RESUMEN
The human Solute Carrier (SLC) family member, monocarboxylate transporter 1 (MCT1), transports lactic and pyruvic acid across biological membranes to regulate cellular pH and metabolism. Proper trafficking of MCT1 from the endoplasmic reticulum to the plasma membrane hinges on its interactions with the membrane-bound chaperone protein, CD147. Here, using AlphaFold2 modeling and copurification, we show how a conserved signature motif located in the flexible N-terminus of MCT1 is a crucial region of interaction between MCT1 and the C-terminus of CD147. Mutations to this motif-namely, the thymic cancer linked G19C and the highly conserved W20A-destabilize the MCT1-CD147 complex and lead to a loss of proper membrane localization and cellular substrate flux. Notably, the monomeric stability of MCT1 remains unaffected in mutants, thus supporting the role of CD147 in mediating the trafficking of the heterocomplex. Using the auxiliary chaperone, GP70, we demonstrated that W20A-MCT1 can be trafficked to the plasma membrane, while G19C-MCT1 remains internalized. Overall, our findings underscore the critical role of the MCT1 transmembrane one signature motif for engaging CD147 and identify altered chaperone binding mechanisms between the CD147 and GP70 glycoprotein chaperones.
Asunto(s)
Secuencias de Aminoácidos , Basigina , Transportadores de Ácidos Monocarboxílicos , Transporte de Proteínas , Simportadores , Basigina/metabolismo , Basigina/genética , Basigina/química , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/química , Humanos , Simportadores/metabolismo , Simportadores/química , Simportadores/genética , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Células HEK293 , Mutación MissenseRESUMEN
XK-related 8 (XKR8), in complex with the transmembrane glycoprotein basigin, functions as a phospholipid scramblase activated by the caspase-mediated cleavage or phosphorylation of its C-terminal tail. It carries a putative phospholipid translocation path of multiple hydrophobic and charged residues in the transmembrane region. It also has a crucial tryptophan at the exoplasmic end of the path that regulates its scrambling activity. We herein investigated the tertiary structure of the human XKR8-basigin complex embedded in lipid nanodiscs at an overall resolution of 3.66 Å. We found that the C-terminal tail engaged in intricate polar and van der Waals interactions with a groove at the cytoplasmic surface of XKR8. These interactions maintained the inactive state of XKR8. Point mutations to disrupt these interactions strongly enhanced the scrambling activity of XKR8, suggesting that the activation of XKR8 is mediated by releasing the C-terminal tail from the cytoplasmic groove. We speculate that the cytoplasmic tail region of XKR8 functions as a plug to prevent the scrambling of phospholipids.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Basigina , Proteínas de la Membrana , Proteínas de Transferencia de Fosfolípidos , Humanos , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Basigina/química , Membrana Celular/metabolismo , Liposomas/química , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Nanopartículas/química , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/genética , Fosfolípidos , Conformación Proteica en Hélice alfa , Imagen Individual de MoléculaRESUMEN
Purpose: Basigin gene products are positioned on adjacent cell types in the neural retina and are thought to compose a lactate metabolon important for photoreceptor cell function. The Ig0 domain of basigin isoform 1 (basigin-1) is highly conserved throughout evolution, which suggests a conserved function. It has been suggested that the Ig0 domain has proinflammatory properties, and it is hypothesized to interact with basigin isoform 2 (basigin-2) for cell adhesion and lactate metabolon formation. Therefore, the purpose of the present study was to determine whether the Ig0 domain of basigin-1 binds to basigin-2 and whether the region of the domain used for binding is also used to stimulate interleukin-6 (IL-6) expression. Methods: Binding was assessed using recombinant proteins corresponding to the Ig0 domain of basigin-1 and endogenously expressed basigin-2 from mouse neural retina and brain protein lysates. The proinflammatory properties of the Ig0 domain were analyzed with exposure of the recombinant proteins to the mouse monocyte RAW 264.7 cell line and subsequent measurement of the IL-6 concentration in the culture medium via enzyme-linked immunosorbent assay (ELISA). Results: The data indicate that the Ig0 domain interacts with basigin-2 through a region within the amino half of the domain and that the Ig0 domain does not stimulate the expression of IL-6 in mouse cells in vitro. Conclusions: The Ig0 domain of basigin-1 binds to basigin-2 in vitro. In addition, contrary to previous reports, there was no evidence that the Ig0 domain potentiates IL-6 expression in a mouse monocyte cell line in vitro. However, it is possible that the Ig0 domain stimulates the expression of proinflammatory cytokines other than IL-6, or that the potential involvement of the Ig0 domain of basigin-1 in the acute inflammatory response is dependent on species.
