INCA, a novel human caspase recruitment domain protein that inhibits interleukin-1beta generation

Using in silico methods for screening the human genome for new caspase recruitment domain (CARD) proteins, we have identified INCA (Inhibitory CARD) as a protein that shares 81% identity with the prodomain of caspase-1. The INCA gene is located on chromosome 11q22 between the genes of COP/Pseudo-ICE and ICEBERG, two other CARD proteins that arose from caspase-1 gene duplications. We show that INCA mRNA is expressed in many tissues. INCA is specifically upregulated by interferon-gamma in the monocytic cell lines THP-1 and U937. INCA physically interacts with procaspase-1 and blocks the release of mature IL-1beta from LPS-stimulated macrophages. Unlike COP/Pseudo-ICE and procaspase-1, INCA does not interact with RIP2 and does not induce NF-kappaB activation. Our data show that INCA is a novel intracellular regulator of procaspase-1 activation, involved in the regulation of pro-IL-1beta processing and its release during inflammation.     

Brucella abortus catalase is a periplasmic protein lacking a standard signal sequence.

A periplasmic catalase has been purified and cloned from Brucella abortus. The functional enzyme is a tetramer with a subunit molecular weight of 55,000. All evidence indicates that a typical N-terminal signal sequence is not associated with the export of this protein to the periplasm.     

The estrogen-responsive B box protein: a novel enhancer of interleukin-1beta secretion

The estrogen-responsive B box protein (EBBP) and Pyrin belong to a family of structurally related proteins. While mutations in the pyrin gene cause an autoinflammatory disease, the biological function of EBBP is unknown. In this study, we identified the proinflammatory cytokine interleukin-1beta (IL-1beta) as an EBBP-binding partner. Furthermore, caspase-1 and NACHT, LRR and Pyrin domain containing protein (NALP) 1, two components of the recently identified inflammasome, a platform for the activation of caspase-1, also interact with EBBP. These proteins bind to the RFP domain of EBBP, suggesting that this domain of so far unknown function is an important protein-binding domain. EBBP was secreted in a caspase-1-dependent manner from cultured cells, and its secretion was enhanced by IL-1beta. Vice versa, endogenous and overerexpressed EBBP increased IL-1beta secretion. These results provide evidence for a role of EBBP in innate immunity by enhancing the alternative secretion pathway of IL-1beta.     

Partial amino acid sequence of human pancreatic stone protein, a novel pancreatic secretory protein.

Pancreatic stone protein (PSP) is the major organic component of human pancreatic stones. With the use of monoclonal antibody immunoadsorbents, five immunoreactive forms (PSP-S) with close Mr values (14,000-19,000) were isolated from normal pancreatic juice. By CM-Trisacryl M chromatography the lowest-Mr form (PSP-S1) was separated from the others and some of its molecular characteristics were investigated. The Mr of the PSP-S1 polypeptide chain calculated from the amino acid composition was about 16,100. The N-terminal sequences (40 residues) of PSP and PSP-S1 are identical, which suggests that the peptide backbone is the same for both of these polypeptides. The PSP-S1 sequence was determined up to residue 65 and was found to be different from all other known protein sequences.     

Inefficient function of the signal sequence of PTHrP for targeting into the secretory pathway

Parathyroid hormone-related peptide (PTHrP) is not only secreted out of cells, but also targeted to the nucleoli due to a nucleolar targeting signal (NTS). We assessed the molecular mechanism underlying the dual targeting of PTHrP by constructing a series of truncated forms of rat PTHrP cDNA and expressing them in CHO cells. Immunostaining was observed in both the Golgi apparatus and nucleoli in the same cell expressing PTHrP with the N-terminal full-length signal sequence. When PTHrP molecules were translated from CUGs downstream of the AUG-initiator codon in the signal sequences, potential alternative initiators of the translation, they were exclusively localized in the nucleoli. In contrast, when a construct containing only the ATG-initiator codon was expressed, PTHrP was found to localize in both the nucleolus and the Golgi apparatus. No nucleolar staining of PTHrP was observed in the CHO cells transfected with PTH/PTHrP receptors even after incubating with a conditioned medium containing PTHrP, ruling out a possibility that PTHrP is, once secreted, internalized via receptor-mediated endocytosis and subsequently conveyed to nucleoli. Compatible with these morphological observations, a preproform of PTHrP was found in the cells expressing PTHrP in addition to proPTHrP, indicative of molecules along the secretory pathway. These results strongly indicate that the signal sequence of PTHrP is not sufficient to direct all the newly synthesized molecules across the endoplasmic reticulum, resulting in part of it being delivered to the nucleoli due to the NTS.     

