Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana

In the past, ultrastructural investigations of Leishmania mexicana amastigotes revealed structures that were tentatively identified as autophagosomes. This study has now provided definitive data that autophagy occurs in the parasite during differentiation both to metacyclic promastigotes and to amastigotes, autophagosomes being particularly numerous during metacyclic to amastigote form transformation. Moreover, the results demonstrate that inhibiting two major lysosomal cysteine peptidases (CPA and CPB) or removing their genes not only interferes with the autophagy pathway but also prevents metacyclogenesis and transformation to amastigotes, thus adding support to the hypothesis that autophagy is required for cell differentiation. The study suggests that L. mexicana CPA and CPB perform similar roles to the aspartic peptidase PEP4 and the serine peptidase PRB1 in Saccharomyces cerevisiae. The results also provide an explanation for why L. mexicana CPA/CPB-deficient mutants transform to amastigotes very poorly and lack virulence in macrophages and mice.     

Cysteine protease B of Leishmania mexicana inhibits host Th1 responses and protective immunity

C3H mice infected with Leishmania mexicana fail to develop a protective Th1 response, and are unable to cure. In this study, we show that L. mexicana cysteine proteases suppress the antileishmanial immune response. Previous studies demonstrated that deletion of the entire multicopy cysteine protease B (CPB) gene array in L. mexicana is associated with decreased parasite virulence, potentially attributable to factors related to parasite fitness rather than to direct effects on the host immune response. We now show that C3H mice infected with the L. mexicana deletion mutant (Deltacpb) initially develop lesions that grow at rates comparable to those of wild-type L. mexicana-infected mice. However, in contrast to controls, Deltacpb-induced lesions heal with an accompanying Th1 immune response. Lesion resolution was Th1 dependent, as Deltacpb-infected IL-12p40(-/-) and STAT4(-/-) mice developed high parasite burdens and progressive disease. Moreover, when L. major was transfected with a cosmid expressing multiple L. mexicana CPB genes, this parasite induced a significantly lower IFN-gamma response compared with wild-type L. major. These data indicate that cysteine proteases of L. mexicana are critical in suppressing protective immune responses and that inhibition of CPB may prove to be a valuable immunomodulatory strategy for chronic forms of leishmaniasis.     

NF-kappaB-inducing kinase restores defective IkappaB kinase activity and NF-kappaB signaling in intestinal epithelial cells

Cytokine-stimulated IkappaBalpha degradation is impaired in HT-29 and primary intestinal epithelial cells. To gain more insight into the mechanism of this defect, we dissected cytokine-induced NF-kappaB signaling pathway in HT-29 cells. IL-1beta and TNF, alone or in combination with IFNgamma, failed to induce IkappaBalpha or IkappaBbeta degradation in HT-29 cells. Despite similar 125I-IL-1beta binding, HT-29 cells displayed no IRAK degradation, a 75% reduction of IKK activity, and decreased IkappaBalpha phosphorylation, NF-kappaB DNA binding activity and IL-8 mRNA accumulation in response to IL-1beta compared to Caco-2 cells. Selective activation of NF-kappaB pathway by adenoviral delivery of NF-kappaB-inducing kinase (Ad5NIK) or IKKbeta (Ad5IKKbeta) strongly activated IKK activity (>20 fold) in HT-29 cells with concomitant endogenous IkappaBalpha serine 32 phosphorylation and total IkappaBalpha degradation. In addition, NF-kappaB DNA binding activity and IL-8 secretion is higher in Ad5NIK-infected than in IL-1beta-stimulated HT-29 cells. These data show that altered NF-kappaB signaling is associated with impaired stimulation of an upstream IKK activator.     

Immunosuppressant FK506 activates NF-kappaB through the proteasome-mediated degradation of IkappaBalpha. Requirement for Ikappabalpha n-terminal phosphorylation but not ubiquitination sites

The immunosuppressant FK506 activates NF-kappaB through IkappaBalpha degradation in nonlymphoid cells. In the present study, we analyzed mechanisms by which FK506 induces IkappaBalpha degradation. We found that FK506 induces the degradation of both IkappaBalpha and IkappaBbeta and that the time courses of the FK506-induced degradation are quite different from degradation induced by interleukin 1 (IL-1). Despite this difference, FK506-induced IkappaBalpha degradation was dependent on the N-terminal Ser-32 and Ser-36 phosphorylation sites and was mediated by proteasomes, as is the case for IL-1-induced IkappaBalpha degradation. We further showed that FK506 induces weak and slow phosphorylation of IkappaBalpha at Ser-32. However, unlike IL-1-induced degradation, IKK-1 and IKK-2 were not activated significantly nor was FK506-induced IkappaBalpha degradation dependent on the N-terminal ubiquitination sites (Lys-21 and Lys-22). These results therefore indicate that FK506 and IL-1 utilize similar but distinct mechanisms to induce the phosphorylation and degradation of IkappaBalpha.     

beta-TrCP mediates the signal-induced ubiquitination of IkappaBbeta.

