The ocular albinism type 1 protein, an intracellular G protein-coupled receptor, regulates melanosome transport in pigment cells

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific G protein-coupled receptor exclusively localized to intracellular organelles, namely lysosomes and melanosomes. Loss of OA1 function leads to the formation of macromelanosomes, suggesting that this receptor is implicated in organelle biogenesis, however the mechanism involved in the pathogenesis of the disease remains obscure. We report here the identification of an unexpected abnormality in melanosome distribution both in retinal pigment epithelium (RPE) and skin melanocytes of Oa1-knock-out (KO) mice, consisting in a displacement of the organelles from the central cytoplasm towards the cell periphery. Despite their depletion from the microtubule (MT)-enriched perinuclear region, Oa1-KO melanosomes were able to aggregate at the centrosome upon disruption of the actin cytoskeleton or expression of a dominant-negative construct of myosin Va. Consistently, quantification of organelle transport in living cells revealed that Oa1-KO melanosomes displayed a severe reduction in MT-based motility; however, this defect was rescued to normal following inhibition of actin-dependent capture at the cell periphery. Together, these data point to a defective regulation of organelle transport in the absence of OA1 and imply that the cytoskeleton might represent a downstream effector of this receptor. Furthermore, our results enlighten a novel function for OA1 in pigment cells and suggest that ocular albinism type 1 might result from a different pathogenetic mechanism than previously thought, based on an organelle-autonomous signalling pathway implicated in the regulation of both membrane traffic and transport

Isolamento del gene responsabile dell'albinismo oculare di tipo 1 e caratterizzazione del suo prodotto proteico

Dottorato di ricerca in scienze dello sviluppo. 8. ciclo. A.a. 1994-95. Relatore F. ZacchelloConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Amino acid deprivation triggers a novel GCN2-independent response leading to the transcriptional reactivation of non-native DNA sequences.

In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan. However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap. We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation. Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome. Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs. The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated. Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2. Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease

GCN2 knockdown does not interfere with transgene reactivation in HepG2 cells.

<p>(A, B) Downregulation of GCN2 protein by RNAi, shown by immunoblotting (A) and quantification (B) of protein extracts from HepG2-OA1 cells, transfected with control or anti-GCN2 siRNAs, and incubated with anti-GCN2 antibody. Arrow, GCN2 specific band; asterisk, non-specific signal detected by the Ab. Ponceau staining was used as loading control. Data are expressed as fold change vs. control siRNA (siRNA CTRL = 1). (C) Relative GCN2 mRNA abundance in HepG2-OA1 cells transfected with control or anti-GCN2 siRNAs. Mean ± SEM of 3 independent experiments. Data are expressed as fold change vs. control siRNA (siRNA CTRL = 1). ***P<0.001 (paired two-tailed Student’s t-test vs. control). (D) Relative transgene (OA1) mRNA abundance in HepG2-OA1 cells transfected with control or anti-GCN2 siRNAs and incubated for 6 h with L-Histidinol (HisOH, GCN2 activator; 4 mM). Mean ± SEM of 3 independent experiments. Data are expressed as fold change vs. untreated control (w/o HisOH = 1). *<i>P</i><0.05, ***<i>P</i><0.001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated). (E) Schematic representation of the experiment.</p

Exogenous transgene and endogenous retroviruses are upregulated in Met/Cys-deprived HeLa cells.

<p>(A,B) Exogenous integrated transgene (OA1) mRNA abundance in HeLa-OA1 cells, cultured in Met/Cys-deprived medium for the indicated time points, and analyzed by RNAseq (A), or RT-qPCR (B), compared to full medium. Data represent RPKM (A), or mean ± SD of 2 technical replicates, expressed as fold change vs. control (full medium at 6 h = 1) (B). (C) Clustering of 172 genomic repeat subfamilies, differentially expressed upon starvation, according to their expression profile. (D) Class distribution of repeat subfamilies belonging to differential expression clusters, compared to all genomic repeat subfamilies (first column). Class DNA includes DNA transposons; SINE includes Alu; LINE includes L1 an L2; LTR includes endogenous retroviruses and solitary LTRs; Satellite includes centromeric acrosomal and telomeric satellites; Others includes SVA, simple repeats, snRNA, and tRNAs. LTR-retroelements are significantly enriched among repeats that are upregulated upon starvation, while LINEs are significantly enriched among repeats that are downregulated. *<i>P</i><0.05, ***<i>P</i><0.001 (Fisher exact test).</p

mTOR inhibition and GCN2 activation differently affect transgene expression in HeLa and HepG2 cells.

