Jade-1S phosphorylation induced by CK1α contributes to cell cycle progression

<p>The PHD zinc finger protein Jade-1S is a component of the HBO1 histone acetyltransferase complex and binds chromatin in a cell cycle-dependent manner. Jade-1S also acts as an E3 ubiquitin ligase for the canonical Wnt effector protein β-catenin and is influenced by CK1α-mediated phosphorylation. To further elucidate the functional impact of this phosphorylation, we used a stable, low-level expression system to express either wild-type or mutant Jade-1S lacking the N-terminal CK1α phosphorylation motif. Interactome analyses revealed that the Jade-1S mutant unable to be phosphorylated by CK1α has an increased binding affinity to proteins involved in chromatin remodelling, histone deacetylation, transcriptional repression, and ribosome biogenesis. Interestingly, cells expressing the mutant displayed an elongated cell shape and a delay in cell cycle progression. Finally, phosphoproteomic analyses allowed identification of a Jade-1S site phosphorylated in the presence of CK1α but closely resembling a PLK1 phosphorylation motif. Our data suggest that Jade-1S phosphorylation at an N-terminal CK1α motif creates a PLK1 phospho-binding domain. We propose CK1α phosphorylation of Jade 1S to serve as a molecular switch, turning off chromatin remodelling functions of Jade-1S and allowing timely cell cycle progression. As Jade-1S protein expression in the kidney is altered upon renal injury, this could contribute to understanding mechanisms underlying epithelial injury repair.</p

Plk1 directly phosphorylates NPHP1 in vitro.

<p><b>A</b> Alignment of NPHP1 protein sequences from multiple species indicates a conserved candidate Plk1 motif at position T87. <b>B</b> An <i>in vitro</i> kinase assay performed with active Plk1 and recombinant His-fused NPHP1 protein indicates phosphorylation within the NPHP1 N-terminal 205 amino acids. CB, Coomassie Blue.</p

Plk1 colocalizes with NPHP1 at the base of the cilium.

<p><b>A</b> Ciliated hTERT-RPE1 (human retinal pigmented epithelial cells and HK2 human kidney cells were stained with antibody to Plk1 (green), acetylated α-tubulin (orange), and γ-tubulin (red), and treated with DAPI to visualize DNA (blue). The scale bar represents 5 µm. <b>B</b> Ciliated hTERT-RPE1 cells and HK2 cells were stained with antibody to acetylated α-tubulin (orange), γ-tubulin (red), to NPHP1 or Plk1 as indicated (green), and with DAPI to visualize DNA (blue). The third row shows merged signals from staining with antibody to Plk1 (orange), NPHP1 (green) acetylated α-tubulin (orange), γ-tubulin (red) and DAPI was used to visualize DNA (blue). The scale bar represents 5 µm.</p

Plk1 associates with NPHP1.

<p><b>A</b> Western blot of immunoprecipitates (IP) or lysates (Lys) from HEK293T cells co-transfected with plasmids expressing V5-tagged NPHP1 and Flag-tagged Plk1 or negative control protein (Eps1–225 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038838#pone.0038838-Habbig1" target="_blank">[13]</a>). β-actin was assessed as a loading control. <b>B</b> Western blot of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells co-transfected with plasmids expressing Myc-tagged Plk1 and Flag-tagged NPHP1 or empty Flag vector. <b>C</b> Western blot of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells transfected with plasmid expressing Flag-tagged NPHP1 or the negative control protein (Eps1–225 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038838#pone.0038838-Habbig1" target="_blank">[13]</a>). Endogenous Plk1 was detected using a specific antibody against Plk1. <b>D</b> A panel of Flag-tagged NPHP1 derivatives, including truncations, internal deletions and a T87A mutant, was analyzed by co-immunoprecipitation with Myc-tagged Plk1. <b>E</b> Western analysis of immunoprecipitates (IP) or cell lysates (Lys) from HEK293T cells co-transfected with plasmids expressing Myc-tagged Plk1 and Flag-tagged NPHP1 constructs as indicated, or the Flag-tagged control protein (Eps1–225). * indicates immunoglobulin heavy chain.</p

Administration of low molecular weight heparin results in elevated urinary sFlt-1 levels.

