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Biomedical subjects

M E Cheetham

Publications and source records attributed to M E Cheetham.

At least 19 recordsLinked to original sources
PubMed2004

Neuronal DnaJ proteins HSJ1a and HSJ1b: a role in linking the Hsp70 chaperone machine to the ubiquitin-proteasome system?

The heat-shock protein 70 chaperone machine is functionally connected to the ubiquitin-proteasome system by the co-chaperone CHIP. In this article, we discuss evidence that the neuronal DnaJ proteins HSJ1a and HSJ1b may represent a further link between the cellular protein folding and degradation machineries. We have demonstrated that HSJ1 proteins contain putative ubiquitin interaction motifs and can modulate the cellular processing of rhodopsin, a protein that is targeted for degradation by the proteasome when it is misfolded.

Amino Acid Motifs↗
PubMed2004

Role of AIP and its homologue the blindness-associated protein AIPL1 in regulating client protein nuclear translocation.

Mutations in the AIPL1 (aryl hydrocarbon receptor interacting protein-like 1) cause the blinding disease Leber's congenital amaurosis. AIPL1 is a homologue of the AIP. AIP functions as part of a chaperone heterocomplex to facilitate signalling by the AhR and plays an important role in regulating the nuclear translocation of the receptor. We review the evidence for the role of AIP in protein translocation and compare the potential functions of AIPL1 in the translocation of its interacting partner the NEDD8 ultimate buster protein 1.

Adaptor Proteins, Signal Transducing↗
PubMed2004

An atypical phenotype of macular and peripapillary retinal atrophy caused by a mutation in the RP2 gene.

AIMS: To determine the molecular basis and describe the phenotype of an atypical retinal dystrophy in a family presenting with bilateral, progressive central visual loss. METHODS: Family members were examined. Investigations included Goldman perimetry, electrophysiology, and autofluorescence imaging. Candidate gene screening was performed using SSCP and sequence analysis. The proband's lymphoblastoid cells were examined for protein expression. RESULTS: Fundal examination of the proband, his mother, and brother revealed peripapillary and macular atrophy. Autosomal dominant retinal dystrophy was suspected, but less severe disease in the mother led to screening for mutations in X linked genes. A 4 bp microdeletion in exon 3 of the RP2 gene, segregating with disease, was identified. No RP2 protein expression was detected. CONCLUSION: The distinct phenotype in this family, caused by this frameshifting mutation in RP2, broadens the phenotypic spectrum of X linked retinitis pigmentosa. The absence of RP2 protein suggests that loss of protein function and not novel gain of function could account for the atypical phenotype. A definitive diagnosis of X linked retinitis pigmentosa permits appropriate genetic counselling with important implications for other family members. Clinicians should have a low threshold for screening RP2 in families with retinal dystrophy, including posterior retinal disease, not immediately suggestive of X linked inheritance.

Adult↗
PubMed2003

Minimising the risk of prion transmission by contact tonometry.

AIMS: The unknown prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the UK population has led to fears of horizontal transmission through routine medical procedures. The potential risk of transmission via contact tonometry was examined. METHODS: The total amount of protein carried over by tonometer tips after applanation of patients was assessed. RESULTS: Tonometer tips had an inherent ability to carry proteinaceous material. There was a large variability in the load carried over between individual patients. Rinsing tonometer tips in water reduced protein carryover. Wiping the tonometer tips also reduced carriage, though less dramatically. CONCLUSION: There is a small theoretical risk of transmission of vCJD by contact tonometry through reuse, but this should be reduced if the prisms are washed and wiped. In the light of these findings a protocol for the management of reusable tonometer prisms is recommended.

Decontamination↗
PubMed2001

Identification and characterization of a human mitochondrial homologue of the bacterial co-chaperone GrpE.

