Novel strategies for targeting the dimerization interface of HIV protease with cross-linked interfacial peptides

As the prevalence of AIDS continues to grow, and current therapeutic agents begin to lose efficacy, the need for alternative treatments to combat HIV has become significantly greater. Targeting the highly conserved dimerization interface of HIV protease (PR) with interfacial peptides has been shown to reduce the activity of the enzyme due to generation of inactive monomers. The potency of these peptide-based inhibitors has been dramatically increased by cross-linking the interfacial sequences derived from HIV PR. This review focuses on a variety of strategies to develop potent, low-molecular-weight dimerization inhibitors of HIV PR.     

Deamidation in human gamma S-crystallin from cataractous lenses is influenced by surface exposure

A major component of human nuclear cataracts is water-insoluble, high molecular weight protein. A significant component of this protein is disulfide bonded gamma S-crystallin that can be reduced to monomers by dithiothreitol. Analysis of this reduced gamma S-crystallin showed that deamidation of glutamine and asparagine residues is a principal modification. Deamidation is one of the modifications of lens crystallins associated with aging and cataractogenesis. One proposed hypothesis of cataractogenesis is that it develops in response to altered surface charges that cause conformational changes, which, in turn, permit formation of disulfide bonds and crystallin insolubility. This report, showing deamidation among the disulfide bonded gamma S-crystallins from cataractous lenses, supports this hypothesis.     

Investigation of the effect of high hydrostatic pressure on proteins and lipidic membranes by dynamic fluorescence spectroscopy

Dynamic fluorescence spectroscopy brings new insight into the functional and structural changes of biological molecules under moderate and high hydrostatic pressure. The principles of time-resolved fluorescence methods are briefly described and the resulting type of information is summarized. A first set of selected applications of the use of dynamic fluorescence on pressure effects on proteins in terms of denaturation, ternary and quaternary structure, aggregation and also interaction with DNA are presented. A second set of applications is devoted to the effect of pressure and of cholesterol on lateral heterogeneity of lipidic membranes.     

The metalloprotease disintegrin ADAM8. Processing by autocatalysis is required for proteolytic activity and cell adhesion

ADAMs (a disintegrin and metalloprotease domains) are metalloprotease and disintegrin domain-containing transmembrane glycoproteins with proteolytic, cell adhesion, cell fusion, and cell signaling properties. ADAM8 was originally cloned from monocytic cells, and its distinct expression pattern indicates possible roles in both immunology and neuropathology. Here we describe our analysis of its biochemical properties. In transfected COS-7 cells, ADAM8 is localized to the plasma membrane and processed into two forms derived either by prodomain removal or as remnant protein comprising the extracellular region with the disintegrin domain at the N terminus. Proteolytic removal of the ADAM8 propeptide was completely blocked in mutant ADAM8 with a Glu(330) to Gln exchange (EQ-A8) in the Zn(2+) binding motif (HE(330)LGHNLGMSHD), arguing for autocatalytic prodomain removal. In co-transfection experiments, the ectodomain but not the entire MP domain of ADAM8 was able to remove the prodomain from EQ-ADAM8. With cells expressing ADAM8, cell adhesion to a substrate-bound recombinant ADAM8 disintegrin/Cys-rich domain was observed in the absence of serum, blocked by an antibody directed against the ADAM8 disintegrin domain. Soluble ADAM8 protease, consisting of either the metalloprotease domain or the complete ectodomain, cleaved myelin basic protein and a fluorogenic peptide substrate, and was inhibited by batimastat (BB-94, IC(50) approximately 50 nm) but not by recombinant tissue inhibitor of matrix metalloproteinases 1, 2, 3, and 4. Our findings demonstrate that ADAM8 processing by autocatalysis leads to a potential sheddase and to a form of ADAM8 with a function in cell adhesion.     

Modulation of the F-actin cytoskeleton by c-Abl tyrosine kinase in cell spreading and neurite extension

The nonreceptor tyrosine kinase encoded by the c-Abl gene has the unique feature of an F-actin binding domain (FABD). Purified c-Abl tyrosine kinase is inhibited by F-actin, and this inhibition can be relieved through mutation of its FABD. The c-Abl kinase is activated by physiological signals that also regulate the actin cytoskeleton. We show here that c-Abl stimulated the formation of actin microspikes in fibroblasts spreading on fibronectin. This function of c-Abl is dependent on kinase activity and is not shared by c-Src tyrosine kinase. The Abl-dependent F-actin microspikes occurred under conditions where the Rho-family GTPases were inhibited. The FABD-mutated c-Abl, which is active in detached fibroblasts, stimulated F-actin microspikes independent of cell attachment. Moreover, FABD-mutated c-Abl stimulated the formation of F-actin branches in neurites of rat embryonic cortical neurons. The reciprocal regulation between F-actin and the c-Abl tyrosine kinase may provide a self-limiting mechanism in the control of actin cytoskeleton dynamics.     

Dynamics of ligand-induced, Rac1-dependent anchoring of cadherins to the actin cytoskeleton

Cadherin receptors are key morphoregulatory molecules during development. To dissect their mode of action, we developed an approach based on the use of myogenic C2 cells and beads coated with an Ncad-Fc ligand, allowing us to mimic cadherin-mediated adhesion. We used optical tweezers and video microscopy to investigate the dynamics of N-cadherin anchoring within the very first seconds of bead-cell contact. The analysis of the bead movement by single-particle tracking indicated that N-cadherin molecules were freely diffusive in the first few seconds after bead binding. The beads rapidly became diffusion-restricted and underwent an oriented rearward movement as a result of N-cadherin anchoring to the actin cytoskeleton. The kinetics of anchoring were dependent on ligand density, suggesting that it was an inducible process triggered by active cadherin recruitment. This anchoring was inhibited by the dominant negative form of Rac1, but not that of Cdc42. The Rac1 mutant had no effect on cell contact formation or cadherin-catenin complex recruitment, but did inhibit actin recruitment. Our results suggest that cadherin anchoring to the actin cytoskeleton is an adhesion-triggered, Rac1-regulated process enabling the transduction of mechanical forces across the cell membrane; they uncover novel aspects of the action of cadherins in cell sorting, cell migration, and growth cone navigation.     

Functional role of epsilon-tubulin in the assembly of the centriolar microtubule scaffold

Centrioles and basal bodies fascinate by their spectacular architecture, featuring an arrangement of nine microtubule triplets into an axial symmetry, whose biogenesis relies on yet elusive mechanisms. However, the recent discovery of new tubulins, such as delta-, epsilon-, or eta-tubulin, could constitute a breakthrough for deciphering the assembly steps of this unconventional microtubule scaffold. Here, we report the functional analysis in vivo of epsilon-tubulin, based on gene silencing in Paramecium, which demonstrates that this protein, which localizes at the basal bodies, is essential for the assembly and anchorage of the centriolar microtubules.