Asunto(s)
Basigina , Interleucina-6 , Ratones , Animales , Basigina/química , Basigina/genética , Basigina/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Monocitos , Retina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Lactatos/metabolismoRESUMEN
Xkr8-Basigin is a plasma membrane phospholipid scramblase activated by kinases or caspases. We combined cryo-EM and X-ray crystallography to investigate its structure at an overall resolution of 3.8 Å. Its membrane-spanning region carrying 22 charged amino acids adopts a cuboid-like structure stabilized by salt bridges between hydrophilic residues in transmembrane helices. Phosphatidylcholine binding was observed in a hydrophobic cleft on the surface exposed to the outer leaflet of the plasma membrane. Six charged residues placed from top to bottom inside the molecule were essential for scrambling phospholipids in inward and outward directions, apparently providing a pathway for their translocation. A tryptophan residue was present between the head group of phosphatidylcholine and the extracellular end of the path. Its mutation to alanine made the Xkr8-Basigin complex constitutively active, indicating that it plays a vital role in regulating its scramblase activity. The structure of Xkr8-Basigin provides insights into the molecular mechanisms underlying phospholipid scrambling.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Basigina/química , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Fosfolípidos/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Basigina/metabolismo , Microscopía por Crioelectrón , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/química , Estructura Terciaria de Proteína , Triptófano/químicaRESUMEN
Monocarboxylate transporter isoforms 1-4, MCT, of the solute carrier SLC16A family facilitate proton-coupled transport of l-lactate. Growth of tumors that exhibit the Warburg effect, that is, high rates of anaerobic glycolysis despite availability of oxygen, relies on swift l-lactate export, whereas oxygenic cancer cells import circulating l-lactate as a fuel. Currently, MCTs are viewed as promising anticancer targets. Small-molecule inhibitors have been found, and, recently, high-resolution protein structures have been obtained. Key questions, however, regarding the exact binding sites of cysteine-modifying inhibitors and the substrate translocation cycle lack a conclusive experimental basis. Here, we report Cys159 of the ubiquitous human MCT1 to reside in a critical hinge region of the alternating access-type transporter. We identified Cys159 as the binding site of the organomercurial pCMBS. The inhibitory effect of pCMBS was proposed to be indirect via modification of the chaperone basigin. We provide evidence that pCMBS locks MCT1 in its outward open conformation in a wedge-like fashion. We corroborated this finding using smaller cysteine-modifying reagents that size-dependently inhibited l-lactate transport. The smallest modifiers targeted additional cysteines as shown by a C159S mutant. We found a Cys399/Cys400 pair to constitute the second hinge of the transporter that tolerated only individual replacement by serine. The hinge cysteines, in particular the selectively addressable Cys159, provide natural anchors for placing probes into MCTs to report, for instance, on the electrostatics or hydration upon binding of the transported l-lactate substrate and the proton cosubstrate.
Asunto(s)
4-Cloromercuribencenosulfonato/farmacología , Basigina/química , Cisteína/química , Inhibidores Enzimáticos/farmacología , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Simportadores/antagonistas & inhibidores , Basigina/genética , Basigina/metabolismo , Humanos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Conformación Proteica , Simportadores/metabolismoRESUMEN
Transmembrane transport of l-lactate by members of the monocarboxylate transporter family, MCT, is vital in human physiology and a malignancy factor in cancer. Interaction with an accessory protein, typically basigin, is required to deliver the MCT to the plasma membrane. It is unknown whether basigin additionally exerts direct effects on the transmembrane l-lactate transport of MCT1. Here, we show that the presence of basigin leads to an intracellular accumulation of l-lactate 4.5-fold above the substrate/proton concentrations provided by the external buffer. Using basigin truncations we localized the effect to arise from the extracellular Ig-I domain. Identification of surface patches of condensed opposite electrostatic potential, and experimental analysis of charge-affecting Ig-I mutants indicated a bivalent harvesting antenna functionality for both, protons and substrate anions. From these data, and determinations of the cytosolic pH with a fluorescent probe, we conclude that the basigin Ig-I domain drives lactate uptake by locally increasing the proton and substrate concentration at the extracellular MCT entry site. The biophysical properties are physiologically relevant as cell growth on lactate media was strongly promoted in the presence of the Ig-I domain. Lack of the domain due to shedding, or misfolding due to breakage of a stabilizing disulfide bridge reversed the effect. Tumor progression according to classical or reverse Warburg effects depends on the transmembrane l-lactate distribution, and this study shows that the basigin Ig-I domain is a pivotal determinant.
Asunto(s)
Basigina/metabolismo , Espacio Intracelular/metabolismo , Ácido Láctico/metabolismo , Basigina/química , Línea Celular Tumoral , Humanos , Dominios Proteicos , Transporte de ProteínasRESUMEN
In the treatment of tumor-targeted small-molecule anti-cancer drugs, antibody-mediated therapies, especially for antibody-drug conjugates (ADCs), have revealed great latent force. However, the therapeutic drugs provided by ADCs possess limitation. Considering that the combination of antibodies and nano-drugs can broaden their applicability in the field of tumor treatment, herein, we developed an antibody conjugated polymeric prodrug nanoparticles SAE-PEG-b-PBYP-ss-CPT for targeted camptothecin (CPT) delivery to liver tumor cells. The diblock copolymer was composed of PEG and biodegradable polyphosphoester (PBYP) containing alkynyl groups in the side chain. A derivative of CPT (CPT-ss-N3) was bonded to the PBYP via "click" reaction. The diethyl squarate (SAE) in the terminal of PEG chain was used as a functional group to bond with CD147 monoclonal antibody (CD147 mAb). The particle size and size distribution of the both nanoparticles, with antibody binding (namely CD147-CPT NPs) and without antibody (abbreviated as CPT-loaded NPs), were measured by dynamic light scattering (DLS). The morphologies of both two kinds of nanoparticles were observed by transmission electron microscope (TEM). The results of X-ray photoelectron spectroscopy (XPS) showed that CD147 mAb had been coupled to the surface of CPT-loaded NPs. Endocytosis test indicated that CD147-CPT NPs had higher uptake rate and accumulation in HepG2 cells than those of CPT-loaded NPs without antibodies, due to CD147 mAb can specifically bind to CD147 protein overexpressed in HepG2 cells. We establish a method to bond monoclonal antibodies to anti-cancer polymeric prodrugs, and endow biodegradable polymeric prodrugs with precise targeting functions to liver cancer cells.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Basigina/química , Materiales Biocompatibles/farmacología , Camptotecina/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Inmunoconjugados/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Antineoplásicos Fitogénicos/química , Basigina/genética , Basigina/metabolismo , Materiales Biocompatibles/química , Camptotecina/química , Carcinoma Hepatocelular/patología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Inmunoconjugados/química , Neoplasias Hepáticas/patología , Ensayo de Materiales , Estructura Molecular , Nanomedicina , Tamaño de la Partícula , Profármacos/química , Profármacos/farmacologíaRESUMEN
Proton-coupled monocarboxylate transporters MCT1-4 catalyze the transmembrane movement of metabolically essential monocarboxylates and have been targeted for cancer treatment because of their enhanced expression in various tumors. Here, we report five cryo-EM structures, at resolutions of 3.0-3.3 Å, of human MCT1 bound to lactate or inhibitors in the presence of Basigin-2, a single transmembrane segment (TM)-containing chaperon. MCT1 exhibits similar outward-open conformations when complexed with lactate or the inhibitors BAY-8002 and AZD3965. In the presence of the inhibitor 7ACC2 or with the neutralization of the proton-coupling residue Asp309 by Asn, similar inward-open structures were captured. Complemented by structural-guided biochemical analyses, our studies reveal the substrate binding and transport mechanism of MCTs, elucidate the mode of action of three anti-cancer drug candidates, and identify the determinants for subtype-specific sensitivities to AZD3965 by MCT1 and MCT4. These findings lay out an important framework for structure-guided drug discovery targeting MCTs.
