6-OHDA-induced dopaminergic neurodegeneration in Caenorhabditis elegans is promoted by the engulfment pathway and inhibited by the transthyretin-related protein TTR-33.

Oxidative stress is linked to many pathological conditions including the loss of dopaminergic neurons in Parkinson's disease. The vast majority of disease cases appear to be caused by a combination of genetic mutations and environmental factors. We screened for genes protecting Caenorhabditis elegans dopaminergic neurons from oxidative stress induced by the neurotoxin 6-hydroxydopamine (6-OHDA) and identified the transthyretin-related gene ttr-33. The only described C. elegans transthyretin-related protein to date, TTR-52, has been shown to mediate corpse engulfment as well as axon repair. We demonstrate that TTR-52 and TTR-33 have distinct roles. TTR-33 is likely produced in the posterior arcade cells in the head of C. elegans larvae and is predicted to be a secreted protein. TTR-33 protects C. elegans from oxidative stress induced by paraquat or H2O2 at an organismal level. The increased oxidative stress sensitivity of ttr-33 mutants is alleviated by mutations affecting the KGB-1 MAPK kinase pathway, whereas it is enhanced by mutation of the JNK-1 MAPK kinase. Finally, we provide genetic evidence that the C. elegans cell corpse engulfment pathway is required for the degeneration of dopaminergic neurons after exposure to 6-OHDA. In summary, we describe a new neuroprotective mechanism and demonstrate that TTR-33 normally functions to protect dopaminergic neurons from oxidative stress-induced degeneration, potentially by acting as a secreted sensor or scavenger of oxidative stress.

Characterisation of the biochemical and cellular roles of native and pathogenic amelogenesis imperfecta mutants of FAM83H.

The majority of mutations identified in patients with amelogenesis imperfecta have been mapped to FAM83H. As FAM83H expression is not limited to the enamel, how FAM83H contributes to amelogenesis is still largely unknown. We previously reported that members of the FAM83 family of proteins interact with and regulate the subcellular distribution of the promiscuous serine-threonine protein kinase CK1 family, through their shared N-terminal DUF1669 domains. FAM83H co-localises with CK1 isoforms to speckle-like structures in both the cytoplasm and nucleus. In this report, we show FAM83H, unlike other FAM83 proteins, interacts and colocalises with NCK1/2 tyrosine kinase adaptor proteins. This interaction is mediated by proline-rich motifs within the C-terminus of FAM83H, specifically interacting with the second and third SH3 domains of NCK1/2. Moreover, FAM83H pathogenic AI mutant proteins, which trigger C-terminal truncations of FAM83H, retain their interactions with CK1 isoforms but lose interaction with NCK1/2. These AI mutant FAM83H proteins acquire a nuclear localisation, and recruit CK1 isoforms to the nucleus where CK1 retains its kinase activity. As understanding the constituents of the FAM83H-localised speckles may hold the key to unravelling potential substrates of FAM83H-associated CK1 substrates, we employed a TurboID-based proximity labelling approach and uncovered several proteins including Iporin and BAG3 as potential constituents of the speckles.

Pathogenic FAM83G palmoplantar keratoderma mutations inhibit the PAWS1:CK1α association and attenuate Wnt signalling.

Background: Two recessive mutations in the FAM83G gene, causing A34E and R52P amino acid substitutions in the DUF1669 domain of the PAWS1 protein, are associated with palmoplantar keratoderma (PPK) in humans and dogs respectively. We have previously reported that PAWS1 associates with the Ser/Thr protein kinase CK1α through the DUF1669 domain to mediate canonical Wnt signalling. Methods: Co-immunoprecipitation was used to investigate possible changes to PAWS1 interactors caused by the mutations. We also compared the stability of wild-type and mutant PAWS1 in cycloheximide-treated cells. Effects on Wnt signalling were determined using the TOPflash luciferase reporter assay in U2OS cells expressing PAWS1 mutant proteins. The ability of PAWS1 to induce axis duplication in Xenopus embryos was also tested. Finally, we knocked-in the A34E mutation at the native gene locus and measured Wnt-induced AXIN2 gene expression by RT-qPCR. Results: We show that these PAWS1 A34E and PAWS1 R52P mutants fail to interact with CK1α but, like the wild-type protein, do interact with CD2AP and SMAD1. Like cells carrying a PAWS1 F296A mutation, which also abolishes CK1α binding, cells carrying the A34E and R52P mutants respond poorly to Wnt signalling to an extent resembling that observed in FAM83G gene knockout cells. Consistent with this observation, these mutants, in contrast to the wild-type protein, fail to induce axis duplication in Xenopus embryos. We also found that the A34E and R52P mutant proteins are less abundant than the native protein and appear to be less stable, both when overexpressed in FAM83G-knockout cells and when knocked-in at the native FAM83G locus. Ala 34 of PAWS1 is conserved in all FAM83 proteins and mutating the equivalent residue in FAM83H (A31E) also abolishes interaction with CK1 isoforms. Conclusions: We propose that mutations in PAWS1 cause PPK pathogenesis through disruption of the CK1α interaction and attenuation of Wnt signalling.

The DUF1669 domain of FAM83 family proteins anchor casein kinase 1 isoforms.

Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain.