RESUMEN
Rheumatoid arthritis (RA) involves several classes of pathogenic autoantibodies, some of which react with type-II collagen (COL2) in articular cartilage. We previously described a subset of COL2 antibodies targeting the F4 epitope (ERGLKGHRGFT) that could be regulatory. Here, using phage display, we developed recombinant antibodies against this epitope and examined the underlying mechanism of action. One of these antibodies, R69-4, protected against cartilage antibody- and collagen-induced arthritis in mice, but not autoimmune disease models independent of arthritogenic autoantibodies. R69-4 was further shown to cross-react with a large range of proteins within the inflamed synovial fluid, such as the complement protein C1q. Complexed R69-4 inhibited neutrophil FCGR3 signaling, thereby impairing downstream IL-1ß secretion and neutrophil self-orchestrated recruitment. Likewise, human isotypes of R69-4 protected against arthritis with comparable efficiency. We conclude that R69-4 abrogates autoantibody-mediated arthritis mainly by hindering FCGR3 signaling, highlighting its potential clinical utility in acute RA.
Asunto(s)
Artritis Experimental , Humanos , Animales , Ratones , Artritis Experimental/prevención & control , Neutrófilos , Colágeno , Autoanticuerpos , EpítoposRESUMEN
OBJECTIVE: To investigate the occurrence and frequency of anti-citrullinated protein antibodies (ACPA) to cyclic citrullinated type II collagen (COL2) epitope with a capacity to bind joint cartilage. METHODS: Luminex immunoassay was used to analyze serum antibody reactivity to 10 COL2-citrullinated peptides (ACC10) and corresponding arginine peptide controls in rheumatoid arthritis (RA), osteoarthritis (OA), and healthy individuals' cohorts. Top ten "promiscuous" sera (cross-reactive with all ACC10) and top ten "private" sera (restrictedly reactive with one ACC10 peptide) from RA and OA cohorts were selected. Enzyme-linked immunosorbent assay (ELISA) was used to detect response to native COL2. Sera were analyzed with naive and arthritic joints from DBA/1J mice by immunohistochemistry, using monoclonal ACPAs and COL2 reactive antibodies with human Fc as comparison. Staining specificity was confirmed with C1 (a major antibody epitope on COL2) mutated mice and competitive blocking with epitope-specific antibodies. RESULTS: All patient sera bound ACC10 compared with control peptides but very few (3/40) bound native triple-helical COL2. Most sera (27/40) specifically bound to arthritic cartilage, whereas only one private RA serum bound to healthy cartilage. Despite very low titers, private sera from both RA and OA showed an epitope-specific response, documented by lack of binding to cartilage from C1-mutated mice and blocking binding to wild-type cartilage with a competitive monoclonal antibody. As a comparison, monoclonal ACPAs visualized typical promiscuous, or private reactivity to joint cartilage and other tissues. CONCLUSION: ACPA from RA and OA sera, reactive with citrullinated non-triple-helical COL2 peptides, can bind specifically to arthritic cartilage.
Asunto(s)
Artritis Reumatoide , Osteoartritis , Animales , Humanos , Ratones , Autoanticuerpos , Colágeno Tipo II , Epítopos , Ratones Endogámicos DBA , Mieloblastina , Cartílago/metabolismoRESUMEN
Rheumatoid arthritis (RA) is a chronic autoimmune disorder affecting joints with a hallmark of autoantibody production. Mannan-enhanced collagen type II (COL2) antibody induced arthritis (mCAIA) in neutrophil cytosolic factor 1(Ncf1) mutation mouse is a chronic disease model imitating RA in mice. In this study, we characterize the chronic phase of mCAIA in Ncf1 mutated (BQ.Ncf1m1j/m1j) mice. Arthritis was induced by an intravenous injection of anti-COL2 monoclonal antibodies on day 0 followed by intra-peritoneal injections of mannan (from Saccharomyces cerevisiae) on days 3 and 65 in BQ.Ncf1 m1j/m1j and BQ mice. Bone erosion was analysed by computed tomography (CT) and blood cell phenotypes by flow cytometry. Cytokines and anti-COL2 antibodies were analyzed with multiplex bead-based assays. The arthritis in the Ncf1m1j/m1j mice developed with a chronic and relapsing disease course, which was followed for 200 days and bone erosions of articular joints were evaluated. An increased number of circulating CD11b+ Ly6G+ neutrophils were observed during the chronic phase, together with a higher level of G-CSF (granulocyte colony-stimulating factor) and TNF-α. In conclusion, the chronic relapsing arthritis of mCAIA in the Ncf1m1j/m1j mice develop bone erosions associated with a sustained neutrophil type of inflammatory responses.
RESUMEN
Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen's amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.
Asunto(s)
Adenocarcinoma del Pulmón/fisiopatología , Progresión de la Enfermedad , Glucosiltransferasas/metabolismo , Neoplasias Pulmonares/fisiopatología , Animales , RatonesRESUMEN
Today, it is known that autoimmune diseases start a long time before clinical symptoms appear. Anti-citrullinated protein antibodies (ACPAs) appear many years before the clinical onset of rheumatoid arthritis (RA). However, it is still unclear if and how ACPAs are arthritogenic. To better understand the molecular basis of pathogenicity of ACPAs, we investigated autoantibodies reactive against the C1 epitope of collagen type II (CII) and its citrullinated variants. We found that these antibodies are commonly occurring in RA. A mAb (ACC1) against citrullinated C1 was found to cross-react with several noncitrullinated epitopes on native CII, causing proteoglycan depletion of cartilage and severe arthritis in mice. Structural studies by X-ray crystallography showed that such recognition is governed by a shared structural motif "RG-TG" within all the epitopes, including electrostatic potential-controlled citrulline specificity. Overall, we have demonstrated a molecular mechanism that explains how ACPAs trigger arthritis.
RESUMEN
The design of selective matrix metalloproteinase (MMP) inhibitors that also possess favorable solubility properties has proved to be especially challenging. A prior approach using collagen-model templates combined with transition state analogs produced a first generation of triple-helical peptide inhibitors (THPIs) that were effective in vitro against discrete members of the MMP family. These THPI constructs were also highly water-soluble. The present study sought improvements in the first generation THPIs by enhancing thermal stability and selectivity. A THPI selective for MMP-2 and MMP-9 was redesigned to incorporate non-native amino acids (Flp and mep), resulting in an increase of 18 °C in thermal stability. This THPI was effective in vivo in a mouse model of multiple sclerosis, reducing clinical severity and weight loss. Two other THPIs were developed to be more selective within the collagenolytic members of the MMP family. One of these THPIs was serendipitously more effective against MMP-8 than MT1-MMP and was utilized successfully in a mouse model of sepsis. The THPI targeting MMP-8 minimized lung damage, increased production of the anti-inflammatory cytokine IL-10, and vastly improved mouse survival.
Asunto(s)
Metaloproteinasas de la Matriz/efectos de los fármacos , Péptidos/farmacología , Inhibidores de Proteasas/farmacología , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Humanos , Esclerosis Múltiple/tratamiento farmacológico , Péptidos/química , Péptidos/uso terapéutico , Inhibidores de Proteasas/uso terapéutico , Sepsis/tratamiento farmacológico , Especificidad por SustratoRESUMEN
Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.
Asunto(s)
Metaloproteinasas de la Matriz/metabolismo , Péptidos/química , Péptidos/metabolismo , Metaloproteinasa 1 de la Matriz/química , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 13 de la Matriz/química , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 14 de la Matriz/química , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/química , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/química , Especificidad por SustratoRESUMEN
Matrix metalloproteinases (MMPs) have been implicated in numerous pathologies. An overall lack of selectivity has rendered active-site-targeted MMP inhibitors problematic. The present study describes MMP inhibitors that function by binding both secondary binding sites (exosites) and the active site. Heterotrimeric triple-helical peptide transition-state analogue inhibitors (THPIs) were assembled utilizing click chemistry. Three different heterotrimers were constructed, allowing for the inhibitory phosphinate moiety to be present uniquely in the leading, middle, or trailing strand of the triple helix. All heterotrimeric constructs had sufficient thermally stability to warrant analysis as inhibitors. The heterotrimeric THPIs were effective against MMP-13 and MT1-MMP, with Ki values spanning 100-400 nM. Unlike homotrimeric THPIs, the heterotrimeric THPIs offered complete selectivity between MT1-MMP and MMP-1. Exosite-based approaches such as this provide inhibitors with desired MMP selectivities.
