McSharry, B. P. | bioRxiv Science

bioRxiv2025

A bipartite, mutation-tolerant NLS regulates interaction of ΔNp63α with importin alpha, nuclear transport and transcriptional activity

{Delta}Np63 is a master regulator of epithelial development, driving the expansion of progenitor cells in stratified epithelia. Mutations in {Delta}Np63 are linked to squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs), as well as to ectodermal dysplasia syndromes such as ectrodactyly-ectodermal dysplasia-clefting (EEC) and ankyloblepharon-ectodermal dysplasia-clefting (AEC). Although {Delta}Np63 functions as a nuclear transcription factor, the mechanisms underlying its nuclear import remain incompletely understood. By combining imaging, biochemical, structural and functional assays, we have thoroughly characterized {Delta}Np63 nuclear import, as mediated by the importin (IMP) /{beta}1 heterodimer. We also show here that {Delta}Np63 has evolved a peculiar strategy to ensure mutation tolerant nuclear localization, which is essential for DNA binding and transcriptional regulation. Despite a canonical bipartite NLS formed by two stretches of basic amino acids was identified between the DNA binding and oligomerization domains, each of them in sufficient to bind both IMP binding sites upon homodimerization. Therefore, in contrast to most known bipartite NLSs, only simultaneous substitution of both basic stretches of amino acids ablated nuclear localization, interaction with IMP, and decreased transcriptional activity. Since several {Delta}Np63 isoforms which lack the N-terminal basic stretch of amino acids have been described, and a number of mutations in the {Delta}Np63 NLS region have been identified in the Genome Aggregation Database, {Delta}Np63 has specifically evolved to tolerate mutations in its NLS without significantly compromising its ability to localize in the nucleus. GRAPHICAL ABSTRACT

cell biology↗

bioRxiv2020

Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death

Herpesviruses are known to encode a number of inhibitors of host cell death, including Rip Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein open reading frame (ORF) 20 that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1-expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies. Author SummaryRip homotypic interaction motifs (RHIMs) are found in host proteins that can signal for programmed cell death and in viral proteins that can prevent it. Complexes stabilized by intermolecular interactions involving RHIMs have a fibrillar amyloid structure. We have identified a novel RHIM within the ORF20 protein expressed by Varicella zoster virus (VZV) that forms amyloid-based complexes with human cellular RHIMs. Whereas other herpesvirus RHIMs inhibit TNF-driven necroptosis, this new VZV RHIM targets the host RHIM-containing protein ZBP1 to inhibit apoptosis during infection. This is the first study to demonstrate the importance of the ZBP1 pathway in VZV infection and to identify the role of a viral RHIM in apoptosis inhibition. It broadens our understanding of host defense pathways and demonstrates how a decoy amyloid strategy is employed by pathogens to circumvent the host response.

microbiology↗