Conformational study on glycosylated asparagine-oligopeptides by NMR spectroscopy and molecular dynamics calculations.

The conformational properties of the homo oligomers of increasing chain length Boc-(Asn)(n)-NHMe (n = 2, 4, 5), (GlcNAc-beta-Asn)(n)-NHMe (n = 2, 4, 5, 8) and Boc-[GlcNAc(Ac)(3)-beta-Asn](n)-NHMe (n = 2, 4, 5) were studied by using NOE experiments and molecular dynamic calculations (MD). Sequential NOEs and medium range NOEs, including (i,i+2) interactions, were detected by ROESY experiments and quantified. The calculated inter-proton distances are longer than those characteristic of beta-turn secondary structures. Owing to the large conformational motions expected for linear peptides, MD simulations were performed without NMR constraints, with explicit water and by applying different treatments of the electrostatic interactions. In agreement with the NOE results, the simulations showed, for all peptides, the presence of both folded and unfolded structures. The existence of significant populations of beta-turn structures can be excluded for all the examined compounds, but two families of structures were more often recognized. The first one with sinusoidal or S-shaped forms, and another family of large turns together with some more extended conformations. Only the glycosylated pentapeptide shows in vacuo a large amount of structures with helical shaped form. The results achieved in water and in DMSO are compared and discussed, together with the effect of the glycosylation.     

Conformational investigations on glycosylated asparagine-oligopeptides of increasing chain length.

Stepwise solution syntheses of the homo-oligomers Boc-(Asn)n-NHCH3 (n = 1-5; I1-5), Boc-[[GlcNAc(Ac)3beta]Asn]n-NHCH3 (n = 1-8; II1-8), and Boc-[(GlcNAcbeta)Asn]n-NHCH3 (n = 1-8; III1-8) are described. Members of the series III were obtained by deacetylation of the corresponding members of the series II. The conformational preferences of the N-protected homo-peptides of the three series were investigated by spectroscopic techniques. 1H-NMR measurements were carried out in various solvents; the CD spectra were recorded in water, aqueous SDS and TFE. The poor solubility of the oligomers of the three series prevented FT-IR measurements in solution. NMR and IR measurements indicate the existence of unordered structures containing some gamma-turns in the carbohydrate-free oligomers and the presence of beta-turns in the glycosylated oligopeptides, whether acetylated or not. The CD spectra do not indicate the presence of organized structures. The sugar moieties apparently do not have a structure-inducing effect on the asparagine homo-oligomer main chain.     

Cytological analysis of the effect of treatments visualizing the chromosome core on non-histone nuclear proteins

A silver-stainable chromatid core is visualized under the light microscope on human leukocyte chromosomes by treating fixed chromosomes with 2M NaCl.An aminoacid which is not part of histone composition, 3H-tryptophan, is used to label the synthesis and location of non-histone proteins and to study the effect on these proteins of treatment with 2M NaCl and 0.2N HCl to visualize the core. 2M NaCl is shown to remove from nuclei and chromosomes an appreciable fraction of acid proteins, as well as all those synthesized during the two hours preceding the metaphase. 3H-Try labelling seems to preferentially affect only one chromatid per chromosome. The silver-stainable halocs appearing around numerous nuclei after treatment with 2M NaCl and 0.2N HCl are found to be labelled by 3H-Try and not by 3H-TdR.     

Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

The proapoptotic protein, prostate apoptosis response-4 (Par-4), acts as a tumor suppressor in prostate cancer cells. The serine/threonine kinase casein kinase 2 (CK2) has a well-reported role in prostate cancer resistance to apoptotic agents or anticancer drugs. However, the mechanistic understanding on how CK2 supports survival is far from complete. In this work, we demonstrate both in rat and humans that (i) Par-4 is a new substrate of the survival kinase CK2 and (ii) phosphorylation by CK2 impairs Par-4 proapoptotic functions. We also unravel different levels of CK2-dependent regulation of Par-4 between species. In rats, the phosphorylation by CK2 at the major site, S124, prevents caspase-mediated Par-4 cleavage (D123) and consequently impairs the proapoptotic function of Par-4. In humans, CK2 strongly impairs the apoptotic properties of Par-4, independently of the caspase-mediated cleavage of Par-4 (D131), by triggering the phosphorylation at residue S231. Furthermore, we show that human Par-4 residue S231 is highly phosphorylated in prostate cancer cells as compared with their normal counterparts. Finally, the sensitivity of prostate cancer cells to apoptosis by CK2 knockdown is significantly reversed by parallel knockdown of Par-4. Thus, Par-4 seems a critical target of CK2 that could be exploited for the development of new anticancer drugs.     

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion

Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A₁ (BafA₁), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA₁. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.     

Delivery of Brain-Derived Neurotrophic Factor by 3D Biocompatible Polymeric Scaffolds for Neural Tissue Engineering and Neuronal Regeneration

Biopolymers are increasingly employed for neuroscience applications as scaffolds to drive and promote neural regrowth, thanks to their ability to mediate the upload and subsequent release of active molecules and drugs. Synthetic degradable polymers are characterized by different responses ranging from tunable distension or shrinkage to total dissolution, depending on the function they are designed for. In this paper we present a biocompatible microfabricated poly-ε-caprolactone (PCL) scaffold for primary neuron growth and maturation that has been optimized for the in vitro controlled release of brain-derived neurotrophic factor (BDNF). We demonstrate that the designed morphology confers to these devices an enhanced drug delivery capability with respect to monolithic unstructured supports. After incubation with BDNF, micropillared PCL devices progressively release the neurotrophin over 21 days in vitro. Moreover, the bioactivity of released BDNF is confirmed using primary neuronal cultures, where it mediates a consistent activation of BDNF signaling cascades, increased synaptic density, and neuronal survival. These results provide the proof-of-principle on the fabrication process of micropatterned PCL devices, which represent a promising therapeutic option to enhance neuronal regeneration after lesion and for neural tissue engineering and prosthetics.