ICEBERG: a novel inhibitor of interleukin-1beta generation

ProIL-1beta is a proinflammatory cytokine that is proteolytically processed to its active form by caspase-1. Upon receipt of a proinflammatory stimulus, an upstream adaptor, RIP2, binds and oligomerizes caspase-1 zymogen, promoting its autoactivation. ICEBERG is a novel protein that inhibits generation of IL-1beta by interacting with caspase-1 and preventing its association with RIP2. ICEBERG is induced by proinflammatory stimuli, suggesting that it may be part of a negative feedback loop. Consistent with this, enforced retroviral expression of ICEBERG inhibits lipopolysaccharide-induced IL-1beta generation. The structure of ICEBERG reveals it to be a member of the death-domain-fold superfamily. The distribution of surface charge is complementary to the homologous prodomain of caspase-1, suggesting that charge-charge interactions mediate binding of ICEBERG to the prodomain of caspase-1.     

Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex.

Basic fibroblast growth factor (bFGF) modulates functions of a variety of cell types. Whereas bFGF is known to act extracellularly, the protein lacks a transient signal peptide. No defined mechanism for bFGF secretion has been characterized besides release from dead or injured cells. To study this problem we devised an experimental system to examine bFGF-mediated migration of isolated single cells. Under these conditions individual cells are not affected by bFGF derived from other cells. By this method we have previously shown that bFGF released by NIH 3T3 cells transfected with bFGF cDNA modulates migration in an autocrine manner. We have now examined the effects on cell motility of drugs or treatments known to affect various pathways of protein secretion. Drugs that block secretion via the endoplasmic reticulum (ER)-Golgi complex or via multidrug resistance proteins did not inhibit cell motility. Migration was enhanced by the calcium ionophore A23187, which stimulates exocytosis, and was inhibited by methylamine, serum-free, and low temperature (18 degrees C) conditions, which block endo- and exocytosis. The reversal of these effects by the concomitant addition of affinity-purified anti-bFGF IgG or recombinant bFGF showed that the alterations in cell migration were mediated by changes in bFGF externalization. Thus bFGF can be released via a mechanism of exocytosis independent of the ER-Golgi pathway.     

The trafficking of alpha 1-antitrypsin,a post-Golgi secretory pathway marker,in INS-1 pancreatic beta cells

A sulfated alpha1-antitrypsin (AAT), thought to be a default secretory pathway marker, is not stored in secretory granules when expressed in neuroendocrine PC12 cells. In search of a constitutive secretory pathway marker for pancreatic beta cells, we produced INS-1 cells stably expressing wild-type AAT. Because newly synthesized AAT arrives very rapidly in the Golgi complex, kinetics alone cannot resolve AAT release via distinct secretory pathways, although most AAT is secreted within a few hours and virtually none is stored in mature granules. Nevertheless, from pulse-chase analyses, a major fraction of newly synthesized AAT transiently exhibits secretogogue-stimulated exocytosis and localizes within immature secretory granules (ISGs). This trafficking occurs without detectable AAT polymerization or binding to lipid rafts. Remarkably, in a manner not requiring its glycans, all of the newly synthesized AAT is then removed from granules during their maturation, leading mostly to constitutive-like AAT secretion, whereas a smaller fraction (approximately 10%) goes on to lysosomes. Secretogogue-stimulated ISG exocytosis reroutes newly synthesized AAT directly into the medium and prevents its arrival in lysosomes. These data are most consistent with the idea that soluble AAT abundantly enters ISGs and then is efficiently relocated to the endosomal system, from which many molecules undergo constitutive-like secretion while a smaller fraction advances to lysosomes.     

