Caspase-mediated cleavage of p130cas in etoposide-induced apoptotic Rat-1 cells

Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell-cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD(416)G and DSPD(748)G) in vitro, and point mutations substituting the Asp of DVPD(416)G and DSPD(748)G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD(416)G generated a 74-kDa fragment, which was in turn cleaved at DSPD(748)G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas-paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.     

Resistance to diet-induced hypercholesterolemia and gallstone formation in ACAT2-deficient mice

The importance of cholesterol ester synthesis by acyl CoA:cholesterol acyltransferase (ACAT) enzymes in intestinal and hepatic cholesterol metabolism has been unclear. We now demonstrate that ACAT2 is the major ACAT in mouse small intestine and liver, and suggest that ACAT2 deficiency has profound effects on cholesterol metabolism in mice fed a cholesterol-rich diet, including complete resistance to diet-induced hypercholesterolemia and cholesterol gallstone formation. The underlying mechanism involves the lack of cholesterol ester synthesis in the intestine and a resultant reduced capacity to absorb cholesterol. Our results indicate that ACAT2 has an important role in the response to dietary cholesterol, and suggest that ACAT2 inhibition may be a useful strategy for treating hypercholesterolemia or cholesterol gallstones.     

Identification of a fmtA-like gene that has similarity to other PBPs and beta-lactamases in Staphylococcus aureus

We identified a gene from Staphylococcus aureus, flp (fmtA-like protein), encoding a protein of 489 amino acid residues with a molecular mass of 56.4 kDa. The deduced amino acid sequence shows similarity to previously characterized penicillin binding proteins (PBPs) and FmtA of S. aureus (one of the factors which affect methicillin resistance). FLP protein has three motifs, which are conserved in PBPs and beta-lactamases, suggesting that it might be associated with cell wall synthesis. Recombinant FLP protein, however, lacks penicillin binding activity, and the inactivation of flp in two methicillin-resistant S. aureus strains did not cause a reduction in the methicillin resistance.     

Cytokines secreted by lymphokine-activated killer cells induce endogenous nitric oxide synthesis and apoptosis in DLD-1 colon cancer cells

IL-2-activated killer lymphocytes (LAK cells) secrete inflammatory cytokines such as interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNFalpha) that can induce nitric oxide (NO) synthesis. We evaluated whether LAK cells could activate NO synthesis in human cancer cells. LAK cells and their culture supernatants induced NO synthesis in DLD-1 colon cancer cells in a dose-dependent manner. NO synthesis was inhibited completely by blocking antibodies to IFN-gamma, demonstrating a key role for this LAK cell cytokine in regulating NO synthesis. The addition of TNFalpha antibodies resulted in partial inhibition. Induction of iNOS mRNA and protein expression in DLD-1 cells was detected. Endogenous NO production inhibited DLD-1 cell proliferation and induced apoptosis, processes that were inhibitable by the NO synthase inhibitor N(G)-monomethyl-l-arginine. Our study has identified a novel, non-contact-dependent LAK cell cytotoxic mechanism: induction of growth inhibition and programmed cell death due to endogenous NO synthesis in susceptible human cancer cells.     

Transforming growth factor-beta induces apoptosis in activated murine T cells through the activation of caspase 1-like protease

Transforming growth factor-beta (TGF-beta) has been known as a potent immunosuppressive cytokine that can induce apoptosis in lymphoid cells. We established an IL-2-independent cell line, CTLL-2A, from murine T cell line CTLL-2. CTLL-2A expressed higher levels of CD95, CD69, and CD18 molecules than CTLL-2 did, suggesting a more activated state in CTLL-2A than in the CTLL-2 by phenotype. Exposing both CTLL-2 and CTLL-2A to TGF-beta results in differential apoptosis patterns defined by DNA fragmentation and plasma membrane alteration. Among the bcl-2 family members, bcl-2, bcl-w, and bcl-x(L) were also differently expressed in these two cell lines. In CTLL-2A, bcl-x(L) was amplified as a major anti-apoptotic molecule, and TGF-beta-induced cell death was more enhanced than in the original cell line. Caspase 1-like protease was activated by TGF-beta treatment and consequently it cleaved bcl-x(L) in CTLL-2A. TGF-beta-induced DNA fragmentation and cleavage of bcl-x(L) were inhibited by pretreatment with tetra peptide caspase 1 inhibitor, YVAD.cmk. These findings suggest that TGF-beta induces cell death in activated murine T cells through cleavage of bcl-x(L) via activated caspase 1-like protease, which may act as an important executor in that process.     

