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Michael E Cheetham

Publications and source records attributed to Michael E Cheetham.

At least 19 recordsLinked to original sources
PubMed2006

Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase.

There is no treatment for the neurodegenerative disorder Huntington disease (HD). Cystamine is a candidate drug; however, the mechanisms by which it operates remain unclear. We show here that cystamine increases levels of the heat shock DnaJ-containing protein 1b (HSJ1b) that are low in HD patients. HSJ1b inhibits polyQ-huntingtin-induced death of striatal neurons and neuronal dysfunction in Caenorhabditis elegans. This neuroprotective effect involves stimulation of the secretory pathway through formation of clathrin-coated vesicles containing brain-derived neurotrophic factor (BDNF). Cystamine increases BDNF secretion from the Golgi region that is blocked by reducing HSJ1b levels or by overexpressing transglutaminase. We demonstrate that cysteamine, the FDA-approved reduced form of cystamine, is neuroprotective in HD mice by increasing BDNF levels in brain. Finally, cysteamine increases serum levels of BDNF in mouse and primate models of HD. Therefore, cysteamine is a potential treatment for HD, and serum BDNF levels can be used as a biomarker for drug efficacy.

Aged↗
PubMed2006

The retinitis pigmentosa-mutated RP2 protein exhibits exonuclease activity and translocates to the nucleus in response to DNA damage.

Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. Mutations in the RP2 gene are linked to the second most frequent form of X-linked retinitis pigmentosa. RP2 is a plasma membrane-associated protein of unknown function. The N-terminal domain of RP2 shares amino acid sequence similarity to the tubulin-specific chaperone protein co-factor C. The C-terminus consists of a domain with similarity to nucleoside diphosphate kinases (NDKs). Human NDK1, in addition to its role in providing nucleoside triphosphates, has recently been described as a 3' to 5' exonuclease. Here, we show that RP2 is a DNA-binding protein that exhibits exonuclease activity, with a preference for single-stranded or nicked DNA substrates that occur as intermediates of base excision repair pathways. Furthermore, we show that RP2 undergoes re-localization into the nucleus upon treatment of cells with DNA damaging agents inducing oxidative stress, most notably solar simulated light and UVA radiation. The data suggest that RP2 may have previously unrecognized roles as a DNA damage response factor and 3' to 5' exonuclease.

Active Transport, Cell Nucleus↗
PubMed2005

Inherited ACTH insensitivity illuminates the mechanisms of ACTH action.

Adrenocorticotrophin (ACTH) insensitivity is a potentially lethal inherited disorder of ACTH signalling in the adrenal. Inactivating mutations of the ACTH receptor account for approximately 25% of these cases. A second genetic cause for this syndrome has recently been identified in the MRAP gene. The MRAP protein appears to function in the trafficking and cell surface expression of the ACTH receptor, and might indicate the existence of more widespread G-protein-coupled receptor trafficking mechanisms. Molecular defects underlying other causes of ACTH insensitivity syndromes will probably contribute further to our understanding of these pathways.

Adrenocorticotropic Hormone↗
PubMed2005

Isolation and characterization of murine Cds (CDP-diacylglycerol synthase) 1 and 2.

Phototransduction in Drosophila is a phosphoinositide-mediated signalling pathway. Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in this process, and its levels are tightly regulated. A photoreceptor-specific form of the enzyme CDP-diacylglycerol synthase (CDS), which catalyzes the formation of CDP-diacylglycerol from phosphatidic acid, is a key regulator of the amount of PIP2 available for signalling. cds mutants develop light-induced retinal degeneration. We report here the isolation and characterization of two murine genes encoding this enzyme, Cds1 and Cds2. The genes encode proteins that are 73% identical and 92% similar but exhibit very different expression patterns. Cds1 shows a very restricted expression pattern but is expressed in the inner segments of the photoreceptors whilst Cds2 shows a ubiquitous pattern of expression. Using fluorescent in situ hybridization we have mapped Cds1 and Cds2 to chromosomes 5E3 and 2G1 respectively. These are regions of synteny with the corresponding human gene localization (4q21 and 20p13). Transient transfection experiments with epitope tagged proteins have also demonstrated that both are associated with the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed2005

Predominant rod photoreceptor degeneration in Leber congenital amaurosis.

