RESUMEN
Fasciolosis is a worldwide parasitic disease of ruminants and an emerging human disease caused by the liver fluke Fasciola hepatica. The cystatin superfamily of cysteine protease inhibitors is composed of distinct families of intracellular stefins and secreted true cystatins. FhCyLS-2 from F. hepatica is an unusual member of the superfamily, where our sequence and 3D structure analyses in this study revealed that it combines characteristics of both families. The protein architecture demonstrates its relationship to stefins, but FhCyLS-2 also contains the secretion signal peptide and disulfide bridges typical of true cystatins. The secretion status was confirmed by detecting the presence of FhCyLS-2 in excretory/secretory products, supported by immunolocalization. Our high-resolution crystal structure of FhCyLS-2 showed a distinct disulfide bridging pattern and functional reactive center. We determined that FhCyLS-2 is a broad specificity inhibitor of cysteine cathepsins from both the host and F. hepatica, suggesting a dual role in the regulation of exogenous and endogenous proteolysis. Based on phylogenetic analysis that identified several FhCyLS-2 homologues in liver/intestinal foodborne flukes, we propose a new group within the cystatin superfamily called cystatin-like stefins.
Asunto(s)
Cistatinas , Fasciola hepatica , Animales , Secuencia de Aminoácidos , Cistatinas/genética , Cistatinas/química , Disulfuros , Fasciola hepatica/genética , Filogenia , Proteínas del Helminto/química , Proteínas del Helminto/genéticaRESUMEN
Cystatins encode a high functional variability not only because of their ability to inhibit different classes of proteases but also because of their propensity to form oligomers and amyloid fibrils. Phytocystatins, essential regulators of protease activity in plants, specifically inhibit papain-like cysteine proteases (PLCPs) and legumains through two distinct cystatin domains. Mammalian cystatins can form amyloid fibrils; however, the potential for amyloid fibril formation of phytocystatins remains unknown. In this study, we demonstrate that Arabidopsis thaliana phytocystatin 6 (AtCYT6) exists as a mixture of monomeric, dimeric, and oligomeric forms in solution. Noncovalent oligomerization was facilitated by the N-terminal cystatin domain, while covalent dimerization occurred through disulfide bond formation in the interdomain linker. The noncovalent dimeric form of AtCYT6 retained activity against its target proteases, papain and legumain, albeit with reduced inhibitory potency. Additionally, we observed the formation of amyloid fibrils by AtCYT6 under acidic pH conditions and upon heating. The amyloidogenic potential could be attributed to the AtCYT6's N-terminal domain (AtCYT6-NTD). Importantly, AtCYT6 amyloid fibrils harbored inhibitory activities against both papain and legumain. These findings shed light on the oligomerization and amyloidogenic behavior of AtCYT6, expanding our understanding of phytocystatin biology and its potential functional implications for plant protease regulation.
Asunto(s)
Arabidopsis , Cistatinas , Animales , Papaína/química , Amiloide/química , Cistatinas/química , Cistatinas/farmacología , Péptido Hidrolasas , MamíferosRESUMEN
Phytocystatins are a type of proteinase inhibitor which are extensively studied for their specific inhibitory action against cysteine protease enzymes (CP) of insects and pathogens. Oryzacystatins (OC), a phytocystatin from rice inhibits CP in a reversible manner with its conserved tripartite wedge. OCs have important role in plant innate defense mechanism through phytohormonal signalling pathways. OC are induced in response to both biotic and abiotic stress conditions and are used to develop transgenic plants exhibiting resistance against stress conditions. In this review, we focus on the structure and mechanism of action of oryzacystatins, their possible role in plant physiology, biotic and abiotic stress tolerance mechanism in plants and their potential application strategies for future crop management studies.
