Publications by authors named "Jean Da Silva Correia"

17 Publications

  • Page 1 of 1

Nucleotide insertions and deletions complement point mutations to massively expand the diversity created by somatic hypermutation of antibodies.

J Biol Chem 2014 Nov 15;289(48):33557-67. Epub 2014 Oct 15.

From Anaptysbio Inc., San Diego, California 92121 and.

During somatic hypermutation (SHM), deamination of cytidine by activation-induced cytidine deaminase and subsequent DNA repair generates mutations within immunoglobulin V-regions. Nucleotide insertions and deletions (indels) have recently been shown to be critical for the evolution of antibody binding. Affinity maturation of 53 antibodies using in vitro SHM in a non-B cell context was compared with mutation patterns observed for SHM in vivo. The origin and frequency of indels seen during in vitro maturation were similar to that in vivo. Indels are localized to CDRs, and secondary mutations within insertions further optimize antigen binding. Structural determination of an antibody matured in vitro and comparison with human-derived antibodies containing insertions reveal conserved patterns of antibody maturation. These findings indicate that activation-induced cytidine deaminase acting on V-region sequences is sufficient to initiate authentic formation of indels in vitro and in vivo and that point mutations, indel formation, and clonal selection form a robust tripartite system for antibody evolution.
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http://dx.doi.org/10.1074/jbc.M114.607176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246108PMC
November 2014

Ebola virion attachment and entry into human macrophages profoundly effects early cellular gene expression.

PLoS Negl Trop Dis 2011 Oct 18;5(10):e1359. Epub 2011 Oct 18.

Integrated Research Facility at Fort Detrick, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Maryland, United States of America.

Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP(1,2)) is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP(1,2) (VLP(VP40-GP)) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLP(VP40) (particles lacking GP(1,2)) caused an aberrant response. This suggests that GP(1,2) binding to macrophages plays an important role in the immediate cellular response.
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http://dx.doi.org/10.1371/journal.pntd.0001359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196478PMC
October 2011

An inflammasome-independent role for epithelial-expressed Nlrp3 in renal ischemia-reperfusion injury.

J Immunol 2010 Nov 20;185(10):6277-85. Epub 2010 Oct 20.

Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, CA 92037, USA.

Cytoplasmic innate immune receptors are important therapeutic targets for diseases associated with overproduction of proinflammatory cytokines. One cytoplasmic receptor complex, the Nlrp3 inflammasome, responds to an extensive array of molecules associated with cellular stress. Under normal conditions, Nlrp3 is autorepressed, but in the presence of its ligands, it oligomerizes, recruits apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), and triggers caspase 1 activation and the maturation of proinflammatory cytokines such as IL-1β and IL-18. Because ischemic tissue injury provides a potential source for Nlrp3 ligands, our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in components of the Nlrp3 inflammasome (Nlrp3(-/-) and Asc(-/-) mice). To examine the role of the inflammasome in renal ischemia-reperfusion injury (IRI) we also tested its downstream targets caspase 1, IL-1β, and IL-18. Both Nlrp3 and Asc were highly expressed in renal tubular epithelium of humans and mice, and the absence of Nlrp3, but not Asc or the downstream inflammasome targets, dramatically protected from kidney IRI. We conclude that Nlrp3 contributes to renal IRI by a direct effect on renal tubular epithelium and that this effect is independent of inflammasome-induced proinflammatory cytokine production.
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http://dx.doi.org/10.4049/jimmunol.1002330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020135PMC
November 2010

Nod1 and nod2 are expressed in human and murine renal tubular epithelial cells and participate in renal ischemia reperfusion injury.

J Immunol 2010 Mar 1;184(5):2297-304. Epub 2010 Feb 1.

Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, CA 92037, USA.

Nucleotide-binding oligomerization domain (Nod) 1 and Nod2 are members of a family of intracellular innate sensors that participate in innate immune responses to pathogens and molecules released during the course of tissue injury, including injury induced by ischemia. Ischemic injury to the kidney is characterized by renal tubular epithelial apoptosis and inflammation. Among the best studied intracellular innate immune receptors known to contribute to apoptosis and inflammation are Nod1 and Nod2. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in Nod1, Nod2, Nod(1 x 2), and in their downstream signaling molecule receptor-interacting protein 2. We found that Nod1 and Nod2 were present in renal tubular epithelial cells in both mouse and human kidneys and that the absence of these receptors in mice resulted in protection from kidney ischemia reperfusion injury. Significant protection from kidney injury was seen with a deficiency of Nod2 and receptor-interacting protein 2, and the simultaneous deficiency of Nod1 and Nod2 provided even greater protection. We conclude that the intracellular sensors Nod1 and Nod2 play an important role in the pathogenesis of acute ischemic injury of the kidney, although possibly through different mechanisms.
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http://dx.doi.org/10.4049/jimmunol.0903065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3020136PMC
March 2010

Identification of MAVS splicing variants that interfere with RIGI/MAVS pathway signaling.

