Photofrin binds to procaspase-3 and mediates photodynamic treatment-triggered methionine oxidation and inactivation of procaspase-3.

Diverse death phenotypes of cancer cells can be induced by Photofrin-mediated photodynamic therapy (PDT), which has a decisive role in eliciting a tumor-specific immunity for long-term tumor control. However, the mechanism(s) underlying this diversity remain elusive. Caspase-3 is a critical factor in determining cell death phenotypes in many physiological settings. Here, we report that Photofrin-PDT can modify and inactivate procaspase-3 in cancer cells. In cells exposed to an external apoptotic trigger, high-dose Photofrin-PDT pretreatment blocked the proteolytic activation of procaspase-3 by its upstream caspase. We generated and purified recombinant procaspase-3-D(3)A (a mutant without autolysis/autoactivation activity) to explore the underlying mechanism(s). Photofrin could bind directly to procaspase-3-D(3)A, and Photofrin-PDT-triggered inactivation and modification of procaspase-3-D(3)A was seen in vitro. Mass spectrometry-based quantitative analysis for post-translational modifications using both (16)O/(18)O- and (14)N/(15)N-labeling strategies revealed that Photofrin-PDT triggered a significant oxidation of procaspase-3-D(3)A (mainly on Met-27, -39 and -44) in a Photofrin dose-dependent manner, whereas the active site Cys-163 remained largely unmodified. Site-directed mutagenesis experiments further showed that Met-44 has an important role in procaspase-3 activation. Collectively, our results reveal that Met oxidation is a novel mechanism for the Photofrin-PDT-mediated inactivation of procaspase-3, potentially explaining at least some of the complicated cell death phenotypes triggered by PDT.

The antiapoptotic protein, FLIP, is regulated by heterogeneous nuclear ribonucleoprotein K and correlates with poor overall survival of nasopharyngeal carcinoma patients.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) mediates antiapoptotic activity in part by inducing downstream antiapoptotic genes. To systematically identify hnRNP K targets in nasopharyngeal carcinoma (NPC), affymetrix chips were used to identify genes that were both overexpressed in primary NPC and downregulated by hnRNP K knockdown in NPC-TW02 cells. The resulting gene set included the antiapoptotic gene, FLIP, which was selected for further study. In cells treated with hnRNP K siRNA, TRAIL-induced apoptosis was enhanced and the FLIP protein level was reduced. Promoter, DNA pull-down and chromatin-immunoprecipitation assays revealed that hnRNP K directly interacts with the poly(C) element on the FLIP promoter, resulting in transcriptional activation. Through iTRAQ-mass spectrometric identification of proteins differentially associated with the poly(C) element or its mutant, nucleolin was determined to be a cofactor of hnRNP K for FLIP activation. Furthermore, FLIP was highly expressed in tumor cells, and this high-level expression was significantly correlated with high-level hnRNP K expression (P=0.002) and poor overall survival (P=0.015) as examined in 67 NPC tissues. A multivariate analysis confirmed that FLIP was an independent prognostic factor for NPC. Taken together, these findings indicate that FLIP expression is transcriptionally regulated by hnRNP K and nucleolin, and may be a potential prognostic and therapeutic marker for NPC.

Serine 13 is the site of mitotic phosphorylation of human thymidine kinase.

It has been reported that the polypeptide of thymidine kinase type 1 (TK1) from human and mouse cells can be modified by phosphorylation. Our laboratory has further shown that the level of human TK phosphorylation increases during mitotic arrest in different cell types (Chang, Z.-F., Huang, D.-Y., and Hsue, N.-C. (1994) J. Biol. Chem. 269:21249-21254). In the present study, we demonstrated that a mutation converting Ser13 to Ala abolished the mitotic phosphorylation of native TK1 expressed in Ltk- cells. Furthermore, we expressed recombinant proteins of wild-type and mutated human TK1 with fused FLAG epitope in HeLa cells, and confirmed the occurrence of mitotic phosphorylation on Ser13 of hTK1. By using an in vitro phosphorylation assay, it was shown that wild-type hTK1, but not mutant TK1(Ala13), could serve as a good substrate for Cdc2 or Cdk2 kinase. Coexpression of p21(waf1/cip1), which is a universal inhibitor of Cdk kinases, in Ltk- fibroblasts also suppressed mitotic phosphorylation of hTK1 expressed in this cell line. Thus, Cdc2 or related kinase(s) is probably involved in mitotic phosphorylation on Ser13 of the hTK1 polypeptide. We also found that mutation on Ser13 did not affect the functional activity of hTK1. As the sequences around Ser13 are highly conserved in vertebrate TK1s, we speculate that phosphorylation of Ser13 may play a role in the regulation of TK1 expression in the cell cycle.

Heparin and heparan sulfate bind to snake cardiotoxin. Sulfated oligosaccharides as a potential target for cardiotoxin action.

Cardiotoxins (CTXs) from cobra venom show cytotoxicity toward several cell types. They cause systolic heart arrest and severe tissue necrosis. Their interaction with phospholipids is established but by itself fails to explain the specificity of these toxins; other component(s) of membrane must, therefore, intervene to direct them toward their target. We herein show, for the first time, that sulfated glycosaminoglycans, heparin, heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS), interact with CTX A3, a major component of Taiwan cobra venom, by use of affinity chromatography, circular dichroism, absorbance, and fluorescence intensity and anisotropy measurements. The relative strength of binding, determined by the NaCl concentration required to dissociate the CTX-glycosaminoglycan complex, varied as follows: heparin > DS > CS > HS. In physiological buffer (8 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, 138 mM NaCl, pH 7.4), however, only heparin and HS bound to CTX, with respective dissociation constants of 1.4 and 16 microM, while CS and DS failed to exhibit well defined binding behavior, as indicated by fluorescence measurements. We estimate that CTX makes 3-4 ionic contacts with heparin based on a salt-dependent binding study and that approximately 40% of binding free energy is derived from purely electrostatic interactions under physiological conditions. Sulfated pentasaccharide may be sufficient to bind to CTX. We also found that heparin accentuates the penetration of CTX into phospholipid membranes as analyzed by Langmuir monolayer measurement. In view of these results we propose that heparin-like moieties of the cell surface may modulate the action of CTX.

Expression of glutathione S-transferase-cardiotoxin fusion protein in Escherichia coli.

We report here the construction of cardiotoxin V gene, from cobra snake venom (Naja naja atra), by chemically synthesized oligonucleotides and its expression as a glutathione S-transferase-cardiotoxin fusion protein in the inclusion bodies of Escherichia coli. The expression of cardiotoxin fusion protein in protein with a yield of about 35 mg/liter culture was confirmed by highly specific anti-peptide antibodies generated against the unique amino acid residues located at the tip of loop II of cardiotoxin V. Since the fusion protein can be easily treated by CNBr to free the toxin moiety, as revealed by immunoblotting of the cleaved protein, the results provide an avenue for future structural and functional studies of cardiotoxin molecules.

Hydrolysis of amyloid precursor protein-derived peptides by cysteine proteinases and extracts of rat brain clathrin-coated vesicles.

Amyloid precursor proteins (APPs) and C-terminal fragments were colocalized with cysteine proteinase-like enzymes in purified rat brain clathrin-coated vesicles. Vesicular extracts degraded beta A4(12-28), yielding a product profile similar to that of purified rat brain cathepsin B. Cathepsin B degraded this peptide sequentially, with initial cleavage occurring at Val18-Phe19 and Phe19-Phe20 followed by release of dipeptides. Enzyme also hydrolyzed beta A4(1-40) at Phe19-Phe20 bond but at lower rates, likely due to aggregate formation. An octapeptide analogue of the domain adjacent to beta A4 (N-Ac-Val-Lys-Met-Asp-Ala-Glu-Phe-NH2) was also hydrolyzed by brain cathepsins B and L, and metalloendopeptidase 24.11. Enzymes acted at multiple sites, but only 24.11 cleaved the Met-Asp bond, thus resembling a proposed beta-secretase. Data imply that clathrin-coated vesicles contain cysteine-like proteinases capable of initiating the processing of APP or its fragments.

Freezing of phosphocholine headgroup in fully hydrated sphingomyelin bilayers and its effect on the dynamics of nonfreezable water at subzero temperatures.

Differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) spectroscopy are applied to characterize the nonfreezable water molecules in fully hydrated D2O/sphingomyelin at temperatures below 0 degrees C. Upon cooling, DSC thermogram displays two thermal transitions peaked at -11 and -34 degrees C. The high-temperature exothermic transition corresponds to the freezing of the bulk D2O, and the low-temperature transition, which has not previously been reported, can be ascribed to the freezing of the phosphocholine headgroup in the lipid bilayer. The dynamics of nonfreezable water are also studied by 2H NMR T1 (spin-lattice relaxation time) and T2e (spin-spin relaxation time obtained by two pulse echo) measurements at 30.7 MHz and at temperatures down to -110 degrees C. The temperature dependence of the T1 relaxation time is characterized by a distinct minimum value of 2.1 +/- 0.1 ms at -30 degrees C. T2e is discontinuous at temperature around -70 degrees C, indicating another freezing-like event for the bound water at this temperature. Analysis of the relaxation data suggest that nonfreezable water undergoes both fast and slow motions at characteristic NMR time scales. The slow motions are affected when the lipid headgroup freezes.