PURPOSE: An unusual retinal vascular morphology in an enucleated eye from a patient with Leber congenital amaurosis (LCA) has been associated with a mutation in AIPL1. The AIPL1 protein is expressed in the pineal gland and retinal photoreceptors. In the retina, AIPL1 is expressed in both developing cone and rod photoreceptors, but it is restricted to rod photoreceptors in the adult human retina. Therefore, this study was conducted to determine the photoreceptor phenotype in this LCA patient to determine if photoreceptors were differentially affected. METHODS: Additional genetic screening was performed and the consequences of the H82Y amino acid substitution characterized in an in vitro assay of NUB1 modulation. The morphology of the photoreceptors was examined by light and electron microscopy. Immunohistochemistry and immunofluorescent confocal microscopy was performed using a range of retinal photoreceptor markers. RESULTS: Transfection of the H82Y mutant AIPL1 in SK-N-SH cells revealed a normal subcellular localization and solubility but resulted in an increased ability of AIPL1 to redistribute GFP-NUB1 to the cytoplasm and resolve NUB1 fragment inclusion formation. Morphologically, the LCA retina appeared to be cone-dominant with a single layer of cone-like cells remaining in the central retina. Photoreceptor outer segments were absent and the surviving residual inner segments were severely shortened. Severe degeneration of the LCA retina was associated with upregulation of glial fibrillary acidic protein (GFAP). No signal was detected for AIPL1, rhodopsin, or L/M and S cone opsins in the LCA retina. Double labeling with peanut agglutinin (PNA) and wheat germ agglutinin (WGA) supported a cone-dominant phenotype for the surviving photoreceptors in the LCA retina, as did double labeling for cone arrestin, and rod and cone recoverin. The cone arrestin signal was restricted to the residual photoreceptor inner segments and was not detected in the cell bodies, axons, or axon terminals of the surviving photoreceptors. Recoverin immunoreactivity was most intense in the residual photoreceptor inner segments. CONCLUSIONS: The phenotype in this patient suggests that although AIPL1 is required for the development of normal rod and cone photoreceptor function, it might only be essential for rod and not cone survival in the adult.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Treatment with extracellular HSP70/HSC70 protein can reduce polyglutamine toxicity and aggregation.

The accumulation of insoluble protein aggregates is a feature of neurodegenerative disease. Overexpression of Heat Shock Protein 70 (HSP70) can protect cells with protein aggregates from apoptosis. Another trait of HSP70 is its ability to cross the plasma membrane. Therefore, we purified a preparation of HSP70/HSC70 from bovine muscle and used it in a model of Huntington's disease. Human neuroblastoma SK-N-SH cells were transfected with huntington exon 1 with short (25) or long (103) CAG trinucleotide repeats coupled to green flourescent protein (GFP). Cells transfected with the long polyCAG repeat had insoluble protein aggregates and died through apoptosis. Biotinylated HSP70/HSC70 incorporated into the culture medium appeared inside the cells within 3-6 h of incubation. This incorporation correlated with a reduction in apoptotic cells by 40-50%. Confocal microscopy revealed that labelled internalized HSP70/HSC70 co-localized with the polyglutamine inclusions. The measurement of the number and size of inclusions showed that HSP70/HSC70 was able to reduce both these parameters. A filter trap assay and immunoblotting demonstrated that the introduction of HSP70/HSC70 also decreased protein aggregation. Together with earlier data on the effects of exogenously administered HSP70/HSC70 on cultured cells and on animals, these data show that preparations based on HSP70 may have some potential as therapies for a variety of neurodegenerative pathologies.

Animals↗
PubMed2005

HSJ1 is a neuronal shuttling factor for the sorting of chaperone clients to the proteasome.

Protein degradation in eukaryotic cells usually involves the attachment of a ubiquitin chain to a substrate protein and its subsequent sorting to the proteasome. Molecular mechanisms underlying the sorting process only recently began to emerge and rely on a cooperation of chaperone machineries and ubiquitin-chain recognition factors [1-3]. Here, we identify isoforms of the cochaperone HSJ1 as neuronal shuttling factors for ubiquitylated proteins. HSJ1 combines a J-domain that stimulates substrate loading onto the Hsc70 chaperone with ubiquitin interaction motifs (UIMs) involved in binding ubiquitylated chaperone clients. HSJ1 prevents client aggregation, shields clients against chain trimming by ubiquitin hydrolases, and stimulates their sorting to the proteasome. In this way, HSJ1 isoforms participate in ER-associated degradation (ERAD) and protect neurons against cytotoxic protein aggregation.

Amino Acid Sequence↗
PubMed2005

Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocorticoid deficiency type 2.

Familial glucocorticoid deficiency (FGD), or hereditary unresponsiveness to adrenocorticotropin (ACTH; OMIM 202200), is an autosomal recessive disorder resulting from resistance to the action of ACTH on the adrenal cortex, which stimulates glucocorticoid production. Affected individuals are deficient in cortisol and, if untreated, are likely to succumb to hypoglycemia or overwhelming infection in infancy or childhood. Mutations of the ACTH receptor (melanocortin 2 receptor, MC2R) account for approximately 25% of cases of FGD. FGD without mutations of MC2R is called FGD type 2. Using SNP array genotyping, we mapped a locus involved in FGD type 2 to chromosome 21q22.1. We identified mutations in a gene encoding a 19-kDa single-transmembrane domain protein, now known as melanocortin 2 receptor accessory protein (MRAP). We show that MRAP interacts with MC2R and may have a role in the trafficking of MC2R from the endoplasmic reticulum to the cell surface.

Adrenocorticotropic Hormone↗
PubMed2005

Mechanisms of cell death in rhodopsin retinitis pigmentosa: implications for therapy.

Retinitis pigmentosa (RP) is a group of retinal degenerative diseases that are characterised primarily by the loss of rod photoreceptor cells. Mutations in rhodopsin are the most common cause of autosomal-dominant RP (ADRP). Here, we propose a new classification for rhodopsin mutations based on their biochemical and cellular properties. Several different potential gain-of-function mechanisms for rhodopsin ADRP are described and discussed. Possible dominant-negative mechanisms, which affect the processing, translocation or degradation of wild-type rhodopsin, are also considered. Understanding the molecular and cellular consequences of rod-opsin mutations and the underlying disease mechanisms in ADRP are essential to develop future therapies for this class of retinal dystrophies.

Amino Acid Sequence↗
PubMed2005

Assay and functional analysis of the ARL3 effector RP2 involved in X-linked retinitis pigmentosa.

Mutations in RP2 cause X-linked retinitis pigmentosa and also macular and peripapillary atrophy. RP2 is a functional homologue of the tubulin folding cofactor, cofactor C, as it can replace the beta tubulin GTPase stimulating activity of cofactor C in an in vitro assay. An important difference between RP2 and cofactor C is their subcellular localization. RP2 is targeted to the cytoplasmic face of the plasma membrane by dual N-terminal acylation, and this post-translational modification is important for protein function. The activity of tubulin folding cofactors is modulated by certain ADP ribosylation factor-like (Arl) proteins. It has been shown that RP2 can interact directly with Arl3. Here we describe the methodologies that we have developed to analyze the interaction of RP2 with Arl3 and to investigate the effect of RP2 post-translational modifications on its subcellular and tissue localization.

ADP-Ribosylation Factors↗
PubMed2005

Not all J domains are created equal: implications for the specificity of Hsp40-Hsp70 interactions.

Heat shock protein 40s (Hsp40s) and heat shock protein 70s (Hsp70s) form chaperone partnerships that are key components of cellular chaperone networks involved in facilitating the correct folding of a broad range of client proteins. While the Hsp40 family of proteins is highly diverse with multiple forms occurring in any particular cell or compartment, all its members are characterized by a J domain that directs their interaction with a partner Hsp70. Specific Hsp40-Hsp70 chaperone partnerships have been identified that are dedicated to the correct folding of distinct subsets of client proteins. The elucidation of the mechanism by which these specific Hsp40-Hsp70 partnerships are formed will greatly enhance our understanding of the way in which chaperone pathways are integrated into finely regulated protein folding networks. From in silico analyses, domain swapping and rational protein engineering experiments, evidence has accumulated that indicates that J domains contain key specificity determinants. This review will critically discuss the current understanding of the structural features of J domains that determine the specificity of interaction between Hsp40 proteins and their partner Hsp70s. We also propose a model in which the J domain is able to integrate specificity and chaperone activity.

Adenosine Triphosphatases↗
PubMed2005

Identification of novel RPGR ORF15 mutations in X-linked progressive cone-rod dystrophy (XLCORD) families.

