Cathepsin D is involved in the regulation of transglutaminase 1 and epidermal differentiation.
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We previously demonstrated that the aspartate protease cathepsin D is activated by ceramide derived from acid sphingomyelinase. Increased expression of cathepsin D in the skin has been reported in wound healing, psoriasis and skin tumors. We explored specific functions of cathepsin D during epidermal differentiation. Protein expression and enzymatic activity of cathepsin D increased in differentiated keratinocytes in both stratified organotypic cultures and in mouse skin during epidermal barrier repair. Treatment of cultured keratinocytes with exogenous cathepsin D increased the activity of transglutaminase 1, known to cross-link the cornified envelope proteins involucrin and loricrin during epidermal differentiation. Inhibition of cathepsin D by pepstatin A suppressed the activity of transglutaminase 1. Cathepsin D-deficient mice revealed reduced transglutaminase 1 activity and reduced protein levels of the cornified envelope proteins involucrin and loricrin. Also, amount and distribution of cornified envelope proteins involucrin, loricrin, filaggrin, and of the keratins K1 and K5 were significantly altered in cathepsin D-deficient mice. Stratum corneum morphology in cathepsin D-deficient mice was impaired, with increased numbers of corneocyte layers and faint staining of the cornified envelope only, which is similar to the human skin disease lamellar ichthyosis. Our findings suggest a functional link between cathepsin D activation, transglutaminase 1 activity and protein expression of cornified envelope proteins during epidermal differentiation. (+info)
Anti-endotoxin agents. 1. Development of a fluorescent probe displacement method optimized for the rapid identification of lipopolysaccharide-binding agents.
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Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS, and have shown using animal models of sepsis that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. Assays reported previously in the literature do not lend themselves well to the rapid screening of large numbers of structurally diverse compounds. In this report, we describe a highly sensitive and robust fluorescent displacement assay using BODIPY TR cadaverine (BC), which binds specifically to the toxic center of LPS, lipid A, and is competitively displaced by compounds displaying an affinity for lipid A. The assay clearly discriminates subtle differences in the binding of polymyxin B, and its nonapeptide derivative, with LPS. The spectral properties of the BODIPY fluorophore are ideally suited for screening diverse structural classes of compounds, including those with conjugated aromatic groups, or with chromophores in the 260-500 nm range. The fluorescent probe: LPS complex is stable under physiologically relevant salt concentrations, resulting in the rapid rejection of spurious binders interacting via non-specific electrostatic interactions, and, therefore, in greatly improved dispersion of ED(50)values. (+info)
Rab7 is required for the normal progression of the autophagic pathway in mammalian cells.
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Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called an autophagosome that finally fuses with the lysosome. Rab7 is a member of the Rab family involved in transport to late endosomes and in the biogenesis of the perinuclear lysosome compartment. To assess the role of Rab7 in autophagy we stably transfected CHO cells with wild-type pEGFP-Rab7, and the mutants T22N (GDP form) and Q67L (GTP form). Autophagy was induced by amino acid starvation and the autophagic vacuoles were labeled with monodansylcadaverine. By fluorescence microscopy we observed that Rab7wt and the active mutant Rab7Q67L were associated with ring-shaped vesicles labeled with monodansylcadaverine indicating that these Rab proteins associate with the membrane of autophagic vesicles. As expected, in cells transfected with the negative mutant Rab7T22N the protein was diffusely distributed in the cytosol. However, upon induction of autophagy by amino acid starvation or by rapamycin treatment this mutant clearly decorated the monodansylcadaverine-labeled vesicles. Furthermore, a marked increase in the size of the monodansylcadaverine-labeled vacuoles induced by starvation was observed by overexpression of the inactive mutant T22N. Similarly, there was an increase in the size of vesicles labeled with LC3, a protein that specifically localizes on the autophagosomal membrane. Taken together the results indicate that a functional Rab7 is important for the normal progression of autophagy. (+info)
Protein-tyrosine phosphatase 1B associates with insulin receptor and negatively regulates insulin signaling without receptor internalization.
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Phosphorylated platelet-derived growth factor (PDGF) receptor becomes internalized and then is dephosphorylated by protein-tyrosine phosphatase (PTP) 1B at the endoplasmic reticulum (ER). However, it remains unclear where PTP1B dephosphorylates insulin receptor and inhibits its activity. To clarify how and where PTP1B could interact with insulin receptor, we overexpressed a phosphatase-inactive mutant, PTP1BC/S, in 3T3-L1 adipocytes. Although PDGF receptor was maximally associated with PTP1BC/S at 30 min after PDGF stimulation, the maximal association of insulin receptor with PTP1BC/S was attained at 5 min after insulin stimulation. Furthermore, dansylcadaverine, a blocker of receptor internalization, inhibited this PDGF-induced association of PTP1BC/S with its receptor. However, dansylcadaverine did not affect the insulin-stimulated association of PTP1BC/S with insulin receptor, as well as dephosphorylation of insulin receptor by PTP1B. These results indicate that PTP1B might interact with insulin receptor and deactivate it without internalization. Finally, we overexpressed the wild-type and cytosolic-form of PTP1B to determine the role of ER-anchoring of PTP1B, and found that both inhibited insulin signaling equally. Thus, our data indicate that localization of PTP1B at the ER is not needed for insulin receptor dephosphorylation by PTP1B. (+info)
Interaction of Chlamydia trachomatis serovar L2 with the host autophagic pathway.
