Search Results (13)

The retina is highly sensitive to oxygen and blood supply, and hypoxia plays a key role in retinal diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Müller glial cells, which are essential for retinal homeostasis, respond to injury and hypoxia with reactive gliosis, characterized by the upregulation of the glial fibrillary acidic protein (GFAP) and vimentin, cellular hypertrophy, and extracellular matrix changes, which can impair retinal function and repair. The retinal pigment epithelium (RPE) supports photoreceptors, forms part of the blood–retinal barrier, and protects against oxidative stress; its dysfunction contributes to retinal degenerative diseases such as AMD, retinitis pigmentosa (RP), and Stargardt disease (SD). Extracellular vesicles (EVs) play a crucial role in intercellular communication, protein homeostasis, and immune modulation, and have emerged as promising diagnostic and therapeutic tools. Understanding the role of extracellular vesicles’ (EVs’) signaling machinery of glial cells and the retinal pigment epithelium (RPE) is critical for developing effective treatments for retinal degeneration. In this study, we investigated the bidirectional EV-mediated crosstalk between RPE and Müller cells under hypoxic conditions and its impact on cellular metabolism and retinal cell integrity. Our findings demonstrate that RPE-derived extracellular vesicles (RPE EVs) induce time-dependent metabolic reprogramming in Müller cells. Short-term exposure (24 h) promotes pathways supporting neurotransmitter cycling, calcium and mineral absorption, and glutamate metabolism, while prolonged exposure (72 h) shifts Müller cell metabolism toward enhanced mitochondrial function and ATP production. Conversely, Müller cell-derived EVs under hypoxia influenced RPE metabolic pathways, enhancing fatty acid metabolism, intracellular vesicular trafficking, and the biosynthesis of mitochondrial co-factors such as ubiquinone. Proteomic analysis revealed significant modulation of key regulatory proteins. In Müller cells, hypoxic RPE-EV exposure led to reduced expression of Dyskerin Pseudouridine Synthase 1 (DKc1), Eukaryotic Translation Termination Factor 1 (ETF1), and Protein Ser/Thr phosphatases (PPP2R1B), suggesting alterations in RNA processing, translational fidelity, and signaling. RPE cells exposed to hypoxic Müller cell EVs exhibited elevated Ribosome-binding protein 1 (RRBP1), RAC1/2, and Guanine Nucleotide-Binding Protein G(i) Subunit Alpha-1 (GNAI1), supporting enhanced endoplasmic reticulum (ER) function and cytoskeletal remodeling. Functional assays also revealed the compromised barrier integrity of the outer blood–retinal barrier (oBRB) under hypoxic co-culture conditions. These results underscore the adaptive but time-sensitive nature of retinal cell communication via EVs in response to hypoxia. Targeting this crosstalk may offer novel therapeutic strategies to preserve retinal structure and function in ischemic retinopathies. Full article

Modern drug discovery relies on combinatorial screening campaigns to find drug molecules targeting specific disease-associated proteins. The success of such campaigns often relies on functional and structural information of the selected therapeutic target, only achievable once its purification is mastered. With the aim of bypassing the protein purification process to gain insights on the druggability, ligand binding, and/or characterization of protein–protein interactions, herein, we describe the Extract2Chip method. This approach builds on the immobilization of site-specific biotinylated proteins of interest, directly from cellular extracts, on avidin-coated sensor chips to allow for the characterization of molecular interactions via surface plasmon resonance (SPR). The developed method was initially validated using Cyclophilin D (CypD) and subsequently applied to other drug discovery projects in which the targets of interest were difficult to express, purify, and crystallize. Extract2Chip was successfully applied to the characterization of Yes-associated protein (YAP): Transcriptional enhancer factor TEF (TEAD1) protein–protein interaction inhibitors, in the validation of a ternary complex assembly composed of Dyskerin pseudouridine synthase 1 (DKC1) and RuvBL1/RuvBL2, and in the establishment of a fast-screening platform to select the most suitable NUAK family SNF1-like kinase 2 (NUAK2) surrogate for binding and structural studies. The described method paves the way for a potential revival of the many drug discovery campaigns that have failed to deliver due to the lack of suitable and sufficient protein supply. Full article

