RESUMEN
Heart transplantation offers life-saving treatment for patients with end-stage heart failure; however, ischemia-reperfusion injury (IRI) and subsequent immune responses remain significant challenges. Current therapies primarily target adaptive immunity, with limited options available for addressing IRI and innate immune activation. Although plant-derived vesicle-like nanoparticles show promise in managing diseases, their application in organ transplantation complications is unexplored. Here, this work develops a novel reactive oxygen species (ROS)-responsive multifunctional fusion extracellular nanovesicles carrying rapamycin (FNVs@RAPA) to address early IRI and Ly6C+Ly6G- inflammatory macrophage-mediated rejection in heart transplantation. The FNVs comprise Exocarpium Citri grandis-derived extracellular nanovesicles with anti-inflammatory and antioxidant properties, and mesenchymal stem cell membrane-derived nanovesicles expressing calreticulin with macrophage-targeting ability. A novel ROS-responsive bio-orthogonal chemistry approach facilitates the active targeting delivery of FNVs@RAPA to the heart graft site, effectively alleviating IRI and promoting the polarization of Ly6C+Ly6G- inflammatory macrophages toward an anti-inflammatory phenotype. Hence, FNVs@RAPA represents a promising therapeutic approach for mitigating early transplantation complications and immune rejection. The fusion-targeted delivery strategy offers superior heart graft site enrichment and macrophage-specific targeting, promising improved transplant outcomes.
Asunto(s)
Rechazo de Injerto , Trasplante de Corazón , Macrófagos , Especies Reactivas de Oxígeno , Sirolimus , Especies Reactivas de Oxígeno/metabolismo , Animales , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/prevención & control , Sirolimus/química , Sirolimus/farmacología , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Nanopartículas/química , Calreticulina/metabolismo , Calreticulina/química , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismoRESUMEN
Calreticulin (CRT) is an intrinsically disordered multifunctional protein that plays essential roles intra-and extra-cellularly. The Michalak laboratory has proposed that CRT was initially identified in 1974 by the MacLennan laboratory as the high-affinity Ca2+-binding protein (HACBP) of the sarcoplasmic reticulin (SR). This widely accepted belief has been ingrained in the scientific literature but has never been rigorously tested. In our report, we have undertaken a comprehensive reexamination of this assumption by meticulously examining the majority of published studies that present a proteomic analysis of the SR. These analyses have utilized proteomic analysis of purified SR preparations or purified components of the SR, namely the longitudinal tubules and junctional terminal cisternae. These studies have consistently failed to detect the HACBP or CRT in skeletal muscle SR. We propose that the existence of the HACBP has failed the test of reproducibility and should be retired to the annals of antiquity. Therefore, the scientific dogma that the HACBP and CRT are identical proteins is a non sequitur.
Asunto(s)
Calreticulina , Animales , Calreticulina/química , Calreticulina/historia , Calreticulina/metabolismo , Historia del Siglo XX , Músculo Esquelético/metabolismo , Proteómica , Reproducibilidad de los Resultados , Retículo Sarcoplasmático/metabolismo , ConejosRESUMEN
Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.
Asunto(s)
Anticuerpos , Electroforesis en Gel de Poliacrilamida , Péptidos , Electroforesis en Gel de Poliacrilamida/métodos , Péptidos/química , Péptidos/inmunología , Anticuerpos/química , Anticuerpos/inmunología , Western Blotting/métodos , Humanos , Calreticulina/química , Calreticulina/inmunología , Calreticulina/metabolismo , Immunoblotting/métodos , Especificidad de Anticuerpos , AnimalesRESUMEN
Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases.
Asunto(s)
Calreticulina , Complemento C1q , Evasión Inmune , Trichinella spiralis , Trichinella spiralis/inmunología , Complemento C1q/inmunología , Complemento C1q/metabolismo , Complemento C1q/química , Animales , Calreticulina/inmunología , Calreticulina/química , Calreticulina/metabolismo , Cristalografía por Rayos X , Unión Proteica , Simulación del Acoplamiento Molecular , Proteínas del Helminto/inmunología , Proteínas del Helminto/química , Activación de Complemento/inmunología , Inmunoglobulina G/inmunología , Humanos , Antígenos Helmínticos/inmunología , Antígenos Helmínticos/química , Triquinelosis/inmunología , Triquinelosis/parasitología , Vía Clásica del Complemento/inmunología , Conformación ProteicaRESUMEN
Calreticulin (CALR) is an endoplasmic reticulum (ER)-retained chaperone that assists glycoproteins in obtaining their structure. CALR mutations occur in patients with myeloproliferative neoplasms (MPNs), and the ER retention of CALR mutants (CALR MUT) is reduced due to a lacking KDEL sequence. Here, we investigate the impact of CALR mutations on protein structure and protein levels in MPNs by subjecting primary patient samples and CALR-mutated cell lines to limited proteolysis-coupled mass spectrometry (LiP-MS). Especially glycoproteins are differentially expressed and undergo profound structural alterations in granulocytes and cell lines with homozygous, but not with heterozygous, CALR mutations. Furthermore, homozygous CALR mutations and loss of CALR equally perturb glycoprotein integrity, suggesting that loss-of-function attributes of mutated CALR chaperones (CALR MUT) lead to glycoprotein maturation defects. Finally, by investigating the misfolding of the CALR glycoprotein client myeloperoxidase (MPO), we provide molecular proof of protein misfolding in the presence of homozygous CALR mutations.
Asunto(s)
Calreticulina , Trastornos Mieloproliferativos , Humanos , Calreticulina/genética , Calreticulina/química , Calreticulina/metabolismo , Mutación/genética , Homocigoto , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteoma/metabolismoRESUMEN
Calreticulin (Crt) is a highly conserved and multi-functional protein with lectin-like properties and important immunological activities. In this study, a Crt homolog, namely, ToCrt, was cloned and characterized from the obscure puffer Takifugu obscurus with an open reading frame of 1278 bp encoding a putative protein of 425 amino acids. The deduced amino acid sequence of ToCrt consisted of three conserved structural domains: N-domain, P-domain, and C-terminal domain. In the phylogenetic tree, ToCrt formed a separate cluster with three Crts from other pufferfish species (Takifugu rubripes, Takifugu flavidus, and Takifugu bimaculatus). The mRNA transcript of ToCrt was ubiquitously expressed in all the examined tissues in a decreasing order: liver, spleen, kidney, gills, intestine, and heart. After Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila stimulations, the levels of ToCrt mRNA in the kidney and spleen were significantly upregulated compared with that in the control group. The recombinant calreticulin domain of ToCrt (rToCrt) could bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and polysaccharides from bacterial cell walls such as lipopolysaccharide and peptidoglycan. Meanwhile, rToCrt could agglutinate different kinds of microorganisms and exhibit antimicrobial activity. These results suggested that T. obscurus ToCrt could serve as an antimicrobial effector in the host immune response against invading microorganisms.
Asunto(s)
Antiinfecciosos/inmunología , Calreticulina/metabolismo , Inmunidad , Takifugu/inmunología , Aeromonas hydrophila/efectos de los fármacos , Aglutinación/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antiinfecciosos/farmacología , Secuencia de Bases , Calreticulina/química , Calreticulina/genética , Calreticulina/aislamiento & purificación , Edwardsiella tarda/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Filogenia , Polisacáridos/metabolismo , Unión Proteica/efectos de los fármacos , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Takifugu/microbiología , Factores de Tiempo , Vibrio/efectos de los fármacosRESUMEN
Calreticulin (CALR) is a highly conserved multifunctional chaperone protein primarily present in the endoplasmic reticulum, where it regulates Ca2+ homeostasis. Recently, CALR has gained special interest for its diverse functions outside the endoplasmic reticulum, including the cell surface and extracellular space. Although high-resolution structures of CALR exist, it has not yet been established how different regions and individual amino acid residues contribute to structural stability of the protein. In the present study, we have identified key residues determining the structural stability of CALR. We used a Saccharomyces cerevisiae expression system to express and purify 50 human CALR mutants, which were analysed for several parameters including secretion titer, melting temperature (Tm), stability and oligomeric state. Our results revealed the importance of a previously identified small patch of conserved surface residues, amino acids 166-187 ("cluster 2") for structural stability of the human CALR protein. Two residues, Tyr172 and Asp187, were critical for maintaining the native structure of the protein. Mutant D187A revealed a severe drop in secretion titer, it was thermally unstable, prone to degradation, and oligomer formation. Tyr172 was critical for thermal stability of CALR and interacted with the third free Cys163 residue. This illustrates an unusual thermal stability of CALR dominated by Asp187, Tyr172 and Cys163, which may interact as part of a conserved structural unit. Besides structural clusters, we found a correlation of some measured parameter values in groups of CALR mutants that cause myeloproliferative neoplasms (MPN) and in mutants that may be associated with sudden unexpected death (SUD).