PURPOSE: To test the incidence of mutations in RPGR ORF15 in six families with X-linked progressive retinal degeneration (cone-rod dystrophy [XLCORD], macular or cone dystrophy) and to undertake a detailed phenotypic assessment of families in whom ORF15 mutations were identified. METHODS: To amplify and sequence ORF15 in its entirety, a cloning strategy was developed. Families with mutations in ORF15 underwent electrophysiological testing, color vision assessment, color fundus photography, and fundus autofluorescence (AF) imaging. RESULTS: Novel protein truncation mutations were identified in two families. In family A, a 2-bp mutation was identified in ORF15+A1094C G1095T, predicted to result in a truncated protein (E364D/E365X). In family B, a G-to-T transversion (ORF15+1176G>T) resulted in a nonsense mutation (G392X). Characteristics of phenotype in both families included progressive deterioration of central vision and subsequently night vision, mild photophobia, and moderate to high myopia. Ophthalmoscopic abnormalities were generally confined to the macula. A parafoveal ring of increased AF was observed, and electrophysiological evidence of a greater generalized abnormality in cone than rod responses were consistent with a cone-rod dystrophy phenotype. CONCLUSIONS: The cloning strategy for ORF15 facilitated comprehensive sequence analysis in patients. Two families were identified with nonsense mutations, and clinical evaluation revealed them both to have a similar phenotype. The presence of a parafoveal ring of increased AF was an early indicator of affected status in these families. No disease-causing mutations in ORF15 were detected in four other families, suggesting that ORF15 mutations may not be the most common cause of XLCORD.

Adult↗
PubMed2004

The Leber congenital amaurosis protein AIPL1 modulates the nuclear translocation of NUB1 and suppresses inclusion formation by NUB1 fragments.

Mutations in the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) cause the blinding disease Leber congenital amaurosis (LCA). The similarity of AIPL1 to AIP has led to suggestions that AIPL1 could function in a similar manner to AIP in facilitating protein translocation and as a component of chaperone complexes. AIPL1 interacts with the cell cycle regulator NEDD8 ultimate buster protein 1 (NUB1). As AIPL1 is predominantly cytoplasmic and NUB1 is predominantly nuclear, we tested the hypothesis that AIPL1 could modulate the nuclear translocation of NUB1. Co-transfection of AIPL1 with GFP-NUB1 resulted in a shift of GFP-NUB1 subcellular distribution toward the cytoplasm. Interestingly, AIPL1 was able to act in a chaperone-like fashion to efficiently suppress inclusion formation by NUB1 fragments. Co-transfection of AIPL1 with GFP-NUB1-N and GFP-NUB1-C resulted in an AIPL1-dependent suppression of GFP-NUB1-N perinuclear inclusions and GFP-NUB1-C intranuclear inclusions leading to the redistribution of these fragments in the cytoplasm. This chaperone-like function of AIPL1 was specific for NUB1, since AIPL1 was unable to suppress the inclusion formation by unrelated aggregation-prone proteins and AIP had no effect on NUB1 localization or inclusion formation. We examined the effect of a range of pathogenic and engineered mutations on the ability of AIPL1 to modulate NUB1 localization or inclusion formation. With the exception of W278X, which formed non-functional SDS-insoluble inclusions, all of the pathogenic mutations studied were soluble and could modulate NUB1 with varying efficiency compared with the wild-type protein. The effect of AIPL1 on NUB1 required the C-terminal region of AIPL1, as engineered C-terminal truncation mutations had no effect on NUB1. These data show that AIPL1 can modulate protein translocation and act in a chaperone-like manner and suggest that AIPL1 is an important modulator of NUB1 cellular function.

Active Transport, Cell Nucleus↗
PubMed2004

Nuclear translocation of the Hsp70/Hsp90 organizing protein mSTI1 is regulated by cell cycle kinases.

The co-chaperone murine stress-inducible protein 1 (mSTI1), an Hsp70/Hsp90 organizing protein (Hop) homologue, mediates the assembly of the Hsp70/Hsp90 chaperone heterocomplex. The mSTI1 protein can be phosphorylated in vitro by cell cycle kinases proximal to a putative nuclear localization signal (NLS), which substantiated a predicted casein kinase II (CKII)-cdc2 kinase-NLS (CcN) motif at position 180-239 and suggested that mSTI1 might move between the cytoplasm and the nucleus under certain cell cycle conditions. The mechanism responsible for the cellular localization of mSTI1 was probed using NIH3T3 fibroblasts to investigate the localization of endogenous mSTI1 and enhanced green fluorescent protein (EGFP)-tagged mSTI1 mutants. Localization studies on cell lines stably expressing NLS(mSTI1)-EGFP and EGFP demonstrated that the NLS(mSTI1) was able to promote a nuclear localization of EGFP. The mSTI1 protein was exclusively cytoplasmic in most cells under normal conditions but was present in the nucleus of a subpopulation of cells and accumulated in the nucleus following inhibition of nuclear export (leptomycin B treatment). G1/S-phase arrest (using hydroxyurea) and inhibition of cdc2 kinase (using olomoucine) but not inhibition of casein kinase II (using 5,6-dichlorobenzimidazole riboside), increased the proportion of cells with endogenous mSTI1 nuclear staining. mSTI1-EGFP behaved identically to endogenous mSTI1. The functional importance of key residues was tested using modified mSTI1-EGFP proteins. Inactivation and phosphorylation mimicking of potential phosphorylation sites in mSTI1 altered the nuclear translocation. Mimicking of phosphorylation at the mSTI1 CKII phosphorylation site (S189E) promoted nuclear localization of mSTI1-EGFP. Mimicking phosphorylation at the cdc2 kinase phosphorylation site (T198E) promoted cytoplasmic localization of mSTI1-EGFP at the G1/S-phase transition,whereas removal of this site (T198A) promoted the nuclear localization of mSTI1-EGFP under the same conditions. These data provide the first evidence of nuclear import and export of a major Hsp70/Hsp90 co-chaperone and the regulation of this nuclear-cytoplasmic shuttling by cell cycle status and cell cycle kinases.

