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1.
Cytotherapy ; 19(12): 1426-1437, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29037943

RESUMEN

BACKGROUND AIMS: Light chain (AL) amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. Patients with LC amyloid involvement of the heart have the worst morbidity and mortality. Current treatments target the plasma cells to reduce further production of amyloid proteins. There is dire need to understand the mechanisms of cardiac tissue damage from amyloid to develop novel therapies. We recently reported that LC soluble and fibrillar species cause apoptosis and inhibit cell growth in human cardiomyocytes. Mesenchymal stromal cells (MSCs) can promote wound healing and tissue remodeling. The objective of this study was to evaluate MSCs to protect cardiomyocytes affected by AL amyloid fibrils. METHODS: We used live cell imaging and proteomics to analyze the effect of MSCs in the growth arrest caused by AL amyloid fibrils. RESULTS: We evaluated the growth of human cardiomyocytes (RFP-AC16 cells) in the presence of cytotoxic LC amyloid fibrils. MSCs reversed the cell growth arrest caused by LC fibrils. We also demonstrated that this effect requires cell contact and may be mediated through paracrine factors modulating cell adhesion and extracellular matrix remodeling. To our knowledge, this is the first report of MSC protection of human cardiomyocytes in amyloid disease. CONCLUSIONS: This important proof of concept study will inform future rational development of MSC therapy in cardiac LC amyloid.


Asunto(s)
Amiloide/toxicidad , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/patología , Células Madre Mesenquimatosas/citología , Miocitos Cardíacos/patología , Amiloide/metabolismo , Apoptosis , Células Cultivadas , Técnicas de Cocultivo , Humanos , Cadenas Ligeras de Inmunoglobulina/metabolismo , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/terapia , Células Madre Mesenquimatosas/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo
2.
Am J Hematol ; 92(6): 536-541, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28295502

RESUMEN

Immunoglobulin light chain (AL) amyloidosis is a fatal complication of B-cell proliferation secondary to deposition of amyloid fibrils in various organs. Urinary exosomes (UEX) are the smallest of the microvesicles excreted in the urine. Previously, we found UEX of patients with AL amyloidosis contained immunoglobulin light chain (LC) oligomers that patients with multiple myeloma did not have. To further explore the role of the LC oligomers, UEX was isolated from an AL amyloidosis patient with progressive renal disease despite achieving a complete response. LC oligomers were identified. Mass spectrometry (MS) of the UEX and serum identified two monoclonal lambda LCs. Proteomics of the trypsin digested amyloid fragments in the kidney by laser microdissection and MS analysis identified a λ6 LC. The cDNA from plasma cell clone was from the IGLV- 6-57 family and it matched the amino acid sequences of the amyloid peptides. The predicted mass of the peptide product of the cDNA matched the mass of one of the two LCs identified in the UEX and serum. UEX combined with MS were able to identify 2 monoclonal lambda LCs that current clinical methods could not. It also identified the amyloidogenic LC which holds potential for response assessment in the future.


Asunto(s)
Amiloidosis/complicaciones , Amiloidosis/metabolismo , Exosomas/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Proteinuria/diagnóstico , Proteinuria/etiología , Adulto , Anciano , Secuencia de Aminoácidos , Amiloidosis/genética , Femenino , Humanos , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/orina , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Peso Molecular , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/orina , Análisis de Secuencia de ADN
3.
J Immunol ; 194(9): 4555-66, 2015 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-25825439

RESUMEN

A key component in T cell activation is the endosomal recycling of receptors to the cell surface, thereby allowing continual integration of signaling and Ag recognition. One protein potentially involved in TCR transport is sorting nexin 17 (SNX17). SNX proteins have been found to bind proteins involved in T cell activation, but specifically the role of SNX17 in receptor recycling and T cell activation is unknown. Using immunofluorescence, we find that SNX17 colocalizes with TCR and localizes to the immune synapse in T- conjugates. Significantly, knockdown of the SNX17 resulted in fewer T-APC conjugates, lower CD69, TCR, and LFA-1 surface expression, as well as lower overall TCR recycling compared with control T cells. Lastly, we identified the 4.1/ezrin/radixin/moesin domain of SNX17 as being responsible in the binding and trafficking of TCR and LFA-1 to the cell surface. These data suggest that SNX17 plays a role in the maintenance of normal surface levels of activating receptors and integrins to permit optimum T cell activation at the immune synapse.


