Loss-of-Function Mutations in Rab Escort Protein 1 (REP-1) Affect Intracellular Transport in Fibroblasts and Monocytes of Choroideremia Patients

BACKGROUND: Choroideremia (CHM) is a progressive X-linked retinopathy caused by mutations in the CHM gene, which encodes Rab escort protein-1 (REP-1), an escort protein involved in the prenylation of Rabs. Under-prenylation of certain Rabs, as a result of loss of function mutations in REP-1, could affect vesicular trafficking, exocytosis and secretion in peripheral cells of CHM patients. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate this hypothesis, intracellular vesicle transport, lysosomal acidification and rates of proteolytic degradation were studied in monocytes (CD14+ fraction) and primary skin fibroblasts from the nine age-matched controls and thirteen CHM patients carrying 10 different loss-of-function mutations. With the use of pHrodo BioParticles conjugated with E. coli, collagen I coated FluoSpheres beads and fluorescent DQ ovalbumin with BODYPY FL dye, we demonstrated for the first time that lysosomal pH was increased in monocytes of CHM patients and, as a consequence, the rates of proteolytic degradation were slowed. Microarray analysis of gene expression revealed that some genes involved in the immune response, small GTPase regulation, transcription, cell adhesion and the regulation of exocytosis were significantly up and down regulated in cells from CHM patients compared to controls. Finally, CHM fibroblasts secreted significantly lower levels of cytokine/growth factors such as macrophage chemoattractant protein-1 (MCP-1), pigment epithelial derived factor (PEDF), tumor necrosis factor (TNF) alpha, fibroblast growth factor (FGF) beta and interleukin (lL)-8. CONCLUSIONS/SIGNIFICANCE: We demonstrated for the first time that peripheral cells of CHM patients had increased pH levels in lysosomes, reduced rates of proteolytic degradation and altered secretion of cytokines. Peripheral cells from CHM patients expose characteristics that were not previously recognized and could used as an alternative models to study the effects of different mutations in the REP-1 gene on mechanism of CHM development in human population

Early-Onset Stroke and Vasculopathy Associated with Mutations in ADA2

We observed a syndrome of intermittent fevers, early-onset lacunar strokes and other neurovascular manifestations, livedoid rash, hepatosplenomegaly, and systemic vasculopathy in three unrelated patients. We suspected a genetic cause because the disorder presented in early childhood

Fundus photographs of the control and CHM patients.

<p><b>a.</b> CHM patient 22 y.o. characterized by RPE depigmentation and widespread RPE disruption <b>b.</b> CHM patient 74 y.o. characterized by loss of RPE and choroid, scattered pigment in macula, faint deep choroidal vessels and severely narrowed retinal vessels and optic nerve pallor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008402#pone-0008402-t001" target="_blank"><b>Table 1</b></a>, CHM 9 and 10 respectively). <b>c.</b> Female CHM carrier, age 50 showing patchy RPE hypopigmentation without pigment dispersion and control subject. <b>d.</b> Fundus photograph of the normal eye.</p

Dynamic Effect of Bortezomib on Nuclear Factor-B Activity and Gene Expression in Tumor Cells

ABSTRACT Nuclear factor-B (NF-B) influences the initiation, progression, and maintenance of diverse cancer types. Despite current therapeutic efforts to block hyperactive NF-B in cancer cells, the in vivo effects of a drug upon this complex pathway are unclear. We monitored NF-B activity and a fast-expressing reporter level simultaneously in head and neck squamous carcinoma cells by quantitative live microscopy. The real-time single cell assay revealed the tumor necrosis factor-␣-induced oscillation of NF-B was echoed by equally dynamic reporter expression rate. Bortezomib is a proteasome inhibitor whose anticancer action is partly mediated through inhibition of NF-B. When administered to preactivated cells, the drug gave rise to distinct inhibition dynamics, with discrete pulses of reporter induction remaining for hours. These findings suggest that, contrary to a simplistic presumption for a pathway &quot;blockade,&quot; the network dynamics and the intracellular pharmacokinetics of the inhibitor must be critically evaluated in developing strategies for optimal intervention of oncogenic pathways. Recent trends in clinical investigations clearly tend toward molecularly targeted approaches that are based on mechanisms underlying the pathophysiology of the disease. Often, the goal is to block the activity of a specific pathway that has been implicated in the disease process, by targeting a key component with a small molecule or an antibody, for example. It is seldom known whether the desired &quot;blockade&quot; is achieved in the relevant tissue and why paradoxical outcomes occur in certain cases. Here, we show that NF-B activity in tumor cells is altered by the action of a proteasome inhibitor in a complex way that cannot sufficiently be explained by the simplistic notion of pathway blockade. NF-B/Rel is a master regulator of inflammatory processes and has a growing list of cancers and other common diseases that require its aberrant activit

Dynamic Effect of Bortezomib on NF-κB Activity and Gene Expression in Tumor Cells Running Title: Inhibition dynamics of Bortezomib on NF-κB in living cells

Abstract 523 words in Introduction 710 words in Discussion Abbreviations: NF-κB, nuclear factor kappa B; TNF-α, tumor necrosis factor alpha; IκB, inhibitor of kappa B; IKK, IκB kinase; HNSCC, head and neck squamous cell carcinoma; EGFP, enhanced green fluorescent protein. MOL #49114 3 Abstract NF-κB influences the initiation, progression, and maintenance of diverse cancer types. Despite current therapeutic efforts to block hyperactive NF-κB in cancer cells, the in vivo effects of a drug upon this complex pathway are unclear. We monitored NF-κB activity and a fast-expressing reporter level simultaneously in head and neck squamous carcinoma cells by quantitative live microscopy. The real time single cell assay revealed the TNF-α induced oscillation of NF-κB was echoed by equally dynamic reporter expression rate. Bortezomib is a proteasome inhibitor whose anti-cancer action is partly mediated through inhibition of NF-κB. When administered to pre-activated cells, the drug gave rise to distinct inhibition dynamics, with discrete pulses of reporter induction remaining for hours. These findings suggest that, contrary to a simplistic presumption for a pathway &apos;blockade&apos;, the network dynamics and the intracellular pharmacokinetics of the inhibitor must be critically evaluated in developing strategies for optimal intervention of oncogenic pathways. MOL #49114

Lysosomal acidification and rate of proteolytic degradation in monocytes from CHM and control patients treated with Bafilomycin-A1 (BafA1).

<p><b>a.</b> Lysosomal acidification and rate of proteolytic degradation in monocytes from CHM and control BafA1. Intralysosomal acidification measurements were performed using <i>E. coli</i> BioParticles conjugated with a pH dependent dye (pHrodo). Treatment caused an increase in lysosomal pH as evident by a decrease in the fluorescence of BioParticles (confocal images, left panel vehicle no effect, right panel cells pre- treated with BafA1 for 30 min, decreased fluorescence). <b>b.</b> Decrease in fluorescence levels of BioParticles following the treatment with BafA1 in monocytes from control and patient CHM4 measured by flow cytometry analysis at 1, 3 and 5 hours following the feeding. <b>c.</b> Representative FACS histograms showing a shift in fluorescence intensity of the CHM and control monocytes fed with BioParticles treated with BafA1 at 1, 3 and 5 h. <b>d.</b> Decreased rate of DQ-ovalbumin degradation in CHM (n = 3) and control (n = 3) patients before and after the treatment with BafA1 measured by flow cytometry analysis at 1, 3 and 5 hours following the feeding. Data expressed as a percent of fluorescence reduction in CHM and control cells treated with BafA1, compared to the non-treated (NT) cells.</p

Experimental design.

<p>Collection of monocyte fractions and culture of primary dermal fibroblasts for the evaluation of gene expression and functional differences between CHM patients and age-matched controls.</p

Mutation in REP-1 affects gene expression and secretion in CHM patients.

<p>a. Hierarchical cluster of 47 probe sets in control and CHM samples. Using consistency testing, twenty-six probe sets were found to be significantly over-expressed and 21 under-expressed in monocytes and primary fibroblasts cells from CHM patients 43 (CHM 6-15) compared to control (Cont 1-5)(p<0.0001, FDR30%) b-d. Level of secretion of the cytokines and growth factors by primary fibroblast cultures into conditioned media. MCP-1, TNF-alpha and FGF factors were detected at significantly higher levels in samples collected from the control cells (n = 9) compared to conditioned media samples from 8 CHM patients (p<0.005)</p

Clinical characteristics of CHM patients and expected effect of determined mutations on the structure of REP-1 protein.

*<p>HM, hand motion; LP, light perception; NLP, no light perception.</p>**<p>Brothers carrying the same mutation in Rep-1 protein.</p>†<p>effect of mutations I553X, L550P, Y504X and P179X previously analyzed by Sergeev et al. 2009.</p

Effect of different mutations on the structure and levels of REP-1 mRNA and protein.

<p><b>a</b>, Effect of different nonsense mutations on the structure of REP-1 protein (Q273X, I460X, M1I and K234X). <b>b</b>, Distribution and position of the mutations in the REP-1 protein, note that 4 of 9 mutations localized in the beta sheet of the REP-1 (blue) and 7 of 9 mutations (P179X, K234X, I244X, I460X, Y504 X, L550P and I553X, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008402#pone-0008402-t001" target="_blank"><b>Table 1</b></a>) localized to domain 2 of the REP-1 protein. <b>c</b>. Levels of mRNA determined by the microarray analysis of the expression profiles from monocytes and fibroblasts from CHM and control patients. Control group, n = 5; group CHM1 includes patients with low levels of REP1 mRNA, n = 7; group CHM2 includes patients with REP-1 mRNA similar to the controls, n = 6. <b>d</b>. Expression levels of REP-1 and REP-2 in different cell types derived from CHM and control patients. Lane: 1, 10 ng of rat recombinant REP-1 or 10 ng of rat recombinant REP-2 with HisTag; cell lysates (40 µg of protein for each) 2, ARPE19; 3, human fetal RPE; 4, MO- monocytes from control; 5, MO-monocytes from patient CHM4; 6, cultured human umbilical vein endothelial cells (HUVECs); 7, primary fibroblasts from control; 8, primary fibroblasts from CHM2 patient. β-actin was used as a loading control.</p