Mol Immunol 2008 Apr 22;45(8):2277-87. Epub 2008 Jan 22.

Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.

The mitochondrial anti-viral signaling protein (MAVS), also known as CARDIF, IPS-1, KIAA1271 and VISA, is a mitochondria associated protein that regulates type I interferon production through coordinated activation of NF-kappaB and IRF3. The N-terminal CARD domain of MAVS interacts with RIGI helicase of upcapped RNA detection and the putative TRAF2 and TRAF6 binding motifs modulate protein interaction for NF-kappaB activation. MAVS is encoded by a single gene composed of 6 exons but is generally detected as multiple protein bands after separation by SDS-PAGE. In an effort to identify MAVS variants with diverse biological functions, we isolated three splicing variants and named them MAVS 1a (exon 2 deletion), 1b (exon 3 deletion) and 1c (exon 6 deletion), respectively. MAVS 1a and 1b, due to a frame shift by exon deletion, encode 131 and 124 aa residues, respectively. Except the first 39 aa residues encoded by exon 1, MAVS 1a does not share sequence homology with known proteins, it instead contains a putative TRAF2-binding motif and interacts with TRAF2 and RIP1. MAVS 1b shares the first 97 residues with wt MAVS and 27 aa residues of unknown protein. Unlike MAVS that activates both NF-kappaB and IRF3 pathways, expression of MAVS 1b selectively activates an IFNbeta but not an IL8 promoter. MAVS 1b interacts with RIP1 and FADD and exhibits anti-viral activity against VSV infection. This study uncovers MAVS splicing variants of diverse biological function.
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http://dx.doi.org/10.1016/j.molimm.2007.11.018DOI Listing
April 2008

SGT1 is essential for Nod1 activation.

Proc Natl Acad Sci U S A 2007 Apr 9;104(16):6764-9. Epub 2007 Apr 9.

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The Nod-like receptor family in man contains proteins that recognize invasive bacteria. Nod1, a member of this family, is activated by specific peptidoglycan-derived muropeptides that contain meso-diaminopimelic acid. Plants contain a large family of proteins known as resistance (R) proteins that have common structural features with the Nod-like receptors and are essential for protection against a variety of plant pathogens. Extensive genetic studies have shown that the R protein function is determined by multiple proteins including SGT1, Rar1, and HSP90. Here we show that SGT1 positively regulates Nod1 activation. Depletion of SGT1 with siRNA did not affect stability of Nod1 protein or of downstream signaling molecules but did prevent multiple cellular responses associated with Nod1 activation. In contrast, depletion of the mammalian orthologue of Rar1, Chp1, had no effect on Nod1-dependent cellular activation. Finally, depletion of HSP90 or addition of a pharmacologic inhibitor of HSP90 resulted in loss of Nod1 protein. Thus, we show common regulatory pathways in plant R protein and human Nod1-dependent pathways and provide the basis for understanding the Nod1 pathway.
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http://dx.doi.org/10.1073/pnas.0610926104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1871859PMC
April 2007

MDP-induced interleukin-1beta processing requires Nod2 and CIAS1/NALP3.

J Leukoc Biol 2007 Jul 2;82(1):177-83. Epub 2007 Apr 2.

Department of Immunology, The Scripps Research Institute, La Jolla, California, USA.

Nucleotide-binding oligomerization domain (Nod)2 is a sensor of muramyl dipeptides (MDP) derived from bacterial peptidoglycan. Nod2 also plays a role in some autoinflammatory diseases. Cold-induced autoinflammatory syndrome 1 (CIAS1)/NACHT domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NALP3) has been suggested to be sufficient for MDP-dependent release of mature IL-1beta, but the role of Nod2 in this process is unclear. Using mice bearing selective gene deletions, we provide in vitro and in vivo data showing that MDP-induced IL-1beta release requires Nod2 and CIAS1/NALP3 as well as receptor-interacting protein-2 (Rip2), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. In contrast, MDP-dependent IL-6 production only requires Nod2 and Rip2. Together, our data provide a new understanding of this important pathway of IL-1beta production and allow for further studies of the role of these proteins within the broader context of inflammatory disease.
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http://dx.doi.org/10.1189/jlb.1006627DOI Listing
July 2007

The subunit CSN6 of the COP9 signalosome is cleaved during apoptosis.