Asunto(s)
Integrinas/metabolismo , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Nexinas de Clasificación/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Línea Celular , Membrana Celular/metabolismo , Humanos , Sinapsis Inmunológicas/metabolismo , Células Jurkat , Activación de Linfocitos/genética , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Lisosomas/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteolisis , Alineación de Secuencia , Nexinas de Clasificación/química , Nexinas de Clasificación/genética
4.
J Immunol ; 188(12): 6135-44, 2012 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-22573807

RESUMEN

The Ras GTPase-activating-like protein IQGAP1 is a multimodular scaffold that controls signaling and cytoskeletal regulation in fibroblasts and epithelial cells. However, the functional role of IQGAP1 in T cell development, activation, and cytoskeletal regulation has not been investigated. In this study, we show that IQGAP1 is dispensable for thymocyte development as well as microtubule organizing center polarization and cytolytic function in CD8(+) T cells. However, IQGAP1-deficient CD8(+) T cells as well as Jurkat T cells suppressed for IQGAP1 were hyperresponsive, displaying increased IL-2 and IFN-γ production, heightened LCK activation, and augmented global phosphorylation kinetics after TCR ligation. In addition, IQGAP1-deficient T cells exhibited increased TCR-mediated F-actin assembly and amplified F-actin velocities during spreading. Moreover, we found that discrete regions of IQGAP1 regulated cellular activation and F-actin accumulation. Taken together, our data suggest that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Transducción de Señal/inmunología , Proteínas Activadoras de ras GTPasa/metabolismo , Actinas/inmunología , Actinas/metabolismo , Animales , Western Blotting , Linfocitos T CD8-positivos/citología , Diferenciación Celular/inmunología , Citoesqueleto/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Células Jurkat , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteínas Activadoras de ras GTPasa/inmunología
5.
Biochim Biophys Acta Proteins Proteom ; 1872(3): 140993, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38169170

RESUMEN

Immunoglobulin light chain (AL) amyloidosis involves the deposition of insoluble monoclonal AL protein fibrils in the extracellular space of different organs leading to dysfunction and death. Development of methods to efficiently express and purify AL proteins with acceptable standards of homogeneity and structural integrity has become critical to understand the in vitro and in vivo aspects of AL protein aggregation, and thus the disease progression. In this study, we report the biophysical characterization of His-tagged and untagged versions of AL full-length (FL) κI and λ6 subgroup proteins and their mutants expressed from the Expi293F human cell line. We used an array of biophysical and biochemical methods to analyze the structure and stability of the monomers, oligomerization states, and thermodynamic characteristics of the purified FL proteins and how they compare with the bacterially expressed FL proteins. Our results demonstrate that the tagged and untagged versions of FL proteins have comparable stability to proteins expressed in bacterial cells but exhibit multiple unfolding transitions and reversibility. Non-reducing SDS-PAGE and analytical ultracentrifugation analysis showed presence of monomers and dimers, with an insignificant amount of higher-order oligomers, in the purified fraction of all proteins. Overall, the FL proteins were expressed with sufficient yields for biophysical studies and can replace bacterial expression systems.


Asunto(s)
Anticuerpos Monoclonales , Cadenas Ligeras de Inmunoglobulina , Humanos , Cadenas Ligeras de Inmunoglobulina/genética , Biofisica , Línea Celular , Progresión de la Enfermedad
6.
Front Oncol ; 12: 978198, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36267963

RESUMEN

Light Chain (AL) Amyloidosis is a plasma cell dyscrasia producing amyloidogenic light chains (LC) that misfold and form amyloid deposits that cause damage in vital organs, primarily the heart and kidneys. Urinary extracellular vesicles (uEVs) are nanoparticles produced by renal epithelial cells throughout the nephron. We previously showed that uEVs from active renal AL amyloidosis patients contain LC oligomers that are large (>250kDa), resistant to heat and chemical denaturation, but of low abundance. Renal dysfunction in AL amyloidosis results in high urine protein, compounding technical challenges to use uEVs as analytical tools. In this study, we assess the use of uEVs as analytical diagnostic tools for response and disease progression in AL amyloidosis. Our results suggest that uEV protein concentration, urine volume, and particle concentrations are not directly correlated. Multiple strategies for overcoming non-specific antibody binding in uEV samples were validated in our study. We demonstrated that the sensitivity for pre-clinical testing is improved with a urine sample requirement algorithm that we developed. The findings of our study will provide a pathway toward development of critically needed tools for patient management. Sensitive detection of LC oligomers from a non-invasive urine sample rather than an invasive renal biopsy will reduce patient burden and healthcare costs. The ability to detect LC oligomers in patients with renal progression, despite positive hematologic response; will allow clinicians to confidently treat, but not overtreat, patients at risk of ongoing significant renal injury.

