RESUMEN
Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of ß-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual's collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and ß-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.
Asunto(s)
Linfocitos T CD8-positivos/patología , Diabetes Mellitus Tipo 1/patología , Células Madre Pluripotentes Inducidas/patología , Diferenciación Celular/fisiología , Humanos , Células Secretoras de Insulina/patología , Islotes Pancreáticos/patologíaRESUMEN
Type 1 Diabetes (T1D) is an autoimmune disease marked by direct elimination of insulin-producing ß cells by autoreactive T effectors. Recent T1D clinical trials utilizing autologous Tregs transfers to restore immune balance and improve disease has prompted us to design a novel Tregs-based antigen-specific T1D immunotherapy. We engineered a Chimeric Antigen Receptor (CAR) expressing a single-chain Fv recognizing the human pancreatic endocrine marker, HPi2. Human T cells, transduced with the resultant HPi2-CAR, proliferated and amplified Granzyme B accumulation when co-cultured with human, but not mouse ß cells. Furthermore, following exposure of HPi2-CAR transduced cells to islets, CD8+ lymphocytes demonstrated enhanced CD107a (LAMP-1) expression, while CD4+ cells produced increased levels of IL-2. HPi2-CAR Tregs failed to maintain expansion due to a persistent tonic signaling from the CAR engagement to unexpectantly HPi2 antigen present on Tregs. Overall, we show lack of functionality of HPi2-CAR and highlight the importance of careful selection of CAR recognition driver for the sustainable activity and expandability of engineered T cells.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Protaminas/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Humanos , Tolerancia Inmunológica/inmunología , Inmunoterapia Adoptiva/métodos , Islotes Pancreáticos , Páncreas/citología , Páncreas/metabolismo , Protaminas/metabolismo , Ingeniería de Proteínas/métodos , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismoRESUMEN
Type 1 diabetes (T1D) is characterized by massive destruction of insulin-producing ß cells by autoreactive T lymphocytes, arising via defective immune tolerance. Therefore, effective anti-T1D therapeutics should combine autoimmunity-preventing and insulin production-restoring properties. We constructed a cell-permeable PDX1-FOXP3-TAT fusion protein (FP) composed of two transcription factors: forkhead box P3 (FOXP3), the master regulator of differentiation and functioning of self-tolerance-promoting Tregs, and pancreatic duodenal homeobox-1 (PDX1), the crucial factor supporting ß cell development and maintenance. The FP was tested in vitro and in a non-obese diabetic mouse T1D model. In vitro, FP converted naive CD4+ T cells into a functional "Treg-like" subset, which suppressed cytokine secretion, downregulated antigen-specific responses, and curbed viability of diabetogenic effector cells. In hepatic stem-like cells, FP potentiated endocrine transdifferentiation, inducing expression of Insulin2 and other ß lineage-specific genes. In vivo, FP administration to chronically diabetic mice triggered (1) a significant elevation of insulin and C-peptide levels, (2) the formation of insulin-containing cell clusters in livers, and (3) a systemic anti-inflammatory shift (higher Foxp3+CD4+CD25+ T cell frequencies, elevated rates of IL-10-producing cells, and reduced rates of IFN-γ-secreting cells). Overall, in accordance with its design, PDX1-FOXP3-TAT FP delivered both Treg-stabilizing anti-autoimmune and de novo insulin-producing effects, proving its anti-T1D therapeutic potential.
