The role of Asn14 in the stability and conformation of the reactive-site loop of winged bean chymotrypsin inhibitor: crystal structures of two point mutants Asn14-->Lys and Asn14-->Asp.

A double-headed chymotrypsin inhibitor, WCI, from winged bean seeds was cloned for structural and biochemical studies. The inhibitor was subjected to two point mutations at a conserved position, Asn14. This residue, known to have a pivotal role in stabilizing the first reactive-site loop (Gln63-Phe68) of the inhibitor, is highly conserved in the sequences of the other members of Kunitz (STI) family as well as in the sequences of Kazal family of serine protease inhibitors. The mutants, N14K and N14D, were subjected to biochemical assay and their characteristics were compared with those of the recombinant inhibitor (rWCI). Crystallographic studies of the recombinant and the mutant proteins are discussed. These studies were primarily aimed at understanding the importance of the protein scaffolding towards the conformational rigidity of the reactive-site loop. Our analysis reveals that, as the Lys14 side chain takes an unusual fold in N14K and the Asp14 side chain in N14D interacts with the loop residues by water-mediated hydrogen bonds, the canonical conformation of the loop has remained effectively intact in both the mutant structures. However, minor alterations such as a 2-fold increase in the inhibitory affinity towards the cognate enzyme were observed.     

Graphical representation of the salient conformational features of protein residues.

A composite plot for depicting in two dimensions the conformation and the secondary structural features of protein residues has been developed. Instead of presenting the exact values of the main- and side-chain torsion angles (φ, psi and chi(1)), it indicates the region in the three-dimensional conformational space to which a residue belongs. Other structural aspects, like the presence of a cis peptide bond and disulfide linkages, are also displayed. The plot may be used to recognize patterns in the backbone and side-chain conformation along a polypeptide chain and to compare protein structures derived from X-ray crystallography, NMR spectroscopy or molecular modelling studies and also to highlight the effect of mutation on structure.     

Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-κB and HSP27/p38/AKT Pathways and by Inhibiting ABCB1

Despite advances in cancer detection and prevention, a diagnosis of metastatic disease remains a death sentence due to the fact that many cancers are either resistant to chemotherapy (conventional or targeted) or develop resistance during treatment, and residual chemoresistant cells are highly metastatic. Metastatic cancer cells resist the effects of chemotherapeutic agents by upregulating drug transporters, which efflux the drugs, and by activating proliferation and survival signaling pathways. Previously, we found that c-Abl and Arg non-receptor tyrosine kinases are activated in breast cancer, melanoma, and glioblastoma cells, and promote cancer progression. In this report, we demonstrate that the c-Abl/Arg inhibitor, imatinib (imatinib mesylate, STI571, Gleevec), reverses intrinsic and acquired resistance to the anthracycline, doxorubicin, by inducing G2/M arrest and promoting apoptosis in cancer cells expressing highly active c-Abl and Arg. Significantly, imatinib prevents intrinsic resistance by promoting doxorubicin-mediated NF-κB/p65 nuclear localization and repression of NF-κB targets in a STAT3-dependent manner, and by preventing activation of a novel STAT3/HSP27/p38/Akt survival pathway. In contrast, imatinib prevents acquired resistance by inhibiting upregulation of the ABC drug transporter, ABCB1, directly inhibiting ABCB1 function, and abrogating survival signaling. Thus, imatinib inhibits multiple novel chemoresistance pathways, which indicates that it may be effective in reversing intrinsic and acquired resistance in cancers containing highly active c-Abl and Arg, a critical step in effectively treating metastatic disease. Furthermore, since imatinib converts a master survival regulator, NF-κB, from a pro-survival into a pro-apoptotic factor, our data suggest that NF-κB inhibitors may be ineffective in sensitizing tumors containing activated c-Abl/Arg to anthracyclines, and instead might antagonize anthracycline-induced apoptosis.     

The specificity of fumarate as a switching factor of the bacterial flagellar motor

Fumarate restores to flagella of cytoplasm-free, CheY- containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemo-taxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.     

Complex carbohydrate-lectin interaction at the interface: a model for cellular adhesion. I. Effect of vesicle size on the kinetics of aggregation between a fatty acid conjugate of lectin and a liposomal asialoganglioside

Two types of phospholipid vesicles capable of mutual recognition have been tailor-made to serve as a model system for the study of carbohydrate-mediated cellular adhesion. One of the vesicles contained a fatty acid conjugate of a galactose specific lectin (lectin vesicle) and the other an asialoganglioside with a reactive terminal galactose residue (galactose vesicle). The kinetics of aggregation of these two types of vesicles was followed by monitoring time-dependent change in turbidity. A 10-100-fold enhancement in the forward rate constant (kf ranging from 7.1 x 10(5) to 4.5 x 10(7) M-1.s-1 at 27 degrees C) was observed when compared with that for the lectin-galactose system in solution (kf being 4.5 x 10(5) M-1.s-1), reported in the literature. A study of the influence of vesicle size on the rate of aggregation showed that enhancement depended on the curvature of the galactose vesicle rather than the density of asialoganglioside suggesting a possible diffusion in the plane of the membrane. The ratio, kf/kd is found to be approx. 10(10) M-1 indicating that the formation of multiple bonds plays a role for stable adhesion.     

Multimeric bivalent immunogens from recombinant tetanus toxin H<Subscript>C</Subscript> fragment,synthetic hexasaccharides,and a glycopeptide adjuvant

Using recombinant tetanus toxin H_C fragment (rTT-H_C) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate–carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.     

Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker''s yeast).

The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin.     

Structural investigation of Klebsiella serotype K10 capsular polysaccharide

The primary structure of Klebsiella serotype K10 capsular polysaccharide has been investigated using mainly the techniques of methylation, partial hydrolysis, and 1H and 13C NMR spectroscopy. The polysaccharide was found to consist of hexasaccharide repeating units having the following structure: (formula; see text)