7.
J Immunol ; 182(11): 6933-42, 2009 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-19454690

RESUMEN

The NK cell-activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell-target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D-coated beads is impaired in the absence of CrkL. Ab-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras family GTPase Rap1 is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner and is required for conjugate formation, MTOC (microtubule organizing center) polarization, and NKG2D-dependent cellular cytotoxicity. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization, and granule release.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Asesinas Naturales/citología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinasas/inmunología , Transducción de Señal/inmunología , Adhesión Celular , Línea Celular , Polaridad Celular , Humanos , Células Asesinas Naturales/inmunología , Centro Organizador de los Microtúbulos , Vesículas Secretoras , Proteínas de Unión al GTP rap1
8.
J Immunol ; 181(10): 6995-7001, 2008 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-18981119

RESUMEN

NK cells are innate immune cells that can eliminate their targets through granule release. In this study, we describe a specialized role for the large GTPase Dynamin 2 (Dyn2) in the regulation of these secretory events leading to cell-mediated cytotoxicity. By modulating the expression of Dyn2 using small interfering RNA or by inhibiting its activity using a pharmacological agent, we determined that Dyn2 does not regulate conjugate formation, proximal signaling, or granule polarization. In contrast, during cell-mediated killing, Dyn2 localizes with lytic granules and polarizes to the NK cell-target interface where it regulates the final fusion of lytic granules with the plasma membrane. These findings identify a novel role for Dyn2 in the exocytic events required for effective NK cell-mediated cytotoxicity.


Asunto(s)
Citotoxicidad Inmunológica , Dinamina II/inmunología , Exocitosis/inmunología , Células Asesinas Naturales/inmunología , Vesículas Secretoras/inmunología , Dinamina II/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Células Asesinas Naturales/metabolismo , Lisosomas/inmunología , Lisosomas/metabolismo , Transporte de Proteínas/inmunología , Vesículas Secretoras/metabolismo
9.
Leukemia ; 34(5): 1383-1393, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-31796914

RESUMEN

Light chain (AL) amyloidosis is a progressive, degenerative disease characterized by the misfolding and amyloid deposition of immunoglobulin light chain (LC). The amyloid deposits lead to organ failure and death. Our laboratory is specifically interested in cardiac involvement of AL amyloidosis. We have previously shown that the fibrillar aggregates of LC proteins can be cytotoxic and arrest the growth of human RFP-AC16 cardiomyocytes in vitro. We showed that adipose-derived mesenchymal stromal cells (AMSC) can rescue the cardiomyocytes from the fibril-induced growth arrest through contact-dependent mechanisms. In this study, we examined the transcriptome changes of human cardiomyocytes and AMSC in the presence of AL amyloid fibrils. The presence of fibrils causes a 'priming' immune response in AMSC associated with interferon associated genes. Exposure to AL fibrils induced changes in the pathways associated with immune response and extracellular matrix components in cardiomyocytes. We also observed upregulation of innate immune-associated transcripts (chemokines, cytokines, and complement), suggesting that amyloid fibrils initiate an innate immune response on these cells, possibly due to phenotypic transformation. This study corroborates and expands our previous studies and identifies potential new immunologic mechanisms of action for fibril toxicity on human cardiomyocytes and AMSC rescue effect on cardiomyocytes.


Asunto(s)
Biomarcadores/análisis , Cadenas Ligeras de Inmunoglobulina/inmunología , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/inmunología , Inflamación/inmunología , Células Madre Mesenquimatosas/inmunología , Miocitos Cardíacos/inmunología , Apoptosis , Proliferación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/metabolismo , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/patología , Inflamación/metabolismo , Inflamación/patología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología
10.
Methods Mol Biol ; 1873: 123-153, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30341607

RESUMEN

Common biophysical techniques like absorption and fluorescence spectroscopy, microscopy, and light scattering studies have been in use to investigate fibril assembly for a long time. However, there is sometimes a lack of consensus from the findings of an individual technique when compared in parallel with the other techniques. In this chapter, we aim to provide a concise compilation of techniques that can effectively be used to obtain a comprehensive representation of the structural, aggregation, and toxicity determinants in immunoglobulin light chain amyloidosis. We start by giving a brief introduction on amyloid assembly and the advantages of using simple and readily available techniques to study aggregation. After an overview on preparation of protein to set up parallel experiments, we provide a systematic description of the in vitro techniques used to study aggregation in AL protein. Additionally, we thoroughly discuss the steps needed in our experience during the individual experiments for better reproducibility and data analysis.


