Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling

Bone morphogenetic proteins (BMPs) control multiple cellular processes in embryos and adult tissues. BMPs signal through the activation of type I BMP receptor kinases, which then phosphorylate SMADs 1/5/8. In the canonical pathway, this triggers the association of these SMADs with SMAD4 and their translocation to the nucleus, where they regulate gene expression. BMPs can also signal independently of SMAD4, but this pathway is poorly understood. Here, we report the discovery and characterization of PAWS1/FAM83G as a novel SMAD1 interactor. PAWS1 forms a complex with SMAD1 in a SMAD4-independent manner, and BMP signalling induces the phosphorylation of PAWS1 through BMPR1A. The phosphorylation of PAWS1 in response to BMP is essential for activation of the SMAD4-independent BMP target genes NEDD9 and ASNS. Our findings identify PAWS1 as the first non-SMAD substrate for type I BMP receptor kinases and as a novel player in the BMP pathway. We also demonstrate that PAWS1 regulates the expression of several non-BMP target genes, suggesting roles for PAWS1 beyond the BMP pathway.     

Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

Mutations in PINK1 and Parkin are associated with early-onset Parkinson''s disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser⁶⁵) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser⁶⁵ phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser⁶⁵ by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser⁶⁵ phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin''s ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser⁶⁵ is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson''s disease.     

A recording viscometer for assaying mammalian collagenase.

A recording viscometer for monitoring the action of mammalian collagenase on soluble collagen is described. For this system, where only one peptide bond is cleaved per subunit, it is shown theoretically that the decrease in viscosity is proportional to the fraction of molecules cleaved. Experimental confirmation was obtained by parallel monitoring of hydrolysis by using the fluorescamine assay of liberated amino groups. The initial velocity of reaction is proportional to substrate concentration and enzyme concentration.     

USP45 deubiquitylase controls ERCC1–XPF endonuclease-mediated DNA damage responses

Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF–ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1–XPF. Together, these results establish USP45 as a new regulator of XPF–ERCC1 crucial for efficient DNA repair.     

Measurement of S-phase fractions in lymphoid tissue comparing fresh versus paraffin-embedded tissue and 4',6'-diamidino-2 phenylindole dihydrochloride versus propidium iodide staining

S-phase fractions for 62 lymphoid biopsies were calculated, by means of flow cytometry, from both fresh and paraffin-embedded tissue. The purposes of this study were to determine whether significant differences were seen between S-phase estimates obtained from fresh and fixed tissue and to compare results obtained with two DNA dyes, namely 4'-6'-diamidino-2 phenylindole dihydrochloride (DAPI) and propidium iodide (PI). The 62 cases consisted of 38 cases of non-Hodgkin's lymphoma (NHL), 19 reactive samples, and 5 cases of Hodgkin's disease. Fifty-four of the samples showed DNA diploid profiles. A good agreement between S-phase results from fresh and fixed tissue was seen, with technical factors accounting for around 20% of the total variance. Using a paired t test, no significant difference was seen between fresh and fixed tissue for diploid cases, but there was a trend for S-phase estimates from fixed tissue to be higher. If all cases (including the eight DNA aneuploid samples) were included in the analysis this difference just reached statistical significance (P less than .05). In a subgroup of 19 of the cases, a comparison was performed on both fresh and fixed tissue of staining with DAPI and PI. A good agreement between results with both DNA stains was found on fresh and fixed tissue, with no significant differences being apparent, and stain-related factors accounted for only 10% of the total variance.     

Mapping of a N-terminal α-helix domain required for human PINK1 stabilization,Serine228 autophosphorylation and activation in cells

Autosomal recessive mutations in the PINK1 gene are causal for Parkinson''s disease (PD). PINK1 encodes a mitochondrial localized protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function; however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain is unknown. We have employed mutagenesis studies to define the minimal region of human PINK1 required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical extension (NTE) domain forming an intramolecular interaction with the C-terminal extension (CTE), which we corroborate using hydrogen/deuterium exchange mass spectrometry of recombinant insect PINK1 protein. Cell-based analysis of human PINK1 reveals that PD-associated mutations (e.g. Q126P), located within the NTE : CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228) and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE and CTE domain mutants disrupt PINK1 stabilization at the mitochondrial Translocase of outer membrane complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE : CTE interface towards PINK1 stabilization and activation and show that loss of NTE : CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.     

The ultrastructure of whole cells from two human keratinocyte strains during cell spreading and island formation in vitro

The ultrastructure of subcultured human keratinocytes derived from normal and neoplastic human epidermis is described during various stages of intercellular collision and island formation following trypsin dispersal. Cells are grown for defined times on Formvar-coated electron-microscopy grids and examined intact after fixation, critical point-drying and staining. Transition from rounded to elongated and polygonal forms were observed followed by the formation of simple overlaps between cells, enmeshing of microvilli between adjacent cells and the appearance of a glycocalyx and desmosomes. Marginal cells in islands descended from a squamous carcinoma exhibited a greater morphological diversity than keratinocytes from normal epidermis. This difference may represent a relative deficit in the adhesive properties of the former cell strain.