Chari, R. | bioRxiv Science

bioRxiv2019

Structure of E3 ligase E6AP with a novel proteasome-binding site provided by substrate receptor hRpn10

Regulated proteolysis by the proteasome involves [~]800 enzymes for substrate modification with ubiquitin, of which [~]600 are E3 ligases. We report here that E6AP/UBE3A is distinguished from other ubiquitin E3 ligases by having a 12 nM binding site at the proteasome contributed by substrate receptor hRpn10/PSMD4/S5a. Intrinsically disordered by itself, and previously uncharacterized, this domain in hRpn10 locks into a novel well-defined helical structure to form an intermolecular 4-helix bundle with the E6AP AZUL domain, which is unique to this E3. We thus name the hRpn10 AZUL-binding domain RAZUL. We further find in human cells that loss of RAZUL by CRISPR-based gene editing leads to loss of E6AP at the proteasome, where associated ubiquitin is correspondingly reduced, suggesting that E6AP ubiquitinates substrates at or for the proteasome. Altogether, our findings indicate E6AP to be a privileged E3 for the proteasome, with a dedicated, high affinity binding site contributed by hRpn10.

molecular biology↗

bioRxiv2019

Massively parallel reporter assays combined with cell-type specific eQTL informed multiple melanoma loci and identified a pleiotropic function of HIV-1 restriction gene, MX2, in melanoma promotion

Genome-wide association studies (GWAS) have identified [~]20 melanoma susceptibility loci. To identify susceptibility genes and variants simultaneously from multiple GWAS loci, we integrated massively-parallel reporter assays (MPRA) with cell type-specific epigenomic data as well as melanocyte-specific expression quantitative trait loci (eQTL) profiling. Starting from 16 melanoma loci, we selected 832 variants overlapping active regions of chromatin in cells of melanocytic lineage and identified 39 candidate functional variants displaying allelic transcriptional activity by MPRA. For four of these loci, we further identified four colocalizing melanocyte cis-eQTL genes (CTSS, CASP8, MX2, and MAFF) matching the allelic activity of MPRA functional variants. Among these, we further characterized the locus encompassing the HIV-1 restriction gene, MX2, on chromosome band Chr21q22.3 and validated a functional variant, rs398206, among multiple high LD variants. rs398206 mediates allelic transcriptional activity via binding of the transcription factor, YY1. This allelic transcriptional regulation is consistent with a significant cis-eQTL of MX2 in primary human melanocytes, where the melanoma risk-associated A allele of rs398206 is correlated with higher MX2 levels. Melanocyte-specific transgenic expression of human MX2 in a zebrafish model demonstrated accelerated melanoma formation in a BRAFV600E background. Thus, using an efficient scalable approach to streamline GWAS follow-up functional studies, we identified multiple candidate melanoma susceptibility genes and variants, and uncovered a pleiotropic function of MX2 in melanoma susceptibility.

genomics↗