Avl9p, a member of a novel protein superfamily, functions in the late secretory pathway

The branching of exocytic transport routes in both yeast and mammalian cells has complicated studies of the late secretory pathway, and the mechanisms involved in exocytic cargo sorting and exit from the Golgi and endosomes are not well understood. Because cargo can be sorted away from a blocked route and secreted by an alternate route, mutants defective in only one route do not exhibit a strong secretory phenotype and are therefore difficult to isolate. In a genetic screen designed to isolate such mutants, we identified a novel conserved protein, Avl9p, the absence of which conferred lethality in a vps1Delta apl2Delta strain background (lacking a dynamin and an adaptor-protein complex 1 subunit). Depletion of Avl9p in this strain resulted in secretory defects as well as accumulation of Golgi-like membranes. The triple mutant also had a depolarized actin cytoskeleton and defects in polarized secretion. Overexpression of Avl9p in wild-type cells resulted in vesicle accumulation and a post-Golgi defect in secretion. Phylogenetic analysis indicated evolutionary relationships between Avl9p and regulators of membrane traffic and actin function.     

A novel protein kinase A-independent, beta-arrestin-1-dependent signaling pathway for p38 mitogen-activated protein kinase activation by beta2-adrenergic receptors

A growing body of evidence has demonstrated that p38 mitogen-activated protein kinase (MAPK) has a crucial role in various physiological and pathological processes mediated by beta(2)-adrenergic receptors (beta(2)-ARs). However, the detailed mechanism of beta(2)-ARs-induced p38 MAPK activation has not yet been fully defined. The present study demonstrates a novel kinetic model of p38 MAPK activation induced by beta(2)-ARs in human embryonic kidney 293A cells. The beta(2)-AR agonist isoproterenol induced a time-dependent biphasic phosphorylation of p38 MAPK: the early phase peaked at 10 min, and was followed by a delayed phase that appeared at 90 min and was sustained for 6 h. Interestingly, inhibition of the cAMP/protein kinase A (PKA) pathway failed to affect the early phosphorylation but abolished the delayed activation. By contrast, silencing of beta-arrestin-1 expression by small interfering RNA inhibited the early phase activation of p38 MAPK. Furthermore, the NADPH oxidase complex is a downstream target of beta-arrestin-1, as evidenced by the fact that isoproterenol-induced Rac1 activation was also suppressed by beta-arrestin-1 knockdown. In addition, early phase activation of p38 MAPK was prevented by inactivation of Rac1 and NADPH oxidase by pharmacological inhibitors, overexpression of a dominant negative mutant of Rac1, and p47(phox) knockdown by RNA interference. Of note, we demonstrated that only early activation of p38 MAPK is involved in isoproterenol-induced F-actin rearrangement. Collectively, these data suggest that the classic cAMP/PKA pathway is responsible for the delayed activation, whereas a beta-arrestin-1/Rac1/NADPH oxidase-dependent signaling is a heretofore unrecognized mechanism for beta(2)-AR-mediated early activation of p38 MAPK.     

Nucleotide sequence of ovine macrophage interleukin-1 beta cDNA.

We have cloned and sequenced the cDNA for the coding region of ovine alveolar macrophage interleukin-1 beta. At the nucleotide level, the ovine cDNA shares 95, 74 and 71% homology with the bovine, human and murine cDNA equivalents or homologs. Comparison at the amino acid level revealed 95% homology with bovine IL-1 beta and approximately 57% with the human and murine homologs.     

ESDN, a novel neuropilin-like membrane protein cloned from vascular cells with the longest secretory signal sequence among eukaryotes, is up-regulated after vascular injury.

A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins.