RESUMEN
YKL-40, also known as human cartilage glycoprotein-39 (HC-gp39) or CHI3L1, shares structural similarities with chitotriosidase (CHIT1), an active chitinase, but lacks chitinase activity. Despite being a biomarker for inflammatory disorders and cancer, the reasons for YKL-40's inert chitinase function have remained elusive. This study reveals that the loss of chitinase activity in YKL-40 has risen from multiple sequence modifications influencing its chitin affinity. Contrary to the common belief associating the lack of chitinase activity with amino acid substitutions in the catalytic motif, attempts to activate YKL-40 by creating two amino acid mutations in the catalytic motif (MT-YKL-40) proved ineffective. Subsequent exploration that included creating chimeras of MT-YKL-40 and CHIT1 catalytic domains (CatDs) identified key exons responsible for YKL-40 inactivation. Introducing YKL-40 exons 3, 6, or 8 into CHIT1 CatD resulted in chitinase inactivation. Conversely, incorporating CHIT1 exons 3, 6, and 8 into MT-YKL-40 led to its activation. Our recombinant proteins exhibited properly formed disulfide bonds, affirming a defined structure in active molecules. Biochemical and evolutionary analysis indicated that the reduced chitinase activity of MT-YKL-40 correlates with specific amino acids in exon 3. M61I and T69W substitutions in CHIT1 CatD diminished chitinase activity and increased chitin binding. Conversely, substituting I61 with M and W69 with T in MT-YKL-40 triggered chitinase activity while reducing the chitin-binding activity. Thus, W69 plays a crucial role in a unique subsite within YKL-40. These findings emphasize that YKL-40, though retaining the structural framework of a mammalian chitinase, has evolved to recognize chitin while surrendering chitinase activity.
Asunto(s)
Quitina , Proteína 1 Similar a Quitinasa-3 , Proteína 1 Similar a Quitinasa-3/metabolismo , Proteína 1 Similar a Quitinasa-3/genética , Proteína 1 Similar a Quitinasa-3/química , Humanos , Quitina/metabolismo , Quitina/química , Quitinasas/metabolismo , Quitinasas/genética , Quitinasas/química , Evolución Molecular , Hexosaminidasas/metabolismo , Hexosaminidasas/química , Hexosaminidasas/genética , Dominio Catalítico , Sustitución de Aminoácidos , Exones , Secuencia de AminoácidosRESUMEN
YKL-40, a chitinase-3-like protein 1 (CHI3L1) or human cartilage glycoprotein 39 (HC gp-39), is expressed and secreted by various cell-types including macrophages, chondrocytes, fibroblast-like synovial cells and vascular smooth muscle cells. Its biological function is not well elucidated, but it is speculated to have some connection with inflammatory reactions and autoimmune diseases. Although having important biological roles in autoimmunity, there were only attempts to elucidate relationships of YKL-40 with a single or couple of diseases in the literature. Therefore, in order to analyze the relationship between YKL-40 and the overall diseases, we reviewed 51 articles that discussed the association of YKL-40 with rheumatoid arthritis, psoriasis, systemic lupus erythematosus, Behçet disease and inflammatory bowel disease. Several studies showed that YKL-40 could be assumed as a marker for disease diagnosis, prognosis, disease activity and severity. It is also shown to be involved in response to disease treatment. However, other studies showed controversial results particularly in the case of Behçet disease activity. Therefore, further studies are needed to elucidate the exact role of YKL-40 in autoimmunity and to investigate its potential in therapeutics.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , Proteína 1 Similar a Quitinasa-3/metabolismo , Adipoquinas , Artritis Reumatoide , Síndrome de Behçet , Biomarcadores , Proteína 1 Similar a Quitinasa-3/sangre , Proteína 1 Similar a Quitinasa-3/química , HumanosRESUMEN
Breast regression protein 39 (BRP39) is a 39 kDa protein that is a member of chitolectin class of glycosyl hydrolase family 18 (GH18). High expression levels of BRP39 have been detected in breast carcinoma. It helps in proliferation of cells during the progression of this disease and may act as a signaling factor. BRP39 may act as a potential candidate for rational structure-based drug design against breast carcinoma. In this study, we report the crystal structure of mouse recombinant BRP39 expressed in E. coli. The structure was solved by molecular replacement and refined to 2.6 Å resolution. The overall structure of BRP39 consisted of two globular domains: a large (ß/α)8 triosephosphate isomerase (TIM) barrel domain and a small (α + ß) domain. Three non-proline cis-peptides were detected in the sugar-binding cleft of BRP39, including Ser57-Phe58, Leu141-Tyr142, and Trp353-Ala354. The latter residues were conserved in other GH18 family members. It was notable that the conformation of critical Trp100 residue within the sugar-binding cleft was oriented away from the barrel. The side-chain conformation was found to be similar to that observed in chitinases, however, it was oriented into the barrel in other chitinase-like proteins (CLPs). The conformation of this critical residue may have significant implications in sugar binding. Further, two amino acid substitutions were observed in the sugar-binding groove of BRP39. The conserved Asn100 and Arg263 in Hcgp39 and other CLPs proteins (SPX-40 structures) were substituted by Lys101 and Lys264 in BRP39 which may have a significant impact on the sugar-binding properties.
