Investigating the effects of different natural molecules on the structure and oligomerization propensity of hen egg-white lysozyme.

The formation of amyloid aggregates is the hallmark of systemic and neurodegenerative diseases, also known as amyloidosis. Many proteins have been found to aggregate into amyloid-like fibrils and thus process is recognized as general tendency of polypeptides. Inhibition of protein aggregation and fibril formation is thus one of the important strategies in the prevention and treatment of such disease. There is a growing interest of identification of small molecules mainly natural compounds that can prevent or delay amyloid fibril formation. In this work, we report the effect of various compounds from different groups on the amyloid fibrillation of hen egg white lysozyme, a model protein for amyloid formation. Herein, a range of biophysical techniques have been employed in order to establish a systematic approach to study the effect of candidate inhibitors on amyloid aggregation. Results demonstrated that the strategy used show that the different techniques are complimentary in order to elucidate a complete in vitro picture of the effect of the used compounds on HEWL aggregation. Moreover, compared to the data obtained by other groups for the inhibition of lysozyme fibril formation, this work provides new insights into the structural changes (local, secondary, oligomeric, fibrillar structures) undergone by HEWL during aggregation in the presence and absence of inhibitors.

Insights into Kinetics of Agitation-Induced Aggregation of Hen Lysozyme under Heat and Acidic Conditions from Various Spectroscopic Methods.

Protein misfolding and amyloid formation are an underlying pathological hallmark in a number of prevalent diseases of protein aggregation ranging from Alzheimer's and Parkinson's diseases to systemic lysozyme amyloidosis. In this context, we have used complementary spectroscopic methods to undertake a systematic study of the self-assembly of hen egg-white lysozyme under agitation during a prolonged heating in acidic pH. The kinetics of lysozyme aggregation, monitored by Thioflavin T fluorescence, dynamic light scattering and the quenching of tryptophan fluorescence by acrylamide, is described by a sigmoid curve typical of a nucleation-dependent polymerization process. Nevertheless, we observe significant differences between the values deduced for the kinetic parameters (lag time and aggregation rate). The fibrillation process of lysozyme, as assessed by the attenuated total reflection-Fourier transform infrared spectroscopy, is accompanied by an increase in the ß-sheet conformation at the expense of the α-helical conformation but the time-dependent variation of the content of these secondary structures does not evolve as a gradual transition. Moreover, the tryptophan fluorescence-monitored kinetics of lysozyme aggregation is described by three phases in which the temporal decrease of the tryptophan fluorescence quantum yield is of quasilinear nature. Finally, the generated lysozyme fibrils exhibit a typical amyloid morphology with various lengths (observed by atomic force microscopy) and contain exclusively the full-length protein (analyzed by highly performance liquid chromatography). Compared to the data obtained by other groups for the formation of lysozyme fibrils in acidic pH without agitation, this work provides new insights into the structural changes (local, secondary, oligomeric/fibrillar structures) undergone by the lysozyme during the agitation-induced formation of fibrils.

Clearance of genetic variants of amyloid ß peptide by neuronal and non-neuronal cells.

The presence of senile plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). The biogenesis and clearance of the amyloid ß peptide (A ß ), the main component of the lesions, lie at the center of the pathogenesis of AD. In sporadic AD, the increase of A ß levels seems to be indicative of failure of clearance mechanisms. We previously showed that the clearance of the wild type A ß40 peptide by various neuronal and non-neuronal cells occurs through a same proteolytic process and that A ß degradation was primarily dictated by its conformational state (Panchal et al., 2007). To gain further insights on the role of the peptide conformation in the clearance mechanism of A ß , two A ß40 peptides, known to be associated with amyloid angiopathy (Dutch and Flemish mutations), and the rodent A ß40 peptide were catabolized by several cells by using the same experimental approach. The peptide fragments, generated by proteolytic cleavage of substrates in cell supernatants, were identified by LC-MS and the cleavage sites of proteases were deduced. In parallel, conformational states of wild type A ß 40 peptide and of the three A ß 40 variants were characterized by circular dichroism spectroscopy. We provide data suggesting that discrete conformational changes of A ß 40 peptide regulate its clearance rate by neuronal and non-neuronal cells.

Acetonic extract of Buxus sempervirens induces cell cycle arrest, apoptosis and autophagy in breast cancer cells.

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC(50) ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC(50) of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC(50) did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer.