Asunto(s)
Cistatinas , Proteasas de Cisteína , Cistatinas/química , Cistatinas/genética , Cistatinas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Estrés FisiológicoRESUMEN
The epididymal lumen contains a complex cystatin-rich nonpathological amyloid matrix with putative roles in sperm maturation and sperm protection. Given our growing understanding for the biological function of this and other functional amyloids, the problem still remains: how functional amyloids assemble including their initial transition to early oligomeric forms. To examine this, we developed a protocol for the purification of nondenatured mouse CRES, a component of the epididymal amyloid matrix, allowing us to examine its assembly to amyloid under conditions that may mimic those in vivo. Herein we use X-ray crystallography, solution-state NMR, and solid-state NMR to follow at the atomic level the assembly of the CRES amyloidogenic precursor as it progressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assembles into highly branched amyloid matrices, comparable to those in vivo, by forming ß-sheet assemblies that our data suggest occur via two distinct mechanisms: a unique conformational switch of a highly flexible disulfide-anchored loop to a rigid ß-strand and by traditional cystatin domain swapping. Our results provide key insight into our understanding of functional amyloid assembly by revealing the earliest structural transitions from monomer to oligomer and by showing that some functional amyloid structures may be built by multiple and distinctive assembly mechanisms.
Asunto(s)
Amiloide/química , Proteínas Amiloidogénicas/química , Cistatinas/química , Amiloide/metabolismo , Amiloide/ultraestructura , Proteínas Amiloidogénicas/metabolismo , Animales , Cristalografía por Rayos X , Cistatinas/metabolismo , Epidídimo/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Multimerización de ProteínaRESUMEN
Accumulating evidence shows that amyloids perform biological roles. We previously showed that an amyloid matrix composed of four members of the CRES subgroup of reproductive family 2 cystatins is a normal component of the mouse epididymal lumen. The cellular mechanisms that control the assembly of these and other functional amyloid structures, however, remain unclear. We speculated that cross-seeding between CRES members could be a mechanism to control the assembly of the endogenous functional amyloid. Herein we used thioflavin T assays and negative stain transmission electron microscopy to explore this possibility. We show that CRES3 rapidly formed large networks of beaded chains that possessed the characteristic cross-ß reflections of amyloid when examined by X-ray diffraction. The beaded amyloids accelerated the amyloidogenesis of CRES, a less amyloidogenic family member, in seeding assays during which beads transitioned into films and fibrils. Similarly, CRES seeds expedited CRES3 amyloidogenesis, although less efficiently than the CRES3 seeding of CRES. These studies suggest that CRES and CRES3 hetero-oligomerize and that CRES3 beaded amyloids may function as stable preassembled seeds. The CRES3 beaded amyloids also facilitated assembly of the unrelated amyloidogenic precursor Aß by providing a surface for polymerization though, intriguingly, CRES3 (and CRES) monomer/early oligomer profoundly inhibited Aß assembly. The cross-seeding between the CRES subgroup members is similar to that which occurs between bacterial curli proteins suggesting that it may be an evolutionarily conserved mechanism to control the assembly of some functional amyloids. Further, interactions between unrelated amyloidogenic precursors may also be a means to regulate functional amyloid assembly.
Asunto(s)
Amiloide/genética , Proteínas Amiloidogénicas/genética , Cistatinas/genética , Amiloide/química , Proteínas Amiloidogénicas/química , Animales , Benzotiazoles/química , Benzotiazoles/farmacología , Cistatinas/química , Epidídimo/química , Epidídimo/crecimiento & desarrollo , Masculino , Ratones , Microscopía Electrónica de Transmisión , Difracción de Rayos XRESUMEN
Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.
Asunto(s)
Cistatinas/química , Osteoblastos/citología , beta Catenina/metabolismo , Células 3T3 , Animales , Antiinflamatorios/química , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Citrus sinensis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Citoesqueleto/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ratones , Osteoblastos/metabolismo , Osteogénesis , Fitoquímicos , Cicatrización de HeridasRESUMEN
Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the "lock and key" mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.
