and the αXß2 integrin as well. This was done by performing bioinformatic docking predictions between CD23 and αXß2 integrin αI domain and SPR spectroscopy analysis of the interaction. This revealed that in the absence of cations, the RKC motif is involved in interaction with the integrin αI domain. However, in the presence of divalent metal cations the interaction showed the involvement of a novel acidic motif within the CD23 protein. This same pattern of interaction was seen in docking predictions between CD23 and the ß3I-like domain. This study thus presents an alternative site as a possible contributor to the CD23-integrin interaction exhibiting cation-dependence.


Assuntos
Integrina alfaXbeta2/química , Integrina alfaXbeta2/metabolismo , Receptores de IgE/química , Receptores de IgE/metabolismo , Sítios de Ligação , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese , Ligação Proteica , Conformação Proteica , Mapas de Interação de Proteínas , Receptores de IgE/genética
6.
Immunol Lett ; 189: 73-81, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28577901

RESUMO

The expression and role of CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in B cells are not yet explored in contrast to myeloid cells, where these ß2-integrin type receptors are known to participate in various cellular functions, including phagocytosis, adherence and migration. Here we aimed to reveal the expression and role of CR3 and CR4 in human B cells. In B cells of healthy donors CR3 and CR4 are scarcely expressed. However, two patients with chronic lymphocytic leukemia (CLL) characterized by a peculiar immune-phenotype containing both CD5-positive and CD5-negative B cell populations made possible to study these molecules in distinct B cell subsets. We found that CD11b and CD11c were expressed on both CD5-positive and CD5-negative B cells, albeit to different extents. Our data suggest that these receptors are involved in spreading, since this activity of CpG-activated B cells on fibrinogen could be partially blocked by monoclonal antibodies specific for CD11b or CD11c. CpG-stimulation lead to proliferation of both CD5-positive and CD5-negative B cells of the patients with a less pronounced effect on the CD5-positive cells. In contrast to normal B cells, CLL B cells of both patients reacted to CpG-stimulation with robust IL-10 production. The concomitant, suboptimal stimulus via the BCR and TLR9 exerted either a synergistic enhancing effect or resulted in inhibition of proliferation and IL-10 production of patients' B cells. Our data obtained studying B cells of leukemic patients point to the role of CR3 and probably CR4 in the interaction of tumor cells with the microenvironment and suggest the involvement of IL-10 producing B cells in the pathologic process.


Assuntos
Linfócitos B/fisiologia , Integrina alfaXbeta2/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Antígeno de Macrófago 1/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Idoso , Antígenos CD18/química , Antígenos CD18/metabolismo , Antígenos CD5/metabolismo , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Humanos , Integrina alfaXbeta2/química , Interleucina-10/metabolismo , Antígeno de Macrófago 1/química , Receptor Toll-Like 9/metabolismo , Microambiente Tumoral
7.
Immunol Lett ; 189: 64-72, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28554712

RESUMO

CR3 and CR4 belong to the family of ß2-integrins and play an important role in phagocytosis, cellular adherence and migration. CR3 and CR4 are generally expected to mediate similar functions due to their structural homology, overlapping ligand specificity and parallel expression on human phagocytes. Although the different signalling pathways of these receptors suggest distinct functions, possible differences are just being revealed. Previously we proved that CR3 plays a key role in the uptake of iC3b-opsonized particles by human dendritic cells. Now, besides measuring the overall phagocytic capacity of cells including the assessment of surface bound as well as internalized particles, we extended our investigations and studied the digestion of the iC3b opsonized antigen by various human phagocytes. The participation of CR3 and CR4 was compared in the process of binding, internalization and digestion of iC3b opsonized Staphylococcus aureus by monocytes, monocyte derived macrophages (MDMs), monocyte derived dendritic cells (MDDCs), and neutrophils. Comparing the activity of the two ß2-integrin type complement receptors we found that CR3 plays a dominant role in the phagocytosis of iC3b opsonized S. aureus by all of these cell types. Studying another important integrin-mediated function we demonstrated earlier that CR4 is dominant in the adhesion of monocytes, MDMs and MDDCs to fibrinogen. Here we studied the participation of CR3 and CR4 in podosome formation by human phagocytes, since these structures are known to play an essential role in cell migration. Our confocal microscopy analysis revealed that both CD11b and CD11c concentrate in the podosome adhesion ring. In summary our data highlight differences in the function of human CR3 and CR4 in the process of uptake and digestion of complement opsonized antigen, while in the process of podosome formation, connected to cellular motility, both receptors equally take part.


