RESUMEN
Because of their ability to induce lymphocyte apoptosis, glucocorticoids (GC) are widely used to treat hematological malignancies such as lymphomas and multiple myeloma. Their effectiveness is often limited, however, due to the development of glucocorticoid resistance by a variety of molecular mechanisms. Here we performed an unbiased genome-wide CRISPR screen with the human T-cell leukemia cell line Jurkat to find previously unidentified genes required for GC-induced apoptosis. One such gene was KMT2D (also known as MLL2 or MLL4), which encodes a histone lysine methyltransferase whose mutations are associated with a variety of cancers, blood malignancies in particular, and are considered markers of poor prognosis. Knockout of KMT2D by CRISPR/Cas9 gene editing in Jurkat and several multiple myeloma cell lines downregulated GR protein expression. Surprisingly, this was not due to a reduction in GR transcripts, but rather to a decrease in the protein's half-life, primarily due to proteasomal degradation. Reconstitution of KMT2D expression restored GR levels. In contrast to the known ability of KMT2D to control gene transcription through covalent histone methylation, KMT2D-mediated upregulation of GR levels did not require its methyltransferase activity. Co-immunoprecipitation and proximity ligation assays found constitutive binding of KMT2D to the GR, which was enhanced in the presence of GC. These observations reveal KMT2D to be essential for the stabilization of cellular GR levels, and suggest a possible mechanism by which KMT2D mutations may lead to GC resistance in some malignancies.
RESUMEN
The TMC genes encode a set of homologous transmembrane proteins whose functions are not well understood. Biallelic mutations in either TMC6 or TMC8 are detected in more than half of cases of the pre-malignant skin disease epidermodysplasia verruciformis (EV). It is controversial whether EV induced by mutations in TMC6 or TMC8 originates from keratinocyte or lymphocyte defects. Quantification of TMC6 and TMC8 RNA levels in various organs revealed that lymphoid tissues have the highest levels of expression of both genes, and custom antibodies confirmed protein expression in mouse lymphocytes. To study the function of these proteins we generated mice with targeted deletion mutant alleles of Tmc6 or Tmc8 Either TMC6 or TMC8 deficiency induced a reduction in apparent molecular weight and/or amount of the other TMC molecule. Co-immunoprecipitation experiments indicated that TMC6 and TMC8 formed a protein complex in mouse and human T cells. MS and biochemical analysis demonstrated that TMC6 and TMC8 additionally interacted with the CIB1 protein to form TMC6-TMC8-CIB1 trimers. We demonstrated that TMC6 and TMC8 regulated CIB1 levels by protecting CIB1 from ubiquitination and proteasomal degradation. Reciprocally, CIB1 was needed for stabilizing TMC6 and TMC8 levels. These results suggest why inactivating mutations in any of the three human genes leads to similar clinical presentations. We also demonstrated that TMC6 and TMC8 levels are drastically lower and the proteins are less active in regulating CIB1 in keratinocytes than in T cells. Our study suggests that defects in lymphocytes may contribute to the etiology and pathogenesis of EV.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Linfocitos T/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Humanos , Células Jurkat , Queratinocitos/citología , Queratinocitos/metabolismo , Proteínas de la Membrana/genética , Ratones , Complejos Multiproteicos/genética , Proteolisis , Linfocitos T/citología , UbiquitinaciónRESUMEN
Actin-based stress fiber formation is coupled to microtubule depolymerization through the local activation of RhoA. While the RhoGEF Lfc has been implicated in this cytoskeleton coupling process, it has remained elusive how Lfc is recruited to microtubules and how microtubule recruitment moderates Lfc activity. Here, we demonstrate that the dynein light chain protein Tctex-1 is required for localization of Lfc to microtubules. Lfc residues 139-161 interact with Tctex-1 at a site distinct from the cleft that binds dynein intermediate chain. An NMR-based GEF assay revealed that interaction with Tctex-1 represses Lfc nucleotide exchange activity in an indirect manner that requires both polymerized microtubules and phosphorylation of S885 by PKA. We show that inhibition of Lfc by Tctex-1 is dynein dependent. These studies demonstrate a pivotal role of Tctex-1 as a negative regulator of actin filament organization through its control of Lfc in the crosstalk between microtubule and actin cytoskeletons.
Asunto(s)
Citoesqueleto de Actina/fisiología , Dineínas/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Microtúbulos/fisiología , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Dineínas/fisiología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/ultraestructura , Fibroblastos/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/fisiología , Ratones , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Fosforilación , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
NEMO is the regulatory subunit of the IkappaB kinase (IKK) in NF-kappaB activation, and its CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ, respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal that the binding sites for diubiquitins at LZ are composites of both chains and that each ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail, while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the proximal ubiquitin uses its conserved hydrophobic patch, while the distal ubiquitin mostly employs the C-terminal arm including the K63 linkage residue. These studies uncover the energetics and geometry for mutual recognition of NEMO and diubiquitins.
Asunto(s)
Quinasa I-kappa B/química , Ubiquitinas/química , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Cristalografía por Rayos X , Predisposición Genética a la Enfermedad , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , FN-kappa B/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Relación Estructura-Actividad , Ubiquitinas/metabolismoRESUMEN
Epithelial cell adhesion molecule (EpCAM) (CD326) is a surface glycoprotein expressed by invasive carcinomas and some epithelia. Herein, we report that EpCAM regulates the composition and function of tight junctions (TJ). EpCAM accumulated on the lateral interfaces of human colon carcinoma and normal intestinal epithelial cells but did not co-localize with TJ. Knockdown of EpCAM in T84 and Caco-2 cells using shRNAs led to changes in morphology and adhesiveness. TJ formed readily after EpCAM knockdown; the acquisition of trans-epithelial electroresistance was enhanced, and TJ showed increased resistance to disruption by calcium chelation. Preparative immunoprecipitation demonstrated that EpCAM bound tightly to claudin-7. Co-immunoprecipitation documented associations of EpCAM with claudin-7 and claudin-1 but not claudin-2 or claudin-4. Claudin-1 associated with claudin-7 in co-transfection experiments, and claudin-7 was required for association of claudin-1 with EpCAM. EpCAM knockdown resulted in decreases in claudin-7 and claudin-1 proteins that were reversed with lysosome inhibitors. Immunofluorescence microscopy revealed that claudin-7 and claudin-1 continually trafficked into lysosomes. Although EpCAM knockdown decreased claudin-1 and claudin-7 protein levels overall, accumulations of claudin-1 and claudin-7 in TJ increased. Physical interactions between EpCAM and claudins were required for claudin stabilization. These findings suggest that EpCAM modulates adhesion and TJ function by regulating intracellular localization and degradation of selected claudins.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Claudinas/metabolismo , Proteolisis , Uniones Estrechas/metabolismo , Antígenos de Neoplasias/genética , Células CACO-2 , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/genética , Claudinas/genética , Molécula de Adhesión Celular Epitelial , Técnicas de Silenciamiento del Gen , Humanos , Uniones Estrechas/genética , TransfecciónRESUMEN
The serine/threonine phosphatase calcineurin is a component of the T cell receptor (TCR) signalosome, where it promotes T cell activation by dephosphorylating LckS59. Using small interfering RNA (siRNA)-mediated knockdown and CRISPR-Cas9-targeted genetic disruption of the calcineurin A chain α and ß isoforms, we find that calcineurin also functions as an adaptor in TCR-signaled human T cells. Unlike inhibition of its phosphatase activity, in the absence of calcineurin A, TCR signaling results in attenuated actin rearrangement, markedly reduced TCR-Lck microcluster formation and recruitment of the adaptor RhoH, and diminished phosphorylation of critical targets downstream of Lck such as TCRζ and ZAP-70. Reconstitution of deficient T cells with either calcineurin Aα or Aß restores TCR microcluster formation and signaling, as does reconstitution with a phosphatase-inactive Aα chain. These results assign a non-enzymatic adaptor function to calcineurin in the formation and stabilization of a functional TCR signaling complex.
