PubMed Health⌕ Search

Biomedical subjects

Roy A Quinlan

Publications and source records attributed to Roy A Quinlan.

At least 19 recordsLinked to original sources
PubMed2006

The Alexander disease-causing glial fibrillary acidic protein mutant, R416W, accumulates into Rosenthal fibers by a pathway that involves filament aggregation and the association of alpha B-crystallin and HSP27.

Here, we describe the early events in the disease pathogenesis of Alexander disease. This is a rare and usually fatal neurodegenerative disorder whose pathological hallmark is the abundance of protein aggregates in astrocytes. These aggregates, termed "Rosenthal fibers," contain the protein chaperones alpha B-crystallin and HSP27 as well as glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein found almost exclusively in astrocytes. Heterozygous, missense GFAP mutations that usually arise spontaneously during spermatogenesis have recently been found in the majority of patients with Alexander disease. In this study, we show that one of the more frequently observed mutations, R416W, significantly perturbs in vitro filament assembly. The filamentous structures formed resemble assembly intermediates but aggregate more strongly. Consistent with the heterozygosity of the mutation, this effect is dominant over wild-type GFAP in coassembly experiments. Transient transfection studies demonstrate that R416W GFAP induces the formation of GFAP-containing cytoplasmic aggregates in a wide range of different cell types, including astrocytes. The aggregates have several important features in common with Rosenthal fibers, including the association of alpha B-crystallin and HSP27. This association occurs simultaneously with the formation of protein aggregates containing R416W GFAP and is also specific, since HSP70 does not partition with them. Monoclonal antibodies specific for R416W GFAP reveal, for the first time for any IF-based disease, the presence of the mutant protein in the characteristic histopathological feature of the disease, namely Rosenthal fibers. Collectively, these data confirm that the effects of the R416W GFAP are dominant, changing the assembly process in a way that encourages aberrant filament-filament interactions that then lead to protein aggregation and chaperone sequestration as early events in Alexander disease.

Alexander Disease↗
PubMed2006

Lenticular chaperones suppress the aggregation of the cataract-causing mutant T5P gamma C-crystallin.

The T5P mutation in human gamma C-crystallin produces a lens cataract. Here, we have investigated the effects of the T5P mutation upon the aggregation of gamma C-crystallin in vitro and in transfected cells. By sedimentation assay and sucrose gradient centrifugation, the mutation significantly increased the aggregation of the protein and reduced dramatically its solubility in vitro. Similar effects were seen when T5P gamma C-crystallin was transfected into tissue culture cells, resulting in the formation of cytoplasmic aggregates of T5P gamma C-crystallin. Interestingly, the major lenticular protein chaperones, alpha A- and alpha B-crystallin, increased the solubility of the T5P gamma C-crystallin both in vitro and in transfected cells. More importantly, the size of the T5P gamma C-crystallin aggregates were also significantly reduced in the presence of the lenticular chaperones. These data therefore suggest a dual role for these chaperones in maintaining transparency in the lens. The first is that these protein chaperones increase the proportion of the soluble T5P gamma C-crystallin and the second is that they also reduce light scatter by reducing the aggregate size of T5P gamma C-crystallin. Both activities could modify the cataract phenotype and help explain the observed variability reported for identical gamma-crystallin mutations, which identify cataract as a polygenic disease.

Cataract↗
PubMed2006

The C terminus of lens aquaporin 0 interacts with the cytoskeletal proteins filensin and CP49.

PURPOSE: Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, is a water-permeable channel, has a role in fiber cell adhesion, and is essential for fiber cell structure and organization. The purpose of this study was to identify proteins that interact with the C terminus of AQP0, by using a proteomics approach, and thus further elucidate the role of AQP0 in the human lens. METHODS: AQP0 C-terminal peptides and AQP0 antibody affinity chromatography were used for affinity purification of interacting human lens proteins. Purified proteins were digested with trypsin, analyzed by liquid chromatography (LC)-tandem mass spectrometry and identified after database searching and manual examination of the mass spectral data. Colocalization of AQP0 with filensin and CP49, two proteins identified after mass spectrometric analysis, were examined by immunoconfocal and immunoelectron microscopy of lens sections. RESULTS: The proteomics approach used to identify affinity-purified proteins revealed the lens-specific intermediate filament proteins filensin and CP49. With immunoconfocal microscopy, regions of colocalization of AQP0 with filensin and CP49 at the fiber cell plasma membrane in the lens cortex were defined. Immunoelectron microscopy confirmed that filensin and AQP0 were present in the same membrane compartments. CONCLUSIONS: These studies suggest a novel interaction between an aquaporin water channel and intermediate filaments, an interaction through which AQP0 may maintain lens fiber cell shape and organization.

