No abstract

Tissue deposition of soluble proteins as amyloid fibrils underlies a range of fatal diseases. The two naturally occurring human lysozyme variants are both amyloidogenic, and are shown here to be unstable. They aggregate to form amyloid fibrils with transformation of the mainly helical native fold, observed in crystal structures, to the amyloid fibril cross-beta fold. Biophysical studies suggest that partly folded intermediates are involved in fibrillogenesis, and this may be relevant to amyloidosis generally.

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.

Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

The solution structure and stability of N-terminally truncated b2-microglobulin~DN6b2-m!, the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that DN6b2-m has a free energy of stabilization that is reduced by 2.5 kcal0mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at mM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that DN6b2-m is significantly less protected than its wild-type counterpart. Analysis of DN6b2-m by NMR shows that this loss of protection occurs in b strands I, III, and part of II. At mM concentration gel filtration analysis shows that DN6b2-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 b2-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 DN6b2-m could be a key intermediate of a proteolytic pathway of b2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of b2-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 b-strands far removed from this constrain is greatly perturbed.Keywords: amyloidosis; b2-microglobulin; hydrogen exchange mass spectrometry; limited proteolysis; NMR; protein folding Amyloidoses are diseases caused by tissue deposition of protein aggregate organized in an ordered b-sheet structure. The conversion of globular proteins to insoluble fibrillar aggregates requires significant conformational changes, such as the loss of tertiary and quaternary interactions or conversion of a to b secondary structurẽ Sunde & Blake, 1998!. Of the 17 or so proteins implicated in amyloidoses the fibril morphology is indistinguishable and there does not appear to be any common features that link the soluble precursor proteins. For many of these proteins, the amyloid fibril formation is facilitated by amino acid mutations that destabilize the native state and confer a structural flexibility to the molecule, but other proteins like IAPP, wild-type TTR, and b2-microglobulin

We describe a kindred with slowly progressive gastrointestinal symptoms and autonomic neuropathy caused by autosomal dominant, hereditary systemic amyloidosis. The amyloid consists of Asp76Asn variant β(2)-microglobulin. Unlike patients with dialysis-related amyloidosis caused by sustained high plasma concentrations of wild-type β(2)-microglobulin, the affected members of this kindred had normal renal function and normal circulating β(2)-microglobulin values. The Asp76Asn β(2)-microglobulin variant was thermodynamically unstable and remarkably fibrillogenic in vitro under physiological conditions. Previous studies of β(2)-microglobulin aggregation have not shown such amyloidogenicity for single-residue substitutions. Comprehensive biophysical characterization of the β(2)-microglobulin variant, including its 1.40-Å, three-dimensional structure, should allow further elucidation of fibrillogenesis and protein misfolding.

Accumulation of amyloid fibrils in the viscera and connective tissues causes systemic amyloidosis, which is responsible for about one per thousand deaths in developed countries1. Localised amyloid can also be very serious, for example cerebral amyloid angiopathy is an important cause of haemorrhagic stroke. The clinical presentations of amyloidosis are extremely diverse and the diagnosis is rarely made before significant organ damage is present1. There is therefore a major unmet medical need for therapy which safely promotes the clearance of established amyloid deposits. Over 20 different amyloid fibril proteins are responsible for different forms of clinically significant amyloidosis and treatments that substantially reduce the abundance of the respective amyloid fibril precursor protein can arrest amyloid accumulation1. Unfortunately control of fibril protein production is not possible in some forms of amyloidosis and in others is often slow and hazardous1. There is no therapy that directly targets amyloid deposits for enhanced clearance. However, all amyloid deposits contain the normal, non-fibrillar, plasma glycoprotein, serum amyloid P component (SAP)2, 3. Here we show that administration of anti-human SAP antibodies to mice with amyloid deposits containing human SAP, triggers a potent, complement dependent, macrophage-derived giant cell reaction which swiftly removes massive visceral amyloid deposits without adverse effects. Anti-SAP antibody treatment is clinically feasible because circulating human SAP can be depleted in patients by the bis-D-proline compound, CPHPC4, thereby enabling injected anti-SAP antibodies to reach residual SAP in the amyloid deposits. The unprecedented capacity of this novel combined therapy to eliminate amyloid deposits should be applicable to all forms of systemic and local amyloidosis.

The folding of ␤ 2 -microglobulin (␤ 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 ϳ14 ؎ 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 ␤ 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 ␤ 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 ␤ 2 -m but also to design new pharmaceutical approaches that consider such species as a putative molecular target.Dialysis-related amyloidosis represents an inevitable and severe complication of long term hemodialysis (1-4). Under this pathological condition, protein aggregates known as amyloid fibrils, accumulate in essential tissues, such as the skeletal muscle, interfering with their normal functions. A major constituent of the amyloid fibrils related to this pathological condition is ␤ 2 -microglobulin (␤ 2 -m).1 In its native form, this 99-residue protein is constituted by two ␤-sheets packed against each other to form a fold typical of the immunoglobulin superfamily (5). The two ␤-sheets, constituted by three and four strands, respectively, interact by means of hydrophobic interactions and a disulfide bridge that stabilizes further the ␤-sandwich structure.␤ 2 -m constitutes the light chain of the major histocompatibility complex class I (MHCI). A significant pool of ␤ 2 -m is also normally present in the plasma as a consequence of the constant release from the MHCI to allow the process of catabolic degradation in the kidney (1, 2). In chronic dialysis patients, the artificial membrane induces an inflammatory reaction, which causes the production and release of ␤ 2 -m to increase significantly (6). In addition, ␤ 2 -m cannot be filtered efficiently through the artificial membrane, resulting in an increase of the levels of soluble ␤ 2 -m from 0.3 to 30 g/ml, the range of concentrations observed within healthy individuals, to ϳ40 g/ml (7). The increase of free circulating ␤ 2 -m, the preferential substrate for amyloid deposition by this protein, is responsible, at least in part, for this form of amyloidosis in these patients (1, 2). Big efforts have been expended to improve the biocompatibility and performance of dialysis approaches. ...