Asunto(s)
Antineoplásicos/farmacología , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Transportadores de Ácidos Monocarboxílicos/química , Simportadores/antagonistas & inhibidores , Simportadores/química , Secuencia de Aminoácidos , Animales , Basigina/química , Sitios de Unión , Microscopía por Crioelectrón , Humanos , Ligandos , Modelos Moleculares , Transportadores de Ácidos Monocarboxílicos/ultraestructura , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Protones , Pirimidinonas/química , Pirimidinonas/farmacología , Ratas , Homología Estructural de Proteína , Especificidad por Sustrato , Simportadores/ultraestructura , Tiofenos/química , Tiofenos/farmacologíaRESUMEN
Fluorescent fusion proteins are powerful tools for studying biological processes in living cells, but universal application is limited due to the voluminous size of those tags, which might have an impact on the folding, localization or even the biological function of the target protein. The designed biocatalyst trypsiligase enables site-directed linkage of small-sized fluorescence dyes on the Nâ terminus of integral target proteins located in the outer membrane of living cells through a stable native peptide bond. The function of the approach was tested by using the examples of covalent derivatization of the transmembrane proteins CD147 as well as the EGF receptor, both presented on human HeLa cells. Specific trypsiligase recognition of the site of linkage was mediated by the dipeptide sequence Arg-His added to the proteins' native Nâ termini, pointing outside the cell membrane. The labeling procedure takes only about 5 minutes, as demonstrated for couplings of the fluorescence dye tetramethyl rhodamine and the affinity label biotin as well.
Asunto(s)
Basigina/metabolismo , Receptores ErbB/metabolismo , Colorantes Fluorescentes/metabolismo , Tripsina/metabolismo , Basigina/química , Biocatálisis , Dipéptidos/metabolismo , Receptores ErbB/química , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Microscopía Confocal , Especificidad por Sustrato , Tripsina/genéticaRESUMEN
The world is experiencing one of the most difficult moments in history with the COVID-19 pandemic, a disease caused by SARS-CoV-2, a new type of coronavirus. Virus infectivity is mediated by the binding of Spike transmembrane glycoprotein to specific protein receptors present on cell host surface. Spike is a homotrimer that emerges from the virion, each monomer containing two subunits named S1 and S2, which are related to cell recognition and membrane fusion, respectively. S1 is subdivided in domains S1A (or NTD) and S1B (or RBD), with experimental and in silico studies suggesting that the former binds to sialic acid-containing glycoproteins, such as CD147, whereas the latter binds to ACE2 receptor. Recent findings indicate that the ABO blood system modulates susceptibility and progression of infection, with type-A individuals being more susceptible to infection and/or manifestation of a severe condition. Seeking to understand the molecular mechanisms underlying this susceptibility, we carried out an extensive bibliographic survey on the subject. Based on this survey, we hypothesize that the correlation between the ABO blood system and susceptibility to SARS-CoV-2 infection can be presumably explained by the modulation of sialic acid-containing receptors distribution on host cell surface induced by ABO antigens through carbohydrate-carbohydrate interactions, which could maximize or minimize the virus Spike protein binding to the host cell. This model could explain previous sparse observations on the molecular mechanism of infection and can direct future research to better understand of COVID-19 pathophysiology.