Mechanism of hemolysis of red blood cell mediated by ethanol.

The effects of ethanol on hemolysis of human red blood cells (RBCs) were studied at 21 +/- 1 degrees C in the saline buffer (138 mM NaCl, 6.1 mM Na2HPO4, 1.4 mM NaH2PO4, 5 mM glucose and pH 7.4). The hemolysis process for ethanol-treated RBCs was preceded by the leakage of the small cation K+ from the cells indicating the colloid-osmotic nature of lysis. Since the extent of membrane lesion increased with an increasing ethanol concentration, osmotic protection experiments by using solutes varying in size were carried out to estimate the diameter of the pore. Quantitative analysis of the data by considering the effect of molecular seiving of the protectants with different sizes indicated that ethanol induced formation of membrane pores with a diameter of approximately 13 A. There was no detectable release of membrane fragments as assayed by the acetylcholinesterase activity, but the membrane structures were significantly perturbed, presumably at the membrane cytoskeletal protein, as evidenced by the altered rheological properties of RBC in the presence of ethanol. It is suggested that the creation of membrane pores might involve in the deranged cytoskeletal network of ethanol-treated RBC.

Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement.

The effects of monopalmitoylphosphatidylcholine (MPPC or lysophosphatidylcholine) and a series of short-chain primary alcohols (ethanol, 1-butanol and 1-hexanol) on cell shape, hemolysis, viscoelastic properties and membrane lipid packing of human red blood cells (RBCs) were studied. For MPPC, the effective membrane concentration to induce the formation of stage 3 echinocytes (8 x 10(6) molecules per cell) was one order of magnitude lower than that needed to induce 50% hemolysis (7 x 10(7) molecules per cell). In contrast, short-chain alcohols induced both shape changes and hemolysis within close concentration range (2.5 x 10(8) to 3.5 x 10(8) molecules per cell). Viscoelastic properties of the RBCs were studied by micropipette aspiration and correlated with shape change. Ethanol-treated RBCs showed a decrease in membrane elastic modulus and an increase in membrane viscosity in the recovery phase at the early stage of shape change. MPPC-treated cells showed the same type of viscoelastic changes, but these were not observed until the formation of stage 2 echinocytes. High-resolution solid-state 13C nuclear magnetic resonance technique was applied to study membrane lipid packing in the ghost membrane by following the chemical shift of hydrocarbon chains. Both MPPC and ethanol caused the 13C-NMR chemical shift to move upfield, indicating that membrane lipids were expanded due to the intercalation of these exogenous molecules. Using data obtained from model compounds, we convert values of chemical shift into a lipid packing parameter, i.e., number of gauche bonds for fatty acyl hydrocarbon chains. Approximately 10(8) interacting molecules per cell are required to induce a detectable change of lipid packing by both MPPC and ethanol. The results indicate that homolysis occurs at a smaller surface area for MPPC- than ethanol-treated RBCs. Our findings suggest that progressive changes in the molecular packing in the membrane lead eventually to hemolysis, but the mode responsible for shape transformation varies with these amphipaths.

Comparisons of lipid dynamics and packing in fully interdigitated monoarachidoylphosphatidylcholine and non-interdigitated dipalmitoylphosphatidylcholine bilayers: cross polarization/magic angle spinning 13C-NMR studies.