Assuntos
Integrina alfaXbeta2/metabolismo , Antígeno de Macrófago 1/metabolismo , Macrófagos/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Podossomos/ultraestrutura , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Adesão Celular , Diferenciação Celular , Movimento Celular , Células Cultivadas , Complemento C3b/metabolismo , Humanos , Interleucina-4/metabolismo , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Monócitos/microbiologia , Monócitos/ultraestrutura , Neutrófilos/microbiologia , Neutrófilos/ultraestrutura , Fagocitose , Agregação Patológica de Proteínas
8.
Mol Cells ; 40(5): 355-362, 2017 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-28535664

RESUMO

The ß2 integrins are cell surface transmembrane proteins regulating leukocyte functions, such as adhesion and migration. Two members of ß2 integrin, αMß2 and αXß2, share the leukocyte distribution profile and integrin αXß2 is involved in antigen presentation in dendritic cells and transendothelial migration of monocytes and macrophages to atherosclerotic lesions. Receptor for advanced glycation end products (RAGE), a member of cell adhesion molecules, plays an important role in chronic inflammation and atherosclerosis. Although RAGE and αXß2 play an important role in inflammatory response and the pathogenesis of atherosclerosis, the nature of their interaction and structure involved in the binding remain poorly defined. In this study, using I-domain as a ligand binding motif of αXß2, we characterize the binding nature and the interacting moieties of αX I-domain and RAGE. Their binding requires divalent cations (Mg2+ and Mn2+) and shows an affinity on the sub-micro molar level: the dissociation constant of αX I-domains binding to RAGE being 0.49 µM. Furthermore, the αX I-domains recognize the V-domain, but not the C1 and C2-domains of RAGE. The acidic amino acid substitutions on the ligand binding site of αX I-domain significantly reduce the I-domain binding activity to soluble RAGE and the alanine substitutions of basic amino acids on the flat surface of the V-domain prevent the V-domain binding to αX I-domain. In conclusion, the main mechanism of αX I-domain binding to RAGE is a charge interaction, in which the acidic moieties of αX I-domains, including E244, and D249, recognize the basic residues on the RAGE V-domain encompassing K39, K43, K44, R104, and K107.


Assuntos
Integrina alfaXbeta2/química , Integrina alfaXbeta2/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Alanina/genética , Alanina/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Humanos , Integrina alfaXbeta2/genética , Cinética , Mutação , Domínios Proteicos , Receptor para Produtos Finais de Glicação Avançada/química , Receptor para Produtos Finais de Glicação Avançada/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de Superfície
9.
Sci Rep ; 7: 46532, 2017 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-28513618

RESUMO

Adhesion of monocytes to micro-injuries on arterial walls is an important early step in the occurrence and development of degenerative atherosclerotic lesions. At these injuries, collagen is exposed to the blood stream. We are interested whether age influences monocyte adhesion to collagen under flow, and hence influences the susceptibility to arteriosclerotic lesions. Therefore, we studied adhesion and rolling of human peripheral blood monocytes from old and young individuals on collagen type I coated surface under shear flow. We find that firm adhesion of monocytes to collagen type I is elevated in old individuals. Pre-stimulation by lipopolysaccharide increases the firm adhesion of monocytes homogeneously in older individuals, but heterogeneously in young individuals. Blocking integrin αx showed that adhesion of monocytes to collagen type I is specific to the main collagen binding integrin αxß2. Surprisingly, we find no significant age-dependent difference in gene expression of integrin αx or integrin ß2. However, if all integrins are activated from the outside, no differences exist between the age groups. Altered integrin activation therefore causes the increased adhesion. Our results show that the basal increase in integrin activation in monocytes from old individuals increases monocyte adhesion to collagen and therefore the risk for arteriosclerotic plaques.