Asunto(s)
Amiloide/química , Bioensayo , Cadenas Ligeras de Inmunoglobulina/química , Amiloide/metabolismo , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/metabolismo , Amiloidosis/diagnóstico , Apoptosis , Benzotiazoles/química , Benzotiazoles/metabolismo , Bioensayo/métodos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Dispersión Dinámica de Luz , Cadenas Ligeras de Inmunoglobulina/metabolismo , Tamaño de la Partícula , Espectrometría de Fluorescencia
11.
Chem Commun (Camb) ; 54(76): 10664-10674, 2018 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-30087961

RESUMEN

Light chain (AL) amyloidosis is a devastating, complex, and incurable protein misfolding disease. It is characterized by an abnormal proliferation of plasma cells (fully differentiated B cells) producing an excess of monoclonal immunoglobulin light chains that are secreted into circulation, where the light chains misfold, aggregate as amyloid fibrils in target organs, and cause organ dysfunction, organ failure, and death. In this article, we will review the factors that contribute to AL amyloidosis complexity, the findings by our laboratory from the last 16 years and the work from other laboratories on understanding the structural, kinetics, and thermodynamic contributions that drive immunoglobulin light chain-associated amyloidosis. We will discuss the role of cofactors and the mechanism of cellular damage. Last, we will review our recent findings on the high resolution structure of AL amyloid fibrils. AL amyloidosis is the best example of protein sequence diversity in misfolding diseases, as each patient has a unique combination of germline donor sequences and multiple amino acid mutations in the protein that forms the amyloid fibril.


Asunto(s)
Amiloide/metabolismo , Proteínas Amiloidogénicas/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/fisiopatología , Multimerización de Proteína , Amiloide/química , Amiloide/genética , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/genética , Reordenamiento Génico , Glicosaminoglicanos/metabolismo , Humanos , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Mutación , Células Plasmáticas/metabolismo , Estabilidad Proteica
12.
Mol Biol Cell ; 26(1): 91-103, 2015 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-25355947

RESUMEN

COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling.


Asunto(s)
Citoesqueleto de Actina , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Movimiento Celular , Cobre/metabolismo , ATPasas Transportadoras de Cobre , Citoplasma/metabolismo , Endosomas/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Ratones , Mutación , Proteínas de Neoplasias/metabolismo , Proteínas/genética , Proteínas/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular
13.
Mol Immunol ; 48(9-10): 1149-59, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21439641

RESUMEN

2B4 is a member of the SLAM receptor family capable of activating NK cell cytotoxicity in the context of EBV infection. SAP (SLAM Associated Protein) deficiency causes defective signaling downstream of SLAM family receptors and high susceptibility to EBV. 2B4 costimulates natural cytotoxicity receptor (NCR) and TCR initiated signals to induce cellular cytotoxicity and cytokine release. The 2B4-SAP signal transduction pathway is not predicted to overlap with the TCR-ITAM pathway, although SAP is required for some TCR-induced signals. We therefore examined the functional relationship between SLAM family receptor 2B4 and ITAM-containing adaptor complexes. Removal of FcɛRIγ or CD3ζ-containing complexes, using genetically manipulated cell lines or siRNA specific suppression, significantly reduces 2B4-initiated functions in NK and T cells, respectively. Consistent with this relationship, Syk and ZAP-70 are capable of transducing 2B4 signals for calcium mobilization and cytolysis. Furthermore, ITAM-containing molecules constitutively associate with SAP. These results suggest a potential physical association between 2B4 and the ITAM receptor complexes that is required for 2B4-initiated signaling and cell-mediated killing.