Asunto(s)
Autoinmunidad , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/metabolismo , Secreción de Insulina , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Transactivadores/metabolismo , Animales , Microambiente Celular/inmunología , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/genética , Hepatocitos/metabolismo , Proteínas de Homeodominio/genética , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Fenotipo , Unión Proteica , Proteínas Recombinantes de Fusión , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transactivadores/genéticaRESUMEN
Ag-specific activation of T cells is an essential process in the control of effector immune responses. Defects in T cell activation, particularly in the costimulation step, have been associated with many autoimmune conditions, including type 1 diabetes (T1D). Recently, we demonstrated that the phenotype of impaired negative costimulation, due to reduced levels of V-set domain-containing T cell activation inhibitor 1 (VTCN1) protein on APCs, is shared between diabetes-susceptible NOD mice and human T1D patients. In this study, we show that a similar process takes place in the target organ, as both α and ß cells within pancreatic islets gradually lose their VTCN1 protein during autoimmune diabetes development despite upregulation of the VTCN1 gene. Diminishment of functional islet cells' VTCN1 is caused by the active proteolysis by metalloproteinase N-arginine dibasic convertase 1 (NRD1) and leads to the significant induction of proliferation and cytokine production by diabetogenic T cells. Inhibition of NRD1 activity, alternatively, stabilizes VTCN1 and dulls the anti-islet T cell responses. Therefore, we suggest a general endogenous mechanism of defective VTCN1 negative costimulation, which affects both lymphoid and peripheral target tissues during T1D progression and results in aggressive anti-islet T cell responses. This mechanism is tied to upregulation of NRD1 expression and likely acts in two synergistic proteolytic modes: cell-intrinsic intracellular and cell-extrinsic systemic. Our results highlight an importance of VTCN1 stabilization on cell surfaces for the restoration of altered balance of immune control during T1D.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Islotes Pancreáticos/inmunología , Inhibidor 1 de la Activación de Células T con Dominio V-Set/inmunología , Animales , Presentación de Antígeno , Citocinas/biosíntesis , Diabetes Mellitus Tipo 1/fisiopatología , Células Secretoras de Glucagón/inmunología , Humanos , Células Secretoras de Insulina/inmunología , Islotes Pancreáticos/metabolismo , Activación de Linfocitos , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Ratones Endogámicos NOD , Proteolisis , Linfocitos T/inmunología , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismoRESUMEN
Modulation of the kallikrein-kinin system (KKS) has been shown to have beneficial effects on glucose homeostasis and several other physiological responses relevant to the progression of type 2 diabetes mellitus (T2D). The importance of bradykinin and its receptors in mediating these responses is well documented, but the role of tissue kallikrein-1, the protease that generates bradykinin in situ, is much less understood. We developed and tested DM199, recombinant human tissue kallikrein-1 protein (rhKLK-1), as a potential novel therapeutic for T2D. Hyperinsulinemic-euglycemic clamp studies suggest that DM199 increases whole body glucose disposal in non-diabetic rats. Single-dose administration of DM199 in obese db/db mice and ZDF rats, showed an acute, dose-dependent improvement in whole-body glucose utilization. Sub-acute dosing for a week in ZDF rats improved glucose utilization, with a concomitant rise in fasting insulin levels and HOMA1-%B scores. After cessation of sub-acute dosing, fasting blood glucose levels were significantly lower in ZDF rats during a drug wash-out period. Our studies show for the first time that DM199 administration results in acute anti-hyperglycemic effects in several preclinical models, and demonstrate the potential for further development of DM199 as a novel therapeutic for T2D.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacocinética , Calicreínas de Tejido/farmacología , Animales , Glucemia , Células CHO , Cricetinae , Cricetulus , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 2/sangre , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Masculino , Ratones , Ratones Obesos , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Factores de TiempoRESUMEN