Assuntos
Envelhecimento/metabolismo , Aterosclerose/metabolismo , Colágeno Tipo I/metabolismo , Integrina alfaXbeta2/metabolismo , Migração e Rolagem de Leucócitos , Monócitos/metabolismo , Adulto , Idoso , Envelhecimento/patologia , Aterosclerose/patologia , Adesão Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/patologia
10.
Proc Natl Acad Sci U S A ; 114(13): 3403-3408, 2017 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-28292891

RESUMO

Recognition by the leukocyte integrins αXß2 and αMß2 of complement iC3b-opsonized targets is essential for effector functions including phagocytosis. The integrin-binding sites on iC3b remain incompletely characterized. Here, we describe negative-stain electron microscopy and biochemical studies of αXß2 and αMß2 in complex with iC3b. Despite high homology, the two integrins bind iC3b at multiple distinct sites. αXß2 uses the αX αI domain to bind iC3b on its C3c moiety at one of two sites: a major site at the interface between macroglobulin (MG) 3 and MG4 domains, and a less frequently used site near the C345C domain. In contrast, αMß2 uses its αI domain to bind iC3b at the thioester domain and simultaneously interacts through a region near the αM ß-propeller and ß2 ßI domain with a region of the C3c moiety near the C345C domain. Remarkably, there is no overlap between the primary binding site of αXß2 and the binding site of αMß2 on iC3b. Distinctive binding sites on iC3b by integrins αXß2 and αMß2 may be biologically beneficial for leukocytes to more efficiently capture opsonized pathogens and to avoid subversion by pathogen factors.


Assuntos
Complemento C3b/metabolismo , Integrina alfaXbeta2/metabolismo , Antígeno de Macrófago 1/metabolismo , Sítios de Ligação , Complemento C3b/química , Complemento C3b/genética , Humanos , Integrina alfaXbeta2/química , Integrina alfaXbeta2/genética , Leucócitos/química , Leucócitos/metabolismo , Antígeno de Macrófago 1/química , Antígeno de Macrófago 1/genética , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína
11.
Infect Immun ; 85(1)2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27799334

RESUMO

Integrins αMß2 and αXß2 are homologous adhesive receptors that are expressed on many of the same leukocyte populations and bind many of the same ligands. Although αMß2 was extensively characterized and implicated in leukocyte inflammatory and immune functions, the roles of αXß2 remain largely obscure. Here, we tested the ability of mice deficient in integrin αMß2 or αXß2 to deal with opportunistic infections and the capacity of cells derived from these animals to execute inflammatory functions. The absence of αMß2 affected the recruitment of polymorphonuclear neutrophils (PMN) to bacterial and fungal pathogens as well as to model inflammatory stimuli, and αMß2-deficient PMN displayed defective inflammatory functions. In contrast, deficiency of αXß2 abrogated intraperitoneal recruitment and adhesive functions of monocytes and macrophages (Mϕ) and the ability of these cells to kill/phagocytose Candida albicans or Escherichia coli cells both ex vivo and in vivo During systemic candidiasis, the absence of αXß2 resulted in the loss of antifungal activity by tissue Mϕ and inhibited the production of tumor necrosis factor alpha (TNF-α)/interleukin-6 (IL-6) in infected kidneys. Deficiency of αMß2 suppressed Mϕ egress from the peritoneal cavity, decreased the production of anti-inflammatory IL-10, and stimulated the secretion of IL-6. The absence of αXß2, but not of αMß2, increased survival against a septic challenge with lipopolysaccharide (LPS) by 2-fold. Together, these results suggest that αMß2 plays a primary role in PMN inflammatory functions and regulates the anti-inflammatory functions of Mϕ, whereas αXß2 is central in the regulation of inflammatory functions of recruited and tissue-resident Mϕ.


