RESUMEN
The Atacama Desert in Chile-hyperarid and with high-ultraviolet irradiance levels-is one of the harshest environments on Earth. Yet, dozens of species grow there, including Atacama-endemic plants. Herein, we establish the Talabre-Lejía transect (TLT) in the Atacama as an unparalleled natural laboratory to study plant adaptation to extreme environmental conditions. We characterized climate, soil, plant, and soil-microbe diversity at 22 sites (every 100 m of altitude) along the TLT over a 10-y period. We quantified drought, nutrient deficiencies, large diurnal temperature oscillations, and pH gradients that define three distinct vegetational belts along the altitudinal cline. We deep-sequenced transcriptomes of 32 dominant plant species spanning the major plant clades, and assessed soil microbes by metabarcoding sequencing. The top-expressed genes in the 32 Atacama species are enriched in stress responses, metabolism, and energy production. Moreover, their root-associated soils are enriched in growth-promoting bacteria, including nitrogen fixers. To identify genes associated with plant adaptation to harsh environments, we compared 32 Atacama species with the 32 closest sequenced species, comprising 70 taxa and 1,686,950 proteins. To perform phylogenomic reconstruction, we concatenated 15,972 ortholog groups into a supermatrix of 8,599,764 amino acids. Using two codon-based methods, we identified 265 candidate positively selected genes (PSGs) in the Atacama plants, 64% of which are located in Pfam domains, supporting their functional relevance. For 59/184 PSGs with an Arabidopsis ortholog, we uncovered functional evidence linking them to plant resilience. As some Atacama plants are closely related to staple crops, these candidate PSGs are a "genetic goldmine" to engineer crop resilience to face climate change.
Asunto(s)
Plantas/genética , Altitud , Chile , Cambio Climático , Clima Desértico , Ecosistema , Genómica/métodos , Filogenia , Suelo , Microbiología del SueloRESUMEN
The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral structural protein that is abundant in the circulation of infected individuals. Previous published studies reported controversial data about the role of the N protein in the activation of the complement system. It was suggested that the N protein directly interacts with mannose-binding lectin-associated serine protease-2 (MASP-2) and stimulates lectin pathway overactivation/activity. In order to check these data and to reveal the mechanism of activation, we examined the effect of the N protein on lectin pathway activation. We found that the N protein does not bind to MASP-2 and MASP-1 and it does not stimulate lectin pathway activity in normal human serum. Furthermore, the N protein does not facilitate the activation of zymogen MASP-2, which is MASP-1 dependent. Moreover, the N protein does not boost the enzymatic activity of MASP-2 either on synthetic or on protein substrates. In some of our experiments, we observed that MASP-2 digests the N protein. However, it is questionable, whether this activity is biologically relevant. Although surface-bound N protein did not activate the lectin pathway, it did trigger the alternative pathway in 10% human serum. Additionally, we detected some classical pathway activation by the N protein. Nevertheless, we demonstrated that this activation was induced by the bound nucleic acid, rather than by the N protein itself.
Asunto(s)
Lectina de Unión a Manosa de la Vía del Complemento , Proteínas de la Nucleocápside de Coronavirus , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , SARS-CoV-2 , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , SARS-CoV-2/metabolismo , Proteínas de la Nucleocápside de Coronavirus/metabolismo , COVID-19/virología , COVID-19/metabolismo , COVID-19/inmunología , Fosfoproteínas/metabolismo , Unión Proteica , Activación de ComplementoRESUMEN
Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway-inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.
Asunto(s)
Proteínas de Escherichia coli/química , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/química , Proteínas Periplasmáticas/química , Sitios de Unión , Lectina de Unión a Manosa de la Vía del Complemento , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Lectinas/genética , Lectinas/metabolismo , Lectina de Unión a Manosa/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas Periplasmáticas/metabolismo , Subunidades de ProteínaRESUMEN
Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.
Asunto(s)
Quimasas , Quimotripsina , Inhibidores de Proteasas , Animales , Bovinos , Humanos , Quimasas/antagonistas & inhibidores , Quimasas/química , Quimotripsina/química , Pancreatitis/prevención & control , Inhibidores de Proteasas/química , Inhibidores de Proteasas/aislamiento & purificación , Inhibidores de Proteasas/farmacología , Especificidad por Sustrato , Tripsinógeno , Biblioteca de PéptidosRESUMEN
Factor D (FD) is an essential element of the alternative pathway of the complement system, and it circulates predominantly in cleaved, activated form in the blood. In resting blood, mannose-binding lectin-associated serine protease 3 (MASP-3) is the exclusive activator of pro-FD. Similarly to FD, MASP-3 also circulates mainly in the active form. It was not clear, however, how zymogen MASP-3 is activated. To decipher its activation mechanism, we followed the cleavage of MASP-3 in human hirudin plasma. Our data suggest that neither lectin pathway proteases nor any protease controlled by C1-inhibitor are required for MASP-3 activation. However, EDTA and the general proprotein convertase inhibitor decanoyl-RVKR-chloromethylketone completely prevented activation of exogenous MASP-3 added to blood samples. In this study, we show that proprotein convertase subtilisin/kexin (PCSK) 5 and PCSK6 are able to activate MASP-3 in vitro. Unlike PCSK5, PCSK6 was detected in human serum and plasma, and previously PCSK6 had also been shown to activate corin in the circulation. In all, PCSK6 emerges as the MASP-3 activator in human blood. These findings clarify the very first step of the activation of the alternative pathway and also connect the complement and the proprotein convertase systems in the blood.
Asunto(s)
Vía Alternativa del Complemento/inmunología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunología , Voluntarios Sanos , HumanosRESUMEN
Ecotin is a serine protease inhibitor produced by hundreds of microbial species, including pathogens. Here we show, that ecotin orthologs from Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa and Leishmania major are potent inhibitors of MASP-1 and MASP-2, the two key activator proteases of the complement lectin pathway. Factor D is the key activator protease of another complement activation route, the alternative pathway. We show that ecotin inhibits MASP-3, which is the sole factor D activator in resting human blood. In pathway-specific ELISA tests, we found that all ecotin orthologs are potent lectin pathway inhibitors, and at high concentration, they block the alternative pathway as well. In flow cytometry experiments, we compared the extent of complement-mediated opsonization and lysis of wild-type and ecotin-knockout variants of two E. coli strains carrying different surface lipopolysaccharides. We show, that endogenous ecotin provides significant protections against these microbicidal activities for both bacteria. By using pathway specific complement inhibitors, we detected classical-, lectin- and alternative pathway-driven complement attack from normal serum, with the relative contributions of the activation routes depending on the lipopolysaccharide type. Moreover, in cell proliferation experiments we observed an additional, complement-unrelated antimicrobial activity exerted by heat-inactivated serum. While ecotin-knockout cells are highly vulnerable to these activities, endogenous ecotin of wild-type bacteria provides complete protection against the lectin pathway-related and the complement-unrelated attack, and partial protection against the alternative pathway-related damage. In all, ecotin emerges as a potent, versatile self-defense tool that blocks multiple antimicrobial activities of the serum. These findings suggest that ecotin might be a relevant antimicrobial drug target.
Asunto(s)
Lectina de Unión a Manosa de la Vía del Complemento/fisiología , Proteínas del Sistema Complemento/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Periplasmáticas/metabolismo , Serina Proteasas/sangre , Activación de Complemento/fisiología , Escherichia coli/metabolismo , Humanos , Pseudomonas aeruginosa/metabolismo , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Yersinia pestis/metabolismoRESUMEN
The lectin pathway (LP) of the complement system is an important antimicrobial defense mechanism, but it also contributes significantly to ischemia reperfusion injury (IRI) associated with myocardial infarct, stroke, and several other clinical conditions. Mannan-binding lectin-associated serine proteinase 2 (MASP-2) is essential for LP activation, and therefore, it is a potential drug target. We have previously developed the first two generations of MASP-2 inhibitors by in vitro evolution of two unrelated canonical serine proteinase inhibitors. These inhibitors were selective LP inhibitors, but their nonhuman origin rendered them suboptimal lead molecules for drug development. Here, we present our third-generation MASP-2 inhibitors that were developed based on a human inhibitor scaffold. We subjected the second Kunitz domain of human tissue factor pathway inhibitor 1 (TFPI1 D2) to directed evolution using phage display to yield inhibitors against human and rat MASP-2. These novel TFPI1-based MASP-2 inhibitor (TFMI-2) variants are potent and selective LP inhibitors in both human and rat serum. Directed evolution of the first Kunitz domain of TFPI1 had already yielded the potent kallikrein inhibitor, Kalbitor® (ecallantide), which is an FDA-approved drug to treat acute attacks of hereditary angioedema. Like hereditary angioedema, acute IRI is also related to the uncontrolled activation of a specific plasma serine proteinase. Therefore, TFMI-2 variants are promising lead molecules for drug development against IRI.
Asunto(s)
Lectina de Unión a Manosa de la Vía del Complemento , Evolución Molecular Dirigida , Lipoproteínas , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Inhibidores de Serina Proteinasa , Animales , Humanos , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/antagonistas & inhibidores , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/química , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Péptidos/química , Ratas , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/metabolismoRESUMEN
The complement system is a sophisticated network of proteases. In this article, we describe an unexpected link between two linear activation routes of the complement system: the lectin pathway (LP) and the alternative pathway (AP). Mannose-lectin binding-associated serine protease (MASP)-1 is known to be the initiator protease of the LP. Using a specific and potent inhibitor of MASP-1, SGMI-1, as well as other MASP-1 inhibitors with different mechanisms of action, we demonstrated that, in addition to its functions in the LP, MASP-1 is essential for bacterial LPS-induced AP activation, whereas it has little effect on zymosan-induced AP activation. We have shown that MASP-1 inhibition prevents AP activation, as well as attenuates the already initiated AP activity on the LPS surface. This newly recognized function of MASP-1 can be important for the defense against certain bacterial infections. Our results also emphasize that the mechanism of AP activation depends on the activator surface.
Asunto(s)
Vía Alternativa del Complemento/inmunología , Lectina de Unión a Manosa de la Vía del Complemento/inmunología , Lipopolisacáridos/inmunología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunología , Zimosan/inmunología , Complemento C3/inmunología , Escherichia coli/inmunología , Voluntarios Sanos , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/antagonistas & inhibidores , Pseudomonas aeruginosa/inmunología , Saccharomyces cerevisiae/inmunología , Salmonella typhimurium/inmunologíaRESUMEN
Mannose-binding lectin (MBL)-associated serine proteases (MASPs) are the enzymatic constituents of the lectin pathway of the complement system. They are complexed with large pattern recognition molecules (PRMs) such as MBL, other collectins, and ficolins. The main function of two of the three MASPs has crystallized lately: MASP-1 autoactivates first, then it activates MASP-2, and finally both participate in the formation of the C4b2a convertase. In addition to this, both enzymes are involved in several other processes which are subject to intense research nowadays. Notably, MASP-1, as a promiscuous enzyme, has been implicated in the coagulation cascade, in the kinin generating contact system, and in cellular activation through protease-activated receptor (PAR) cleavage on endothelial cells. The third protease MASP-3 has emerged recently as the protease responsible for pro-factor D activation in resting blood, providing a fundamental link between two complement pathways. At present all three MASPs have at least one well-defined role and several other possible functions were implicated. Defect or more likely over-activation of MASPs may culminate into diseases such as ischemia-reperfusion injury (IRI); hence, MASPs are all potential targets of drug development.
Asunto(s)
Lectina de Unión a Manosa de la Vía del Complemento , Inmunidad Innata , Lectina de Unión a Manosa/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Daño por Reperfusión/inmunología , Animales , Humanos , Terapia Molecular DirigidaRESUMEN
Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reasons for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) to select reversible high affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage display-selected inhibitors, we recombinantly expressed and purified 12 SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B, and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception that was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable, whereas CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.
