Sandro Montefusco

Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB

The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signalling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release through mucolipin 1 (MCOLN1) activates calcineurin, which binds and dephosphorylates TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis through TFEB required MCOLN1-mediated calcineurin activation. These data link lysosomal calcium signalling to both calcineurin regulation and autophagy induction and identify the lysosome as a hub for the signalling pathways that regulate cellular homeostasis.

FGF signalling regulates bone growth through autophagy

Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34–beclin-1. Autophagy is completely suppressed in growth plates from Fgf18−/− embryos, while Fgf18+/− heterozygous and Fgfr4−/− mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18+/− and Fgfr4−/− phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.

The Phytoestrogen Genistein Modulates Lysosomal Metabolism and Transcription Factor EB (TFEB) Activation

Background: Genistein is a potential drug for certain inherited lysosomal disorders. Results: Genistein influences molecular cross-talk in the cell responsible for lysosomal enhancement. Conclusion: Genistein potentiates lysosomal metabolism by activating transcription factor EB (TFEB). Significance: The explanation of genistein action offers more adequate therapeutic procedures for the treatment of some lysosomal storage diseases. Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) has been previously proposed as a potential drug for use in substrate reduction therapy for mucopolysaccharidoses, a group of inherited metabolic diseases caused by mutations leading to inefficient degradation of glycosaminoglycans (GAGs) in lysosomes. It was demonstrated that this isoflavone can cross the blood-brain barrier, making it an especially desirable potential drug for the treatment of neurological symptoms present in most lysosomal storage diseases. So far, no comprehensive genomic analyses have been performed to elucidate the molecular mechanisms underlying the effect elicited by genistein. Therefore, the aim of this work was to identify the genistein-modulated gene network regulating GAG biosynthesis and degradation, taking into consideration the entire lysosomal metabolism. Our analyses identified over 60 genes with known roles in lysosomal biogenesis and/or function whose expression was enhanced by genistein. Moreover, 19 genes whose products are involved in both GAG synthesis and degradation pathways were found to be remarkably differentially regulated by genistein treatment. We found a regulatory network linking genistein-mediated control of transcription factor EB (TFEB) gene expression, TFEB nuclear translocation, and activation of TFEB-dependent lysosome biogenesis to lysosomal metabolism. Our data indicate that the molecular mechanism of genistein action involves not only impairment of GAG synthesis but more importantly lysosomal enhancement via TFEB. These findings contribute to explaining the beneficial effects of genistein in lysosomal storage diseases as well as envisage new therapeutic approaches to treat these devastating diseases.

AAV-mediated tyrosinase gene transfer restores melanogenesis and retinal function in a model of oculo-cutaneous albinism type I (OCA1).

Oculo-cutaneous albinism type 1 (OCA1) is characterized by congenital hypopigmentation and is due to mutations in the TYROSINASE gene (TYR). In this study, we have characterized the morpho-functional consequences of the lack of tyrosinase activity in the spontaneous null mouse model of OCA1 (Tyr(c-2j)). Here, we show that adult Tyr(c-2j) mice have several retinal functional anomalies associated with photoreceptor loss. To test whether these anomalies are reversible upon TYR complementation, we performed intraocular administration of an adeno-associated virus (AAV)-based vector, encoding the human TYR gene, in adult Tyr(c-2j) mice. This resulted in melanosome biogenesis and ex novo synthesis of melanin in both neuroectodermally derived retinal pigment epithelium (RPE) and in neural crest-derived choroid and iris melanocytes. Ocular melanin accumulation prevented progressive photoreceptor degeneration and resulted in restoration of retinal function. Our results reveal novel properties of pigment cells and show that the developmental anomalies of albino mice are associated with defects occurring in postnatal life, adding novel insights on OCA1 disease pathogenesis. In addition, we provide proof-of-principle of an effective gene-based strategy relevant for future application in albino patients.

