EVC-EVC2 complex stability and ciliary targeting are regulated by modification with ubiquitin and SUMO.

Ellis van Creveld syndrome and Weyers acrofacial dysostosis are two rare genetic diseases affecting skeletal development. They are both ciliopathies, as they are due to malfunction of primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae and are required for Hedgehog signaling, a key pathway during skeletal morphogenesis. These ciliopathies are caused by mutations affecting the EVC-EVC2 complex, a transmembrane protein heterodimer that regulates Hedgehog signaling from inside primary cilia. Despite the importance of this complex, the mechanisms underlying its stability, targeting and function are poorly understood. To address this, we characterized the endogenous EVC protein interactome in control and Evc-null cells. This proteomic screen confirmed EVC's main known interactors (EVC2, IQCE, EFCAB7), while revealing new ones, including USP7, a deubiquitinating enzyme involved in Hedgehog signaling. We therefore looked at EVC-EVC2 complex ubiquitination. Such ubiquitination exists but is independent of USP7 (and of USP48, also involved in Hh signaling). We did find, however, that monoubiquitination of EVC-EVC2 cytosolic tails greatly reduces their protein levels. On the other hand, modification of EVC-EVC2 cytosolic tails with the small ubiquitin-related modifier SUMO3 has a different effect, enhancing complex accumulation at the EvC zone, immediately distal to the ciliary transition zone, possibly via increased binding to the EFCAB7-IQCE complex. Lastly, we find that EvC zone targeting of EVC-EVC2 depends on two separate EFCAB7-binding motifs within EVC2's Weyers-deleted peptide. Only one of these motifs had been characterized previously, so we have mapped the second herein. Altogether, our data shed light on EVC-EVC2 complex regulatory mechanisms, with implications for ciliopathies.

Phylogenetic and genetic linkage between novel atypical dual-specificity phosphatases from non-metazoan organisms.

Dual-specificity phosphatases (DSPs) constitute a large protein tyrosine phosphatase (PTP) family, with examples in distant evolutive phyla. PFA-DSPs (Plant and Fungi Atypical DSPs) are a group of atypical DSPs present in plants, fungi, kinetoplastids, and slime molds, the members of which share structural similarity with atypical- and lipid phosphatase DSPs from mammals. The analysis of the PFA-DSPs from the plant Arabidopsis thaliana (AtPFA-DSPs) showed differential tissue mRNA expression, substrate specificity, and catalytic activity for these proteins, suggesting different functional roles among plant PFA-DSPs. Bioinformatic analysis revealed the existence of novel PFA-DSP-related proteins in fungi (Oca1, Oca2, Oca4 and Oca6 in Saccharomyces cerevisiae) and protozoa, which were segregated from plant PFA-DSPs. The closest yeast homolog for these proteins was the PFA-DSP from S. cerevisiae ScPFA-DSP1/Siw14/Oca3. Oca1, Oca2, Siw14/Oca3, Oca4, and Oca6 were involved in the yeast response to caffeine and rapamycin stresses. Siw14/Oca3 was an active phosphatase in vitro, whereas no phosphatase activity could be detected for Oca1. Remarkably, overexpression of Siw14/Oca3 suppressed the caffeine sensitivity of oca1, oca2, oca4, and oca6 deleted strains, indicating a genetic linkage and suggesting a functional relationship for these proteins. Functional studies on mutations targeting putative catalytic residues from the A. thaliana AtPFA-DSP1/At1g05000 protein indicated the absence of canonical amino acids acting as the general acid/base in the phosphor-ester hydrolysis, which suggests a specific mechanism of reaction for PFA-DSPs and related enzymes. Our studies demonstrate the existence of novel phosphatase protein families in fungi and protozoa, with active and inactive enzymes linked in common signaling pathways. This illustrates the catalytic and functional complexity of the expanding family of atypical dual-specificity phosphatases in non-metazoans, including parasite organisms responsible for infectious human diseases.

Microbiomic Analysis on Low Abundant Respiratory Biomass Samples; Improved Recovery of Microbial DNA From Bronchoalveolar Lavage Fluid.

In recent years the study of the commensal microbiota is driving a remarkable paradigm shift in our understanding of human physiology. However, intrinsic technical difficulties associated with investigating the Microbiomics of some body niches are hampering the development of new knowledge. This is particularly the case when investigating the functional role played by the human microbiota in modulating the physiology of key organ systems. A major hurdle in investigating specific Microbiome communities is linked to low bacterial density and susceptibility to bias caused by environmental contamination. To prevent such inaccuracies due to background processing noise, harmonized tools for Microbiomic and bioinformatics practices have been recommended globally. The fact that the impact of this undesirable variability is negatively correlated with the DNA concentration in the sample highlights the necessity to improve existing DNA isolation protocols. In this report, we developed and tested a protocol to more efficiently recover bacterial DNA from low volumes of bronchoalveolar lavage fluid obtained from infants and adults. We have compared the efficiency of the described method with that of a commercially available kit for microbiome analysis in body fluids. We show that this new methodological approach performs better in terms of extraction efficiency. As opposed to commercial kits, the DNA extracts obtained with this new protocol were clearly distinguishable from the negative extraction controls in terms of 16S copy number and Microbiome community profiles. Altogether, we described a cost-efficient protocol that can facilitate microbiome research in low-biomass human niches.

Arabidopsis thaliana AtGppl and AtGpp2: two novel low molecular weight phosphatases involved in plant glycerol metabolism.

We have isolated two Arabidopsis thaliana genes, AtGppl and AtGpp2, showing homology with the yeast low molecular weight phosphatases GPP1 and GPP2, which have a high specificity for DL-glycerol-3-phosphate, and moreover homology with DOG1 and DOG2 that dephosphorylate 2-deoxyglucose-6-phosphate. Using a comparative genomic approach, the corresponding genes were identified as conceptual translated haloacid dehalogenase-like hydrolase proteins. AtGppl (gi 18416631) and AtGpp2 (gi 18423981), encode proteins that share 95% identity, with a predicted Mw of 33 and 27 kDa and a pI of 7.8 and 5.6, respectively. Both isoforms have a high specificity for DL-glycerol-3-phosphate, pH optima at 7.0, and Km in the range of 3.5-5.2 mM. AtGppl and AtGpp2 are expressed throughout development in all plant organs, most strongly in siliqua, and expression is not affected by osmotic, ionic or oxidative stress. A putative chloroplast transit peptide cTP-containing sequence is appended to the AtGppl N-terminus while AtGpp2, devoid of this tail, is predicted to be in the extraplastidial cytosol; this compartmenting was further confirmed by subcellular fractionation. An immunohystochemical localization study, using anti-AtGpp2 antibodies, indicates that the AtGpp proteins are mainly restricted to the meristem of immature flower and vascular elements of the root, shoot, leave, siliqua and developing embryo. Considerable immunoreaction was observed in the cytoplasm as well as in plastid compartments of distinct cells types from different heterotrophic Arabidopsis tissues, and particularly localised within phloem companion cells. Transgenic Arabidopsis plants, with gain of AtGpp2 function, show altered phosphatase activity rates and improved tolerance to salt, osmotic and oxidative stress.