Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.     

Isoform-specific membrane insertion of secretory phospholipase A2 and functional implications

Despite increasing evidence that the membrane-binding mode of interfacial enzymes including the depth of membrane insertion is crucial for their function, the membrane insertion of phospholipase A(2) (PLA(2)) enzymes has not been studied systematically. Here, we analyze the membrane insertion of human group IB PLA(2) (hIBPLA(2)) and compare it with that of a structurally homologous V3W mutant of human group IIA PLA(2) (V3W-hIIAPLA(2)) and with a structurally divergent group III bee venom PLA(2) (bvPLA(2)). Increasing the anionic charge of membranes results in a blue shift of the fluorescence of Trp(3) of hIBPLA(2), a decrease in quenching by acrylamide, and an increase in enzyme activity, reflecting an enhancement in the membrane binding of PLA(2). Fluorescence quenching by brominated lipids indicates significant penetration of Trp(3) into fluid POPC/POPG membranes but little insertion into the solid DPPC/DPPG membranes. Increased membrane fluidity also supports hIBPLA(2) activity, suggesting that membrane insertion of hIBPLA(2) is controlled by membrane fluidity and is necessary for the full activity of the enzyme. Trp fluorescence quenching of the V3W-hIIAPLA(2) and bvPLA(2) by water- and membrane-soluble quenchers indicates substantial membrane insertion of Trp(3) of V3W-hIIAPLA(2), similar to that found for hIBPLA(2), and no insertion of tryptophans of bvPLA(2). Our results provide evidence that (a) structurally similar group IB and IIA PLA(2)s, but not structurally diverse group III PLA(2), significantly penetrate into membranes; (b) membrane insertion is controlled by membrane fluidity and facilitates activation of IB and IIA PLA(2)s; and (c) structurally distinct PLA(2) isoforms may employ different tactics of substrate accession/product release during lipid hydrolysis.     

Mechanism of the membrane interaction of polynuclear platinum anticancer agents. Implications for cellular uptake

The interaction between phospholipids and polynuclear platinum drugs was studied as a mechanism model for cellular uptake of anticancer drugs. The interaction was studied by differential scanning calorimetry (DSC), 31P nuclear magnetic resonance spectroscopy (NMR), inductively coupled plasma optical emission spectroscopy (ICP-OES), and electrospray ionization mass spectrometry (ESI-MS). The transition temperature, enthalpy, and entropy of negatively charged phospholipids DPPS, DPPA, and DPPG were changed upon reaction with the trinuclear platinum complex [{trans-PtCl(NH3)2}2mu-Pt(NH3)2{H2N(CH2)6NH2}2](NO3)4 (I, BBR3464) and the dinuclear analogue [{trans-PtCl(NH3)2}mu-{(NH2)(CH2)3NH2(CH2)4(NH2)}Cl3 (II, BBR3571). This suggests that these platinum complexes interacted not only with the phosphate headgroup but also with the region of the fatty acid tail of liposomes and finally changed the fluidity of the membrane. Both noncovalent (presumably electrostatic and hydrogen bonding) and covalent interactions were involved in the reactions of the negatively charged phospholipids DPPA, DPPS, and DPPG with the highly positively charged platinum complexes. In contrast, few differences were seen for the zwitterionic phospholipids DPPC and DPPE. The binding ratio of BBR3464 to DPPA liposomes was higher than the ratio of BBR3464 to DPPS liposomes, and similar differences were seen for BBR3571. The binding ratios of the platinum complexes to negatively charged phospholipids DPPA, DPPS, and DPPG were slightly lower in a 100 mM chloride solution than in a chloride-free solution. The binding of BBR3464 and BBR3571 with the liposomes was significantly stronger than that with cis-[PtCl2(NH3)2], cisplatin. ESI-MS confirmed that the products of the incubation of BBR3464 with DPPA and DPPS correspond to chloride displacement and formation of [Pt3(NH3)6{NH2(CH2)6NH2}2(DPPA)2]2+ (1) and [Pt3(NH3)6{NH2(CH2)6NH2}2(DPPS)2]2+ (2), respectively. Similar observations were made for BBR3571. 31P NMR spectra confirmed that the site of binding for DPPA was the phosphate oxygen, whereas for DPPS, a binding site of the nitrogen of the serine side chain is indicated. Noncovalent interactions were also confirmed by use of the analogue [{Pt(NH3)3}2mu-Pt(NH3)2{H2N(CH2)6NH2}2](NO3)6 (III, 0,0,0/t,t,t). The implications of these results for the mechanism of cellular uptake of polynuclear platinum complexes are discussed.     

