Jayne, T. | bioRxiv Science

bioRxiv2020

Developing in vivo assays for investigation of p75NTR and NRH1 transmembrane domain cleavage events using zebrafish embryos

{gamma}-secretase is an important protease complex responsible for the cleavage of over 100 substrates within their transmembrane domains. {gamma}-secretase acts in Alzheimers disease by cleavage of AMYLOID BETA (A4) PRECURSOR PROTEIN to produce aggregation-prone Amyloid beta peptide. Other {gamma}-secretase substrates such as p75NTR are also relevant to Alzheimers disease. How {gamma}-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 {gamma}-secretase inhibitor DAPT, suggesting that it is not processed by {gamma}-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 {gamma}-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 {gamma}-secretase while Nrh1 is cleaved by a separate {gamma}-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 {gamma}-secretase cleavage. Our results from analysis of this chimeric protein revealed that the p75NTR transmembrane domain alone is insufficient to confer {gamma}-secretase cleavage susceptibility. This is not completely unexpected, as there is evidence to suggest that other factors are crucial for selection/cleavage by the {gamma}-secretase complex. We have established a system in which we can now attempt to dissect the structural basis for {gamma}-secretase cleavage specificity and evolution.

neuroscience↗

bioRxiv2018

HMGA1 zebrafish co-orthologue hmga1b can modulate p53-dependent cellular responses but is unable to control the alternative splicing of psen1

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 Alzheimers 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 p53-dependent responses and illuminate the evolutionary subfunctionalisation.

developmental biology↗