RESUMEN
Methods to engineer the genomes of human cells for therapeutic intervention continue to advance at a remarkable pace. Chimeric antigen receptor-engineered T lymphocytes have pioneered the way for these therapies, initially beginning with insertions of chimeric antigen receptor transgenes into T-cell genomes using classical gene therapy vectors. The broad use of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies to edit endogenous genes has now opened the door to a new era of precision medicine. To add complexity, many engineered cellular therapies under development integrate gene therapy with genome editing to introduce novel biological functions and enhance therapeutic efficacy. Here, we review the current state of scientific, translational, and regulatory oversight of gene-edited cell products.
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Chimeric antigen receptor (CAR)-based therapies have pioneered synthetic cellular immunity but remain limited in their long-term efficacy. Emerging data suggest that dysregulated CAR-driven T cell activation causes T cell dysfunction and therapeutic failure. To re-engage the precision of the endogenous T cell response, we designed MHC-independent T cell receptors (miTCRs) by linking antibody variable domains to TCR constant chains. Using predictive modeling, we observed that this standard "cut and paste" approach to synthetic protein design resulted in myriad biochemical conflicts at the hybrid variable-constant domain interface. Through iterative modeling and sequence modifications we developed structure-enhanced miTCRs which significantly improved receptor-driven T cell function across multiple tumor models. We found that 41BB costimulation specifically prolonged miTCR T cell persistence and enabled improved leukemic control in vivo compared to classic CAR T cells. Collectively, we have identified core features of hybrid receptor structure responsible for regulating function.
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Genome editing technologies have seen remarkable progress in recent years, enabling precise regulation of exogenous and endogenous genes. These advances have been extensively applied to the engineering of human T lymphocytes, leading to the development of practice changing therapies for patients with cancer and the promise of synthetic immune cell therapies for a variety of non-malignant diseases. Many distinct conceptual and technical approaches have been used to edit T-cell genomes, however targeted assessments of which techniques are most effective for manufacturing, gene editing and transgene expression are rarely reported. Through extensive comparative evaluation, we identified methods that most effectively enhance engineering of research-scale and pre-clinical T-cell products at critical stages of manufacturing.
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T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B-cell cancers yet fail to induce durable remissions for nearly half of all patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven antitumor T-cell function. We developed and validated an in vitro model that drives T-cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T-cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T-cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation using CAR T cells from a patient with progressive lymphoma confirmed the activation of this novel program in a failing clinical product. Furthermore, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T-cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T-cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.
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Linfoma , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Receptores de Antígenos de Linfocitos T/genética , Inmunoterapia Adoptiva/métodos , Linfocitos T , Linfoma/etiología , Antígenos CD19RESUMEN
Chimeric antigen receptor (CAR) engineered T cells often fail to enact effector functions after infusion into patients. Understanding the biological pathways that lead CAR T cells to failure is of critical importance in the design of more effective therapies. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains contribute to T cell failure. We found that dysfunctional CD28-based CARs targeting CD19 bear hallmarks of classical T cell exhaustion while dysfunctional 41BB-based CARs are phenotypically, transcriptionally and epigenetically distinct. We confirmed activation of this unique transcriptional program in CAR T cells that failed to control clinical disease. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is a significant contributor to this dysfunction and disruption of FOXO3 improves CAR T cell function. These findings identify that chronic activation of 41BB leads to novel state of T cell dysfunction that can be alleviated by genetic modification of FOXO3. Summary: Chronic stimulation of CARs containing the 41BB costimulatory domain leads to a novel state of T cell dysfunction that is distinct from T cell exhaustion.
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While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy.
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Antígenos CD19 , Inmunoterapia Adoptiva , Antígenos CD19/metabolismo , Linfocitos B , Glicosilación , Humanos , Inmunoterapia Adoptiva/métodos , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos TRESUMEN
Chimeric antigen receptor (CAR) T cells have revolutionized the management of B cell malignancies. These synthetic molecules are composed of peptide fragments from several distinct immune cell proteins and link highly-specific antigen recognition with potent T cell activation. Despite impressive results in many, less than half of patients treated will achieve durable remission after CAR therapy. Recent studies have identified the central role that each structural component of the CAR molecule plays in regulating T cell function. Significant effort has been dedicated to exploring strategies to improve the design of CARs themselves or integrate their activity with other regulatory circuits to enable more precise function. In this review, we will summarize recent pre-clinical and clinical studies that have evaluated novel CAR design formats.