T-cell responses directed against insulin-secreting pancreatic ß-cells are the key events highlighting type 1 diabetes (T1D). Therefore, a defective control of T-cell activation is thought to underlie T1D development. Recent studies implicated a B7-like negative costimulatory protein, V-set domain-containing T-cell activation inhibitor-1 (VTCN1), as a molecule capable of inhibiting T-cell activation and, potentially, an important constituent in experimental models of T1D. Here, we unravel a general deficiency within the VTCN1 pathway that is shared between diabetes-prone mice and a subset of T1D patients. Gradual loss of membrane-tethered VTCN1 from antigen-presenting cells combined with an increased release of soluble VTCN1 (sVTCN1) occurs in parallel to natural T1D development, potentiating hyperproliferation of diabetogenic T cells. Mechanistically, we demonstrate that the loss of membrane-tethered VTCN1 is linked to proteolytic cleavage mediated by the metalloproteinase nardilysin. The cleaved sVTCN1 fragment was detected at high levels in the peripheral blood of 53% T1D patients compared with only 9% of the healthy subjects. Elevated blood sVTCN1 levels appeared early in the disease progression and correlated with the aggressive pace of disease, highlighting the potential use of sVTCN1 as a new T1D biomarker, and identifying nardilysin as a potential therapeutic target.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Metaloendopeptidasas/metabolismo , Transducción de Señal/fisiología , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismo , Adolescente , Adulto , Animales , Biomarcadores/metabolismo , Niño , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Linfocitos T/metabolismo , Adulto JovenRESUMEN
Because of their important function, matrix metalloproteinases (MMPs) are promising drug targets in multiple diseases, including malignancies. The structure of MMPs includes a catalytic domain, a hinge, and a hemopexin domain (PEX), which are followed by a transmembrane and cytoplasmic tail domains or by a glycosylphosphatidylinositol linker in membrane-type MMPs (MT-MMPs). TIMPs-1, -2, -3, and -4 are potent natural regulators of the MMP activity. These are the inhibitory N-terminal and the non-inhibitory C-terminal structural domains in TIMPs. Based on our structural modeling, we hypothesized that steric clashes exist between the non-inhibitory C-terminal domain of TIMPs and the PEX of MMPs. Conversely, a certain mobility of the PEX relative to the catalytic domain is required to avoid these obstacles. Because of its exceedingly poor association constant and, in contrast with TIMP-2, TIMP-1 is inefficient against MT1-MMP. We specifically selected an MT1-MMP·TIMP-1 pair to test our hypothesis, because any improvement of the inhibitory potency would be readily recorded. We characterized the domain-swapped MT1-MMP chimeras in which the PEX of MMP-2 (that forms a complex with TIMP-2) and of MMP-9 (that forms a complex with TIMP-1) replaced the original PEX in the MT1-MMP structure. In contrast with the wild-type MT1-MMP, the diverse proteolytic activities of the swapped-PEX chimeras were then inhibited by both TIMP-1 and TIMP-2. Overall, our studies suggest that the structural parameters of both domains of TIMPs have to be taken into account for their re-engineering to harness the therapeutic in vivo potential of the novel TIMP-based MMP antagonists with constrained selectivity.
Asunto(s)
Colagenasas/química , Inhibidores de la Metaloproteinasa de la Matriz , Modelos Moleculares , Inhibidores Tisulares de Metaloproteinasas/química , Animales , Células CHO , Colagenasas/genética , Colagenasas/metabolismo , Cricetinae , Cricetulus , Humanos , Ratones , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Inhibidores Tisulares de Metaloproteinasas/genética , Inhibidores Tisulares de Metaloproteinasas/metabolismoRESUMEN
PTK7 is an essential component of the Wnt/planar cell polarity (PCP) pathway. We provide evidence that the Wnt/PCP pathway converges with pericellular proteolysis in both normal development and cancer. Here, we demonstrate that membrane type-1 matrix metalloproteinase (MT1-MMP), a key proinvasive proteinase, functions as a principal sheddase of PTK7. MT1-MMP directly cleaves the exposed PKP(621)↓LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generates, as a result, an N-terminal, soluble PTK7 fragment (sPTK7). The enforced expression of membrane PTK7 in cancer cells leads to the actin cytoskeleton reorganization and the inhibition of cell invasion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our data also demonstrate that a fine balance between the metalloproteinase activity and PTK7 levels is required for normal development of zebrafish (Danio rerio). Aberration of this balance by the proteinase inhibition or PTK7 silencing results in the PCP-dependent convergent extension defects in the zebrafish. Overall, our data suggest that the MT1-MMP-PTK7 axis plays an important role in both cancer cell invasion and normal embryogenesis in vertebrates. Further insight into these novel mechanisms may promote understanding of directional cell motility and lead to the identification of therapeutics to treat PCP-related developmental disorders and malignancy.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Embrión no Mamífero/embriología , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Sitios de Unión/genética , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Polaridad Celular , Citoesqueleto/metabolismo , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Hibridación in Situ , Metaloproteinasa 14 de la Matriz/genética , Datos de Secuencia Molecular , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Proteínas Wnt/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD/L(50) site initiates the MT1-MMP activation, whereas the (108)RRKR(111)/Y(112) cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.