J Biol Chem 2007 Apr 2;282(17):12557-65. Epub 2007 Mar 2.

Department of Immunology, Scripps Research Institute, La Jolla, California 92037, USA.

The COP9 signalosome is a large multiprotein complex that consists of eight subunits termed CSN1-CSN8. The diverse functions of the COP9 complex include regulation of several important intracellular pathways, including the ubiquitin/proteasome system, DNA repair, cell cycle, developmental changes, and some aspects of immune responses. Nod1 is also thought to be an important cytoplasmic receptor involved in innate immune responses. It detects specific motifs of bacterial peptidoglycan, and this results in activation of multiple signaling pathways and changes in cell function. In this report, we performed a yeast two-hybrid screening and discovered that Nod1 interacts with several components of the COP9 signalosome through its CARD domain. Moreover, we observed that activation of the Nod1 apoptotic pathway leads to specific cleavage of the subunit CSN6. This cleavage is concomitant with caspase processing and generates a short amino-terminal peptide of 3 kDa. A complete inhibition of this cleavage was achieved in the presence of the broad spectrum pharmacological inhibitor of apoptosis, Z-VAD. Furthermore, overexpression of CLARP, a specific caspase 8 inhibitor, completely blocked cleavage of CSN6. Taken together, these results suggest a critical role of caspase 8 in the processing of CSN6. Moreover, these findings suggest that CSN6 cleavage may result in modifications of functions of the COP9 complex that are involved in apoptosis.
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http://dx.doi.org/10.1074/jbc.M609587200DOI Listing
April 2007

Cleavage of p65/RelA of the NF-kappaB pathway by Chlamydia.

Proc Natl Acad Sci U S A 2007 Feb 14;104(8):2933-8. Epub 2007 Feb 14.

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Chlamydia trachomatis is a bacterial pathogen that infects the eyes and urogenital tract. Ocular infection by this organism is the leading cause of preventable blindness worldwide. The infection is also a leading cause of sexually transmitted disease in the United States. As obligate intracellular pathogens, chlamydiae have evolved sophisticated, yet undefined, mechanisms to maintain a favorable habitat for intracellular growth while avoiding harm to the host. We show here that chlamydiae have the ability to interfere with the NF-kappaB pathway of host inflammatory response. We found that Chlamydia infection did not promote IkappaBalpha degradation, a prerequisite for NF-kappaB nuclear translocation/activation, nor induce p65/RelA nuclear redistribution. Instead, it caused p65 cleavage into an N terminus-derived p40 fragment and a p22 of the C terminus. The activity was specific because no protein cleavage or degradation of NF-kappaB pathway components was detected. Moreover, murine p65 protein was resistant to cleavage by both human and mouse biovars. The chlamydial protein that selectively cleaved p65 was identified as a tail-specific protease (CT441). Importantly, expression of either this protease or the p40 cleavage product could block NF-kappaB activation. A hallmark of chlamydial STD is its asymptomatic nature, although inflammatory cellular response and chronic inflammation are among the underlying mechanisms. The data presented here demonstrate that chlamydiae have the ability to convert a regulatory molecule of host inflammatory response to a dominant negative inhibitor of the same pathway potentially to minimize inflammation.
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http://dx.doi.org/10.1073/pnas.0608393104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1815284PMC
February 2007

Nod1-dependent control of tumor growth.

Proc Natl Acad Sci U S A 2006 Feb 30;103(6):1840-5. Epub 2006 Jan 30.

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Nod1, a cytosolic protein that senses meso-diaminopimelic acid-containing ligands derived from peptidoglycan, plays a role in host responses to invasive bacteria. Here we describe a function for Nod1, whereby it controls tumor formation. Cell lines derived from the human breast cancer epithelial cell line MCF-7 were used in a severe combined immune deficiency (SCID) mouse xenograft model to characterize a pathway linking Nod1 to the growth of estrogen-sensitive tumors. In MCF-7 cells, the absence of Nod1 correlates with tumor growth, an increased sensitivity to estrogen-induced cell proliferation, and a failure to undergo Nod1-dependent apoptosis. Conversely, overexpression of Nod1 in MCF-7 cells results in inhibition of estrogen-dependent tumor growth and reduction of estrogen-induced proliferative responses in vitro.
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http://dx.doi.org/10.1073/pnas.0509228103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1413646PMC
February 2006

A synthetic TLR4 antagonist has anti-inflammatory effects in two murine models of inflammatory bowel disease.

