A strategy for the production of soluble human senescence marker protein-30 in Escherichia coli.

Senescence marker protein-30 (SMP30) has been reported to hydrolyze diisopropyl fluorophosphate (DFP), a surrogate compound of chemical warfare nerve agents. Thus, SMP30 has the potential to be useful as a prophylactic against chemical warfare nerve agent toxicity. Our efforts to generate human SMP30 in bacteria using a variety of expression vectors invariably resulted in insoluble and inactive preparations. In this study, properly folded and active recombinant human SMP30 (rHuSMP30) was produced in Escherichia coli by coexpressing it with molecular chaperones in a combined strategy. The coexpression of rHuSMP30 with GroES/GroEL/Tf at 15 degrees C, combined with the addition of a membrane fluidizer, increased osmolytes, and a two-step expression resulted in the highest enhancement of solubility and DFPase activity. Our results pave the way for exploring the use of rHuSMP30 against organophosphate and nerve agent toxicity.

Protective effects of recombinant kunitz-domain 1 of human tissue factor pathway inhibitor-2 against 2-chloroethyl ethyl sulfide toxicity in vitro.

OBJECTIVE: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P(1) residue (arginine at position 24) responsible for protease inhibitory activity. METHODS: Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay. RESULTS: Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain1 was stable for 4 weeks at 42 degrees C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide-treated cells but to a much lesser degree. CONCLUSION: These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

Production of lysophosphatidylcholine by cPLA2 in the brain of mice lacking PPT1 is a signal for phagocyte infiltration.

In the majority of neurodegenerative storage disorders, neuronal death in the brain is followed by infiltration of phagocytic cells (e.g. activated microglia, astroglia and macrophages) for the efficient removal of cell corpses. However, it is increasingly evident that these phagocytes may also cause death of adjoining viable neurons contributing to rapid progression of neurodegeneration. Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating, neurodegenerative, lysosomal storage disorder caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1 catalyzes the cleavage of thioester linkages in S-acylated (palmitoylated) proteins and its deficiency leads to abnormal accumulation of thioesterified polypeptides (ceroid) in lysosomes causing INCL pathogenesis. PPT1-knockout (PPT1-KO) mice mimic the clinical and pathological features of human INCL including rapid neuronal death by apoptosis and phagocyte infiltration. We previously reported that in PPT1-KO mice, the neurons undergo endoplasmic reticulum stress activating unfolded protein response, which mediates caspase-12 activation and apoptosis. However, the molecular mechanism(s) by which the phagocytic cells are recruited in the PPT1-KO mouse brain remains poorly understood. We report here that increased production of lysophosphatidylcholine (LPC), catalyzed by the activation of cytosolic phospholipase A(2) (cPLA(2)) in the PPT1-KO mouse brain, is a 'lipid signal' for phagocyte recruitment. We also report that an age-dependent increase in LPC levels in the PPT1-KO mouse brain positively correlates with elevated expression of the genes characteristically associated with phagocytes. We propose that increased cPLA(2)-catalyzed LPC production in the brain is at least one of the mechanisms that mediate phagocyte infiltration contributing to INCL neuropathology.

Protective effects of recombinant kunitz-domain 1 of human tissue factor pathway inhibitor-2 against 2-chloroethyl ethyl sulfide toxicity in vitro.

OBJECTIVE: Sulfur mustard is a well-known blistering chemical warfare agent that has been investigated for its toxicological mechanisms and an efficacious antidote. Since sulfur mustard injury involves dermal:epidermal separation, proteolytic enzymes were suspected to be involved for this separation and eventual blister development. Therefore, protease inhibitors could be of therapeutic utility against sulfur mustard injury. In this study, the effects of Kunitz-domain 1 of human tissue factor pathway inhibitor-2 were evaluated against the toxic effects of 2-chloroethyl ethyl sulfide, a surrogate agent of sulfur mustard. Tissue factor pathway inhibitor-2 is a 32-kDa serine protease inhibitor produced by a variety of cell types including human epidermal keratinocytes, fibroblasts, and endothelial cells. It consists of 3 Kunitz-domains and the first Kunitz-domain contains the putative P(1) residue (arginine at position 24) responsible for protease inhibitory activity. METHODS: Recombinant wild-type and R24Q mutant Kunitz-domain 1s were expressed in Escherichia coli and purified. The purified proteins were refolded, and their effects were tested in an in vitro human epidermal keratinocyte cell wounding assay. RESULTS: Wild-type but not R24Q Kunitz-domain 1 inhibited the amidolytic activity of trypsin and plasmin. Wild-type Kunitz-domain 1 was stable for 4 weeks at 42 degrees C and for more than 8 weeks at room temperature. Wild-type Kunitz-domain 1 significantly improved wound healing of unexposed and 2-chloroethyl ethyl sulfide-exposed cells without influencing cell proliferation. Although R24Q Kunitz-domain 1 lacked trypsin and plasmin inhibitory activity, it promoted wound closure of untreated and 2-chloroethyl ethyl sulfide-treated cells but to a much lesser degree. CONCLUSION: These data suggest that wild-type Kunitz-domain 1 of human tissue factor pathway inhibitor-2 can be developed as a medical countermeasure against sulfur mustard cutaneous injury.

Palmitoyl-protein thioesterase-1 deficiency mediates the activation of the unfolded protein response and neuronal apoptosis in INCL.