Asunto(s)
Proteasas de Cisteína/química , Inhibidores de Cisteína Proteinasa/química , Animales , Cistatinas/química , Cistatinas/metabolismo , Cistatinas/farmacología , Proteasas de Cisteína/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Unión Proteica , Securina/química , Securina/metabolismo , Securina/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/química , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/farmacologíaRESUMEN
Cystatins are natural inhibitors of cysteine peptidases. Recently, cystatins derived from plants, named phytocystatins, have been extensively studied. Among them, CsinCPI-2 proteins from Citrus sinensis were identified and recombinantly produced by our group. Thus, this study described the recombinant expression, purification, and inhibitory activity of this new phytocystatin against human cathepsins K and B and assessed the anti-inflammatory effect of CsinCPI-2 in vitro in mouse and in vivo in rats. In addition, the pro-osteogenic effect of CsinCPI-2 was investigated in vitro. The inflammatory response of mouse macrophage cells stimulated with P. gingivalis was modulated by CsinCPI-2. The in vitro results showed an inhibitory effect (pâ¯<â¯0.05) on cathepsin K, cathepsin B, IL-1ß, and TNF-α gene expression. In addition, CsinCPI-2 significantly inhibited in vivo the activity of TNF-α (pâ¯<â¯0.05) in the blood of rats, previously stimulated by E. coli lipopolysaccharide (LPS). CsinCPI-2 had a pro-osteogenic effect in human dental pulp cells, demonstrated by the increase in alkaline phosphatase (ALP) activity, deposition of mineralized nodules, and the gene expression of the osteogenic markers as bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (Runx-2), ALP, osteocalcin, and bone sialoprotein (BSP). These preliminary studies suggested that CsinCPI-2 has a potential anti-inflammatory, and at the same time, a pro-osteogenic effect. This may lead to new therapies for the control of diseases where inflammation plays a key role, such as periodontal disease and apical periodontitis.
Asunto(s)
Antígenos de Diferenciación/biosíntesis , Citrus/química , Cistatinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/metabolismo , Osteogénesis/efectos de los fármacos , Proteínas de Plantas/farmacología , Animales , Cistatinas/química , Humanos , Macrófagos/patología , Masculino , Ratones , Proteínas de Plantas/química , Células RAW 264.7 , Ratas , Ratas WistarRESUMEN
Cystein protease plays a critical role as a virulence factor in the development and progression of various diseases. Cystatin is a superfamily of cysteine protease inhibitors that participates in various physiological and pathological processes. The cysteine protease inhibitor CsStein-1 isolated from Clonorchis sinensis belongs to the type 1 stefin of cystatins. This inhibitor regulates the activity and processing of CsCF (Cathepsin F of Clonorchis sienesis), which plays an important role in parasite nutrition and host-parasite interaction. CsStefin-1 has also been proposed as a host immune modulator and a participant in the mechanism associated with anti-inflammatory ability. Here, we report the first crystal structure of CsStefin-1 determined by the multi-wavelength anomalous diffraction (MAD) method to 2.3â¯Å. There are six molecules of CsStefin-1 per asymmetric unit, with a solvent content of 36.5%. The structure of CsStefin-1 is composed of twisted four-stranded antiparallel ß-sheets, a central α-helix, and a short α-helix. We also demonstrate that CsStefin-1 binds to CsCF-8 cysteine protease and inhibits its activity. In addition, a molecular docking model of CsStefin-1 and CsCF-8 was developed using homology modeling based on their structures. The structural information regarding CsStefin-1 and molecular insight into its interaction with CsCF-8 are important to understanding their biological function and to design of inhibitors that modulate cysteine protease activity.
Asunto(s)
Clonorchis sinensis/química , Cistatinas/química , Inhibidores de Cisteína Proteinasa/química , Secuencia de Aminoácidos , Animales , Catepsina F/antagonistas & inhibidores , Catepsina F/metabolismo , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión ProteicaRESUMEN
Reactive dicarbonyl species such as methylglyoxal (MGO) and glyoxal (GO) have recently received extensive attention due to their high reactivity and ability to modify biological substances such as proteins, phospholipids, and DNA. In case of proteins these reactive species mainly react with lysine and arginine residues to form AGEs, oxidative products, and aggregates. Chickpea cystatin (CPC) was incubated with varying concentrations of glyoxal and methylglyoxal which caused, along with altered secondary and tertiary structures, glycation, functional inactivation, altered redox state, cross-linking and high-molecular-mass aggregation. All these processes were examined and characterized by UV-Vis, fluorescence, and CD spectroscopies. Further characterization of CPC modified by reactive dicarbonyls was done by polyacrylamide gel electrophoresis which also showed alterations in the CPC molecules. Thus, in addition to describing the effects of GO and MGO on structure, conformation and function of CPC, this study also shows the relatively superior modifying effect of methylglyoxal for CPC in terms of glycation, oxidation and aggregation. This model system could shed some more light on the role of the reactive dicarbonyls in the specific alterations of proteins with different biological consequences having implications to ageing and disease such as diabetes.
Asunto(s)
Cicer/metabolismo , Cistatinas/metabolismo , Glioxal/metabolismo , Proteínas de Plantas/metabolismo , Piruvaldehído/metabolismo , Arginina/metabolismo , Cicer/química , Cistatinas/química , Cistatinas/ultraestructura , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Lisina/metabolismo , Oxidación-Reducción , Proteínas de Plantas/química , Agregado de ProteínasRESUMEN
Cystatins are a family of cysteine protease inhibitors which are associated with a variety of physiological and pathological processes in vivo. In the present study, the cDNA sequence of a cystatin F homologue called Lm-cystatin F was cloned from the buccal glands of Lampetra morii. Although Lm-cystatin F shares a lower homology with cystatin superfamily members, it is also composed of a signal peptide and three highly conserved motifs, including the G in the N-terminal, QXVXG, as well as the PW in the C-terminal of the sequence. After sequence optimization and recombination, the recombinant protein was expressed as a soluble protein in Escherichia coli with a molecular weight of 19.85 kDa. Through affinity chromatography and mass spectrometry analysis, the purified protein was identified as a recombinant Lm-cystatin F (rLm-cystatin F). Additionally, rLm-cystatin F could inhibit the activity of papain. Based on MTT assay, rLm-cystatin F inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) dose dependently with an IC50 of 5 µM. In vitro studies show that rLm-cystatin F suppressed the adhesion, migration, invasion, and tube formation of HUVECs, suggesting that rLm-cystatin F possesses anti-angiogenic activity, which provides information on the feeding mechanisms of Lampetra morii and insights into the application of rLm-cystatin F as a potential drug in the future.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Cistatinas/farmacología , Lampreas , Neovascularización Fisiológica/efectos de los fármacos , Papaína/antagonistas & inhibidores , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/genética , Inhibidores de la Angiogénesis/aislamiento & purificación , Animales , Proliferación Celular/efectos de los fármacos , Cistatinas/química , Cistatinas/genética , Cistatinas/aislamiento & purificación , ADN Complementario , Células Endoteliales de la Vena Umbilical Humana , Humanos , Concentración 50 Inhibidora , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Homología de Secuencia de AminoácidoRESUMEN
Pesticides are chemical substances that eliminate or control a variety of agricultural pests that damage crops and livestock. They not only affect the targeted pests but also affect the nontargeted systems, raising more concerns for their effect on both plant and animal systems. Cystatins (cysteine protease inhibitor) are ubiquitously present in all living cells and show a variety of important physiological functions. The present study shows the effect of different pesticides (pendimethalin, methoxyfenozide, and CuII hydroxide) on purified almond cystatin. Almond cystatin showed concentration-dependent loss in papain inhibitory activity on interaction with the pesticides, showing maximum loss in the presence of Cu(II) hydroxide and minimum in the case of methoxyfenozide. Native polyacrylamide gel electrophoresis showed maximum degradation of purified cystatin in the presence of Cu(II) hydroxide with insignificant effect in the presence of methoxyfenozide. Structural alterations were significant in the case of Cu(II) hydroxide and less in the case of methoxyfenozide as revealed by UV and fluorescence spectral studies. Secondary structural alterations were further conformed by circular dichroism and Fourier transform infrared spectroscopy. The α-helix content of almond cystatin decreases from 35.64% (native) to 34.83%, 30.79%, and 29.62% for methoxyfenozide-, pendimethalin-, and Cu(II) hydroxide-treated cystatin, respectively. A Fourier transform infrared study shows an amide I band shift for almond cystatin from 1649.15 ± 0.5 to 1646.48 ± 0.6, 1640.44 ± 0.6, and 1635.11 ± 0.3 cm-1 for methoxyfenozide, pendimethalin, and Cu(II) hydroxide, respectively. Values obtained for different thermodynamic parameters (ΔH0 , ΔG0 , N, and ΔS0 ) by isothermal titration calorimetric experiments reveal maximum binding of almond cystatin with Cu(II) hydroxide followed by pendimethalin and little interaction with methoxyfenozide.
