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1.
Cell ; 185(19): 3533-3550.e27, 2022 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-36113427

RESUMEN

Integrins are validated drug targets with six approved therapeutics. However, small-molecule inhibitors to three integrins failed in late-stage clinical trials for chronic indications. Such unfavorable outcomes may in part be caused by partial agonism, i.e., the stabilization of the high-affinity, extended-open integrin conformation. Here, we show that the failed, small-molecule inhibitors of integrins αIIbß3 and α4ß1 stabilize the high-affinity conformation. Furthermore, we discovered a simple chemical feature present in multiple αIIbß3 antagonists that stabilizes integrins in their bent-closed conformation. Closing inhibitors contain a polar nitrogen atom that stabilizes, via hydrogen bonds, a water molecule that intervenes between a serine residue and the metal in the metal-ion-dependent adhesion site (MIDAS). Expulsion of this water is a requisite for transition to the open conformation. This change in metal coordination is general to integrins, suggesting broad applicability of the drug-design principle to the integrin family, as validated with a distantly related integrin, α4ß1.


Asunto(s)
Diseño de Fármacos , Integrina alfa4beta1 , Conformación Proteica , Serina , Agua
2.
Cell ; 182(3): 545-562.e23, 2020 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-32621799

RESUMEN

Scar tissue size following myocardial infarction is an independent predictor of cardiovascular outcomes, yet little is known about factors regulating scar size. We demonstrate that collagen V, a minor constituent of heart scars, regulates the size of heart scars after ischemic injury. Depletion of collagen V led to a paradoxical increase in post-infarction scar size with worsening of heart function. A systems genetics approach across 100 in-bred strains of mice demonstrated that collagen V is a critical driver of postinjury heart function. We show that collagen V deficiency alters the mechanical properties of scar tissue, and altered reciprocal feedback between matrix and cells induces expression of mechanosensitive integrins that drive fibroblast activation and increase scar size. Cilengitide, an inhibitor of specific integrins, rescues the phenotype of increased post-injury scarring in collagen-V-deficient mice. These observations demonstrate that collagen V regulates scar size in an integrin-dependent manner.


Asunto(s)
Cicatriz/metabolismo , Colágeno Tipo V/deficiencia , Colágeno Tipo V/metabolismo , Lesiones Cardíacas/metabolismo , Contracción Miocárdica/genética , Miofibroblastos/metabolismo , Animales , Cicatriz/genética , Cicatriz/fisiopatología , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Colágeno Tipo V/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Femenino , Fibrosis/genética , Fibrosis/metabolismo , Regulación de la Expresión Génica/genética , Integrinas/antagonistas & inhibidores , Integrinas/genética , Integrinas/metabolismo , Isoproterenol/farmacología , Masculino , Mecanotransducción Celular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía de Fuerza Atómica/instrumentación , Microscopía Electrónica de Transmisión , Contracción Miocárdica/efectos de los fármacos , Miofibroblastos/citología , Miofibroblastos/patología , Miofibroblastos/ultraestructura , Análisis de Componente Principal , Proteómica , RNA-Seq , Análisis de la Célula Individual
3.
Cell ; 174(1): 156-171.e16, 2018 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-29909984

RESUMEN

Extracellular proTGF-ß is covalently linked to "milieu" molecules in the matrix or on cell surfaces and is latent until TGF-ß is released by integrins. Here, we show that LRRC33 on the surface of microglia functions as a milieu molecule and enables highly localized, integrin-αVß8-dependent TGF-ß activation. Lrrc33-/- mice lack CNS vascular abnormalities associated with deficiency in TGF-ß-activating integrins but have microglia with a reactive phenotype and after 2 months develop ascending paraparesis with loss of myelinated axons and death by 5 months. Whole bone marrow transplantation results in selective repopulation of Lrrc33-/- brains with WT microglia and halts disease progression. The phenotypes of WT and Lrrc33-/- microglia in the same brain suggest that there is little spreading of TGF-ß activated from one microglial cell to neighboring microglia. Our results suggest that interactions between integrin-bearing cells and cells bearing milieu molecule-associated TGF-ß provide localized and selective activation of TGF-ß.


Asunto(s)
Proteínas Portadoras/metabolismo , Microglía/metabolismo , Sistema Nervioso/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Axones/metabolismo , Trasplante de Médula Ósea , Encéfalo/metabolismo , Proteínas Portadoras/clasificación , Proteínas Portadoras/genética , Células Cultivadas , Integrinas/metabolismo , Estimación de Kaplan-Meier , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/citología , Mutagénesis Sitio-Dirigida , Enfermedades Neurodegenerativas/mortalidad , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/terapia , Filogenia , Unión Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Factor de Crecimiento Transformador beta/genética
4.
Immunity ; 52(3): 513-527.e8, 2020 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-32187519

RESUMEN

Intrinsic complement C3 activity is integral to human T helper type 1 (Th1) and cytotoxic T cell responses. Increased or decreased intracellular C3 results in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression remain undefined. We identified complement, including C3, as among the most significantly enriched biological pathway in tissue-occupying cells. We generated C3-reporter mice and confirmed that C3 expression was a defining feature of tissue-immune cells, including T cells and monocytes, occurred during transendothelial diapedesis, and depended on integrin lymphocyte-function-associated antigen 1 (LFA-1) signals. Immune cells from patients with leukocyte adhesion deficiency type 1 (LAD-1) had reduced C3 transcripts and diminished effector activities, which could be rescued proportionally by intracellular C3 provision. Conversely, increased C3 expression by T cells from arthritis patients correlated with disease severity. Our study defines integrins as key controllers of intracellular complement, demonstrates that perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency, and identifies intracellular C3 as biomarker of severity in autoimmunity.


Asunto(s)
Complemento C3/inmunología , Integrinas/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Linfocitos/inmunología , Monocitos/inmunología , Migración Transendotelial y Transepitelial/inmunología , Adulto , Anciano , Animales , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Niño , Preescolar , Complemento C3/genética , Complemento C3/metabolismo , Femenino , Humanos , Integrinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Linfocitos/metabolismo , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Monocitos/metabolismo , Transducción de Señal/inmunología
5.
Immunity ; 50(1): 137-151.e6, 2019 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-30650373

RESUMEN

Fever is an evolutionarily conserved response that confers survival benefits during infection. However, the underlying mechanism remains obscure. Here, we report that fever promoted T lymphocyte trafficking through heat shock protein 90 (Hsp90)-induced α4 integrin activation and signaling in T cells. By inducing selective binding of Hsp90 to α4 integrins, but not ß2 integrins, fever increased α4-integrin-mediated T cell adhesion and transmigration. Mechanistically, Hsp90 bound to the α4 tail and activated α4 integrins via inside-out signaling. Moreover, the N and C termini of one Hsp90 molecule simultaneously bound to two α4 tails, leading to dimerization and clustering of α4 integrins on the cell membrane and subsequent activation of the FAK-RhoA pathway. Abolishment of Hsp90-α4 interaction inhibited fever-induced T cell trafficking to draining lymph nodes and impaired the clearance of bacterial infection. Our findings identify the Hsp90-α4-integrin axis as a thermal sensory pathway that promotes T lymphocyte trafficking and enhances immune surveillance during infection.


Asunto(s)
Fiebre/inmunología , Proteínas HSP90 de Choque Térmico/metabolismo , Integrina alfa4/metabolismo , Infecciones por Salmonella/inmunología , Salmonella typhimurium/inmunología , Linfocitos T/inmunología , Animales , Carga Bacteriana , Adhesión Celular , Movimiento Celular , Dimerización , Quinasa 1 de Adhesión Focal/metabolismo , Vigilancia Inmunológica , Integrina alfa4/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Transducción de Señal , Proteína de Unión al GTP rhoA/metabolismo
6.
EMBO J ; 42(18): e113987, 2023 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-37577760

RESUMEN

Dysregulation of the PI3K/AKT pathway is a common occurrence in high-grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P3 -rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor ß1-integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss-associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active ß1-integrin to maintain invasive activity into the ECM. The expression of the CYTH2-ARF6-AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN-depleted HGSOC.


Asunto(s)
Proteínas de Unión al GTP Monoméricas , Neoplasias Ováricas , Humanos , Femenino , Integrinas/metabolismo , Proteómica , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo
7.
EMBO J ; 41(2): e105531, 2022 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-34904718

RESUMEN

Recessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.Cys57Tyr or C57Y) mutation in the gene coding for protein disulfide isomerase A3 (PDIA3, also known as ERp57), an enzyme that catalyzes formation of disulfide bonds in the endoplasmic reticulum, to be associated with syndromic intellectual disability. Experiments in zebrafish embryos show that PDIA3C57Y expression is pathogenic and causes developmental defects such as axonal disorganization as well as skeletal abnormalities. Expression of PDIA3C57Y in the mouse hippocampus results in impaired synaptic plasticity and memory consolidation. Proteomic and functional analyses reveal that PDIA3C57Y expression leads to dysregulation of cell adhesion and actin cytoskeleton dynamics, associated with altered integrin biogenesis and reduced neuritogenesis. Biochemical studies show that PDIA3C57Y has decreased catalytic activity and forms disulfide-crosslinked aggregates that abnormally interact with chaperones in the endoplasmic reticulum. Thus, rare disease gene variant can provide insight into how perturbations of neuronal proteostasis can affect the function of the nervous system.


Asunto(s)
Discapacidades del Desarrollo/genética , Retículo Endoplásmico/metabolismo , Proteína Disulfuro Isomerasas/genética , Proteostasis , Adolescente , Adulto , Animales , Axones/metabolismo , Axones/patología , Adhesión Celular , Células Cultivadas , Niño , Citoesqueleto/metabolismo , Discapacidades del Desarrollo/metabolismo , Discapacidades del Desarrollo/patología , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Integrinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación Missense , Proyección Neuronal , Plasticidad Neuronal , Linaje , Proteína Disulfuro Isomerasas/metabolismo , Pez Cebra
8.
J Cell Sci ; 137(10)2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38813860

RESUMEN

WW domain-containing transcription regulator 1 (WWTR1, referred to here as TAZ) and Yes-associated protein (YAP, also known as YAP1) are transcriptional co-activators traditionally studied together as a part of the Hippo pathway, and are best known for their roles in stem cell proliferation and differentiation. Despite their similarities, TAZ and YAP can exert divergent cellular effects by differentially interacting with other signaling pathways that regulate stem cell maintenance or differentiation. In this study, we show in mouse neural stem and progenitor cells (NPCs) that TAZ regulates astrocytic differentiation and maturation, and that TAZ mediates some, but not all, of the effects of bone morphogenetic protein (BMP) signaling on astrocytic development. By contrast, both TAZ and YAP mediate the effects on NPC fate of ß1-integrin (ITGB1) and integrin-linked kinase signaling, and these effects are dependent on extracellular matrix cues. These findings demonstrate that TAZ and YAP perform divergent functions in the regulation of astrocyte differentiation, where YAP regulates cell cycle states of astrocytic progenitors and TAZ regulates differentiation and maturation from astrocytic progenitors into astrocytes.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Astrocitos , Diferenciación Celular , Proliferación Celular , Células-Madre Neurales , Transducción de Señal , Transactivadores , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAP , Animales , Astrocitos/metabolismo , Astrocitos/citología , Proteínas Señalizadoras YAP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Ratones , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Transactivadores/metabolismo , Transactivadores/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Integrina beta1/metabolismo , Integrina beta1/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Morfogenéticas Óseas/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Proteínas Serina-Treonina Quinasas
9.
J Cell Sci ; 137(10)2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38639242

