RESUMEN
Collagen posttranslational processing is crucial for its proper assembly and function. Disruption of collagen processing leads to tissue development and structure disorders like osteogenesis imperfecta (OI). OI-related collagen processing machinery includes prolyl 3-hydroxylase 1 (P3H1), peptidyl-prolyl cis-trans isomerase B (PPIB), and cartilage-associated protein (CRTAP), with their structural organization and mechanism unclear. We determine cryo-EM structures of the P3H1/CRTAP/PPIB complex. The active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled modification mechanism. The structure of the P3H1/CRTAP/PPIB/collagen peptide complex reveals multiple binding sites, suggesting a substrate interacting zone. Unexpectedly, a dual-ternary complex is observed, and the balance between ternary and dual-ternary states can be altered by mutations in the P3H1/PPIB active site and the addition of PPIB inhibitors. These findings provide insights into the structural basis of collagen processing by P3H1/CRTAP/PPIB and the molecular pathology of collagen-related disorders.
Asunto(s)
Colágeno , Microscopía por Crioelectrón , Ciclofilinas , Proteínas de la Matriz Extracelular , Humanos , Colágeno/metabolismo , Colágeno/química , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Ciclofilinas/metabolismo , Ciclofilinas/química , Ciclofilinas/genética , Dominio Catalítico , Isomerasa de Peptidilprolil/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/genética , Procesamiento Proteico-Postraduccional , Sitios de Unión , Unión Proteica , Autoantígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Modelos Moleculares , Mutación , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/química , Glicoproteínas de Membrana , Proteoglicanos , Chaperonas Moleculares , Prolil HidroxilasasRESUMEN
Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.
Asunto(s)
Dipéptidos , Isoenzimas , Prolil Hidroxilasas , Prolil Hidroxilasas/genética , Isoenzimas/genética , Colágeno Tipo I/genética , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/metabolismo , Colágeno/genética , Colágeno/metabolismo , Prolina/metabolismoRESUMEN
The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen-dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C-terminal HIF2α-ODD in the presence of its 2-oxoglutarate cosubstrate or N-oxalylglycine inhibitor. Combined with the reported PHD2.HIFα-ODD structures and biochemical studies, the results inform on the different PHD.HIFα-ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform-selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/química , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Oxígeno/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Prolil Hidroxilasas , Isoformas de ProteínasRESUMEN
Molecular oxygen is essential for all multicellular life forms. In humans, the hypoxia-inducible factor (HIF) prolyl hydroxylase domain-containing enzymes (PHDs) serve as important oxygen sensors by regulating the activity of HIF, the master regulator that mediates cellular oxygen homeostasis, in an oxygen-dependent manner. In normoxia, PHDs catalyze the prolyl hydroxylation of HIF, which leads to its degradation and prevents cellular hypoxic response to be triggered. PHDs are current inhibition targets for the potential treatments of a number of diseases. In this chapter, we discuss in vitro and cell-based methods to study the modulation of PHD2, the most important human PHD isoform in normoxia and mild hypoxia. These include the production and purification of recombinant PHD2, the use of mass spectrometry to follow PHD2-catalyzed reactions and the studies of HIF stabilization in cells by immunoblotting.
Asunto(s)
Prolina Dioxigenasas del Factor Inducible por Hipoxia , Oxígeno , Humanos , Oxígeno/metabolismo , Hipoxia/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/metabolismo , Isoformas de ProteínasRESUMEN
Collagen, the most abundant organic compound of vertebrate organisms, is a supramolecular, protein-made polymer. Details of its post-translational maturation largely determine the mechanical properties of connective tissues. Its assembly requires massive, heterogeneous prolyl-4-hydroxylation (P4H), catalyzed by Prolyl-4-hydroxylases (P4HA1-3), providing thermostability to its elemental, triple helical building block. So far, there was no evidence of tissue-specific regulation of P4H, nor of a differential substrate repertoire of P4HAs. Here, the post-translational modifications of collagen extracted from bone, skin, and tendon were compared, revealing lower hydroxylation of most GEP/GDP triplets, together with fewer other residue positions along collagen a chains, in the tendon. This regulation is mostly conserved in two distant homeotherm species, mouse and chicken. The comparison of detailed P4H patterns in both species suggests a two-step mechanism of specificity. P4ha2 expression is low in tendon and its genetic invalidation in the ATDC5 cellular model of collagen assembly specifically mimics the tendon-related P4H profile. Therefore, P4HA2 has a better ability than other P4HAs to hydroxylate the corresponding residue positions. Its local expression participates in determining the P4H profile, a novel aspect of the tissue specificities of collagen assembly.