Assuntos
Anti-Infecciosos/metabolismo , Inflamação/metabolismo , Integrina alfaXbeta2/metabolismo , Leucócitos/metabolismo , Antígeno de Macrófago 1/metabolismo , Animais , Candida albicans/metabolismo , Candidíase/metabolismo , Candidíase/microbiologia , Adesão Celular/fisiologia , Escherichia coli/metabolismo , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Inflamação/microbiologia , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Leucócitos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Fagocitose/fisiologia , Fator de Necrose Tumoral alfa/metabolismo
12.
Cell Rep ; 13(9): 1937-48, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26628365

RESUMO

Multinucleated giant cells (MGCs) form by fusion of macrophages and are presumed to contribute to the removal of debris from tissues. In a systematic in vitro analysis, we show that IL-4-induced MGCs phagocytosed large and complement-opsonized materials more effectively than their unfused M2 macrophage precursors. MGC expression of complement receptor 4 (CR4) was increased, but it functioned primarily as an adhesion integrin. In contrast, although expression of CR3 was not increased, it became functionally activated during fusion and was located on the extensive membrane ruffles created by excess plasma membrane arising from macrophage fusion. The combination of increased membrane area and activated CR3 specifically equips MGCs to engulf large complement-coated targets. Moreover, we demonstrate these features in vivo in the recently described complement-dependent therapeutic elimination of systemic amyloid deposits by MGCs. MGCs are evidently more than the sum of their macrophage parts.


Assuntos
Células Gigantes/metabolismo , Interleucina-4/farmacologia , Fagocitose/efeitos dos fármacos , Amiloide/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Antígeno CD11c/metabolismo , Antígenos CD18/metabolismo , Complemento C3/deficiência , Complemento C3/genética , Complemento C3/metabolismo , Cricetinae , Células Gigantes/imunologia , Humanos , Integrina alfaXbeta2/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ratos , Receptores de IgG/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Regulação para Cima/efeitos dos fármacos
13.
Immunol Lett ; 168(1): 13-21, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26306739

RESUMO

The opportunistic pathogenic yeast Candida albicans employs several mechanisms to interfere with the human complement system. This includes the acquisition of host complement regulators, the release of molecules that scavenge complement proteins or block cellular receptors, and the secretion of proteases that inactivate complement components. Secreted aspartic protease 2 (Sap2) was previously shown to cleave C3b, C4b and C5. C. albicans also recruits the complement inhibitor factor H (FH), but yeast-bound FH can enhance the antifungal activity of human neutrophils via binding to complement receptor type 3 (CR3). In this study, we characterized FH binding to human monocyte-derived macrophages. Inhibition studies with antibodies and siRNA targeting CR3 (CD11b/CD18) and CR4 (CD11c/CD18), as well as analysis of colocalization of FH with these integrins indicated that both function as FH receptors on macrophages. Preincubation of C. albicans yeast cells with FH induced increased production of IL-1ß and IL-6 in macrophages. Furthermore, FH enhanced zymosan-induced production of these cytokines. C. albicans Sap2 cleaved FH, diminishing its complement regulatory activity, and Sap2-treatment resulted in less detectable CR3 and CR4 on macrophages. These data show that FH enhances the activation of human macrophages when bound on C. albicans. However, the fungus can inactivate both FH and its receptors on macrophages by secreting Sap2, which may represent an additional means for C. albicans to evade the host innate immune system.


