ABSTRACT
Elastin, a key structural protein essential for the elasticity of the skin and elastogenic tissues, degrades with age. Replenishing elastin holds promise for anti-aging cosmetics and the supplementation of elastic activities of the cardiovascular system. We employed RiboScreenTM, a technology for identifying molecules that enhance the production of specific proteins, to target the production of tropoelastin. We make use of RiboScreenTM in two crucial steps: first, to pinpoint a target ribosomal protein (TRP), which acts as a switch to increase the production of the protein of interest (POI), and second, to identify small molecules that activate this ribosomal protein switch. Using RiboScreenTM, we identified ribosomal protein L40, henceforth eL40, as a TRP switch to boost tropoelastin production. Drug discovery identified a small-molecule hit that binds to eL40. In-cell treatment demonstrated activity of the eL40 ligand and delivered increased tropoelastin production levels in a dose-dependent manner. Thus, we demonstrate that RiboScreenTM can successfully identify a small-molecule hit capable of selectively enhancing tropoelastin production. This compound has the potential to be developed for topical or systemic applications to promote skin rejuvenation and to supplement elastic functionality within the cardiovascular system.
Subject(s)
Elastin , Ribosomal Proteins , Ribosomes , Tropoelastin , Tropoelastin/metabolism , Tropoelastin/genetics , Humans , Ribosomal Proteins/metabolism , Ribosomal Proteins/genetics , Elastin/metabolism , Elastin/genetics , Ribosomes/metabolism , Ribosomes/drug effects , Ligands , Small Molecule Libraries/pharmacologyABSTRACT
Severe junctional epidermolysis bullosa is a rare genetic, postpartum lethal skin disease, predominantly caused by nonsense/premature termination codon (PTC) sequence variants in LAMB3 gene. LAMB3 encodes LAMB3, the ß subunit of epidermal-dermal skin anchor laminin 332. Most translational reads of a PTC mRNA deliver truncated, nonfunctional proteins, whereas an endogenous PTC readthrough mechanism produces full-length protein at minimal and insufficient levels. Conventional translational readthrough-inducing drugs amplify endogenous PTC readthrough; however, translational readthrough-inducing drugs are either proteotoxic or nonselective. Ribosome editing is a more selective and less toxic strategy. This technique identified ribosomal protein L35/uL29 (ie, RpL35) and RpL35-ligands repurposable drugs artesunate and atazanavir as molecular tools to increase production levels of full-length LAMB3. To evaluate ligand activity in living cells, we monitored artesunate and atazanavir treatment by dual luciferase reporter assays. Production levels of full-length LAMB3 increased up to 200% upon artesunate treatment, up to 150% upon atazanavir treatment, and up to 170% upon combinatorial treatment of RpL35 ligands at reduced drug dosage, with an unrelated PTC reporter being nonresponsive. Proof of bioactivity of RpL35 ligands in selective increase of full-length LAMB3 provides the basis for an alternative, targeted therapeutic route to replenish LAMB3 in severe junctional epidermolysis bullosa.
ABSTRACT
INTRODUCTION: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. METHODS: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. RESULTS: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. CONCLUSION: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB.
Subject(s)
Cell Adhesion Molecules/genetics , Epidermolysis Bullosa, Junctional/genetics , RNA, Messenger/metabolism , Ribosomal Proteins/metabolism , Artesunate/chemistry , Atazanavir Sulfate/chemistry , Epidermolysis Bullosa, Junctional/pathology , Humans , Molecular Docking Simulation , Protein Binding/physiology , Skin/pathology , Skin Physiological Phenomena , KalininABSTRACT
The care of patients with epidermolysis bullosa (EB) poses a major challenge due to the rarity, heterogeneity and complexity of the disease as well as the occurrence of numerous primary and secondary extracutaneous manifestations, causing a significant morbidity and mortality. Specialized treatment centers are essential for offering these patients adequate care, including individual, interdisciplinary coordinated treatments according to current medical standards, and access to innovative therapeutic options. Against this background, the EB House Austria was founded in 2005 and designated the first national center of expertise for genodermatoses with a focus on EB in 2017. In the same year, it became a member of the European Reference Network for Rare Skin Diseases (ERN Skin). The pillars of this institution (outpatient clinic, research unit, academy, clinical study center) interact closely with each other, with numerous national and international clinical and scientific partners, as well as with patients and their relatives via the DEBRA Austria patient group. The development of the EB House Austria as a reference center is characterized by a long-term pioneering work, which in turn could pave the way for the optimization of care for comparable diseases as well as general care structures.