Asunto(s)
Cistatinas/química , Cistatinas/efectos de los fármacos , Plaguicidas/farmacología , Prunus dulcis/metabolismo , Compuestos de Anilina/farmacología , Calorimetría , Dicroismo Circular , Cobre/farmacología , Hidrazinas/farmacología , Hidróxidos/farmacología , Hormonas Juveniles/farmacología , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion-secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.
Asunto(s)
Cistatinas/genética , Inhibidores de Cisteína Proteinasa/farmacología , Fasciola hepatica/genética , Proteínas del Helminto/genética , Secuencia de Aminoácidos , Animales , Catepsina B/metabolismo , Catepsina C/metabolismo , Catepsina L/metabolismo , Bovinos , Cistatinas/química , Cistatinas/metabolismo , Escherichia coli/genética , Fasciola hepatica/enzimología , Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Humanos , Organismos Modificados Genéticamente , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1ß (IL-1ß) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.
Asunto(s)
Anaplasma phagocytophilum/inmunología , Anexina A2/inmunología , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas de Unión al Calcio/inmunología , Cistatinas/inmunología , Ehrlichiosis/microbiología , Evasión Inmune , Secuencia de Aminoácidos , Anaplasma phagocytophilum/genética , Animales , Anexina A2/química , Anexina A2/genética , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Vectores Arácnidos/química , Vectores Arácnidos/genética , Vectores Arácnidos/inmunología , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Caspasa 1/genética , Caspasa 1/inmunología , Caspasas/genética , Caspasas/inmunología , Caspasas Iniciadoras , Cistatinas/química , Cistatinas/genética , Ehrlichiosis/inmunología , Ehrlichiosis/patología , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamasomas/genética , Inflamasomas/inmunología , Interleucina-18/genética , Interleucina-18/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Ixodes/química , Ixodes/genética , Ixodes/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Modelos Moleculares , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transducción de SeñalRESUMEN
This study describes the isolation and purification of a phytocystatin from seeds of Brassica juncea (Indian mustard; cultivar RoAgro 5444), which is an important oilseed crop both agriculturally and economically. The protein was purified by gel filtration chromatography with 24.3% yield and 204-fold purification, and visualised by 2D gel electrophoresis. The 18.1 kDa mustard cystatin was highly specific for cysteine proteinases. The plant cystatin inhibited cathepsin B, confirming its role in conferring pest resistance. The inhibitor was highly stable over a pH range of 3-10 and retained significant inhibitory potential up to 70 °C. The stoichiometry of its interaction with papain, determined by isothermal calorimetry, suggests a 1:1 complex. Secondary structural elements calculated by far-UV circular dichroism (CD) spectroscopy show an 18.8% α-helical and 21% ß-sheet structure. The protein was a non-competitive inhibitor of thiol proteinases. The Stokes radius and frictional co-efficient were used to describe the shape and size of the protein. Homology modelling and docking studies proposed a prototype illustrating the Brassica phytocystatin mediated papain inhibition. Molecular dynamics (MD) study revealed the excellent stability of the papain-phytocystatin complex during a simulation for 100 ns. Detailed results identify the mustard cystatin as an important member of the phytocystatin family.