Assuntos
Ácido Aspártico Endopeptidases/imunologia , Candida albicans/imunologia , Fator H do Complemento/imunologia , Proteínas Fúngicas/imunologia , Integrina alfaXbeta2/imunologia , Antígeno de Macrófago 1/imunologia , Ácido Aspártico Endopeptidases/metabolismo , Western Blotting , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Antígenos CD18/genética , Antígenos CD18/imunologia , Antígenos CD18/metabolismo , Candida albicans/enzimologia , Candida albicans/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Fator H do Complemento/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/metabolismo , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/metabolismo , Interferência de RNA
14.
Biochim Biophys Acta ; 1854(8): 930-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25839998

RESUMO

Integrin α(X)ß(2) (also known as complement receptor 4, p150,95, or CD11c/CD18) is expressed in the cell membrane of myeloid leukocytes. α(X)ß(2) has been reported to bind a large number of structurally unrelated ligands, often with a shared molecular character in the presence of polyanionic stretches in poorly folded proteins or glucosaminoglycans. Nevertheless, it is unclear what chemical sources of polyanionicity enable the binding by α(X)ß(2). Osteopontin (OPN) is an intrinsically disordered protein, which facilitates phagocytosis via the integrin α(X)ß(2). Unlike for other integrins, neither the RGD nor the SVVYGLR motifs account for this binding, and the molecular basis of OPN binding by α(X)ß(2) remains uncharacterized. Here, we show that the monovalent interactions between the ligand-binding domain of α(X)ß(2) and OPN, its fragments, or caseins are weak, with dissociation constants higher than 10(-5)M but with high apparent stoichiometries. From comparison with cell adhesion studies, the discrimination between α(X)ß(2) ligands and non-ligands appears to rely on these apparent stoichiometries in a way, which involves glutamate rather than aspartate side chains. Surprisingly, the extensive, negatively charged phosphorylation of OPN is not contributing to α(X)ß(2) binding. Furthermore, synchrotron radiation circular spectroscopy excludes that the phosphorylation affects the general folding of OPN. Taken together, our quantitative analyses reveal a mode of ligand recognition by integrin α(X)ß(2), which seem to differ in principles considerably from other OPN receptors.


Assuntos
Integrina alfaXbeta2/química , Osteopontina/química , Dobramento de Proteína , Motivos de Aminoácidos , Adesão Celular , Humanos , Integrina alfaXbeta2/metabolismo , Leucócitos/química , Leucócitos/metabolismo , Osteopontina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína
15.
J Biol Chem ; 289(46): 32230-32242, 2014 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-25278023

RESUMO

The regulation of integrins expressed on leukocytes must be controlled precisely, and members of different integrin subfamilies have to act in concert to ensure the proper traffic of immune cells to sites of inflammation. The activation of ß2 family integrins through the T cell receptor or by chemokines leads to the inactivation of very late antigen 4. The mechanism(s) of this cross-talk has not been known. We have now elucidated in detail how the signals are transmitted from leukocyte function-associated antigen 1 and show that, after its activation, the signaling involves specific phosphorylations of ß2 integrin followed by interactions with cytoplasmic signaling proteins. This results in loss of ß1 phosphorylation and a decrease in very late antigen 4 binding to its ligand vascular cell adhesion molecule 1. Our results show how a member of one integrin family regulates the activity of another integrin. This is important for the understanding of integrin-mediated processes.


Assuntos
Antígenos CD18/metabolismo , Integrina alfa4beta1/metabolismo , Integrina alfaXbeta2/metabolismo , Integrina beta1/metabolismo , Leucócitos/citologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Adesão Celular , Movimento Celular , Células Cultivadas , Citoplasma/metabolismo , Filaminas/metabolismo , Regulação da Expressão Gênica , Humanos , Células K562 , Ligantes , Fosforilação , Transdução de Sinais
16.
J Antimicrob Chemother ; 69(4): 995-1004, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24275118

