RESUMO
Mammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. By employing a tactic termed bump- and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells. The versatility of our approach allows pinpointing glycosylation sites by tandem mass spectrometry, and exploiting the chemical handle to manufacture proteoglycans with defined GAG chains for cellular applications. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.
RESUMO
Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most prevalent circulating autoantibodies in membranous nephropathy are against phospholipase A2 receptor (PLA2R), a cell surface receptor. The dominant epitope in PLA2R is located within the cysteine-rich domain, yet high-resolution structure-based mapping is lacking. In this study, we define the key nonredundant amino acids in the dominant epitope of PLA2R involved in autoantibody binding. We further describe two essential regions within the dominant epitope and spacer requirements for a synthetic peptide of the epitope for drug discovery. In addition, using cryo-electron microscopy, we have determined the high-resolution structure of PLA2R to 3.4 Å resolution, which shows that the dominant epitope and key residues within the cysteine-rich domain are accessible at the cell surface. In addition, the structure of PLA2R not only suggests a different orientation of domains but also implicates a unique immunogenic signature in PLA2R responsible for inducing autoantibody formation and recognition.
Assuntos
Apresentação de Antígeno , Autoanticorpos , Glomerulonefrite Membranosa , Epitopos Imunodominantes , Receptores da Fosfolipase A2 , Autoanticorpos/química , Sítios de Ligação , Microscopia Crioeletrônica , Cisteína/química , Glomerulonefrite Membranosa/imunologia , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Domínios Proteicos , Receptores da Fosfolipase A2/química , Receptores da Fosfolipase A2/imunologiaRESUMO
Mutations of the rearranged during transfection (RET) kinase are frequently reported in cancer, which make it as an attractive therapeutic target. Herein, we discovered a series of N-trisubstituted pyrimidine derivatives as potent inhibitors for both wild-type (wt) RET and RETV804M, which is a resistant mutant for several FDA-approved inhibitors. The X-ray structure of a representative inhibitor with RET revealed that the compound binds in a unique pose that bifurcates beneath the P-loop and confirmed the compound as a type I inhibitor. Through the structure-activity relationship (SAR) study, compound 20 was identified as a lead compound, showing potent inhibition of both RET and RETV804M. Additionally, compound 20 displayed potent antiproliferative activity of CCDC6-RET-driven LC-2/ad cells. Analysis of RET phosphorylation indicated that biological activity was mediated by RET inhibition. Collectively, N-trisubstituted pyrimidine derivatives could serve as scaffolds for the discovery and development of potent inhibitors of type I RET and its gatekeeper mutant for the treatment of RET-driven cancers.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Pirimidinas/química , Adenocarcinoma de Pulmão/patologia , Apoptose , Proliferação de Células , Humanos , Neoplasias Pulmonares/patologia , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-ret/genética , Relação Estrutura-Atividade , Células Tumorais Cultivadas , CicatrizaçãoRESUMO
RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.
Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Caderinas/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Complexos Multiproteicos/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-ret/química , Proteínas de Peixe-Zebra/químicaRESUMO
Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.
Assuntos
Acetilgalactosamina/metabolismo , Glicoproteínas/metabolismo , Acetilgalactosamina/química , Regulação Enzimológica da Expressão Gênica , Glicosilação , Humanos , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Especificidade por Substrato , Uridina Difosfato N-Acetilgalactosamina/químicaRESUMO
RET receptor tyrosine kinase is a driver oncogene in human cancer. We recently identified the clinical drug candidate Pz-1, which targets RET and VEGFR2. A key in vivo metabolite of Pz-1 is its less active demethylated pyrazole analogue. Using bioisosteric substitution methods, here, we report the identification of NPA101.3, lacking the structural liability for demethylation. NPA101.3 showed a selective inhibitory profile and an inhibitory concentration 50 (IC50) of <0.003 µM for both RET and VEGFR2. NPA101.3 inhibited phosphorylation of all tested RET oncoproteins as well as VEGFR2 and proliferation of cells transformed by RET. Oral administration of NPA101.3 (10 mg/kg/day) completely prevented formation of tumors induced by RET/C634Y-transformed cells, while it weakened, but did not abrogate, formation of tumors induced by a control oncogene (HRAS/G12V). The balanced synchronous inhibition of both RET and VEGFR2, as well the resistance to demethylation, renders NPA101.3 a potential clinical candidate for RET-driven cancers.