Molecular and Clinical Characterization of Albinism in a Large Cohort of Italian Patients

PURPOSE The purpose of this study was to identify the molecular basis of albinism in a large cohort of Italian patients showing typical ocular landmarks of the disease and to provide a full characterization of the clinical ophthalmic manifestations. METHODS DNA samples from 45 patients with ocular manifestations of albinism were analyzed by direct sequencing analysis of five genes responsible for albinism: TYR, P, TYRP1, SLC45A2 (MATP), and OA1. All patients studied showed a variable degree of skin and hair hypopigmentation. Eighteen patients with distinct mutations in each gene associated with OCA were evaluated by detailed ophthalmic analysis, optical coherence tomography (OCT), and fundus autofluorescence. RESULTS Disease-causing mutations were identified in more than 95% of analyzed patients with OCA (28/45 [62.2%] cases with two or more mutations; 15/45 [33.3%] cases with one mutation). Thirty-five different mutant alleles were identified of which 15 were novel. Mutations in TYR were the most frequent (73.3%), whereas mutations in P occurred more rarely (13.3%) than previously reported. Novel mutations were also identified in rare loci such as TYRP1 and MATP. Mutations in the OA1 gene were not detected. Clinical assessment revealed that patients with iris and macular pigmentation had significantly higher visual acuity than did severe hypopigmented phenotypes. CONCLUSIONS TYR gene mutations represent a relevant cause of oculocutaneous albinism in Italy, whereas mutations in P present a lower frequency than that found in other populations. Clinical analysis revealed that the severity of the ocular manifestations depends on the degree of retinal pigmentation.

Identification of p38 MAPK and JNK as new targets for correction of Wilson disease‐causing ATP7B mutants

Wilson disease (WD) is an autosomal recessive disorder that is caused by the toxic accumulation of copper (Cu) in the liver. The ATP7B gene, which is mutated in WD, encodes a multitransmembrane domain adenosine triphosphatase that traffics from the trans‐Golgi network to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. Several ATP7B mutations, including H1069Q and R778L that are two of the most frequent variants, result in protein products, which, although still functional, remain in the endoplasmic reticulum. Thus, they fail to reach Cu excretion sites, resulting in the toxic buildup of Cu in the liver of WD patients. Therefore, correcting the location of these mutants by leading them to the appropriate functional sites in the cell should restore Cu excretion and would be beneficial to help large cohorts of WD patients. However, molecular targets for correction of endoplasmic reticulum‐retained ATP7B mutants remain elusive. Here, we show that expression of the most frequent ATP7B mutant, H1069Q, activates p38 and c‐Jun N‐terminal kinase signaling pathways, which favor the rapid degradation of the mutant. Suppression of these pathways with RNA interference or specific chemical inhibitors results in the substantial rescue of ATP7BH1069Q (as well as that of several other WD‐causing mutants) from the endoplasmic reticulum to the trans‐Golgi network compartment, in recovery of its Cu‐dependent trafficking, and in reduction of intracellular Cu levels. Conclusion: Our findings indicate p38 and c‐Jun N‐terminal kinase as intriguing targets for correction of WD‐causing mutants and, hence, as potential candidates, which could be evaluated for the development of novel therapeutic strategies to combat WD. (Hepatology 2016;63:1842‐1859)

Copper binds the carboxy-terminus of trefoil protein 1 (TFF1), favoring its homodimerization and motogenic activity

Trefoil protein 1 (TFF1) is a small secreted protein belonging to the trefoil factor family of proteins, that are present mainly in the gastrointestinal (GI) tract and play pivotal roles as motogenic factors in epithelial restitution, cell motility, and other incompletely characterized biological processes. We previously reported the up-regulation of TFF1 gene in copper deficient rats and the unexpected property of the peptide to selectively bind copper. Following the previous evidence, here we report the characterization of the copper binding site by fluorescence quenching spectroscopy and mass spectrometric analyses. We demonstrate that Cys58 and at least three Glu surrounding residues surrounding it, are essential to efficiently bind copper. Moreover, copper binding promotes the TFF1 homodimerization, thus increasing its motogenic activity in in vitro wound healing assays. Copper levels could then modulate the TFF1 functions in the GI tract, as well as its postulated role in cancer progression and invasion.

Copper promotes TFF1-mediated Helicobacter pylori colonization.