Asunto(s)
Evolución Molecular , Isoenzimas/metabolismo , Elastasa Pancreática/metabolismo , Secuencia de Aminoácidos , Animales , Bacteriófagos/genética , Humanos , Proteínas de Insectos/metabolismo , Isoenzimas/genética , Cinética , Elastasa Pancreática/genética , Péptidos/metabolismo , Especificidad por Sustrato , PorcinosRESUMEN
Protein-protein interactions (PPIs) formed between short linear motifs and globular domains play important roles in many regulatory and signaling processes but are highly underrepresented in current protein-protein interaction databases. These types of interactions are usually characterized by a specific binding motif that captures the key amino acids shared among the interaction partners. However, the computational proteome-level identification of interaction partners based on the known motif is hindered by the huge number of randomly occurring matches from which biologically relevant motif hits need to be extracted. In this work, we established a novel bioinformatic filtering protocol to efficiently explore interaction network of a hub protein. We introduced a novel measure that enabled the optimization of the elements and parameter settings of the pipeline which was built from multiple sequence-based prediction methods. In addition, data collected from PPI databases and evolutionary analyses were also incorporated to further increase the biological relevance of the identified motif hits. The approach was applied to the dynein light chain LC8, a ubiquitous eukaryotic hub protein that has been suggested to be involved in motor-related functions as well as promoting the dimerization of various proteins by recognizing linear motifs in its partners. From the list of putative binding motifs collected by our protocol, several novel peptides were experimentally verified to bind LC8. Altogether 71 potential new motif instances were identified. The expanded list of LC8 binding partners revealed the evolutionary plasticity of binding partners despite the highly conserved binding interface. In addition, it also highlighted a novel, conserved function of LC8 in the upstream regulation of the Hippo signaling pathway. Beyond the LC8 system, our work also provides general guidelines that can be applied to explore the interaction network of other linear motif binding proteins or protein domains.
Asunto(s)
Dineínas Citoplasmáticas/química , Dineínas Citoplasmáticas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Biología Computacional , Secuencia Conservada , Dineínas Citoplasmáticas/genética , Bases de Datos de Proteínas/estadística & datos numéricos , Evolución Molecular , Vía de Señalización Hippo , Humanos , Filogenia , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Transducción de SeñalRESUMEN
It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin-associated serine protease (MASP)-1/3(-/-) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of â¼ 3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator.
Asunto(s)
Vía Alternativa del Complemento/inmunología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunología , Factor D del Complemento/inmunología , Inhibidores Enzimáticos/farmacología , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Espectrometría de MasasRESUMEN
Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRMs) from the lectin pathway bind CC and function as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using recombinant proteins, specific inhibitors, as well as deficient and normal sera. Additionally, we examined the deposition of ficolin-2 and MBL in human carotid plaques by immunohistochemistry and fluorescence microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL in vitro, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium-dependent manner whereas ficolin-2 binding was calcium-independent. No binding was observed for ficolin-1 or ficolin-3. MBL and ficolin-2 were present in human carotid plaques, and binding of MBL to CC was confirmed in vivo by immunohistochemistry, showing localization of MBL around CC clefts. Moreover, we demonstrated that IgM, but not IgG, bound to CC in vitro and that C1q binding was facilitated by IgM. In conclusion, our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis.
Asunto(s)
Aterosclerosis/inmunología , Aterosclerosis/fisiopatología , Activación de Complemento , Progresión de la Enfermedad , Lectinas/metabolismo , Lectina de Unión a Manosa/metabolismo , Calcio/metabolismo , Estenosis Carotídea/inmunología , Colesterol/química , Colesterol/inmunología , Colesterol/metabolismo , Colesterol/farmacología , Complemento C4/metabolismo , Cristalización , Técnica del Anticuerpo Fluorescente , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Inflamación , Lectinas/inmunología , Lectina de Unión a Manosa/inmunología , Microscopía Fluorescente , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Proteínas Recombinantes/metabolismo , FicolinasRESUMEN
The lectin pathway of complement activation is an important component of the innate immune defense. The initiation complexes of the lectin pathway consist of a recognition molecule and associated serine proteases. Until now the autoactivating mannose-binding lectin-associated serine protease (MASP)-2 has been considered the autonomous initiator of the proteolytic cascade. The role of the much more abundant MASP-1 protease was controversial. Using unique, monospecific inhibitors against MASP-1 and MASP-2, we corrected the mechanism of lectin-pathway activation. In normal human serum, MASP-2 activation strictly depends on MASP-1. MASP-1 activates MASP-2 and, moreover, inhibition of MASP-1 prevents autoactivation of MASP-2. Furthermore we demonstrated that MASP-1 produces 60% of C2a responsible for C3 convertase formation.
