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Find video protocols related to scientific articles indexed in Pubmed.
Rab GTPase Prenylation Hierarchy and Its Potential Role in Choroideremia Disease.
PLoS ONE
PUBLISHED: 01-01-2013
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Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transferase and Rab escort protein (REP). Human genetic defects resulting in the loss one of two REP isoforms REP-1, lead to underprenylation of RabGTPases that manifests in retinal degradation and blindness known as choroideremia. In this study we used a combination of microinjections and chemo-enzymatic tagging to establish whether Rab GTPases are prenylated and delivered to their target cellular membranes with the same rate. We demonstrate that although all tested Rab GTPases display the same rate of membrane delivery, the extent of Rab prenylation in 5 hour time window vary by more than an order of magnitude. We found that Rab27a, Rab27b, Rab38 and Rab42 display the slowest prenylation in vivo and in the cell. Our work points to possible contribution of Rab38 to the emergence of choroideremia in addition to Rab27a and Rab27b.
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Cellular FRET-Biosensors to Detect Membrane Targeting Inhibitors of N-Myristoylated Proteins.
PLoS ONE
PUBLISHED: 01-01-2013
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Hundreds of eukaryotic signaling proteins require myristoylation to functionally associate with intracellular membranes. N-myristoyl transferases (NMT) responsible for this modification are established drug targets in cancer and infectious diseases. Here we describe NANOMS (NANOclustering and Myristoylation Sensors), biosensors that exploit the FRET resulting from plasma membrane nanoclustering of myristoylated membrane targeting sequences of G?i2, Yes- or Src-kinases fused to fluorescent proteins. When expressed in mammalian cells, NANOMS report on loss of membrane anchorage due to chemical or genetic inhibition of myristoylation e.g. by blocking NMT and methionine-aminopeptidase (Met-AP). We used Yes-NANOMS to assess inhibitors of NMT and a cherry-picked compound library of putative Met-AP inhibitors. Thus we successfully confirmed the activity of DDD85646 and fumagillin in our cellular assay. The developed assay is unique in its ability to identify modulators of signaling protein nanoclustering, and is amenable to high throughput screening for chemical or genetic inhibitors of functional membrane anchorage of myristoylated proteins in mammalian cells.
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AAV-mediated gene therapy for choroideremia: preclinical studies in personalized models.
PLoS ONE
PUBLISHED: 01-01-2013
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Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1(st) or 2(nd) decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.
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Design and application of in vivo FRET biosensors to identify protein prenylation and nanoclustering inhibitors.
Chem. Biol.
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Protein prenylation is required for membrane anchorage of small GTPases. Correct membrane targeting is essential for their biological activity. Signal output of the prenylated proto-oncogene Ras in addition critically depends on its organization into nanoscale proteolipid assemblies of the plasma membrane, so called nanoclusters. While protein prenylation is an established drug target, only a handful of nanoclustering inhibitors are known, partially due to the lack of appropriate assays to screen for such compounds. Here, we describe three cell-based high-throughput screening amenable Förster resonance energy transfer NANOclustering and Prenylation Sensors (NANOPS) that are specific for Ras, Rho, and Rab proteins. Rab-NANOPS provides the first evidence for nanoclustering of Rab proteins. Using NANOPS in a cell-based chemical screen, we now identify macrotetrolides, known ionophoric antibiotics, as submicromolar disruptors of Ras nanoclustering and MAPK signaling.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.