Faculty Bibliography

Familial Danish dementia (FDD), also known as heredopathia ophthalmo-oto-encephalica, is an autosomal dominant disorder characterized by cataracts, deafness, progressive ataxia, and dementia. Neuropathological findings include severe widespread cerebral amyloid angiopathy, hippocampal plaques, and neurofibrillary tangles, similar to Alzheimer's disease. N-terminal sequence analysis of isolated leptomeningeal amyloid fibrils revealed homology to ABri, the peptide originated by a point mutation at the stop codon of gene BRI in familial British dementia. Molecular genetic analysis of the BRI gene in the Danish kindred showed a different defect, namely the presence of a 10-nt duplication (795-796insTTTAATTTGT) between codons 265 and 266, one codon before the normal stop codon 267. The decamer duplication mutation produces a frame-shift in the BRI sequence generating a larger-than-normal precursor protein, of which the amyloid subunit (designated ADan) comprises the last 34 C-terminal amino acids. This de novo-created amyloidogenic peptide, associated with a genetic defect in the Danish kindred, stresses the importance of amyloid formation as a causative factor in neurodegeneration and dementia

Expression of the breast and ovarian cancer gene BRCA1 is regulated at both the transcriptional and post-transcriptional levels. We found that the expression of the BRCA1 protein may also be regulated at the translational level. In addition to an AUG start codon at position 1, BRCA1 mRNA has a second in-frame AUG (+17) that acts as an alternative start codon to generate a novel BRCA1 protein that lacks the first 17 amino acids (DeltaBRCA1(17aa)). We fused cDNAs encoding the second exon of BRCA1 of the wild-type BRCA1 gene (wt-BRCA1) and a mutated BRCA1 gene (mt-BRCA1), in which the first initiation site and its Kozak consensus sequence were abolished, with the nucleophosmin (NPM) reporter gene and used them for in vitro and in vivo translation assays. In both systems, the wt-BRCA1-NPM constructs produced two distinct proteins (18 and 16 kD) begun from the first and second AUGs. The mt-BRCA1-NPM constructs produced only the shorter 16-kD protein lacking the first 17 amino acids of the BRCA1 gene. Next, we analysed the N-terminal protein sequence of purified BRCA1 protein from normal thymocytes and found two different BRCA1 proteins, derived from translation of the first and second in-frame AUGs. Thus, BRCA1 protein expression can be regulated at the translation level in normal cells. Characterization of DeltaBRCA1(17aa) may shed light on the function and regulation of BRCA1 in normal cells as well as the pathogenesis of breast and ovarian cancers. Oncogene (2000)

Familial British dementia (FBD) is a disorder characterized by the presence of amyloid deposits in cerebral blood vessels and brain parenchyma coexisting with neurofibrillary tangles in limbic areas. The amyloid subunit (ABri) is a 4 kDa fragment of a 266 amino acid type II single-spanning transmembrane precursor protein encoded by the BRI gene located on chromosome 13. In FBD patients, a single base substitution at the stop codon of this gene generates a larger 277-residue precursor (ABriPP-277). Proteolytic processing by a furin-like enzyme at the C-terminus of the elongated precursor generates the 34 amino acid ABri that undergoes rapid aggregation and fibrillization. ABri is structually unrelated to all known amyloids including A beta, the main component of the amyloid lesions in Alzheimer's disease (AD), indicating that cerebral deposition of amyloid molecules other than A beta can trigger similar neuropathological changes leading to neuronal loss and dementia. These data support the concept that amyloid deposition in the vascular wall and brain parenchyma is of primary importance in the initiation of neurogeneration

Familial British dementia (FBD) is an autosomal dominant neurodegenerative disorder clinically characterized by progressive dementia, spastic tetraparesis and cerebellar ataxia, with an age of onset in the fourth decade. The neuropathology of FBD (amyloid angiopathy, parenchymal plaques and neurofibrillary tangles) is similar to that of AD. Amyloid deposits in cerebral blood vessels and parenchymal plaques are mainly composed of a 4 kDa subunit, ^ABri, derived from a type II transmembrane precursor molecule mutated at the stop codon. The mutation produces a longer open reading frame that generates a larger 277 aa precursor. ^ABri is a 34 amino acids peptide released by proteolytic processing of the C-terminus of the mutated precursor protein (ABriPP277). We have identified soluble ABri (sABri) in serum and CSF of patients with FBD using a combination of immunoprecipitation, mass spectrometry and western blot analysis. The 4 kDa component was present in all tested carriers of the Stop-to-Arg mutation and consistently absent in non-carrier family members and normal controls. In contrast to the CNS-deposited ^ABri, sABri was monomeric and devoided of N-terminal pyroglutamate. In view of these findings, we tested systemic organs in an autopsy case of FBD for amyloid deposits. Antibodies specific to the ^ABri peptide labeled Congo red positive vascular lesions in several peripheral organs; in addition, parenchymal immunoreactivity was also seen in many of the tissues tested. Biochemical analysis of the deposited material isolated from pancreas, uterus and skeletal muscle identified ^ABri species similar to those found in amyloid lesions in the CNS (Nature, 399:776,1999). The main component was full-length ABri featuring N-terminus pyroglutamate. The data indicate that amyloid formation in FBD occurs not only in the brain but also peripherally. This is the first case of cerebral amyloidosis associated with neurodegeneration in demented patients where the amyloid deposition is also systemic

