alpha-chymotrypsin and Amyloidosis

Amyloid fibrils of patients treated with regular haemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment shows a higher propensity to self-aggregate and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology and the conformational analysis of native beta2-m and the truncated DeltaN6beta2-m species both in the soluble and in the fibrillar forms were investigated by the limited proteolysis/mass spectrometry strategy. The conformation in solution of a further truncated mutant DeltaN3beta2-m lacking three residues at the N-terminus was also examined. This approach appeared particularly suited to investigate the regions that are solvent-exposed, or flexible enough to be accessible to protein-protein interactions and to describe the conformation of transient intermediates. Moreover, proteolysis experiments can also be tailored to investigate amyloid fibrils by discriminating the protein regions constituting the unaccessible core of the fibrils and those still flexible and exposed to the solvent. Although native beta2-m and DeltaN3beta2-m shared essentially the same conformation, significative structural differences exist between the native and the DeltaN6beta2-m proteins in solution with major differences located at the end moiety of strand V and subsequent loop with strand VI and at both the N- and C-termini of the proteins. On the contrary, an identical distribution of preferential proteolytic sites was observed in both proteins in the fibrillar state, which was nearly superimposible to that observed for the soluble form of DeltaN6beta2-m. These data revealed that synthetic fibrils essentially consists of an unaccessible core comprising residues 20-87 of the beta2-m protein with exposed and flexible N- and C-terminal ends. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to take place in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these results, a molecular mechanism for fibril formation has been proposed.

Topics: Amyloid; Amyloidosis; beta 2-Microglobulin; Chromatography, High Pressure Liquid; Chymotrypsin; Humans; Metalloendopeptidases; Peptide Fragments; Peptide Hydrolases; Protein Conformation; Protein Structure, Quaternary; Renal Dialysis; Spectrometry, Mass, Electrospray Ionization; Trypsin

Topics: Amyloidosis; Bromelains; Chymotrypsin; Dimethyl Sulfoxide; Humans; Intestinal Absorption; Molecular Weight; Muramidase; Nervous System Diseases

Topics: Amyloidosis; Animals; Arteriosclerosis; Arthritis, Rheumatoid; Blood Coagulation Disorders; Blood Coagulation Factors; Chymotrypsin; Chymotrypsinogen; Diabetes Mellitus; Emphysema; Fibrinolysis; Gastrointestinal Diseases; Humans; Kallikreins; Kidney Diseases; Muscular Dystrophies; Oligopeptides; Peptide Hydrolases; Substrate Specificity; Swine

Cerebrovascular amyloidosis belongs to the pathological hallmarks of Alzheimer's disease brains. Although definite proof is still lacking, it is very well possible that the amyloid and its associated proteins are produced locally in the brain. In this paper we describe the development of a model system of cultured human brain pericytes to study the mechanisms of microvascular amyloid formation in vitro. These cultured cells may serve to study several aspects of cerebrovascular amyloidosis, which include the production of the amyloid precursor protein and of amyloid beta-protein-associated proteins as well as cytotoxic effects of amyloid beta-protein on perivascular cells. We demonstrated that pericytes produce and metabolize the amyloid precursor protein, and that they produce amyloid beta-protein-associated proteins, such as heparan sulfate proteoglycans, apolipoprotein E, and complement factor C1q. They are also prone to cellular degeneration after treatment with amyloid beta-protein, which is accompanied by increased expression of a number of amyloid beta-protein-associated proteins. This may be an important mechanism to explain the cell death observed in vivo. Our data indicate that this cell culture model of human brain pericytes provides a useful and pathophysiologically relevant tool to study cerebrovascular amyloidosis.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloidosis; Apolipoproteins; Blotting, Western; Brain; Cell Survival; Cells, Cultured; Cerebrovascular Disorders; Chymotrypsin; Heparan Sulfate Proteoglycans; Humans; Immunohistochemistry; In Vitro Techniques; Pericytes; Time Factors

