Autophagy appears to be an important mechanism in the pathogenesis of TRAPS, an observation that provides a rationale for the most effective therapy in this autoinflammatory disorder. Our findings also suggest that autophagy could be proposed as a novel therapeutic target for TRAPS and possibly other similar diseases.

Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials. While the activity of wild-type TMEM16E depended on elevated cytosolic Ca2+ levels, a mutant form carrying the GDD-causing T513I substitution showed PLS and large time-dependent ion currents even at low cytosolic Ca2+ concentrations. Contrarily, mutation of the homologous position in the Ca2+-activated Cl− channel TMEM16B paralog hardly affected its function. In summary, these data provide the first direct demonstration of Ca2+-dependent PLS activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-017-2704-9) contains supplementary material, which is available to authorized users.

GDP-D-mannose 4,6-dehydratase (GMD) is a key enzyme involved in the synthesis of 6-deoxyhexoses in prokaryotes and eukaryotes. Paramecium bursaria chlorella virus-1 (PBCV-1) encodes a functional GMD, which is unique among characterized GMDs because it also has a strong stereospecific NADPHdependent reductase activity leading to GDP-D-rhamnose formation (Tonetti, M., Zanardi, D., Gurnon, J., Fruscione, F., Armirotti, A., Damonte, G., Sturla, L., De Flora, A., and Van Etten, J.L. (2003) J. Biol. Chem. 278, 21559 -21565). In the present study we characterized a recombinant GMD encoded by another chlorella virus, Acanthocystis turfacea chlorella virus 1 (ATCV-1), demonstrating that it has the expected dehydratase activity. However, it also displayed significant differences when compared with PBCV-1 GMD. In particular, ATCV-1 GMD lacks the reductase activity present in the PBCV-1 enzyme. Using recombinant PBCV-1 and ATCV-1 GMDs, we determined that the enzymatically active proteins contain tightly bound NADPH and that NADPH is essential for maintaining the oligomerization status as well as for the stabilization and function of both enzymes. Phylogenetic analysis indicates that PBCV-1 GMD is the most evolutionary diverged of the GMDs. We conclude that this high degree of divergence was the result of the selection pressures that led to the acquisition of new reductase activity to synthesize GDP-D-rhamnose while maintaining the dehydratase activity in order to continue to synthesize GDP-L-fucose.GDP-D-mannose 4,6-dehydratase (GMD, 2 EC 4.2.1.47) is the first enzyme in the biosynthetic pathways that leads to the formation of several deoxyhexoses, such as GDP-L-fucose (1), GDP-D-rhamnose (2), GDP-6-deoxy-D-talose (3), and GDP-Dperosamine (4). Genes encoding GMDs have been identified in most prokaryotes and eukaryotes (5-8). Recently, we characterized the first GMD encoded by a virus, Paramecium bursaria chlorella virus 1 (PBCV-1) (9). PBCV-1 is the prototype of a group (family Phycodnaviridae) of large, icosahedral, plaqueforming, double-stranded DNA viruses that replicate in certain unicellular, eukaryotic chlorella-like green algae (10). In nature, the chlorella host is a hereditary endosymbiont in the ciliated protozoan P. bursaria. PBCV-1 has ϳ365 protein-encoding genes, including genes that encode several proteins that resemble enzymes involved in the metabolism and processing of sugars; these proteins include five putative glycosyltransferases believed to be involved in the glycosylation of the virus major capsid protein (11). PBCV-1 also encodes the two enzymes involved in GDP-Lfucose biosynthesis, GMD and GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER) (Fig. 1) (9).GMD is a member of the short-chain dehydrogenase/reductase family of proteins (12) and in general has structural features compatible with enzymes belonging to the reductase-epimerase-dehydrogenase superfamily (13). GMD sequences are well conserved among organisms; they all contain three conserved amino acids, Ser/Thr, Tyr, and Lys, involved in ca...

Heterozygous mutations in the PHOX2B gene are causative of congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by defective autonomic control of breathing due to the impaired differentiation of neural crest cells. Among PHOX2B mutations, polyalanine (polyAla) expansions are almost exclusively associated with isolated CCHS, whereas frameshift variants, although less frequent, are often more severe than polyAla expansions and identified in syndromic CCHS. This article provides a complete review of all the frameshift mutations identified in cases of isolated and syndromic CCHS reported in the literature as well as those identified by us and not yet published. These were considered in terms of both their structure, whether the underlying indels induced frameshifts of either 1 or 2 steps (“frame 2” and “frame 3” mutations respectively), and clinical associations. Furthermore, we evaluated the structural and functional effects of one “frame 3” mutation identified in a patient with isolated CCHS, and one “frame 2” mutation identified in a patient with syndromic CCHS, also affected with Hirschsprung's disease and neuroblastoma. The data thus obtained confirm that the type of translational frame affects the severity of the transcriptional dysfunction and the predisposition to isolated or syndromic CCHS.

Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P) is a low-abundance signaling lipid associated with endo-lysosomal and vacuolar membranes in eukaryotic cells. Recent studies on indicated a critical role of PI(3,5)P in vacuolar acidification and morphology during ABA-induced stomatal closure, but the molecular targets in plant cells remained unknown. By using patch-clamp recordings on vacuoles, we show here that PI(3,5)P does not affect the activity of vacuolar H-pyrophosphatase or vacuolar H-ATPase. Instead, PI(3,5)P at low nanomolar concentrations inhibited an inwardly rectifying conductance, which appeared upon vacuolar acidification elicited by prolonged H pumping activity. We provide evidence that this novel conductance is mediated by chloride channel a (CLC-a), a member of the anion/H exchanger family formerly implicated in stomatal movements in H-dependent currents were absent in knock-out vacuoles, and canonical CLC-a-dependent nitrate/H antiport was inhibited by low concentrations of PI(3,5)P Finally, using the pH indicator probe BCECF, we show that CLC-a inhibition contributes to vacuolar acidification. These data provide a mechanistic explanation for the essential role of PI(3,5)P and advance our knowledge about the regulation of vacuolar ion transport.

No abstract

No abstract

HSCR is a congenital disorder of the enteric nervous system, characterized by the absence of neurons along a variable length of the gut resulting from loss-of-function RET mutations. Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy characterized by impaired response to hypercapnia and hypoxemia caused by heterozygous mutations of the PHOX2B gene, mostly polyalanine (polyA) expansions but also missense, nonsense, and frameshift mutations, while polyA contractions are common in the population and believed neutral. HSCR associated CCHS can present in patients carrying PHOX2B mutations. Indeed, RET expression is orchestrated by different transcriptional factors among which PHOX2B, thus suggesting its possible role in HSCR pathogenesis. Following the observation of HSCR patients carrying in frame trinucleotide deletions within the polyalanine stretch in exon 3 (polyA contractions), we have verified the hypothesis that these PHOX2B variants do reduce its transcriptional activity, likely resulting in a down-regulation of RET expression and, consequently, favouring the development of the HSCR phenotype. Using proper reporter constructs, we show here that the in vitro transactivation of the RET promoter by different HSCR-associated PHOX2B polyA variants has resulted significantly lower compared to the effect of PHOX2B wild type protein. In particular, polyA contractions do induce a reduced transactivation of the RET promoter, milder compared to the severe polyA expansions associated with CCHS+HSCR, and correlated with the length of the deleted trait, with a more pronounced effect when contractions are larger.