Asunto(s)
Colágeno , Procolágeno-Prolina Dioxigenasa , Ratones , Animales , Hidroxilación , Colágeno/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Prolil Hidroxilasas/química , Matriz Extracelular/metabolismoRESUMEN
Prolyl hydroxylase domain protein 2 (PHD2)-catalyzed modification of hypoxia-inducible factor (HIF)-α is a key event in oxygen sensing. We previously showed that the zinc finger of PHD2 binds to a Pro-Xaa-Leu-Glu (PXLE) motif. Here, we show that the zinc finger binds to this motif in the ribosomal chaperone nascent polypeptide complex-α (NACA). This recruits PHD2 to the translation machinery to cotranslationally modify HIF-α. Importantly, this cotranslational modification is enhanced by a translational pause sequence in HIF-α. Mice with a knock-in Naca gene mutation that abolishes the PXLE motif display erythrocytosis, a reflection of HIF pathway dysregulation. In addition, human erythrocytosis-associated mutations in the zinc finger of PHD2 ablate interaction with NACA. Tibetans, who have adapted to the hypoxia of high altitude, harbor a PHD2 variant that we previously showed displays a defect in zinc finger binding to p23, a PXLE-containing HSP90 cochaperone. We show here that Tibetan PHD2 maintains interaction with NACA, thereby showing differential interactions with PXLE-containing proteins and providing an explanation for why Tibetans are not predisposed to erythrocytosis.
Asunto(s)
Policitemia , Humanos , Ratones , Animales , Policitemia/genética , Policitemia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Dedos de Zinc , Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
Human protein disulfide isomerase A1 (PDIA1) shows both catalytic (i.e., oxidoreductase) and non-catalytic (i.e., chaperone) activities and plays a crucial role in the oxidative folding of proteins within the endoplasmic reticulum. PDIA1 dysregulation is a common trait in numerous pathophysiological conditions, including neurodegenerative disorders and cancerous diseases. The 1178A>G mutation of the human PDIA1-encoding gene is a non-synonymous single nucleotide polymorphism detected in patients with Cole-Carpenter syndrome type 1 (CSS1), a particularly rare bone disease. In vitro studies showed that the encoded variant (PDIA1 Y393C) exhibits limited oxidoreductase activity. To gain knowledge on the structure-function relationship, we undertook a molecular dynamics (MD) approach to examine the structural stability of PDIA1 Y393C. Results showed that significant conformational changes are the structural consequence of the amino acid substitution Tyr>Cys at position 393 of the PDIA1 protein. This structure-based study provides further knowledge about the molecular origin of CCS1.
Asunto(s)
Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/genética , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Craneosinostosis/genética , Retículo Endoplásmico/metabolismo , Anomalías del Ojo/genética , Humanos , Hidrocefalia/genética , Simulación de Dinámica Molecular , Osteogénesis Imperfecta/genética , Oxidación-ReducciónRESUMEN
Accumulated unfolded proteins in the endoplasmic reticulum (ER) trigger the unfolded protein response (UPR) to increase ER protein folding capacity. ER proteostasis and UPR signaling need to be regulated in a precise and timely manner. Here, we identify phosphorylation of protein disulfide isomerase (PDI), one of the most abundant and critical folding catalysts in the ER, as an early event during ER stress. The secretory pathway kinase Fam20C phosphorylates Ser357 of PDI and responds rapidly to various ER stressors. Phosphorylation of Ser357 induces an open conformation of PDI and turns it from a "foldase" into a "holdase", which is critical for preventing protein misfolding in the ER. Phosphorylated PDI also binds to the lumenal domain of IRE1α, a major UPR signal transducer, and attenuates excessive IRE1α activity. Importantly, PDI-S359A knock-in mice display enhanced IRE1α activation and liver damage under acute ER stress. We conclude that the Fam20C-PDI axis constitutes a post-translational response to maintain ER proteostasis and plays a vital role in protecting against ER stress-induced cell death.