Extracellular deposition of amyloid fibrils is responsible for the pathology in the systemic amyloidoses and probably also in Alzheimer disease [Haass, C. & Selkoe, D. J. (1993) Cell 75, 1039-1042] and type II diabetes mellitus [Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760]. The fibrils themselves are relatively resistant to proteolysis in vitro but amyloid deposits do regress in vivo, usually with clinical benefit, if new amyloid fibril formation can be halted. Serum amyloid P component (SAP) binds to all types of amyloid fibrils and is a universal constituent of amyloid deposits, including the plaques, amorphous amyloid beta protein deposits and neurofibrillary tangles of Alzheimer disease [Coria, F., Castano, E., Prelli, F., Larrondo-Lillo, M., van Duinen, S., Shelanski, M. L. & Frangione, B. (1988) Lab. Invest. 58, 454-458; Duong, T., Pommier, E. C. & Scheibel, A. B. (1989) Acta Neuropathol. 78, 429-437]. Here we show that SAP prevents proteolysis of the amyloid fibrils of Alzheimer disease, of systemic amyloid A amyloidosis and of systemic monoclonal light chain amyloidosis and may thereby contribute to their persistence in vivo. SAP is not an enzyme inhibitor and is protective only when bound to the fibrils. Interference with binding of SAP to amyloid fibrils in vivo is thus an attractive therapeutic objective, achievement of which should promote regression of the deposits.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Protein Precursor; Amyloidosis; Chymotrypsin; Galactose; Humans; Kinetics; Neurofibrillary Tangles; Protein Binding; Serum Amyloid A Protein; Spleen; Trypsin

The effects of 4 proteolytic enzymes, alpha-chymotrypsin, bromeline, collagenase, and lysozyme on amyloid tissue sections from a patient with familial amyloidotic polyneuropathy (FAP) were evaluated. Degradation of amyloid fibrils was significant with alpha-chymotrypsin, moderate with bromeline and collagenase, and slight with lysozyme. All of these proteases except collagenase are used as oral mucolytics in humans. The possibility of their clinical usefulness in the treatment or prevention of the development of FAP is discussed.

Topics: Amyloid; Amyloidosis; Bromelains; Chymotrypsin; Humans; In Vitro Techniques; Kidney; Microbial Collagenase; Muramidase; Peptide Hydrolases; Peripheral Nervous System Diseases

Human serum contains enzyme(s) able to degrade serum amyloid A protein (SAA) and amyloid A (AA) fibrils. On the basis of inhibition tests these enzymes are regarded as serine proteases, but further characterization of the enzymes has, however, so far not been done. Chymotrypsin, trypsin, elastase, collagenase and kallikrein, when added to SAA-containing serum, all degraded SAA to peptides within 2 h at 37 degrees C. With the exception of collagenase these enzymes also destroyed the Sirius-Red-binding ability of amyloidotic tissue and that of isolated AA fibrils. Hence, they altered the conformation of the beta-pleated structure and possibly also degraded the fibrils. These results suggest that any of these serine proteases could be responsible of the degradation of SAA in serum. The enzyme concentrations needed to degrade amyloid fibrils, however, were much higher than normally found in serum. Thus, it is unlikely that the amyloid-fibril-degrading activity in serum could be due to any of these enzymes.

Topics: Amyloidosis; Chymotrypsin; Endopeptidases; Humans; Kallikreins; Microbial Collagenase; Pancreatic Elastase; Serine Endopeptidases; Staining and Labeling; Trypsin

Topics: Amino Acid Sequence; Amino Acids; Ammonium Sulfate; Amyloid; Amyloidosis; Aspartic Acid; Autoanalysis; Bence Jones Protein; Carboxypeptidases; Chromatography, Ion Exchange; Chymotrypsin; Glutamates; Humans; Hydrogen-Ion Concentration; Hydrolysis; Isoantigens; Leucyl Aminopeptidase; Pepsin A; Peptides; Thiocyanates; Trypsin

Topics: Adult; Amyloidosis; Antimetabolites; Chymotrypsin; Female; Glomerulonephritis; Humans; Kidney Diseases; Lupus Erythematosus, Systemic; Male; Middle Aged; Nephrotic Syndrome; Prednisolone; Pyelonephritis; Trypsin; Trypsin Inhibitors