Subject(s)
Epidermolysis Bullosa , Rare Diseases , Austria , Humans , SkinABSTRACT
Accurately addressing the diverse and complex issues of rare diseases (RD) in terms of prevention, recognition, diagnosis, treatment, care and research along key RD specificities, such as great heterogeneity, a limited number of patients, scarcity of relevant knowledge and expertise as well as enormous costs for patient care is a challenging task for healthcare providers and authorities that makes a supranational approach particularly feasible. The European Union has acknowledged RD matters by several initiatives, including efforts to implement national centres of expertise and European reference networks as well as a cross-border referral mechanism to foster access to expert services and to boost dissemination of clinical expertise and research activities. Exemplified by the EB House Austria, a centre of expertise for epidermolysis bullosa cross-linked with international reference partner institutions, this strategy proves its potential to be translated into optimized patient care and to meet the major medical, scientific, social and health-economic impact of RD.
Subject(s)
Epidermolysis Bullosa/diagnosis , Epidermolysis Bullosa/therapy , European Union/organization & administration , Hospitals, General/organization & administration , Models, Organizational , Patient Care Team/organization & administration , Austria , Evidence-Based Medicine , Humans , Interinstitutional Relations , Patient Care Management/organization & administration , Rare Diseases/diagnosis , Rare Diseases/therapyABSTRACT
This retrospective multicentre analysis from the Psoriasis Registry Austria (PsoRA) was conducted to determine drug effectiveness and survival of anti-tumour necrosis factor alpha (anti-TNF-α) agents in patients with moderate-to-severe chronic plaque psoriasis over a 9-year period. Data on 1,019 treatment cycles with adalimumab (n = 460), etanercept (n = 501), and/or infliximab (n = 58) administered to 827 patients (272 women, 555 men) were available for analysis. Compared with etanercept, adalimumab and infliximab showed superior short-term effectiveness. Intention-to-treat-calculated median drug survivals for adalimumab (1,264 days) and etanercept (1,438 days) were similar to each other (p = 0.74), but significantly superior to that of infliximab (477 days) (p = 7.0e-07 vs. adalimumab and p=2.2e-07 vs. etanercept, respectively). Their drug survival rates at 36 months were 51.6%, 56.0%, and 22.6%, respectively. Survival rates correlated significantly with effectiveness for adalimumab and etanercept, but not for infliximab.
Subject(s)
Activities of Daily Living , Biological Products/therapeutic use , Immunosuppressive Agents/therapeutic use , Psoriasis/drug therapy , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Adolescent , Adult , Aged , Aged, 80 and over , Austria , Biological Products/adverse effects , Female , Humans , Immunosuppressive Agents/adverse effects , Intention to Treat Analysis , Kaplan-Meier Estimate , Male , Middle Aged , Proportional Hazards Models , Psoriasis/diagnosis , Psoriasis/immunology , Registries , Retrospective Studies , Severity of Illness Index , Time Factors , Treatment Outcome , Tumor Necrosis Factor-alpha/immunology , Young AdultABSTRACT
BACKGROUND: Several new targeted genes and clinical subtypes have been identified since publication in 2008 of the report of the last international consensus meeting on diagnosis and classification of epidermolysis bullosa (EB). As a correlate, new clinical manifestations have been seen in several subtypes previously described. OBJECTIVE: We sought to arrive at an updated consensus on the classification of EB subtypes, based on newer data, both clinical and molecular. RESULTS: In this latest consensus report, we introduce a new approach to classification ("onion skinning") that takes into account sequentially the major EB type present (based on identification of the level of skin cleavage), phenotypic characteristics (distribution and severity of disease activity; specific extracutaneous features; other), mode of inheritance, targeted protein and its relative expression in skin, gene involved and type(s) of mutation present, and--when possible--specific mutation(s) and their location(s). LIMITATIONS: This classification scheme critically takes into account all published data through June 2013. Further modifications are likely in the future, as more is learned about this group of diseases. CONCLUSION: The proposed classification scheme should be of value both to clinicians and researchers, emphasizing both clinical and molecular features of each EB subtype, and has sufficient flexibility incorporated in its structure to permit further modifications in the future.
