Biomedical subjects

Vittorio Bellotti

Publications and source records attributed to Vittorio Bellotti.

At least 19 recordsLinked to original sources

PubMed2006

Molecular dynamics simulation suggests possible interaction patterns at early steps of beta2-microglobulin aggregation.

Early events in aggregation of proteins are not easily accessible by experiments. In this work, we perform a 5-ns molecular dynamics simulation of an ensemble of 27 copies of beta(2)-microglobulin in explicit solvent. During the simulation, the formation of intermolecular contacts is observed. The simulation highlights the importance of apical residues and, in particular, of those at the N-terminus end of the molecule. The most frequently found pattern of interaction involves a head-to-head contact arrangement of molecules. Hydrophobic contacts appear to be important for the establishment of long-lived (on the simulation timescale) contacts. Although early events on the pathway to aggregation and fibril formation are not directly related to the end-state of the process, which is reached on a much longer timescale, simulation results are consistent with experimental data and in general with a parallel arrangement of intermolecular beta-strand pairs.

Computer Simulation↗

PubMed2006

Recombinant amyloidogenic domain of ApoA-I: analysis of its fibrillogenic potential.

A variety of amyloid diseases are associated with fibrillar aggregates from N-terminal fragments of ApoA-I generated through a largely unexplored multi-step process. The understanding of the molecular mechanism is impaired by the lack of suitable amounts of the fibrillogenic polypeptides that could not be produced by recombinant methods so far. We report the production and the conformational analysis of recombinant ApoA-I 1-93 fragment. Similarly to the polypeptide isolated ex vivo, a pH switch from 7 to 4 induces a fast and reversible conformational transition to a helical state and leads to the identification of a key intermediate in the fibrillogenesis process. Limited proteolysis experiments suggested that the C-terminal region is involved in helix formation. The recombinant polypeptide generates fibrils at pH 4 on a time scale comparable with that of the native fragment. These findings open the way to studies on structural, thermodynamic, and kinetic aspects of ApoA-I fibrillogenesis.

Amyloid↗

PubMed2006

Targeting C-reactive protein for the treatment of cardiovascular disease.

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement, increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively. Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement. Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

Animals↗

PubMed2006

Collagen plays an active role in the aggregation of beta2-microglobulin under physiopathological conditions of dialysis-related amyloidosis.

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils of beta(2)-microglobulin (beta(2)-m) in the musculoskeletal system. Atomic force microscopy inspection of ex vivo amyloid material reveals the presence of bundles of fibrils often associated to collagen fibrils. Aggregation experiments were undertaken in vitro with the aim of reproducing the physiopathological fibrillation process. To this purpose, atomic force microscopy, fluorescence techniques, and NMR were employed. We found that in temperature and pH conditions similar to those occurring in periarticular tissues in the presence of flogistic processes, beta(2)-m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology of beta(2)-m fibrils obtained in vitro in the presence of collagen is extremely similar to that observed in the ex vivo sample. This result indicates that collagen plays a crucial role in beta(2)-m amyloid deposition under physiopathological conditions and suggests an explanation for the strict specificity of dialysis-related amyloidosis for the tissues of the skeletal system. We hypothesize that positively charged regions along the collagen fiber could play a direct role in beta(2)-m fibrillogenesis. This hypothesis is sustained by aggregation experiments performed by replacing collagen with a poly-L-lysine-coated mica surface. As shown by NMR measurements, no similar process occurs when poly-L-lysine is dissolved in solution with beta(2)-m. Overall, the findings are consistent with the estimates resulting from a simplified collagen model whereby electrostatic effects can lead to high local concentrations of oppositely charged species, such as beta(2)-m, that decay on moving away from the fiber surface.

Amyloid↗

PubMed2006

Micro-heterogeneity and aggregation in beta2-microglobulin solutions: effects of temperature, pH, and conformational variant addition.

We show that beta(2)-microglobulin solutions in physiological conditions contain a tiny fraction of aggregates, which can hardly be filtered out and tend to re-form spontaneously. At physiological pH the fractional amount and size distribution of the latter aggregates do not depend on temperature. Conversely, in the pH range typical of the peri-articular tissue acidosis that often occurs in hemodialysis, temperature increase leads to fast and irreversible growth of the aggregates. Quite similar, but strongly enhanced aggregation effects can be induced even in physiological conditions by adding a very small amount of DeltaN6, a naturally occurring truncated isoform of beta(2)-m known to promote fibrillogenesis.

Computer Simulation↗

PubMed2006

Structure, function and amyloidogenic propensity of apolipoprotein A-I.