Asunto(s)
Sistema del Grupo Sanguíneo ABO , COVID-19/sangre , COVID-19/diagnóstico , Carbohidratos/química , Susceptibilidad a Enfermedades , Glicoproteína de la Espiga del Coronavirus/química , Enzima Convertidora de Angiotensina 2/química , Animales , Basigina/química , Sitios de Unión , COVID-19/epidemiología , Humanos , Modelos Teóricos , Simulación de Dinámica Molecular , Unión Proteica , Dominios Proteicos , SARS-CoV-2 , Internalización del VirusAsunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Basigina/antagonistas & inhibidores , Basigina/fisiología , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/uso terapéutico , Basigina/química , Basigina/inmunología , Humanos , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendencias , Neoplasias/patologíaRESUMEN
Tumor cells rely on glycolysis to meet their elevated demand for energy. Thereby they produce significant amounts of lactate and protons, which are exported via monocarboxylate transporters (MCTs), supporting the formation of an acidic microenvironment. The present study demonstrates that carbonic anhydrase IX (CAIX), one of the major acid/base regulators in cancer cells, forms a protein complex with MCT1 and MCT4 in tissue samples from human breast cancer patients, but not healthy breast tissue. Formation of this transport metabolon requires binding of CAIX to the Ig1 domain of the MCT1/4 chaperon CD147 and is required for CAIX-mediated facilitation of MCT1/4 activity. Application of an antibody, directed against the CD147-Ig1 domain, displaces CAIX from the transporter and suppresses CAIX-mediated facilitation of proton-coupled lactate transport. In cancer cells, this "metabolon disruption" results in a decrease in lactate transport, reduced glycolysis, and ultimately reduced cell proliferation. Taken together, the study shows that carbonic anhydrases form transport metabolons with acid/base transporters in human tumor tissue and that these interactions can be exploited to interfere with tumor metabolism and proliferation.
Asunto(s)
Neoplasias de la Mama/patología , Anhidrasa Carbónica IX/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Simportadores/metabolismo , Basigina/química , Basigina/metabolismo , Humanos , Células MCF-7 , Modelos Moleculares , Dominios ProteicosRESUMEN
Many important infectious diseases are the result of zoonoses, in which pathogens that normally infect animals acquire mutations that enable the breaching of species barriers to permit the infection of humans. Our understanding of the molecular events that enable host switching are often limited, and yet this is a fundamentally important question. Plasmodium falciparum, the etiological agent of severe human malaria, evolved following a zoonotic transfer of parasites from gorillas. One gene-rh5-which encodes an essential ligand for the invasion of host erythrocytes, is suspected to have played a critical role in this host switch. Genome comparisons revealed an introgressed sequence in the ancestor of P. falciparum containing rh5, which likely allowed the ancestral parasites to infect both gorilla and human erythrocytes. To test this hypothesis, we resurrected the ancestral introgressed reticulocyte-binding protein homologue 5 (RH5) sequence and used quantitative protein interaction assays to demonstrate that this ancestral protein could bind the basigin receptor from both humans and gorillas. We also showed that this promiscuous receptor binding phenotype of RH5 was shared with the parasite clade that transferred its genome segment to the ancestor of P. falciparum, while the other lineages exhibit host-specific receptor binding, confirming the central importance of this introgression event for Plasmodium host switching. Finally, since its transfer to humans, P. falciparum, and also the RH5 ligand, have evolved a strong human specificity. We show that this subsequent restriction to humans can be attributed to a single amino acid mutation in the RH5 sequence. Our findings reveal a molecular pathway for the origin and evolution of human P. falciparum malaria and may inform molecular surveillance to predict future zoonoses.
Asunto(s)
Basigina/genética , Proteínas Portadoras/genética , Genoma de Protozoos , Malaria Falciparum/transmisión , Malaria Falciparum/veterinaria , Plasmodium falciparum/genética , Sustitución de Aminoácidos , Animales , Basigina/química , Basigina/metabolismo , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Eritrocitos/parasitología , Expresión Génica , Introgresión Genética , Gorilla gorilla/parasitología , Historia Antigua , Especificidad del Huésped , Humanos , Malaria Falciparum/epidemiología , Malaria Falciparum/historia , Modelos Moleculares , Mutación , Filogenia , Plasmodium falciparum/clasificación , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Unión Proteica , Estructura Secundaria de Proteína , ZoonosisRESUMEN
An increased surface level of CIE (clathrin-independent endocytosis) proteins is a new feature of malignant neoplasms. CD147 is a CIE glycoprotein highly up-regulated in hepatocellular carcinoma (HCC). The ability to sort out the early endosome and directly target the recycling pathway confers on CD147 a prolonged surface half-life. However, current knowledge on CD147 trafficking to and from the cell-surface is limited. In this study, an MSP (membrane and secreted protein)-cDNA library was screened against EpoR/LR-F3/CD147EP-expressed cells by MAPPIT (mammalian protein-protein interaction trap). CD147 co-expressing with the new binder was investigated by GEPIA (gene expression profiling interactive analysis). The endocytosis, ER-Golgi trafficking and recycling of CD147 were measured by confocal imaging, flow cytometry, and biotin-labeled chase assays, respectively. Rab GTPase activation was checked by GST-RBD pull-down and MMP activity was measured by gelatin zymography. HCC malignant phenotypes were determined by cell adhesion, proliferation, migration, Transwell motility, and invasion assays. An ER-Golgi-resident transmembrane protein YIPF2 was identified as an intracellular binder to CD147. YIPF2 correlated and co-expressed with CD147, which is a survival predictor for HCC patients. YIPF2 is critical for CD147 glycosylation and trafficking functions in HCC cells. YIPF2 acts as a Rab-GDF (GDI-displacement factor) regulating three independent trafficking steps. First, YIPF2 recruits and activates Rab5 and Rab22a GTPases to the endomembrane structures. Second, YIPF2 modulates the endocytic recycling of CD147 through distinctive regulation on Rab5 and Rab22a. Third, YIPF2 mediates the mature processing of CD147 via the ER-Golgi trafficking route. Decreased YIPF2 expression induced a CD147 efficient delivery to the cell-surface, promoted MMP secretion, and enhanced the adhesion, motility, migration, and invasion behaviors of HCC cells. Thus, YIPF2 is a new trafficking determinant essential for CD147 glycosylation and transport. Our findings revealed a novel YIPF2-controlled ER-Golgi trafficking signature that promotes CD147-medated malignant phenotypes in HCC.
