RESUMEN
The practical realization of disease modulation by catalytic degradation of a therapeutic target protein suffers from the difficulty to identify candidate proteases, or to engineer their specificity. We identified 23 measurable, specific, and new protease activities using combinatorial screening of 27 human proteases against 24 therapeutic protein targets. We investigate the cleavage of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-13 by matrix metalloproteinases (MMPs) and serine proteases, and demonstrate that cleavage of IL-13 leads to potent inhibition of its biological activity in vitro. MMP-8 degraded human IL-13 most efficiently in vitro and ex vivo in human IL-13 transgenic mouse bronchoalveolar lavage. Hence, MMP-8 is a therapeutic protease lead against IL-13 for inflammatory conditions whereby reported genetic and genomics data suggest an involvement of MMP-8. This work describes the first exploitation of human enzyme promiscuity for therapeutic applications, and reveals both starting points for protease-based therapies and potential new regulatory networks in inflammatory disease.
Asunto(s)
Interleucina-13/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Animales , Dominio Catalítico , Línea Celular , Quimiocina CCL2/metabolismo , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patología , Dermatitis Atópica/terapia , Electroforesis en Gel de Poliacrilamida , Humanos , Interleucina-6/metabolismo , Cinética , Metaloproteinasa 8 de la Matriz/química , Metaloproteinasa 8 de la Matriz/genética , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/química , Ratones , Ratones Transgénicos , Ingeniería de Proteínas , Proteolisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) ligand superfamily and has a proliferative effect on both normal and tumor cells. The TNF family receptors (B-cell maturation antigen (BCMA), transmembrane activator and CAML-interactor (TACI), and BAFF receptor-3 (BR3)) for APRIL and the closely related ligand, B-cell activating factor of the TNF family (BAFF), bind these ligands through a highly conserved six residue DXL motif ((F/Y/W)-D-X-L-(V/T)-(R/G)). Panning peptide phage display libraries led to the identification of several novel classes of APRIL-binding peptides, which could be grouped by their common sequence motifs. Interestingly, only one of these ten classes consisted of peptides containing the DXL motif. Nevertheless, all classes of peptides prevented APRIL, but not BAFF, from binding BCMA, their shared receptor. Synthetic peptides based on selected sequences inhibited APRIL binding to BCMA with IC(50) values of 0.49-27 microM. An X-ray crystallographic structure of APRIL bound to one of the phage-derived peptides showed that the peptide, lacking the DXL motif, was nevertheless bound in the DXL pocket on APRIL. Our results demonstrate that even though a focused, highly conserved motif is required for APRIL-receptor interaction, remarkably, many novel and distinct classes of peptides are also capable of binding APRIL at the ligand receptor interface.
Asunto(s)
Biblioteca de Péptidos , Péptidos/clasificación , Péptidos/aislamiento & purificación , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/antagonistas & inhibidores , Alanina/metabolismo , Secuencia de Aminoácidos , Animales , Antígeno de Maduración de Linfocitos B/química , Antígeno de Maduración de Linfocitos B/metabolismo , Proteínas Inmovilizadas/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis/genética , Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Solubilidad , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/química , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismoRESUMEN
BR3, which is expressed on all mature B cells, is a specific receptor for the B-cell survival and maturation factor BAFF (B-cell-activating factor belonging to the tumor necrosis factor [TNF] family). In order to investigate the consequences of targeting BR3 in murine models and to assess the potential of BR3 antibodies as human therapeutics, synthetic antibody phage libraries were employed to identify BAFF-blocking antibodies cross-reactive to murine and human BR3, which share 52% identity in their extracellular domains. We found an antibody, CB1, which exhibits muM affinity for murine BR3 and very weak affinity for the human receptor. CB3s, an affinity-matured variant of CB1, has sub-nM affinity for BR3 from both species. Alanine scanning and crystallographic structural analysis of the CB3s/BR3 complex reveal that CB3s mimics BAFF by interacting with a similar region of the BR3 surface. Despite this similarity in binding epitopes, CB1 variants antagonize BAFF-dependent human B-cell proliferation in vitro and are effective at reducing murine B-cell populations in vivo, showing significant promise as therapeutics for human B-cell-mediated diseases.
Asunto(s)
Factor Activador de Células B/inmunología , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/inmunología , Linfocitos B/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/química , Sitios de Unión , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Activación de Linfocitos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. TACI binds two ligands, APRIL and BAFF, with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular region; in contrast, BCMA and BR3, the other known high affinity receptors for APRIL and BAFF, respectively, contain only a single or partial CRD. However, another form of TACI exists wherein the N-terminal CRD is removed by alternative splicing. We find that this shorter form is capable of ligand-induced cell signaling and that the second CRD alone (TACI_d2) contains full affinity for both ligands. Furthermore, we report the solution structure and alanine-scanning mutagenesis of TACI_d2 along with co-crystal structures of APRIL.TACI_d2 and APRIL.BCMA complexes that together reveal the mechanism by which TACI engages high affinity ligand binding through a single CRD, and we highlight sources of ligand-receptor specificity within the APRIL/BAFF system.
