Abstract. The majority of mutations causing familial Alzheimer's disease (fAD) have been found in the gene PRESENILIN1 (PSEN1) with additional mutations in the related gene PRESENILIN2 (PSEN2). The best characterized function of PRESE-NILIN (PSEN) proteins is in ␥-secretase enzyme activity. One substrate of ␥-secretase is encoded by the gene AMYLOID BETA A4 PRECURSOR PROTEIN (APP/APP) that is a fAD mutation locus. APP is the source of the amyloid- (A) peptide enriched in the brains of people with fAD or the more common, late onset, sporadic form of AD, sAD. These observations have resulted in a focus on ␥-secretase activity and A as we attempt to understand the molecular basis of AD pathology. In this paper we briefly review some of the history of research on ␥-secretase in AD. We then discuss the main ideas regarding the role of ␥-secretase and the PSEN genes in this disease. We examine the significance of the "fAD mutation reading frame preservation rule" that applies to PSEN1 and PSEN2 (and APP) and look at alternative roles for APP and A in fAD. We present a case for an alternative interpretation of published data on the role of ␥-secretase activity and fAD-associated mutations in AD pathology. Evidence supports a "PSEN holoprotein multimer hypothesis" where PSEN
The HIGH MOBILITY GROUP AT-HOOK 1 (HMGA1) family of chromatin-binding proteins plays important roles in cellular responses to low oxygen. HMGA1 proteins regulate gene activity both in the nucleus and within mitochondria. They are expressed mainly during embryogenesis and their upregulation in cancerous cells indicates poor prognosis. The human HMGA1a isoform is upregulated under hypoxia via oxidative stress-dependent signalling and can then bind nascent transcripts of the familial Alzheimer's disease gene PSEN2 to regulate alternative splicing to produce the truncated PSEN2 protein isoform PS2V. Zebrafish where hmga1a expression is induced by hypoxia to control splicing of the psen1 gene to produce the PS2V-equivalent isoform PS1IV. Zebrafish possess a second gene with apparent HMGA1 orthology, hmga1b. Here we investigate the predicted structure of Hmga1b protein and demonstrate it to be co-orthologous to human HMGA1 and most similar in structure to human isoform HMGA1c. We show that forced over-expression of either hmga1a or hmga1b mRNA can suppress the action of the cytotoxin hydroxyurea in stimulating cell death and transcription of the genes mdm2 and cdkn1a that, in humans, are controlled by p53. Our experimental data support an important role for HMGA1 proteins in modulation of p53dependent responses and illuminate the evolutionary subfunctionalisation.
Abstractγ-secretase is an important protease complex responsible for the cleavage of over 100 substrates within their transmembrane domains. γ-secretase acts in Alzheimer’s disease by cleavage of AMYLOID BETA (A4) PRECURSOR PROTEIN to produce aggregation-prone Amyloid beta peptide. Other γ-secretase substrates such as p75NTR are also relevant to Alzheimer’s disease. How γ-secretase cleavage site specificity is determined is still unclear. A previous study using Xenopus laevis to investigate the proteolytic processing of p75NTR and its homolog NRH1 found that transmembrane cleavage of NRH1 was not sensitive to the γ-secretase inhibitor DAPT, suggesting that it is not processed by γ-secretase. To investigate this further, we identified zebrafish orthologues of the genes p75NTR and NRH1 and developed in vivo assays to assess cleavage of the resultant p75NTR and Nrh1 proteins. Our observations from these assays in zebrafish are consistent with the Xenopus laevis study. Inhibition of γ-secretase by DAPT treatment results in accumulation of uncleaved p75NTR substrate, while cleavage of Nrh1 is not affected. This supports that p75NTR is cleaved by γ-secretase while Nrh1 is cleaved by a separate γ-secretase-like activity. We extended our approach by generating a chimeric Nrh1 protein in which the Nrh1 transmembrane domain was replaced by that of p75NTR, in an attempt to determine whether it is the p75NTR TMD that confers susceptibility for γ-secretase cleavage. Our results from analysis of this chimeric protein revealed that the p75NTR transmembrane domain alone is insufficient to confer γ-secretase cleavage susceptibility. This is not completely unexpected, as there is evidence to suggest that other factors are crucial for selection/cleavage by the γ-secretase complex. We have established a system in which we can now attempt to dissect the structural basis for γ-secretase cleavage specificity and evolution.
Objective NGFR/p75NTR and NRADD/NRH proteins are closely related structurally and are encoded by genes that arose from a duplication event early in vertebrate evolution. The transmembrane domain (TMD) of NGFR is cleaved by γ-secretase but there is conflicting data around the susceptibility to γ-secretase cleavage of NRADD proteins. If NGFR and NRADD show differential susceptibility to γ-secretase, then they can be used to dissect the structural constraints determining substrate susceptibility. We sought to test this differential susceptibility. Results We developed labelled, lumenally-truncated forms of zebrafish Ngfrb and Nradd and a chimeric protein in which the TMD of Nradd was replaced with the TMD of Ngfrb. We expressed these in zebrafish embryos to test their susceptibility to γ-secretase cleavage by monitoring their stability using western immunoblotting. Inhibition of γ-secretase activity using DAPT increased the stability of only the Ngfrb construct. Our results support that only NGFR is cleaved by γ-secretase. Either NGFR evolved γ-secretase-susceptibility since its creation by gene duplication, or NRADD evolved to be refractory to γ-secretase. Protein structure outside of the TMD of NGFR is likely required for susceptibility to γ-secretase.
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