Arsenic trioxide induces gallbladder carcinoma cell apoptosis via downregulation of Bcl-2

Gallbladder carcinoma (GBC), an aggressive and mostly lethal malignancy, is known to be resistant to a number of apoptotic stimuli. Here, we report for the first time the pro-apoptosis role of arsenic trioxide (As2O3) in gallbladder carcinoma and identify the contribution of Bcl-2 in the As2O3-induced apoptosis. The treatment of As2O3 in gallbladder carcinoma cells could induce apoptosis in a dose-dependent manner and downregulate the expression of anti-apoptotic protein Bcl-2 at mRNA level. Moreover, Bcl-2 overexpression could protect gallbladder carcinoma cells from As2O3-induced apoptosis, indicating the contribution of Bcl-2 in As2O3-induced apoptosis. Taken together, these results suggest that arsenic trioxide induces gallbladder carcinoma cell apoptosis via downregulation of Bcl-2, which may have important therapeutic implications in gallbladder carcinoma patients.     

A new cell line from larval fat bodies of the bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Summary A new cell line, designated IOZCAS-Ha-I, was initiated from the fat body of larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. Spherical cells were predominant among the various cell types. The cell line showed a typical lepidopteran chromosome pattern ranging from 58 to 239 chromosomes in the majority of the cells, it was confirmed to have originated from the H. armigera by the DNA amplification-fingerprinting polymerase chain reaction (DAF-PCR) technique. The new cell line was only slightly susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses (NPV) from H. armigera.     

Carotenoid biosynthesis in cyanobacteria: structural and evolutionary scenarios based on comparative genomics

Carotenoids are widely distributed pigments in nature and their biosynthetic pathway has been extensively studied in various organisms. The recent access to the overwhelming amount genomic data of cyanobacteria has given birth to a novel approach called comparative genomics. The putative enzymes involved in the carotenoid biosynthesis among the cyanobacteria were determined by similarity-based tools. The reconstruction of biosynthetic pathway was based on the related enzymes. It is interesting to find that nearly all the cyanobacteria share quite similar pathway to synthesize beta-carotene except for Gloeobacter violaceus PCC 7421. The enzymes, crtE-B-P-Qb-L, involved in the upstream pathway are more conserved than the subsequent ones (crtW-R). In addition, many carotenoid synthesis enzymes exhibit diversity in structure and function. Such examples in the families of zeta -carotene desaturase, lycopene cylases and carotene ketolases were described in this article. When we mapped these crt genes to the cyanobacterial genomes, the crt genes showed great structural variation among species. All of them are dispersed on the whole chromosome in contrast to the linear adjacent distribution of the crt gene cluster in other eubacteria. Moreover, in unicellular cyanobacteria, each step of the carotenogenic pathway is usually catalyzed by one gene product, whereas multiple ketolase genes are found in filamentous cyanobacteria. Such increased numbers of crt genes and their correlation to the ecological adaptation were carefully discussed.     

Prediction of protein structural class with Rough Sets

Background  

A new method for the prediction of protein structural classes is constructed based on Rough Sets algorithm, which is a rule-based data mining method. Amino acid compositions and 8 physicochemical properties data are used as conditional attributes for the construction of decision system. After reducing the decision system, decision rules are generated, which can be used to classify new objects.     

Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2

Background  

Francisella tularensis is a zoonotic intracellular bacterial pathogen that causes tularemia. The subspecies tularensis is highly virulent and is classified as a category A agent of biological warfare because of its low infectious dose by an aerosol route, and its ability to cause severe disease. In macrophages F. tularensis exhibits a rather novel intracellular lifestyle; after invasion it remains in a phagosome for three to six hours before escaping to, and replicating in the cytoplasm. The molecular mechanisms that allow F. tularensis to invade and replicate within a host cell have not been well defined.