<p>Urinary sFlt-1 levels (pg/mg Crea) are increased 2 hours after administration of enoxaparin in 7 out of 10 patients (<b>A</b>). Subgroup analysis according to proteinuria at presentation (cut-off of 300 mg protein/g creatinine) reveals that patients with a proteinuria <300 mg/g respond with a significantly higher increase of urinary sFlt-1 after heparin treatment (p = 0.0365) (<b>B</b>).</p

Heparin does not affect sFlt-1 protein complex size but interferes with sFlt-1 binding to negatively charged surfaces.

<p>Serum samples before and after low molecular weight heparin treatment were subjected to velocity gradient centrifugation. Western blotting of fractions 5–18 was performed using Flt-1 specific antibody (<b>A</b>). Serum samples before and after addition of 10 mg/l enoxaparin were subjected to cation exchange chromatography. In non-treated serum samples 70.14% of sFlt-1 binds to the column, whereas the remaining 29. 86% are found in the flow through. After enoxaparin treatment only 56.88% of sFlt-1 is bound and 43.11% appear in the flow through (p = 0.0386) (<b>B</b>).</p

Patient characteristics on admission.

<p>Physical parameters and laboratory values were assessed upon admission in all patients. DM1 = diabetes mellitus type 1; IUGR = intra uterine growth restriction; PE = preeclampsia; PIH = pregnancy induced hypertension.</p

sFlt-1 and PlGF serum levels increase after the administration of low molecular weight heparin.

<p>2-1 levels increase to 1.26 fold on average (95% CI (interval before LMWH) 0,9627–1,040; 95% CI (interval after LMWH) 1.103–1.418; p = 0.0045) (<b>A</b>), PlGF levels increase to 1.15 fold on average (p = 0.0126) (<b>B</b>), the resulting increase of sFlt-1/PlGF ratio does not reach statistical significance (<b>C</b>).</p

Dysregulated Autophagy Contributes to Podocyte Damage in Fabry’s Disease

<div><p>Fabry’s disease results from an inborn error of glycosphingolipid metabolism that is due to deficiency of the lysosomal hydrolase α-galactosidase A. This X-linked defect results in the accumulation of enzyme substrates with terminally α-glycosidically bound galactose, mainly the neutral glycosphingolipid Globotriaosylceramide (Gb3) in various tissues, including the kidneys. Although end-stage renal disease is one of the most common causes of death in hemizygous males with Fabry’s disease, the pathophysiology leading to proteinuria, hematuria, hypertension, and kidney failure is not well understood. Histological studies suggest that the accumulation of Gb3 in podocytes plays an important role in the pathogenesis of glomerular damage. However, due to the lack of appropriate animal or cellular models, podocyte damage in Fabry’s disease could not be directly studied yet. As murine models are insufficient, a human model is needed. Here, we developed a human podocyte model of Fabry’s disease by combining RNA interference technology with lentiviral transduction of human podocytes. Knockdown of α-galactosidase A expression resulted in diminished enzymatic activity and slowly progressive accumulation of intracellular Gb3. Interestingly, these changes were accompanied by an increase in autophagosomes as indicated by an increased abundance of LC3-II and a loss of mTOR kinase activity, a negative regulator of the autophagic machinery. These data suggest that dysregulated autophagy in α-galactosidase A-deficient podocytes may be the result of deficient mTOR kinase activity. This finding links the lysosomal enzymatic defect in Fabry’s disease to deregulated autophagy pathways and provides a promising new direction for further studies on the pathomechanism of glomerular injury in Fabry patients.</p></div

Risk factors for TMA-associated death.

<p>IQR: Interquartile range.</p