13C-NMR spectra have been obtained at 50.3 MHz for monoarachidoylphosphatidylcholine (MAPC) and dipalmitoylphosphatidylcholine (DPPC) dispersions from 25 degrees C to 55 degrees C and for DPPC polycrystals at 25 degrees C using the cross polarization/magic angle spinning technique. Differential scanning calorimetric studies on DPPC and MAPC dispersions show comparable lipid phase transitions with transition temperatures at 41 degrees C and 45 degrees C, respectively, and thus enable the comparison of thermal, structural and dynamic differences between these two systems at corresponding temperatures. Conformational-dependent 13C chemical shift studies on DPPC dispersions demonstrate not only the coexistence of the tilted gel (L beta') and liquid-crystalline (L alpha) phases in the rippled gel (P beta') phase, but also the presence of an intermediate third microscopic phase as evidenced by three resolvable peaks for omega - 1 methylene carbon signals at the temperature interval between Tp and Tm. By comparing chemical shifts of MAPC in the hydrocarbon chain region with those of DPPC at similar reduced temperatures, it can be concluded that the packings are perturbed markedly in the middle segment of the fatty acyl chain during the lamellar to micellar transition. However, terminal methylene and methyl groups of interdigitated MAPC lamellae were found to be more ordered than those of non-interdigitated DPPC bilayers in the gel state. Interestingly, the terminal methyl groups of MAPC in the micelles remain to be relatively ordered; in fact, they are more ordered than the corresponding acyl chain end of DPPC in the liquid-crystalline state. Analysis of data obtained from rotating frame proton spin-lattice relaxation measurements shows a highly mobile phosphocholine headgroup, a rigid carbonyl group and an ordered hydrocarbon chain for lamellar MAPC in the interdigitated state. Furthermore, results suggest that free rotations of the glycerol C2-C3 bond within MAPC molecules may occur in the interdigitated bilayer, whereas intramolecular exchange between two conformations of the glycerol backbone in DPPC become possible at temperatures close to the pretransition temperature. Without isotope enrichment, we conclude that high-resolution solid-state 13C-NMR is indeed a useful technique which can be employed to study the packing and dynamics of phospholipids.

Effective bilayer expansion and erythrocyte shape change induced by monopalmitoyl phosphatidylcholine. Quantitative light microscopy and nuclear magnetic resonance spectroscopy measurements.

When human erythrocytes are treated with exogenous monopalmitoyl phosphatidylcholine (MPPC), the normal biconcave disk shape red blood cells (RBC) become spiculate echinocytes. The present study examines the quantitative aspect of the relationship between effective bilayer expansion and erythrocyte shape change by a newly developed method. This method is based on the combination of direct surface area measurement of micropipette and relative bilayer expansion measurement of 13C crosspolarization/magic angle spinning nuclear magnetic resonance (NMR). Assuming that 13C NMR chemical shift of fatty acyl chain can be used as an indicator of lateral packing of membrane bilayers, it is possible for us to estimate the surface area expansion of red cell membrane induced by MPPC from that induced by ethanol. Partitions of lipid molecules into cell membrane were determined by studies of shape change potency as a function of MPPC and red cell concentration. It is found that 8(+/- 0.5) x 10(6) molecules of MPPC per cell will effectively induce stage three echinocytes and yield 3.2(+/- 0.2)% expansion of outer monolayer surface area. Surface area of normal cells determined by direct measurements from fixed geometry of red cells aspirated by micropipette was 118.7 +/- 8.5 microns2. The effective cross-sectional area of MPPC molecules in the cell membrane therefore was determined to be 48(+/- 4) A2, which is in agreement with those determined by x-ray from model membranes and crystals of lysophospholipids. We concluded that surface area expansion of RBC can be explained by a simple consideration of cross-sectional area of added molecules and that erythrocyte shape changes correspond quantitatively to the incorporated lipid molecules.

Diversity of rat brain cysteine proteinase inhibitors: isolation of low-molecular-weight cystatins and a higher-molecular weight T-kininogen-like glycoprotein.

Conditions for extraction of rat brain soluble and particulate cysteine proteinase inhibitors (CPIs) were compared and an optimal one was selected to isolate low- and high-molecular-weight forms active toward papain or brain cathepsins B/L. The different forms were purified by affinity chromatography on alkylated papain, and identified on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by use of Schiff's reagent, or by immunoblots using antisera to monomer or polymeric forms of human urinary cystatin c, to a human plasma histidine-rich glycoprotein (HRG), or to rat plasma T-kininogen. In particulates containing nuclei (P1) or synaptosomes (P2) the predominant CPI was an 80-kDa glycoprotein cross-reacting to anti-HRG and shown to be a T-kininogen by treatment with TPCK-trypsin, and subsequent bioassay of the released kinins. The levels found in rat brain were approximately 0.5 nmol/g wet weight. The higher-molecular-weight CPI potently inhibited cathepsin L hydrolysis of Leu-enkephalin at the Gly2-Gly3 bond with a Ki 10(-10) M. In contrast the low-molecular-weight CPIs were present in postmicrosomal fractions (S3) and cross-reacted with anti-cystatin c, but not with anti-HRG, anti-lysozyme, anti-beta protein amyloid peptide, or anti-T-kininogen. The low-molecular-weight forms were present at approximately 1-1.5 nmol/g wet weight and resembled "cerebrocystatin" purified previously from rat brain cytosol by M. Kopitar, F. Stern, and N. Marks [1983) Biochem. Biophys. Res. Commun. 112, 1000-1006.).