Animals↗
PubMed2005

Alexander-disease mutation of GFAP causes filament disorganization and decreased solubility of GFAP.

Alexander disease is a fatal neurological illness characterized by white-matter degeneration and the formation of astrocytic cytoplasmic inclusions called Rosenthal fibers, which contain the intermediate filament glial fibrillary acidic protein (GFAP), the small heat-shock proteins HSP27 and alphaB-crystallin, and ubiquitin. Many Alexander-disease patients are heterozygous for one of a set of point mutations in the GFAP gene, all of which result in amino acid substitutions. The biological effects of the most common alteration, R239C, were tested by expressing the mutated protein in cultured cells by transient transfection. In primary rat astrocytes and Cos-7 cells, the mutant GFAP was incorporated into filament networks along with the endogenous GFAP and vimentin, respectively. In SW13Vim(-) cells, which have no endogenous cytoplasmic intermediate filaments, wild-type human GFAP frequently formed filamentous bundles, whereas the R239C GFAP formed 'diffuse' and irregular patterns. Filamentous bundles of R239C GFAP were sometimes formed in SW13Vim(-) cells when wild-type GFAP was co-transfected. Although the presence of a suitable coassembly partner (vimentin or GFAP) reduced the potential negative effects of the R239C mutation on GFAP network formation, the mutation affected the stability of GFAP in cells in a dominant fashion. Extraction of transfected SW13Vim(-) cells with Triton-X-100-containing buffers showed that the mutant GFAP was more resistant to solubilization at elevated KCl concentrations. Both wild-type and R239C GFAP assembled into 10 nm filaments with similar morphology in vitro. Thus, although the R239C mutation does not appear to affect filament formation per se, the mutation alters the normal solubility and organization of GFAP networks.

Actin Cytoskeleton↗
PubMed2005

Seeing is believing! The optical properties of the eye lens are dependent upon a functional intermediate filament cytoskeleton.

Beaded filaments are the major cytoskeletal element of the eye lens and they are essential to the optical properties of the eye lens. They were discovered in 1972 by Harry Maisel and Margaret Perry and have since been found to comprise two novel intermediate filament proteins, CP49 and filensin. These proteins possess unique structure features and unusual assembly characteristics, which distinguish them from canonical IF proteins. Whilst CP49 is completely tailless, filensin has a rather short rod domain and extremely large C-terminal tail domain. In vitro, CP49 and filensin do not form IFs on their own. In vitro studies suggest that CP49 and filensin have a distinct coassembly mechanism. Whilst CP49 self-assembles into thick bundles of filaments, filensin only forms short fibrils, but when combined together they form filaments. The generation of gene knockouts by the targeted deletion of Bfsp1 and Bfsp2 that encode filensin and CP49, respectively, have been made to explore the function of beaded filaments in the lens. Our results suggest that the lens-specific beaded filaments are the key cytoskeletal element in organising and maintaining lens fibre cell architecture and are a key factor in determining the optical properties of the lens. We have also found that some common mouse strains contain a natural mutation in Bfsp2 that will effectively generate a CP49 knockout. This finding has important implications for lens research involving other gene knockouts maintained on a 129 background. It has also been observed that mutations in Bfsp2 are the genetic basis of inherited human cataract. Collectively, these data demonstrate that beaded filaments are fundamental to lens function.

Amino Acid Sequence↗
PubMed2005

Antimycin A induced cardioprotection is dependent on pre-ischemic p38-MAPK activation but independent of MKK3.

To examine the role of mitogen-activated protein kinase kinase 3 (MKK3) and p38 mitogen-activated protein kinase (p38-MAPK) in the cardioprotection afforded by antimycin A. Langendorff perfused murine hearts exposed to antimycin A or vehicle prior to global ischemia with p38-MAPK and HSP27 phosphorylation examined in the presence and absence of SB203580 or the presence (mkk3(+/+)) and absence (mkk3(-/-)) of MKK3. Infarct size was determined after 30 or 40 min of global ischemia and 2 h reperfusion. p38-MAPK dual phosphorylation in response to antimycin A was attenuated by co-administration of the antioxidant mercaptopropyonyl-glycine but unaffected by the absence of MKK3 or the presence of SB203580 at a concentration that inhibited the downstream phosphorylation of HSP27. Pre-ischemic exposure to antimycin A caused a significant reduction in subsequent infarction (I:R%) compared to vehicle on both the mkk3(-/-) and mkk3(+/+) background (23.7+/-2.9 and 22.8+/-4.6 compared to 50.7+/-4.0 and 49.6+/-5.4 P=0.001, respectively). In C57Bl6 mice, antimycin A prior to ischemia reduced infarct size compared to vehicle (22.8 +/- 6.1 vs. 48.3+/-5.2 P=0.01, respectively), an effect abolished by coincident SB203580. The cardiac protection initiated by antimycin A is dependent on the activation of p38-MAPK which occurs, at least in part, in response to oxygen-derived free radicals. The mechanism of this protective form of p38-MAPK activation is independent of the upstream kinase MKK3 and does not involve autophosphorylation.