Asunto(s)
Proteína 1 Similar a Quitinasa-3/química , Proteína 1 Similar a Quitinasa-3/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Dominio Catalítico , Proteína 1 Similar a Quitinasa-3/genética , Proteína 1 Similar a Quitinasa-3/aislamiento & purificación , Quitinasas/química , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Azúcares/metabolismo , Triptófano/químicaRESUMEN
Dis3L2 is a highly conserved 3'-5' exoribonuclease which is mutated in the human overgrowth disorders Perlman syndrome and Wilms' tumour of the kidney. Using Drosophila melanogaster as a model system, we have generated a new dis3L2 null mutant together with wild-type and nuclease-dead genetic lines in Drosophila to demonstrate that the catalytic activity of Dis3L2 is required to control cell proliferation. To understand the cellular pathways regulated by Dis3L2 to control proliferation, we used RNA-seq on dis3L2 mutant wing discs to show that the imaginal disc growth factor Idgf2 is responsible for driving the wing overgrowth. IDGFs are conserved proteins homologous to human chitinase-like proteins such as CHI3L1/YKL-40 which are implicated in tissue regeneration as well as cancers including colon cancer and non-small cell lung cancer. We also demonstrate that loss of DIS3L2 in human kidney HEK-293T cells results in cell proliferation, illustrating the conservation of this important cell proliferation pathway. Using these human cells, we show that loss of DIS3L2 results in an increase in the PI3-Kinase/AKT signalling pathway, which we subsequently show to contribute towards the proliferation phenotype in Drosophila. Our work therefore provides the first mechanistic explanation for DIS3L2-induced overgrowth in humans and flies and identifies an ancient proliferation pathway controlled by Dis3L2 to regulate cell proliferation and tissue growth.
Asunto(s)
Proliferación Celular , Discos Imaginales/metabolismo , Animales , Proteína 1 Similar a Quitinasa-3/química , Proteína 1 Similar a Quitinasa-3/metabolismo , Secuencia Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Discos Imaginales/crecimiento & desarrollo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Chitinase 3 like 1 protein (Chi3L1) is expressed in several cancers, and a few evidences suggest that the secreted Chi3L1 contributes to tumor development. However, the molecular mechanisms of intracellular Chi3L1 are unknown in the lung tumor development. METHODS: In the present study, we generated Chi3L1 knockout mice (Chi3L1KO(-/-)) using CRISPR/Cas9 system to investigate the role of Chi3L1 on lung tumorigenesis. RESULTS: We established lung metastasis induced by i.v. injections of B16F10 in Chi3L1KO(-/-). The lung tumor nodules were significantly reduced in Chi3L1KO(-/-) and protein levels of p53, p21, BAX, and cleaved-caspase 3 were significantly increased in Chi3L1KO(-/-), while protein levels of cyclin E1, CDK2, and phsphorylation of STAT3 were decreased in Chi3L1KO(-/-). Allograft mice inoculated with B16F10 also suppressed tumor growth and increased p53 and its target proteins including p21 and BAX. In addition, knockdown of Chi3L1 in lung cancer cells inhibited lung cancer cell growth and upregulated p53 expression with p21 and BAX, and a decrease in phosphorylation of STAT3. Furthermore, we found that intracellular Chi3L1 physically interacted and colocalized with p53 to inhibit its protein stability and transcriptional activity for target genes related with cell cycle arrest and apoptosis. In lung tumor patient, we clinically found that Chi3L1 expression was upregulated with a decrease in p53 expression, as well as we validated that intracellular Chi3L1 was colocalized, reversely expressed, and physically interacted with p53, which results in suppression of the expression and function of p53 in lung tumor patient. CONCLUSIONS: Our studies suggest that intracellular Chi3L1 plays a critical role in the lung tumorigenesis by regulating its novel target protein, p53 in both an in vitro and in vivo system.
