Lysosomal Targeting of Cystinosin Requires AP‐3

Cystinosin is a lysosomal cystine transporter defective in cystinosis, an autosomal recessive lysosomal storage disorder. It is composed of seven transmembrane (TM) domains and contains two lysosomal targeting motifs: a tyrosine‐based signal (GYDQL) in its C‐terminal tail and a non‐classical motif in its fifth inter‐TM loop. Using the yeast two‐hybrid system, we showed that the GYDQL motif specifically interacted with the μ subunit of the adaptor protein complex 3 (AP‐3). Moreover, cell surface biotinylation and total internal reflection fluorescence microscopy revealed that cystinosin was partially mislocalized to the plasma membrane (PM) in AP‐3‐depleted cells. We generated a chimeric CD63 protein to specifically analyze the function of the GYDQL motif. This chimeric protein was targeted to lysosomes in a manner similar to cystinosin and was partially mislocalized to the PM in AP‐3 knockdown cells where it also accumulated in the trans‐Golgi network and early endosomes. Together with the fact that the surface levels of cystinosin and of the CD63‐GYDQL chimeric protein were not increased when clathrin‐mediated endocytosis was impaired, our data show that the tyrosine‐based motif of cystinosin is a ‘strong’ AP‐3 interacting motif responsible for lysosomal targeting of cystinosin by a direct intracellular pathway.      

Escape of Sgs1 from Rad9 inhibition reduces the requirement for Sae2 and functional MRX in DNA end resection

Homologous recombination requires nucleolytic degradation (resection) of DNA double‐strand break (DSB) ends. In Saccharomyces cerevisiae, the MRX complex and Sae2 are involved in the onset of DSB resection, whereas extensive resection requires Exo1 and the concerted action of Dna2 and Sgs1. Here, we show that the checkpoint protein Rad9 limits the action of Sgs1/Dna2 in DSB resection by inhibiting Sgs1 binding/persistence at the DSB ends. When inhibition by Rad9 is abolished by the Sgs1‐ss mutant variant or by deletion of RAD9, the requirement for Sae2 and functional MRX in DSB resection is reduced. These results provide new insights into how early and long‐range resection is coordinated.     

Covalent cell surface functionalization of human fetal osteoblasts for tissue engineering

The chemical functionalization of cell-surface proteins of human primary fetal bone cells with hydrophilic bioorthogonal intermediates was investigated. Toward this goal, chemical pathways were developed for click reaction-mediated coupling of alkyne derivatives with cellular azido-expressing proteins. The incorporation via a tetraethylene glycol linker of a dipeptide and a reporter biotin allowed the proof of concept for the introduction of cell-specific peptide ligands and allowed us to follow the reaction in living cells. Tuning the conditions of the click reaction resulted in chemical functionalization of living human fetal osteoblasts with excellent cell survival.     

Atg1 allows second-signaled autophagic cell death in Dictyostelium

We investigated the role of Atg1 in autophagic cell death (ACD) in a Dictyostelium monolayer model. The model is especially propitious, not only because of genetic tractability and absence of apoptosis machinery, but also because induction of ACD requires two successive exogenous signals, first the combination of starvation and cAMP, second the differentiation factor DIF-1. This enables one to analyze separately first-signal-induced autophagy and subsequent second-signal-induced ACD. We used mutants of atg1, a gene that plays an essential role in the initiation of autophagy. Upon starvation/cAMP, in contrast to parental cells, atg1 mutant cells showed irreversible lesions, clearly establishing a protective role for Atg1. Upon subsequent exposure to DIF-1 or to more ACD-specific second signals, starved parental cells progressed to ACD, but starved atg1 mutant cells did not, showing that Atg1 was required for ACD. Thus, in the same cells Atg1 was required in two apparently opposite ways, upon first-signaling for cell survival and upon second-signaling for ACD. Our findings strongly suggest that Atg1, thus presumably autophagy, protects the cells from starvation-induced cell death, allowing subsequent induction of ACD by the second signal. ACD is therefore not only "with" autophagy (since it showed signs of autophagy throughout), but is also "allowed by" autophagy. This does not exclude a role for autophagy also after second signaling. These results may account for discrepancies reported in the literature, encourage searches for second signals in different developmental models of ACD, and incite caution in autophagy-related therapeutic attempts.     

Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination

Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.     

Structural insights into the inhibition of cytosolic 5'-nucleotidase II (cN-II) by ribonucleoside 5'-monophosphate analogues

Cytosolic 5'-nucleotidase II (cN-II) regulates the intracellular nucleotide pools within the cell by catalyzing the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates. Beside this physiological function, high level of cN-II expression is correlated with abnormal patient outcome when treated with cytotoxic nucleoside analogues. To identify its specific role in the resistance phenomenon observed during cancer therapy, we screened a particular class of chemical compounds, namely ribonucleoside phosphonates to predict them as potential cN-II inhibitors. These compounds incorporate a chemically and enzymatically stable phosphorus-carbon linkage instead of a regular phosphoester bond. Amongst them, six compounds were predicted as better ligands than the natural substrate of cN-II, inosine 5'-monophosphate (IMP). The study of purine and pyrimidine containing analogues and the introduction of chemical modifications within the phosphonate chain has allowed us to define general rules governing the theoretical affinity of such ligands. The binding strength of these compounds was scrutinized in silico and explained by an impressive number of van der Waals contacts, highlighting the decisive role of three cN-II residues that are Phe 157, His 209 and Tyr 210. Docking predictions were confirmed by experimental measurements of the nucleotidase activity in the presence of the three best available phosphonate analogues. These compounds were shown to induce a total inhibition of the cN-II activity at 2 mM. Altogether, this study emphasizes the importance of the non-hydrolysable phosphonate bond in the design of new competitive cN-II inhibitors and the crucial hydrophobic stacking promoted by three protein residues.     

Weather, Water Quality and Infectious Gastrointestinal Illness in Two Inuit Communities in Nunatsiavut, Canada: Potential Implications for Climate Change

Climate change is expected to cause changes in precipitation quantity, intensity, frequency and duration, which will subsequently alter environmental conditions and might increase the risk of waterborne disease. The objective of this study was to describe the seasonality of and explore associations between weather, water quality and occurrence of infectious gastrointestinal illnesses (IGI) in two communities in Nunatsiavut, Canada. Weather data were obtained from meteorological stations in Nain (2005–2008) and Rigolet (2008). Free-chlorine residual levels in drinking water were extracted from municipal records (2005–2008). Raw surface water was tested weekly for total coliform and E. coli counts. Daily counts of IGI-related clinic visits were obtained from health clinic registries (2005–2008). Analysis of weather and health variables included seasonal-trend decomposition procedures based on Loess. Multivariable zero-inflated Poisson regression was used to examine potential associations between weather events (considering 0–4 week lag periods) and IGI-related clinic visits. In Nain, water volume input (rainfall + snowmelt) peaked in spring and summer and was positively associated with levels of raw water bacteriological variables. The number of IGI-related clinic visits peaked in the summer and fall months. Significant positive associations were observed between high levels of water volume input 2 and 4 weeks prior, and IGI-related clinic visits (P < 0.05). This study is the first to systematically gather, analyse and compare baseline data on weather, water quality and health in Nunatsiavut, and illustrates the need for high quality temporal baseline information to allow for detection of future impacts of climate change on regional Inuit human and environmental health.