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Chlamydiae are obligate intracellular pathogens that replicate within a membrane-bound compartment (the inclusion) and are associated with important human diseases, such as trachoma, pneumonia, and atherosclerosis. We have examined the interaction of the host autophagic pathway with Chlamydia trachomatis serovar L2 by using the specific autophagosomal stain monodansylcadaverine, antibodies to autophagosome-associated markers, and traditionally used autophagic inhibitors, particularly 3-methyladenine and amino acids. Chlamydial inclusions did not sequester monodansylcadaverine, suggesting absence of fusion with autophagosomes. Interestingly, exposure of cultures infected for 19 h to 3-methyladenine or single amino acids until the end of infection (44 h) caused various degrees of abnormalities in the inclusion maturation and in the progeny infectivity. Incubation of host cells with chemicals throughout the entire period of infection modulated the growth of Chlamydia even more dramatically. Remarkably, autophagosomal markers MAP-LC3 and calreticulin were redistributed to the inclusion of Chlamydia, a process that appears to be sensitive to 3-methyladenine and some amino acids. The present data indicate the lack of autophagosomal fusion with the inclusion because it was devoid of monodansylcadaverine and no distinct rim of autophagosomal protein-specific staining around the inclusion could be observed. However, high sensitivity of Chlamydia to conditions that could inhibit host autophagic pathway and the close association of MAP-LC3 and calreticulin with the inclusion membrane still suggest a potential role of host autophagy in the pathogenesis of Chlamydia. (+info)
Nuclear localisation of the G-actin sequestering peptide thymosin beta4.
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Thymosin beta4 is regarded as the main G-actin sequestering peptide in the cytoplasm of mammalian cells. It is also thought to be involved in cellular events like cancerogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. Thymosin beta4 has been previously reported to localise intracellularly to the cytoplasm as detected by immunofluorescence. It can be selectively labelled at two of its glutamine-residues with fluorescent Oregon Green cadaverine using transglutaminase; however, this labelling does not interfere with its interaction with G-actin. Here we show that after microinjection into intact cells, fluorescently labelled thymosin beta4 has a diffuse cytoplasmic and a pronounced nuclear staining. Enzymatic cleavage of fluorescently labelled thymosin beta4 with AsnC-endoproteinase yielded two mono-labelled fragments of the peptide. After microinjection of these fragments, only the larger N-terminal fragment, containing the proposed actin-binding sequence exhibited nuclear localisation, whereas the smaller C-terminal fragment remained confined to the cytoplasm. We further showed that in digitonin permeabilised and extracted cells, fluorescent thymosin beta4 was solely localised within the cytoplasm, whereas it was found concentrated within the cell nuclei after an additional Triton X100 extraction. Therefore, we conclude that thymosin beta4 is specifically translocated into the cell nucleus by an active transport mechanism, requiring an unidentified soluble cytoplasmic factor. Our data furthermore suggest that this peptide may also serve as a G-actin sequestering peptide in the nucleus, although additional nuclear functions cannot be excluded. (+info)
Protective role of tissue transglutaminase in the cell death induced by TNF-alpha in SH-SY5Y neuroblastoma cells.
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Tissue transglutaminase (tTGase) regulates various biological processes, including extracellular matrix organization, cellular differentiation, and apoptosis. Here we report the protective role of tTGase in the cell death that is induced by the tumor necrosis factor alpha (TNF-alpha) and ceramide, a product of the TNF-alpha signaling pathway, in human neuroblastoma SH-SY5Y cells. Treatment with retinoic acid (RA) induced the differentiation of the neuroblastoma cells with the formation of extended neurites. Immunostaining and Western blot analysis showed the tTGase expression by RA treatment. TNF-alpha or C(2) ceramide, a cell permeable ceramide analog, induced cell death in normal cells, but cell death was largely inhibited by the RA treatment. The inhibition of tTGase by the tTGase inhibitors, monodansylcadaverine and cystamine, eliminated the protective role of RA-treatment in the cell death that is caused by TNF-alpha or C(2)-ceramide. In addition, the co-treatment of TNF-alpha and cycloheximide decreased the protein level of tTGase and cell viability in the RA-treated cells, supporting the role of tTGase in the protection of cell death. DNA fragmentation was also induced by the co-treatment of TNF-alpha and cycloheximide. These results suggest that tTGase expressed by RA treatment plays an important role in the protection of cell death caused by TNF-alpha and ceramide. (+info)
Small artery remodeling depends on tissue-type transglutaminase.
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Remodeling of small arteries is essential in the long-term regulation of blood pressure and blood flow to specific organs or tissues. A large part of the change in vessel diameter may occur through non-growth-related reorganization of vessel wall components. The hypothesis was tested that tissue-type transglutaminase (tTG), a cross-linking enzyme, contributes to the inward remodeling of small arteries. The in vivo inward remodeling of rat mesenteric arteries, induced by low blood flow, was attenuated by inhibition of tTG. Rat skeletal muscle arteries expressed tTG, as identified by Western blot and immunostaining. In vitro, activation of these arteries with endothelin-1 resulted in inward remodeling, which was blocked by tTG inhibitors. Small arteries obtained from rats and pigs both showed inward remodeling after exposure to exogenous transglutaminase, which was inhibited by addition of a nitric oxide donor. Enhanced expression of tTG, induced by retinoic acid, increased inward remodeling of porcine coronary arteries kept in organ culture for 3 days. The activity of tTG was dependent on pressure. Inhibition of tTG reversed remodeling, causing a substantial increase in vessel diameter. In a collagen gel contraction assay, tTG determined the compaction of collagen by smooth muscle cells. Collectively, these data show that small artery remodeling associated with chronic vasoconstriction depends on tissue-type transglutaminase. This mechanism may reveal a novel therapeutic target for pathologies associated with inward remodeling of the resistance arteries. (+info)