Asunto(s)
Inhibidores de la Metaloproteinasa de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Metaloproteinasas de la Matriz/metabolismo , Péptidos/química , Péptidos/farmacología , Multimerización de Proteína , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
Skeletal development and invasion by tumor cells depends on proteolysis of collagen by the pericellular metalloproteinase MT1-MMP. Its hemopexin-like (HPX) domain binds to collagen substrates to facilitate their digestion. Spin labeling and paramagnetic nuclear magnetic resonance (NMR) detection have revealed how the HPX domain docks to collagen I-derived triple helix. Mutations impairing triple-helical peptidase activity corroborate the interface. Saturation transfer difference NMR suggests rotational averaging around the longitudinal axis of the triple-helical peptide. Part of the interface emerges as unique and potentially targetable for selective inhibition. The triple helix crosses the junction of blades I and II at a 45° angle to the symmetry axis of the HPX domain, placing the scissile Glyâ¼Ile bond near the HPX domain and shifted â¼25 Å from MMP-1 complexes. This raises the question of the MT1-MMP catalytic domain folding over the triple helix during catalysis, a possibility accommodated by the flexibility between domains suggested by atomic force microscopy images.
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Colágeno/química , Colágeno/metabolismo , Metaloproteinasa 14 de la Matriz/química , Metaloproteinasa 14 de la Matriz/metabolismo , Modelos Moleculares , Invasividad Neoplásica/fisiopatología , Secuencia de Aminoácidos , Cristalografía , Humanos , Espectroscopía de Resonancia Magnética , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Proteolisis , Marcadores de SpinRESUMEN
The adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active adipokines, including adiponectin. Recently we developed and characterized a first-in-class peptide-based adiponectin receptor agonist by using in vitro and in vivo models of glioblastoma and breast cancer (BC). In the current study, we further explored the effects of peptide ADP355 in additional cellular models and found that ADP355 inhibited chronic myeloid leukemia (CML) cell proliferation and renal myofibroblast differentiation with mid-nanomolar IC50 values. According to molecular modeling calculations, ADP355 was remarkably flexible in the global minimum with a turn present in the middle of the peptide. Considering these structural features of ADP355 and the fact that adiponectin normally circulates as multimeric complexes, we developed and tested the activity of a linear branched dimer (ADP399). The dimer exhibited approximately 20-fold improved cellular activity inhibiting K562 CML and MCF-7 cell growth with high pM-low nM relative IC50 values. Biodistribution studies suggested superior tissue dissemination of both peptides after subcutaneous administration relative to intraperitoneal inoculation. After screening of a 397-member adiponectin active site library, a novel octapeptide (ADP400) was designed that counteracted 10-1000 nM ADP355- and ADP399-mediated effects on CML and BC cell growth at nanomolar concentrations. ADP400 induced mitogenic effects in MCF-7 BC cells perhaps due to antagonizing endogenous adiponectin actions or acting as an inverse agonist. While the linear dimer agonist ADP399 meets pharmacological criteria of a contemporary peptide drug lead, the peptide showing antagonist activity (ADP400) at similar concentrations will be an important target validation tool to study adiponectin functions.
RESUMEN
Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382-393 and α1(IV)531-543 sequences, which are binding sites for the α2ß1 and α3ß1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl(393) in α1(IV)382-393 and Hyl(540) and Hyl(543) in α1(IV)531-543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3ß1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2ß1 integrin interaction with α1(IV)382-393 but right in the middle of α3ß1 integrin interaction with α1(IV)531-543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no ß-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.
Asunto(s)
Colágeno Tipo IV/metabolismo , Integrina alfa2beta1/metabolismo , Integrina alfa3beta1/metabolismo , Melanoma/metabolismo , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Glicosilación , Humanos , Modelos Moleculares , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
ADAM proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off target toxicity. Targeting secondary substrate binding sites (exosites) can potentially work as an alternative strategy for drug discovery; however, there are only a few reports of potential exosites in ADAM protease structures. In the study presented here, we utilized a series of TNFα-based substrates to probe ADAM10 and 17 interactions with its canonical substrate to identify the structural features that determine ADAM protease substrate specificity. We found that noncatalytic domains of ADAM17 did not directly bind the substrates used in the study but affected the binding nevertheless, most likely because of steric hindrance. Additionally, noncatalytic domains of ADAM17 affected the size/shape of the carbohydrate-binding pocket contained within the catalytic domain of ADAM17. This suggests that noncatalytic domains of ADAM17 play a role in substrate specificity and might help explain differences in substrate repertoires of ADAM17 and its closest homologue, ADAM10. We also addressed the question of which substrate features can affect ADAM protease specificity. We found that all ADAM proteases tested (i.e., ADAM10, 12, and 17) significantly decreased activity when the TNFα-derived sequence was induced into α-helical conformation, suggesting that conformation plays a role in determining ADAM protease substrate specificity. These findings can help in the discovery of ADAM isoform- and substrate-specific inhibitors.