Asunto(s)
Quinasa de la Caseína I/metabolismo , Endorribonucleasas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Estrés del Retículo Endoplásmico , Femenino , Células HeLa , Células Hep G2 , Humanos , Masculino , Ratones , Modelos Moleculares , Fosforilación , Conformación Proteica , Proteostasis , Respuesta de Proteína DesplegadaRESUMEN
Optimization of protein formulations at subzero temperatures is required for many applications such as storage, transport, and lyophilization. Using isochoric cooling (constant volume) is possible to reach subzero temperatures without freezing aqueous solutions. This accelerates protein damage as protein may unfold by cold denaturation and diffusional and conformational freedom is still present. The use of isochoric cooling to faster protein formulations was first demonstrated for the biomedical relevant protein disulfide isomerase A1. Three osmolytes, sucrose, glycerol, and l-arginine, significantly increased the stability of protein disulfide isomerase A1 at -20°C with all tested under isochoric cooling within the short time frame of 700 h. The redox green fluorescent protein 2 was used to evaluate the applicability of isochoric cooling for stability analysis of highly stable proteins. This derivative of GFP is 2.6-fold more stable than the highly stable GFP ß-barrel structure. Nevertheless, it was possible to denature a fraction of roGFP2 at -20°C and to assign a stabilizing effect to sucrose. Isochoric cooling was further applied to insulin. Protein damage was evaluated through a signaling event elicited on human hepatocyte carcinoma cells. Insulin at -20°C under isochoric cooling lost 22% of its function after 15 days and 0.6M sucrose prevented insulin deactivation.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Proteínas Recombinantes/química , Frío , Composición de Medicamentos , Estabilidad de Medicamentos , Congelación , Hemoglobinas/química , Células Hep G2 , Humanos , Insulina/química , Insulina/farmacología , Transición de Fase , Procolágeno-Prolina Dioxigenasa/química , Desnaturalización Proteica , Proteína Disulfuro Isomerasas/química , Replegamiento Proteico , Estabilidad Proteica , TermodinámicaRESUMEN
Osteogenesis imperfecta (OI) is a rare heritable bone disorder characterized by low bone mineral density (BMD), recurrent bone fractures, and progressive bone deformities. P4HB encodes protein disulfide isomerase (PDI) and is identified as a novel candidate gene of OI. The purposes of the present study are to detect pathogenic mutation, to evaluate the phenotypes of a Chinese family with mild OI, and to investigate the effects of bisphosphonates on bone of the proband. We detected the pathogenic mutation by next generation sequencing and Sanger sequencing. Laboratory and radiological investigations were conducted to evaluate the phenotypes. The proband was a 12-year-old girl with low BMD, history of recurrent non-traumatic fractures, slight scoliosis, with bluish grey sclera and ligamentous laxity. Her father suffered from one fragility fracture and slight wedge changes of vertebras, with bluish grey sclera. We identified a novel heterozygous missense mutation (c.692A>C, p.His231Pro) in P4HB in the proband and her father. This mutation was predicted to affect the combination of PDI with type I procollagen and lead to the disorder of its triple helix formation. Bisphosphonates were effective in reducing bone resorption and increasing BMD of the proband with well tolerance. In conclusion, we identified a novel mutation in P4HB in a Chinese family with mild OI, which expanded the genotypic and phenotypic spectrum of OI. Bisphosphonates were effective to this extremely rare OI induced by P4HB mutation.
Asunto(s)
Mutación Missense , Osteogénesis Imperfecta/genética , Procolágeno-Prolina Dioxigenasa/genética , Proteína Disulfuro Isomerasas/genética , Adulto , Niño , Femenino , Heterocigoto , Humanos , Masculino , Modelos Moleculares , Osteogénesis Imperfecta/patología , Linaje , Procolágeno-Prolina Dioxigenasa/química , Proteína Disulfuro Isomerasas/químicaRESUMEN
The hydroxylation of prolyl-residues in eukaryotes is important in collagen biosynthesis and in hypoxic signalling. The hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are drug targets for the treatment of anaemia, while the procollagen prolyl hydroxylases and other 2-oxoglutarate dependent oxygenases are potential therapeutic targets for treatment of cancer, fibrotic disease, and infection. We describe assay development and inhibition studies for a procollagen prolyl hydroxylase from Paramecium bursaria chlorella virus 1 (vCPH). The results reveal HIF PHD inhibitors in clinical trials also inhibit vCPH. Implications for the targeting of the human PHDs and microbial prolyl hydroxylases are discussed.
Asunto(s)
Procolágeno-Prolina Dioxigenasa/química , Inhibidores de Prolil-Hidroxilasa/química , Pruebas de Enzimas , Hidroxilación , Prolina Dioxigenasas del Factor Inducible por Hipoxia/química , Ácidos Cetoglutáricos/química , Oligopéptidos/química , Phycodnaviridae/enzimología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Protein disulfide isomerase A1 (PDIA1) is an endoplasmic reticulum (ER)-localized thiol-disulfide oxidoreductase that is an important folding catalyst for secretory pathway proteins. PDIA1 contains two active-site domains (a and a'), each containing a Cys-Gly-His-Cys (CGHC) active-site motif. The two active-site domains share 37% sequence identity and function independently to perform disulfide-bond reduction, oxidation, and isomerization. Numerous inhibitors for PDIA1 have been reported, yet the selectivity of these inhibitors toward the a and a' sites is poorly characterized. Here, we identify a potent and selective PDIA1 inhibitor, KSC-34, with 30-fold selectivity for the a site over the a' site. KSC-34 displays time-dependent inhibition of PDIA1 reductase activity in vitro with a kinact/ KI of 9.66 × 103 M-1 s-1 and is selective for PDIA1 over other members of the PDI family, and other cellular cysteine-containing proteins. We provide the first cellular characterization of an a-site selective PDIA1 inhibitor and demonstrate that KSC-34 has minimal sustained effects on the cellular unfolded protein response, indicating that a-site inhibition does not induce global protein folding-associated ER stress. KSC-34 treatment significantly decreases the rate of secretion of a destabilized, amyloidogenic antibody light chain, thereby minimizing pathogenic amyloidogenic extracellular proteins that rely on high PDIA1 activity for proper folding and secretion. Given the poor understanding of the contribution of each PDIA1 active site to the (patho)physiological functions of PDIA1, site selective inhibitors like KSC-34 provide useful tools for delineating the pathological role and therapeutic potential of PDIA1.
Asunto(s)
Inhibidores Enzimáticos/química , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Procolágeno-Prolina Dioxigenasa/química , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/química , Pliegue de Proteína , Dominio Catalítico , Humanos , Procolágeno-Prolina Dioxigenasa/genética , Proteína Disulfuro Isomerasas/genéticaRESUMEN
Protein-disulfide isomerase (PDI) is a ubiquitous dithiol-disulfide oxidoreductase that performs an array of cellular functions, such as cellular signaling and responses to cell-damaging events. PDI can become dysfunctional by post-translational modifications, including those promoted by biological oxidants, and its dysfunction has been associated with several diseases in which oxidative stress plays a role. Because the kinetics and products of the reaction of these oxidants with PDI remain incompletely characterized, we investigated the reaction of PDI with the biological oxidant peroxynitrite. First, by determining the rate constant of the oxidation of PDI's redox-active Cys residues (Cys53 and Cys397) by hydrogen peroxide (k = 17.3 ± 1.3 m-1 s-1 at pH 7.4 and 25 °C), we established that the measured decay of the intrinsic PDI fluorescence is appropriate for kinetic studies. The reaction of these PDI residues with peroxynitrite was considerably faster (k = (6.9 ± 0.2) × 104 m-1 s-1), and both Cys residues were kinetically indistinguishable. Limited proteolysis, kinetic simulations, and MS analyses confirmed that peroxynitrite preferentially oxidizes the redox-active Cys residues of PDI to the corresponding sulfenic acids, which reacted with the resolving thiols at the active sites to produce disulfides (i.e. Cys53-Cys56 and Cys397-Cys400). A fraction of peroxynitrite, however, decayed to radicals that hydroxylated and nitrated other active-site residues (Trp52, Trp396, and Tyr393). Excess peroxynitrite promoted further PDI oxidation, nitration, inactivation, and covalent oligomerization. We conclude that these PDI modifications may contribute to the pathogenic mechanism of several diseases associated with dysfunctional PDI.