Animals↗
PubMed2005

R120G alphaB-crystallin promotes the unfolding of reduced alpha-lactalbumin and is inherently unstable.

alpha-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, alphaB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced alpha-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G alphaB-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G alphaB-crystallin exists as a larger oligomer than wild-type alphaB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of alphaB-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G alphaB-crystallin with human disease states.

Animals↗
PubMed2004

Comparison of the small heat shock proteins alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP in heart and skeletal muscle.

Seven members of the small heat shock protein (sHSP) family are exceptional with respect to their constitutive high abundance in muscle tissue. It has been suggested that sHSPs displaying chaperone-like properties may stabilize myofibrillar proteins during stress conditions and prevent them from loss of function. In the present study five sHSPs (alphaB-crystallin, MKBP, HSP25, HSP20, and cvHSP) were investigated with respect to similarities and differences of their expression in heart and skeletal muscle under normal and ischemic conditions. In ischemic heart and skeletal muscle these five sHSPs translocated from cytosol to the Z-/I-area of myofibrils. Myofibrillar binding of all sHSPs was very tight and resisted for the most part extraction with 1 M NaSCN or 1 M urea. MKBP and HSP20 became extracted by 1 M NaSCN to a significant extent indicating that these two sHSPs may bind partially to actin-associated proteins which were completely extracted by this treatment. Ultrastructural localization of alphaB-crystallin showed diffuse distribution of immunogold label throughout the entire I-band in skeletal muscle fibers whereas in cardiomyocytes alphaB-crystallin was preferentially located at the N-line position of the I-band. These observations indicate different myofibrillar binding sites of alphaB-crystallin in cardiomyocytes versus skeletal muscle fibers. Further differences of the properties of sHSPs could be observed regarding fiber type distribution of sHSPs. Thus sHSPs form a complex stress-response system in striated muscle tissue with some common as well as some distinct functions in different muscle types.

Animals↗
PubMed2004

Desmin aggregate formation by R120G alphaB-crystallin is caused by altered filament interactions and is dependent upon network status in cells.

The R120G mutation in alphaB-crystallin causes desmin-related myopathy. There have been a number of mechanisms proposed to explain the disease process, from altered protein processing to loss of chaperone function. Here, we show that the mutation alters the in vitro binding characteristics of alphaB-crystallin for desmin filaments. The apparent dissociation constant of R120G alphaB-crystallin was decreased while the binding capacity was increased significantly and as a result, desmin filaments aggregated. These data suggest that the characteristic desmin aggregates seen as part of the disease histopathology can be caused by a direct, but altered interaction of R120G alphaB-crystallin with desmin filaments. Transfection studies show that desmin networks in different cell backgrounds are not equally affected. Desmin networks are most vulnerable when they are being made de novo and not when they are already established. Our data also clearly demonstrate the beneficial role of wild-type alphaB-crystallin in the formation of desmin filament networks. Collectively, our data suggest that R120G alphaB-crystallin directly promotes desmin filament aggregation, although this gain of a function can be repressed by some cell situations. Such circumstances in muscle could explain the late onset characteristic of the myopathies caused by mutations in alphaB-crystallin.

Cell Line↗
PubMed2004

Inhibition of p38 MAPK activity fails to attenuate contractile dysfunction in a mouse model of low-flow ischemia.

OBJECTIVE: The basal activity of p38 MAPK has recently been shown to impair myocardial contractility. This kinase is activated by ischemia and short-term hibernation. We hypothesized that p38 MAPK activation may contribute to the contractile deficit that characterizes low-flow ischemia. METHODS: In Langendorff-perfused isolated C57BL/6 mouse hearts, perfusion pressure was reduced from 85 to 15 or 30 mm Hg for 120 min to induce ischemic left ventricular dysfunction. The effect of the p38 MAPK inhibitor SB203580 (1 microM/l) on contractile function and p38 MAPK activation was assessed. RESULTS: Reduction in perfusion pressure to 15 or 30 mm Hg was accompanied by stable reductions in coronary flow (83+/-2% and 66+/-2%, respectively) and developed pressure (84+/-2% and 61+/-3%), with minimal infarction (15.6+/-0.69% and 10.6+/-0.98% of LV myocardium, respectively), but marked activation of p38 MAPK (reflected in pHSP27 1092+/-326% basal and 996+/-301% basal, respectively). The p38 MAPK inhibitor SB203580, present during the last 60 min of reduced pressure perfusion, prevented p38 MAPK activation (pHSP27 281+/-92% basal, p=0.01 and 186+/-72% basal, p=0.01) but, despite the presence of a contractile reserve, had no effect on developed pressure. Similarly, early treatment with SB203580 started 5 min after the onset of reduced flow also failed to attenuate contractile dysfunction. CONCLUSION: The p38 MAPK activation that accompanies short-term hibernation does not appear to contribute to the contractile deficit.

Animals↗
PubMed2004

Bfsp2 mutation found in mouse 129 strains causes the loss of CP49 and induces vimentin-dependent changes in the lens fibre cell cytoskeleton.

Here we report the first natural mutation in the mouse Bfsp2 gene. Characterisation of mouse Bfsp2 in the 129X1/SvJ revealed a mutation that deleted the acceptor site of exon 2. This results in exon 1 being erroneously spliced to exon 3 causing a frameshift in the reading frame and the introduction of a stop codon at position 2 of exon 3 in the Bfsp2 transcript. RT-PCR studies of lens RNA isolated from 129S1/SvImJ, 129S2/SvPas and 129S4/SvJae strains confirmed the presence of this mutation in these diverse 129 strains and similar mutations were found in both CBA and 101 strains, but not in C3H or C57BL/6J mouse strains. This mutation is predicted to result in a severely truncated protein product called CP49, comprising essentially only exon 1, but polyclonal antibodies to CP49 failed to detect either full length or fragments of CP49 in extracts made from either 129S1/SvImJ or 129S4/SvJae suggesting that these 129 strains lack CP49 protein. Like the knockout of Bfsp2 reported recently, filensin protein levels and its proteolytic processing were altered also in the 129S1/SvImJ and 129S4/SvJae strains compared to C57BL/6J. Electron microscopy of the lens cytoskeleton from 129S2/SvPas revealed similar morphological changes in the cytoskeleton as compared to the CP49 knockout, with beaded and intermediate filaments being apparently replaced by poorly defined filament-like material. Vimentin was a key component of this residual material as shown by immunoelectron microscopy and by the generation of a CP49/vimentin double knockout mouse. This report of a natural mutation in Bfsp2 in the 129 and other mouse strains also has important implications for lens studies that have used the 129X1/SvJ strain in knockout strategies.

Animals↗
PubMed2004

Bfsp2 mutation found in mouse 129 strains causes the loss of CP49' and induces vimentin-dependent changes in the lens fibre cell cytoskeleton.

Here we report the first natural mutation in the mouse Bfsp2 gene. Characterisation of mouse Bfsp2 in the 129X1/SvJ revealed a mutation that deleted the acceptor site of exon 2. This results in exon 1 being erroneously spliced to exon 3 causing a frameshift in the reading frame and the introduction of a stop codon at position 2 of exon 3 in the Bfsp2 transcript. RT-PCR studies of lens RNA isolated from 129S1/SvImJ, 129S2/SvPas and 129S4/SvJae strains confirmed the presence of this mutation in these diverse 129 strains and similar mutations were found in both CBA and 101 strains, but not in C3H or C57BL/6J mouse strains. This mutation is predicted to result in a severely truncated protein product called CP49, comprising essentially only exon 1, but polyclonal antibodies to CP49 failed to detect either full length or fragments of CP49 in extracts made from either 129S1/SvImJ or 129S4/SvJae suggesting that these 129 strains lack CP49 protein. Like the knockout of Bfsp2 reported recently, filensin protein levels and its proteolytic processing were altered also in the 129S1/SvImJ and 129S4/SvJae strains compared to C57BL/6J. Electron microscopy of the lens cytoskeleton from 129S2/SvPas revealed similar morphological changes in the cytoskeleton as compared to the CP49 knockout, with beaded and intermediate filaments being apparently replaced by poorly defined filament-like material. Vimentin was a key component of this residual material as shown by immunoelectron microscopy and by the generation of a CP49/vimentin double knockout mouse. This report of a natural mutation in Bfsp2 in the 129 and other mouse strains also has important implications for lens studies that have used the 129X1/SvJ strain in knockout strategies.

Animals↗
PubMed2003

Nuclear speckle localisation of the small heat shock protein alpha B-crystallin and its inhibition by the R120G cardiomyopathy-linked mutation.

