1,721,045 research outputs found
Identification of a TGF-beta responsive element in the human elastin promoter.
No full textIn a previous report (Marigo, V., Volpin, D., and Bressan, G. M. (1993) Biochim. Biophys. Acta 1172, 31-36) it was shown that the elastin promoter contains a region mediating transcriptional activation by TGF-beta in aorta cells, but not in tendon fibroblasts from chick embryos. In this paper we have identified the sequence responsible for this effect by a combination of CAT assays with mutant constructs, DNase I footprinting and electrophoretic mobility shift assays. This TGF-beta responsive element binds different nuclear proteins in chick embryo aorta and tendon cells. Whereas association of the aorta protein(s) to the element is necessary for TGF-beta activation, binding of the tendon protein(s) has apparently no effect on promoter stimulation by the cytokine
Identification of a TGF-beta responsive element in the human elastin promoter
No full textIn a previous report (Marigo, V., Volpin, D., and Bressan, G. M. (1993) Biochim. Biophys. Acta 1172, 31-36) it was shown that the elastin promoter contains a region mediating transcriptional activation by TGF-β in aorta cells, but not in tendon fibroblasts from chick embryos. In this paper we have identified the sequence responsible for this effect by a combination of CAT assays with mutant constructs, DNase I footprinting and electrophoretic mobility shift assays. This TGF-β responsive element binds different nuclear proteins in chick embryo aorta and tendon cells. Whereas association of the aorta protein(s) to the element is necessary for TGF-β activation, binding of the tendon protein(s) has apparently no effect on promoter stimulation by the cytokine
Structural aspects of rod opsin and their implication in genetic diseases
No full textVision in dim-light conditions is triggered by photoactivation of rhodopsin, the visual pigment of rod photoreceptor cells. Rhodopsin is made of a protein, the G protein coupled receptor (GPCR) opsin, and the chromophore 11-cis-retinal. Vertebrate rod opsin is the GPCR best characterized at the atomic level of detail. Since the release of the first crystal structure 20 years ago, a huge number of structures have been released that, in combination with valuable spectroscopic determinations, unveiled most aspects of the photobleaching process. A number of spontaneous mutations of rod opsin have been found linked to vision-impairing diseases like autosomal dominant or autosomal recessive retinitis pigmentosa (adRP or arRP, respectively) and autosomal congenital stationary night blindness (adCSNB). While adCSNB is mainly caused by constitutive activation of rod opsin, RP shows more variegate determinants affecting different aspects of rod opsin function. The vast majority of missense rod opsin mutations affects folding and trafficking and is linked to adRP, an incurable disease that awaits light on its molecular structure determinants. This review article summarizes all major structural information available on vertebrate rod opsin conformational states and the insights gained so far into the structural determinants of adCSNB and adRP linked to rod opsin mutations. Strategies to design small chaperones with therapeutic potential for selected adRP rod opsin mutants will be discussed as well
Targeting molecular pathways for the treatment of inherited retinal degeneration
Full text linkInherited retinal degeneration is a major cause of incurable blindness characterized by loss of retinal photoreceptor cells. Inherited retinal degeneration is characterized by high genetic and phenotypic heterogeneity with several genes mutated in patients affected by these genetic diseases. The high genetic heterogeneity of these diseases hampers the development of effective therapeutic interventions for the cure of a large cohort of patients. Common cell demise mechanisms can be envisioned as targets to treat patients regardless the specific mutation. One of these targets is the increase of intracellular calcium ions, that has been detected in several murine models of inherited retinal degeneration. Recently, neurotrophic factors that favor the efflux of calcium ions to concentrations below toxic levels have been identified as promising molecules that should be evaluated as new treatments for retinal degeneration. Here, we discuss therapeutic options for inherited retinal degeneration and we will focus on neuroprotective approaches, such as the neuroprotective activity of the Pigment epithelium-derived factor. The characterization of specific targets for neuroprotection opens new perspectives together with many questions that require deep analyses to take advantage of this knowledge and develop new therapeutic approaches. We believe that minimizing cell demise by neuroprotection may represent a promising treatment strategy for retinal degeneration