Asunto(s)
Sustitución de Aminoácidos , Calreticulina/química , Simulación de Dinámica Molecular , Calreticulina/genética , Humanos , Dominios Proteicos , Estabilidad ProteicaRESUMEN
In recent years, 5-pyrazolyl-ureas have mostly been known for their attractive poly-pharmacological outline and, in particular, ethyl 1-(2-hydroxypentyl)-5-(3-(3-(trifluoromethyl) phenyl) ureido)-1H-pyrazole-4-carboxylate (named GeGe-3) has emerged as a capable anti-angiogenic compound. This paper examines its interactome by functional proteomics using a label-free mass spectrometry based platform, coupling Drug Affinity Responsive Target Stability and targeted Limited Proteolysis-Multiple Reaction Monitoring. Calreticulin has been recognized as the GeGe-3 principal target and this evidence has been supported by immunoblotting and in silico molecular docking. Furthermore, cell studies have shown that GeGe-3 lowers cell calcium mobilization, cytoskeleton organization and focal adhesion kinase expression, thus linking its biological potential to calreticulin binding and, ultimately, shedding light on the reasonable action mechanism of this molecule as an anti-angiogenic factor.
Asunto(s)
Inhibidores de la Angiogénesis/química , Proteoma/metabolismo , Proteómica/métodos , Pirazoles/química , Urea/química , Inhibidores de la Angiogénesis/metabolismo , Inhibidores de la Angiogénesis/farmacología , Sitios de Unión , Calcio/metabolismo , Calreticulina/química , Calreticulina/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Quinasa 1 de Adhesión Focal/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ionomicina/farmacología , Simulación del Acoplamiento Molecular , Proteoma/efectos de los fármacosRESUMEN
INTRODUCTION: Calreticulin (CALR) mutations in myeloproliferative neoplasms (MPN) have been reported to be key markers in the molecular diagnosis, particularly in patients lacking JAK2 V617F mutation. In most current reports, CALR mutations were analysed by either allele-specific PCR (AS-PCR), or the more expensive quantitative real-time PCR, pyrosequencing and next-generation sequencing. Hence, we report the use of an alternative method, the conformation sensitive gel electrophoresis (CSGE) for the detection of CALR mutations in BCR-ABL1-negative MPN patients. METHODS: Forty BCR-ABL1-negative MPN patients' DNA: 19 polycythemia vera (PV), 7 essential thrombocytosis (ET) and 14 primary myelofibrosis (PMF), were screened for CALR mutations by CSGE. PCR primers were designed to amplify sequences spanning between exons 8 and 9 to target the mutation hotspots in CALR. Amplicons displaying abnormal CSGE profiles by electrophoresis were directly sequenced, and results were analysed by BioEdit Sequence Alignment Editor v7.2.6. CSGE results were compared with AS-PCR and confirmed by Sanger sequencing. RESULTS: CSGE identified 4 types of mutations; 2 PMF patients with either CALR type 1 (c.1099_1150del52) or type 2 (c.1155_1156insTTGTC), 1 ET patient with nucleotide deletion (c.1121delA) and insertion (c.1190insA) and 1 PV patient with p.K368del (c.1102_1104delAAG) and insertion (c.1135insA) inframe mutations. Three patients have an altered KDEL motif at the C-terminal of CALR protein. In comparison, AS-PCR only able to detect two PMF patients with mutations, either type 1 and type 2. CONCLUSION: CSGE is inexpensive, sensitive and reliable alternative method for the detection of CALR mutations in BCR-ABL1-negative MPN patients.
Asunto(s)
Biomarcadores , Calreticulina/genética , Calreticulina/metabolismo , Electroforesis/métodos , Mutación , Trastornos Mieloproliferativos/etiología , Trastornos Mieloproliferativos/metabolismo , Alelos , Calreticulina/química , Análisis Mutacional de ADN , Diagnóstico Diferencial , Susceptibilidad a Enfermedades , Proteínas de Fusión bcr-abl/genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Trastornos Mieloproliferativos/diagnóstico , Reacción en Cadena de la Polimerasa , PronósticoRESUMEN
BACKGROUND: The relevance of the cancer immune cycle in therapy response implies that successful treatment may trigger the exposure or the release of immunogenic signals. Previous results with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals released in vitro by GB cells. METHODS: GL261 GB cells were treated with TMZ and CX-4945 at different concentrations (25 µM-4 mM) and time frames (12-72 h). Cell viability was measured with Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, and ATP release was measured with bioluminescence. RESULTS: TMZ showed cytostatic rather than cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. Calreticulin exposure after 24 h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at high concentrations, as well as with TMZ/CX-4945. CONCLUSIONS: combined treatment may produce the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able to mimic pharmacokinetics observed in vivo fully.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Naftiridinas/administración & dosificación , Fenazinas/administración & dosificación , Temozolomida/administración & dosificación , Adenosina Trifosfato/química , Antineoplásicos Alquilantes/administración & dosificación , Calreticulina/química , Quinasa de la Caseína II/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Terapia Combinada , Humanos , Inflamación , Microscopía Fluorescente , Propidio/química , Transducción de Señal , Resultado del TratamientoRESUMEN
N-glycans are attached to newly synthesised polypeptides and are involved in the folding, secretion, and degradation of N-linked glycoproteins. In particular, the calnexin/calreticulin cycle, which is the central mechanism of the entry and release of N-linked glycoproteins depending on the folding sates, has been well studied. In addition to biological studies on the calnexin/calreticulin cycle, several studies have revealed complementary roles of in vitro chemistry-based research in the structure-based understanding of the cycle. In this mini-review, we summarise chemistry-based results and highlight their importance for further understanding of the cycle.
Asunto(s)
Calnexina/metabolismo , Calreticulina/metabolismo , Glicoproteínas/metabolismo , Polisacáridos/metabolismo , Calnexina/química , Calreticulina/química , Conformación de Carbohidratos , Glicoproteínas/química , Polisacáridos/químicaRESUMEN
Molecular chaperones are crucial for the correct folding of newly synthesized polypeptides, in particular, under stress conditions. Various studies have revealed the involvement of molecular chaperones, such as heat shock proteins, in diapause maintenance and starvation; however, the role of other chaperones in diapause and starvation relatively is unknown. In the current study, we identified two lectin-type chaperones with calcium affinity, a calreticulin (LdCrT) and a calnexin (LdCnX), that were present in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) during diapause. Both proteins possessed an N-globular domain, a P-arm domain, and a highly charged C-terminal domain, while an additional transmembrane domain was present in LdCnX. Phylogenetic analysis revealed distinction at the order level. Both genes were expressed in multiple tissues in larval and adult stages, and constitutively throughout development, though a starvation response was detected only for LdCrT. In females, diapause-related expression analysis in the whole body revealed an upregulation of both genes by post-diapause, but a downregulation by diapause only for LdCrT. By contrast, males revealed no alteration in their diapause-related expression pattern in the entire body for both genes. Fat body-specific expression analysis of both genes in relation to diapause revealed the same expression pattern with no alteration in females and downregulation in males by post-diapause. This study suggests that calcium-binding chaperones play similar and possibly gender-specific roles during diapause.
Asunto(s)
Calnexina , Calreticulina , Escarabajos/metabolismo , Diapausa de Insecto/fisiología , Cuerpo Adiposo/metabolismo , Animales , Calcio/metabolismo , Calnexina/química , Calnexina/genética , Calnexina/metabolismo , Calreticulina/química , Calreticulina/genética , Calreticulina/metabolismo , Escarabajos/genética , Femenino , Genes de Insecto , Masculino , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Filogenia , Caracteres Sexuales , InaniciónRESUMEN
Somatic mutations of calreticulin (CALR) have been identified as a main disease driver of myeloproliferative neoplasms, suggesting that development of drugs targeting mutant CALR is of great significance. Site-directed mutagenesis in the N-glycan binding domain (GBD) abolishes the ability of mutant CALR to oncogenically activate the thrombopoietin receptor (MPL). We therefore hypothesized that a small molecule targeting the GBD might inhibit the oncogenicity of the mutant CALR. Using an in silico molecular docking study, we identified candidate binders to the GBD of CALR. Further experimental validation of the hits identified a group of catechols inducing a selective growth inhibitory effect on cells that depend on oncogenic CALR for survival and proliferation. Apoptosis-inducing effects by the compound were significantly higher in the CALR-mutated cells than in CALR wild-type cells. Additionally, knockout or C-terminal truncation of CALR eliminated drug hypersensitivity in CALR-mutated cells. We experimentally confirmed the direct binding of the selected compound to CALR, disruption of the mutant CALR-MPL interaction, inhibition of the JAK2-STAT5 pathway, and reduction at the intracellular level of mutant CALR upon drug treatment. Our data indicate that small molecules targeting the GBD of CALR can selectively kill CALR-mutated cells by disrupting the CALR-MPL interaction and inhibiting oncogenic signaling.
Asunto(s)
Calreticulina/metabolismo , Hematoxilina/farmacología , Mapas de Interacción de Proteínas/efectos de los fármacos , Receptores de Trombopoyetina/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Calreticulina/química , Calreticulina/genética , Línea Celular , Descubrimiento de Drogas , Humanos , Ratones , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Unión Proteica/efectos de los fármacos , Receptores de Trombopoyetina/químicaRESUMEN
Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.
Asunto(s)
Proteína Disulfuro Isomerasas/metabolismo , Pliegue de Proteína , Multimerización de Proteína , Animales , Calnexina/química , Calnexina/metabolismo , Calreticulina/química , Calreticulina/metabolismo , Humanos , Proteína Disulfuro Isomerasas/química , ProteostasisRESUMEN
Calreticulin (CRT) is a multifunctional ubiquitous protein that is widely presented in all cells in eukaryotes except erythrocytes. CRT is well known for diverse cellular functions such as endoplasmic reticulum (ER)-specialized protein quality control during protein synthesis and folding, in-vivo Ca2+ homeostasis, antigen presentation, phagocytosis, wound-healing, proliferation, adhesion, and migration of cells. In the current study, we identified CRT from Hippocampus abdominalis (HaCRT) and analyzed expression profiles and functional properties. The cDNA sequence of HaCRT was identified with an open reading frame of 1226 bp. The molecular weight of HaCRT was estimated as 49 kDa. The in-silico study revealed conserved sequence arrangements such as two CRT signature motifs (5'-KHEQSIDCGGGYVKVF-3' and 5'-LMFGPDICG-3'), triplicate repeats (5'-IKDPEAKKPEDWD-3', 5'-IPDPDDTKPEDWD-3', 5'-IPDPDAKKPDDWD-3'), signal peptide and an ER-targeting 5'-KDEL-3' sequence of HaCRT. Close sequence similarity of HaCRT was observed with Hippocampus comes from phylogenetic analysis and pairwise sequence comparison. From quantitative polymerase chain reaction (qPCR) results, HaCRT was ubiquitously distributed in all tested tissues and expression levels of HaCRT were significantly modulated in blood, liver and gill tissues after stimulation with Streptococcus iniae, Edwardsiella tarda, polyinosinic:polycytidylic acid, and lipopolysaccharides. Bacterial- and pathogen-associated molecular patterns-binding activities were observed with recombinant HaCRT (rHaCRT). The treatment of murine macrophages with rHaCRT induced the expression of immune genes, such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and interleukin-1ß (IL-1ß). Furthermore, rHaCRT exhibited wound-healing ability. Based on the results from the above study, we suggest that HaCRT play an indispensable role in the immunity of big-belly seahorses by recognition and elimination of pathogens as well as the tissue repairing process.