RESUMEN

WW domain-containing transcription regulator 1 (WWTR1, referred to here as TAZ) and Yes-associated protein (YAP, also known as YAP1) are transcriptional co-activators traditionally studied together as a part of the Hippo pathway, and are best known for their roles in stem cell proliferation and differentiation. Despite their similarities, TAZ and YAP can exert divergent cellular effects by differentially interacting with other signaling pathways that regulate stem cell maintenance or differentiation. In this study, we show in mouse neural stem and progenitor cells (NPCs) that TAZ regulates astrocytic differentiation and maturation, and that TAZ mediates some, but not all, of the effects of bone morphogenetic protein (BMP) signaling on astrocytic development. By contrast, both TAZ and YAP mediate the effects on NPC fate of ß1-integrin (ITGB1) and integrin-linked kinase signaling, and these effects are dependent on extracellular matrix cues. These findings demonstrate that TAZ and YAP perform divergent functions in the regulation of astrocyte differentiation, where YAP regulates cell cycle states of astrocytic progenitors and TAZ regulates differentiation and maturation from astrocytic progenitors into astrocytes.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Astrocitos , Diferenciación Celular , Proliferación Celular , Células-Madre Neurales , Transducción de Señal , Transactivadores , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAP , Animales , Astrocitos/metabolismo , Astrocitos/citología , Proteínas Señalizadoras YAP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Ratones , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Transactivadores/metabolismo , Transactivadores/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Integrina beta1/metabolismo , Integrina beta1/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Morfogenéticas Óseas/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Proteínas Serina-Treonina Quinasas
10.
J Cell Sci ; 137(20)2024 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-38738282

RESUMEN

Advances in imaging, segmentation and tracking have led to the routine generation of large and complex microscopy datasets. New tools are required to process this 'phenomics' type data. Here, we present 'Cell PLasticity Analysis Tool' (cellPLATO), a Python-based analysis software designed for measurement and classification of cell behaviours based on clustering features of cell morphology and motility. Used after segmentation and tracking, the tool extracts features from each cell per timepoint, using them to segregate cells into dimensionally reduced behavioural subtypes. Resultant cell tracks describe a 'behavioural ID' at each timepoint, and similarity analysis allows the grouping of behavioural sequences into discrete trajectories with assigned IDs. Here, we use cellPLATO to investigate the role of IL-15 in modulating human natural killer (NK) cell migration on ICAM-1 or VCAM-1. We find eight behavioural subsets of NK cells based on their shape and migration dynamics between single timepoints, and four trajectories based on sequences of these behaviours over time. Therefore, by using cellPLATO, we show that IL-15 increases plasticity between cell migration behaviours and that different integrin ligands induce different forms of NK cell migration.


Asunto(s)
Movimiento Celular , Interleucina-15 , Células Asesinas Naturales , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/inmunología , Interleucina-15/metabolismo , Programas Informáticos , Molécula 1 de Adhesión Intercelular/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo
11.
Trends Immunol ; 44(11): 917-931, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37858490

RESUMEN

Following stimulation, the T cell receptor (TCR) and its coreceptors integrate multiple intracellular signals to initiate T cell proliferation, migration, gene expression, and metabolism. Among these signaling molecules are the small GTPases RAS and RAP1, which induce MAPK pathways and cellular adhesion to activate downstream effector functions. Although many studies have helped to elucidate the signaling intermediates that mediate T cell activation, the molecules and pathways that keep naive T cells in check are less understood. Several recent studies provide evidence that RASA2 and RASA3, which are GAP1-family GTPase-activating proteins (GAPs) that inactivate RAS and RAP1, respectively, are crucial molecules that limit T cell activation and adhesion. In this review we describe recent data on the roles of RASA2 and RASA3 as gatekeepers of T cell activation and migration.