Asunto(s)
Basigina/metabolismo , Carcinoma Hepatocelular/metabolismo , Endocitosis , Neoplasias Hepáticas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Células 3T3 , Animales , Basigina/química , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidad , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Endocitosis/genética , Retículo Endoplásmico/metabolismo , Glicosilación , Aparato de Golgi/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Metaloproteinasas de la Matriz/metabolismo , Proteínas de la Membrana/genética , Ratones , Transporte de Proteínas/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
PURPOSE: CD147 is a tumor-associated antigen that plays a key regulatory role in tumor invasion and distant metastasis. However, the exact role of CD147 phosphorylation, which is deregulated during cancer progression, is unknown. Here, the effects of CD147 phosphorylation on the malignant behavior of hepatocellular carcinoma (HCC) cells and its possible underlying mechanisms are explored. METHODS: An in situ Duolink-proximity ligation assay (PLA) was used to detect CD147 phosphorylation. Tandem mass spectrometry was employed to identify the phosphorylation sites of CD147. The effects of CD147 phosphorylation on the malignant behavior of HCC cells were evaluated using scratch wound healing assays, transwell invasion assays and cell cycle assays. The genes regulated by CD147 phosphorylation were detected by RNA sequencing. RESULTS: We identified phosphorylated serine-246 in the C terminus of CD147 in primary HCC tissues, whereas serine to alanine substitution mutation analysis suggested that CD147 is phosphorylated mainly at serine-252 in HCC-derived Huh-7 cells. Recovery expression of S246A/S252A mutants in CD147 knockout cells revealed significantly increased migration and invasion capacities compared to wildtype CD147 expressing cells. Cyclophilin A (CyPA) treatment decreased the phosphorylation level of CD147, whereas NIMA-related kinase 6 (NEK6) increased the CD147 phosphorylation level. Moreover, the CD147 phosphorylation level was found to be dramatically decreased in HCC tissues in patients with distant metastases, and a low phosphorylation level of CD147 was found to be associated with a high serum AFP level, recurrence and a poor overall survival. CONCLUSIONS: From our data we conclude that hypo-phosphorylated CD147 promotes the migration and invasion of HCC cells and correlates with an unfavorable prognosis in HCC patients, indicating that targeting the aberrantly hypo-phosphorylated form of CD147 may be instrumental for the development of novel therapeutic modalities directed against HCC metastasis.
Asunto(s)
Basigina/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Movimiento Celular , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Secuencia de Aminoácidos , Basigina/química , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Ciclofilina A/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Hepáticas/genética , Masculino , Quinasas Relacionadas con NIMA/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Pronóstico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The transmembrane glycoprotein basigin, a member of the immunoglobulin superfamily, stimulates matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation and thereby drives cancer cell invasion. Basigin is proteolytically shed from the cell surface and high concentrations of soluble basigin in the blood dictates poor prognosis in cancer patients. A positive correlation between basigin and a disintegrin and metalloproteinase (ADAM)-12 in serum from prostate cancer patients has been reported. Yet, the functional relevance of this correlation is unknown. Here, we show that ADAM12 interacts with basigin and cleaves it in the juxtamembrane region. Specifically, overexpression of ADAM12 increases ectodomain shedding of an alkaline phosphatase-tagged basigin reporter protein from the cell surface. Moreover, CRISPR/Cas9-mediated knockout of ADAM12 in human HeLa carcinoma cells results in reduced shedding of the basigin reporter, which can be rescued by ADAM12 re-expression. We detected endogenous basigin fragments, corresponding to the expected size of the ADAM12-generated ectodomain, in conditioned media from ADAM12 expressing cancer cell-lines, as well as serum samples from a healthy pregnant donor and five bladder cancer patients, known to contain high ADAM12 levels. Supporting the cancer relevance of our findings, we identified several cancer-associated mutations in the basigin membrane proximal region. Subsequent in vitro expression showed that some of these mutants are more prone to ADAM12-mediated shedding and that the shed ectodomain can enhance gelatin degradation by cancer cells. In conclusion, we identified ADAM12 as a novel basigin sheddase with a potential implication in cancer.
Asunto(s)
Proteína ADAM12/metabolismo , Basigina/metabolismo , Proteína ADAM12/química , Proteína ADAM12/genética , Secuencia de Aminoácidos , Basigina/química , Basigina/genética , Sistemas CRISPR-Cas , Línea Celular , Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Reporteros , Humanos , Mutación , Especificidad por SustratoRESUMEN
CD147/Basigin/EMMPRIN (Extracellular Matrix Metalloproteinase inducer) is a single pass type1 transmembrane protein playing a central role in developmental process, wound healing, nutrient transport, inflammation, arthritis and also in microbial pathologies. It is also found to be a potent stimulator of MMP (matrix metalloproteinases) and has been considered as a prognostic marker in cancer. Dysregulation of CD147 is reported in several types of cancer. It activates cell proliferation, invasion, metastasis and inhibits tumor cell apoptosis under hypoxic condition. Thus, CD147 serves as a hub protein in cancer, as it is involved in several homophilic and heterophilic cellular interactions spanning the major hallmarks of cancer. Targeting these interactions is considered to be an efficient therapeutic modality in cancerous conditions. Hence, by this review we intend to collate the structure-function relationships of CD147, with an exclusive thrust on potential druggable hotspots based on its intra and inter molecular interactions.