Asunto(s)
Calreticulina/genética , Calreticulina/inmunología , Proteínas de Peces/genética , Smegmamorpha/genética , Smegmamorpha/inmunología , Secuencia de Aminoácidos , Animales , Calreticulina/química , Proteínas de Peces/química , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , FilogeniaRESUMEN
My coworkers and I have used animal viruses and their interaction with host cells to investigate cellular processes difficult to study by other means. This approach has allowed us to branch out in many directions, including membrane protein characterization, endocytosis, secretion, protein folding, quality control, and glycobiology. At the same time, our aim has been to employ cell biological approaches to expand the fundamental understanding of animal viruses and their pathogenic lifestyles. We have studied mechanisms of host cell entry and the uncoating of incoming viruses as well as the synthesis, folding, maturation, and intracellular movement of viral proteins and molecular assemblies. I have had the privilege to work in institutions in four different countries. The early years in Finland (the University of Helsinki) were followed by 6 years in Germany (European Molecular Biology Laboratory), 16 years in the United States (Yale School of Medicine), and 16 years in Switzerland (ETH Zurich).
Asunto(s)
Calnexina/genética , Calreticulina/genética , Interacciones Huésped-Patógeno/genética , Virus de la Influenza A/genética , Picornaviridae/genética , Proteínas Virales/genética , Virología/historia , Animales , Calnexina/química , Calnexina/metabolismo , Calreticulina/química , Calreticulina/metabolismo , Línea Celular , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Endosomas/metabolismo , Endosomas/virología , Regulación de la Expresión Génica , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Virus de la Influenza A/metabolismo , Picornaviridae/metabolismo , Pliegue de Proteína , Virus de los Bosques Semliki/genética , Virus de los Bosques Semliki/metabolismo , Vesiculovirus/genética , Vesiculovirus/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Internalización del VirusRESUMEN
Calnexin (CNX) and calreticulin (CRT) are ER-resident lectin-like molecular chaperones involved in the quality control of secretory or membrane glycoproteins. They can exert molecular chaperone functions via specific binding to the early processing intermediates of Glc1Man9GlcNAc2 oligosaccharides of N-glycoproteins. CNX and CRT have similar N-terminal luminal domains and share the same jelly roll tertiary structure as legume lectins. In addition to the lectin-like interactions, CNX and CRT also suppress the aggregation of non-glycosylated substrates through interaction with hydrophobic peptide parts, suggesting a general chaperone function in glycan-dependent and glycan-independent manners. This chapter describes the isolation and purification of CRT produced in a bacterial expression system. We also introduce in vitro assays to estimate the molecular chaperone functions of CRT via the interaction with monoglucosylated N-glycans using Jack bean α-mannosidase as a target substrate. These assays are valuable in assessing quality control events related to the CNX/CRT chaperone cycle and lectin functions.
Asunto(s)
Bacterias/crecimiento & desarrollo , Calreticulina/genética , Calreticulina/metabolismo , Polisacáridos/metabolismo , Animales , Bacterias/genética , Calreticulina/química , Retículo Endoplásmico/metabolismo , Ratones , Unión Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/metabolismo , alfa-Manosidasa/metabolismoRESUMEN
Calreticulin (CRT) is localized to and has functions in multiple cellular compartments, including the cell surface, the endoplasmic reticulum, and the extracellular matrix. Mutagenesis studies have identified several residues on a concave ß-sheet surface of CRT critical for CRT binding to carbohydrate and other proteins/peptides. How the mutations of these key residues in CRT affect the conformation and dynamics of CRT, further influencing CRT binding to carbohydrates and other proteins to signal the important biological activities remain unknown. In this study, we investigated the effect of three key point mutations (C105A, C137A and W319A) on CRT conformation and dynamics via atomistic molecular dynamics simulations. Results show that these three key residues mutations induced the changes of CRT local backbone flexibility and secondary structure of CRT N-domain, which could further affect CRT's binding activity. C137A mutation led to dramatic decrease of the overall size of CRT due to the P-domain fold back to the globular domain and formed new inter-domain contacts, which can cause blockage of CRT's binding with other large substrates. Furthermore, for CRT concave ß-strand surface patch containing lectin binding site, CRT C105A, C137A and W319A point mutation resulted in the changes in solvent accessible surface area, key residues' side chain atom positions and dynamical correlated motions between residues. All these changes could directly affect CRT binding behavior. Results of this study provide molecular and structural insights into understanding the role of key residues of CRT in its binding behavior.