Asunto(s)
Calcineurina , Receptores de Antígenos de Linfocitos T , Transducción de Señal , Calcineurina/metabolismo , Humanos , Receptores de Antígenos de Linfocitos T/metabolismo , Células Jurkat , Linfocitos T/metabolismo , Fosforilación , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
The transcription factor NF-kappaB is sequestered in the cytoplasm in a complex with IkappaB. Almost all NF-kappaB activation pathways converge on IkappaB kinase (IKK), which phosphorylates IkappaB resulting in Lys 48-linked polyubiquitination of IkappaB and its degradation. This allows migration of NF-kappaB to the nucleus where it regulates gene expression. IKK has two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma or NEMO. NEMO is essential for NF-kappaB activation, and NEMO dysfunction in humans is the cause of incontinentia pigmenti and hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). The recruitment of IKK to occupied cytokine receptors, and its subsequent activation, are dependent on the attachment of Lys 63-linked polyubiquitin chains to signalling intermediates such as receptor-interacting protein (RIP). Here, we show that NEMO binds to Lys 63- but not Lys 48-linked polyubiquitin, and that single point mutations in NEMO that prevent binding to Lys 63-linked polyubiquitin also abrogates the binding of NEMO to RIP in tumour necrosis factor (TNF)-alpha-stimulated cells, the recruitment of IKK to TNF receptor (TNF-R) 1, and the activation of IKK and NF-kappaB. RIP is also destabilized in the absence of NEMO binding and undergoes proteasomal degradation in TNF-alpha-treated cells. These results provide a mechanism for NEMO's critical role in IKK activation, and a key to understanding the link between cytokine-receptor proximal signalling and IKK and NF-kappaB activation.
Asunto(s)
Técnicas Biosensibles , Quinasa I-kappa B/genética , Lisina/metabolismo , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Células HeLa , Humanos , Quinasa I-kappa B/metabolismo , Inmunoprecipitación , Lisina/genética , FN-kappa B/genética , Mutación Puntual , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Saccharomyces cerevisiae , Transducción de Señal , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Técnicas del Sistema de Dos HíbridosRESUMEN
OBJECTIVE: MFG-E8 (also called lactadherin and SED1) is a secreted glycoprotein that has been previously implicated in enhancement of vascular endothelial growth factor-dependent angiogenesis. Major sources of MFG-E8 in vivo and precise mechanisms of MFG-E8 action remain undetermined. The objective of this study was to identify important sources of MFG-E8 in vivo and further elucidate the role(s) of MFG-E8 in the regulation of angiogenesis. METHODS AND RESULTS: We used knockout mice and anti-MFG-E8 antibodies to study MFG-E8 function in vivo. In melanomas and in retinas of mice with oxygen-induced retinopathy, MFG-E8 colocalized with pericytes rather than endothelial cells, and platelet-derived growth factor receptor ß+ pericytes/pericyte precursors purified from tumors contained large amounts of MFG-E8 mRNA. Tumor- and retinopathy-associated angiogenesis was diminished in MFG-E8 knockout mice, and pericyte coverage of neovessels was reduced. Inhibition of MFG-E8 production by 10T1/2 cells (surrogate pericyte/pericyte precursors) using small interfering RNAs and short hairpin RNAs, or inhibition of MFG-E8 action with some anti-MFG-E8 antibodies, selectively attenuated migration in vitro. Significantly, the anti-MFG-E8 antibodies that inhibited 10T1/2 cell migration in vitro also inhibited pathological angiogenesis in vivo. CONCLUSIONS: These studies strongly implicate MFG-E8 in pericyte/pericyte precursor function and indicate that MFG-E8-directed therapeutics may merit further development.