Light chain of natural antibody plays a dominant role in protein antigen binding

Examinations of the contribution and the specificity of heavy (H) and light (L) chains of natural antibodies to antigen binding may help us to better understand antigen recognition and the development of naive B cells. We previously generated natural Fab antibody fragments reactive to preS1 of HBV using a naive, non-immunized Fab antibody library derived from peripheral B cells of a normal healthy volunteer. We now constructed expression vectors for the Fd (VH + CH1), L chain, and scFv fragments using the sequences encoding parental Fabs as a source of natural antibody genes. The recombinant antibody fragments were expressed as inclusion bodies in E. coli BL21 (DE) cells. When denatured and then refolded, the antibody fragments retained their binding properties. Recombinant L chains and scFvs exhibited three- to 40-fold higher affinities (in the order of 10(7) M(-1)) over the parental Fabs, whereas the affinities of Fds (in the order of 10(5) M(-1)) were much lower compared to the parental Fabs. The results obtained from sandwich ELISA revealed that the L chains bound the virus more efficiently than Fds. Additional experiments were performed to evaluate the specificity of the recombinant fragments for surface proteins of HBV. Fds and L chains were reactive towards HBsAg and the preS2 peptide as well as preS1 and showed patterns of epitope recognition quite different from those of parental Fabs. The data presented here demonstrate that the prominence of the L chain in determining protein binding activity is a property of natural antibodies and is quite unlike the antibodies induced by immunization, and that the specificity of Fab is not determined by the individual antibody chain but by the correct pairing of H and L chain.     

Lysozyme microencapsulation within biodegradable PLGA microspheres: urea effect on protein release and stability

Lysozyme was encapsulated within biodegradable poly(D, L-lactide-co-glycolide) microspheres by a double emulsion solvent evaporation method for studying its release mechanism associated with protein stability problems. When urea, a protein unfolding agent, was added into the incubation medium lysozyme release rate from the microspheres increased with the increase in urea concentration. The enhanced lysozyme release was attributed to the suppression of protein aggregation, to the facilitated diffusion of unfolded lysozyme by an efficient reptile motion of unfolded protein molecules through porous channels in microspheres, and to the largely decreased extent of nonspecific protein adsorption onto the enlarged surface area of degrading polymer microspheres in the presence of urea. Encapsulating lysozyme in an unfolded form within PLGA microspheres was attempted by using urea as an excipient. This new urea-based formulation exhibited a more sustained lysozyme release profile than the control formulation, and released lysozyme from the microspheres showed a much less amount of lysozyme dimer population while maintaining a correct conformation after refolding in the incubation medium. This study provides new insights for the formulation of protein encapsulated PLGA microspheres.     

Regulation of plasmin activity by annexin II tetramer

Annexin II tetramer (AIIt) is a major Ca(2+)-binding protein of the endothelial cell surface which has been shown to stimulate the tissue plasminogen activator (t-PA)-dependent conversion of plasminogen to plasmin. In the present report, we have examined the regulation of plasmin activity by AIIt. The incubation of plasmin with AIIt resulted in a 95% loss in plasmin activity. SDS-PAGE analysis established that AIIt stimulated the autoproteolytic digestion of plasmin heavy and light chains. The kinetics of AIIt-stimulated plasmin autoproteolysis were first-order, suggesting that binding of plasmin to AIIt resulted in the spontaneous autoproteolysis of the bound plasmin. AIIt did not affect the activity of other serine proteases such as t-PA or urokinase-type plasminogen activator. Furthermore, other annexins such as annexin I, II, V, or VI did not stimulate plasmin autoproteolysis. Increasing the concentration of AIIt on the surface of human 293 epithelial cells increased cell-mediated plasmin autoproteolysis. Thus, in addition to stimulating the formation of plasmin, AIIt also promotes plasmin inactivation. These results therefore suggest that AIIt may function to provide the cell surface with a transient pulse of plasmin activity.     

Temperature and salt effects on proteolytic function of turnip mosaic potyvirus nuclear inclusion protein a exhibiting a low-temperature optimum activity

The nuclear inclusion protein a (NIa) of turnip mosaic potyvirus is a protease responsible for processing the viral polyprotein into functional proteins. The NIa protease exhibits an unusual optimum proteolytic activity at about 16 degrees C. In order to understand the origin of the low-temperature optimum activity, the effects of temperature and salt ions on the catalytic activity and the structure of the NIa protease have been investigated. The analysis of the temperature dependence of k(cat) and K(m) revealed that K(m) decreases more drastically than k(cat) as temperature decreases. The thermodynamic analysis showed that the decrease of K(m) is driven entropically, suggesting a possibility that the substrate binding might need a large entropy cost. The secondary structure of the NIa protease was significantly perturbed at temperatures between 20 and 40 degrees C and the protease was unfolded at very low concentrations of guanidine hydrochloride with a transition midpoint of 0.8 M. These results suggest that the NIa protease is highly flexible in structure. Interestingly, salt ions including NaCl, KCl, CaCl(2) and MgCl(2) stimulated the proteolytic activity by 2-6-fold and increased the optimum temperature to 20-25 degrees C. This stimulatory effect of the salt ions was due to the lowering of K(m). The salt ions promoted the structural rigidity as evidenced in the higher resistance to the heat-induced unfolding in the presence of the salt ions. The increase in rigidity may lead to the lowering of K(m) possibly by reducing the entropic cost for substrate binding. Taken together, these results suggest that the NIa protease is highly flexible in structure and the low-temperature optimum activity might possibly be attributed to lowered entropy cost for substrate binding at lower temperatures.