Asunto(s)
Ácido Peroxinitroso/química , Procolágeno-Prolina Dioxigenasa/química , Proteína Disulfuro Isomerasas/química , Tolueno/análogos & derivados , Secuencias de Aminoácidos , Humanos , Oxidación-Reducción , Tolueno/químicaRESUMEN
In native tissues, cellular organization is predominantly anisotropic. Yet, it remains a challenge to engineer anisotropic scaffolds that promote anisotropic cellular organization at macroscopic length scales. To overcome this challenge, an innovative, cheap and easy method to align clinically approved non-woven surgical microfibrillar scaffolds is presented. The method involves a three-step process of coating, unidirectional stretching of scaffolds after heating them above glass transition temperature, and cooling back to room temperature. Briefly, a polymer coating is applied to a non-woven mesh that results in a partial welding of randomly oriented microfibers at their intersection points. The coated scaffold is then heated above the glass transition temperature of the coating and the scaffold polymer. Subsequently, the coated scaffold is stretched to produce aligned and three dimentional (3D) porous fibrillar scaffolds. In a proof of concept study, a polyglycolic acid (PGA) micro-fibrillar scaffold was coated with poly(4-hydroxybutirate) (P4HB) acid and subsequently aligned. Fibroblasts were cultured in vitro within the scaffold and results showed an increase in cellular alignment along the direction of the PGA fibers. This method can be scaled up easily for industrial production of polymeric meshes or directly applied to small pieces of scaffolds at the point of care.
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Procedimientos de Cirugía Plástica/métodos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Animales , Humanos , Ácido Poliglicólico/química , Porosidad , Procolágeno-Prolina Dioxigenasa/química , Proteína Disulfuro Isomerasas/químicaRESUMEN
Essentials Inhibitors of protein disulfide isomerase (PDI) have been considered a new antithrombotic class. CxxC is a PDI-targeted peptide that has been previously shown to inhibit its reductase activity. CxxC binds to surface PDI and inhibits ADP- and thrombin-evoked platelet activation and aggregation. CxxC binds to Cys400 on CGHC redox motif of PDI a' domain, a site for PDI prothrombotic activity. SUMMARY: Background Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI's actions. Conclusion In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.
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Inhibidores de Agregación Plaquetaria/química , Procolágeno-Prolina Dioxigenasa/química , Proteína Disulfuro Isomerasas/química , Alanina/química , Secuencias de Aminoácidos , Plaquetas/metabolismo , Dominio Catalítico , Cisteína/química , Disulfuros , Humanos , Oxidación-Reducción , Péptidos/química , Activación Plaquetaria , Agregación Plaquetaria , Unión Proteica , Dominios Proteicos , Pliegue de ProteínaRESUMEN
Collagen prolyl 4-hydroxylases (CP4Hs) catalyze a prevalent posttranslational modification, the hydroxylation of (2S)-proline residues in protocollagen strands. The ensuing (2S,4R)-4-hydroxyproline residues are necessary for the conformational stability of the collagen triple helix. Prolyl peptide bonds isomerize between cis and trans isomers, and the preference of the enzyme is unknown. We synthesized alkene isosteres of the cis and trans isomers to probe the conformational preferences of human CP4H1. We discovered that the presence of a prolyl peptide bond is necessary for catalysis. The cis isostere is, however, an inhibitor with a potency greater than that of the trans isostere, suggesting that the cis conformation of a prolyl peptide bond is recognized preferentially. Comparative studies with a Chlamydomonas reinhardtii P4H, which has a similar catalytic domain but lacks an N-terminal substrate-binding domain, showed a similar preference for the cis isostere. These findings support the hypothesis that the catalytic domain of CP4Hs recognizes the cis conformation of the prolyl peptide bond and inform the use of alkenes as isosteres for peptide bonds.
