RESUMO
G protein-coupled receptor associated sorting protein 1 (GPRASP1) belongs to a family of 10 proteins that display sequence homologies in their C-terminal region. Several members including GPRASP1 also display a short repeated sequence called the GASP motif that is critically involved in protein-protein interactions with G protein-coupled receptors (GPCRs). Here, we characterized anti-GASP motif antibodies and investigated their potential inhibitory functions. We first showed that our in-house anti-GPRASP1 rabbit polyclonal serum contains anti-GASP motif antibodies and purified them by affinity chromatography. We further showed that these antibodies can detect GPRASP1 and GPRASP2 in Western blot, immunoprecipitation and immunofluorescence experiments while a mutant of GPRASP2, in which the most conserved hydrophobic core of the GASP motifs is mutated, was no more detected. Further characterization of anti-GASP motif antibodies by ELISA and Surface Plasmon Resonance assays suggests that GASP motifs function as multivalent epitopes. Finally, we set-up an Amplified Luminescent Proximity Homogeneous AlphaScreen® assay to detect the interaction between purified ADRB2 receptor and the central domain of GPRASP1 and showed that anti-GASP motif antibodies efficiently inhibit this interaction. Altogether, our results suggest that anti-GASP motif antibodies could represent a valuable tool to neutralize the interaction of GPRASP1 and GPRASP2 with different GPCRs.
Assuntos
Proteínas de Transporte , Receptores Acoplados a Proteínas G , Animais , Coelhos , Transporte Proteico/fisiologiaRESUMO
Genetic loss of collagen VII causes recessive dystrophic epidermolysis bullosa (RDEB), a skin fragility disorder that, unexpectedly, manifests also with elevated colonization of commensal bacteria and frequent wound infections. Here, we describe an unprecedented systemic function of collagen VII as a member of a unique innate immune-supporting multiprotein complex in spleen and lymph nodes. In this complex, collagen VII specifically binds and sequesters the innate immune activator cochlin in the lumen of lymphoid conduits. In genetic mouse models, loss of collagen VII increased bacterial colonization by diminishing levels of circulating cochlin LCCL domain. Intraperitoneal injection of collagen VII, which restored cochlin in the spleen, but not in the skin, reactivated peripheral innate immune cells via cochlin and reduced bacterial skin colonization. Systemic administration of the cochlin LCCL domain was alone sufficient to diminish bacterial supercolonization of RDEB mouse skin. Human validation demonstrated that RDEB patients displayed lower levels of systemic cochlin LCCL domain with subsequently impaired macrophage response in infected wounds. This study identifies an intrinsic innate immune dysfunction in RDEB and uncovers a unique role of the lymphoid extracellular matrix in systemic defense against bacteria.
Assuntos
Colágeno Tipo VII/fisiologia , Epidermólise Bolhosa Distrófica/imunologia , Proteínas da Matriz Extracelular/metabolismo , Imunidade Inata , Tecido Linfoide/metabolismo , Animais , Modelos Animais de Doenças , Matriz Extracelular/imunologia , Humanos , Camundongos Knockout , Pele/microbiologiaRESUMO
The extracellular matrix protein collagen VII is part of the microenvironment of stratified epithelia and critical in organismal homeostasis. Mutations in the encoding gene COL7A1 lead to the skin disorder dystrophic epidermolysis bullosa (DEB), are linked to skin fragility and progressive inflammation-driven fibrosis that facilitates aggressive skin cancer. So far, these changes have been linked to mesenchymal alterations, the epithelial consequences of collagen VII loss remaining under-addressed. As epithelial dysfunction is a principal initiator of fibrosis, we performed a comprehensive transcriptome and proteome profiling of primary human keratinocytes from DEB and control subjects to generate global and detailed images of dysregulated epidermal molecular pathways linked to loss of collagen VII. These revealed downregulation of interaction partners of collagen VII on mRNA and protein level, but also increased abundance of S100 pro-inflammatory proteins in primary DEB keratinocytes. Increased TGF-ß signaling because of loss of collagen VII was associated with enhanced activity of lysosomal proteases in both keratinocytes and skin of collagen VII-deficient individuals. Thus, loss of a single structural protein, collagen VII, has extra- and intracellular consequences, resulting in inflammatory processes that enable tissue destabilization and promote keratinocyte-driven, progressive fibrosis.
Assuntos
Colágeno Tipo VII/genética , Queratinócitos/metabolismo , Lisossomos/metabolismo , Células Cultivadas , Colágeno Tipo VII/metabolismo , Homeostase , Humanos , Mutação , Proteoma , TranscriptomaRESUMO
Genetic disorders affecting the skin, genodermatoses, constitute a large and heterogeneous group of diseases, for which treatment is generally limited to management of symptoms. RNA-based therapies are emerging as a powerful tool to treat genodermatoses. In this review, we discuss in detail RNA splicing modulation by antisense oligonucleotides and RNA trans-splicing, transcript replacement and genome editing by in vitro-transcribed mRNAs, and gene knockdown by small interfering RNA and antisense oligonucleotides. We present the current state of these therapeutic approaches and critically discuss their opportunities, limitations and the challenges that remain to be solved. The aim of this review was to set the stage for the development of new and better therapies to improve the lives of patients and families affected by a genodermatosis.
Assuntos
Terapia Genética/métodos , RNA/uso terapêutico , Dermatopatias Genéticas/terapia , Animais , Técnicas de Silenciamento de Genes , Humanos , Oligonucleotídeos Antissenso/uso terapêutico , RNA Mensageiro/uso terapêutico , Trans-SplicingRESUMO
Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)-a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB.
Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Éxons , Marcação de Genes , Deleção de Sequência , Processamento Alternativo , Animais , Adesão Celular/genética , Linhagem Celular , Movimento Celular/genética , Colágeno Tipo VII/química , Epidermólise Bolhosa Distrófica/metabolismo , Epidermólise Bolhosa Distrófica/patologia , Epidermólise Bolhosa Distrófica/terapia , Humanos , Camundongos , Oligonucleotídeos Antissenso/genética , Dobramento de Proteína , Estabilidade Proteica , Fases de Leitura , Pele/metabolismo , Relação Estrutura-AtividadeRESUMO
Fibronectin serves as a platform to guide and facilitate deposition of collagen and fibrillin microfibrils. During development of fibrotic diseases, altered fibronectin deposition in the extracellular matrix (ECM) is generally an early event. After this, dysregulated organization of fibrillins and fibrillar collagens occurs. Because fibronectin is an essential orchestrator of healthy ECM, perturbation of its ECM-organizational capacity may be involved in development of fibrosis. To investigate this, we employed recessive dystrophic epidermolysis bullosa as a disease model with progressive, severe dermal fibrosis. Fibroblasts from donors with recessive dystrophic epidermolysis bullosa in 2-dimensional and 3-dimensional cultures displayed dysregulated fibronectin deposition. Our analyses revealed that increase of profibrotic dipeptidyl peptidase-4-positive fibroblasts coincides with altered fibronectin deposition. Dipeptidyl peptidase-4 inhibitors normalized deposition of fibronectin and subsequently of fibrillin microfibrils and collagen I. Intriguingly, proteomics and inhibitor and mutagenesis studies disclosed that dipeptidyl peptidase-4 modulates ECM deposition through the proteolysis of the fibronectin N-terminus. Our study provides mechanistic insights into the observed profibrotic activities of dipeptidyl peptidase-4 and extends the understanding of fibronectin-guided ECM assembly in health and disease.
RESUMO
Astacin-like proteinases (ALPs) are regulators of tissue and extracellular matrix (ECM) homeostasis. They convey this property through their ability to convert ECM protein pro-forms to functional mature proteins and by regulating the bioavailability of growth factors that stimulate ECM synthesis. The most studied ALPs in this context are the BMP-1/tolloid-like proteinases. The other subclass of ALPs in vertebrates - the meprins, comprised of meprin α and meprin ß - are emerging as regulators of tissue and ECM homeostasis but have so far been only limitedly investigated. Here, we functionally assessed the roles of meprins in skin wound healing using mice genetically deficient in one or both meprins. Meprin deficiency did not change the course of macroscopic wound closure. However, subtle but distinct contributions of meprins to the healing process and dermal homeostasis were observed. Loss of both meprins delayed re-epithelialization and reduced macrophage infiltration. Abnormal dermal healing and ECM regeneration was observed in meprin deficient wounds. Our analyses also revealed meprin α as one proteinase responsible for maturation of pro-collagen VII to anchoring fibril-forming-competent collagen VII in vivo. Collectively, our study identifies meprins as subtle players in skin wound healing.
RESUMO
Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive DEB (RDEB) or dominant DEB (DDEB) and is associated with a high wound burden. Perpetual cycles of wounding and healing drive fibrosis in DDEB and RDEB, as well as the formation of a tumor-permissive microenvironment. Prolonging wound-free episodes by improving the quality of wound healing would therefore confer substantial benefit for individuals with DEB. The collagenous domain of collagen VII is encoded by 82 in-frame exons, which makes splice-modulation therapies attractive for DEB. Indeed, antisense oligonucleotide-based exon skipping has shown promise for RDEB. However, the suitability of antisense oligonucleotides for treatment of DDEB remains unexplored. Here, we developed QR-313, a clinically applicable, potent antisense oligonucleotide specifically targeting exon 73. We show the feasibility of topical delivery of QR-313 in a carbomer-composed gel for treatment of wounds to restore collagen VII abundance in human RDEB skin. Our data reveal that QR-313 also shows direct benefit for DDEB caused by exon 73 mutations. Thus, the same topically applied therapeutic could be used to improve the wound healing quality in RDEB and DDEB.