<p>Relative transgene (OA1) and CHOP mRNA abundance in HeLa-OA1 (A) and HepG2-OA1 (B) cells, cultured in Met/Cys-deprived medium, or in the presence of PP242 (mTOR inhibitor; 1–3 μM) or L-Histidinol (HisOH, GCN2 activator; 4–16 mM), either alone or in combination for 24–48 h, compared to full medium. Mean ± SEM of 4 (A) or 3 (B) independent experiments. Data are expressed as fold change vs. control (full medium = 1). *P<0.05, **P<0.01, ***P<0.001 (one way ANOVA, followed by Dunnett’s post-test vs. full medium). PP-1 and PP-3, PP242 at 1 and 3 μM, respectively; HisOH-4 and HisOH-16, L-Histidinol at 4 and 16 mM, respectively.</p

Transgene reactivation is abolished by MAPK inhibitors, and is induced by ribosomal inhibitors.

<p>(A) Relative transgene (OA1) mRNA abundance in HeLa-OA1 and HepG2-OA1 cells cultured in full medium or Met/Cys-deprived medium, in the presence or absence of inhibitors for MEK1/2 (U0126; 50 μM), and JNK1/2/3 (SP600125; 20–50 μM) for 24 h. For HeLa-OA1 cells data represent the mean ± SEM of 4 (CTRL and SP600125), or the mean ± range of 2 (U0126) independent experiments; for HepG2-OA1 cells, data represent the mean ± SEM of 3 independent experiments. Results are expressed as fold change vs. control (full medium = 1). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated). Reference genes for qPCR: ACTB (actin beta; HeLa) and ARPC2 (HepG2). (B) Relative transgene (OA1) mRNA abundance in HeLa-OA1 and HepG2-OA1 cells cultured in the presence of CHX (protein elongation inhibitor; 50–100 ug/ml) for different time points, as indicated, compared to untreated control. For HeLa-OA1 cells, data represent the mean ± SEM of 3 independent experiments; for HepG2-OA1 cells, data represent the mean ± SEM of 4 (6 h), or the mean ± range of 2 (16 h) independent experiments. Results are expressed as fold change vs. control (mock = 1). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated). Since the expression of reference genes used for normalizations change considerably upon CHX treatment (particularly at 15–16 h), the data presented are not normalized. However, comparable results were obtained by ARPC2 normalization. (C) Model for the transgene reactivation response to EAA starvation. EAA deficiency inhibits mTORC1 and activates GCN2, which attenuate general translation at different initiation steps. We propose the presence of an additional pathway (red arrows and text), thereby EAA limitation may directly lead to ribosomal stalling or delay during translation initiation (Met deficiency) and/or elongation (all EAA deficiencies), eventually resulting in epigenetic/transcriptional changes. The ERK and JNK branches of MAPKs are known to be activated during EAA starvation by yet unclear mechanisms.</p

The ISR is neither sufficient nor necessary to induce transgene reactivation in HepG2 cells.

<p>(A) Schematic representation of GCN2 activation by AA starvation, resulting in phosphorylation of eIF2a and initiation of the downstream ISR. In addition to GCN2, the ISR may be activated by other eIF2a kinases (PKR, HRI and PERK; not shown in the picture). (B) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 24 h with Salubrinal (a drug that induces the ISR by inhibiting the dephosphorylation of eIF2α; 75 μM), compared to full medium. Mean ± range of two experiments. Data are expressed as fold change vs. control (DMEM = 1). *P<0.05 (paired two-tailed Student’s t-test vs. control). (C) Relative transgene (OA1) and CHOP mRNA abundance in HepG2-OA1 cells treated for 6 h with L-Histidinol (HisOH, GCN2 activator; 4 mM), in the absence or presence of ISRIB (a drug that bypasses the phosphorylation of eIF2α, inhibiting triggering of the ISR; 100 nM). Mean ± range of two experiments. Data are expressed as fold change vs. control (DMEM = 1). **P<0.01, ***P<0.001 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated). (D) Relative transgene (OA1) and ATF4 mRNA abundance in HepG2-OA1 cells transfected with control (CTRL) or anti-ATF4 siRNAs, and incubated in the presence or absence of L-Histidinol (HisOH, GCN2 activator; 4 mM) for 6 h. Mean ± range of two experiments. Data are expressed as fold change vs. control (w/o HisOH = 1, top; control siRNA = 1, bottom). *P<0.05 (one way ANOVA, followed by Tukey’s post-test; P values refer to comparisons vs. control, unless otherwise indicated).</p