Asunto(s)
Antígenos CD/metabolismo , Citotoxicidad Inmunológica/inmunología , Espacio Intracelular/inmunología , Receptores Inmunológicos/química , Receptores Inmunológicos/metabolismo , Transducción de Señal/inmunología , Secuencias de Aminoácidos , Animales , Calcio/metabolismo , Señalización del Calcio , Línea Celular , Membrana Celular/metabolismo , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Asesinas Naturales/citología , Células Asesinas Naturales/enzimología , Ratones , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Fosfolipasa C gamma/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes/metabolismo , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Quinasa Syk
14.
J Immunol ; 179(6): 3397-401, 2007 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-17785771

RESUMEN

Little is known about the regulatory roles of specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in cytotoxic lymphocytes. Recent information suggests that mutations in the SNARE protein syntaxin 11 result in a form of familial hemophagocytic lymphohistiocytosis (FHL). Because genetic abnormalities in key granule components (e.g., perforin) or in regulators of secretion (e.g., Munc13-4) underlie the other identified forms of FHL, we assessed whether syntaxin 11 might also serve a related regulatory role. We determined that syntaxin 11 is expressed in NK cells and activated CTLs and is located in discrete membrane-associated structures in the cytoplasm. Enhanced expression of syntaxin 11 augments the secretion and killing of tumor targets, and suppression of syntaxin 11 expression inhibits these functions. Our data identify and characterize a role for syntaxin 11 in granule exocytosis and in the generation of cell-mediated killing. These results also provide new insights on the mechanisms of hemopoietic dysregulation in FHL.


Asunto(s)
Pruebas Inmunológicas de Citotoxicidad , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Proteínas Qa-SNARE/fisiología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Línea Celular , Membrana Celular/metabolismo , Células Clonales , Citoplasma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Exocitosis/inmunología , Humanos , Células Jurkat , Células K562 , Proteínas Qa-SNARE/biosíntesis , Linfocitos T Citotóxicos/citología
15.
J Immunol ; 178(6): 3575-82, 2007 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-17339454

RESUMEN

NK cells are effector lymphocytes that can recognize and eliminate virally infected and transformed cells. NK cells express distinct activating receptors, including an ITAM-containing FcR complex that recognizes Ab-coated targets, and the DNAX-activating protein of 10 kDa-containing NKG2D receptor complex that recognizes stress-induced ligands. The regulatory role of specific tyrosine kinases in these pathways is incompletely understood. In this study, we show that, in activated human NK cells, the tyrosine kinase IL-2-inducible T cell kinase (Itk), differentially regulates distinct NK-activating receptors. Enhanced expression of Itk leads to increases in calcium mobilization, granule release, and cytotoxicity upon stimulation of the ITAM-containing FcR, suggesting that Itk positively regulates FcR-initiated cytotoxicity. In contrast, enhanced Itk expression decreases cytotoxicity and granule release downstream of the DNAX-activating protein of 10 kDa-containing NKG2D receptor, suggesting that Itk is involved in a pathway of negative regulation of NKG2D-initiated granule-mediated killing. Using a kinase mutant, we show that the catalytic activity of Itk is required for both the positive and negative regulation of these pathways. Complementary experiments where Itk expression was suppressed also showed differential regulation of the two pathways. These findings suggest that Itk plays a complex role in regulating the functions initiated by distinct NK cell-activating receptors. Moreover, understanding how these pathways may be differentially regulated has relevance in the setting of autoimmune diseases and antitumor immune responses where NK cells play key regulatory roles.


Asunto(s)
Señalización del Calcio/inmunología , Regulación Enzimológica de la Expresión Génica , Células Asesinas Naturales/inmunología , Proteínas Tirosina Quinasas/inmunología , Animales , Enfermedades Autoinmunes/enzimología , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Señalización del Calcio/genética , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Inmunidad Celular/genética , Células Asesinas Naturales/citología , Células Asesinas Naturales/metabolismo , Ratones , Mutación , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/inmunología , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/genética , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/inmunología
16.
Nat Immunol ; 7(5): 524-32, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16582911

RESUMEN

NKG2D is an important immunosurveillance receptor that responds to stress-induced ligand expression on tumors and virus-infected cells. Human natural killer cells express NKG2D and require the transmembrane adaptor DAP10 to initiate their full cytotoxic activation. However, DAP10 has no immunoreceptor tyrosine-based activation motif and thus the mechanism of recruiting 'downstream' effector proteins is unclear. We show here that binding of the p85 subunit of phosphatidylinositol-3- kinase to DAP10 could not by itself trigger cell-mediated cytotoxicity and that binding of an intermediate consisting of the DAP10 binding partner Grb2 and the effector molecule Vav1 (Grb2-Vav1) to DAP10 was sufficient to initiate tyrosine-phosphorylation events. For full calcium release and cytotoxicity to occur, both Grb2-Vav1 and p85 had to bind to DAP10. These findings identify a previously unknown mechanism by which NKG2D-DAP10 mediates cytotoxicity and provides a framework for evaluating activation by other receptor complexes that lack immunoreceptor tyrosine-based activation motifs.