Assuntos
Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/terapia , Terapia Genética/métodos , Oligonucleotídeos Antissenso/administração & dosagem , Cicatrização/genética , Animais , Biópsia , Linhagem Celular , Modelos Animais de Doenças , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/patologia , Éxons/genética , Fibroblastos , Fibrose , Humanos , Queratinócitos , Camundongos , Camundongos Transgênicos , Mutação , Oligonucleotídeos Antissenso/genética , Cultura Primária de Células , Pele/efeitos dos fármacosRESUMO
The size and relatively high GC content of cDNAs are challenges for efficient targeted engineering of large collagens. There are both basic biological and therapeutic interests in the ability to modify collagens, as this would allow for studies precisely describing interactions of collagens with specific interaction partners, addressing consequences of individual disease-causing mutations, and assessing therapeutic applicability of precision medicine approaches. Using collagen VII as an example, we will here describe a strategy for rapid and simple modification of cDNAs encoding large collagens. The method is flexible and can be used for the creation of point mutations, small or large deletions, and insertion of DNA.
Assuntos
Clonagem Molecular/métodos , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Mutagênese , Humanos , Deleção de SequênciaRESUMO
High conservation of extracellular matrix proteins often makes the generation of potent species-specific antibodies challenging. For collagen VII there is a particular preclinical interest in the ability to discriminate between human and murine collagen VII. Deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB) - a genetic skin blistering disease, which in its most severe forms is highly debilitating. Advances in gene and cell therapy approaches have made curative therapies for genetic diseases a realistic possibility. DEB is one disorder for which substantial progress has been made toward curative therapies and improved management of the disease. However, to increase their efficacy further preclinical studies are needed. The early neonatal lethality of complete collagen VII deficient mice, have led researches to resort to using models maintaining residual collagen VII expression or grafting of DEB model skin on wild-type mice for preclinical therapy studies. These approaches are challenged by collagen VII expression by the murine host. Thus, the ability to selectively visualize human and murine collagen VII would be a substantial advantage. Here, we describe a novel resource toward this end. By immunization with homologous peptides we generated rabbit polyclonal antibodies that recognize either human or murine collagen VII. Testing on additional species, including rat, sheep, dog, and pig, combined sequence alignment and peptide competition binding assays enabled identification of the major antisera recognizing epitopes. The species-specificity was maintained after denaturation and the antibodies allowed us to simultaneously, specifically visualize human and murine collagen VII in situ.
RESUMO
A specialized, highly developed dermal extracellular matrix (ECM) provides the skin with its unique mechano-resilient properties and is vital for organ function. Accordingly, genetically acquired deficiency of dermal ECM proteins or proteins essential for the post-translational modification and homeostasis of the dermal ECM, results in diseases affecting the skin. Some of these diseases are lethal or lead to severe complications for the affected individuals. At present limited efficient and evidence-based treatment options exist for genetic ECM diseases of the skin. There is thus a high unmet medical need, creating an urgent demand to develop improved care for these diseases. Here, by drawing examples from the wealth of research on epidermolysis bullosa, we present the current status of biological and small molecule therapies for genetic ECM diseases with skin manifestations. We discuss challenges, and using existing data to propose strategies and future directions allowing development of more efficacious therapies and advancement of them into clinical practice.
Assuntos
Epidermólise Bolhosa/terapia , Matriz Extracelular/genética , Pele/patologia , Terapia Baseada em Transplante de Células e Tecidos , Epidermólise Bolhosa/genética , Epidermólise Bolhosa/patologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Predisposição Genética para Doença , Humanos , Processamento de Proteína Pós-Traducional , Proteostase , Pele/efeitos dos fármacos , Pele/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
For most disorders caused by mutations in genes encoding basement membrane (BM) proteins, there are at present only limited treatment options available. Genetic BM-linked disorders can be viewed as especially suited for treatment with cell-based therapy approaches because the proteins that need to be restored are located in the extracellular space. In consequence, complete and permanent engraftment of cells does not necessarily have to occur to achieve substantial causal therapeutic effects. For these disorders cells can be used as transient vehicles for protein replacement. In addition, it is becoming evident that BM-linked genetic disorders are modified by secondary diseases mechanisms. Cell-based therapies have also the ability to target such disease modifying mechanisms. Thus, cell therapies can simultaneously provide causal treatment and symptomatic relief, and accordingly hold great potential for treatment of BM-linked disorders. However, this potential has for most applications and diseases so far not been realized. Here, we will present the state of cell therapies for BM-linked diseases. We will discuss use of both pluripotent and differentiated cells, the limitation of the approaches, their challenges, and the way forward to potential wider implementation of cell therapies in the clinics.