The highly conserved family of cyclophilins comprises multifunctional chaperones that interact with proteins and RNAs, facilitating the dynamic assembly of multimolecular complexes involved in various cellular processes. Cyclophilin A (CypA), the predominant member of this family, exhibits peptidyl–prolyl cis–trans isomerase activity. This enzymatic function aids with the folding and activation of protein structures and often serves as a molecular regulatory switch for large multimolecular complexes, ensuring appropriate inter- and intra-molecular interactions. Here, we investigated the involvement of CypA in the nucleus, where it plays a crucial role in supporting the assembly and trafficking of heterogeneous ribonucleoproteins (RNPs). We reveal that CypA is enriched in the nucleolus, where it colocalizes with the pseudouridine synthase dyskerin, the catalytic component of the multifunctional H/ACA RNPs involved in the modification of cellular RNAs and telomere stability. We show that dyskerin, whose mutations cause the X-linked dyskeratosis (X-DC) and the Hoyeraal–Hreidarsson congenital ribosomopathies, can directly interact with CypA. These findings, together with the remark that substitution of four dyskerin prolines are known to cause X-DC pathogenic mutations, lead us to indicate this protein as a CypA client. The data presented here suggest that this chaperone can modulate dyskerin activity influencing all its partecipated RNPs. Full article

Dyskerin is an evolutionarily conserved nucleolar protein implicated in a wide range of fundamental biological roles, including telomere maintenance and ribosome biogenesis. Germline mutations of DKC1, the human gene encoding dyskerin, cause the hereditary disorders known as X-linked dyskeratosis congenita (X-DC). Moreover, dyskerin is upregulated in several cancers. Due to the pleiotropic functions of dyskerin, the X-DC clinical features overlap with those of both telomeropathies and ribosomopathies. In this paper, we evaluate the telomerase-independent effects of dyskerin depletion on cellular physiology by using inducible DCK1 knockdown. This system allows the downregulation of DKC1 expression within a short timeframe. We report that, in these cellular systems, dyskerin depletion induces the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum, which in turn induces the activation of the PERK branch of the unfolded protein response. We also demonstrate that the PERK-eIF2a-ATF4-CHOP signaling pathway, activated by dyskerin downregulation, triggers a functional autophagic flux through the inhibition of the PI3K/AKT/mTOR pathway. By revealing a novel unpredicted connection between the loss of dyskerin, autophagy and UPR, our results establish a firm link between the lowering of dyskerin levels and the activation of the ER stress response, that plays a key role in the pathogenesis of several diseases. Full article

Dyskerin is a core-component of the telomerase holo-enzyme, which elongates telomeres. Telomerase is involved in endometrial epithelial cell proliferation. Most endometrial cancers (ECs) have high telomerase activity; however, dyskerin expression in human healthy endometrium or in endometrial pathologies has not been investigated yet. We aimed to examine the expression, prognostic relevance, and functional role of dyskerin in human EC. Endometrial samples from a cohort of 175 women were examined with immunohistochemistry, immunoblotting, and qPCR. The EC cells were transfected with Myc-DDK-DKC1 plasmid and the effect of dyskerin overexpression on EC cell proliferation was assessed by flow cytometry. Human endometrium expresses dyskerin (DKC1) and dyskerin protein levels are significantly reduced in ECs when compared with healthy postmenopausal endometrium. Low dyskerin immunoscores were potentially associated with worse outcomes, suggesting a possible prognostic relevance. Cancer Genome Atlas (TCGA) ECs dataset (n = 589) was also interrogated. The TCGA dataset further confirmed changes in DKC1 expression in EC with prognostic significance. Transient dyskerin overexpression had a negative effect on EC cell proliferation. Our data demonstrates a role for dyskerin in normal endometrium for the first time and confirms aberrant expression with possible prognostic relevance in EC. Interventions aimed at modulating dyskerin levels may provide novel therapeutic options in EC. Full article