Asunto(s)
Antígenos de Superficie/fisiología , Melanoma Experimental/irrigación sanguínea , Neovascularización Patológica/etiología , Pericitos/metabolismo , Animales , Movimiento Celular , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Leche , Enfermedades de la Retina/etiologíaRESUMEN
The mechanism by which the Carma1-Bcl10-MALT1 (CBM) complex couples T cell antigen receptor (TCR) signaling to IkappaB kinase (IKK) and NF-kappaB activation is not known. Here, we show that Bcl10 undergoes K63-linked polyubiquitination in response to T cell activation and subsequently binds NEMO, the regulatory subunit of IKK. This interaction requires the ubiquitin-binding activity of NEMO. The sites of Bcl10 ubiquitination were mapped to K31 and K63. Mutation of these residues did not affect TCR signaling-induced CBM complex assembly but prevented Bcl10 ubiquitination, NEMO binding, and NF-kappaB activation. Therefore, the regulated ubiquitination of Bcl10 and its recognition by NEMO are a critical link between the CBM complex, IKK recruitment, and NF-kappaB activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quinasa I-kappa B/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Proteína 10 de la LLC-Linfoma de Células B , Sitios de Unión/genética , Escherichia coli , Humanos , Immunoblotting , Inmunoprecipitación , Células Jurkat , Luciferasas , Mutación/genética , UbiquitinaciónRESUMEN
NF-kappaB essential modulator (NEMO), the regulatory subunit of the IkappaB kinase (IKK) that activates NF-kappaB, is essential for NF-kappaB activation. NEMO was recently found to contain a region that preferentially binds Lys (K)63-linked but not K48-linked polyubiquitin (polyUb) chains, and the ability of NEMO to bind to K63-linked polyUb RIP (receptor-interacting protein) is necessary for efficient tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. Optineurin is a homolog of NEMO, and mutations in the optineurin gene are found in a subset of patients with glaucoma, a neurodegenerative disease involving the loss of retinal ganglion cells. Although optineurin shares considerable homology with NEMO, in resting cells, it is not present in the high-molecular-weight complex containing IKKalpha and IKKbeta, and optineurin cannot substitute for NEMO in lipopolysaccharide (LPS)-induced NF-kappaB activation. On the other hand, the overexpression of optineurin blocks the protective effect of E3-14.7K on cell death caused by the overexpression of TNFalpha receptor 1 (TNFR1). Here we show that optineurin has a K63-linked polyUb-binding region similar to that of NEMO, and like NEMO, it bound K63- but not K48-linked polyUb. Optineurin competitively antagonized NEMO's binding to polyUb RIP, and its overexpression inhibited TNFalpha-induced NF-kappaB activation. This competition occurs at physiologic protein levels because microRNA silencing of optineurin resulted in markedly enhanced TNFalpha-induced NF-kappaB activity. These results reveal a physiologic role for optineurin in dampening TNFalpha signaling, and this role might provide an explanation for its association with glaucoma.
Asunto(s)
Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Transcripción TFIIIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas de Ciclo Celular , Glaucoma/metabolismo , Humanos , Proteínas de Transporte de Membrana , Ubiquitina/metabolismoRESUMEN
The homologs EpCAM and TROP2, which both interact with claudin-1 and claudin-7, are frequently coexpressed in epithelia including skin. Intestine uniquely expresses high levels of EpCAM but not TROP2. We previously identified EpCAM as a substrate of the membrane-anchored protease matriptase and linked HAI-2, matriptase, EpCAM and claudin-7 in a pathway that is pivotal for intestinal epithelial cells (IEC) homeostasis. Herein, we reveal that TROP2 is also a matriptase substrate. Matriptase cleaved TROP2 when purified recombinant proteins were mixed in vitro. TROP2, like EpCAM, was also cleaved after co-transfection of matriptase in 293T cells. Neither EpCAM nor TROP2 cleavage was promoted by protease-disabled matriptase or matriptase that harbored the ichthyosis-associated G827R mutation. We confirmed that EpCAM and TROP2 are both expressed in skin and detected cleavage of these proteins in human keratinocytes (HaCaT cells) after the physiologic inhibition of matriptase by HAI proteins was relieved by siRNA knockdown. Knockdown of EpCAM