RESUMEN
Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.
Asunto(s)
Proteínas Bacterianas/inmunología , Glicosiltransferasas/inmunología , Interacciones Huésped-Patógeno/inmunología , Staphylococcus aureus Resistente a Meticilina/fisiología , Infecciones Estafilocócicas/inmunología , Staphylococcus epidermidis/fisiología , Factores de Virulencia/inmunología , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana/genética , Adhesión Bacteriana/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catepsina G/genética , Catepsina G/inmunología , Catepsina G/metabolismo , Línea Celular Tumoral , Pared Celular/enzimología , Pared Celular/genética , Pared Celular/inmunología , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Ratones , Secuencias Repetitivas de Aminoácido , Infecciones Estafilocócicas/enzimología , Infecciones Estafilocócicas/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The zona pellucida (ZP) domain is a bipartite protein structural element comprised of ZP-N and ZP-C regions. Most notable for its ability to mediate protein polymerization, many ZP proteins polymerize and assemble into long fibrils that form specialized extracellular matrices. Other ZP proteins (namely, betaglycan and endoglin) do not polymerize but serve as important membrane coreceptors for ligands in the transforming growth factor-ß (TGF-ß) superfamily. Here, we present the 2.0-Å resolution crystal structure of the betaglycan ZP-C region in combination with a downstream region known as the external hydrophobic patch (EHP). Similar to the ZP-N region, the ZP-C region also adopts an immunoglobulin-like fold, despite sharing no sequence homology and possessing different disulfide linkages. The EHP region, which was previously thought to be external to the ZP region, is integral to the ZP-C domain and corresponds to the ZP-C G strand. Our structure also indicates that the critical maturation cleavage of ZP proteins, a process that activates nascent ZP proteins for polymerization, occurs within the immunoglobulin domain at the FG loop. Nonpolymerizing ZP proteins such as betaglycan and endoglin do not contain this cleavage site. Finally, our structure suggests that the AB loop and the convex surface pocket are regions important for TGF-ß ligand binding.
Asunto(s)
Estructura Terciaria de Proteína , Proteoglicanos/química , Receptores de Factores de Crecimiento Transformadores beta/química , Factor de Crecimiento Transformador beta/metabolismo , Zona Pelúcida/metabolismo , Animales , Cristalografía por Rayos X , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Polimerizacion , Unión Proteica , Estructura Secundaria de Proteína , Proteoglicanos/genética , Proteoglicanos/metabolismo , Ratas , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
Delta-like ligand 3 (DLL3) is expressed in more than 70% of small cell lung cancers (SCLCs) and other neuroendocrine-derived tumor types. SCLC is highly aggressive and limited therapeutic options lead to poor prognosis for patients. HPN328 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: a CD3 binder for T cell engagement, an albumin binder for half-life extension, and a DLL3 binder for tumor cell engagement. In vitro assays, rodent models and non-human primates were used to assess the activity of HPN328. HPN328 induces potent dose-dependent killing of DLL3-expressing SCLC cell lines in vitro concomitant with T cell activation and cytokine release. In an NCI-H82 xenograft model with established tumors, HPN328 treatment led to T cell recruitment and anti-tumor activity. In an immunocompetent mouse model expressing a human CD3ε epitope, mice previously treated with HPN328 withstood tumor rechallenge, demonstrating long-term anti-tumor immunity. When repeat doses were administered to cynomolgus monkeys, HPN328 was well tolerated up to 10 mg/kg. Pharmacodynamic changes, such as transient cytokine elevation, were observed, consistent with the expected mechanism of action of T cell engagers. HPN328 exhibited linear pharmacokinetic in the given dose range with a serum half-life of 78 to 187 hours, supporting weekly or less frequent administration of HPN328 in humans. Preclinical and nonclinical characterization suggests that HPN328 is a highly efficacious, safe, and novel therapeutic candidate. A phase 1/2 clinical trial is currently underway testing safety and efficacy in patients with DLL3 expressing malignancies.