Asunto(s)
Proteínas Activadoras de GTPasa , Transducción de Señal , Humanos , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Transducción de Señal/fisiología , Adhesión Celular/fisiología , Linfocitos T/metabolismo , Proteínas Activadoras de ras GTPasa
12.
EMBO Rep ; 25(2): 832-852, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38191874

RESUMEN

BRD4, a bromodomain and extraterminal (BET) protein, is deregulated in multiple cancers and has emerged as a promising drug target. However, the function of the two main BRD4 isoforms (BRD4-L and BRD4-S) has not been analysed in parallel in most cancers. This complicates determining therapeutic efficacy of pan-BET inhibitors. In this study, using functional and transcriptomic analysis, we show that BRD-L and BRD4-S isoforms play distinct roles in fusion negative embryonal rhabdomyosarcoma. BRD4-L has an oncogenic role and inhibits myogenic differentiation, at least in part, by activating myostatin expression. Depletion of BRD4-L in vivo impairs tumour progression but does not impact metastasis. On the other hand, depletion of BRD4-S has no significant impact on tumour growth, but strikingly promotes metastasis in vivo. Interestingly, BRD4-S loss results in the enrichment of BRD4-L and RNA Polymerase II at integrin gene promoters resulting in their activation. In fusion positive alveolar rhabdomyosarcoma, BRD4-L is unrestricted in its oncogenic role, with no evident involvement of BRD4-S. Our work unveils isoform-specific functions of BRD4 in rhabdomyosarcoma.


Asunto(s)
Rabdomiosarcoma , Factores de Transcripción , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Isoformas de Proteínas/genética , Rabdomiosarcoma/genética , Proteínas que Contienen Bromodominio
13.
Proc Natl Acad Sci U S A ; 120(6): e2218915120, 2023 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-36730200

RESUMEN

Alzheimer's disease (AD) is the most common form of incurable dementia and represents a critical public health issue as the world's population ages. Although microglial dysregulation is a cardinal feature of AD, the extensive heterogeneity of these immunological cells in the brain has impeded our understanding of their contribution to this disease. Here, we identify a pathogenic microglial subset which expresses the CD11c surface marker as the sole producer of Osteopontin (OPN) in the 5XFAD mouse model of AD. OPN production divides Disease-Associated Microglia (DAM) into two functionally distinct subsets, i.e., a protective CD11c+OPN- subset that robustly ingests amyloid ß (Aß) in a noninflammatory fashion and a pathogenic CD11c+OPN+ subset that produces proinflammatory cytokines and fails to ingest significant amounts of Aß. Genetic ablation of OPN or administration of monoclonal anti-OPN antibody to 5XFAD mice reduces proinflammatory microglia, plaque formation, and numbers of dystrophic neurites and results in improved cognitive function. Analysis of brain tissue from AD patients indicates that levels of OPN-producing CD11c+ microglia correlate strongly with the degree of cognitive deficit and AD neuropathology. These findings define an OPN-dependent pathway to disease driven by a distinct microglial subset, and identify OPN as a novel therapeutic target for potentially effective immunotherapy to treat AD.


Asunto(s)
Enfermedad de Alzheimer , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Microglía/metabolismo , Péptidos beta-Amiloides/metabolismo , Osteopontina/metabolismo , Ratones Transgénicos , Modelos Animales de Enfermedad , Placa Amiloide/metabolismo
14.
Proc Natl Acad Sci U S A ; 120(26): e2218116120, 2023 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-37339195

RESUMEN

Integrin-mediated adhesion is essential for metazoan life. Integrin binding to ligand requires an activation step prior to binding ligand that depends on direct binding of talin and kindlin to the ß-integrin cytoplasmic tail and the transmission of force from the actomyosin via talin to the integrin-ligand bonds. However, the affinity of talin for integrin tails is low. It is therefore still unclear how such low-affinity bonds are reinforced to transmit forces up to 10 to 40 pN. In this study, we use single-molecule force spectroscopy by optical tweezers to investigate the mechanical stability of the talin•integrin bond in the presence and absence of kindlin. While talin and integrin alone form a weak and highly dynamic slip bond, the addition of kindlin-2 induces a force-independent, ideal talin•integrin bond, which relies on the steric proximity of and the intervening amino acid sequences between the talin- and kindlin-binding sites in the ß-integrin tail. Our findings show how kindlin cooperates with talin to enable transmission of high forces required to stabilize cell adhesion.


