N-terminally truncated amyloid-b (Ab) peptides are present in early and diffuse plaques of individuals with Alzheimer's disease (AD), are overproduced in early onset familial AD and their amount seems to be directly correlated to the severity and the progression of the disease in AD and Down's syndrome (DS). The pyroglutamate-containing isoforms at position 3 [AbN3(pE))40/42] represent the prominent form among the N-truncated species, and may account for more than 50% of Ab accumulated in plaques. In this study, we compared the toxic properties, fibrillogenic capabilities, and in vitro degradation profile of Ab1-40, Ab1-42, AbN3(pE))40 and AbN3(pE))42. Our data show that fibre morphology of Ab peptides is greatly influenced by the C-terminus while toxicity, interaction with cell membranes and degradation are influenced by the N-terminus. AbN3(pE))40 induced significantly more cell loss than the other species both in neuronal and glial cell cultures. Aggregated AbN3(pE) peptides were heavily distributed on plasma membrane and within the cytoplasm of treated cells. AbN3(pE))40/42 peptides showed a significant resistance to degradation by cultured astrocytes, while fulllength peptides resulted partially degraded. These findings suggest that formation of N-terminally modified peptides may enhance b-amyloid aggregation and toxicity, likely worsening the onset and progression of the disease.

A good deal of information on the thermodynamic properties of chromatin was derived in the last few years from optical melting experiments. The structural domains of the polynucleosomal chain, the linker, and the core particle denature as independent units. The differential scanning calorimetry profile of isolated chromatin is made up of three endotherms, at approximately 74, 90, and 107 degrees C, having an almost Gaussian shape. Previous work on this matter, however, was mainly concerned with the dependence of the transition enthalpy on external parameters, such as the ionic strength, or with the melting of nuclei from different sources. In this paper we report the structural assignment of the transitions of rat liver nuclei, observed at 58, 66, 75, 92, and 107 degrees C. They are representative of the quiescent state of the cell. The strategy adopted in this work builds on the method developed for the investigation of complex biological macromolecules. The heat absorption profile of the nucleus was related to the denaturation of isolated nuclear components; electron microscopy and electrophoretic techniques were used for their morphological and molecular characterization. The digestion of chromatin by endogenous nuclease mimics perfectly the decondensation of the higher order structure and represented the source of several misinterpretations. This point was carefully examined in order to define unambiguously the thermal profile of native nuclei. The low-temperature transitions, centered around 58 and 66 degrees C, arise from the melting of scaffolding structures and of the proteins associated with heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

We present a detailed thermodynamic investigation of the conformational transitions of chromatin in calf thymus nuclei. Differential scanning calorimetry was used as the leading method, in combination with infrared spectroscopy, electron microscopy, and techniques for the molecular characterization of chromatin components. The conformational transitions were induced by changes in the counterion concentration. In this way, it was possible to discriminate between the interactions responsible for the folding of the higher order structure and for the coiling of nucleosomal DNA. Our experiments confirm that the denaturation of nuclear chromatin at physiological ionic strength occurs at the level of discrete structural domains, the linker and the core particle, and we were able to rule out that the actual denaturation pattern might be determined by dissociation of the nucleohistone complex and successive migration of free histones toward native regions, as recently suggested. The sequence of the denaturation events is (1) the conformational change of the histone complement at 66 degrees C, (2) the unstacking of the linker DNA at 74 degrees C, and (3) the unstacking of the core particle DNA, that can be observed either at 90 or at 107 degrees C, depending on the degree of condensation of chromatin. Nuclear chromatin unfolds in low-salt buffers, and can be refolded by increasing the ionic strength, in accordance with the well-known behavior of short fragments. The process is athermal, therefore showing that the stability of the higher order structure depends on electrostatic interactions. The transition between the folded conformation and the unfolded one proceeds through an intermediate condensation state, revealed by an endotherm at 101 degrees C. The analysis of the thermodynamic parameters of denaturation of the polynucleosomal chain demonstrates that the wrapping of the DNA around the histone octamer involves a large energy change. The most striking observation concerns the linker segment, which melts a few degrees below the peak temperature of naked DNA. This finding is in line with previous thermal denaturation investigations on isolated chromatin at low ionic strength, and suggests that a progressive destabilization of the linker occurs in the course of the salt-induced coiling of DNA in the nucleosome.