The trefoil peptides (TFF1, TFF2 and TFF3) are a family of small highly conserved proteins that play an essential role in epithelial regeneration within the gastrointestinal tract, where they are mainly expressed. TFF1 expression is strongly induced after mucosal injury and it has been proposed that tff1 functions as a gastric tumor suppressor gene. Several studies confirm that tff1 expression is frequently lost in gastric cancer because of deletions, mutations or methylation of the tff1 promoter. Infection by Helicobacter pylori (H. pylori) results in chronic gastritis and it can lead to the development of gastric or duodenal ulcers. Moreover, it is known that there is a strong link to the development of gastric cancer. It has been shown that H. pylori interacts with the dimeric form of TFF1 and that the rough form of lipopolysaccharide mediates this interaction. We have previously reported that the carboxy-terminus of TFF1 is able to specifically bind copper ions (Cu) and that Cu binding favours the homodimerization of the peptide, thus enhancing its motogenic activity. Here, we report that the Cu-TFF1 cuprocomplex promotes adherence of H. pylori to epithelial cells. Adherence of H. pylori to gastric adenocarcinoma cells, AGS AC1 cells, induced to hyper-express TFF1 was enhanced compared to noninduced cells. Copper further promoted this interaction. A H. pylori mutant unable to bind TFF1 did not show enhanced infection of induced cells. Cu treatment induced a thickening of the mucus layer produced by the colorectal adenocarcinoma mucus secreting, goblet cells, HT29-E12 and promoted H. pylori colonisation. Finally, SPR analysis shows that the C-terminus of TFF1, involved in the binding of copper, is also able to selectively bind H. pylori RF-LPS.

A reverse-engineering approach to dissect post-translational modulators of transcription factor’s activity from transcriptional data

BackgroundTranscription factors (TFs) act downstream of the major signalling pathways functioning as master regulators of cell fate. Their activity is tightly regulated at the transcriptional, post-transcriptional and post-translational level. Proteins modifying TF activity are not easily identified by experimental high-throughput methods.ResultsWe developed a computational strategy, called Differential Multi-Information (DMI), to infer post-translational modulators of a transcription factor from a compendium of gene expression profiles (GEPs). DMI is built on the hypothesis that the modulator of a TF (i.e. kinase/phosphatases), when expressed in the cell, will cause the TF target genes to be co-expressed. On the contrary, when the modulator is not expressed, the TF will be inactive resulting in a loss of co-regulation across its target genes. DMI detects the occurrence of changes in target gene co-regulation for each candidate modulator, using a measure called Multi-Information. We validated the DMI approach on a compendium of 5,372 GEPs showing its predictive ability in correctly identifying kinases regulating the activity of 14 different transcription factors.ConclusionsDMI can be used in combination with experimental approaches as high-throughput screening to efficiently improve both pathway and target discovery. An on-line web-tool enabling the user to use DMI to identify post-transcriptional modulators of a transcription factor of interest che be found at http://dmi.tigem.it.

Trefoil Factor 1 is involved in gastric cell copper homeostasis.

Trefoil Factor 1 belongs to a group of small secreted proteins (the Trefoil Factor Family proteins), that are localized within the mucous granules and are expressed and secreted by epithelial cells that line mucous membranes. Trefoil factors are mainly expressed in the gastrointestinal tract, where they normally contribute to maintain the integrity of the mucosa. We recently demonstrated a selective binding ability of Trefoil Factor 1 for copper ions, through its carboxy-terminal tail, and we also observed that copper levels influence the equilibrium between the monomeric and homodimeric forms of Trefoil Factor 1, thus modulating its biological activity. Here we report that transcriptional regulation of Trefoil Factor 1 is also affected by copper levels, through the modulated binding of the copper-sensing transcription factor Sp1 onto the responsive elements present in the regulatory region of the gene. In addition we demonstrate that copper overload causes an accumulation of the trefoil protein in the Trans-Golgi Network and that Trefoil Factor 1 levels can influence copper excretion and copper related toxicity. These findings suggest that the protein might play a role in the overall complex mechanisms of copper homeostasis in the gastrointestinal tissues.