Asunto(s)
Antígenos de Diferenciación de Linfocitos T/metabolismo , Proteína Adaptadora GRB2/metabolismo , Células Asesinas Naturales/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptores Inmunológicos/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Citotoxicidad Inmunológica , Humanos , Modelos Biológicos , Subfamilia K de Receptores Similares a Lectina de Células NK , Fosforilación , Receptores de Células Asesinas Naturales , Transducción de Señal/inmunología
17.
J Immunol ; 175(1): 213-8, 2005 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-15972651

RESUMEN

The two isoforms of phospholipase C (PLC)-gamma couple immune recognition receptors to important calcium- and protein kinase C-dependent cellular functions. It has been assumed that PLC-gamma1 and PLC-gamma2 have redundant functions and that the receptors can use whichever PLC-gamma isoform is preferentially expressed in a cell of a given hemopoietic lineage. In this study, we demonstrate that ITAM-containing immune recognition receptors can use either PLC-gamma1 or PLC-gamma2, whereas the novel NK cell-activating receptor NKG2D preferentially couples to PLC-gamma2. Experimental models evaluating signals from either endogenous receptors (FcR vs NKG2D-DAP10) or ectopically expressed chimeric receptors (with ITAM-containing cytoplasmic tails vs DAP10-containing cytoplasmic tails) demonstrate that PLC-gamma1 and PLC-gamma2 both regulate the functions of ITAM-containing receptors, whereas only PLC-gamma2 regulates the function of DAP10-coupled receptors. These data suggest that specific immune recognition receptors can differentially couple to the two isoforms of PLC-gamma. More broadly, these observations reveal a basis for selectively targeting the functions initiated by distinct immune recognition receptors.


Asunto(s)
Células Asesinas Naturales/enzimología , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Señalización del Calcio , Línea Celular Tumoral , Células Clonales , Citotoxicidad Inmunológica , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Subfamilia K de Receptores Similares a Lectina de Células NK , Fosfolipasa C gamma , Fosforilación , Receptores Fc/metabolismo , Receptores Inmunológicos/química , Receptores de Células Asesinas Naturales , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T Citotóxicos/inmunología , Fosfolipasas de Tipo C/química , Tirosina/química
18.
Nat Immunol ; 4(6): 557-64, 2003 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12740575

RESUMEN

The immune recognition receptor complex NKG2D-DAP10 on natural killer cells is stimulated by specific ligands carried on virus-infected and malignant cells. Because DAP10 does not have an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail, its ability to trigger killing has been debated. Here we show that a crucial Tyr-Ile-Asn-Met amino acid motif in the cytoplasmic tail of DAP10 couples receptor stimulation to the downstream activation of phosphatidylinositol 3-kinase, Vav1, Rho family GTPases and phospholipase C. Unlike that of ITAM-containing receptors, the activation of NKG2D-DAP10 proceeds independently of Syk family protein tyrosine kinases. Yet the signals initiated by NKG2D-DAP10 are fully capable of inducing killing. Our findings identify a previously unknown mechanism by which receptor complexes that lack ITAM motifs can trigger lymphocyte activation.


Asunto(s)
Proteínas de Ciclo Celular , Precursores Enzimáticos/inmunología , Células Asesinas Naturales/inmunología , Proteínas de la Membrana/inmunología , Proteínas Tirosina Quinasas/inmunología , Receptores Inmunológicos/inmunología , Secuencias de Aminoácidos/inmunología , Animales , Citotoxicidad Inmunológica/inmunología , Precursores Enzimáticos/metabolismo , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intracelular , Células Jurkat , Ratones , Subfamilia K de Receptores Similares a Lectina de Células NK , Fosfatidilinositol 3-Quinasas/inmunología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas c-vav , Receptores de Células Asesinas Naturales , Transducción de Señal/inmunología , Quinasa Syk , Fosfolipasas de Tipo C/inmunología , Proteínas de Unión al GTP rho/inmunología , Dominios Homologos src/inmunología
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