RESUMEN
Inhibin is a heterodimeric peptide hormone produced in the ovary that antagonizes activin signaling and FSH synthesis in the pituitary. The inhibin ß-subunit interacts with the activin type II receptor (ActRII) to functionally antagonize activin. The inhibin α-subunit mature domain (N terminus) arose relatively early during the evolution of the hormone, and inhibin function is decreased by an antibody directed against the α-subunit N-terminal extension region or by deletion of the N-terminal region. We hypothesized that the α-subunit N-terminal extension region interacts with the activin type I receptor (ALK4) to antagonize activin signaling in the pituitary. Human or chicken free α-subunit inhibited activin signaling in a pituitary gonadotrope-derived cell line (LßT2) in a dose-dependent manner, whereas an N-terminal extension deletion mutant did not. An α-subunit N-terminal peptide, but not a control peptide, was able to inhibit activin A signaling and decrease activin-stimulated FSH synthesis. Biotinylated inhibin A, but not activin A, bound ALK4. Soluble ALK4-ECD bioneutralized human free α-subunit in LßT2 cells, but did not affect activin A function. Competitive binding ELISAs with N-terminal mutants and an N-terminal region peptide confirmed that this region is critical for direct interaction of the α-subunit with ALK4. These data expand our understanding of how endocrine inhibin achieves potent antagonism of local, constitutive activin action in the pituitary, through a combined mechanism of competitive binding of both ActRII and ALK4 by each subunit of the inhibin heterodimer, in conjunction with the co-receptor betaglycan, to block activin receptor-ligand binding, complex assembly, and downstream signaling.
Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Inhibinas/metabolismo , Transducción de Señal/fisiología , Receptores de Activinas Tipo I/genética , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Animales , Células CHO , Pollos , Cricetinae , Cricetulus , Humanos , Inhibinas/genética , Masculino , Ratones , Mutación , Péptidos/genética , Péptidos/metabolismoRESUMEN
Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.
Asunto(s)
Colesterol/sangre , Furina/metabolismo , Proproteína Convertasas/metabolismo , Proteolisis , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Anticuerpos Monoclonales de Origen Murino/química , Colesterol/genética , Furina/genética , Células Hep G2 , Humanos , Hígado/metabolismo , Ratones , Ratones Noqueados , Proproteína Convertasa 9 , Proproteína Convertasas/genética , Estructura Secundaria de Proteína , Receptores de LDL/genética , Serina Endopeptidasas/genéticaRESUMEN
Vascular leakage, or edema, is a serious complication of acute allergic reactions. Vascular leakage is triggered by the release of histamine and serotonin from granules within tissue-resident mast cells. Here, we show that expression of Neutrophil Serine Protease 4 (NSP4) during the early stages of mast cell development regulates mast cell-mediated vascular leakage. In myeloid precursors, the granulocyte-macrophage progenitors (GMPs), loss of NSP4 results in the decrease of cellular levels of histamine, serotonin and heparin/heparan sulfate. Mast cells that are derived from NSP4-deficient GMPs have abnormal secretory granule morphology and a sustained reduction in histamine and serotonin levels. Consequently, in passive cutaneous anaphylaxis and acute arthritis models, mast cell-mediated vascular leakage in the skin and joints is substantially reduced in NSP4-deficient mice. Our findings reveal that NSP4 is required for the proper storage of vasoactive amines in mast cell granules, which impacts mast cell-dependent vascular leakage in mouse models of immune complex-mediated diseases.
