Biomedical subjects

Sofia Giorgetti

Publications and source records attributed to Sofia Giorgetti.

17 recordsLinked to original sources

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

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

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↗

PubMed2006

Variants of beta-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature.

Cleavage of the small amyloidogenic protein beta2-microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related beta2-microglobulin amyloidosis, since elevated levels of cleaved beta2-microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved beta2-microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine beta2-microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of beta2-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type beta2-microglobulin and is strongly dependent on temperature and protein concentration. 1H-NMR spectroscopy and amide hydrogen (1H/2H) exchange monitored by MS show that the overall three-dimensional structure of the variants is similar to that of wild-type beta2-microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E beta-strands close to the D-E loop cleavage site at lysine-58, and the experiments suggest conformational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered beta2-microglobulin molecules. This is the first single-residue resolution study of a specific beta2-microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in beta2-microglobulin amyloidogenicity in vivo.

Electrophoresis, Capillary↗

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

Purification and characterization of soluble Cichorium intybusVar. silvestre lipoxygenase.

A water-soluble lipoxygenase enzyme (EC 1.13.11.12; LOX) occurring in the red cultivar produced in the geographical area of Chioggia (Italy) of Cichorium intybus var. silvestre was isolated and characterized. The molecular mass of the enzyme was estimated to be 74,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. The isoelectric point was pH 6.85. The optimum values of pH, ionic strength, and temperature, shown by isoresponse surface calculated by a randomized multilevel factorial design, were 7.58, 30 mM, and 38.5 degrees C, respectively. The enzyme showed high specificity toward linoleic acid, and the study of the variation of linoleic acid concentration between 30 and 300 microM, in the presence of Tween 20 at a concentration lower than the critical micelle concentration (0.01 v/v), resulted in a typical Michaelis-Mentem curve with KM and Vmax values of 1.49 x 10(-4) M and 2.049 microM min(-1) mg(-1), respectively. The biochemical properties, the kinetic parameters found, and the carotene-bleaching activity shown in aerobic conditions seem to indicate that the isolated enzyme is a lipoxygenase type III according to the indications given for soybean isoenzymes.

Cichorium intybus↗

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

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

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↗

PubMed2003

Structural and folding dynamic properties of the T70N variant of human lysozyme.

Definition of the transition mechanism from the native globular protein into fibrillar polymer was greatly improved by the biochemical and biophysical studies carried out on the two amyloidogenic variants of human lysozyme, I56T and D67H. Here we report thermodynamic and kinetic data on folding as well as structural features of a naturally occurring variant of human lysozyme, T70N, which is present in the British population at an allele frequency of 5% and, according to clinical and histopathological data, is not amyloidogenic. This variant is less stable than the wild-type protein by 3.7 kcal/mol, but more stable than the pathological, amyloidogenic variants. Unfolding kinetics in guanidine are six times faster than in the wild-type, but three and twenty times slower than in the amyloidogenic variants. Enzyme catalytic parameters, such as maximal velocity and affinity, are reduced in comparison to the wild-type. The solution structure, determined by 1H NMR and modeling calculations, exhibits a more compact arrangement at the interface between the beta-sheet domain and the subsequent loop on one side and part of the alpha domain on the other side, compared with the wild-type protein. This is the opposite of the conformational variation shown by the amyloidogenic variant D67H, but it accounts for the reduced stability and catalytic performance of T70N.

Alleles↗

PubMed2002

Conformational switching and fibrillogenesis in the amyloidogenic fragment of apolipoprotein a-I.

The N-terminal portion of apolipoprotein A-I corresponding to the first 93 residues has been identified as the main component of apolipoprotein A-I fibrils in a form of systemic amyloidosis. We have been able to characterize the process of conformational switching and fibrillogenesis in this fragment of apolipoprotein A-I purified directly from ex vivo amyloid material. The peptide exists in an unstructured form in aqueous solution at neutral pH. The acidification of the solution provokes a collapse into a more compact, intermediate state and the transient appearance of a helical conformation that rapidly converts to a stable, mainly beta-structure in the fibrils. The transition from helical to sheet structure occurs concomitantly with peptide self-aggregation, and fibrils are detected after 72 h. The alpha-helical conformation is induced by the addition of trifluoroethanol and phospholipids. Interaction of the amyloidogenic polypeptide with phospholipids prevents the switching from helical to beta-sheet form and inhibits fibril formation. The secondary structure propensity of the apolipoprotein A-I fragment appears poised between helix and the beta-sheet. These findings reinforce the idea of a delicate balance between natively stabilizing interactions and fatally stabilizing interactions and stress the importance of cellular localization and environment in the maintenance of protein conformation.

Amino Acid Sequence↗

PubMed2002

Capillary electrophoresis investigation of a partially unfolded conformation of beta(2)-microglobulin.

Dialysis-related amyloidosis is a disease in which partial unfolding of beta(2)-microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of beta(2)-microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full-length beta(2)-microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full-length beta(2)-microglobulin.

Amino Acid Sequence↗

PubMed2002

Topological investigation of amyloid fibrils obtained from beta2-microglobulin.

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact beta2-m and truncated DeltaN6beta2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of beta2-m within the fibrillar polymer. The distribution of prefential proteolytic sites observed in both fibrils revealed that the central region of the protein, which had been easily cleaved in the full-length globular beta2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of beta2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that beta2-m molecules in the fibrils consist of an unaccessible core comprising residues 20-87 with the strands I and VIII being not constrained in the fibrillar polymer and exposed to the proteases. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to occur in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native beta2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.

Amyloid↗

PubMed2002

The solution structure of human beta2-microglobulin reveals the prodromes of its amyloid transition.

The solution structure of human beta2-microglobulin (beta2-m), the nonpolymorphic component of class I major histocompatibility complex (MHC-I), was determined by (1)H NMR spectroscopy and restrained modeling calculations. Compared to previous structural data obtained from the NMR secondary structure of the isolated protein and the crystal structure of MHC-I, in which the protein is associated to the heavy-chain component, several differences are observed. The most important rearrangements were observed for (1) strands V and VI (loss of the C-terminal and N-terminal end, respectively), (2) interstrand loop V-VI, and (3) strand I, including the N-terminal segment (displacement outward of the molecular core). These modifications can be considered as the prodromes of the amyloid transition. Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches at the interface with heavy chain and the nearby region at the surface charge cluster of the C-terminal segment. As a result, the molecule is placed in a state in which even minor charge and solvation changes in response to pH or ionic-strength variations can easily compromise the hydrophobic/hydrophilic balance and trigger the transition into a partially unfolded intermediate that starts with unpairing of strand I and leads to polymerization and precipitation into fibrils or amorphous aggregates. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu(2+) binding, which is shown to occur primarily at His 31 and involve partially also His 13, the next available His residue along the partial unfolding pathway.

Amino Acid Sequence↗