RD Genes Associated with High Photoreceptor cGMP-Levels (Mini-Review)
No full textMany RD-causing mutations lead to a dysregulation of cyclic guanosine monophosphate (cGMP), making cGMP signalling a prime target for the development of new treatment approaches. We showed previously that an analogue of cGMP, which inhibited cGMP signalling targets, increased photoreceptor viability in three rodent RD models carrying different genetic defects, in different RD genes. This raises the question of the possible generality of this approach as a treatment for RD. Here, we review RD genes that can be associated with high cGMP and discuss which RD genes might be amenable to a treatment aimed at inhibiting excessive cGMP signalling
CRISPR/Cas9 gene editing in vitro and in retinal cells in vivo
No full textCRISPR/Cas9 is an efficient tool to knock down specific genes in various organisms. In this chapter, we describe how to assess knock-down of human Rhodopsin (RHO) gene carrying the P23H mutation in vitro, in engineered HeLa cells and in vivo, in P23H RHO transgenic mice. To this aim, we report two molecular assays: site-specific PCR on P23H RHO cells treated with CRISPR/Cas9 and Western blotting analysis on retinal cells prepared from P23H RHO transgenic mice electroporated with CRISPR/Cas9 and GFP plasmids
Identification of a recognition element for CAAT-enhancer binding proteins (C/EBPs) in the elastin promoter.
No full textDNase I footprinting experiments with a DNA fragment of the human elastin promoter have revealed a protected segment comprised between -156 and -172 nucleotides from the translation start site. Various types of gel retardation experiments indicate that the protected element binds different members of the C/EBP family of transcription factors. CAT (chloramphenicol acetyltransferase) fusion constructs carrying the wild type or a mutated promoter sequence were transfected into NIH3T3 and chick embryo aorta cells. The mutation significantly lowered CAT expression in NIH3T3 cells, but was ineffective in aorta cells. Cotransfection of the CAT promoter constructs with eucaryotic vectors expressing C/EBPs, did not affect the production of the reporter gene in NIH3T3 cells; on the contrary a several-fold increase of CAT activity was observed in aortic cells. This increase, however, was identical for the wild type and the mutated constructs. Taken together the data indicate that the elastin promoter contains a recognition site for proteins of the C/EBP family and that the function of this cis-acting element on basal elastin transcription varies with the cell type
Calpain Activation Is the Major Cause of Cell Death in Photoreceptors Expressing a Rhodopsin Misfolding Mutation
Full text linkThe majority of mutations in rhodopsin (RHO) cause misfolding of the protein and has been linked to degeneration of photoreceptor cells in the retina. A lot of attention has been set on targeting ER stress for the development of new therapies for inherited retinal degeneration caused by mutations in the RHO gene. Nevertheless, the cell death pathway activated by RHO misfolded protein is still debated. In this study, we analyzed the retina of the knock-in mouse expressing the P23H misfolded mutant RHO. We found persistent unfolded protein response (UPR) during degeneration. Interestingly, long-term stimulation of the PERK branch of ER stress had a protective effect by phosphorylating nuclear factor erythroid 2–related factor 2 (NRF2) transcription factor, associated with antioxidant responses. Otherwise, we provide evidence that increased intracellular calcium and activation of calpains strongly correlated with rod photoreceptor cell death. By blocking calpain activity, we significantly decreased the activation of caspase-7 and apoptosis-inducing factor (AIF), two cell death effectors, and cell demise, and effectively protected the retina from degeneration caused by the P23H dominant mutation in RHO
Protective effects by inhibition of CNG channels and PKG activity in rd1 mutant photoreceptors
No full textProtective effects by inhibition of CNG channels and PKG activity in rd1 mutant photoreceptor
Identification of novel substrates for cGMP dependent protein kinase (PKG) through kinase activity profiling to understand its putative role in inherited retinal degeneration
Full text linkInherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3',5'-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3',5'-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs
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