We have examined the role of beta-TrCP (beta-transducin repeat-containing protein) in the ubiquitination and degradation of IkappaBbeta, one of the two major IkappaB isoforms in mammalian cells. We demonstrate that beta-TrCP interacts specifically with IkappaBbeta, and such interaction is dependent on prior phosphorylation of IkappaBbeta on serines 19 and 23. Interaction with beta-TrCP is also necessary for ubiquitination of IkappaBbeta upon stimulation of cells, and deletion of the F-box in beta-TrCP abolishes its ability to ubiquitinate IkappaBbeta. Therefore, these results indicate that beta-TrCP plays a critical role in the activation of NF-kappaB by assembling the ubiquitin ligase complex for both phosphorylated IkappaBalpha and IkappaBbeta.     

IkappaBbeta, but not IkappaBalpha, functions as a classical cytoplasmic inhibitor of NF-kappaB dimers by masking both NF-kappaB nuclear localization sequences in resting cells

NF-kappaB dimers, inhibitor IkappaB proteins, and NF-kappaB.IkappaB complexes exhibit distinct patterns in partitioning between nuclear and cytoplasmic cellular compartments. IkappaB-dependent modulation of NF-kappaB subcellular localization represents one of the more poorly understood processes in the NF-kappaB signaling pathway. In this study, we have combined in vitro biochemical and cell-based methods to elucidate differences in NF-kappaB regulation exhibited by the inhibitors IkappaBbeta and IkappaBalpha. We show that although both IkappaBalpha and IkappaBbeta bind to NF-kappaB with similar global architecture and stability, significant differences exist that contribute to their unique functional roles. IkappaBbeta derives its high affinity toward NF-kappaB dimers by binding to both NF-kappaB subunit nuclear localization signals. In contrast, IkappaBalpha contacts only one NF-kappaB NLS and employs its carboxyl-terminal proline, glutamic acid, serine, and threonine-rich region for high affinity NF-kappaB binding. We show that the presence of one free NLS in the NF-kappaB.IkappaBalpha complex renders it a dynamic nucleocytoplasmic complex, whereas NF-kappaB.IkappaBbeta complexes are localized to the cytoplasm of resting cells.     

RelA/p65 regulation of IkappaBbeta

IkappaB inhibitor proteins are the primary regulators of NF-kappaB. In contrast to the defined regulatory interplay between NF-kappaB and IkappaBalpha, much less is known regarding the regulation of IkappaBbeta by NF-kappaB. Here, we describe in detail the regulation of IkappaBbeta by RelA/p65. Using p65(-/-) fibroblasts, we show that IkappaBbeta is profoundly reduced in these cells, but not in other NF-kappaB subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65(-/-) cells and tissues, IkappaBalpha is also reduced, but not nearly to the same extent as IkappaBbeta, thus highlighting the degree to which IkappaBbeta is dependent on p65. This dependence is based on the ability of p65 to stabilize IkappaBbeta protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IkappaBbeta was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65(-/-) fibroblasts, expression of a proteolysis-resistant form of IkappaBbeta, but not IkappaBalpha, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IkappaBbeta protein by p65 is necessary for the maintenance of cellular homeostasis.     

Distinct roles of IkappaB proteins in regulating constitutive NF-kappaB activity

The inhibitor of NF-kappaB (IkappaB) family of proteins is believed to regulate NF-kappaB activity by cytoplasmic sequestration. We show that in cells depleted of IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins, a small fraction of p65 binds DNA and leads to constitutive activation of NF-kappaB target genes, even without stimulation, whereas most of the p65 remains cytoplasmic. These results indicate that although IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins could be dispensable for cytoplasmic retention of NF-kappaB, they are essential for preventing NF-kappaB-dependent gene expression in the basal state. We also show that in the absence of IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins, cytoplasmic retention of NF-kappaB by other cellular proteins renders the pathway unresponsive to activation.     

Elafin prevents lipopolysaccharide-induced AP-1 and NF-kappaB activation via an effect on the ubiquitin-proteasome pathway

The serine anti-protease elafin is expressed by monocytes, alveolar macrophages, neutrophils, and at mucosal surfaces and possesses antimicrobial activity. It is also known to reduce lipopolysaccharide-induced neutrophil influx into murine alveoli as well as to abrogate lipopolysaccharide-induced production of matrix metalloprotease 9, macrophage inhibitory protein 2, and tumor necrosis factor-alpha by as-yet unidentified mechanisms. In this report we have shown that elafin inhibits the lipopolysaccharide-induced production of monocyte chemoattractant protein-1 in monocytes by inhibiting AP-1 and NF-kappaB activation. Elafin prevented lipopolysaccharide-induced phosphorylation of AP-1, c-Jun, and JNK but had no effect on phosphorylation of p38. The lipopolysaccharide-induced degradation of IL-1R-associated kinase 1, IkappaBalpha, and IkappaBbeta was inhibited by elafin but phosphorylation of IkappaBalpha was unaffected. Polyubiquitinated protein including polyubiquitinated IkappaBalpha was shown to accumulate in the presence of elafin. These results suggest that inhibition by elafin of lipopolysaccharide-induced AP-1 and NF-kappaB activation occurs via an effect on the ubiquitin-proteasome pathway.     

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.