Subject(s)
Epidermolysis Bullosa/classification , Epidermolysis Bullosa/genetics , Genetic Predisposition to Disease/epidemiology , Consensus , Epidermolysis Bullosa/diagnosis , Female , Gene Expression Regulation , Humans , Incidence , Male , Prognosis , Sensitivity and Specificity , Severity of Illness IndexABSTRACT
BACKGROUND: Known genetic variants can account for 10% to 20% of all cases with autism spectrum disorders (ASD). Overlapping cellular pathomechanisms common to neurons of the central nervous system (CNS) and in tissues of peripheral organs, such as immune dysregulation, oxidative stress and dysfunctions in mitochondrial and protein synthesis metabolism, were suggested to support the wide spectrum of ASD on unifying disease phenotype. Here, we studied in patient-derived lymphoblastoid cell lines (LCLs) how an ASD-specific mutation in ribosomal protein RPL10 (RPL10[H213Q]) generates a distinct protein signature. We compared the RPL10[H213Q] expression pattern to expression patterns derived from unrelated ASD patients without RPL10[H213Q] mutation. In addition, a yeast rpl10 deficiency model served in a proof-of-principle study to test for alterations in protein patterns in response to oxidative stress. METHODS: Protein extracts of LCLs from patients, relatives and controls, as well as diploid yeast cells hemizygous for rpl10, were subjected to two-dimensional gel electrophoresis and differentially regulated spots were identified by mass spectrometry. Subsequently, Gene Ontology database (GO)-term enrichment and network analysis was performed to map the identified proteins into cellular pathways. RESULTS: The protein signature generated by RPL10[H213Q] is a functionally related subset of the ASD-specific protein signature, sharing redox-sensitive elements in energy-, protein- and redox-metabolism. In yeast, rpl10 deficiency generates a specific protein signature, harboring components of pathways identified in both the RPL10[H213Q] subjects' and the ASD patients' set. Importantly, the rpl10 deficiency signature is a subset of the signature resulting from response of wild-type yeast to oxidative stress. CONCLUSIONS: Redox-sensitive protein signatures mapping into cellular pathways with pathophysiology in ASD have been identified in both LCLs carrying the ASD-specific mutation RPL10[H213Q] and LCLs from ASD patients without this mutation. At pathway levels, this redox-sensitive protein signature has also been identified in a yeast rpl10 deficiency and an oxidative stress model. These observations point to a common molecular pathomechanism in ASD, characterized in our study by dysregulation of redox balance. Importantly, this can be triggered by the known ASD-RPL10[H213Q] mutation or by yet unknown mutations of the ASD cohort that act upstream of RPL10 in differential expression of redox-sensitive proteins.
ABSTRACT
Targeting tumor marker genes by RNA trans-splicing is a promising means to induce tumor cell-specific death. Using a screening system we designed RNA trans-splicing molecules (RTM) specifically binding the pre-mRNA of SLCO1B3, a marker gene in epidermolysis bullosa associated squamous cell carcinoma (EB-SCC). Specific trans-splicing, results in the fusion of the endogenous target mRNA of SLCO1B3 and the coding sequence of the suicide gene, provided by the RTM. SLCO1B3-specific RTMs containing HSV-tk were analyzed regarding their trans-splicing potential in a heterologous context using a SLCO1B3 expressing minigene (SLCO1B3-MG). Expression of the chimeric SLCO1B3-tk was detected by semi-quantitative RT-PCR and Western blot analysis. Cell viability and apoptosis assays confirmed that the RTMs induced suicide gene-mediated apoptosis in SLCO1B3-MG expressing cells. The lead RTM also showed its potential to facilitate a trans-splicing reaction into the endogenous SLCO1B3 pre-mRNA in EB-SCC cells resulting in tk-mediated apoptosis. We assume that the pre-selection of RTMs by our inducible cell-death system accelerates the design of optimal RTMs capable to induce tumor specific cell death in skin cancer cells.
