Publications by authors named "Roy A Quinlan"

69 Publications

Joining European Scientific Forces to Face Pandemics.

Trends Microbiol 2021 02 4;29(2):92-97. Epub 2020 Dec 4.

Department of Oncology, University of Turin, IT-10126, Turin, Italy. Electronic address:

Despite the international guidelines on the containment of the coronavirus disease 2019 (COVID-19) pandemic, the European scientific community was not sufficiently prepared to coordinate scientific efforts. To improve preparedness for future pandemics, we have initiated a network of nine European-funded Cooperation in Science and Technology (COST) Actions that can help facilitate inter-, multi-, and trans-disciplinary communication and collaboration.
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http://dx.doi.org/10.1016/j.tim.2020.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716745PMC
February 2021

The genetic landscape of crystallins in congenital cataract.

Orphanet J Rare Dis 2020 11 26;15(1):333. Epub 2020 Nov 26.

Department of Genetics, UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK.

Background: The crystalline lens is mainly composed of a large family of soluble proteins called the crystallins, which are responsible for its development, growth, transparency and refractive index. Disease-causing sequence variants in the crystallins are responsible for nearly 50% of all non-syndromic inherited congenital cataracts, as well as causing cataract associated with other diseases, including myopathies. To date, more than 300 crystallin sequence variants causing cataract have been identified.

Methods: Here we aimed to identify the genetic basis of disease in five multi-generation British families and five sporadic cases with autosomal dominant congenital cataract using whole exome sequencing, with identified variants validated using Sanger sequencing. Following bioinformatics analysis, rare or novel variants with a moderate to damaging pathogenicity score, were filtered out and tested for segregation within the families.

Results: We have identified 10 different heterozygous crystallin variants. Five recurrent variants were found: family-A, with a missense variant (c.145C>T; p.R49C) in CRYAA associated with nuclear cataract; family-B, with a deletion in CRYBA1 (c.272delGAG; p.G91del) associated with nuclear cataract; and family-C, with a truncating variant in CRYGD (c.470G>A; W157*) causing a lamellar phenotype; individuals I and J had variants in CRYGC (c.13A>C; T5P) and in CRYGD (c.418C>T; R140*) causing unspecified congenital cataract and nuclear cataract, respectively. Five novel disease-causing variants were also identified: family D harboured a variant in CRYGC (c.179delG; R60Qfs*) responsible for a nuclear phenotype; family E, harboured a variant in CRYBB1 (c.656G>A; W219*) associated with lamellar cataract; individual F had a variant in CRYGD (c.392G>A; W131*) associated with nuclear cataract; and individuals G and H had variants in CRYAA (c.454delGCC; A152del) and in CRYBB1 (c.618C>A; Y206*) respectively, associated with unspecified congenital cataract. All novel variants were predicted to be pathogenic and to be moderately or highly damaging.

Conclusions: We report five novel variants and five known variants. Some are rare variants that have been reported previously in small ethnic groups but here we extend this to the wider population and record a broader phenotypic spectrum for these variants.
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http://dx.doi.org/10.1186/s13023-020-01613-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691105PMC
November 2020

Three-dimensional data capture and analysis of intact eye lenses evidences emmetropia-associated changes in epithelial cell organization.

Sci Rep 2020 10 9;10(1):16898. Epub 2020 Oct 9.

Department of Biosciences, Durham University, South Road Science Site, Durham, DH1 3LE, UK.

Organ and tissue development are highly coordinated processes; lens growth and functional integration into the eye (emmetropia) is a robust example. An epithelial monolayer covers the anterior hemisphere of the lens, and its organization is the key to lens formation and its optical properties throughout all life stages. To better understand how the epithelium supports lens function, we have developed a novel whole tissue imaging system using conventional confocal light microscopy and a specialized analysis software to produce three-dimensional maps for the epithelium of intact mouse lenses. The open source software package geometrically determines the anterior pole position, the equatorial diameter, and three-dimensional coordinates for each detected cell in the epithelium. The user-friendly cell maps, which retain global lens geometry, allow us to document age-dependent changes in the C57/BL6J mouse lens cell distribution characteristics. We evidence changes in epithelial cell density and distribution in C57/BL6J mice during the establishment of emmetropia between postnatal weeks 4-6. These epithelial changes accompany a previously unknown spheroid to lentoid shape transition of the lens as detected by our analyses. When combined with key findings from previous mouse genetic and cell biological studies, we suggest a cytoskeleton-based mechanism likely underpins these observations.
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http://dx.doi.org/10.1038/s41598-020-73625-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547080PMC
October 2020

Whole Exome Sequencing Reveals Novel and Recurrent Disease-Causing Variants in Lens Specific Gap Junctional Protein Encoding Genes Causing Congenital Cataract.

Genes (Basel) 2020 05 6;11(5). Epub 2020 May 6.

UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK.

Pediatric cataract is clinically and genetically heterogeneous and is the most common cause of childhood blindness worldwide. In this study, we aimed to identify disease-causing variants in three large British families and one isolated case with autosomal dominant congenital cataract, using whole exome sequencing. We identified four different heterozygous variants, three in the large families and one in the isolated case. Family A, with a novel missense variant (c.178G>C, p.Gly60Arg) in with lamellar cataract; family B, with a recurrent variant in (c.262C>T, p.Pro88Ser) associated with nuclear cataract; and family C, with a novel variant in (c.771dupC, p.Ser258GlnfsTer68) causing a lamellar phenotype. Individual D had a novel variant in (c.82G>T, p.Val28Leu) associated with congenital cataract. Each sequence variant was found to co-segregate with disease. Here, we report three novel and one recurrent disease-causing sequence variant in the gap junctional protein encoding genes causing autosomal dominant congenital cataract. Our study further extends the mutation spectrum of these genes and further facilitates clinical diagnosis. A recurrent p.P88S variant in causing isolated nuclear cataract provides evidence of further phenotypic heterogeneity associated with this variant.
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http://dx.doi.org/10.3390/genes11050512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7288463PMC
May 2020

Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity.

