Jean da Silva Correia

IAP Suppression of Apoptosis Involves Distinct Mechanisms: the TAK1/JNK1 Signaling Cascade and Caspase Inhibition

ABSTRACT 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-α-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-α- 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 XIAPs antiapoptotic activity is achieved by two separate mechanisms: one requiring TAK1-dependent JNK1 activation and the second involving caspase inhibition.

A Synthetic TLR4 Antagonist Has Anti-Inflammatory Effects in Two Murine Models of Inflammatory Bowel Disease

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-α 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.

RSK-B, a Novel Ribosomal S6 Kinase Family Member, Is a CREB Kinase under Dominant Control of p38α Mitogen-activated Protein Kinase (p38αMAPK)

A novel ribosomal S6 kinase (RSK) family member, RSK-B, was identified in a p38αMAPK-baited intracellular interaction screen. RSK-B presents two catalytic domains typical for the RSK family. The protein kinase C-like N-terminal and the calcium/calmodulin kinase-like C-terminal domains both contain conserved ATP-binding and activation consensus sequences. RSK-B is a p38αMAPK substrate, and activated by p38αMAPK and, more weakly, by ERK1. RSK-B phosphorylates the cAMP response element-binding protein (CREB) and c-Fos peptides. In intracellular assays, RSK-B drives cAMP response element- and AP1-dependent reporter expression. RSK-B locates to the cell nucleus and co-translocates p38αMAPK. In conclusion, RSK-B is a novel CREB kinase under dominant p38αMAPKcontrol, also phosphorylating additional substrates.

An Inflammasome-Independent Role for Epithelial-Expressed Nlrp3 in Renal Ischemia-Reperfusion Injury

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.

MDP‐induced interleukin‐1β processing requires Nod2 and CIAS1/NALP3

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‐1β, 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‐1β 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‐1β production and allow for further studies of the role of these proteins within the broader context of inflammatory disease.

SGT1 is essential for Nod1 activation

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.

Cleavage of p65/RelA of the NF-κB pathway by Chlamydia

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-κB pathway of host inflammatory response. We found that Chlamydia infection did not promote IκBα degradation, a prerequisite for NF-κB 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-κB 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-κB 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.

Nod1 and Nod2 Are Expressed in Human and Murine Renal Tubular Epithelial Cells and Participate in Renal Ischemia Reperfusion Injury

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 × 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.

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

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.

Ebola Virion Attachment and Entry into Human Macrophages Profoundly Effects Early Cellular Gene Expression

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 (GP1,2) 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 GP1,2 (VLPVP40-GP) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLPVP40 (particles lacking GP1,2) caused an aberrant response. This suggests that GP1,2 binding to macrophages plays an important role in the immediate cellular response.