Asunto(s)
Cistatinas/química , Cistatinas/metabolismo , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/metabolismo , Planta de la Mostaza/metabolismo , Animales , Formación de Anticuerpos , Cromatografía en Gel , Simulación por Computador , Cistatinas/inmunología , Cistatinas/aislamiento & purificación , Inhibidores de Cisteína Proteinasa/inmunología , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Inmunoglobulina G/inmunología , Cinética , Masculino , Modelos Moleculares , Simulación de Dinámica Molecular , Planta de la Mostaza/crecimiento & desarrollo , Papaína/metabolismo , Proteínas de Plantas/inmunología , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , ConejosRESUMEN
Phytocystatins belong to the family of cysteine proteinases inhibitors. They are ubiquitously found in plants and carry out various significant physiological functions. These plant derived inhibitors are gaining wide consideration as potential candidate in engineering transgenic crops and in drug designing. Hence it is crucial to identify these inhibitors from various plant sources. In the present study a phytocystatin has been isolated and purified by a simple two-step procedure using ammonium sulfate saturation and gel filtration chromatography on Sephacryl S-100HR from Brassica alba seeds (yellow mustard seeds).The protein was purified to homogeneity with 60.3% yield and 180-fold of purification. The molecular mass of the mustard seed cystatin was estimated to be nearly 26,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as by gel filtration chromatography. The stokes radius and diffusion coefficient of the mustard cystatin were found to be 23A° and 9.4 × 10(-7) cm(2) s(-1) respectively. The isolated phytocystatin was found to be stable in the pH range of 6-8 and is thermostable up to 60 °C. Kinetic analysis revealed that the phytocystatin exhibited non-competitive type of inhibition and inhibited papain more efficiently (K(i) = 3 × 10(-7) M) than ficin (K(i) = 6.6 × 10(-7) M) and bromelain (K(i) = 7.7 × 10(-7) M respectively). CD spectral analysis shows that it possesses 17.11% alpha helical content.
Asunto(s)
Cistatinas/aislamiento & purificación , Cistatinas/farmacología , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Inhibidores de Cisteína Proteinasa/farmacología , Sinapis/metabolismo , Bromelaínas/antagonistas & inhibidores , Cromatografía en Gel , Dicroismo Circular , Cistatinas/química , Inhibidores de Cisteína Proteinasa/química , Ficaína/antagonistas & inhibidores , Peso Molecular , Papaína/antagonistas & inhibidores , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Estructura Secundaria de Proteína , Semillas/metabolismoRESUMEN
Potato (Solanum tuberosum) multicystatin (PMC) is a unique cystatin composed of eight repeating units, each capable of inhibiting cysteine proteases. PMC is a composite of several cystatins linked by trypsin-sensitive (serine protease) domains and undergoes transitions between soluble and crystalline forms. However, the significance and the regulatory mechanism or mechanisms governing these transitions are not clearly established. Here, we report the 2.2-Å crystal structure of the trypsin-resistant PMC core consisting of the fifth, sixth, and seventh domains. The observed interdomain interaction explains PMC's resistance to trypsin and pH-dependent solubility/aggregation. Under acidic pH, weakening of the interdomain interactions exposes individual domains, resulting in not only depolymerization of the crystalline form but also exposure of cystatin domains for inhibition of cysteine proteases. This in turn allows serine protease-mediated fragmentation of PMC, producing â¼ 10-kD domains with intact inhibitory capacity and faster diffusion, thus enhancing PMC's inhibitory ability toward cysteine proteases. The crystal structure, light-scattering experiments, isothermal titration calorimetry, and site-directed mutagenesis confirmed the critical role of pH and N-terminal residues in these dynamic transitions between monomer/polymer of PMC. Our data support a notion that the pH-dependent structural regulation of PMC has defense-related implications in tuber physiology via its ability to regulate protein catabolism.
Asunto(s)
Cistatinas/química , Proteínas de Plantas/química , Solanum tuberosum/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Cristalografía por Rayos X , Cistatinas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Cystatin F, a member of the family II cystatins, plays important roles in immune response-related processes through inhibiting specific enzyme targets. In this study, a cystatin F homologue, LycCysF, was identified and characterized from large yellow croaker (Larimichthys crocea). The deduced LycCysF protein exhibits a typical structural feature of type II cystatins, including three evolutionally conserved motifs, Gly(35), QVVRG(79-83) and PW(130-131). Tissue expression analysis showed that LycCysF mRNA was expressed in all tissues examined, albeit at different levels. Recombinant LycCysF (rLycCysF) produced in Pichia pastoris could inhibit the activity of multiple cysteine proteases, including papain, legumain and recombinant large yellow croaker cathepsin B, L and S. Moreover, rLycCysF could inhibit the Ii chain processing by recombinant cathepsin S in vitro. These data suggest that LycCysF may participate in regulation of cathepsins and MHC-II associated Ii chain processing. In addition, mammalian cystatin F is produced as an inactive dimer, becoming activated by proteolysis in the endo/lysosome of immune cells and then exerts its function of regulating downstream proteases activity. However, the N-terminal extension and two additional cysteine residues responsible for dimer formation are absent in LycCysF and cystatin F from other fish species, reptiles and Aves, indicating that these proteins can not form dimer and may regulate the proteases activity via an alternate pathway distinct from mammalian cystatin F. To our knowledge, this is the first report on molecular characteristics of a teleost cystatin F and its role in Ii chain processing.