Asunto(s)
Alquenos/química , Hidroxiprolina/química , Péptidos/química , Procolágeno-Prolina Dioxigenasa/química , Prolina/química , Prolil Hidroxilasas/química , Alquenos/metabolismo , Dominio Catalítico , Chlamydomonas reinhardtii/enzimología , Humanos , Hidroxilación , Hidroxiprolina/metabolismo , Isomerismo , Cinética , Modelos Químicos , Estructura Molecular , Péptidos/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Prolina/metabolismo , Prolil Hidroxilasas/metabolismo , Unión Proteica , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Proline hydroxylation is the most prevalent post-translational modification in collagen. The resulting product trans-4-hydroxyproline (Hyp) is of critical importance for the stability and thus function of collagen, with defects leading to several diseases. Prolyl 4-hydroxylases (P4Hs) are mononuclear non-heme iron α-ketoglutarate (αKG)-dependent dioxygenases that catalyze Hyp formation. Although animal and plant P4Hs target peptidyl proline, prokaryotes have been known to use free l-proline as a precursor to form Hyp. The P4H from Bacillus anthracis (BaP4H) has been postulated to act on peptidyl proline in collagen peptides, making it unusual within the bacterial clade, but its true physiological substrate remains enigmatic. Here we use mass spectrometry, fluorescence binding, x-ray crystallography, and docking experiments to confirm that BaP4H recognizes and acts on peptidyl substrates but not free l-proline, using elements characteristic of an Fe(II)/αKG-dependent dioxygenases. We further show that BaP4H can hydroxylate unique peptidyl proline sites in collagen-derived peptides with asymmetric hydroxylation patterns. The cofactor-bound crystal structures of BaP4H reveal active site conformational changes that define open and closed forms and mimic "ready" and "product-released" states of the enzyme in the catalytic cycle. These results help to clarify the role of BaP4H as well as provide broader insights into human collagen P4H and proteins with poly-l-proline type II helices.
Asunto(s)
Bacillus anthracis/enzimología , Proteínas Bacterianas/química , Procolágeno-Prolina Dioxigenasa/química , Secuencia de Aminoácidos , Dominio Catalítico , Colágeno/química , Cristalografía por Rayos X , Humanos , Hidroxilación , Cinética , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Prolina/química , Unión Proteica , Estructura Secundaria de Proteína , Especificidad por SustratoRESUMEN
Collagen is the most abundant protein in animals. Its overproduction is associated with fibrosis and cancer metastasis. The stability of collagen relies on post-translational modifications, the most prevalent being the hydroxylation of collagen strands by collagen prolyl 4-hydroxylases (CP4Hs). Catalysis by CP4Hs enlists an iron cofactor to convert proline residues to 4-hydroxyproline residues, which are essential for the conformational stability of mature collagen. Ethyl 3,4-dihydroxybenzoate (EDHB) is commonly used as a "P4H" inhibitor in cells, but suffers from low potency, poor selectivity, and off-target effects that cause iron deficiency. Dicarboxylates of 2,2'-bipyridine are among the most potent known CP4H inhibitors but suffer from a high affinity for free iron. A screen of biheteroaryl compounds revealed that replacing one pyridyl group with a thiazole moiety retains potency and enhances selectivity. A diester of 2-(5-carboxythiazol-2-yl)pyridine-5-carboxylic acid is bioavailable to human cells and inhibits collagen biosynthesis at concentrations that neither cause general toxicity nor disrupt iron homeostasis. These data anoint a potent and selective probe for CP4H and a potential lead for the development of a new class of antifibrotic and antimetastatic agents.