Asunto(s)
Integrinas , Talina , Animales , Talina/metabolismo , Ligandos , Proteínas de la Membrana/metabolismo , Adhesión Celular
15.
Proc Natl Acad Sci U S A ; 120(43): e2304288120, 2023 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-37844244

RESUMEN

Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM-integrin-cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, "stiff-like" spreading on soft is determined by a balance of a cell's contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates.


Asunto(s)
Matriz Extracelular , Integrinas , Humanos , Adhesión Celular , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Citoesqueleto/metabolismo , Transducción de Señal
16.
Proc Natl Acad Sci U S A ; 120(24): e2304874120, 2023 06 13.
Artículo en Inglés | MEDLINE | ID: mdl-37279271

RESUMEN

Activation of latent transforming growth factor (TGF)-ß2 is incompletely understood. Unlike TGF-ß1 and ß3, the TGF-ß2 prodomain lacks a seven-residue RGDLXX (L/I) integrin-recognition motif and is thought not to be activated by integrins. Here, we report the surprising finding that TGF-ß2 contains a related but divergent 13-residue integrin-recognition motif (YTSGDQKTIKSTR) that specializes it for activation by integrin αVß6 but not αVß8. Both classes of motifs compete for the same binding site in αVß6. Multiple changes in the longer motif underlie its specificity. ProTGF-ß2 structures define interesting differences from proTGF-ß1 and the structural context for activation by αVß6. Some integrin-independent activation is also seen for proTGF-ß2 and even more so for proTGF-ß3. Our findings have important implications for therapeutics to αVß6 in clinical trials for fibrosis, in which inhibition of TGF-ß2 activation has not been anticipated.


Asunto(s)
Integrinas , Factor de Crecimiento Transformador beta2 , Humanos , Integrinas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Antígenos de Neoplasias/metabolismo , Fibrosis , Factor de Crecimiento Transformador beta/metabolismo
17.
Immunol Rev ; 311(1): 224-233, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35451082

RESUMEN

Osteopontin (OPN) also known by its official gene designation secreted phosphoprotein-1 (SPP1) is a fascinating, multifunctional protein expressed in a number of cell types that functions not only in intercellular communication, but also in the extracellular matrix (ECM). OPN/SPP1 possesses cytokine, chemokine, and signal transduction functions by virtue of modular structural motifs that provide interaction surfaces for integrins and CD44-variant receptors. In humans, there are three experimentally verified splice variants of OPN/SPP1 and CD44's ten exons are also alternatively spiced in a cell/tissue-specific manner, although very little is known about how this is regulated in the central nervous system (CNS). Post-translational modifications of phosphorylation, glycosylation, and localized cleavage by specific proteases in the cells and tissues where OPN/SPP1 functions, provides additional layers of specificity. However, the former make elucidating the exact molecular mechanisms of OPN/SPP1 function more complex. Flexibility in OPN/SPP1 structure and its engagement with integrins having the ability to transmit signals in inside-out and outside-in direction, is likely why OPN/SPP1 can serve as an early detector of inflammation and ongoing tissue damage in response to cancer, stroke, traumatic brain injury, pathogenic infection, and neurodegeneration, processes that impair tissue homeostasis. This review will focus on what is currently known about OPN/SPP1 function in the brain.