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Descoberta de Drogas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação , Células NIH 3T3 , Polifarmacologia , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The structure-specific endonuclease XPF-ERCC1 participates in multiple DNA damage repair pathways including nucleotide excision repair (NER) and inter-strand crosslink repair (ICLR). How XPF-ERCC1 is catalytically activated by DNA junction substrates is not currently understood. Here we report cryo-electron microscopy structures of both DNA-free and DNA-bound human XPF-ERCC1. DNA-free XPF-ERCC1 adopts an auto-inhibited conformation in which the XPF helical domain masks the ERCC1 (HhH)2 domain and restricts access to the XPF catalytic site. DNA junction engagement releases the ERCC1 (HhH)2 domain to couple with the XPF-ERCC1 nuclease/nuclease-like domains. Structure-function data indicate xeroderma pigmentosum patient mutations frequently compromise the structural integrity of XPF-ERCC1. Fanconi anaemia patient mutations in XPF often display substantial in-vitro activity but are resistant to activation by ICLR recruitment factor SLX4. Our data provide insights into XPF-ERCC1 architecture and catalytic activation.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Anemia de Fanconi/enzimologia , Anemia de Fanconi/genética , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Relação Estrutura-Atividade , Xeroderma Pigmentoso/enzimologia , Xeroderma Pigmentoso/genéticaRESUMO
The lymphatic vessel endothelial receptor LYVE-1 is implicated in the uptake of hyaluronan (HA) and trafficking of leukocytes to draining lymph nodes. Yet LYVE-1 has only weak affinity for hyaluronan and depends on receptor clustering and higher order ligand organization for durable binding in lymphatic endothelium. An unusual feature of LYVE-1 not found in other HA receptors is the potential to form disulfide-linked homodimers. However, their influence on function has not been investigated. Here we show LYVE-1 homodimers are the predominant configuration in lymphatic endothelium in vitro and in vivo, and formation solely requires the unpaired cysteine residue Cys-201 within the membrane-proximal domain, yielding a 15-fold higher HA binding affinity and an â¼67-fold slower off-rate than the monomer. Moreover, we show non-dimerizing LYVE-1 mutants fail to bind HA even when expressed at high densities in lymphatic endothelial cells or artificially cross-linked with antibody. Consistent with these findings, small angle X-ray scattering (SAXS) indicates the Cys-201 interchain disulfide forms a hinge that maintains the homodimer in an "open scissors" conformation, likely allowing arrangement of the two HA binding domains for mutual engagement with ligand. Finally, we demonstrate the Cys-201 interchain disulfide is highly labile, and selective reduction with TCEP-HCl disrupts LYVE-1 homodimers, ablating HA binding. These findings reveal binding is dependent not just on clustering but also on the biochemical properties of LYVE-1 homodimers. They also mark LYVE-1 as the first Link protein superfamily member requiring covalent homodimerization for function and suggest the interchain disulfide acts as a redox switch in vivo.
Assuntos
Células Endoteliais/metabolismo , Endotélio Linfático/metabolismo , Ácido Hialurônico/metabolismo , Multimerização Proteica/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/metabolismo , Células Endoteliais/citologia , Endotélio Linfático/citologia , Humanos , Ácido Hialurônico/genética , Oxirredução , Proteínas de Transporte Vesicular/genéticaRESUMO
The C-type mannose receptor and its homolog Endo180 (or uPARAP, for urokinase plasminogen activator receptor-associated protein) mediate the endocytic uptake of collagen by macrophages and fibroblasts. This process is required for normal tissue remodeling, but also facilitates the growth and dissemination of tumors. We have determined the crystal structure at 2.5 Å resolution of the N-terminal region of Endo180, consisting of a ricin-like domain, a fibronectin type II (FN2) domain, and two C-type lectin (CTL) domains. The L-shaped arrangement of these domains creates a shallow trench spanning the FN2 and CTL1 domains, which was shown by mutagenesis to bind triple-helical and denatured collagen. Small-angle X-ray scattering showed that the L-shaped structure is maintained in solution at neutral and acidic pH, irrespective of calcium ion loading. Collagen binding was equally unaffected by acidic pH, suggesting that collagen release in endosomes is not regulated by changes within the Endo180 N-terminal region.
Assuntos
Lectinas Tipo C/química , Lectinas de Ligação a Manose/química , Receptores de Superfície Celular/química , Receptores Mitogênicos/química , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Colágeno/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Mitogênicos/genética , Receptores Mitogênicos/metabolismoRESUMO
Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was (13)C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a D-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.