J Immunol 2005 May;174(10):6416-23

Corixa Corporation, Seattle, WA 98101, USA.

Current evidence indicates that the chronic inflammation observed in the intestines of patients with inflammatory bowel disease is due to an aberrant immune response to enteric flora. We have developed a lipid A-mimetic, CRX-526, which has antagonistic activity for TLR4 and can block the interaction of LPS with the immune system. CRX-526 can prevent the expression of proinflammatory genes stimulated by LPS in vitro. This antagonist activity of CRX-526 is directly related to its structure, particularly secondary fatty acyl chain length. In vivo, CRX-526 treatment blocks the ability of LPS to induce TNF-alpha release. Importantly, treatment with CRX-526 inhibits the development of moderate-to-severe disease in two mouse models of colonic inflammation: the dextran sodium sulfate model and multidrug resistance gene 1a-deficient mice. By blocking the interaction between enteric bacteria and the innate immune system, CRX-526 may be an effective therapeutic molecule for inflammatory bowel disease.
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http://dx.doi.org/10.4049/jimmunol.174.10.6416DOI Listing
May 2005

Innate immune responses during infection.

Vaccine 2004 Dec;22 Suppl 1:S25-30

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The innate immune system senses bacteria in the environment and defends against infection. Here we will discuss two types of sensor protein families. The plasma membrane receptors that comprise the Toll-like receptor (TLRs) family and the intracellular proteins termed NOD1 and NOD2. These proteins directly bind bacterial products such as lipopolysaccharides (LPS), peptidoglycan fragments, bacterial DNA, and receptor binding leads to intracellular signaling and gene expression. TLR signaling involves members of the MyD88 family of adaptor proteins. In contrast NOD1 or NOD2 utilize pathways that do not depend on the MyD88 family members.
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http://dx.doi.org/10.1016/j.vaccine.2004.08.013DOI Listing
December 2004

Mechanism of p21-activated kinase 6-mediated inhibition of androgen receptor signaling.

J Biol Chem 2004 Jan 22;279(3):1922-31. Epub 2003 Oct 22.

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

PAK6 was first identified as an androgen receptor (AR)-interacting protein able to inhibit AR-mediated transcriptional responses. PAK6 is a serine/threonine kinase belonging to the p21-activated kinase (PAK) family implicated in actin reorganization and cell motility, gene transcription, apoptosis, and cell transformation. We investigated the biochemical basis for inhibition of AR signaling by PAK6. We compared the kinase activity of PAK6 with two other well characterized members of the PAK family, PAK1 and PAK4. Like PAK4, PAK6 possesses a constitutive basal kinase activity that, unlike PAK1, is not modulated by the binding of active Rac or Cdc42 GTPases. In order to test the involvement of PAK6 kinase activity in suppression of AR-mediated transcription, we generated kinase-dead (K436A) and kinase-active (S531N) mutants of PAK6. We show that PAK6 kinase activity is required for effective PAK6-induced repression of AR signaling. Suppression does not depend upon GTPase binding to PAK6 and is not mimicked by the closely related PAK1 and PAK4 isoforms. Kinase-dependent inhibition by PAK6 extended to the enhanced AR-mediated transcription seen in the presence of coactivating molecules and to the action of AR coinhibitors. Active PAK6 inhibited nuclear translocation of the stimulated AR, suggesting a possible mechanism for inhibition of AR responsiveness. Finally, we observe that autophosphorylated, active PAK6 protein is differently expressed among prostate cancer cell lines. Modulation of PAK6 activity may be responsible for regulation of AR signaling in various forms of prostate cancer.
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http://dx.doi.org/10.1074/jbc.M311145200DOI Listing
January 2004

Structure-activity relationship of synthetic toll-like receptor 4 agonists.

J Biol Chem 2004 Feb 21;279(6):4440-9. Epub 2003 Oct 21.

Corixa Corporation, Seattle, Washington 98104, USA.