RESUMO

OBJECTIVES: The potential cardiovascular (CV) toxicity associated with combined antiretroviral therapy (cART) has been attributed mainly to the nucleoside reverse transcriptase inhibitors abacavir and didanosine. However, the other two components of cART--non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs)--may also be implicated, either directly or by influencing the action of the other drugs. This study evaluates the acute direct effects of the NNRTIs efavirenz and nevirapine and one of the most widely employed PIs, lopinavir, on leucocyte-endothelium interactions, a hallmark of CV disease. METHODS: Drugs were analysed in vitro in human cells (interactions of peripheral blood polymorphonuclear or mononuclear cells with human umbilical vein endothelial cells) using a flow chamber system, and in vivo in rat mesenteric vessels by means of intravital microscopy. The expression of adhesion molecules in leucocytes and endothelial cells was studied by flow cytometry, and the role of these molecules in white cell recruitment was evaluated by pre-treating human cells or rats with blocking antibodies. RESULTS: Efavirenz and nevirapine, but not lopinavir, increased the rolling flux and adhesion of leucocytes in vitro and in vivo while inducing emigration in rat venules. Efavirenz, but not nevirapine, augmented the levels of CD11b, CD11c and CD18 in neutrophils and monocytes. The actions of efavirenz, but not of nevirapine, were reversed by antibodies against Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18) or ICAM-1 (CD54). CONCLUSIONS: NNRTIs, but not PIs, interfere with leucocyte-endothelial interactions. However, differences between efavirenz and nevirapine suggest a specific CV profile for each compound.


Assuntos
Fármacos Anti-HIV/metabolismo , Benzoxazinas/metabolismo , Adesão Celular , Endotélio/fisiologia , Integrina alfaXbeta2/metabolismo , Leucócitos/fisiologia , Antígeno de Macrófago 1/metabolismo , Alcinos , Animais , Células Cultivadas , Ciclopropanos , Endotélio/efeitos dos fármacos , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Leucócitos/efeitos dos fármacos , Lopinavir/metabolismo , Masculino , Nevirapina/metabolismo , Ratos , Ratos Sprague-Dawley
17.
Int J Biochem Cell Biol ; 45(7): 1204-11, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23542015

RESUMO

Integrins αLß2, αMß2 and αXß2 are expressed on leukocytes. Their primary ligands are counter transmembrane receptors or plasma proteins, such as intercellular cell adhesion molecule-1 (ICAM-1) or components of complement system (iC3b, iC4b), respectively. Function blocking antibodies for these integrins may also reduce cell adhesion to collagens. To make the first systematical comparison of human α(L)ß2, α(M)ß2 and α(X)ß2 as collagen receptors, we produced the corresponding integrin αI domains both in wild-type and activated form and measured their binding to collagens I-VI. In the "closed" (wild-type) conformation, the α(L)I and α(M)I domains bound with low avidity to their primary ligands, and the interaction with collagens was also very weak. Gain-of-function mutations α(L) I306G, α(L) K287C/K294C and α(M) I316G are considered to mimic "open", activated αI domains. The binding of these activated αI domains to the primary ligands was clearly stronger and they also recognized collagens with moderate avidity (K(d)400 nM). After activation, the αLI domain favored collagen I (K(d )≈ 80 nM) when compared to collagen IV. The integrin αXI domain acted in a very different manner since already in native, wild-type form it bound to collagen IV and iC3b (K(d) ≈ 200-400 nM). Antibodies against αXß2 and αMß2 blocked promyelocytic leukemia cell adhesion to the collagenous GFOGER motif, a binding site for the ß1 integrin containing collagen receptors. In brief, leukocyte ß2 integrins may act as collagen receptors in a heterodimer specific manner.


Assuntos
Colágeno/metabolismo , Integrina alfaXbeta2/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Antígeno de Macrófago 1/metabolismo , Motivos de Aminoácidos , Anticorpos Bloqueadores/imunologia , Sítios de Ligação , Adesão Celular , Linhagem Celular Tumoral , Células HL-60 , Humanos , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/imunologia , Leucemia Promielocítica Aguda/metabolismo , Antígeno-1 Associado à Função Linfocitária/genética , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Colágeno
18.
J Cell Biol ; 203(4): 629-42, 2013 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-24385486