Subject(s)
Biomarkers, Tumor/genetics , Genetic Therapy/methods , Neoplasm Proteins/genetics , Organic Anion Transporters, Sodium-Independent/genetics , RNA, Neoplasm/genetics , Skin Neoplasms/genetics , Skin Neoplasms/therapy , Trans-Splicing/genetics , Apoptosis/genetics , Biomarkers, Tumor/biosynthesis , Epidermolysis Bullosa/genetics , Epidermolysis Bullosa/metabolism , HEK293 Cells , Humans , Neoplasm Proteins/biosynthesis , Organic Anion Transporters, Sodium-Independent/biosynthesis , RNA, Neoplasm/metabolism , Skin Neoplasms/metabolism , Solute Carrier Organic Anion Transporter Family Member 1B3ABSTRACT
Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered 'specialized' ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin ß3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.
Subject(s)
Protein Biosynthesis , Ribosomes/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Genes, Reporter , Luciferases/metabolism , Models, Molecular , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Ribosome Subunits, Small, Eukaryotic/metabolism , Transformation, GeneticABSTRACT
Steady improvements in proteomics present a bioinformatic challenge to retrieve, store, and process the accumulating and often redundant amount of information. In particular, a large-scale comparison and analysis of protein-protein interaction (PPI) data requires tools for data interpretation as well as validation. At this juncture, the Protein Interaction and Molecule Search (PRIMOS) platform represents a novel web portal that unifies six primary PPI databases (BIND, Biomolecular Interaction Network Database; DIP, Database of Interacting Proteins; HPRD, Human Protein Reference Database; IntAct; MINT, Molecular Interaction Database; and MIPS, Munich Information Center for Protein Sequences) into a single consistent repository, which currently includes more than 196,700 redundancy-removed PPIs. PRIMOS supports three advanced search strategies centering on disease-relevant PPIs, on inter- and intra-organismal crosstalk relations (e.g., pathogen-host interactions), and on highly connected protein nodes analysis ("hub" identification). The main novelties distinguishing PRIMOS from other secondary PPI databases are the reassessment of known PPIs, and the capacity to validate personal experimental data by our peer-reviewed, homology-based validation. This article focuses on definite PRIMOS use cases (presentation of embedded biological concepts, example applications) to demonstrate its broad functionality and practical value. PRIMOS is publicly available at http://primos.fh-hagenberg.at.
Subject(s)
Database Management Systems , Databases, Protein , Information Storage and Retrieval/methods , Internet , Protein Interaction Mapping/methods , Proteome/chemistry , Proteome/metabolism , Systems Integration , User-Computer InterfaceABSTRACT
The yeast two-hybrid (Y2H) system is one of the most technically straightforward, effective, and widely used tools for the discovery of the binary peptide or protein interactions. However, its exceptional detection sensitivity poses a serious challenge for affinity ranking and hence prioritizing the resultant large number of putative interactors for follow-up analyses. To overcome this apparent bottleneck, we describe here a novel yeast growth curve-based interaction-monitoring approach that permits semiautomatic quantification, comparison, and statistically ascertained scoring of a large collection of Y2H interactions under real-time conditions. Initially, we conducted a proof-of-concept test of five literature-validated peptide-protein interactions with known affinities in the low µM range, and subsequently used the method to classify 88 novel vitamin D receptor-binding peptides derived from high-throughput screening of a highly diverse artificial peptide aptamer library. Based on our in-depth data evaluation, we conclude that real-time monitoring of clone growth as a measure of relative binding strength offers a facile, cost-effective, accurate, reproducible, and further adaptable complement to standard Y2H-derived clone management.