or TROP2 individually had only small effects on claudin-1 and claudin-7 levels, whereas elimination of both markedly diminished claudin levels. HAI-1 knockdown promoted EpCAM and TROP2 cleavage accompanied by reductions in claudins, whereas HAI-2 knockdown had little impact. Double knockdown of HAI-1 and HAI-2 induced nearly complete cleavage of EpCAM and TROP2 and drastic reductions of claudins. These effects were eliminated by concurrent matriptase knockdown. Decreases in claudin levels were also diminished by the lysosomal inhibitor chloroquine and cleaved EpCAM/TROP2 fragments accumulated preferentially. We demonstrate that TROP2 and EpCAM exhibit redundancies with regard to regulation of claudin metabolism and that an HAI, matriptase, EpCAM and claudin pathway analogous to what we described in IECs exists in keratinocytes. This study may offer insights into the mechanistic basis for matriptase dysregulation-induced ichthyosis.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Claudinas/metabolismo , Molécula de Adhesión Celular Epitelial/metabolismo , Queratinocitos/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Células HaCaT , Humanos , Intestinos/fisiología , Lisosomas/metabolismo , Ratones Endogámicos C57BL , Proteínas Mutantes/metabolismo , Estabilidad Proteica , Proteolisis , Piel/metabolismoRESUMEN
TROP1 (EpCAM) and TROP2 are homologous cell surface proteins that are widely expressed, and often co-expressed, in developing and adult epithelia. Various functions have been ascribed to EpCAM and TROP2, but responsible mechanisms are incompletely characterized and functional equivalence has not been examined. Adult intestinal epithelial cells (IEC) express high levels of EpCAM, while TROP2 is not expressed. EpCAM deficiency causes congenital tufting enteropathy (CTE) in humans and a corresponding lethal condition in mice. We expressed TROP2 and EpCAM in the IEC of EpCAM-deficient mice utilizing a villin promoter to assess EpCAM and TROP2 function. Expression of EpCAM or TROP2 in the IEC of EpCAM knockout mice prevented CTE. TROP2 rescue (T2R) mice were smaller than controls, while EpCAM rescue (EpR) mice were not. Abnormalities were observed in the diameters and histology of T2R small intestine, and Paneth and stem cell markers were decreased. T2R mice also exhibited enlarged mesenteric lymph nodes, enhanced permeability to 4 kDa FITC-dextran and increased sensitivity to detergent-induced colitis, consistent with compromised barrier function. Studies of IEC organoids and spheroids revealed that stem cell function was also compromised in T2R mice. We conclude that EpCAM and TROP2 exhibit functional redundancy, but they are not equivalent.
Asunto(s)
Antígenos de Neoplasias/fisiología , Moléculas de Adhesión Celular/fisiología , Diarrea Infantil/genética , Molécula de Adhesión Celular Epitelial/fisiología , Células Epiteliales/metabolismo , Síndromes de Malabsorción/genética , Animales , Células HEK293 , Humanos , Mucosa Intestinal/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras GenéticasRESUMEN
Congenital tufting enteropathy (CTE) is a severe autosomal recessive human diarrheal disorder with characteristic intestinal epithelial dysplasia. CTE can be caused by mutations in genes encoding EpCAM, a putative adhesion molecule, and HAI-2, a cell surface protease inhibitor. A similar phenotype occurs in mice whose intestinal epithelial cells (IECs) fail to express the tight junction-associated protein claudin-7. EpCAM stabilizes claudin-7 in IECs, and HAI-2 regulates the cell surface serine protease matriptase, a known modifier of intestinal epithelial physiology. Therefore, we hypothesized that HAI-2, matriptase, EpCAM, and claudin-7 were functionally linked. Herein we have demonstrated that active matriptase cleaves EpCAM after Arg80 and that loss of HAI-2 in IECs led to unrestrained matriptase activity and efficient cleavage of EpCAM. Cleavage of EpCAM decreased its ability to associate with claudin-7 and targeted it for internalization and lysosomal degradation in conjunction with claudin-7. CTE-associated HAI-2 mutant proteins exhibited reduced ability to inhibit matriptase and also failed to efficiently stabilize claudin-7 in IECs. These results identify EpCAM as a substrate of matriptase and link HAI-2, matriptase, EpCAM, and claudin-7 in a functionally important pathway that causes disease when it is dysregulated.