RNA modifications are essential for proper RNA processing, quality control, and maturation steps. In the last decade, some eukaryotic DNA repair enzymes have been shown to have an ability to recognize and process modified RNA substrates and thereby contribute to RNA surveillance. Single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1) is a base excision repair enzyme that not only recognizes and removes uracil and oxidized pyrimidines from DNA but is also able to process modified RNA substrates. SMUG1 interacts with the pseudouridine synthase dyskerin (DKC1), an enzyme essential for the correct assembly of small nucleolar ribonucleoproteins (snRNPs) and ribosomal RNA (rRNA) processing. Here, we review rRNA modifications and RNA quality control mechanisms in general and discuss the specific function of SMUG1 in rRNA metabolism. Cells lacking SMUG1 have elevated levels of immature rRNA molecules and accumulation of 5-hydroxymethyluridine (5hmU) in mature rRNA. SMUG1 may be required for post-transcriptional regulation and quality control of rRNAs, partly by regulating rRNA and stability. Full article

Telomeres protect chromosomal ends and they are maintained by the specialised enzyme, telomerase. Endometriosis is a common gynaecological disease and high telomerase activity and higher hTERT levels associated with longer endometrial telomere lengths are characteristics of eutopic secretory endometrial aberrations of women with endometriosis. Our ex-vivo study examined the levels of hTERC and DKC1 RNA and dyskerin protein levels in the endometrium from healthy women and those with endometriosis (n = 117). The in silico study examined endometriosis-specific telomere- and telomerase-associated gene (TTAG) transcriptional aberrations of secretory phase eutopic endometrium utilising publicly available microarray datasets. Eutopic secretory endometrial hTERC levels were significantly increased in women with endometriosis compared to healthy endometrium, yet dyskerin mRNA and protein levels were unperturbed. Our in silico study identified 10 TTAGs (CDKN2A, PML, ZNHIT2, UBE3A, MCCC2, HSPC159, FGFR2, PIK3C2A, RALGAPA1, and HNRNPA2B1) to be altered in mid-secretory endometrium of women with endometriosis. High levels of hTERC and the identified other TTAGs might be part of the established alteration in the eutopic endometrial telomerase biology in women with endometriosis in the secretory phase of the endometrium and our data informs future research to unravel the fundamental involvement of telomerase in the pathogenesis of endometriosis. Full article

Dyskerin is a nucleolar protein involved in the small nucleolar RNA (snoRNA)-guided pseudouridylation of specific uridines on ribosomal RNA (rRNA), and in the stabilization of the telomerase RNA component (hTR). Loss of function mutations in DKC1 causes X-linked dyskeratosis congenita, which is characterized by a failure of proliferating tissues and increased susceptibility to cancer. However, several tumors show dyskerin overexpression. We observed that patients with primary breast cancers with high dyskerin levels are more frequently characterized by shorter survival rates and positive lymph node status than those with tumors with a lower dyskerin expression. To functionally characterize the effects of high dyskerin expression, we generated stably overexpressing DKC1 models finding that increased dyskerin levels conferred a more aggressive cellular phenotype in untransformed immortalized MCF10A cells. Contextually, DKC1 overexpression led to an upregulation of some snoRNAs, including SNORA67 and a significantly increased U1445 modification on 18S rRNA, the known target of SNORA67. Lastly, we found that dyskerin overexpression strongly enhanced the synthetic activity of ribosomes increasing translational efficiency in MCF10A. Altogether, our results indicate that dyskerin may sustain the neoplastic phenotype from an early stage in breast cancer endowing ribosomes with an augmented translation efficiency. Full article

Dyskeratosis congenita (DKC) is a rare inherited disease of impaired telomere maintenance that progressively leads to multi-organ failure, including the bone marrow. By enhancing telomerase activity, androgen derivatives (ADs) are a potential therapeutic option able to re-elongate previously shortened telomeres. Danazol, oxymetholone, and nandrolone are ADs most frequently used to treat DKC. However, no direct in vitro analyses comparing the efficacy of these ADs have been conducted so far. We therefore treated mononuclear cells derived from peripheral blood and bone marrow of four patients with mutations in telomerase reverse transcriptase (TERT, n = 1),in the telomerase RNA component (TERC, n = 2) and in dyskerin pseudouridine synthase 1 (DKC1, n = 1) and found no substantial differences in the activity of these three agents in patients with TERC/TERT mutations. All AD studied produced comparable improvements of proliferation rates as well as degrees of telomere elongation. Increased TERT expression levels were shown with danazol and oxymetholone. The beneficial effects of all ADs on proliferation of bone marrow progenitors could be reversed by tamoxifen, an estrogen antagonist abolishing estrogen receptor-mediated TERT expression, thereby underscoring the involvement of TERT in AD mechanism of action. In conclusion, no significant differences in the ability to functionally enhance telomerase activity could be observed for the three AD studied in vitro. Physicians therefore might choose treatment based on patients’ individual co-morbidities, e.g., pre-existing liver disease and expected side-effects. Full article