Asunto(s)
Mastocitos/enzimología , Serina Proteasas/metabolismo , Traslado Adoptivo , Animales , Complejo Antígeno-Anticuerpo , Regulación Enzimológica de la Expresión Génica , Histamina/metabolismo , Ratones , Ratones Noqueados , Neutrófilos , Serina Proteasas/genética , Serotonina/metabolismoRESUMEN
The recently discovered neutrophil serine protease 4 (NSP4) is the fourth member of the NSP family, which includes the well-studied neutrophil elastase, proteinase 3 and cathepsin G. Like the other three NSP members, NSP4 is synthesized by myeloid precursors in the bone marrow and, after cleavage of the two-amino acid activation peptide, is stored as an active protease in azurophil granules of neutrophils. Based on its primary amino acid sequence, NSP4 is predicted to have a shallow S1 specificity pocket with elastase-like substrate specificity. However, NSP4 was found to preferentially cleave after an arginine residue. Structural studies resolved this paradox by revealing an unprecedented mechanism of P1-arginine recognition. In contrast to the canonical mechanism in which the P1-arginine residue points down into a deep S1 pocket, the arginine side chain adopts a surface-exposed 'up' conformation in the NSP4 active site. This conformation is stabilized by the Phe190 residue, which serves as a hydrophobic platform for the aliphatic portion of the arginine side chain, and a network of hydrogen bonds between the arginine guanidium group and the NSP4 residues Ser192 and Ser216. This unique configuration allows NSP4 to cleave even after naturally modified arginine residues, such as citrulline and methylarginine. This non-canonical mechanism, characterized by the hallmark 'triad' Phe190-Ser192-Ser216, is largely preserved throughout evolution starting with bony fish, which appeared about 400 million years ago. Although the substrates and physiological role of NSP4 remain to be determined, its remarkable evolutionary conservation, restricted tissue expression and homology to other neutrophil serine proteases anticipate a function in immune-related processes.
Asunto(s)
Arginina/química , Neutrófilos/enzimología , Proteolisis , Serina Endopeptidasas/química , Animales , Dominio Catalítico , Humanos , Cinética , Ratones , Especificidad por SustratoRESUMEN
Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs.
Asunto(s)
Neutrófilos/metabolismo , Serina Proteasas/metabolismo , Especificidad por Sustrato/inmunología , Aminoácidos/metabolismo , Humanos , Péptidos/metabolismo , Especificidad por Sustrato/fisiologíaRESUMEN
Trypsin-fold proteases, the largest mammalian protease family, are classified by their primary substrate specificity into one of three categories, trypsin-like, chymotrypsin-like, and elastase-like, based on key structural features of their active site. However, the recently discovered neutrophil serine protease 4 (NSP4, also known as PRSS57) presents a paradox: NSP4 exhibits a trypsin-like specificity for cleaving substrates after arginine residues, but it bears elastase-like specificity determining residues in the active site. Here we show that NSP4 has a fully occluded S1 pocket and that the substrate P1-arginine adopts a noncanonical "up" conformation stabilized by a solvent-exposed H-bond network. This uncommon arrangement, conserved in all NSP4 orthologs, enables NSP4 to process substrates after both arginine as well as post-translationally modified arginine residues, such as methylarginine and citrulline. These findings establish a distinct paradigm for substrate recognition by a trypsin-fold protease and provide insights into the function of NSP4.