Asunto(s)
Furina/metabolismo , Metaloproteinasa 14 de la Matriz/química , Metaloproteinasa 14 de la Matriz/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Activación Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Metaloproteinasa 14 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Estructura Terciaria de ProteínaRESUMEN
Ubiquitously expressed membrane type-1 matrix metalloproteinase (MT1-MMP), an archetype member of the MMP family, binds tissue inhibitor of metalloproteinases-2 (TIMP-2), activates matrix metalloproteinase-2 (MMP-2), and stimulates cell migration in various cell types. In contrast with MT1-MMP, the structurally similar MT6-MMP associates with the lipid raft compartment of the plasma membrane using a GPI anchor. As a result, MT6-MMP is functionally distinct from MT1-MMP. MT6-MMP is insufficiently characterized as yet. In addition, a number of its biochemical features are both conflicting and controversial. To reassess the biochemical features of MT6-MMP, we have expressed the MT6-MMP construct tagged with a FLAG tag in breast carcinoma MCF-7 and fibrosarcoma HT1080 cells. We then used phosphatidylinositol-specific phospholipase C to release MT6-MMP from the cell surface and characterized the solubilized MT6-MMP fractions. We now are confident that cellular MT6-MMP partially exists in its complex with TIMP-2. Both TIMP-1 and TIMP-2 are capable of inhibiting the proteolytic activity of MT6-MMP. MT6-MMP does not stimulate cell migration. MT6-MMP, however, generates a significant level of gelatinolysis of the fluorescein isothiocyanate-labeled gelatin and exhibits an intrinsic, albeit low, ability to activate MMP-2. As a result, it is exceedingly difficult to record the activation of MMP-2 by cellular MT6-MMP. Because of its lipid raft localization, cellular MT6-MMP is inefficiently internalized. MT6-MMP is predominantly localized in the cell-to-cell junctions. Because MT6-MMP has been suggested to play a role in disease, including cancer and autoimmune multiple sclerosis, the identity of its physiologically relevant cleavage targets remains to be determined.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Uniones Intercelulares/enzimología , Metaloproteinasas de la Matriz Asociadas a la Membrana/metabolismo , Microdominios de Membrana/enzimología , Complejos Multiproteicos/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Activación Enzimática , Proteínas Ligadas a GPI , Glicosilfosfatidilinositoles/genética , Humanos , Uniones Intercelulares/genética , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana/genética , Microdominios de Membrana/genética , Esclerosis Múltiple/enzimología , Esclerosis Múltiple/genética , Complejos Multiproteicos/genética , Neoplasias/enzimología , Neoplasias/genética , Inhibidor Tisular de Metaloproteinasa-2/genética , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
There is a need to develop inhibitors of mosquito-borne flaviviruses, including WNV (West Nile virus). In the present paper, we describe a novel and efficient recombinant-antibody technology that led us to the isolation of inhibitory high-affinity human antibodies to the active-site region of a viral proteinase. As a proof-of-principal, we have successfully used this technology and the synthetic naive human combinatorial antibody library HuCAL GOLD(R) to isolate selective and potent function-blocking active-site-targeting antibodies to the two-component WNV NS (non-structural protein) 2B-NS3 serine proteinase, the only proteinase encoded by the flaviviral genome. First, we used the wild-type enzyme in antibody screens. Next, the positive antibody clones were counter-screened using an NS2B-NS3 mutant with a single mutation of the catalytically essential active-site histidine residue. The specificity of the antibodies to the active site was confirmed by substrate-cleavage reactions and also by using proteinase mutants with additional single amino-acid substitutions in the active-site region. The selected WNV antibodies did not recognize the structurally similar viral proteinases from Dengue virus type 2 and hepatitis C virus, and human serine proteinases. Because of their high selectivity and affinity, the identified human antibodies are attractive reagents for both further mutagenesis and structure-based optimization and, in addition, for studies of NS2B-NS3 activity. Conceptually, it is likely that the generic technology reported in the present paper will be useful for the generation of active-site-specific antibody probes for multiple enzymes.