In this study, the small heat shock protein (sHSP) chaperones, alpha B-crystallin and HSP27, are identified as nuclear speckle components in unstressed cells in tissue culture. This new finding suggests a constitutive function for these sHSP chaperones in the nucleus and suggests a new perspective on the cardiomyopathy-causing mutation for alpha B-crystallin that could involve transcriptional splicing effects. Both alpha B-crystallin and HSP27 were immunolocalised to nuclear speckles (interchromatin granule clusters). While alpha B-crystallin was preferentially localised to speckles as shown by colocalisation with non-snRNP, SC35, as well as the snRNP components Sm and U1A, HSP27 was also seen associated with the nucleolar compartment, indicating a subtle difference between these closely related sHSPs. Actinomycin D treatment caused the relocalisation of alpha B-crystallin along with Sm and SC35 to a smaller number of more distinct spots, suggesting a link between speckle localisation and the transcriptional status of the cells. We then examined several transformed, immortalised, and primary cells expressing endogenous alpha B-crystallin as well as some cells with ectopic alpha B-crystallin expression. All consistently showed alpha B-crystallin in nuclear speckles. The nuclear localisation of the sHSPs was also confirmed biochemically and 2D gel electrophoresis revealed that there was only one major nuclear alpha B-crystallin isoform. This suggested that phosphorylation was not required for nuclear localisation of alpha B-crystallin. This was confirmed by the transient transfection of HeLa cells with a phosphorylation-defective alpha B-crystallin. In contrast, the transfection of R120G alpha B-crystallin, the mutation that causes cardiomyopathy, inhibited the nuclear speckle localisation of alpha B-crystallin. These data suggest that the cardiomyopathy-causing mutation for alpha B-crystallin has nuclear as well as cytoplasmic consequences, suggesting an explanation for the difference in severity of the desmin and alpha B-crystallin transgenic models of their respective cardiomyopathies.

Antibiotics, Antineoplastic↗
PubMed2003

Tumor necrosis factor-induced protection of the murine heart is independent of p38-MAPK activation.

Brief exposure to tumor necrosis factor (TNF) is known to trigger subsequent cardioprotection. TNF activates multiple downstream signaling cascades including p38-MAPK, a kinase known to initiate ischemic preconditioning. However, it is not known whether this kinase is similarly involved in TNF-induced cardioprotection. In isolated perfused murine hearts, subjected to 30-min global ischemia/2-h reperfusion, infarction/risk volume was significantly reduced by pretreatment with TNF for 15 min at 0.5 ng/ml, but not at 5 or 10 ng/ml, followed by 10-min washout vs. control (% I/R = 31 +/- 3, 46 +/- 5 or 54 +/- 3 vs. 48 +/- 5; P = 0.01, 0.80 and 0.25, respectively). This was in direct contrast to the concentration dependence of myocardial p38-MAPK phosphorylation, as measured by dual phosphorylated p38-MAPK, which was apparent at TNF concentrations of 5 and 10 ng/ml but not at 0.5 ng/ml vs. time-matched control (as % basal 315 +/- 25, 422 +/- 94 and 97 +/- 25 vs. 95 +/- 10; P < 0.01, 0.01 and =0.86, respectively). However, phosphorylation of p38-MAPK at 10 min of ischemia was similar among groups (as % basal 393 +/- 98, 410 +/- 67 and 369 +/- 49 for time-matched control, 0.5 and 5 ng/ml, respectively). These patterns were also reflected in the phosphorylation of the downstream substrate HSP27. Furthermore, the effects of TNF on infarct size were not affected by SB203580 (1 micromol/l). These findings suggest that the pre-ischemic activation of p38-MAPK by TNF does not contribute to cardioprotection afforded by this agent.

Animals↗
PubMed2003

Knockout of the intermediate filament protein CP49 destabilises the lens fibre cell cytoskeleton and decreases lens optical quality, but does not induce cataract.

In this report, the phenotype associated with the first targeted knockout of the lens specific intermediate filament gene CP49 is described. Several surprising observations have been made. The first was that no cataract was observed despite the fact that the beaded filaments of the lens fibre cells had been disrupted. Light scatter and the lens optical properties had, however, deteriorated in the CP49 knockout lenses compared to litter mate controls. These changes were accompanied by dramatic changes in plasma membrane organisation of the fibre cells as revealed by detailed morphological examinations and providing the second surprising result. The CP49 knockout mouse is therefore an important model to study the functional link between lens transparency, the cytoskeleton and plasma membrane organisation.

Animals↗