Numerous proteins undergo modification by palmitic acid (S-acylation) for their biological functions including signal transduction, vesicular transport and maintenance of cellular architecture. Although palmitoylation is an essential modification, these proteins must also undergo depalmitoylation for their degradation by lysosomal proteases. Palmitoyl-protein thioesterase-1 (PPT1), a lysosomal enzyme, cleaves thioester linkages in S-acylated proteins and removes palmitate residues facilitating the degradation of these proteins. Thus, inactivating mutations in the PPT1 gene cause infantile neuronal ceroid lipofuscinosis (INCL), a devastating neurodegenerative storage disorder of childhood. Although rapidly progressing brain atrophy is the most dramatic pathological manifestation of INCL, the molecular mechanism(s) remains unclear. Using PPT1-knockout (PPT1-KO) mice that mimic human INCL, we report here that the endoplasmic reticulum (ER) in the brain cells of these mice is structurally abnormal. Further, we demonstrate that the level of growth-associated protein-43 (GAP-43), a palmitoylated neuronal protein, is elevated in the brains of PPT1-KO mice. Moreover, forced expression of GAP-43 in PPT1-deficient cells results in the abnormal accumulation of this protein in the ER. Consistent with these results, we found evidence for the activation of unfolded protein response (UPR) marked by elevated levels of phosphorylated translation initiation factor, eIF2alpha, increased expression of chaperone proteins such as glucose-regulated protein-78 and activation of caspase-12, a cysteine proteinase in the ER, mediating caspase-3 activation and apoptosis. Our results, for the first time, link PPT1 deficiency with the activation of UPR, apoptosis and neurodegeneration in INCL and identify potential targets for therapeutic intervention in this uniformly fatal disease.

Uteroglobin represses allergen-induced inflammatory response by blocking PGD2 receptor-mediated functions.

Uteroglobin (UG) is an antiinflammatory protein secreted by the epithelial lining of all organs communicating with the external environment. We reported previously that UG-knockout mice manifest exaggerated inflammatory response to allergen, characterized by increased eotaxin and Th2 cytokine gene expression, and eosinophil infiltration in the lungs. In this study, we uncovered that the airway epithelia of these mice also express high levels of cyclooxygenase (COX)-2, a key enzyme for the production of proinflammatory lipid mediators, and the bronchoalveolar lavage fluid (BALF) contain elevated levels of prostaglandin D2. These effects are abrogated by recombinant UG treatment. Although it has been reported that prostaglandin D2 mediates allergic inflammation via its receptor, DP, neither the molecular mechanism(s) of DP signaling nor the mechanism by which UG suppresses DP-mediated inflammatory response are clearly understood. Here we report that DP signaling is mediated via p38 mitogen-activated protein kinase, p44/42 mitogen-activated protein kinase, and protein kinase C pathways in a cell type-specific manner leading to nuclear factor-kappaB activation stimulating COX-2 gene expression. Further, we found that recombinant UG blocks DP-mediated nuclear factor-kappaB activation and suppresses COX-2 gene expression. We propose that UG is an essential component of a novel innate homeostatic mechanism in the mammalian airways to repress allergen-induced inflammatory responses.

IFN-gamma stimulates the expression of a novel secretoglobin that regulates chemotactic cell migration and invasion.

IFNs are a family of cytokines that alert the immune system against viral infections of host cells. The IFNs (IFN-alpha, IFN-beta, and IFN-gamma) interact with specific cellular receptors and stimulate the production of second messengers, leading to the expression of antiviral and immunomodulatory proteins. We report in this study that IFN-gamma stimulates the expression of a novel gene that encodes a protein with 30% amino acid sequence identity with uteroglobin, the founding member of the newly formed Secretoglobin (SCGB) superfamily. We named this protein IFN-gamma-inducible SCGB (IIS), because its expression in lymphoblast cells is augmented by IFN-gamma treatment. IIS is expressed in virtually all tissues, and the highest level of expression is detectable in lymph nodes, tonsil, cultured lymphoblasts, and the ovary. Interestingly, although the expression of IIS mRNA is not significantly different in resting lymphoid cells, it is markedly elevated in activated CD8(+) and CD19(+) cells. Furthermore, treatment of lymphoblast cells with IIS antisense phosphorothioate (S)-oligonucleotides prevents chemotactic migration and invasion. Taken together, these results raise the possibility that this novel SCGB has immunological functions.

Neutral ceramidase gene: role in regulating ceramide-induced apoptosis.

The sphingolipid, ceramide, is a natural dietary constituent and a potent mediator of apoptosis. If left undegraded, it may induce apoptosis and cause disruption of cellular integrity. A potential mechanism to prevent ceramide-induced apoptosis in various organs may involve ceramidases that facilitate the degradation of ceramide. In this study, we first isolated and characterized the murine neutral ceramidase (N-CDase) gene, mapped its chromosomal location and determined its developmental and organ-specific expression. Then we used cultured mesangial cells as our in vitro model and mouse gastrointestinal (GI) tract as the in vivo model to determine the effects of an inhibitor of N-CDase, D-erythro-MAPP, to delineate whether N-CDase plays a role in preventing ceramide-induced apoptosis. Our results show that: (i) the structure of the murine neutral ceramidase gene is virtually identical to that of the human gene; (ii) it is localized on chromosome 19 at bands C2-C3 that is syntenic to human chromosome 10q24-26; (iii) N-CDase expression is developmentally regulated and it is expressed at high levels in cultured mesangial cells and in specific regions of the mouse small intestine; (iv) inhibition of N-CDase by D-erythro-MAPP leads to increased ceramide levels and consequent apoptosis in cultured mesangial cells; (v) mice treated with D-erythro-MAPP alone also caused apoptosis in the small intestine; and (vi) mice treated with D-erythro-MAPP prior to feeding C2 ceramide manifest markedly elevated levels of apoptosis in the GI tract raising the possibility that neutral ceramidase plays a detoxifying role against inadvertent stimulation of ceramide-induced apoptosis in organs that come in contact with this sphingolipid. We propose that N-CDase is an essential component of an innate detoxifying mechanism to prevent ceramide-induced apoptosis.