Asunto(s)
Cistatinas , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas de Peces , Perciformes , Aeromonas hydrophila , Animales , Presentación de Antígeno , Encéfalo/metabolismo , Cistatinas/química , Cistatinas/genética , Cistatinas/inmunología , Proteasas de Cisteína/metabolismo , ADN Complementario/genética , Enfermedades de los Peces/genética , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Genes MHC Clase II/inmunología , Branquias/metabolismo , Infecciones por Bacterias Gramnegativas/genética , Infecciones por Bacterias Gramnegativas/veterinaria , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Músculos/metabolismo , Perciformes/genética , Perciformes/inmunología , Perciformes/metabolismo , ARN Mensajero/sangre , ARN Mensajero/metabolismo , Piel/metabolismo , Bazo/metabolismoRESUMEN
The increased level of reducing sugars and their derivatives in a diabetic condition has been the main cause of protein related complications. The changes in native state of proteins upon glycation induce loss in the function and structure of proteins. This further leads to cell damage and accumulation of immune system inducing AGE formation. Here in the present study cystatin was purified from liver (BLC) through affinity chromatography and was incubated with glucose, fructose and ribose. Changes were observed in the intensity of Trp absorption at 280 nm as well as AGE's specific fluorescence at 435 nm upon excitation at 370 nm to monitor the formation of BLC-sugar adducts. Protein intrinsic fluorescence showed marked conformational changes when BLC was incubated with D-ribose, glucose and fructose. Glycation with D-ribose induces BLC to misfold rapidly into an intermediate state retaining a low percentage of α-helical content compared to fructose and glucose as revealed by far-UV CD data. Furthermore, a caseinolytic assay of papain in presence of glycated liver cystatin showed decreased activity in the protein induced by these reducing sugars. Ribose had more effect on the structure as well as the function of liver cystatin followed by fructose and least for glucose. Absorption spectroscopy shows change in BLC and formation of AGE's. These results shows that liver cystatin-cathepsin imbalance is compromised in diabetic state which may lead to improper balance of proteinases leading to cirrhosis or liver damage.
Asunto(s)
Cistatinas/química , Cistatinas/metabolismo , Hígado/metabolismo , Animales , Búfalos , Dicroismo Circular , Fluorescencia , Fructosa/metabolismo , Glucosa/metabolismo , Glicosilación , Espectrometría de FluorescenciaRESUMEN
Hereditary cystatin C amyloid angiopathy is an autosomal dominant disorder in which a variant form of cystatin C (L68Q) readily forms amyloid deposits in cerebral arteries in affected individuals resulting in early death. L68Q protein deposits in human cystatin C amyloid angiopathy patients have also been found in tissues outside of the brain including the testis, suggesting possible effects on fertility. Heterozygous transgenic mice (L68Q) that express the human L68Q variant of cystatin C under the control of the mouse cystatin C promoter were unable to generate offspring, suggesting the presence of L68Q cystatin C amyloid affected sperm function. In vitro studies showed that epididymal spermatozoa from L68Q mice were unable to fertilize oocytes and exhibited poor sperm motility. Furthermore, spermatozoa from L68Q mice exhibited reduced cell viability compared with wild type (WT) spermatozoa and often were detected in large agglutinated clumps. Examination of the epididymal fluid and spermatozoa from L68Q mice showed increased levels and distinct forms of cystatin C amyloid that were not present in WT mice. The addition of epididymal fluid from L68Q mice to WT spermatozoa resulted in a recapitulation of the L68Q phenotype in that WT spermatozoa showed reduced cell viability and motility compared with WT spermatozoa incubated in epididymal fluid from WT mice. L68Q epididymal fluid that was depleted of cystatin C amyloids, however, did not impair the motility of WT spermatozoa. Taken together these studies suggest that amyloids in the epididymal fluid can be cytotoxic to the maturing spermatozoa resulting in male infertility.