Reduction of WDR81 impairs autophagic clearance of aggregated proteins and cell viability in neurodegenerative phenotypes

Neurodegenerative diseases are characterized by neuron loss and accumulation of undegraded protein aggregates. These phenotypes are partially due to defective protein degradation in neuronal cells. Autophagic clearance of aggregated proteins is critical to protein quality control, but the underlying mechanisms are still poorly understood. Here we report the essential role of WDR81 in autophagic clearance of protein aggregates in models of Huntington’s disease (HD), Parkinson’s disease (PD) and Alzheimer’s disease (AD). In hippocampus and cortex of patients with HD, PD and AD, protein level of endogenous WDR81 is decreased but autophagic receptor p62 accumulates significantly. WDR81 facilitates the recruitment of autophagic proteins onto Htt polyQ aggregates and promotes autophagic clearance of Htt polyQ subsequently. The BEACH and MFS domains of WDR81 are sufficient for its recruitment onto Htt polyQ aggregates, and its WD40 repeats are essential for WDR81 interaction with covalent bound ATG5-ATG12. Reduction of WDR81 impairs the viability of mouse primary neurons, while overexpression of WDR81 restores the viability of fibroblasts from HD patients. Notably, in Caenorhabditis elegans, deletion of the WDR81 homolog (SORF-2) causes accumulation of p62 bodies and exacerbates neuron loss induced by overexpressed α-synuclein. As expected, overexpression of SORF-2 or human WDR81 restores neuron viability in worms. These results demonstrate that WDR81 has crucial evolutionarily conserved roles in autophagic clearance of protein aggregates and maintenance of cell viability under pathological conditions, and its reduction provides mechanistic insights into the pathogenesis of HD, PD, AD and brain disorders related to WDR81 mutations.

Regulation of gene expression by repression condensates during development

There is emerging evidence for transcription condensates in the activation of gene expression1–3. However, there is considerably less information regarding transcriptional repression, despite its pervasive importance in regulating gene expression in development and disease. Here, we explore the role of liquid-liquid phase separation (LLPS) in the organization of the Groucho/TLE (Gro) family of transcriptional corepressors, which interact with a variety of sequence-specific repressors such as Hes/Hairy4. Gro-dependent repressors have been implicated in a variety of developmental processes, including segmentation of the Drosophila embryo and somitogenesis in vertebrates. These repressors bind to specific recognition sequences, but instead of interacting with coactivators (e.g., Mediator) they recruit Gro corepressors5. Gro contains a series of WD40 repeats that are thought to mediate oligomerization6. How putative Hes/Gro oligomers repress transcription has been the subject of numerous studies5, 6. Here we show that Hes/Gro complexes form discrete puncta within nuclei of living Ciona embryos. These puncta rapidly dissolve during the onset of mitosis and reappear in the ensuing cell cycle. Modified Hes/Gro complexes that are unable to bind DNA exhibit the properties of viscous liquid droplets, similar to those underlying the biogenesis of P-granules in C. elegans7 and nucleoli in Xenopus oocytes8. These observations provide vivid evidence for LLPS in the control of gene expression and suggest a simple physical exclusion mechanism for transcriptional repression. WD40 repeats have been implicated in a wide variety of cellular processes in addition to transcriptional repression9. We suggest that protein interactions using WD40 motifs might be a common feature of processes reliant on LLPS.

ARABIDOPSIS POLLEN ABORTION MUTANT 2, Encoding an Unknown Protein with Three WD40-Repeats, Is Essential for Late Pollen Mature in Arabidopsis thaliana

Background In flowering plants, pollen formation is a very complex process. It is strictly regulated by various genetic factors. Some of these factors have been identified, but the regulatory mechanism concerning the process of late pollen maturing remains unknown.Results Previously we identified and reported an ARABIDOPSIS POLLEN ABORTION MUTANT 2 (APAM2) gene which was required for pollen formation in Arabidopsis thaliana. In this study, we further analysed the phenotype of atapam2 and the expression pattern of AtAPAM2. Mutation in AtAPAM2 drastically affected male gametophytic function, impacted the late pollen mature process, and resulted in mature pollen grains from atapam2/+ plants being shrivelled and dead. It was expressed among seedlings, roots, stems, leaves, siliques, flowers, and pollen grains. However, its expression levels in flowers and pollen grains were much higher than in other tissues. AtAPAM2 encodes an unknown protein with three WD40-repeats with localisation in plasma membrane and nucleus, and homologous proteins exist in many species.Conclusions We determined that AtAPAM2 may play a role in pollen formation and other development processes by interacting with other proteins, but we did not identify the protein that interacts with it.