We have identified a novel human cDNA with a predicted protein sequence that has 28% amino acid identity with the E. coli Hsp70 co-chaperone GrpE and designated it HMGE. Even with this low level of amino acid identity the human sequence could be efficiently modelled on the X-ray structure of the E. coli protein, suggesting that there may be significant functional conservation. Indeed, HMGE expressed in E. coli as a GST fusion protein co-purified with the E. coli Hsp70 protein DnaK in the absence of ATP. DnaK could be released from the GST-HMGE with a Mg-ATP wash. Subcellular fractionation and immunocytochemistry studies using antisera raized against HMGE show that it is a mitochondrial protein. In contrast to studies of rat GrpE, however, HMGE also appears to bind the constitutive cytosolic Hsp70, Hsc70, in addition to mitochondrial Hsp70, Mt-Hsp70. We have previously shown that Hsc70 nucleotide-exchange is rate limiting in the presence of the DnaJ-protein, HSJ1b. However, HMGE was found to inhibit the HSJ1b-enhanced Hsc70 ATPase activity and may mediate this inhibition by binding the DnaJ-protein, HSJ1b. This is the first description of a direct interaction between a DnaJ protein and GrpE-like protein. These studies suggest that the structure of GrpE has been conserved throughout evolution and that the conserved structure can interact with several forms of Hsp70, but that HMGE cannot form part of the reaction cycle for cytosolic Hsc70.

Amino Acid Sequence↗
PubMed2001

The cochaperone murine stress-inducible protein 1: overexpression, purification, and characterization.

Murine stress-inducible protein 1 (mSTI1) is a cochaperone that is homologous with the human heat shock cognate protein 70 (Hsc70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). To analyze the biochemical properties of mSTI1 and the stoichiometry of the Hsc70.mSTI1.Hsp90 association, recombinant mSTI1 was produced in untagged, histidine (His)-tagged, and glutathione S-transferase (GST)-tagged forms. His-mSTI1 was detected either as a dimer during size-exclusion-high-performance liquid chromatography (SE-HPLC) or as a monomer during Superdex 200 gel filtration chromatography. SE-HPLC on GST-mSTI1 and untagged mSTI1 suggested that mSTI1 existed as a monomer. Cross-linking of His-mSTI1 detected a compact monomeric species and a dimeric species. Gel filtration on the association of bovine STI1 or His-mSTI1 with Hsc70 detected species of molecular mass consistent with a dimeric STI1 species or a 1:1 complex of STI1 and Hsc70. Our data and that of others suggest that mSTI1 and its homologues exist as either a monomer or a dimer and that this facilitates its proposed function as an Hsc70/Hsp90 organizing protein.

Animals↗
PubMed2001

Unfolding retinal dystrophies: a role for molecular chaperones?

Inherited retinal dystrophy is a major cause of blindness worldwide. Recent molecular studies have suggested that protein folding and molecular chaperones might play a major role in the pathogenesis of these degenerations. Incorrect protein folding could be a common consequence of causative mutations in retinal degeneration disease genes, particularly mutations in the visual pigment rhodopsin. Furthermore, several retinal degeneration disease genes have recently been identified as putative facilitators of correct protein folding, molecular chaperones, on the basis of sequence homology. We also consider whether manipulation of chaperone levels or chaperone function might offer potential novel therapies for retinal degeneration.

Animals↗
PubMed2000

Mutations in the N-terminus of the X-linked retinitis pigmentosa protein RP2 interfere with the normal targeting of the protein to the plasma membrane.