Important questions remain regarding the impact of variations in the structure of the lipid A portion of lipopolysaccharide on activation of cells via the Toll-like receptor 4 complex. We have studied a series of synthetic lipid A mimetic compounds known as aminoalkyl glucosaminide phosphates in which the length of the secondary acyl chain has been systematically varied. Using transcriptional profiling of human monocytes and responses of Toll-like receptor 4 complex cell transfectants, we demonstrate a clear dependence of length on secondary acyl chain on Toll-like receptor 4 activation. Compounds with secondary acyl chains less than eight carbons in length have dramatically reduced activity, and substitutions of the left-sided secondary acyl chain had the most important effect on the Toll-like receptor 4 agonist activity of these molecules. The structure-function relationships of these compounds assessed via the induction of chemokines and cytokines following in vivo administration closely mirrored those seen with cell-based studies. This novel set of synthetic lipid A mimetics will be useful for Toll-like receptor 4-based investigations and may have clinical utility as stand-alone immunomodulators.
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http://dx.doi.org/10.1074/jbc.M310760200DOI Listing
February 2004

ILPIP, a novel anti-apoptotic protein that enhances XIAP-mediated activation of JNK1 and protection against apoptosis.

J Biol Chem 2002 Aug 4;277(34):30454-62. Epub 2002 Jun 4.

Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

We have previously described a new aspect of the Inhibitor of Apoptosis (IAP) family of proteins anti-apoptotic activity that involves the TAK1/JNK1 signal transduction pathway (1,2). Our findings suggest the existence of a novel mechanism that regulates the anti-apoptotic activity of IAPs that is separate from caspase inhibition but instead involves TAK1-mediated activation of JNK1. In a search for proteins involved in the XIAP/TAK1/JNK1 signaling pathway we isolated by yeast two-hybrid screening a novel X chromosome-linked IAP (XIAP)-interacting protein that we called ILPIP (hILP-Interacting Protein). Whereas ILPIP moderately activates JNK family members when expressed alone, it strongly enhances XIAP-mediated activation of JNK1, JNK2, and JNK3. The expression of a catalytically inactive mutant of TAK1 blocked XIAP/ILPIP synergistic activation of JNK1 thereby implicating TAK1 in this signaling pathway. ILPIP moderately protects against interleukin-1beta converting enzyme- or Fas-induced apoptosis and significantly potentiates the anti-apoptotic activity of XIAP. In vivo co-precipitation experiments show that both ILPIP and XIAP interact with TAK1 and tumor necrosis factor receptor-associated factor 6. Finally, expression of ILPIP did not affect the ability of XIAP to inhibit caspase activation, further supporting the idea that XIAP protection against apoptosis is achieved by two separate mechanisms: one requiring JNK1 activation and a second involving caspase inhibition.
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http://dx.doi.org/10.1074/jbc.M203312200DOI Listing
August 2002

IAP suppression of apoptosis involves distinct mechanisms: the TAK1/JNK1 signaling cascade and caspase inhibition.

Mol Cell Biol 2002 Mar;22(6):1754-66

Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

The antiapoptotic properties of the inhibitor of apoptosis (IAP) family of proteins have been linked to caspase inhibition. We have previously described an alternative mechanism of XIAP inhibition of apoptosis that depends on the selective activation of JNK1. Here we report that two other members of the IAP family, NAIP and ML-IAP, both activate JNK1. Expression of catalytically inactive JNK1 blocks NAIP and ML-IAP protection against ICE- and TNF-alpha-induced apoptosis, indicating that JNK1 activation is necessary for the antiapoptotic effect of these proteins. The MAP3 kinase, TAK1, appears to be an essential component of this antiapoptotic pathway since IAP-mediated activation of JNK1, as well as protection against TNF-alpha- and ICE-induced apoptosis, is inhibited when catalytically inactive TAK1 is expressed. In addition, XIAP, NAIP, and JNK1 bind to TAK1. Importantly, expression of catalytically inactive TAK1 did not affect XIAP inhibition of caspase activity. These data suggest that XIAP's antiapoptotic activity is achieved by two separate mechanisms: one requiring TAK1-dependent JNK1 activation and the second involving caspase inhibition.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC135597PMC
http://dx.doi.org/10.1128/mcb.22.6.1754-1766.2002DOI Listing
March 2002

MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor.

J Biol Chem 2002 Jan 12;277(3):1845-54. Epub 2001 Nov 12.

Scripps Research Institute, Department of Immunology, La Jolla, California 92037, USA.

The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins, CD14, TLR4, and MD-2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn(26) and Asn(114), whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site, whereas in contrast, TLR4 mutants carrying substitutions in Asn(526) or Asn(575) failed to be transported to the cell surface. Using a UV-activated derivative of Re595 LPS (ASD-Re595 LPS) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 (IL-8) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase (JNK). Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins, CD14, MD-2, and TLR4, and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor.
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http://dx.doi.org/10.1074/jbc.M109910200DOI Listing
January 2002