Familial British dementia (FBD), previously designated familial cerebral amyloid angiopathy-British type, is an autosomal dominant disorder of undetermined origin characterized by progressive dementia, spasticity, and cerebellar ataxia, with onset at around the fifth decade of life. Cerebral amyloid angiopathy, non-neuritic and perivascular plaques and neurofibrillary tangles are the predominant pathological lesions. Here we report the identification of a unique 4K protein subunit named ABri from isolated amyloid fibrils. This highly insoluble peptide is a fragment of a putative type-II single-spanning transmembrane precursor that is encoded by a novel gene, BRI, located on chromosome 13. A single base substitution at the stop codon of this gene generates a longer open reading frame, resulting in a larger, 277-residue precursor. Release of the 34 carboxy-terminal amino acids from the mutated precursor generates the ABri amyloid subunit. The mutation creates a cutting site for the restriction enzyme XbaI, which is useful for detecting asymptomatic carriers. Antibodies against the amyloid or homologous synthetic peptides recognize both parenchymal and vascular lesions in FBD patients. A point mutation at the stop codon of BRI therefore results in the generation of the ABri peptide, which is deposited as amyloid fibrils causing neuronal disfunction and dementia

Fibronectin (Fn) binds to fibrin in clots by covalent and non-covalent interactions. The N- and C-termini of Fn each contain one non-covalent fibrin-binding site, which are composed of type 1 (F1) structural repeats. We have previously localized the N-terminal site to the fourth and fifth F1 repeats (4F1.5F1). In the current studies, using proteolytic and recombinant proteins representing both the N- and C-terminal fibrin-binding regions, we localized and characterized the C-terminal fibrin-binding site, compared the relative fibrin-binding activities of both sites and determined the contribution of each site to the fibrin-binding activity of intact Fn. By fibrin-affinity chromatography, a protein composed of the 10F1 repeat through to the C-terminus of Fn (10F1-COOH), expressed in COS-1 cells, and 10F1-12F1, produced in Saccharomyces cerevisiae, displayed fibrin-binding activity. However, since 10F1 and 10F1.11F1 were not active, the presence of 12F1 is required for fibrin binding. A proteolytic fragment of 14.4 kDa, beginning 14 residues N-terminal to 10F1, was isolated from the fibrin-affinity matrix. Radio-iodinated 14.4 kDa fibrin-binding peptide/protein (FBP) demonstrated a dose-dependent and saturable binding to fibrin-coated wells that was both competitively inhibited and reversed by unlabelled 14.4 kDa FBP. Comparison of the fibrin-binding affinities of proteolytic FBPs from the N-terminus (25.9 kDa FBP), the C-terminus (14.4 kDa) and intact Fn by ELISA yielded estimated Kd values of 216, 18 and 2.1 nM, respectively. The higher fibrin-binding affinity of the N-terminus was substantiated by the ability of both a recombinant 4F1.5F1 and a monoclonal antibody (mAb) to this site to maximally inhibit biotinylated Fn binding to fibrin by 80%, and by blocking the 90% inhibitory activity of a polyclonal anti-Fn, by absorption with the 25.9 kDa FBP. We propose that whereas the N-terminal site appears to contribute to most of the binding activity of native Fn to fibrin, the specific binding of the C-terminal site may strengthen this interaction

Disorders of immunoglobulin (Ig) synthesis that occur in malignant plasma-cell proliferation may result in either granular (LCDD) or fibrillar (AL) tissue deposition of light-chain monoclonal components. The structural features that govern the transition from soluble polypeptides to either fibrillar or granular conformational states remain undefined. Among the many factors presumed to play a role in these transitions the net charge of the molecule has been associated with folding conformation changes. The majority of the proteins involved in AL amyloidosis show acidic isoelectric points (pI 3.8-5.2), whereas most L chains with basic pIs deposit in granular patterns. In our studies a 12 kD VkappaIII fragment was purified as the main component of the fibrils isolated from myocardium and adipose tissue of the pericardium obtained post-mortem from an individual with systemic AL amyloidosis. An apparently identical 12 kD VL fragment with the same N-terminal sequence constituted the BJ protein present in the urine. This urinary protein exhibited strikingly cathodic electrophoretic mobility on agarose gels and lacked retention by anionic exchange chromatography matrices, indicative of a highly basic pI (>10). When it was subjected to in vitro fibril-formation experiments, the BJ protein adopted a fibrillar conformation only at acidic pHs, remaining aggregated but not fibrillar at physiological pH. The data indicate that a specific tissue deposition pattern involves not only structural properties of the protein but rather more complex mechanisms in which acidic micro-environments may contribute to the stabilization of amyloidogenic conformations