Subject(s)
High-Throughput Screening Assays/methods , Peptides/chemistry , Proteins/chemistry , Two-Hybrid System Techniques , Binding Sites , Computer Systems , Protein BindingABSTRACT
An important characteristic of epidermolysis bullosa simplex Dowling-Meara (EBS-DM) keratinocytes is the increased level of Jun N-terminal kinase (JNK) stress signalling, which is thought to contribute to the disease phenotype. In this work, we report on the dramatic up-regulation of cytokeratin 14 (K14) in the EBS-DM model cell line KEB7 at both the transcriptional and translational levels, which is noteworthy because KEB7 patient cells are heterozygous for a missense mutation (R125P) in K14. By performing functional assays, we show a direct link between overexpressed wild-type K14 and increased JNK signalling in healthy, immortalized keratinocytes. This observation led us to hypothesize a positive feedback model in which mutant (R125P) K14 triggers JNK signalling, leading to increased AP1-dependent expression of K14, which in turn amplifies JNK signalling further. We therefore suggest that an imbalance of cytoplasmic K14 monomers and K14 incorporated into the intermediate filament (IF) network leads to elevated stress signalling, potentially altering IF dynamics by phosphorylation, which as a side effect, weakens EBS-DM keratinocytes.
Subject(s)
Epidermolysis Bullosa Simplex/metabolism , Keratin-14/metabolism , Cell Line , Epidermolysis Bullosa Simplex/genetics , Humans , Intermediate Filaments/metabolism , Keratin-14/genetics , Keratinocytes/metabolism , MAP Kinase Signaling System , Models, Biological , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense , Stress, Physiological , Transcription Factor AP-1/metabolismSubject(s)
Epidermolysis Bullosa Simplex/etiology , Epidermolysis Bullosa Simplex/physiopathology , Interleukin-1beta/physiology , MAP Kinase Signaling System/physiology , Severity of Illness Index , Signal Transduction/physiology , Alleles , Anthraquinones/pharmacology , Anti-Inflammatory Agents/pharmacology , Antibodies/pharmacology , Cells, Cultured , Epidermolysis Bullosa Simplex/pathology , Humans , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/drug effects , Keratin-14/genetics , Keratin-14/metabolism , Keratinocytes/metabolism , Keratinocytes/pathology , Mutation/geneticsABSTRACT
Numerous studies to date have been aimed at unraveling the large suite of calcitriol (1α,25-dihydroxyvitamin D(3)) response genes in diverse tissues including skin, where this hormone is involved in regulating keratinocyte proliferation, differentiation, permeability barrier formation, innate immunity promotion, antimicrobial peptide production, and wound healing. However, the various approaches differ considerably in probed cell types, scale, throughput, and statistical reliability and do, of note, not reveal much overlap. To further expand our knowledge on presently elusive targets and characterize the extent of fragmentation of existing datasets, we have performed whole-transcriptome microarray examinations of calcitriol-treated human primary keratinocytes. Out of 28,869 genes investigated, we uncovered 86 differentially expressed (67 upregulated and 19 downregulated) candidates that were functionally clustered into five annotation categories: response to wounding, protease inhibition, secondary metabolite biosynthesis, cellular migration, and amine biosynthetic processes. A complementary RTq-PCR study of 78 nominees selected thereof demonstrated significant differential expression of 55 genes (48 upregulated and seven downregulated) within biological replicates. Our hit list contains nine previously authenticated targets (16.36%, proof of concept) and 46 novel genes (83.6%) that have not yet been explicitly described as being differentially regulated within human primary keratinocytes. Direct vitamin D receptor response element predictions within the regulatory promoter regions of 50 of the RTq-PCR-validated targets agreed with known biological functionality and corroborated our stringent data validation pipeline. Altogether, our results indicate the value of continuing these kinds of gene expression studies, which contribute to an enhanced comprehension of calcitriol-mediated processes that may be dysregulated in human skin pathophysiology.