Asunto(s)
Enfermedades Neuroinflamatorias , Osteopontina , Comunicación Celular , Citocinas , Humanos , Integrinas/metabolismo , Ligandos , Osteopontina/genética , Osteopontina/metabolismo , Péptido Hidrolasas , Fosfoproteínas
18.
Immunol Rev ; 306(1): 25-42, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35147233

RESUMEN

Spleen dendritic cells (DC) are critical for initiation of adaptive immune responses against blood-borne invaders. Key to DC function is their positioning at sites of pathogen entry, and their abilities to selectively capture foreign antigens and promptly engage T cells. Focusing on conventional DC2 (cDC2), we discuss the contribution of chemoattractant receptors (EBI2 or GPR183, S1PR1, and CCR7) and integrins to cDC2 positioning and function. We give particular attention to a newly identified role in cDC2 for adhesion G-protein coupled receptor E5 (Adgre5 or CD97) and its ligand CD55, detailing how this mechanosensing system contributes to splenic cDC2 positioning and homeostasis. Additional roles of CD97 in the immune system are reviewed. The ability of cDC2 to be activated by circulating missing self-CD47 cells and to integrate multiple red blood cell (RBC)-derived inputs is discussed. Finally, we describe the process of activated cDC2 migration to engage and prime helper T cells. Throughout the review, we consider the insights into cDC function in the spleen that have emerged from imaging studies.


Asunto(s)
Células Dendríticas , Bazo , Antígenos , Homeostasis , Humanos , Ligandos
19.
Trends Biochem Sci ; 46(2): 124-137, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33020011

RESUMEN

Integrins are transmembrane receptors that transduce biochemical and mechanical signals across the plasma membrane and promote cell adhesion and migration. In addition, integrin adhesion complexes are functionally and structurally linked to components of the intracellular trafficking machinery and accumulating data now reveal that they are key regulators of endocytosis and exocytosis in a variety of cell types. Here, we highlight recent insights into integrin control of intracellular trafficking in processes such as degranulation, mechanotransduction, cell-cell communication, antibody production, virus entry, Toll-like receptor signaling, autophagy, and phagocytosis, as well as the release and uptake of extracellular vesicles. We discuss the underlying molecular mechanisms and the implications for a range of pathophysiological contexts, including hemostasis, immunity, tissue repair, cancer, and viral infection.


Asunto(s)
Integrinas , Mecanotransducción Celular , Adhesión Celular , Membrana Celular , Endocitosis
20.
J Biol Chem ; 300(2): 105631, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38199575

RESUMEN

Integrins are cell adhesion receptors that dimerize to mediate cell-cell interactions and regulate processes, including proliferation, inflammation, and tissue repair. The role of integrins in regulating insulin signaling is incompletely understood. We have previously shown that binding of the integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) to the αvß5 integrin promotes termination of insulin receptor signaling in mice. Upon ligation of MFGE8, integrin ß5 complexes with the insulin receptor beta (IRß) in skeletal muscle, resulting in dephosphorylation of IRß and reduction of insulin-stimulated glucose uptake. Here, we investigate the mechanism by which the interaction between ß5 and IRß impacts IRß phosphorylation status. We show in in vitro and in vivo in skeletal muscle in mice that antibody-mediated blockade of the ß5 integrin inhibits and recombinant MFGE8 promotes PTP1B binding to and dephosphorylation of IRß resulting in increased or reduced insulin-stimulated glucose uptake, respectively. The ß5-PTP1B complex is recruited by MFGE8 to IRß leading to termination of canonical insulin signaling. ß5 blockade enhances insulin-stimulated glucose uptake in wildtype but not Ptp1b KO mice indicating that PTP1B functions downstream of MFGE8 in modulating insulin receptor signaling. Furthermore, in a human cohort, we report serum MFGE8 levels correlate with indices of insulin resistance. These data provide mechanistic insights into the role of MFGE8 and ß5 in regulating insulin signaling.


Asunto(s)
Insulina , Receptor de Insulina , Animales , Humanos , Ratones , Antígenos de Superficie/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Cadenas beta de Integrinas , Proteínas de la Leche/metabolismo , Receptor de Insulina/genética , Ratones Endogámicos C57BL , Masculino , Línea Celular
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