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Inmunoterapia Adoptiva , Neoplasias , Linfocitos B , Humanos , Receptores de Antígenos de Linfocitos T/genética , Linfocitos TRESUMEN
BACKGROUND/PURPOSE: Positional obstructive sleep apnea (OSA) is defined as an apnea hypopnea index at least twice as high in the supine position as in the lateral position. Whether a positional OSA patient persistently has positional OSA in the follow-up period is unknown. This study was conducted to investigate the maintenance of the positional effect on OSA patients and the predictors of changing from positional OSA to nonpositional OSA. METHODS: Patients who were diagnosed to have positional OSA were screened for a follow-up polysomnography (PSG), which evaluated the effect of the same lateral position as baseline PSG on the severity of OSA. Those who met the criteria of positional OSA in both PSGs were classified as the unchanged group, the others were classified as the changed group. RESULTS: Seventy-eight positional OSA patients were enrolled in the final analyses. Twenty-seven of the enrolled patients (35%) were changed to nonpositional OSA patients in the second PSG. A higher apnea index in the lateral position was found in the changed group compared with that in the unchanged group (p = 0.02). Logistic regression also showed that the apnea index in the lateral position was the only independent predictor of changing from positional OSA to nonpositional OSA in the follow-up PSG (odds ratio = 1.13, p = 0.004). CONCLUSION: One-third of positional OSA patients who had a high apnea index in the lateral position tends to become nonpositional OSA patients in the follow-up PSG and must be closely monitored if receiving positional therapy only.
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Indicadores de Salud , Postura/fisiología , Apnea Obstructiva del Sueño/fisiopatología , Sueño/fisiología , Anciano , Femenino , Estudios de Seguimiento , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Polisomnografía , Estudios Retrospectivos , Apnea Obstructiva del Sueño/terapia , Posición Supina/fisiologíaRESUMEN
Cellular metabolism alters patterns of gene expression through a variety of mechanisms, including alterations in histone modifications and transcription factor activity. Nicotinamide adenine dinucleotide (NAD)-dependent proteins such as poly(ADP ribose) polymerases (PARPs) and sirtuin deacetylases play important roles in this regulation, thus NAD provides a crucial link between metabolism and these cellular signaling processes. Here, we found that lowering NAD levels in mouse primary cortical neurons led to decreased activity-dependent BDNF expression. The altered BDNF transcription occurred independently of Sirt or Parp activities; instead, low NAD levels promoted increased DNA methylation of the activity-dependent BDNF promoter. Increased methylation at this promoter triggered the dissociation of the insulator protein CTCF as well as the accompanying cohesin from the BDNF locus. The loss of these proteins resulted in histone acetylation and methylation changes at this locus consistent with chromatin compaction and gene silencing. Because BDNF is critical for neuronal function, these results suggest that age- or nutrition-associated declines in NAD levels as well as deficits in cohesin function associated with disease modulate BDNF expression and could contribute to cognitive impairment.
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Factor Neurotrófico Derivado del Encéfalo/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , NAD/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Metilación de ADN , Regulación de la Expresión Génica , Ratones , Neuronas/citología , Neuronas/fisiología , Regiones Promotoras Genéticas , Proteínas Represoras/genética , CohesinasRESUMEN
Axonal degeneration is a hallmark of many debilitating neurological disorders and is thought to be regulated by mechanisms distinct from those governing cell body death. Recently, caspase 6 activation via amyloid precursor protein (APP) cleavage and activation of DR6 was discovered to induce axon degeneration after NGF withdrawal. We tested whether this pathway is involved in axonal degeneration caused by withdrawal of other trophic support, axotomy or vincristine exposure. Neurturin deprivation, like NGF withdrawal activated this APP/DR6/caspase 6 pathway and resulted in axonal degeneration, however, APP cleavage and caspase 6 activation were not involved in axonal degeneration induced by mechanical or toxic insults. However, loss of surface APP (sAPP) and caspase 6 activation were observed during axonal degeneration induced by dynactin 1(Dctn1) dysfunction, which disrupts axonal transport. Mutations in Dctn1 are associated with motor neuron disease and frontal temporal dementia, thus suggesting that the APP/caspase 6 pathway could be important in specific types of disease-associated axonal degeneration. The NGF deprivation paradigm, with its defined molecular pathway, was used to examine the context of Nmnat-mediated axonal protection. We found that although Nmnat blocks axonal degeneration after trophic factor withdrawal, it did not prevent loss of axon sAPP or caspase 6 activation within the axon, suggesting it acts downstream of caspase 6. These results indicate that diverse insults induce axonal degeneration via multiple pathways and that these degeneration signals converge on a common, Nmnat-sensitive program that is uniquely involved in axonal, but not cell body, degeneration.