Processing of peptide and hormone precursors at the dibasic cleavage sites.

Many functionally important cellular peptides and proteins, including hormones, neuropeptides, and growth factors, are synthesized as inactive precursor polypeptides, which require post-translational proteolytic processing to become biologically active polypeptides. This is achieved by the action of a relatively small number of proteases that belong to a family of seven subtilisin-like proprotein convertases (PCs) including furin. In view of this, this review focuses on the importance of privileged secondary structures and of given amino acid residues around basic cleavage sites in substrate recognition by these endoproteases. In addition to their participation in normal cell functions, PCs are crucial for the initiation and progress of many important diseases. Hence, these proteases constitute potential drug targets in medicine. Accordingly, this review also discusses the approaches used to shed light on the cleavage preference and the substrate specificity of the PCs, a prerequisite to select which PCs are promising drug targets in each disease.

Clearance of amyloid-beta peptide by neuronal and non-neuronal cells: proteolytic degradation by secreted and membrane associated proteases.

Deposition of amyloid-beta peptide (Abeta) in the brain is an early and invariant feature of all forms of Alzheimer's disease (AD). As for all proteins or peptides, the steady-state level of Abeta peptide is determined not only by its production, but also by its degradation. So, overactive proteases involved in generating Abeta from amyloid precursor protein or underactive Abeta-degrading enzymes could lead to abnormal Abeta deposition in the brain. Since in the sporadic forms of AD (90% of all AD cases) an impaired clearance of Abeta appears to be at the origin of its aggregation and tissue deposition, we have investigated its proteolytic degradation by several neuronal and non-neuronal cells. In this report, we show that these cell types exhibit a similar profile of Abeta-degradation by cell-surface and secreted proteases which were respectively characterized as metallo- and serine proteases. By using a combination of the liquid chromatography/on-line mass spectrometry, we demonstrate that: (i)-the membrane associated protease(s) hydrolizes Abeta40 essentially at Lys(28) Gly(29), Phe(19) Phe(20) and Val(18) Phe(19) bonds; and (ii)-the secreted protease(s) cleaves the generating fragments Abeta (1-28), Abeta (1-19), Abeta (1-18) at His(14) Gln(15) bond and also Abeta (1-28) at Phe(20) Ala(21) and Asp(23) Val(24) sites. This is the first time our results define a proteolytic degradation process of Abeta40 that appears to be independent of the cell type and may represent a general pattern of its enzymatic clearance.

Reactivity of basic amino acid pairs in prohormone processing: model of pro-ocytocin/neurophysin processing domain.

Statistical analysis of several potential dibasic cleavage sites reveals differences in the distribution of basic doublets when the in vivo cleaved sites were compared to those which are not cleaved. Analysis of the substrate specificity of protease Kex2 towards the pro-ocytocin/neurophysin processing domain (pro-OT/Np(7-15) with altered basic pairs shows a cleavage efficiency order in accord with the statistical data. Structural analysis of these substrates indicates that each basic pair is associated with a local and specific conformational change. Thus, the in vivo cleavage hierarchy of dibasic sites is encoded by both the nature of basic pairs and the plasticity of proteolytic processing domains.

Endogenous C-terminal fragments of beta-amyloid precursor protein from Xenopus laevis skin exudate.

Using a monoclonal antibody against the entire C-terminal end of human APP(695) (643-695 sequence) and a monoclonal antibody directed against human beta[1-40] amyloid peptide (betaA), we show the existence of endogenous peptides proteolytically derived from APP in skin exudate of the non transgenic Xenopus laevis frog. The majority of the immunoreactivity is found associated with a 30 kDa molecular species. Biochemical fractionation followed by mass spectrometry identification allowed us to assign this molecular species to C-terminal APP fragments containing all or part of betaA. According to the nature of N- and C-terminal amino acids we identified endogenous beta-, gamma-, epsilon-secretase-like activities, caspase-like activity and numerous endogenous cleavage sites within the beta-amyloid sequence at same sites as those observed in human betaA sequence. All these homologies with human indicate that X. laevis skin exudate is a good natural model to study betaA metabolism. In this way, interestingly, we identified endogenous cleavages at prohormone convertase-like sites not yet described at the same sites in human. Finally, all identified peptide fragments were stably associated with a 20.2 kDa protein. These new observed features suggest new research pathways concerning human betaA metabolism and carriage of hydrophobic peptide fragments issued from APP processing.