RESUMO

How is massive conformational change in integrins achieved on a rapid timescale? We report crystal structures of a metastable, putative transition state of integrin αXß2. The αXß2 ectodomain is bent; however, a lattice contact stabilizes its ligand-binding αI domain in a high affinity, open conformation. Much of the αI α7 helix unwinds, loses contact with the αI domain, and reshapes to form an internal ligand that binds to the interface between the ß propeller and ßI domains. Lift-off of the αI domain above this platform enables a range of extensional and rotational motions without precedent in allosteric machines. Movements of secondary structure elements in the ß2 ßI domain occur in an order different than in ß3 integrins, showing that integrin ß subunits can be specialized to assume different intermediate states between closed and open. Mutations demonstrate that the structure trapped here is metastable and can enable rapid equilibration between bent and extended-open integrin conformations and up-regulation of leukocyte adhesiveness.


Assuntos
Integrina alfaXbeta2/química , Integrina alfaXbeta2/metabolismo , Leucócitos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Complemento C3b/metabolismo , Cristalografia por Raios X , Epitopos/química , Eritrócitos/metabolismo , Células HEK293 , Humanos , Integrina alfaXbeta2/genética , Ligantes , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Ovinos
19.
J Immunol ; 189(5): 2468-77, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22844116

RESUMO

The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin α(X)ß2 in the pathogenesis of fungal infection was assessed. Both purified α(X)ß2 and α(X)ß2-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by ß-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of α(M)ß2. Mice deficient in α(X)ß2 were more prone to systemic infection with the LD50 fungal inoculum decreasing 3-fold in α(X)ß2-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of α(X)ß2-deficient mice within 9 d. Organs taken from α(X)ß2-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that α(X)ß2 is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use α(X)ß2 to control fungal invasion.


Assuntos
Candida albicans/patogenicidade , Candidíase/imunologia , Candidíase/prevenção & controle , Integrina alfaXbeta2/metabolismo , Leucócitos/metabolismo , Receptores Imunológicos/metabolismo , Animais , Candidíase/microbiologia , Adesão Celular/imunologia , Linhagem Celular , Movimento Celular/imunologia , Citofagocitose/imunologia , Células HEK293 , Humanos , Integrina alfaXbeta2/genética , Integrina alfaXbeta2/fisiologia , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores Imunológicos/fisiologia
20.
PLoS One ; 7(7): e41924, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22844534

RESUMO

BACKGROUND: Integrins are signal transducer proteins involved in a number of vital physiological processes including cell adhesion, proliferation and migration. Integrin molecules are hetero-dimers composed of two distinct subunits, α and ß. In humans, 18 α and 8 ß subunits are combined into 24 different integrin molecules. Each of the subunit comprises a large extracellular domain, a single pass transmembrane segment and a cytosolic tail (CT). The CTs of integrins are vital for bidirectional signal transduction and in maintaining the resting state of the receptors. A large number of intracellular proteins have been found to interact with the CTs of integrins linking integrins to the cytoskeleton. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we have investigated structure and interactions of CTs of the leukocyte specific integrin αXß2. We determined the atomic resolution structure of a myristoylated CT of αX in perdeuterated dodecylphosphocholine (DPC) by NMR spectroscopy. Our results reveal that the 35-residue long CT of αX adopts an α-helical conformation for residues F4-N17 at the N-terminal region. The remaining residues located at the C-terminal segment of αX delineate a long loop of irregular conformations. A segment of the loop maintains packing interactions with the helical structure by an extended non-polar surface of the αX CT. Interactions between αX and ß2 CTs are demonstrated by (15)N-(1)H HSQC NMR experiments. We find that residues constituting the polar face of the helical conformation of αX are involved in interactions with the N-terminal residues of ß2 CT. A docked structure of the CT complex indicates that a network of polar and/or salt-bridge interactions may sustain the heteromeric interactions. CONCLUSIONS/SIGNIFICANCE: The current study provides important insights into the conservation of interactions and structures among different CTs of integrins.


Assuntos
Citosol/metabolismo , Integrina alfaXbeta2/química , Integrina alfaXbeta2/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Ácido Mirístico/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Modificação Traducional de Proteínas , Estrutura Terciária de Proteína
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