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Precursor de Proteína beta-Amiloide/metabolismo , Axones/metabolismo , Ganglios Espinales/metabolismo , Degeneración Nerviosa/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Axones/patología , Axotomía , Caspasa 6/metabolismo , Células Cultivadas , Ganglios Espinales/citología , Inmunohistoquímica , Ratones , Degeneración Nerviosa/patología , Neuronas/patología , Nicotinamida-Nucleótido Adenililtransferasa/genética , ARN Interferente Pequeño , Transducción de SeñalRESUMEN
Head and neck cancers usually present with advanced disease and novel therapies are urgently needed. Genetic therapy aims at restoring malfunctioned tumor suppressor gene(s) or introducing proapoptotic genes. Oncolytic virotherapeutics induce multiple cycles of cancer-specific virus replication, followed by oncolysis, virus spreading and infection of adjacent cancer cells. Oncolytic viruses can also be armed to express therapeutic transgene(s). Recent advances in preclinical and clinical studies are revealing the potential of both therapeutic classes for advanced head and neck cancers, including the approval of two products (Gendicine and H101) by a governmental agency. This review summarizes the available clinical data to date and discusses the challenges and future directions.
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Marcación de Gen/métodos , Terapia Genética/métodos , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/terapia , Viroterapia Oncolítica/métodos , Animales , Ensayos Clínicos como Asunto/métodos , Ensayos Clínicos como Asunto/tendencias , Marcación de Gen/tendencias , Terapia Genética/tendencias , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Neoplasias de Cabeza y Cuello/patología , Humanos , Viroterapia Oncolítica/tendenciasRESUMEN
NIMA-related kinases (Neks) belong to a large family of Ser/Thr kinases that have critical roles in coordinating microtubule dynamics during ciliogenesis and mitotic progression. The Nek kinases are also expressed in neurons, whose axonal projections are, similarly to cilia, microtubule-abundant structures that extend from the cell body. We therefore investigated whether Nek kinases have additional, non-mitotic roles in neurons. We found that Nek3 influences neuronal morphogenesis and polarity through effects on microtubules. Nek3 is expressed in the cytoplasm and axons of neurons and is phosphorylated at Thr475 located in the C-terminal PEST domain, which regulates its catalytic activity. Although exogenous expression of wild-type or phosphomimic (T475D) Nek3 in cultured neurons has no discernible impact, expression of a phospho-defective mutant (T475A) or PEST-truncated Nek3 leads to distorted neuronal morphology with disturbed polarity and deacetylation of microtubules via HDAC6 in its kinase-dependent manner. Thus, the phosphorylation at Thr475 serves as a regulatory switch that alters Nek3 function. The deacetylation of microtubules in neurons by unphosphorylated Nek3 raises the possibility that it could have a role in disorders where axonal degeneration is an important component.