Apolipoprotein A-I, the major structural apolipoprotein of high-density lipoproteins, efficiently protects humans from cholesterol accumulation in tissues; however, it can cause systemic amyloidosis in the presence of peculiar amino acid replacements. The wild-type molecule also has an intrinsic tendency to generate amyloid fibrils that localise within the atherosclerotic plaques. The structure, folding and metabolism of normal apolipoprotein A-I are extremely complex and as yet not completely clarified, but their understanding appears essential for the elucidation of the amyloid transition. We reviewed present knowledge on the structure, function and amyloidogenic propensity of apolipoprotein A-I with the aim of highlighting the possible molecular mechanisms that might contribute to the pathogenesis of this disease. Important clues on apolipoprotein A-I amyloidogenesis may be obtained from classical comparative studies of the properties of the wild-type versus the amyloidogenic counterpart. Additionally, in the case of apoA-I, further insights on the molecular mechanisms underlying its amyloidogenic propensity may derive from comparative studies between amyloidogenic variants and other mutations associated with hypoalphalipoproteinemia without amyloidosis.

Amino Acid Sequence↗

PubMed2005

Proteomics of beta2-microglobulin amyloid fibrils.

Knowledge on the chemical structure of beta2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of beta2-microglobulin extracted from non-osteotendinous tissues. beta2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to beta2-microglobulin derived from fibrils but is not observed in the beta2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar beta2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1-2% of the intact molecule.

Amino Acid Sequence↗

PubMed2005

Lysozyme: a paradigmatic molecule for the investigation of protein structure, function and misfolding.

BACKGROUND: The term amyloidosis encompasses a wide group of conditions characterised by the tissue deposition of autologous proteins assembled in homogeneous regularly spaced antiparallel beta strands. The mechanism by which the different proteins gain a conformation, allowing monomers to bind to each other to form the regular amyloid fibril, is under intensive investigation. The discovery that human lysozyme, a protein thoroughly structurally and functionally characterised, can form amyloid fibrils has offered unique opportunities to unveil the molecular mechanisms involved in amyloid formation. Four amyloidogenic mutations have been identified and an apparently non-amyloidogenic polymorphism has been recently described. RESULTS AND CONCLUSIONS: Lysozyme is well characterised for structure, function, folding dynamics and metabolism and comparative studies are becoming available that highlight pathogenic differences between the wild-type and the amyloidogenic variants. The chemical structure of lysozyme in natural amyloid fibrils was characterised in high detail in the early cases, but it is still lacking in the cases most recently discovered. Amyloidogenic lysozymes represent a prototypic molecule for new pharmaceutical approaches in which the formation of amyloid fibrils is abrogated through a stabilisation of the precursor.

Amyloid↗

PubMed2005

Solution structure of beta(2)-microglobulin and insights into fibrillogenesis.

The solution structure of human beta(2)-microglobulin (beta(2)-m) was determined by (1)H NMR spectroscopy and restrained modeling calculations. Compared to the crystal structure of type I major histocompatibility complex (MHC-I), where the protein is associated to the heavy-chain component, several differences are observed, i.e., increased separation between strands A and B, displacements of strand C' and loop DE, shortening of strands D and E. These modifications can be considered as the prodromes of the amyloid transition. Even minor charge changes in response to pH, as is the case with H31 imidazole protonation, trigger the transition that starts with unpairing of strand A. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu(2+) binding which is shown to occur primarily at H31. Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches via surface charge cluster. Mutants or truncated forms of beta(2)-m can be designed to remove the instability from H31 titration or to enhance the instability through surface charge suppression. By monitoring the conformational evolution of wild-type protein and variants thereof, either in response or absence of external perturbation, valuable insights into intermediate structure and fibrillogenesis mechanisms are gained.

Amyloid↗

PubMed2005

Beta2-microglobulin isoforms display an heterogeneous affinity for type I collagen.

It has been claimed that beta2-microglobulin (beta2-m) interacts with type I and type II collagen, and this property has been linked to the tissue specificity of the beta2-m amyloid deposits that target the osteo-articular system. The binding parameters of the interaction between collagen and beta2-m were determined by band shift electrophoresis and surface plasma resonance by using bovine collagen of type I and type II and various isoforms of beta2-m. Wild-type beta2-m binds collagen type I with a Kd of 4.1 x 10(-4) M and type II with 2.3 x 10(-3) M. By the BIAcore system we monitored the binding properties of the conformers of the slow phase of folding of beta2-m. The folding intermediates during the slow phase of folding do not display any significant difference with respect to the binding properties of the fully folded molecule. The affinity of beta2-m truncated at the third N-terminal residue does not differ from that reported for the wild-type protein. Increased affinity for collagen type I is found in the case of N-terminal truncated species lacking of six residues. The Kd of this species is 3.4 x 10 (-5) M at pH 7.4 and its affinity increases to 4.9 x 10(-6) M at pH 6.4. Fluctuations of the affinity caused by beta2-m truncation and pH change can cause modifications of protein concentration in the solvent that surrounds the collagen, and could contribute to generate locally a critical protein concentration able to prime the protein aggregation.