Asunto(s)
Antineoplásicos/farmacología , Basigina/química , Basigina/metabolismo , Neoplasias/tratamiento farmacológico , Animales , Diseño de Fármacos , Humanos , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Within the "seed and soil" theory of organ tropic cancer metastasis is a growing compilation of evidence that S100A8/A9 functions as a soil signal that attracts cancer cells to certain organs, which prove beneficial to their growth. S100A8/A9-sensing receptors including Toll-like receptor 4 (TLR4), advanced glycation end products (RAGE), and also important receptors we recently succeeded in identifying (EMMPRIN, NPTNß, MCAM, and ALCAM) have the potential to become promising therapeutic targets. In our study, we prepared extracellular regions of these novel molecules and fused them to human IgG2-Fc to extend half-life expectancy, and we evaluated the anti-metastatic effects of the purified decoy proteins on metastatic cancer cells. The purified proteins markedly suppressed S100A8/A9-mediated lung tropic cancer metastasis. We hence expect that our novel biologics may become a prominent medicine to prevent cancer metastasis in clinical settings through cutting the linkage between "seed and soil".
Asunto(s)
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Melanoma Experimental/prevención & control , Melanoma Experimental/secundario , Proteínas Recombinantes de Fusión/farmacología , Animales , Basigina/química , Basigina/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/farmacología , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/farmacología , Inmunoglobulina G/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Dominios Proteicos , Receptor para Productos Finales de Glicación Avanzada/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Monocarboxylate transporters (MCTs) mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation of MCT transport activity requires direct binding of CAIV to the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme's intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. In human CD147, binding of CAIV was mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 in CAIV can either act as H donor or H acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase in MCT transport activity requires direct binding between CAIV-His-88 and a charged amino acid in the extracellular domain of the transporter's chaperone.
Asunto(s)
Basigina/metabolismo , Anhidrasa Carbónica IV/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Mapas de Interacción de Proteínas , Secuencia de Aminoácidos , Animales , Basigina/química , Células HEK293 , Humanos , Proteínas de la Membrana , Modelos Moleculares , Dominios Proteicos , Ratas , Alineación de Secuencia , Simportadores/metabolismo , XenopusRESUMEN
The interaction of the proteins, tumor necrosis factor receptor-associated factor6 (TRAF6) and Basigin (CD147), is known to be associated with the over-expression of matrix metalloproteinases (MMPs) in melanoma cells. MMPs are known to be responsible for melanoma metastasis. Hence, the TRAF6-Basigin complex can act as a potential therapeutic target. In previous studies, amino acid residues Lys340, Lys 384, Glu417 and Glu511 of TRAF6 were identified as the most vital residues on the basis of their contributions to interaction energy, relative solvent accessibility and electrostatic interactions in the TRAF6-Basigin protein-protein interaction (PPI) scheme. In our current work, we performed structure-based virtual screenings of some natural compounds obtained from ZINC database (nâ¯=â¯14509) to search for molecules which can act as inhibitors against the formation of TRAF6-Basigin complex. Three potential inhibitors were identified which were observed to make intermolecular interactions with Lys384 and Glu511 of TRAF6. Molecular dynamics simulation results suggested the substantial pharmacological importance of the ligand molecules as it was observed that there was total destabilization of TRAF6-Basigin complex upon binding of the molecule ZINC02578057. From our studies, we could conclude that the ligands termed as ZINC49048033, ZINC02578057 and ZINC72320240 could have great potentials to act as inhibitors to prevent melanoma metastasis.