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The transition of prion protein from a mainly a-structured isoform (PrPC) to a~sheet-containing protein (PrPSC) represents a major pathogenetic mechanism in prion diseases. To study the role of PrP structural conformation in prion-dependent neurodegeneration, we analysed the neurotoxicity of PrP in a and~conformations, using a recombinant protein encompassing amino acids 90-231 of the human PrP (hPrP90-231). Using controlled thermal denaturation (53°C, Ih) we converted hPrP90-231 in a structural isoform displaying Prpsc-related characteristics: high~sheet content, increased aggregability and a slight increase in the resistance to protease K. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing a caspase-3 and p38-dependent apoptosis. Conversely, in the native a-helix-rich conformation, hPrP90-231 did not show significant cell toxicity. The relationship between the structural state of hPrP90-231 and its neurotoxicity was demonstrated, inducing the thermal denaturation of the peptide in the presence of Congo red that prevented both the transition of hPrP90-231 into a~-rich isoform and the acquisition of toxic properties. In conclusion, we report that the toxicity of hPrP90-231 is dependent on its three-dimensional structure, as is supposed to occur for the pathogen PrP during TSE.Transmissible spongiform encephalopathies (TSE) represent a group of fatal neurodegenerative disorders that affect both animals and humans, occurring through infective, genetic or sporadic mechanisms. TSE share a common histopathology with a pathognomonic triad: spongiform vacuolation of the gray matter, neuronal death and glial proliferation, with amyloid deposition less constantly observed. In contrast to other transmissible diseases in which the infectious particles are viruses or bacteria, the only agent currently associated with TSE is a structural isoform of the cellular prion protein (PrP C ) known as the scrapie conformation (PrPSC) (1).In Prpsc, large portions of the N-terminus of the protein adopt a~-sheet conformation, this feature being the main difference between the cellular and the pathogenic protein (2). Thus, the conversion of

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The process of carcinogenesis is characterized by definite changes in the protein composition of the nuclear matrix. We have recently found that lamins form, in addition to the nuclear lamina, an intranuclear web of thin fibrils. This finding prompted us to address the question of whether changes in the expression of lamins occur in the course of tumor development. In prostate cancer, lamin B undergoes a significant increase; interestingly, its nuclear content strongly correlates with tumor differentiation. Moreover, all the lamins show reproducible alterations in the distribution of the isoelectric variants, suggesting that dephosphorylation events could trigger changes in the pattern of gene expression by inducing structural rearrangements of the nuclear scaffold.

Chromatin condensation and DNA cleavage at internucleosomal sites have been recognized early as hallmarks of apoptosis, and it has been suggested that extensive DNA chain scission could directly result in the formation of dense chromatin bodies. Here we have shown that no causal relationship exists between DNA degradation and chromatin condensation in glucocorticoid-induced thymocyte apoptosis. The chromatin rearrangement occurred independent of as well as prior to DNA cleavage and involved a specific conformational change at the nucleosome level. In the early stages of the process, the core particles appeared to be tightly packed face-to-face in smooth 11-nm filaments that progressively folded to generate a closely woven network. The network finally collapsed, producing dense apoptotic bodies. Since trypsin digestion relaxed condensed chromatin and histone H4 underwent appreciable deacetylation in the apoptotic cell, we suggest that changes in the DNA-histone interactions represented a major modulating factor of condensation.Although the term "apoptosis" was originally derived from the Greek to emphasize cytoplasmic and nuclear alterations peculiar to the process of programmed cell death (1), no attempt has been made thus far to search for the molecular events underlying these changes, particularly the collapse of the bulk of chromatin into dense domains. The reasons for this delay in the development of a fundamental approach are manifold. In the first place, the unique condensed appearance of the apoptotic nucleus is closely associated with the cleavage of chromatin at internucleosomal sites (2), a circumstance that supports the hypothesis of a causal relationship between extensive chromatin digestion and condensation (3). This early view has recently been challenged on the basis of more refined determinations of the chain length of the DNA isolated from apoptotic cells (4 -6) but has long distracted from the search for the molecular mechanisms involved in the process of condensation. Moreover, since apoptosis plays a key regulatory role in several physiological and pathological processes, major efforts are currently being directed to the elucidation of the biochemical aspects and to the identification of the genes involved in the activation of the cell death program. The onset of chromatin condensation might direct the orderly turning off of genes required for the execution of metabolic suicide, therefore warranting a detailed structural characterization of apoptotic chromatin and a search for terminal modulating factors.Bearing in mind the spatial distribution of interphase chromatin, the appearance of the apoptotic nucleus immediately suggests the occurrence of a structural change involving extremely large domains, and the question arises whether a specific conformational transition at the nucleosome level might account for such a catastrophic phenomenon. In the first place, is chromatin in apoptosis characterized by a three-dimensional array of nucleosomes different from that prevailing in the interphase 3...