Asunto(s)
Serina Endopeptidasas/química , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Unión Proteica , Especificidad por SustratoRESUMEN
The serine protease hepsin is highly upregulated in prostate cancer and is implicated in tumor progression. Therefore, specific inhibition of hepsin enzymatic activity by an antibody constitutes an attractive therapeutic approach. Here, we report the identification of the anti-hepsin antibody Fab25 by screening of a Fab phage display library with a restricted chemical diversity at the complementary determining regions. Hepsin with its S1 pocket occupied by 3,4-dichloro-isocoumarin was used as the 'bait' for library screening. Fab25 was highly specific and it potently inhibited hepsin activity toward a panel of synthetic and macromolecular substrates. Biochemical and enzymatic studies with synthetic substrates of variable length suggested that Fab25 acts as an allosteric inhibitor based on non-competitive inhibition kinetics. Isothermal titration calorimetric experiments showed that the high-affinity (K(D) 6.1 nM) binding of Fab25 with hepsin is enthalpically driven. Despite an unusually long CDR-H3 loop with several potential hepsin cleavage sites (Lys, Arg residues), Fab25 was not processed by hepsin. Antibody-25 should be valuable for investigating hepsin's role in cancer progression and for potential therapeutic applications. Furthermore, the herein presented phage display strategy using an active site-modified protease should be widely applicable for identifying potential allosteric anti-protease antibodies.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/inmunología , Biblioteca de Péptidos , Serina Endopeptidasas/inmunología , Adenocarcinoma/patología , Regulación Alostérica , Animales , Anticuerpos Antineoplásicos/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Sitios de Unión de Anticuerpos , Biotinilación , Dominio Catalítico , Línea Celular Tumoral , Movimiento Celular , Regiones Determinantes de Complementariedad , Humanos , Masculino , Ratones , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Neoplasias de la Próstata/patología , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/inmunología , Serina Endopeptidasas/química , Serina Endopeptidasas/genéticaRESUMEN
The homotrimeric human serine protease HtrA1 is homologous to bacterial HtrA proteases regarding the trypsin-like catalytic and PDZ domains but differs by the presence of an N-terminal domain with IGFBP and Kazal homology. The crystal structures and SAXS analysis presented herein reveal the rare tandem of IGFBP- and Kazal-like modules, a protease active site that adopts a competent conformation in the absence of substrate or inhibitor and a model for the intact protein in solution. Highly sensitive enzymatic assays and binding studies demonstrate that the N-terminal tandem has no apparent effect on protease activity, and in accordance with the structure-based predictions, neither the IGFBP- nor Kazal-like module retains the function of their prototype proteins. Our structures of the unliganded HtrA1 active site suggest two-state equilibrium and a "conformational selection" model, in which substrate binds to the active conformer.
Asunto(s)
Serina Endopeptidasas/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Apoproteínas/química , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Activación Enzimática , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Enlace de Hidrógeno , Isoflurofato/química , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Dispersión del Ángulo Pequeño , Serina Endopeptidasas/genética , Inhibidores de Serina Proteinasa/química , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. The MSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive single-chain form (pro-MSP) into the disulfide-linked α/ß heterodimer. The pro-MSP cleavage sequence (Ser-Lys-Leu-Arg(483)↓Val(484)) closely matches the substrate recognition sequences of hepsin, a type II transmembrane serine protease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at the consensus site Arg(483)-Val(484) with superior efficiency compared with the known activators MT-SP1 and hepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsin concentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variant of MSP weakly bound to a RON-Fc fusion protein, whereas hepsin-cleaved MSP bound with a K(D) of 10.3 nmol/L, suggesting that the high-affinity binding site in MSP ß-chain was properly formed. LNCaP prostate cancer cells overexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsin antibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activated protein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RON protein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependent production of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated by hepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders.
Asunto(s)
Neoplasias Ováricas/metabolismo , Neoplasias de la Próstata/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Serina Endopeptidasas/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Macrófagos/metabolismo , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Precursores de Proteínas/genética , Proteolisis , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/genética , Transducción de SeñalRESUMEN
Transforming growth factor-beta (TGFbeta) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFbeta superfamily that share a common beta-subunit. The evolution of the inhibin alpha-subunit created the only antagonist within the TGFbeta superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin alpha-subunit appear to reflect specific insertion-deletion changes within the inhibin beta-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFbeta superfamily.
Asunto(s)
Inhibinas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Dimerización , Evolución Molecular , Genómica , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The transforming growth factor-beta (TGF-beta) superfamily is a large group of structurally related growth factors that play prominent roles in a variety of cellular processes. The importance and prevalence of TGF-beta signaling are also reflected by the complex network of check points that exist along the signaling pathway, including a number of extracellular antagonists and membrane-level signaling modulators. Recently, a number of important TGF-beta crystal structures have emerged and given us an unprecedented clarity on several aspects of the signal transduction process. This review will highlight these latest advances and present our current understanding on the mechanisms of specificity and regulation on TGF-beta signaling outside the cell.