Asunto(s)
Activación de Complemento , Lectinas/metabolismo , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Coagulación Sanguínea , Convertasas de Complemento C3-C5/metabolismo , Activación Enzimática , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/antagonistas & inhibidoresRESUMEN
Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is â¼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.
Asunto(s)
Lectina de Unión a Manosa de la Vía del Complemento , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/química , Catálisis , Proteínas del Sistema Complemento , Humanos , Inmunidad Innata , Cinética , Lectinas/química , Lectinas de Unión a Manosa/química , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Mutación , Péptidos/química , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 Å resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme.
Asunto(s)
Lectina de Unión a Manosa de la Vía del Complemento , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/química , Inhibidores de Proteasas/química , Cristalografía por Rayos X , Humanos , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/antagonistas & inhibidores , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Proteases play important roles in human physiology and pathology. The complement system is a proteolytic cascade, where serine proteases activate each other by limited proteolysis in a strictly ordered manner. Serine proteases are essential in both the initiation and the amplification of the cascade. Since uncontrolled complement activation contributes to the development of serious disease conditions, inhibition of the complement serine proteases could be an attractive therapeutic approach. In this chapter, we give a brief overview of the major types of natural serine protease inhibitors and their role in controlling the complement cascade. A special emphasis is laid on C1-inhibitor, a natural complement protease inhibitor, which is approved for clinical use in hereditary angioedema (HAE). We also examine the potential of developing artificial complement protease inhibitors. Synthetic small-molecule drugs can be very efficient serine protease inhibitors, but they usually lack sufficient specificity. A promising approach to yield more specific compounds is the alteration of natural protease inhibitors through engineering or directed evolution resulting in new variants with fine-tuned specificity and enhanced affinity.
Asunto(s)
Proteínas del Sistema Complemento/fisiología , Inhibidores de Serina Proteinasa/farmacología , Animales , Proteína Inhibidora del Complemento C1/farmacología , Humanos , Ingeniería de Proteínas , Serina Proteasas/fisiología , Inhibidores de Serina Proteinasa/uso terapéutico , Bibliotecas de Moléculas PequeñasRESUMEN
Background: Haemostasis is a crucial process by which the body stops bleeding. It is achieved by the formation of a platelet plug, which is strengthened by formation of a fibrin mesh mediated by the coagulation cascade. In proinflammatory and prothrombotic conditions, multiple interactions of the complement system and the coagulation cascade are known to aggravate thromboinflammatory processes and increase the risk of arterial and venous thrombosis. Whether those interactions also play a relevant role during the physiological process of haemostasis is not yet completely understood. The aim of this study was to investigate the potential role of complement components and activation during the haemostatic response to mechanical vessel injury. Methods: We used a microvascular bleeding model that simulates a blood vessel, featuring human endothelial cells, perfusion with fresh human whole blood, and an inducible mechanical injury to the vessel. We studied the effects of complement inhibitors against components of the lectin (MASP-1, MASP-2), classical (C1s), alternative (FD) and common pathways (C3, C5), as well as a novel triple fusion inhibitor of all three complement pathways (TriFu). Effects on clot formation were analysed by recording of fibrin deposition and the platelet activation marker CD62P at the injury site in real time using a confocal microscope. Results: With the inhibitors targeting MASP-2 or C1s, no significant reduction of fibrin formation was observed, while platelet activation was significantly reduced in the presence of the FD inhibitor. Both common pathway inhibitors targeting C3 or C5, respectively, were associated with a substantial reduction of fibrin formation, and platelet activation was also reduced in the presence of the C3 inhibitor. Triple inhibition of all three activation pathways at the C3-convertase level by TriFu reduced both fibrin formation and platelet activation. When several complement inhibitors were directly compared in two individual donors, TriFu and the inhibitors of MASP-1 and C3 had the strongest effects on clot formation. Conclusion: The observed impact of complement inhibition on reducing fibrin clot formation and platelet activation suggests a role of the complement system in haemostasis, with modulators of complement initiation, amplification or effector functions showing distinct profiles. While the interactions between complement and coagulation might have evolved to support haemostasis and protect against bleeding in case of vessel injury, they can turn harmful in pathological conditions when aggravating thromboinflammation and promoting thrombosis.