Asunto(s)
Cisteína , Proteínas de la Membrana/química , Receptores del Factor de Necrosis Tumoral/química , Factor de Necrosis Tumoral alfa/química , Empalme Alternativo , Animales , Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Cristalización , Cristalografía por Rayos X , Humanos , Ligandos , Proteínas de la Membrana/genética , Ratones , Mutagénesis , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Terciaria de Proteína , Receptores del Factor de Necrosis Tumoral/genética , Transducción de Señal , Soluciones , Proteína Activadora Transmembrana y Interactiva del CAML , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis TumoralRESUMEN
B cell maturation antigen (BCMA) is a tumor necrosis factor receptor family member whose physiological role remains unclear. BCMA has been implicated as a receptor for both a proliferation-inducing ligand (APRIL) and B cell-activating factor (BAFF), tumor necrosis factor ligands that bind to multiple tumor necrosis factor receptor and have been reported to play a role in autoimmune disease and cancer. The results presented herein provide a dual perspective analysis of BCMA binding to both APRIL and BAFF. First, we characterized the binding affinity of monomeric BCMA for its ligands; BAFF binding affinity (IC50 = 8 +/- 5 microm) is about 1000-fold reduced compared with the high affinity interaction of APRIL (IC50 = 11 +/- 3 nm). Second, shotgun alanine scanning of BCMA was used to map critical residues for either APRIL or BAFF binding. In addition to a previously described "DXL" motif (Gordon, N. C., Pan, B., Hymowitz, S. G., Yin, J., Kelley, R. F., Cochran, A. G., Yan, M., Dixit, V. M., Fairbrother, W. J., and Starovasnik, M. A. (2003) Biochemistry 42, 5977-5983), the alanine scanning results predicted four amino acid positions in BCMA (Tyr13, Ile22, Gln25, and Arg27) that could impart ligand specificity. Substitution of Tyr13 was tolerated for BAFF binding but not APRIL binding. Arg27 was required for high affinity binding to APRIL, whereas substitutions of this residue had minimal effect on affinity for BAFF. Further phage display experiments suggested the single mutations of I22K, Q25D, and R27Y as providing the greatest difference in APRIL versus BAFF binding affinity. Incorporation of the Q25D and R27Y substitutions into BCMA produced a dual specificity variant, since it has comparable binding affinity for both APRIL and BAFF, IC50 = 350 and 700 nm, respectively. Binding of the I22K mutant of monomeric BCMA to BAFF was undetectable (IC50 > 100 microm), but affinity for binding to APRIL was similar to wild-type BCMA. Based on these results, a BCMA-Fc fusion with the single I22K mutation was produced that binds APRIL, IC50 = 12 nm, and has no measurable affinity for BAFF. These results suggest that APRIL is the preferred ligand for BCMA and show that specificity can be further modified through amino acid substitutions.
Asunto(s)
Proteínas de la Membrana/metabolismo , Ingeniería de Proteínas , Receptores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/metabolismo , Sustitución de Aminoácidos , Factor Activador de Células B , Antígeno de Maduración de Linfocitos B , Linfocitos B/inmunología , Linfocitos B/metabolismo , Sitios de Unión/genética , Humanos , Ligandos , Proteínas de la Membrana/inmunología , Mutación , Mapeo Peptídico , Unión Proteica , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
BAFF/BLyS, a member of the tumor necrosis family (TNF) superfamily of ligands, is a crucial survival factor for B cells. BAFF binds three receptors, TACI, BCMA, and BR3, with signaling through BR3 being essential for promoting B cell function. Typical TNF receptor (TNFR) family members bind their cognate ligands through interactions with two cysteine-rich domains (CRDs). However, the extracellular domain (ECD) of BR3 consists of only a partial CRD, with cysteine spacing distinct from other modules described previously. Herein, we report the solution structure of the BR3 ECD. A core region of only 19 residues adopts a stable structure in solution. The BR3 fold is analogous to the first half of a canonical TNFR CRD but is stabilized by an additional noncanonical disulfide bond. BAFF-binding determinants were identified by shotgun alanine-scanning mutagenesis of the BR3 ECD expressed on phage. Several of the key BAFF-binding residues are presented from a beta-turn that we have shown previously to be sufficient for ligand binding when transferred to a structured beta-hairpin scaffold [Kayagaki, N., Yan, M., Seshasayee, D., Wang, H., Lee, W., French, D. M., Grewal, I. S., Cochran, A. G., Gordon, N. C., Yin, J., Starovasnik, M. A, and Dixit, V. M. (2002) Immunity 10, 515-524]. Outside of the turn, mutagenesis identifies additional hydrophobic contacts that enhance the BAFF-BR3 interaction. The crystal structure of the minimal hairpin peptide, bhpBR3, in complex with BAFF reveals intimate packing of the six-residue BR3 turn into a cavity on the ligand surface. Thus, BR3 binds BAFF through a highly focused interaction site, unprecedented in the TNFR family.
Asunto(s)
Proteínas de la Membrana/química , Receptores del Factor de Necrosis Tumoral/química , Factor de Necrosis Tumoral alfa/química , Secuencia de Aminoácidos , Factor Activador de Células B , Receptor del Factor Activador de Células B , Sitios de Unión , Cisteína/química , Humanos , Técnicas In Vitro , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Soluciones , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The TNF-like ligand BAFF/BLyS is a potent survival factor for B cells. It binds three receptors: TACI, BCMA, and BR3. We show that BR3 signaling promotes processing of the transcription factor NF-kappaB2/p100 to p52. NF-kappaB2/p100 cleavage was abrogated in B cells from A/WySnJ mice possessing a mutant BR3 gene, but not in TACI or BCMA null B cells. Furthermore, wild-type mice injected with BAFF-neutralizing BR3-Fc protein showed reduced basal NF-kappaB2 activation. BR3-Fc treatment of NZB/WF1 mice, which develop a fatal lupus-like syndrome, inhibited NF-kappaB2 processing and attenuated the disease process. Since inhibiting the BR3-BAFF interaction has therapeutic ramifications, the ligand binding interface of BR3 was investigated and found to reside within a 26 residue core domain. When stabilized within a structured beta-hairpin peptide, six of these residues were sufficient to confer binding to BAFF.