Amyloidosis↗

PubMed2005

A novel AbetaPP mutation exclusively associated with cerebral amyloid angiopathy.

Mutations in AbetaPP cause deposition of Abeta amyloid fibrils in brain parenchyma and cerebral vessels, resulting in Alzheimer's disease (AD) and/or cerebral amyloid angiopathy (CAA). We report a novel mutation (L705V) within the Abeta sequence of AbetaPP in a family with autosomal dominant, recurrent intracerebral hemorrhages. Pathological examination disclosed severe CAA, without parenchymal amyloid plaques or neurofibrillary tangles. This variant highlights the vascular tropism of mutated Abeta, resulting in CAA instead of the pathological hallmarks of AD.

Amyloid beta-Peptides↗

PubMed2005

Search of ligands for the amyloidogenic protein beta2-microglobulin by capillary electrophoresis and other techniques.

Beta2-microglobulin (beta2-m) is a small amyloidogenic protein normally present on the surface of most nucleated cells and responsible for dialysis-related amyloidosis, which represents a severe complication of long-term hemodialysis. A therapeutic approach for this amyloidosis could be based on the stabilization of beta2-m through the binding to a small molecule, and consequent inhibition of protein misfolding and amyloid fibril formation. A few compounds have been described to weakly bind beta2-m, including the drug suramin. The lack of a binding site for nonpolypeptidic ligands on the beta2-m structure makes it difficult for both the identification of functional groups responsible for the binding and the search of hits to be optimized. The characterization of the binding properties of suramin for beta2-m by using three different techniques (surface plasmon resonance, affinity CE (ACE), ultrafiltration) is here described and the results obtained are compared. The common features of the chemical structures of the compounds known to bind the protein led us to select 200 sulfonated/suramin-like molecules from a wider chemical library on the basis of similarity rules, so as to possibly single out some interesting hits and to gain more information on the functional groups involved in the binding. The development of screening methods to test the compounds by using ultrafiltration and ACE is described.

Amyloid↗

PubMed2004

Ultrastructural organization of ex vivo amyloid fibrils formed by the apolipoprotein A-I Leu174Ser variant: an atomic force microscopy study.

Atomic force microscopy was employed to study ex vivo amyloid material isolated from the transplanted hearts of two patients affected by systemic amyloidosis caused by the Leu174Ser apolipoprotein A-I variant. The purified material consists of fibrils and globular aggregates. For both patients the same morphological patterns are observed; in addition, fibril diameters obtained for the two patients turn out to be compatible, both in air (2.00+/-0.02 and 2.04+/-0.04 nm) and under liquid (10.7+/-0.4 and 11.3+/-0.5 nm). Fibrils display heterogeneous morphologies, occasionally showing a left-handed twist. Inspection of fibril ends, the study of fibril contour shape and the analysis of partially unfolded fibrils yield independent evidences suggesting that most twisted fibrils are composed of three protofilaments. The size of globular aggregates is the same for both patients (4.4+/-0.4 and 5.1+/-0.5 nm, measured under liquid) and is compatible with the protofilament expected diameter, suggesting that globules may represent protofilament precursors.

Amyloid↗

PubMed2004

Capillary electrophoresis studies on the aggregation process of beta-amyloid 1-42 and 1-40 peptides.

The possibility to monitor, in solution, the steps of beta-amyloid (Abeta) nucleation and therefore to describe this dynamic process by using capillary electrophoresis and under optimized experimental conditions is described. Striking differences in the electrophoretic patterns of Abeta 1-42 and Abeta 1-40 over time are here shown, and different aggregation states are elucidated, which reflect the very diverse oligomerization behavior of two very similar peptides. The isolation of one aggregated species of high molecular weight by ultracentrifugation allowed us to assess its role as toxic oligomer. The perturbation of the existing equilibrium among the identified species by the addition of small molecules can in principle interfere with the aggregation process of the peptides and ultimately prevent the plaque formation in vitro.

Amyloid beta-Peptides↗

PubMed2004

Neurodegenerative diseases caused by protein aggregation: a phenomenon at the borderline between molecular evolution and ageing.