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Microtúbulos/metabolismo , Neuronas , Proteínas Serina-Treonina Quinasas/metabolismo , Acetilación , Anilidas/metabolismo , Animales , Polaridad Celular , Células Cultivadas , Ganglios Espinales/citología , Células HeLa , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/metabolismo , Ratones , Quinasas Relacionadas con NIMA , Neuronas/citología , Neuronas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Treonina/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Cornelia de Lange syndrome (CdLS), a disorder caused by mutations in cohesion proteins, is characterized by multisystem developmental abnormalities. PDS5, a cohesion protein, is important for proper chromosome segregation in lower organisms and has two homologues in vertebrates (PDS5A and PDS5B). Pds5B mutant mice have developmental abnormalities resembling CdLS; however the role of Pds5A in mammals and the association of PDS5 proteins with CdLS are unknown. To delineate genetic interactions between Pds5A and Pds5B and explore mechanisms underlying phenotypic variability, we generated Pds5A-deficient mice. Curiously, these mice exhibit multiple abnormalities that were previously observed in Pds5B-deficient mice, including cleft palate, skeletal patterning defects, growth retardation, congenital heart defects and delayed migration of enteric neuron precursors. They also frequently display renal agenesis, an abnormality not observed in Pds5B(-/-) mice. While Pds5A(-/-) and Pds5B(-/-) mice die at birth, embryos harboring 3 mutant Pds5 alleles die between E11.5 and E12.5 most likely of heart failure, indicating that total Pds5 gene dosage is critical for normal development. In addition, characterization of these compound homozygous-heterozygous mice revealed a severe abnormality in lens formation that does not occur in either Pds5A(-/-) or Pds5B(-/-) mice. We further identified a functional missense mutation (R1292Q) in the PDS5B DNA-binding domain in a familial case of CdLS, in which affected individuals also develop megacolon. This study shows that PDS5A and PDS5B functions other than those involving chromosomal dynamics are important for normal development, highlights the sensitivity of key developmental processes on PDS5 signaling, and provides mechanistic insights into how PDS5 mutations may lead to CdLS.
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Proteínas de Unión al ADN/genética , Síndrome de Cornelia de Lange/embriología , Síndrome de Cornelia de Lange/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/fisiología , Modelos Animales de Enfermedad , Desarrollo Embrionario/genética , Femenino , Dosificación de Gen , Heterocigoto , Homocigoto , Humanos , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Mutación Missense , Linaje , Fenotipo , Embarazo , Homología de Secuencia de Aminoácido , Factores de Transcripción/deficiencia , Factores de Transcripción/fisiologíaRESUMEN
Primary and metastatic neoplasms of the liver account for more than a million deaths per year worldwide. Despite decades of research, effective novel therapies for these cancers are urgently needed. Oncolytic virotherapeutics represent a novel class of pharmacophore that holds promise for the treatment of hepatic neoplasms. Cancer-specific replication is followed by oncolysis, virus spreading and infection of adjacent cancer cells. This process is then repeated. Virotherapeutics target multiple genetic pathways involved in carcino-genesis, and demonstrate activity against apoptosis-resistant tumour cells. This platform can also exploit the advantage of multiple intrinsic anti-cancer therapeutic mechanisms, combining direct viral oncolysis with therapeutic transgene expression. Recent advances in pre-clinical and clinical studies are revealing the potential of this unique therapeutic class, in particular for liver cancers. This review summarizes the available data on applying oncolytic virotherapeutics to hepatic neoplasms to date, and discusses the challenges and future directions for virotherapy.
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Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Viroterapia Oncolítica/métodos , Animales , Ensayos Clínicos como Asunto , Ensayos de Selección de Medicamentos Antitumorales , HumanosRESUMEN
In vitro culturing of primary neurons is a mainstay of neurobiological research. Many of these culture paradigms have taken advantage of defined culture media rather than serum additives that contain undefined survival factors to facilitate experimental manipulations and interpretation of the results. To culture neurons in the absence of serum, defined supplements such as B27 are now widely used. However, commercially available supplements exhibit large variability in their capabilities to support neurons in culture. We re-optimized and modified earlier published formulations of B27 using 21 different ingredients (NS21). NS21 supports neuronal cultures of high quality as manifested by their morphological characteristics, formation of synapses, and postsynaptic responses. Much of the variability in the quality of B27/NS21 was due to variability in the quality of different sources of bovine serum albumin. Furthermore, we found that holo-transferrin used in NS21 is preferable over apo-transferrin used in B27 for the quality of neuronal cultures.
