Molecular functions of ANKLE2 and its implications in human disease

Abstract

Ankyrin repeat and LEM domain-containing 2 (ANKLE2) is a scaffolding protein with established roles in cell division and development, the dysfunction of which is increasingly implicated in human disease. ANKLE2 regulates nuclear envelope disassembly at the onset of mitosis and its reassembly after chromosome segregation. ANKLE2 dysfunction is associated with abnormal nuclear morphology and cell division. It regulates the nuclear envelope by mediating protein-protein interactions with barrier to autointegration factor (BANF1; also known as BAF) and with the kinase and phosphatase that modulate the phosphorylation state of BAF. In brain development, ANKLE2 is crucial for proper asymmetric division of neural progenitor cells. In humans, pathogenic loss-of-function mutations in ANKLE2 are associated with primary congenital microcephaly, a condition in which the brain is not properly developed at birth. ANKLE2 is also linked to other disease pathologies, including congenital Zika syndrome, cancer and tauopathy. Here, we review the molecular roles of ANKLE2 and the recent literature on human diseases caused by its dysfunction.

Keywords: Cell division; Microcephaly; Neurodevelopment.

Fig. 1.

Conservation of ANKLE2 structural domains. (A) Prediction of human ANKLE2 (UniProt Q86XL3) protein structure by using the AlphaFold database, with annotated and uncharacterized (Unc) structural regions highlighted. C, C-terminus; N, N-terminus. (B) Protein structures of ANKLE2 and its orthologs in different species according to the National Center for Biotechnology Information (NCBI). Structured domains not previously annotated (such as LEM or ankyrin-repeat domain) were identified by using AlphaFold (Jumper et al., 2021; Varadi et al., 2022). Regions were considered structured when the AlphaFold per-residue estimate of its confidence (pLDDT) was >70 (on a scale from 0-100) for each given amino acid (aa) residue. Transmembrane domains were annotated using DeepTMHMM (https://dtu.biolib.com/DeepTMHMM) (Hallgren et al., 2022 preprint). The Caulimovirus domain in D. melanogaster Ankle2 appears to maintain a similar structure but is broken into two segments with a β-sheet-disordered region–α-helix-β sheet-α-helix organization. The region in D. melanogaster Ankle2 between aa residues 713-894 represents an uncharacterized structured region between region 5 and 6 without a defined orthologous region among other evaluated orthologs. (C-E) Conservation of aa residues among ANKLE2 orthologs as well as for specific protein domains (as shown in B) determined using Clustal Omega multiple sequence alignment (Sievers et al., 2011). Conservation of aa residues in the entire ANKLE2 protein sequence (C), the ankyrin-repeat domain (D) and the LEM domain (E), showing that these vital domains have higher degrees of conservation.

Fig. 2.

Role of ANKLE2 in nuclear envelope disassembly and reassembly via its regulation of BAF phosphorylation. (A) During interphase, LEM-domain proteins contribute to chromatin organization by acting as a bridge to DNA, BAF, nuclear lamina and the inner nuclear membrane. Non-phosphorylated BAF is diffusely spread throughout the nucleoplasm and may interact with other proteins, while some phosphorylated BAF exists in the cytoplasm. ANKLE2 is localized to the inner nuclear membrane and to the ER where it is thought to interact with other host proteins and may have other roles in the cell. (B) During early mitosis, ANKLE2 facilitates the phosphorylation of BAF through VRK1. Phosphorylated BAF loses its affinity for DNA and relocalizes to the cytoplasm, allowing nuclear envelope (NE) disassembly and chromosome condensation. (C) During late mitosis, ANKLE2 inhibits VRK1 phosphorylation of BAF and facilitates BAF dephosphorylation through the PP2A-complex. This reinstates BAF DNA-binding, leading to nuclear envelope reassembly by recruitment of membrane-bound LEM proteins.

Fig. 3.

Model of ANKLE2-mediated dysregulation of asymmetric division in neural progenitor cells, leading to microcephaly. (A) During brain development, neural progenitor cells (NPCs) divide asymmetrically, giving rise to two daughter cells: one cell acquires a neuronal fate (neuron) and the other is retained as a NPC. These different daughter cell fates are brought about by the polarization of fate-determining factors in NPCs across the metaphase plate. Thus, this process depends on the alignment of cell polarity with the mitotic spindle and is required for proper neurogenesis and brain development. (B) In ANKLE2-deficient cells the polarity of cell fate factors becomes misaligned relative to the mitotic spindle. The resulting dysregulation of NPC polarity leads to a reduction in neuronal cell numbers generated during neurogenesis, an effect that is compounded by increased apoptosis and decreased cell division, resulting in microcephaly.

Fig. 4.

Pathogenic mutations in human ANKLE2 associated with primary congenital microcephaly. Schematic of human ANKLE2 protein structure showing sites of known pathogenic mutations, as originally described (Yamamoto et al., 2014; Link et al., 2019; Shaheen et al., 2019; Masih et al., 2022; Thomas et al., 2022). Point mutations leading to missense mutations are shown in black, mutations leading to protein truncations are shown in red. G510* indicates a hypothetical splicing mutation that leads to a premature protein termination (c.1421-1G>C), as identified in a compound heterozygous individual together with the A109P point mutation (Link et al., 2019). Allele combinations are listed in Table 1.

Fig. 5.

Zika virus (ZIKV) NS4A inhibits ANKLE2 and causes similar pathogenic outcomes. During mitosis, ANKLE2 interacts with BAF, VRK1 and PP2A to regulate nuclear envelope dynamics. During asymmetric cell division the ANKLE2-VRK1 pathway is crucial for establishing proper cell polarity. (A) Zika virus (ZIKV) NS4A interacts with ANKLE2 and inhibits its function to cause microcephaly. (B) Top row: Simplified schematic showing normal development of human brain (left) compared to microcephaly arising from dysfunction ANKLE2 (middle) or from congenital Zika syndrome (top). Middle and bottom rows: The mirrored ‘small brain’ phenotype in larval Drosophila brain (middle) and respective defects in asymmetric neuroblast division (bottom).