The X-linked retinitis pigmentosa (XLRP) gene, RP2, codes for a novel 350 amino acid protein of unknown function. We have identified putative sites for N-terminal acyl modification by myristoylation and palmitoylation in the RP2 protein. The RP2 protein is expressed ubiquitously in human tissues at relatively low levels (0.01% of total protein) and has a predominantly plasma membrane localization in cultured cells, as would be expected if the protein was subject to dual N-terminal acylation. Furthermore, mutagenesis of residues potentially required for N-terminal acylation prevents targeting of RP2 to the plasma membrane and the N-terminal 15 amino acids of the protein appear to be sufficient for this targeting. Our data suggest that the protein is dually acylated and that the palmitoyl moiety is responsible for targeting of the myristoylated protein from intracellular membranes to the plasma membrane. The effect of two mutations, which have been reported as causes of XLRP, R118H and DeltaS6, were investigated. The R118H mutation does not affect the normal plasma membrane localization of RP2; in contrast, the DeltaS6 mutation interferes with the targeting of the protein to the plasma membrane. Therefore, the DeltaS6 mutation may cause XLRP because it prevents normal amounts of RP2 reaching the correct cellular locale, whereas the R118H mutation is in a region of the protein that is vital for another aspect of RP2 function in the retina.

Acylation↗
PubMed2000

Interaction of the human DnaJ homologue, HSJ1b with the 90 kDa heat shock protein, Hsp90.

The 90 kDa heat shock protein (Hsp90) is a major cytoplasmic molecular chaperone associating with numerous other proteins. Both genetic and in vitro refolding experiments using reticulocyte lysate have suggested a functional interaction of Hsp90 with yeast human homologues of E. coli DnaJ. Here we present direct evidence using surface plasmon resonance that Hsp90 and the human DnaJ homologue, HSJ1b, bind to each other. We also show that Hsp90 and HSJ1b transfer alpha-lactalbumin to each other in an ATP-dependent manner. The two chaperones have additive effects in preventing rhodanese aggregation.

HSP40 Heat-Shock Proteins↗
PubMed2000

Analysis of the levels of conservation of the J domain among the various types of DnaJ-like proteins.

DnaJ-like proteins are defined by the presence of an approximately 73 amino acid region termed the J domain. This region bears similarity to the initial 73 amino acids of the Escherichia coli protein DnaJ. Although the structures of the J domains of E coli DnaJ and human heat shock protein 40 have been solved using nuclear magnetic resonance, no detailed analysis of the amino acid conservation among the J domains of the various DnaJ-like proteins has yet been attempted. A multiple alignment of 223 J domain sequences was performed, and the levels of amino acid conservation at each position were established. It was found that the levels of sequence conservation were particularly high in 'true' DnaJ homologues (ie, those that share full domain conservation with DnaJ) and decreased substantially in those J domains in DnaJ-like proteins that contained no additional similarity to DnaJ outside their J domain. Residues were also identified that could be important for stabilizing the J domain and for mediating the interaction with heat shock protein 70.

Amino Acid Sequence↗
PubMed1999

Domain requirements of DnaJ-like (Hsp40) molecular chaperones in the activation of a steroid hormone receptor.

DnaJ-like proteins function in association with Hsp70 molecular chaperones to facilitate protein folding. We previously demonstrated that a yeast DnaJ-like protein, Ydj1p, was important for activation of heterologously expressed steroid hormone receptors (Caplan, A. J., Langley, E., Wilson, E. M., and Vidal, J. (1995) J. Biol. Chem. 270, 5251-5257). In the present study, we analyzed Ydj1p function by assaying hormone binding to the human androgen receptor (AR) heterologously expressed in yeast. We analyzed hormone binding in strains that were wild type or deleted for the YDJ1 gene. In the deletion mutant, the AR did not bind hormone to the same extent as the wild type. Introduction of mutant forms of Ydj1p to the deletion strain revealed that the J-domain is necessary but not sufficient for Ydj1p action, and that other domains of the protein are also functionally important. Of three human DnaJ-like proteins introduced into the deletion mutant, only Hdj2, which displays full domain conservation with Ydj1p, suppressed the hormone binding defect of the deletion mutant. By comparison of the domains shared by these three human proteins, and with mutants of Ydj1p that were functional, it was deduced that the cysteine-rich zinc binding domain is important for Hdj2/Ydj1p action in hormone receptor function. A model for the mechanism of DnaJ-like protein action is discussed.

Binding Sites↗
PubMed1996

Inhibition of hsc70-catalysed clathrin uncoating by HSJ1 proteins.