Asunto(s)
Carcinogénesis/patología , Proteína 1 Similar a Quitinasa-3/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteína p53 Supresora de Tumor/metabolismo , Aloinjertos , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Proteína 1 Similar a Quitinasa-3/química , Regulación hacia Abajo , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Metástasis de la Neoplasia , Unión Proteica , Estabilidad Proteica , Transcripción Genética , UbiquitinaciónRESUMEN
The heparin-binding glycoprotein YKL-40 (CHI3L1) is intimately associated with microvascularization in multiple human diseases including cancer and inflammation. However, the heparin-binding domain(s) pertinent to the angiogenic activity have yet been identified. YKL-40 harbors a consensus heparin-binding motif that consists of positively charged arginine (R) and lysine (K) (RRDK; residues 144-147); but they don't bind to heparin. Intriguingly, we identified a separate KR-rich domain (residues 334-345) that does display strong heparin binding affinity. A short synthetic peptide spanning this KR-rich domain successfully competed with YKL-40 and blocked its ability to bind heparin. Three individual point mutations, where alanine (A) substituted for K or R (K337A, K342A, R344A), led to remarkable decreases in heparin-binding ability and angiogenic activity. In addition, a neutralizing anti-YKL-40 antibody that targets these residues and prevents heparin binding impeded angiogenesis in vitro. MDA-MB-231 breast cancer cells engineered to express ectopic K337A, K342A or R344A mutants displayed reduced tumor development and compromised tumor vessel formation in mice relative to control cells expressing wild-type YKL-40. These data reveal that the KR-rich heparin-binding motif is the functional heparin-binding domain of YKL-40. Our findings shed light on novel molecular mechanisms underlying endothelial cell angiogenesis promoted by YKL-40 in a variety of diseases.
Asunto(s)
Arginina/metabolismo , Neoplasias de la Mama/patología , Proteína 1 Similar a Quitinasa-3/química , Proteína 1 Similar a Quitinasa-3/metabolismo , Heparina/metabolismo , Lisina/metabolismo , Neovascularización Patológica/patología , Animales , Apoptosis , Arginina/química , Arginina/genética , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Movimiento Celular , Proliferación Celular , Proteína 1 Similar a Quitinasa-3/genética , Femenino , Heparina/química , Humanos , Lisina/química , Lisina/genética , Ratones , Ratones SCID , Mutación , Neovascularización Patológica/metabolismo , Unión Proteica , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
YKL-40 is a mammalian glycoprotein associated with progression, severity, and prognosis of chronic inflammatory diseases and a multitude of cancers. Despite this well documented association, identification of the lectin's physiological ligand and, accordingly, biological function has proven experimentally difficult. YKL-40 has been shown to bind chito-oligosaccharides; however, the production of chitin by the human body has not yet been documented. Possible alternative ligands include proteoglycans, polysaccharides, and fibers like collagen, all of which makeup the extracellular matrix. It is likely that YKL-40 is interacting with these alternative polysaccharides or proteins within the body, extending its function to cell biological roles such as mediating cellular receptors and cell adhesion and migration. Here, we consider the feasibility of polysaccharides, including cello-oligosaccharides, hyaluronan, heparan sulfate, heparin, and chondroitin sulfate, and collagen-like peptides as physiological ligands for YKL-40. We use molecular dynamics simulations to resolve the molecular level recognition mechanisms and calculate the free energy of binding the hypothesized ligands to YKL-40, addressing thermodynamic preference relative to chito-oligosaccharides. Our results suggest that chitohexaose and hyaluronan preferentially bind to YKL-40 over collagen, and hyaluronan is likely the preferred physiological ligand, because the negatively charged hyaluronan shows enhanced affinity for YKL-40 over neutral chitohexaose. Collagen binds in two locations at the YKL-40 surface, potentially related to a role in fibrillar formation. Finally, heparin non-specifically binds at the YKL-40 surface, as predicted from structural studies. Overall, YKL-40 likely binds many natural ligands in vivo, but its concurrence with physical maladies may be related to associated increases in hyaluronan.