Biomedical subjects

Isam Qahwash

Publications and source records attributed to Isam Qahwash.

4 recordsLinked to original sources

PubMed2004

Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation.

Inhibiting the activity of the beta-amyloid converting enzyme 1 (BACE1) or reducing levels of BACE1 in vivo decreases the production of amyloid-beta. The reticulon family of proteins has four members, RTN1, RTN2, RTN3 and RTN4 (also known as Nogo), the last of which is well known for its role in inhibiting neuritic outgrowth after injury. Here we show that reticulon family members are binding partners of BACE1. In brain, BACE1 mainly colocalizes with RTN3 in neurons, whereas RTN4 is more enriched in oligodendrocytes. An increase in the expression of any reticulon protein substantially reduces the production of Abeta. Conversely, lowering the expression of RTN3 by RNA interference increases the secretion of Abeta, suggesting that reticulon proteins are negative modulators of BACE1 in cells. Our data support a mechanism by which reticulon proteins block access of BACE1 to amyloid precursor protein and reduce the cleavage of this protein. Thus, changes in the expression of reticulon proteins in the human brain are likely to affect cellular amyloid-beta and the formation of amyloid plaques.

Amyloid Precursor Protein Secretases↗

PubMed2004

Processing amyloid precursor protein at the beta-site requires proper orientation to be accessed by BACE1.

Membrane-bound BACE1 naturally cleaves its transmembrane substrate amyloid precursor protein (APP) at the two adjacent beta- and beta'-sites. Cleavage at these two sites generates the heterogeneous N-terminal end of APP C-terminal fragments that are further processed by gamma-secretase to release Abeta-(1-40/42) or Abeta-(11-40/42). The significance underlying Abeta-(11-40/42) in Alzheimer's disease pathogenesis has remained to be experimentally elucidated, but increased production of Abeta-(1-40/42) has been broadly demonstrated to contribute to amyloid depositions in senile plaques. In this study, we show that the cleavage of APP at the beta-site by BACE1 is readily disrupted through limited structural twists, whereas the beta'-site is relatively better positioned to gain access to the BACE1 catalytic cavity. Radical insertion or deletion of residues between beta- and beta'-site also favors cleavage of APP at the beta'-site. On the other hand, either lengthening or shortening the loop region of BACE1 has a minor impact on the selective cleavage of APP at these two adjacent sites, but significantly shortening the loop region impairs the ability of BACE1 to process APP at both sites. Thus, processing of APP by BACE1 is clearly dependent on a mutual structural compatibility in addition to the sequence feature. The knowledge gained from this study will potentially offer an opportunity for rational design of small molecule drugs to block the cleavage of APP specifically at the beta-site while not disturbing the functions of other cellular aspartyl proteases.

Amino Acid Sequence↗

PubMed2003

Identification of a mutant amyloid peptide that predominantly forms neurotoxic protofibrillar aggregates.

The amyloid peptide (Abeta), derived from the proteolytic cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases, undergoes multistage assemblies to fibrillar depositions in the Alzheimer's brains. Abeta protofibrils were previously identified as an intermediate preceding insoluble fibrils. While characterizing a synthetic Abeta variant named EV40 that has mutations in the first two amino acids (D1E/A2V), we discerned unusual aggregation profiles of this variant. In comparison of the fibrillogenesis and cellular toxicity of EV40 to the wild-type Abeta peptide (Abeta40), we found that Abeta40 formed long fibrillar aggregates while EV40 formed only protofibrillar aggregates under the same in vitro incubation conditions. Cellular toxicity assays indicated that EV40 was slightly more toxic than Abeta40 to human neuroblastoma SHEP cells, rat primary cortical, and hippocampal neurons. Like Abeta40, the neurotoxicity of the protofibrillar EV40 could be partially attributed to apoptosis since multiple caspases such as caspase-9 were activated after SHEP cells were challenged with toxic concentrations of EV40. This suggested that apoptosis-induced neuronal loss might occur before extensive depositions of long amyloid fibrils in AD brains. This study has been the first to show that a mutated Abeta peptide formed only protofibrillar species and mutations of the amyloid peptide at the N-terminal side affect the dynamic amyloid fibrillogenesis. Thus, the identification of EV40 may lead to further understanding of the structural perturbation of Abeta to its fibrillation.

Amino Acid Substitution↗

PubMed2003

Employing a superior BACE1 cleavage sequence to probe cellular APP processing.

The involvement of beta-secretase (BACE1; beta-site APP-cleaving enzyme) in producing the beta-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2' sequence, ...VKM--DA..., was replaced by ...ISY--EV... (-- = beta site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL--DAEFR. The expanded peptide, GLTNIKTEEISEISY--EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these beta-site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the beta-site. Insertion of the optimized ISY--EV motif at either the beta-site (Asp1) or beta'-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.

Alzheimer Disease↗