Assuntos
Membrana Basal/patologia , Encefalopatias/terapia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Oftalmopatias/terapia , Cardiopatias/terapia , Nefropatias/terapia , Doenças Musculares/terapia , Dermatopatias/terapia , Membrana Basal/metabolismo , Encefalopatias/metabolismo , Encefalopatias/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Oftalmopatias/metabolismo , Oftalmopatias/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/transplante , Cardiopatias/metabolismo , Cardiopatias/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinócitos/transplante , Nefropatias/metabolismo , Nefropatias/patologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Dermatopatias/metabolismo , Dermatopatias/patologia , Transplante de Células-TroncoRESUMO
Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies.
Assuntos
Detergentes/química , Proteínas de Membrana/química , Detergentes/metabolismo , Lipossomos , Proteínas de Membrana/metabolismo , Micelas , Modelos Moleculares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normasRESUMO
Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors--a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption.
Assuntos
Bicamadas Lipídicas/química , Nanoestruturas/química , Receptores Opioides kappa/química , Receptores Opioides kappa/metabolismo , Detergentes/química , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Humanos , Ligantes , Bicamadas Lipídicas/metabolismo , Pichia/genética , Ligação Proteica , Estabilidade Proteica , Receptores Opioides kappa/genética , Receptores Opioides kappa/isolamento & purificação , SolubilidadeRESUMO
The "generalized severe" form of recessive dystrophic epidermolysis bullosa (RDEB-gen sev) is caused by bi-allelic null mutations in COL7A1, encoding type VII collagen. The absence of type VII collagen leads to blistering of the skin and mucous membranes upon the slightest trauma. Because most patients carry exonic point mutations or small insertions/deletions, most exons of COL7A1 are in-frame, and low levels of type VII collagen already drastically improve the disease phenotype, this gene seems a perfect candidate for antisense oligonucleotide (AON)-mediated exon skipping. In this study, we examined the feasibility of AON-mediated exon skipping in vitro in primary cultured keratinocytes and fibroblasts, and systemically in vivo using a human skin-graft mouse model. We show that treatment with AONs designed against exon 105 leads to in-frame exon 105 skipping at the RNA level and restores type VII collagen protein production in vitro. Moreover, we demonstrate that systemic delivery in vivo induces de novo expression of type VII collagen in skin grafts generated from patient cells. Our data demonstrate strong proof-of-concept for AON-mediated exon skipping as a systemic therapeutic strategy for RDEB.
Assuntos
Colágeno Tipo VII/genética , DNA/genética , Epidermólise Bolhosa Distrófica/genética , Deleção de Sequência , Sequência de Aminoácidos , Colágeno Tipo VII/metabolismo , Epidermólise Bolhosa Distrófica/diagnóstico , Epidermólise Bolhosa Distrófica/metabolismo , Feminino , Humanos , Masculino , LinhagemRESUMO
GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure-function relationship analysis of the molecular interaction between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence of a small, highly conserved and repeated "GASP motif" of 15 amino acids. We further showed using GST-pull down, surface plasmon resonance and co-immunoprecipitation experiments that the central domain of GASP-1, which contains 22 GASP motifs, is essential for the interaction with GPCRs. We then used site directed mutagenesis and competition experiments with synthetic peptides to demonstrate that the GASP motif, and particularly its highly conserved core sequence SWFW, is critically involved in the interaction with GPCRs. Overall, our data show that several members of the GASP family interact with GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Humanos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Proteínas de Transporte Vesicular/químicaRESUMO
Among the small number of expression systems validated for the mass production of eukaryotic membrane proteins (EMPs), the methylotrophic yeast Pichia pastoris stands as one of the most efficient hosts. This system has been used to produce crystallization-grade proteins for a variety of EMPs, from which high-resolution 3D structures have been determined. This unit describes a set of guidelines and instructions to overexpress membrane proteins using the P. pastoris system. Using a G protein-coupled receptor (GPCR) as a model EMP, these protocols illustrate the necessary steps, starting with the design of the DNA sequence to be expressed, through the preparation and analysis of samples containing the corresponding membrane protein of interest. In addition, recommendations are given on a series of experimental parameters that can be optimized to substantially improve the amount and/or the functionality of the expressed EMPs.