Asunto(s)
Claudinas/metabolismo , Molécula de Adhesión Celular Epitelial/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Células CACO-2 , Claudinas/genética , Molécula de Adhesión Celular Epitelial/genética , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones , Estabilidad Proteica , Serina Endopeptidasas/genéticaRESUMEN
In order to enhance the thermostability of D-Glucose isomerase (GI), its mutants, GIK253R and GIN184V, were obtained with the double primer method of site-directed mutagenesis. Then their biochemical properties were assayed and compared with wild-type GI. The results showed that: (1) The mutant K253R was less stable than the wild-type GI at 70 degrees and 80 degrees. But in 1 M L-Rhaminose at 70 degrees, they had similar rate of heat inactivation. Furthermore, the mutant K253R has higher specific activity than the wild-type GI. (2) The mutant N184V had much less thermostability and specific activity as compared with the wild-type GI. These results were explained by their kinetic parameters and crystal structure model.
RESUMEN
In order to enhance the thermostability of D-glucose isomerase (GI), Gly 138 was decided to be the target to be replaced by molecular design. The mutant G138P was obtained by in vitro site-directed mutagenesis of GI gene. The recombinant plasmid pTKD-GI containing mutant site was expressed in E. coli K38 strain. The comparison experiments of GIG138P with wild-type GI showed that: (1) The half time of GIG138P was as about two times as that of the wild type. (2) The optimum temperature of GIG138P was increased by 10-12 degrees. (3) The specific-activity of GIG138P was similar to the wild-type GI. We supposed, based on the above facts, that the substitution of Pro for Gly at position 138 introduced a pyrrolidine ring, which could just fill perfectly the empty hole leaved by Gly-138 which has no side chain and could make the protein structure more rigid, therefore the mutant G138P enhanced the thermostability of SM33GI.
RESUMEN
Stimulation through the interleukin-1 receptor (IL-1R) and some Toll-like receptors (TLRs) induces ubiquitination of TRAF6 and IRAK-1, signaling components required for NF-kappaB and mitogen-activated protein kinase activation. Here we show that although TRAF6 and IRAK-1 acquired Lys63 (K63)-linked polyubiquitin chains upon IL-1 stimulation, only ubiquitinated IRAK-1 bound NEMO, the regulatory subunit of IkappaB kinase (IKK). The sites of IRAK-1 ubiquitination were mapped to Lys134 and Lys180, and arginine substitution of these residues impaired IL-1R/TLR-mediated IRAK-1 ubiquitination, NEMO binding, and NF-kappaB activation. K63-linked ubiquitination of IRAK-1 required enzymatically active TRAF6, indicating that it is the physiologically relevant E3. Thus, K63-linked polyubiquitination of proximal signaling proteins is a common mechanism used by diverse innate immune receptors for recruiting IKK and activating NF-kappaB.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , FN-kappa B/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores Toll-Like/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Células Cultivadas , Cartilla de ADN/genética , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/genética , Lisina/química , Ratones , Receptores de Interleucina-1/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptores Toll-Like/genética , Transfección , UbiquitinaciónRESUMEN
Signaling through tumor necrosis factor receptor 2 (TNF-R2) results in ubiquitination of TRAF2 by the E3 c-IAP1. In this report, we confirm that TRAF2 translocates to a Triton X-100 (TX)-insoluble compartment upon TNF-R2 engagement. Moreover, TRAF2 ubiquitination occurs in this compartment, from which TRAF2 is degraded in a proteasome-dependent manner. Confocal microscopy demonstrated that the TX-insoluble compartment is perinuclear and co-localizes with endoplasmic reticulum (ER) markers. The ER transmembrane Ubc6 bound to c-IAP1 and served as a cognate E2 for c-IAP1's E3 activity in vitro. Furthermore, Ubc6 co-localized with translocated TRAF2/c-IAP1 in the ER-associated compartment in vivo, and a catalytically inactive Ubc6 mutant inhibited TNF-alpha-induced, TNF-R2-dependent TRAF2 degradation. These results indicate that upon TNF-R2 signaling, translocation of TRAF2 and c-IAP1 to an ER-associated, Ubc6-containing perinuclear compartment is required for the ubiquitination of TRAF2 by c-IAP1. Therefore, the ER plays a key role in the TNF-R-mediated signal transduction cascade by acting as a site of assembly for E2/E3/substrate complexes.