ARABIDOPSIS POLLEN ABORTION MUTANT 2, Encoding an Unknown Protein with Three WD40-Repeats, Is Essential for Late Pollen Mature in Arabidopsis thaliana

Background In flowering plants, pollen formation is a very complex process. It is strictly regulated by various genetic factors. Some of these factors have been identified, but the regulatory mechanism concerning the process of late pollen maturing remains unknown. Results Previously we identified and reported an ARABIDOPSIS POLLEN ABORTION MUTANT 2 (APAM2) gene which was required for pollen formation in Arabidopsis thaliana. In this study, we further analysed the phenotype of atapam2 and the subcellular localisation of AtAPAM2. Mutation in AtAPAM2 drastically affected male gametophytic function, impacted the late pollen mature process, and resulted in mature pollen grains from atapam2/+ plants being shrivelled and dead. It was expressed among seedlings, roots, stems, leaves, siliques, flowers, and pollen grains. However, its expression levels in flowers and pollen grains were much higher than in other tissues. Gene AtAPAM2 encodes an unknown protein with three WD40-repeats with localisation in plasma membrane and nucleus, and homologous proteins exist in many species. Conclusions We determined that AtAPAM2 may play a role in pollen formation and other development processes by interacting with other proteins, but we did not identify the protein that interacts with it.

Targeting Human Retinoblastoma Binding Protein 4 (RBBP4) and 7 (RBBP7)

RBBP4 and RBBP7 (RBBP4/7) are highly homologous nuclear WD40 motif containing proteins widely implicated in various cancers and are valuable drug targets. They interact with multiple proteins within diverse complexes such as NuRD and PRC2, as well as histone H3 and H4 through two distinct binding sites. FOG-1, PHF6 and histone H3 bind to the top of the donut shape seven-bladed β-propeller fold, while SUZ12, MTA1 and histone H4 bind to a pocket on the side of the WD40 repeats. Here, we briefly review these six interactions and present binding assays optimized for medium to high throughput screening. These assays enable screening of RBBP4/7 toward the discovery of novel cancer therapeutics.

Molecular analysis of anthocyanin-related genes in ornamental cabbage

Ornamental cabbage (Brassica oleracea var. acephala) is a winter-grown and important decorative plant of the family Brassicaceae, which displays an exceptional coloration in the central leaves of the rosette. Anthocyanins are the key determinant of the red, purple, and blue colors of vegetative and reproductive parts of many plant species including ornamental cabbage. Total anthocyanin content was measured spectrophotometrically, and the highest anthocyanin content was detected in the red followed by light-red and white ornamental cabbage lines. Anthocyanin biosynthesis is controlled by members of three different transcription factor (TF) families, such as MYB, basic helix-loop-helix (bHLH), and WD40 repeats (WDR), which function as a MBW complex. We identified three MYB, six bHLH, and one WDR TFs that regulate anthocyanin biosynthesis in ornamental cabbage. The expression of the regulatory and biosynthetic genes for anthocyanin synthesis was determined by qPCR. The tested structural genes of the anthocyanin pathway were shown to be up-regulated in the red followed by light-red ornamental cabbage lines; however, the expression levels of the late biosynthetic genes were barely detected in the white ornamental cabbage lines. Among the regulatory genes, BoPAP2 (MYB), BoTT8, BoEGL3.1, and BoMYC1.2 (bHLH), and BoTTG1 (WDR) were identified as candidates for the regulation of anthocyanin biosynthesis. This work could be useful for the breeding of novel colorful ornamental cabbage cultivars.

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain

Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1–4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4–ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4–ALK and EML1–ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4–ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein–protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4–ALK NSCLC.