Assuntos
Moléculas de Adesão Celular/química , Ácido Hialurônico/química , Modelos Moleculares , Oligossacarídeos/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Feminino , Humanos , Receptores de Hialuronatos/química , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/genética , Ácido Hialurônico/metabolismo , Inflamação/genética , Inflamação/metabolismo , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Ovulação/genética , Ovulação/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Under inflammatory conditions and in the matrix of the cumulus-oocyte complex, the polysaccharide hyaluronan (HA) becomes decorated covalently with heavy chains (HCs) of the serum glycoprotein inter-α-inhibitor (IαI). This alters the functional properties of the HA as well as its structural role within extracellular matrices. The covalent transfer of HCs from IαI to HA is catalyzed by TSG-6 (tumor necrosis factor-stimulated gene-6), but TSG-6 is also known as a HA cross-linker that induces condensation of the HA matrix. Here, we investigate the interplay of these two distinct functions of TSG-6 by studying the ternary interactions of IαI and TSG-6 with well defined films of end-grafted HA chains. We demonstrate that TSG-6-mediated cross-linking of HA films is impaired in the presence of IαI and that this effect suppresses the TSG-6-mediated enhancement of HA binding to CD44-positive cells. Furthermore, we find that the interaction of TSG-6 and IαI in the presence of HA gives rise to two types of complexes that independently promote the covalent transfer of heavy chains to HA. One type of complex interacts very weakly with HA and is likely to correspond to the previously reported covalent HC·TSG-6 complexes. The other type of complex is novel and binds stably but noncovalently to HA. Prolonged incubation with TSG-6 and IαI leads to HA films that contain, in addition to covalently HA-bound HCs, several tightly but noncovalently bound molecular species. These findings have important implications for understanding how the biological activities of TSG-6 are regulated, such that the presence or absence of IαI will dictate its function.
Assuntos
alfa-Globulinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , alfa-Globulinas/química , Animais , Sítios de Ligação , Ligação Competitiva , Western Blotting , Moléculas de Adesão Celular/química , Linhagem Celular , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Humanos , Receptores de Hialuronatos/química , Ácido Hialurônico/química , Cinética , Microscopia de Interferência , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein. Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation).
Assuntos
Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Inflamação/metabolismo , Multimerização Proteica/efeitos dos fármacos , Humanos , Ácido Hialurônico/metabolismo , Modelos Moleculares , Concentração Osmolar , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized "orphan" Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with ß-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65(Arp7A)·LIM2-3(Tes)·EVH1(Mena) complex reveals that residues 28-49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.
Assuntos
Citoesqueleto/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas do Citoesqueleto , Citoesqueleto/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Domínio LIM , Masculino , Proteínas dos Microfilamentos/genética , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Ratos , Proteínas Supressoras de Tumor/genéticaRESUMO
The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility, and vascular biology (e.g. it inhibits FGF2 (fibroblast growth factor 2)-mediated angiogenesis). PTX3 is composed of multiple protomers, each composed of distinct N- and C-terminal domains; however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer), giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer, explaining both its quaternary organization and how this is required for its antiangiogenic function.
Assuntos
Proteína C-Reativa/fisiologia , Fator 2 de Crescimento de Fibroblastos/química , Neovascularização Patológica , Componente Amiloide P Sérico/fisiologia , Inibidores da Angiogênese/farmacologia , Animais , Sítios de Ligação , Proteína C-Reativa/química , Células CHO , Cricetinae , Cricetulus , Dissulfetos/química , Humanos , Ligantes , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Componente Amiloide P Sérico/químicaRESUMO
OBJECTIVE: SS is a chronic inflammatory condition characterized by systemic and tissue-specific autoimmune features. In view of recent findings indicating a role for killer cell immunoglobulin-like receptors (KIRs) in the pathogenesis of other autoimmune rheumatic disorders such as SSc, and the autoimmune disorders RA and PsA, we sought to determine whether KIRs predict general or specific susceptibility in SS. METHODS: Eleven separate KIR genes were typed using PCR sequence-specific primers on genomic DNA from 72 patients diagnosed with primary SS and a control panel consisting of 223 blood donors. RESULTS: We found no individual KIR genes to be associated with SS. In contrast, 11 patients with primary SS (15%) and 9 control blood donors (4%) had KIR genotypes with the activating KIR2DS2 in the absence of its corresponding inhibitory homologue KIR2DL2 (P = 0.01). Further analysis of these individuals showed that seven SS patients were positive for HLA-C ligand for KIR2DS2 only compared with one control sample (P = 0.00026). CONCLUSION: The genetic combination of KIR2DS2+ and KIR2DL2- in the presence of HLA-C ligand specific for activating KIR2DS2 is associated with primary SS. This implies that autologous KIR-ligand interaction is a contributory factor to predisposition for this disease.