Asunto(s)
Anticuerpos Antivirales/aislamiento & purificación , Serina Proteasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/enzimología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/metabolismo , Western Blotting , Dominio Catalítico/genética , Dominio Catalítico/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , Serina Proteasas/química , Serina Proteasas/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Virus del Nilo Occidental/genéticaRESUMEN
Multiple sclerosis (MS) is a disease of the central nervous system with autoimmune etiology. Susceptibility to MS is linked to viral and bacterial infections. Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination. The splice variants of the single MBP gene are expressed in the oligodendrocytes of the central nervous system (classic MBP) and in the immune cells (Golli-MBPs). Our data suggest that persistent inflammation caused by environmental risk factors is a step to MS. We have discovered biochemical evidence suggesting the presence of the inflammatory proteolytic pathway leading to MS. The pathway involves the self-activated furin and PC2 proprotein convertases and membrane type-6 MMP (MT6-MMP/MMP-25) that is activated by furin/PC2. These events are followed by MMP-25 proteolysis of the Golli-MBP isoforms in the immune system cells and stimulation of the specific autoimmune T cell clones. It is likely that the passage of these autoimmune T cell clones through the disrupted blood-brain barrier to the brain and the recognition of neuronal, classic MBP causes inflammation leading to the further up-regulation of the activity of the multiple individual MMPs, the massive cleavage of MBP in the brain, demyelination, and MS. In addition to the cleavage of Golli-MBPs, MMP-25 proteolysis readily inactivates crystallin alphaB that is a suppressor of MS. These data suggest that MMP-25 plays an important role in MS pathology and that MMP-25, especially because of its restricted cell/tissue expression pattern and cell surface/lipid raft localization, is a promising drug target in MS.
Asunto(s)
Células Presentadoras de Antígenos/patología , Encéfalo/inmunología , Inflamación/enzimología , Metaloproteinasas de la Matriz Asociadas a la Membrana/metabolismo , Esclerosis Múltiple/etiología , Proproteína Convertasas/metabolismo , Células Presentadoras de Antígenos/metabolismo , Encéfalo/enzimología , Encéfalo/patología , Proteínas Ligadas a GPI , Humanos , Inflamación/etiología , Metaloproteinasas de la Matriz Asociadas a la Membrana/genética , Microdominios de Membrana , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Proteína Básica de Mielina/metabolismo , Transducción de Señal , Distribución Tisular , Regulación hacia ArribaRESUMEN
Invasion-promoting MT1-MMP (membrane type-1 matrix metalloproteinase) is a key element in cell migration processes. To identify the proteins that interact and therefore co-precipitate with this proteinase from cancer cells, we used the proteolytically active WT (wild-type), the catalytically inert E240A and the C-end truncated (tailless; DeltaCT) MT1-MMP-FLAG constructs as baits. The identity of the pulled-down proteins was determined by LC-MS/MS (liquid chromatography tandem MS) and then confirmed by Western blotting using specific antibodies. We determined that, in breast carcinoma MCF cells (MCF-7 cells), ANT (adenine nucleotide translocator) efficiently interacted with the WT, E240A and DeltaCT constructs. The WT and E240A constructs also interacted with alpha-tubulin, an essential component of clathrin-mediated endocytosis. In turn, tubulin did not co-precipitate with the DeltaCT construct because of the inefficient endocytosis of the latter, thus suggesting a high level of selectivity of our test system. To corroborate these results, we then successfully used the ANT2-FLAG construct as a bait to pull-down MT1-MMP, which was naturally produced by fibrosarcoma HT1080 cells. We determined that the presence of the functionally inert catalytic domain alone was sufficient to cause the proteinase to interact with ANT2, thus indicating that there is a non-proteolytic mode of these interactions. Overall, it is tempting to hypothesize that by interacting with pro-invasive MT1-MMP, ANT plays a yet to be identified role in a coupling mechanism between energy metabolism and pericellular proteolysis in migrating cancer cells.