Active Transport, Cell Nucleus↗
PubMed2004

Oxidative stress affects the junctional integrity of retinal pigment epithelial cells.

PURPOSE: Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration. The cell line ARPE-19 was therefore examined for response to oxidative stress and its effect on stress protein induction and junctional integrity. METHODS: ARPE-19 cell viability after 1 week or 5 weeks in culture was assessed in response to different concentrations of hydrogen peroxide. The response to sublethal doses was assessed by examination of heme oxygenase (HO)-1, Hsp27 and Hsp70 by immunofluorescence and Western blot analysis. Immunofluorescence was used to investigate the localization of the junctional proteins zonula occludens (ZO)-1, occludin, and N-cadherin, and beta-catenin. Subcellular fractionation was used to assess any redistribution of beta-catenin. Monolayer integrity was examined by measurement of flux of rhodamine-conjugated dextrans from the apical to basal aspect of cells. RESULTS: ARPE-19 cells cultured for 5 weeks were less sensitive to chronic oxidative stress induced by hydrogen peroxide than those cultured for 1 week. The more differentiated ARPE-19 cells had higher steady state levels of Hsp27 and Hsp70. The response to stress also differed with time in culture. The localization of junctional proteins, which became strongly peripheral after 5 weeks in culture, became disrupted after oxidative stress, and cytosolic beta-catenin increased. Chronic oxidative stress also increased paracellular flux across the monolayer. CONCLUSIONS: Increased resistance to chronic oxidative stress with differentiation in ARPE-19 cells correlated with higher steady state levels of Hsp27 and Hsp70. Oxidative stress disrupted RPE cell junction and barrier integrity, which may contribute to the pathogenesis of diseases related to RPE through disruption of the blood-retinal barrier.

Blood-Retinal Barrier↗
PubMed2004

Modulation of Sub-RPE deposits in vitro: a potential model for age-related macular degeneration.

PURPOSE: Sub-RPE deposits form in a variety of conditions most notably in age-related macular degeneration. The purpose of this study was to generate sub-RPE deposits in vitro and to test the hypotheses that high protein concentrations or retinal homogenate increase deposit formation and that a challenge with tumor necrosis factor (TNF)-alpha or metalloproteinase (MMP)-2 decreases such deposits. METHODS: ARPE-19 cells were grown on plastic and on collagen type I-coated membrane inserts in media containing various concentrations of fetal calf serum (FCS), bovine serum albumin, or porcine retinal homogenate. In addition, cells grown on membrane inserts were treated with TNF-alpha or MMP-2. Sub-RPE deposits were assessed by electron microscopy and classified into fibrillar, condensed, banded, and membranous subtypes. The area of the micrograph occupied by each type was estimated with a point-counting technique. MMP-2 activity was assessed in tissue culture supernatants by zymography. RESULTS: With increasing time in culture, total deposit formation did not change, but the amount of condensed material deposited by ARPE-19 cells increased while the fibrillar component decreased. Albumin challenge resulted in an increased amount of deposit, predominantly of the membranous type. Challenge with retinal homogenate led to a greater net deposit formation with significant increases in the condensed and banded forms. Cells treated with TNF-alpha or MMP-2 showed a dramatic reduction in all types of sub-RPE deposit. Zymography demonstrated that unchallenged cells produced predominantly MMP-2. Retinal homogenate challenge reduced the total amount of active MMP-2 produced, and TNF-alpha stimulated MMP-9 production. CONCLUSIONS: Sub-RPE deposits formed in vitro share ultrastructural features with those seen in vivo. Deposit formation can be modulated by challenge with retinal homogenate, TNF-alpha, or MMP-2. Significantly, the results provide proof of the principle that sub-RPE deposits can be formed and modified in vitro.

Animals↗