Asunto(s)
Calreticulina/química , Biología Computacional , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Sitios de Unión , Calreticulina/genética , Humanos , Mutación Puntual , Conformación Proteica , Alineación de SecuenciaRESUMEN
Cell surface calreticulin (CRT) can mediate apoptotic cells removal by binding and activating LDL receptor-related protein (LRP1). Phosphatidylserine (PS) lipids in the inner leaflet of the cell membrane are externalized and become exposed in cholesterol (CHOL)-rich membrane raft-like microdomain during apoptosis and co-localized with cell surface CRT. How the apoptotic raft-like membrane microdomain affects the structure and dynamics of CRT, further affecting CRT binding with LRP1 to signal apoptotic-cell clearance, remains unknown. In this study, we investigate the interactions between CRT and raft-like bilayers with or without POPS lipids with molecular dynamics simulations. In addition, the effect of an apoptotic raft bilayer on the binding between CRT and thrombospondin-1 (TSP1), a ligand of CRT on the cell surface to signal focal adhesion disassembly, was also investigated. Results of single CRT interactions with raft-like bilayers show that PS lipids in apoptotic raft-like bilayer increased the interactions between CRT and lipid bilayer, which enhanced the conformational stability and increased dynamical motion of CRT. The microscopic and mesoscopic properties of apoptotic raft-like bilayer were altered by the binding of CRT with lipid bilayer. Results of CRT-TSP1 complex interactions with raft-like bilayers show that the binding free energy between TSP1 and CRT was reduced in apoptotic raft-like bilayer environment. This study provides molecular and structural insight into the effect of an apoptotic raft-like bilayer on the conformation and dynamics of CRT, which could enrich our understanding of CRT-mediated apoptotic-cell clearance and focal adhesion disassembly.
Asunto(s)
Apoptosis , Calreticulina/química , Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Simulación de Dinámica Molecular , Sitios de Unión , Calreticulina/metabolismo , Microdominios de Membrana/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Unión Proteica , Trombospondinas/química , Trombospondinas/metabolismoRESUMEN
Microbes have developed mechanisms to resist the host immune defenses and some elicit antitumor immune responses. About 6 million people are infected with Trypanosoma cruzi, the protozoan agent of Chagas' disease, the sixth neglected tropical disease worldwide. Eighty years ago, G. Roskin and N. Klyuyeva proposed that T. cruzi infection mediates an anti-cancer activity. This observation has been reproduced by several other laboratories, but no molecular basis has been proposed. We have shown that the highly pleiotropic chaperone calreticulin (TcCalr, formerly known as TcCRT), translocates from the parasite ER to the exterior, where it mediates infection. Similar to its human counterpart HuCALR (formerly known as HuCRT), TcCalr inhibits C1 in its capacity to initiate the classical pathway of complement activation. We have also proposed that TcCalr inhibits angiogenesis and it is a likely mediator of antitumor effects. We have generated several in silico structural TcCalr models to delimit a peptide (VC-TcCalr) at the TcCalr N-domain. Chemically synthesized VC-TcCalr did bind to C1q and was anti-angiogenic in Gallus gallus chorioallantoic membrane assays. These properties were associated with structural features, as determined in silico. VC-TcCalr, a strong dipole, interacts with charged proteins such as collagen-like tails and scavenger receptors. Comparatively, HuCALR has less polarity and spatial stability, probably due to at least substitutions of Gln for Gly, Arg for Lys, Arg for Asp and Ser for Arg that hinder protein-protein interactions. These differences can explain, at least in part, how TcCalr inhibits the complement activation pathway and has higher efficiency as an antiangiogenic and antitumor agent than HuCALR.