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P Mangione

Publications and source records attributed to P Mangione.

At least 19 recordsLinked to original sources
PubMed2004

beta2-microglobulin H31Y variant 3D structure highlights the protein natural propensity towards intermolecular aggregation.

beta2-Microglobulin (beta2m) is the non-covalently bound light chain of the human class I major histocompatibility complex (MHC-I). The natural turnover of MHC-I gives rise to the release of beta2m into plasmatic fluids and to its catabolism in the kidney. beta2m dissociation from the heavy chain of the complex is a severe complication in patients receiving prolonged hemodialysis. As a consequence of renal failure, the increasing beta2m concentrations can lead to deposition of the protein as amyloid fibrils. Here we characterize the His31-->Tyr human beta2m mutant, a non-natural form of beta2m that is more stable than the wild-type protein, displaying a ten-fold acceleration of the slow phase of folding. We report the 2.9A resolution crystal structure and the NMR characterization of the mutant beta2m, focussing on selected structural features and on the molecular packing observed in the crystals. Juxtaposition of the four mutant beta2m molecules contained in the crystal asymmetric unit, and specific hydrogen bonds, stabilize a compact protein assembly. Conformational heterogeneity of the four independent molecules, some of their mutual interactions and partial unpairing of the N-terminal beta-strand in one protomer are in keeping with the amyloidogenic properties displayed by the mutant beta2m.

Amino Acid Substitution↗
PubMed2001

A partially structured species of beta 2-microglobulin is significantly populated under physiological conditions and involved in fibrillogenesis.

The folding of beta(2)-microglobulin (beta(2)-m), the protein forming amyloid deposits in dialysis-related amyloidosis, involves formation of a partially folded conformation named I(2), which slowly converts into the native fold, N. Here we show that the partially folded species I(2) can be separated from N by capillary electrophoresis. Data obtained with this technique and analysis of kinetic data obtained with intrinsic fluorescence indicate that the I(2) conformation is populated to approximately 14 +/- 8% at equilibrium under conditions of pH and temperature close to physiological. In the presence of fibrils extracted from patients, the I(2) conformer has a 5-fold higher propensity to aggregate than N, as indicated by the thioflavine T test and light scattering measurements. A mechanism of aggregation of beta(2)-m in vivo involving the association of the preformed fibrils with the fraction of I(2) existing at equilibrium is proposed from these results. The possibility of isolating and quantifying a partially folded conformer of beta(2)-m involved in the amyloidogenesis process provides new opportunities to monitor hemodialytic procedures aimed at the reduction of such species from the pool of circulating beta(2)-m but also to design new pharmaceutical approaches that consider such species as a putative molecular target.

Benzothiazoles↗
PubMed2001

Detection of two partially structured species in the folding process of the amyloidogenic protein beta 2-microglobulin.

beta 2-Microglobulin is a small, major histocompatibility complex class I-associated protein that undergoes aggregation and accumulates as amyloid deposits in human tissues as a consequence of long-term haemodialysis. The folding process of this amyloidogenic protein has been studied in vitro by diluting the guanidine hydrochloride-denatured protein in refolding buffer at pH 7.4 and monitoring the folding process by means of a number of spectroscopic probes that allow the native structure of the protein to be detected as it develops. These techniques include fluorescence spectroscopy, far and near-UV circular dichroism, 8-anilino-1-naphthalenesulfonic acid binding and double jump assays. All spectroscopic probes indicate that a significant amount of structure forms within the dead-time of stopped-flow measurements (<5 ms). The folding reaction goes to completion through a fast phase followed by a slow phase, whose rate constants are ca 5.1 and 0.0030 s(-1) in water, respectively. Unfolding-folding double jump experiments, together with the use of peptidyl prolyl isomerase, reveal that the slow phase of folding of beta 2-microglobulin is not fundamentally determined by cis/trans isomerisation of X-Pro peptide bonds. Other folding-unfolding double jump experiments also suggest that the fast and slow phases of folding are not related to independent folding of different populations of protein molecules. Rather, we provide evidence for a sequential mechanism of folding where denatured beta 2-microglobulin collapses to an ensemble of partially folded conformations (I(1)) which fold subsequently to a more highly structured species (I(2)) and, finally, attain the native state. The partially folded species I(2) appears to be closely similar to previously studied amyloidogenic forms of beta 2-microglobulin, such as those adopted by the protein at mildly acid pH values and by a variant with six residues deleted at the N terminus. Since amyloid formation in vivo originates from partial denaturation of beta 2-microglobulin under conditions favouring the folding process, the long-lived, partially structured species detected here might be significantly populated under some physiological conditions and hence might play an important role in the process of amyloid formation.

Amyloidosis↗
PubMed2001

Amyloid fibrils derived from the apolipoprotein A1 Leu174Ser variant contain elements of ordered helical structure.

We recently described a new apolipoprotein A1 variant presenting a Leu174Ser replacement mutation that is associated with a familial form of systemic amyloidosis displaying predominant heart involvement. We have now identified a second unrelated patient with very similar clinical presentation and carrying the identical apolipoprotein A1 mutation. In this new patient the main protein constituent of the amyloid fibrils is the polypeptide derived from the first 93 residues of the protein, the identical fragment to that found in the patient previously described to carry this mutation. The X-ray fiber diffraction pattern obtained from preparations of partially aligned fibrils displays the cross-beta reflections characteristic of all amyloid fibrils. In addition to these cross-beta reflections, other reflections suggest the presence of well-defined coiled-coil helical structure arranged with a defined orientation within the fibrils. In both cases the fibrils contain a trace amount of full-length apolipoprotein A1 with an apparent prevalence of the wild-type species over the variant protein. We have found a ratio of full-length wild-type to mutant protein in plasma HDL of three to one. The polypeptide 1--93 purified from natural fibrils can be solubilized in aqueous solutions containing denaturants, and after removal of denaturants it acquires a monomeric state that, based on CD and NMR studies, has a predominantly random coil structure. The addition of phospholipids to the monomeric form induces the formation of some helical structure, thought most likely to occur at the C-terminal end of the polypeptide.

Amino Acid Substitution↗
PubMed2000

Affinity capillary electrophoresis is a powerful tool to identify transthyretin binding drugs for potential therapeutic use in amyloidosis.

In this work we used affinity capillary electrophoresis (ACE) to investigate the extent of interaction between a pool of drugs and wild-type transthyretin. After qualitative preliminary screening, attention was focused on the most promising molecules, flufenamic acid and flurbiprofen, which underwent a further stage of investigation, the determination of the binding constants, and, when possible, the assessment of the number of binding sites by ACE, frontal analysis (FA) capillary electrophoresis (CE) and parallel ultrafiltration (UF) experiments. Furthermore, our data demonstrate that FA CE is a suitable technique for identifying fibril ligands. This represents a novel CE application of pharmaceutical interest.

Amyloidosis↗
PubMed2000

Review: immunoglobulin light chain amyloidosis--the archetype of structural and pathogenic variability.

AL amyloidosis is caused by deposition in target tissue of amyloid fibrils constituted by monoclonal immunoglobulin light chains. The amyloidogenic plasma cells derive from a transformed memory B cell that can be identified by anti-idiotype monoclonal antibodies. Comparison of the primary structures of amyloidogenic and nonamyloidogenic light chains does not show any common structural motif in the amyloidogenic variants but reveals peculiar replacements which can destabilize the folding state. Reduced folding stability now appears to be a unifying property of amyloidogenic light chains. The tendency of these proteins to populate a partially unfolded intermediate state is a key event in the self-association that progresses to the formation of oligomers and fibrils. The mechanism of organ damage caused by AL amyloid deposition is not known, but clinical findings suggest that the process of amyloid fibril formation itself exerts tissue toxic effects independently of the amount of amyloid deposited. Since the disease is caused by the neoplastic expansion of the plasma cell population synthesizing the amyloidogenic light chains, the clone represents the prime therapeutic target of conventional chemotherapy and experimental immunotherapy. In common with other types of amyloidosis the therapeutic strategy can take advantage of drugs able to improve the reabsorption of the amyloid deposits or able to bind and stabilize the light chain in the native-like folded state.

Amyloidosis↗
PubMed2000

Conformational dynamics of the beta2-microglobulin C terminal in the cell-membrane-anchored major histocompatibility complex type I.

We have recently described an anti-beta2-microglobulin (beta2-m) monoclonal antibody (mAb 14H3) capable of recognizing the epitope 92-99 of the protein in the monomeric native state as well as in the fibrillar polymeric state, but not in the major histocompatibility complex type I (MHCI) anchored to the cell membrane. In the present study, we investigated the molecular basis for the inaccessibility of the C-terminal end of beta2-m in the MHCI complex, and demonstrated that mAb 14H3 binds the soluble fraction of the MHCI complex with a Kd of 0.3 microM. An interaction between the complex and the membrane protects beta2-m from immunological recognition at the MHCI level. This protection from antibody recognition can be weakened by procedures such as heat shock or gamma irradiation that perturb the membrane structure and commit the cell to the apoptotic pathway. mAb 14H3 can recognize MHCI in a transient state that most likely precedes beta2-m shedding and may be proposed as a useful tool for dynamic analysis of MHCI conformational modifications.