Abnormalities of peptide metabolism in Alzheimer disease.

The steady-state level of peptide hormones represents a balance between their biosynthesis and proteolytic processing by convertases and their catabolism by proteolytic enzymes. Low levels of neuropeptide Y, somatostatin and corticotropin-releasing factor, described in Alzheimer disease (AD), were related to a defect in proteolytic processing of their protein precursors. In contrast the abundance of beta-amyloid peptides, the major protein constituents of senile plaques is likely related to inefficient catabolism. Therefore, attention is mainly focused on convertases that generate active peptides and counter-regulatory proteases that are involved in their catabolism. Some well-described proteases such as NEP are thought to be involved in beta-amyloid catabolism. The search of other possible candidates represents a primary effort in the field. A variety of vascular risk factors such as diabetes, hypertension and arteriosclerosis suggest that the functional vascular defect contributes to AD pathology. It has also been described that beta-amyloid peptides potentiate endothelin-1 induced vasoconstriction. In this review, we will critically evaluate evidence relating proteases implicated in amyloid protein precursor proteolytic processing and beta-amyloid catabolism.

Specific cleavage of beta-amyloid peptides by a metallopeptidase from Xenopus laevis skin secretions.

Dactylysin (EC 3.5.24.60) is a metalloendopeptidase first isolated from the skin granular gland secretions of Xenopus laevis. This peptidase hydrolyzes bonds on the amino-terminus of singlets and between doublets of hydrophobic amino acids and was considered to play a role in the in vivo inactivation of biologically active regulatory peptides. Here, we show that dactylysin has also the ability to cleave human beta[1-40]-amyloid peptide and related peptides. Cleavage of the wild type beta[1-40]-amyloid peptide form, and to a lesser extent Flemish and Dutch mutants, occurred predominantly at the His14-Glu15 bond. We demonstrate that frog skin exudate contains a full-length amyloid protein precursor detected by immunochemical cross-reactivity with monoclonal antibody against C-terminal human amyloid protein precursor. The possibility that dactylysin, might be involved in normal catabolism of beta amyloid peptide of Xenopus laevis is discussed.

Kinetics of precursor cleavage at the dibasic sites. Involvement of peptide dynamics.

The presence in the P'1 position relative to the LysArg doublet of either Phe, Tyr or Trp residues affects only pro-OT/Np(7-15) flexibility. This has a measurable effect on the dynamics of the peptide. Since the same modifications have a major influence on the K(m) and V(max) values of the peptide cleavage, these kinetic parameters should depend on the peptide substrate motions. Therefore, the primary kinetic contribution of substrate cleavage should arise from substrate dynamics rather than from the enzyme.

The somatostatin-28(1-12)-NPAMAP sequence: an essential helical-promoting motif governing prosomatostatin processing at mono- and dibasic sites.

Proline residues, known to have special structural properties, induce particular conformations which participate in some biological functions. Two prolines (Pro(-9), Pro(-5)) located near the processing sites (Arg(-15) and Arg(-2)Lys(-)(1)) of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin-28 (S-28) and somatostatin-14 (S-14) [Gomez et al. (1989) EMBO J. 8, 2911-2916]. In this study, the importance of the pentapeptide P-A-M-A-P sequence (P-(X)(3)-P pattern), located in the S-28(1-12) segment connecting the mono- and dibasic cleavage sites, was investigated by using site-directed mutagenesis. Analysis of prosomatostatin-derived peptides produced by expression of mutated cDNA species in Neuro2A cells indicated that (i) deletion of PAMAP decreased S-14 production, (ii) deletion of the two Pro residues almost abolished the cleavage at the dibasic site, and (iii) Pro displacement generating the AMAPP motif resulted in a decrease of S-28 production. Moreover, both theoretical and spectroscopic studies of synthetic peptides reproducing the S-28(1-12) sequence bearing critical mutations showed that this sequence can organize as an alpha helical structure. These observations demonstrate that NPAMAP constitutes an accurate alpha-helix nucleation motif allowing for the generation of equal amounts of S-28 and S-14 from their common precursor in Neuro2A cells. Moreover, they emphasize the importance of the S-28(1-12) segment joining Arg(-15) and Arg(-2)Lys(-1) cleavage sites whose conformational organization is essential for controlling their accessibility to the appropriate processing proteases.