A process of protein aggregation that causes intracellular or extracellular accumulation of insoluble protein deposits causes many important neurodegenerative diseases associated with the ageing. The recognition that protein aggregation plays a prominent role in pathogenesis of important pathologies such as Alzheimer's and Parkinson's diseases prompted the scientific community to focus on the molecular mechanism of protein aggregation. Many proteins with sophisticated functions can self-aggregate because their folding is complicate and abnormal intermolecular contacts can predominate over the normal intramolecular interactions. The review of biochemical functional and pathogenic implications attributed to alpha synuclein, A beta peptide, presenilin and apoE highlights for these proteins a common conformational plasticity and the capacity to adapt their secondary structure to surrounding solvent as well as to the contacted ligands. Their functions are not fully elucidated but there is an elevated number of metabolic pathways in which apparently they are involved as well as they generate functional contact with a remarkable number of other proteins. The mechanism by which alpha synuclein and A beta protein make fibrils is an example of conformational plasticity because both these polypeptides can visit a coil or helical structure, but otherwise they convert into a pathogenic beta sheet structure highly suitable for polymerisation and fibril formation. The emerging question in the puzzling pathogenic basis of these diseases is if protein aggregation associated with ageing has a role in molecular evolution of the species or if it just represents a calculated drawback.

Aging↗

PubMed2004

Liver biopsy discloses a new apolipoprotein A-I hereditary amyloidosis in several unrelated Italian families.

BACKGROUND & AIMS: Hereditary systemic amyloidoses are autosomal dominant, late-onset disorders caused by mutations in the genes for a group of plasma proteins including transthyretin, lysozyme, fibrinogen Aalpha chain, gelsolin, apolipoprotein A-I, and apolipoprotein A-II. We investigated both phenotypic and genotypic aspects of apolipoprotein A-I amyloidosis unexpectedly disclosed by liver biopsy in 13 unrelated individuals with asymptomatic, persistent elevation of alkaline phosphatase and gamma-glutamyltransferase levels. METHODS: Immunoelectron microscopy was used for in situ characterization of amyloid deposits on liver biopsy specimens. Mutation analysis was performed by sequencing of the apolipoprotein A-I gene in all patients. Wild-type/variant apolipoprotein A-I ratio in plasma high-density lipoproteins was assessed by a peptide mass fingerprinting approach after purification of total apolipoprotein A-I of 2 patients. RESULTS: Family history was informative in 5 cases. Renal failure developed in 9 cases. Hypogonadism due to testicular involvement was observed. Amyloid fibrils specifically stained with anti-apolipoprotein A-I antibody. A novel (Leu75Pro) heterozygous mutation in the apolipoprotein A-I gene was present in affected individuals but not in controls. Variant apolipoprotein A-I was about 10% of the total protein in high-density lipoproteins. CONCLUSIONS: The high number of individuals with apparently sporadic disease might reflect widespread occurrence of this mutation in the population and a milder phenotype of this variant compared with other apolipoprotein A-I amyloidogenic mutants. These findings suggest that specific staining for amyloid should be performed on liver biopsy of individuals with asymptomatic chronic elevation of alkaline phosphatase and gamma-glutamyltransferase levels.

Aged↗

PubMed2004

Pharmaceutical strategies against amyloidosis: old and new drugs in targeting a "protein misfolding disease".

The group of diseases caused by abnormalities of the process of protein folding and unfolding is rapidly growing and includes diseases caused by loss of function as well as diseases caused by gain of function of misfolded proteins. Amyloidoses are caused by gain of function of certain proteins that lose their native structure and self-assemble into toxic insoluble, extracellular fibrils. This process requires the contribution of multiple factors of which only a few are established, namely the conformational modification of the amyloidogenic protein, protein's post-translational modifications and the co-deposition of glycosaminoglicans and of serum amyloid P component. In parallel with the exponential growth of biochemical data regarding the key events of the fibrillogenic process, several reports have shown that small molecules, through the interaction with either the amyloidogenic proteins or with the common constituents, can modify the kinetics of formation of amyloid fibrils or can facilitate amyloid reabsorption. These small molecules can be classified on the basis of their protein target and mechanism of action, according to the following properties. 1) molecules that stabilize the amyloidogenic protein precursor 2) molecules that prevent fibrillogenesis by acting on partially folded intermediates of the folding process as well as on low molecular weight oligomers populating the initial phase of fibril formation 3) molecules that interact with mature amyloid fibrils and weaken their structural stability 4) molecules that displace fundamental co-factors of the amyloid deposits like glycosaminoglycans and serum amyloid P component and favor the dissolution of the fibrillar aggregate.

Amyloid↗

PubMed2003

Properties of some variants of human beta2-microglobulin and amyloidogenesis.

Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Ybeta(2)-m and DeltaN3beta(2)-m. Precipitation and unfolding were observed over time periods shorter than 4-6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for beta(2)-m fibrillogenesis. The mechanism is consistent with the previous and present results on beta(2)-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer.

Amyloid↗