Antibodies, Monoclonal↗
PubMed2000

Removal of the N-terminal hexapeptide from human beta2-microglobulin facilitates protein aggregation and fibril formation.

The solution structure and stability of N-terminally truncated beta2-microglobulin (deltaN6beta2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that deltaN6beta2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at microM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that deltaN6beta2-m is significantly less protected than its wild-type counterpart. Analysis of deltaN6beta2-m by NMR shows that this loss of protection occurs in beta strands I, III, and part of II. At mM concentration gel filtration analysis shows that deltaN6beta2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of beta2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that deltaN6beta2-m could be a key intermediate of a proteolytic pathway of beta2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of beta2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between beta-strands far removed from this constrain is greatly perturbed.

Amino Acid Sequence↗
PubMed1999

Biological activity and pathological implications of misfolded proteins.

The physiological metabolism of proteins guarantees that different cellular compartments contain the appropriate concentration of proteins to perform their biological functions and, after a variable period of wear and tear, mediates their natural catabolism. The equilibrium between protein synthesis and catabolism ensures an effective turnover, but hereditary or acquired abnormalities of protein structure can provoke a premature loss of biological function, an accelerated catabolism and diseases caused by the loss of an irreplaceable function. In certain proteins, abnormal structure and metabolism are associated with a strong tendency to self-aggregation into a polymeric fibrillar structure, and in these cases the disease is not principally caused by the loss of an irreplaceable function but by the action of this new biological entity. Amyloid fibrils are an apparently inert, insoluble, mainly extracellular protein polymer that kills the cell without tissue necrosis but by activation of the apoptotic mechanism. We analyzed the data reported so far on the structural and functional properties of four prototypic proteins with well-known biological functions (lysozyme, transthyretin, beta 2-microglobulin and apolipoprotein AI) that are able to create amyloid fibrils under certain conditions, with the perspective of evaluating whether the achievement of biological function favors or inhibits the process of fibril formation. Furthermore, studying the biological functions carried out by amyloid fibrils reveals new types of protein-protein interactions in the transmission of messages to cells and may provide new ideas for effective therapeutic strategies.

Alzheimer Disease↗
PubMed1999

The new apolipoprotein A-I variant leu(174) --> Ser causes hereditary cardiac amyloidosis, and the amyloid fibrils are constituted by the 93-residue N-terminal polypeptide.

We identified a novel missense mutation in the apolipoprotein A-I gene, T2069C Leu(174) --> Ser, in a patient affected by familial systemic nonneuropathic amyloidosis. The amyloid deposits mostly affected the heart of the proband, who underwent transplantation for end-stage congestive heart failure. Amyloid fibrils of myocardial and periumbilical fat samples immunoreacted exclusively with anti-ApoA-I antibodies. Amyloid fibrils extracted from the heart were constituted, according to amino acid sequencing and mass spectrometry analysis, by an amino-terminal polypeptide ending at Val(93) of apolipoprotein A-I (apoA-I); no other significant fragments were detected. The mutation segregates with the disease; it was demonstrated in the proband and in an affected uncle and excluded in three healthy siblings. The plasma levels of high-density lipoprotein and apoA-I were significantly lower in the patient than in unaffected individuals. This represents the first case of familial apoA-I amyloidosis in which the mutation is outside the polypeptide fragment deposited as fibrils. Visualization of the mutation in the three-dimensional structure of lipid-free apoA-I, composed of four identical polypeptide chains, indicates that position 174 of one chain is located near position 93 of an adjacent chain and suggests that the amino acid replacement in position 174 is permissive for a proteolytic split at the C-terminal of Val(93).

Amino Acid Substitution↗
PubMed1999

[Thoracoscopy versus thoracotomy in spinal surgery: comparison of 2 paired series].

PURPOSE OF THE STUDY: This study was carried out to compare the intraoperative and postoperative results of conventional surgery of the thoracic spine by thoracotomy with those of the thoracoscopic technique to determine the advantages of this new approach. MATERIAL AND METHODS: A series of twenty-nine patients operated by thoracoscopy for a spinal disorder was matched regarding the etiology of spine disease and type of surgical procedure with twenty-four patients operated by thoracotomy. This matching procedure yielded two similar groups of twenty patients. The criteria used for evaluation were the duration of the procedure, blood loss, intraoperative complications, the duration of stay in postoperative intensive care, the duration and yield of pleural drainage, the time until return to the upright position, duration of use of WHO grade-three analgesics (morphine derivatives), the postoperative complications, and the length of hospitalization. RESULTS: There was a significant difference in three parameters: the duration of the procedure (thoracotomy, 172 min; thoracoscopy, 246 min; p < 0.006), intraoperative bleeding (thoracotomy, 837 mL; thoracoscopy, 447 mL; p < 0.0009), and duration of use of WHO grade-three analgesics (thoracotomy, 4.5 days; thoracoscopy, 2.3 days; p = 0.011). There was no difference in the intra- or postoperative complication rates of the two methods. DISCUSSION: The better view provided by thoracoscopy and its preservation of the wall structures probably explain why there was less bleeding and postoperative pain with this technique. The insufficiency of the current thoracoscopic instrumentation and the learning curve account for the longer duration of these interventions. CONCLUSION: These data confirm the usefulness of thoracoscopy which is less traumatizing, less hemorrhagic, and causes no more complications than thoracotomy. The longer operative duration is currently a minor drawback, and should shorten with experience and the development of specific instrumentation.

Adolescent↗
PubMed1998

Beta2-microglobulin can be refolded into a native state from ex vivo amyloid fibrils.

Beta2-microglobulin fibrils have been extracted from the femoral head of a patient who has been under chronic haemodialysis for 11 years. The primary structure of the N-terminal portion of the protein and mass determination by electrospray mass spectrometry demonstrate that beta2-microglobulin, extracted as fibrils by the water extraction procedure, was not glycated and that Asn17 was not deamidated. Limited proteolysis was observed in more than 20% of beta2-microglobulin molecules and the main cleavage sites were at the C-terminus of Lys6 and Tyr10. Beta2-microglobulin from fibrils has been purified by gel filtration in 6 M Gdn/HCl and submitted to a refolding procedure. The refolding conditions have been determined through the study of the unfolding pathway of the native protein. Beta2-microglobulin is stable at neutral pH where it displays a lower tendency to self-aggregate than in acidic conditions. Pulse dilution and extensive dialysis in refolding buffer at pH 7.5 yields beta2-microglobulin with a tertiary structure identical to that of the native form. The CD spectrum in the near-ultraviolet region and the spectrum of the intrinsic fluorescence of Trp overlap those of the native protein, but the CD spectrum in the far-ultraviolet region is affected by the contribution of oligomers created by beta2-microglobulin fragments that reduce the positive light polarisation at 205 nm typical of native beta2-microglobulin.

Amino Acid Sequence↗
PubMed1998

Evidence that amyloidogenic light chains undergo antigen-driven selection.

AL amyloidosis is characterized by fibrillar tissue deposits (amyloid) composed of monoclonal light chains secreted by small numbers of indolent bone marrow plasma cells whose ontogenesis is unknown. To address this issue and to provide insights into the processes that accompanied pathogenic light chain formation, we isolated the complete variable (V) regions of 14 light (VL) and 3 heavy (VH) chains secreted by amyloid clones at diagnosis (10 Bence Jones and 4 with complete Igs, 9 lambda and 5 kappa) by using an inverse polymerase chain reaction-based approach free of primer-induced biases. Amyloid V regions were found to be highly mutated compared with the closest germline genes in the databases or those isolated from the patients' DNA, and mutations were not associated with intraclonal diversification. Apparently high usage of the lambdaIII family germline gene V lambdaIII.1 was observed (4 of 9 lambda light chains). Analysis of the nature and distribution of somatic mutations in amyloid V regions showed that there was statistical evidence of antigen selection in 8 of 14 clones (7 in VL and 1 in VH). These results indicate that a substantial proportion of the amyloid clones developed from B cells selected for improved antigen binding properties and that pathogenic light chains show evidence of this selection.

Aged↗
PubMed1997

Use of anti-(beta2 microglobulin) mAb to study formation of amyloid fibrils.

Three mAbs, IgG1k 1F11, 7B6 and 14H3, were raised against in vitro-self-aggregated beta2-microglobulin. They recognize the native and unfolded forms of the protein and bind its fibrillar form that is present in amyloid tissue. When assayed in fibrillogenesis tests in vitro, mAb 14H3 inhibited fibril formation from beta2-microglobulin. This mAb recognizes a sequential epitope corresponding to the C-terminal octapeptide, residues 92-99, of beta2-microglobulin. By using synthetic peptides it has been found that the integrity of the sequence is essential for the formation of the immunocomplex: the binding affinity is lowered by one order of magnitude (Kd from 10(-7) M to 10(-6) M) by removal of Met99 and completely abolished when both Asp98 and Met99 are lost or Arg98 is substituted with Lys. The other two mAbs, 1F11 and 7B6, which bind sequences 20-41 and 63-75, respectively, are without effect on beta2-microglobulin fibrillogenesis. These two mAbs recognize beta2-microglobulin bound to the heavy chain in the major histocompatibility complex of type I located in the cell membrane, a property which is not shared by mAb 14H3.

Amyloid↗