Dyskerin is a protein involved in the formation of small nucleolar and small Cajal body ribonucleoproteins. These complexes participate in RNA pseudouridylation and are also components of the telomerase complex required for telomere elongation. Dyskerin mutations cause a rare disease, X-linked dyskeratosis congenita, with no curative treatment. The social amoeba Dictyostelium discoideum contains a gene coding for a dyskerin homologous protein. In this article D. discoideum mutant strains that have mutations corresponding to mutations found in dyskeratosis congenita patients are described. The phenotype of the mutant strains has been studied and no alterations were observed in pseudouridylation activity and telomere structure. Mutant strains showed increased proliferation on liquid culture but reduced growth feeding on bacteria. The results obtained indicated the existence of increased DNA damage response and reactive oxygen species, as also reported in human Dyskeratosis congenita cells and some other disease models. These data, together with the haploid character of D. discoideum vegetative cells, that resemble the genomic structure of the human dyskerin gene, located in the X chromosome, support the conclusion that D. discoideum can be a good model system for the study of this disease. Full article

Immortality is one of the main features of cancer cells. Tumor cells have an unlimited replicative potential, principally due to the holoenzyme telomerase. Telomerase is composed mainly by dyskerin (DKC1), a catalytic retrotranscriptase (hTERT) and an RNA template (hTR). The aim of this work is to develop new inhibitors of telomerase, selecting the interaction between hTR–DKC1 as a target. We designed two models of the human protein DKC1: homology and ab initio. These models were evaluated by different procedures, revealing that the homology model parameters were the most accurate. We selected two hydrophobic pockets contained in the PUA (pseudouridine synthase and archaeosine transglycosylase) domain, using structural and stability analysis. We carried out a docking-based virtual screen on these pockets, using the reported mutation K314 as the center of the docking. The hDKC1 model was tested against a library of 450,000 drug-like molecules. We selected the first 10 molecules that showed the highest affinity values to test their inhibitory activity on the cell line MDA MB 231 (Monroe Dunaway Anderson Metastasis Breast cancer 231), obtaining three compounds that showed inhibitory effect. These results allowed us to validate our design and set the basis to continue with the study of telomerase inhibitors for cancer treatment. Full article

Pseudouridine is the most abundant modification found in RNA. Today, thanks to next-generation sequencing techniques used in the detection of RNA modifications, pseudouridylation sites have been described in most eukaryotic RNA classes. In the present review, we will first consider the available information on the functional roles of pseudouridine(s) in different RNA species. We will then focus on how alterations in the pseudouridylation process may be connected with a series of human pathologies, including inherited disorders, cancer, diabetes, and viral infections. Finally, we will discuss how the availability of novel technical approaches are likely to increase the knowledge in this field. Full article

Pseudouridine (Ψ) is the most abundant post-transcriptionally modified ribonucleoside. Different Ψ modifications correlate with stress responses and are postulated to coordinate the distinct biological responses to a diverse panel of cellular stresses. With the help of different guide RNAs, the dyskerin complex pseudouridylates ribosomal RNA, small nuclear RNA and selective messenger RNAs. To monitor Ψ levels quantitatively, a previously reported high performance liquid chromatography method coupled with ultraviolet detection (HPLC-UV) was modified to determine total Ψ levels in different cellular RNA fractions. Our method was validated to be accurate and precise within the linear range of 0.06–15.36 pmol/μL and to have absolute Ψ quantification levels as low as 3.07 pmol. Using our optimized HPLC assay, we found that 1.20% and 1.94% of all ribonucleosides in nuclear-enriched RNA and small non-coding RNA pools from the HEK293 cell line, and 1.77% and 0.98% of ribonucleosides in 18S and 28S rRNA isolated from the HeLa cell line, were pseudouridylated. Upon knockdown of dyskerin expression, a consistent and significant reduction in total Ψ levels in nuclear-enriched RNA pools was observed. Our assay provides a fast and accurate quantification method to measure changes in Ψ levels of different RNA pools without sample derivatization. Full article