Asunto(s)
Proteínas ADAM/metabolismo , Proteínas ADAM/química , Proteína ADAM17 , Secuencia de Aminoácidos , Dominio Catalítico , Dicroismo Circular , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Especificidad por SustratoRESUMEN
Bacterial infections are becoming increasingly difficult to treat due to the development and spread of antibiotic resistance. Therefore, identifying novel antibacterial targets and new antibacterial agents capable of treating infections by drug-resistant bacteria is of vital importance. The structurally simple yet potent fusaricidin or LI-F class of natural products represents a particularly attractive source of candidates for the development of new antibacterial agents. We synthesized 18 fusaricidin/LI-F analogues and investigated the effects of structure modification on their conformation, serum stability, antibacterial activity, and toxicity toward human cells. Our findings show that substitution of an ester bond in depsipeptides with an amide bond may afford equally potent analogues with improved stability and greatly decreased cytotoxicity. The lower overall hydrophobicity/amphiphilicity of amide analogues in comparison with their parent depsipeptides, as indicated by HPLC retention times, may explain the dissociation of antibacterial activity and human cell cytotoxicity. These results indicate that amide analogues may have significant advantages over fusaricidin/LI-F natural products and their depsipeptide analogues as lead structures for the development of new antibacterial agents.
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Amidas/síntesis química , Depsipéptidos/síntesis química , Diseño de Fármacos , Péptidos Cíclicos/síntesis química , Amidas/química , Amidas/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Depsipéptidos/química , Depsipéptidos/farmacología , Farmacorresistencia Bacteriana , Estabilidad de Medicamentos , Humanos , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Estructura Molecular , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacologíaRESUMEN
A simple and practical general synthetic protocol towards orthogonally protected tHyAsp derivatives fully compatible with Fmoc solid-phase peptide synthetic methodology is reported. Our approach includes enantioresolution of commercially available D: ,L: -tHyAsp racemic mixture by co-crystallization with L: -Lys, followed by ion exchange chromatography yielding enantiomerically pure L: -tHyAsp and D: -tHyAsp, and their selective orthogonal protection. In this way N ( α )-Fmoc protected tHyAsp derivatives were prepared ready for couplings via either α- or ß-carboxylic group onto the resins or the growing peptide chain. In addition, coupling of tHyAsp via ß-carboxylic group onto amino resins allows preparation of peptides containing tHyAsn sequences, further increasing the synthetic utility of prepared tHyAsp derivatives.
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Ácido Aspártico/análogos & derivados , Ácido Aspártico/síntesis química , Ácido Aspártico/química , Estructura Molecular , EstereoisomerismoRESUMEN
The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Matrix metalloproteinase 13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). In the present study, a triple-helical FRET substrate has been utilized for high throughput screening (HTS) of MMP-13 with the MLSCN compound library (n approximately 65,000). Thirty-four compounds from the HTS produced pharmacological dose-response curves. A secondary screen using RP-HPLC validated 25 compounds as MMP-13 inhibitors. Twelve of these compounds were selected for counter-screening with 6 representative MMP family members. Five compounds were found to be broad-spectrum MMP inhibitors, 3 inhibited MMP-13 and one other MMP, and 4 were selective for MMP-13. One of the selective inhibitors was more active against MMP-13 triple-helical peptidase activity compared with single-stranded peptidase activity. Since the THP FRET substrate has distinct conformational features that may interact with MMP secondary binding sites (exosites), novel non-active site-binding inhibitors may be identified via HTS protocols utilizing such assays.
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Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/química , Sitios de Unión , Evaluación Preclínica de Medicamentos , Transferencia Resonante de Energía de Fluorescencia , Humanos , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Péptidos/química , Inhibidores de Proteasas/farmacología , Bibliotecas de Moléculas Pequeñas , Especificidad por SustratoRESUMEN
Research in the field of protease inhibitors is focused on obtaining potent, specific and protease-resistant inhibitors. To our knowledge, there are no reports in the literature that consider the application of N-substituted glycine residues (peptoid monomers) for the design of peptidomimetic protease inhibitors. We hereby present the chemical synthesis and kinetic properties of two new analogues of the trypsin inhibitor SFTI-1 modified at the P1 position. Substitution of Lys5 in SFTI-1 by N-(4-aminobutyl)-glycine and N-benzylglycine, which mimic Lys and Phe, respectively, made these analogues completely protease-resistant at their P1-P1' reactive sites. The analogues synthesised appeared to be potent inhibitors of bovine beta-trypsin and alpha-chymotrypsin. These noncovalent, competitive and selective peptide-peptoid hybrid (peptomeric) inhibitors might open the way to targeting unwanted proteolysis.