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Técnicas de Cultivo de Célula , Medios de Cultivo/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Animales , Animales Recién Nacidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero , Homólogo 4 de la Proteína Discs Large , Agonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Ganglios Espinales/citología , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Técnicas de Placa-Clamp/métodos , Ratas , Ratas Sprague-Dawley , Retina/citología , Sinaptotagminas/metabolismo , Factores de Tiempo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
The control of myelination by oligodendrocytes in the CNS is poorly understood. Here we show that LINGO-1 is an important negative regulator of this critical process. LINGO-1 is expressed in oligodendrocytes. Attenuation of its function by dominant-negative LINGO-1, LINGO-1 RNA-mediated interference (RNAi) or soluble human LINGO-1 (LINGO-1-Fc) leads to differentiation and increased myelination competence. Attenuation of LINGO-1 results in downregulation of RhoA activity, which has been implicated in oligodendrocyte differentiation. Conversely, overexpression of LINGO-1 leads to activation of RhoA and inhibition of oligodendrocyte differentiation and myelination. Treatment of oligodendrocyte and neuron cocultures with LINGO-1-Fc resulted in highly developed myelinated axons that have internodes and well-defined nodes of Ranvier. The contribution of LINGO-1 to myelination was verified in vivo through the analysis of LINGO-1 knockout mice. The ability to recapitulate CNS myelination in vitro using LINGO-1 antagonists and the in vivo effects seen in the LINGO-1 knockout indicate that LINGO-1 signaling may be critical for CNS myelination.
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Sistema Nervioso Central/embriología , Regulación hacia Abajo/genética , Vaina de Mielina/metabolismo , Glicoproteína Asociada a Mielina/genética , Fibras Nerviosas Mielínicas/metabolismo , Oligodendroglía/metabolismo , Receptores de Superficie Celular/genética , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Técnicas de Cocultivo , Regulación hacia Abajo/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Proteínas de la Membrana , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Vaina de Mielina/genética , Vaina de Mielina/ultraestructura , Glicoproteína Asociada a Mielina/antagonistas & inhibidores , Glicoproteína Asociada a Mielina/metabolismo , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fibras Nerviosas Mielínicas/ultraestructura , Proteínas del Tejido Nervioso , Oligodendroglía/efectos de los fármacos , Oligodendroglía/ultraestructura , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fyn , Interferencia de ARN/efectos de los fármacos , Interferencia de ARN/fisiología , Nódulos de Ranvier/genética , Nódulos de Ranvier/metabolismo , Nódulos de Ranvier/ultraestructura , Ratas , Ratas Long-Evans , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína de Unión al GTP rhoA/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
A major obstacle for successful axon regeneration in the adult central nervous system (CNS) arises from inhibitory molecules in CNS myelin, which signal through a common receptor complex on neurons consisting of the ligand-binding Nogo-66 receptor (NgR) and two transmembrane coreceptors, p75 and LINGO-1. However, p75 expression is only detectable in subpopulations of mature neurons, raising the question of how these inhibitory signals are transduced in neurons lacking p75. In this study, we demonstrate that TROY (also known as TAJ), a TNF receptor family member selectively expressed in the adult nervous system, can form a functional receptor complex with NgR and LINGO-1 to mediate cellular responses to myelin inhibitors. Also, both overexpressing a dominant-negative TROY or presence of a soluble TROY protein can efficiently block neuronal response to myelin inhibitors. Our results implicate TROY in mediating myelin inhibition, offering new insights into the molecular mechanisms of regeneration failure in the adult nervous system.
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Sistema Nervioso Central/metabolismo , Inhibidores de Crecimiento/metabolismo , Proteínas de la Mielina/metabolismo , Regeneración Nerviosa/fisiología , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factores de Edad , Animales , Células COS , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Regulación hacia Abajo/fisiología , Proteínas Ligadas a GPI , Proteínas de la Membrana , Ratones , Mutación/fisiología , Glicoproteína Asociada a Mielina/metabolismo , Proteínas del Tejido Nervioso , Receptor Nogo 1 , Ratas , Receptor de Factor de Crecimiento Nervioso , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The yeast silent information regulators Sir2, Sir3, and Sir4 physically interact with one another to establish a transcriptionally silent state by forming repressive chromatin structures. The Sir4 protein contains binding sites for both Sir2 and Sir3, and these protein-protein interactions are required for gene silencing. Here, we report the X-ray structure of the coiled-coil dimerization motif within the C-terminus of Sir4 and show that it forms a stable 1:1 complex with a dimeric fragment of Sir3 (residues 464-978). We have identified a cluster of residues on the surface of the Sir4 coiled coil required for specific interactions with Sir3. The histone deacetylase Sir2 can also bind to this complex, forming a ternary complex with the truncated Sir3 and Sir4 proteins. The dual interactions of Sir4 with Sir3 and Sir2 suggest a physical basis for recruiting Sir3 to chromatin by virtue of its interactions with Sir4 and with deacetylated histones in chromatin.