Asunto(s)
Ácidos Carboxílicos/farmacología , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Ácidos Carboxílicos/química , Ácidos Carboxílicos/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Procolágeno-Prolina Dioxigenasa/química , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
Angiogenesis-osteogenesis coupling processes are vital in bone tissue engineering. Normal biomaterials implanted in bone defects have issues in the sufficient formation of blood vessels, especially in the central part. Single delivery of vascular endothelial growth factors (VEGF) to foci in previous studies did not show satisfactory results due to low loading doses, a short protein half-life and low efficiency. Development of a hypoxia-mimicking microenvironment for cells by local prolyl-4-hydroxylase inhibitor release, which can stabilize hypoxia-inducible factor 1α (HIF-1α) expression, is an alternative method. The aim of this study was to design a dimethyloxallyl glycine (DMOG) delivering scaffold composed of mesoporous bioactive glasses and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) polymers (MPHS scaffolds), so as to investigate whether the sustained release of DMOG promotes local angiogenesis and bone healing. The morphology and microstructure of composite scaffolds were characterized. The DMOG release patterns from scaffolds loaded with different DMOG dosages were evaluated, and the effects of DMOG delivery on human bone marrow stromal cell (hBMSC) adhesion, viability, proliferation, osteogenic differentiation and angiogenic-relative gene expressions with scaffolds were also investigated. In vivo studies were carried out to observe vascular formations and new bone ingrowth with DMOG-loaded scaffolds. The results showed that DMOG could be released in a sustained manner over 4 weeks from MPHS scaffolds and obviously enhance the angiogenesis and osteogenesis in the defects. Microfil perfusion showed a significantly increased formation of vessels in the defects with DMOG delivery. Furthermore, micro-CT imaging and fluorescence labeling indicated larger areas of bone formation for DMOG-loaded scaffolds. It is concluded that MPHS-DMOG scaffolds are promising for enhancing bone healing of osseous defects.
Asunto(s)
Ácido 3-Hidroxibutírico/química , Inductores de la Angiogénesis/química , Materiales Biocompatibles/química , Células de la Médula Ósea/efectos de los fármacos , Huesos/química , Caproatos/química , Diferenciación Celular/efectos de los fármacos , Glicina/análogos & derivados , Subunidad alfa del Factor 1 Inducible por Hipoxia/química , Neovascularización Patológica/metabolismo , Osteogénesis/efectos de los fármacos , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/metabolismo , Ingeniería de Tejidos/métodos , Factores de Crecimiento Endotelial Vascular/química , Factores de Crecimiento Endotelial Vascular/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Inductores de la Angiogénesis/metabolismo , Células de la Médula Ósea/química , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Huesos/metabolismo , Caproatos/metabolismo , Glicina/química , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neovascularización Fisiológica , Impresión TridimensionalRESUMEN
Collagen is the most abundant protein in animals. A variety of indications are associated with the overproduction of collagen, including fibrotic diseases and cancer metastasis. The stability of collagen relies on the posttranslational modification of proline residues to form (2S,4R)-4-hydroxyproline. This modification is catalyzed by collagen prolyl 4-hydroxylases (CP4Hs), which are Fe(II)- and α-ketoglutarate (AKG)-dependent dioxygenases located in the lumen of the endoplasmic reticulum. Human CP4Hs are validated targets for treatment of both fibrotic diseases and metastatic breast cancer. Herein, we report on 2,2'-bipyridinedicarboxylates as inhibitors of a human CP4H. Although most 2,2'-bipyridinedicarboxylates are capable of inhibition via iron sequestration, the 4,5'- and 5,5'-dicarboxylates were found to be potent competitive inhibitors of CP4H, and the 5,5'-dicarboxylate was selective in its inhibitory activity. Our findings clarify a strategy for developing CP4H inhibitors of clinical utility.