The uncoating of clathrin-coated vesicles can be mediated in vitro by the 'uncoating ATPase' that has been identified as the constitutive 70 kDa heat shock protein (hsp70), hsc70. It is now established that the activity of hsp70 proteins can be regulated by another family of molecular chaperones, the DnaJ family. In this study, we have investigated the effects of DnaJ-like proteins (the human neuron-specific proteins HSJ1a and HSJ1b) on clathrin uncoating. In order to measure the kinetics of clathrin release from coated vesicles, we have developed a quantitative, two-site ELISA for clathrin triskelions and demonstrated that stoichiometric amounts of HSJ1 proteins inhibit the initial burst of hsc70-mediated clathrin uncoating by over 40%. This inhibition is not a consequence of ADP binding by hsc70 or the aggregation of hsc70, but correlates with an increase in the hsc70 associated with the coated vesicle fraction, suggesting that the inhibition is a consequence of a non-productive stabilization of hsc70 with a component of the coated vesicle fraction. These results strongly suggest that HSJ1 proteins interfere with an endogenous DnaJ-like protein that is involved in uncoating. Recent evidence suggests that the brain-specific vesicle-associated protein auxilin could play such a role. Although we find no evidence for auxilin in our coated vesicle preparation, our results predict that an auxilin-like protein will be a general factor in clathrin uncoating.

Adaptor Proteins, Vesicular Transport↗
PubMed1994

Regulation of 70-kDa heat-shock-protein ATPase activity and substrate binding by human DnaJ-like proteins, HSJ1a and HSJ1b.

The DnaJ family of molecular chaperones is characterized by the presence of a highly conserved 70-amino-acid J domain. Escherichia coli DnaJ interacts with the 70-kDa heat-shock protein (DnaK), in vitro, to stimulate the 70-kDa heat-shock protein ATPase activity and modify substrate binding. The conservation of the interaction of DnaJ-like proteins with the 70-kDa heat-shock proteins has been demonstrated for the yeast protein YDJ1, a protein that shows full domain conservation with E. coli DnaJ. Human neurone-specific DnaJ-like proteins, HSJ1a and HSJ1b, possess a J domain and a glycine/phenylalanine-rich region in common with E. coli DnaJ, although the overall amino acid identity is less than 23%. We have investigated, in vitro, the interaction of HSJ1a and HSJ1b with the mammalian brain constitutive 70-kDa heat-shock protein (hsc70). The weak intrinsic ATPase activity of the constitutive 70-kDa heat-shock protein is enhanced more than fivefold by stoichiometric amounts of both HSJ1a and HSJ1b. This enhancement is mediated by an increase in the rate of bound ATP hydrolysis, whereas the rate of ADP release is unaffected. HSJ1 proteins appear to regulate the affinity of the 70-kDa constitutive heat-shock protein for the permanently unfolded substrate, carboxymethylated alpha-lactalbumin. A recent report [Palleros, D. R., Reid, K. L., Shi, L., Welch, W. J. & Fink, A. L. (1993) Nature 365, 664-666] has suggested that substrate release by 70-kDa heat-shock proteins requires a conformational change in these proteins induced by K+ in concert with ATP binding. In the presence of ATP, HSJ1 proteins reduce 70-kDa constitutive heat-shock protein/carboxymethylated alpha-lactalbumin complex formation both in the presence and absence of K+. This suggests that HSJ1 proteins induce a conformational change in the 70-kDa constitutive heat-shock protein that can mimic the effect mediated by K+ and therefore modulate 70-kDa heat-shock protein substrate release by another mechanism rather than merely stimulating the 70-kDa heat-shock protein ATPase activity. As HSJ1 proteins have limited similarity to DnaJ, we suggest that this action is being mediated by the J domain alone, and that this modulation of 70-kDa heat-shock-protein substrate binding will be common to all proteins that contain a J domain.

Adenosine Triphosphatases↗