Assuntos
Receptores KIR/genética , Síndrome de Sjogren/genética , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Feminino , Frequência do Gene , Genótipo , Antígenos HLA-C/análise , Heterozigoto , Humanos , Reação em Cadeia da Polimerase/métodos , Receptores KIR/metabolismo , Receptores KIR2DL2/genética , Receptores KIR2DL2/metabolismo , Síndrome de Sjogren/imunologiaRESUMO
The intracellular targeting of Ena/VASP family members is achieved via the interaction of their EVH1 domain with FPPPP sequence motifs found in a variety of cytoskeletal proteins, including lamellipodin, vinculin, and zyxin. Here we show that the LIM3 domain of Tes, which lacks the FPPPP motif, binds to the EVH1 domain of Mena, but not to those of VASP or Evl. The structure of the LIM3:EVH1 complex reveals that Tes occludes the FPPPP-binding site and competes with FPPPP-containing proteins for EVH1 binding. Structure-based gain-of-function experiments define the molecular basis for the specificity of the Tes-Mena interaction. Consistent with in vitro observations, the LIM3 domain displaces Mena, but not VASP, from the leading edge and focal adhesions. It also regulates cell migration through a Mena-dependent mechanism. Our observations identify Tes as an atypical EVH1 binding partner and a regulator specific to a single Ena/VASP family member.
Assuntos
Proteínas de Homeodomínio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Movimento Celular/genética , Movimento Celular/fisiologia , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Immunoblotting , Proteínas com Domínio LIM , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
Killer immunoglobulin-like receptors (KIRs) regulate cell activity of natural killer (NK) cells and some T cells. The predominant ligand for inhibitory KIRs is HLA-C, which subdivides into 2 groups based on the specificity of inhibitory KIRs. The ligands for activatory KIRs are unknown. Following hematopoietic stem cell transplantation (HSCT), recipient tissues may not express a ligand for KIRs present within the graft, and the combination of donor KIR and recipient HLA-C types could influence outcome. HLA and KIR genotypes were determined in 220 donor-recipient pairs from HLA-matched sibling HSCTs performed for myeloid (n = 112) and lymphoid (n = 108) diseases. In HSCTs performed for myeloid disease, overall survival was worse in patients homozygous for group 2 HLA-C (C2) than in patients who carried a group 1 HLA-C (C1) allele (P <.005). Moreover, this effect is seen only when the donor additionally carries the activating KIR gene KIR2DS2 (P =.045). No effect was seen in patients with lymphoid disease. Thus, in HLA-matched sibling HSCT for myeloid leukemia, patients homozygous for C2 alleles receiving a graft from a donor carrying the KIR gene KIR2DS2 have a significantly reduced chance of survival.
Assuntos
Antígenos HLA-C/genética , Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide/genética , Leucemia Mieloide/terapia , Receptores Imunológicos/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Genótipo , Teste de Histocompatibilidade , Humanos , Leucemia Mieloide/mortalidade , Masculino , Pessoa de Meia-Idade , Receptores KIR , Análise de Sobrevida , Resultado do TratamentoRESUMO
Killer immunoglobulinlike receptors (KIRs) are expressed on natural killer and T cells. Both inhibitory and noninhibitory forms have been described, leading to inhibition or continuation of cellular killing activity. The natural ligands identified so far of KIRs are class I human leukocyte antigens (HLA). In particular, the interaction of some KIRs with HLA-Cw has been well characterized. Recent work has implicated KIRs in affecting the outcome of hematopoietic stem-cell transplant (HSCT). This may well lead to a requirement for prospective KIR typing of donor and recipient. We have utilized different typing systems (two using polymerase chain reaction-sequence-specific primers, and one using polymerase chain reaction-sequence-specific oligonucleotide probes) in three separate laboratories to characterize the KIR gene complement of 25 cell lines from the 10th International Histocompatibility Workshop. There were consistent results in 22, and minor differences in 3. When compared with previous results for some of these cell lines, no further differences were found. The differences are due to typing of KIRs KIR2DL1 and KIR2DS5, and may be explained by technical differences or the inability to type new variants. Further improvements in typing may be required if population and clinical studies are to produce accurate results.