Elife 2019 11 4;8. Epub 2019 Nov 4.

Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, United States.

Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.
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http://dx.doi.org/10.7554/eLife.47789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927689PMC
November 2019

Cataractogenic load - A concept to study the contribution of ionizing radiation to accelerated aging in the eye lens.

Mutat Res 2019 Jan - Mar;779:68-81. Epub 2019 Feb 15.

Department of Biosciences, University of Durham, Mountjoy Science Site, South Road, Durham, DH1 3LE, UK; Biophysical Sciences Institute, University of Durham, Mountjoy Science Site, South Road, Durham, DH1 3LE, UK. Electronic address:

Ionizing radiation (IR) damages DNA and other macromolecules, including proteins and lipids. Most cell types can repair DNA damage and cycle continuously their macromolecules as a mechanism to remove defective proteins and lipids. In those cells that lack nuclei and other organelles, such as lens fiber cells and mammalian erythrocytes, IR-induced damage to macromolecules is retained because they cannot be easily replenished. Whilst the life span for an erythrocyte is several months, the life span of a human lens is decades. There is very limited turnover in lens macromolecules, therefore the aging process greatly impacts lens structure and function over its lifetime. The lens is a tissue where biomolecular longevity, lifelong retention of its components and continued growth are integral to its homeostasis. These characteristics make the lens an excellent model to study the contribution of retained macromolecular damage over time. Epidemiological data have revealed a significant association between exposure to IR, the loss of lens optical function and the formation of cataracts (cataractogenesis) later in life. Lifestyle, genetic and environmental factors all contribute to cataractogenesis due to their effect on the aging process. Cataract is an iconic age-related disease in humans. IR is a recognised cause of cataract and the occupational lens dose limit is reduced from 150 to 20 mGy / year averaged over 5 years (ICRP Publication 118). Understanding the effects of low dose IR on the lens and its role in cataractogenesis is therefore very important. So we redefine "cataractogenic load" as a term to account for the combined lifestyle, genetic and environmental processes that increase biomolecular damage to lens macromolecules leading to cataract formation. These processes weaken metabolic defenses, increase post-translational protein modifications, and alter the lipid structure and content of the lens. IR exposure is a significant insult to the lens because of free radical generation and the ensuing oxidative stress. We support the concept that damage caused by IR compounds the aging process by increasing the cataractogenic load, hereby accelerating lens aging and its loss of function.
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http://dx.doi.org/10.1016/j.mrrev.2019.02.004DOI Listing
March 2020

BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQP0 water permeability.

Exp Eye Res 2019 08 18;185:107585. Epub 2019 Feb 18.

Department of Biosciences, The University of Durham, South Road, Durham, DH1 3LE, UK; Biophysical Sciences Institute, The University of Durham, South Road, Durham, DH1 3LE, UK. Electronic address:

BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434-440). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 is not required for membrane association. Biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalized with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various C-terminal domain fragments of BFSP1 generated by caspase cleavage. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca. The complete C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments can enhance regulation by elevated calcium or reverse the response, indicative of the regulatory potential of BFSP1 with respect to AQP0. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca concentrations.
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http://dx.doi.org/10.1016/j.exer.2019.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713518PMC
August 2019

γ-Crystallin redox-detox in the lens.

J Biol Chem 2018 11;293(46):18010-18011

Centenary Institute, NHMRC Clinical Trials Center, Sydney Medical School, University of Sydney, NSW 2006, Australia. Electronic address:

In the vertebrate eye, limiting oxidation of proteins and lipids is key to maintaining lens function and avoiding cataract formation. A study by Serebryany identifies a surprising contributor to the eye's oxidative defense in their demonstration that γD-crystallin (HγD) functions as an oxidoreductase and uses disulfide exchange to initiate aggregation of mutant crystallins that mimic oxidative damage. These insights suggest a mechanism by which a dynamic pool of closely packed proteins might avoid oxidation-driven protein-folding traps, providing new avenues to understand the basis of a human disease with global impact.
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http://dx.doi.org/10.1074/jbc.H118.006240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240857PMC
November 2018

Non-invasive quantification of the developing optical properties and graded index of the embryonic eye lens using SPIM.

Biomed Opt Express 2018 May 10;9(5):2176-2188. Epub 2018 Apr 10.

Department of Physics, Durham University, South Road, Durham, DH1 3LE, UK.

Graded refractive index lenses are inherent to advanced visual systems in animals. By understanding their formation and local optical properties, significant potential for improved ocular healthcare may be realized. We report a novel technique measuring the developing optical power of the eye lens, in a living animal, by exploiting the orthogonal imaging modality of a selective plane illumination microscope (SPIM). We have quantified the maturation of the lenticular refractive index at three different visible wavelengths using a combined imaging and ray tracing approach. We demonstrate that the method can be used with transgenic and vital dye labeling as well as with both fixed and living animals. Using a key eye lens morphogen and its inhibitor, we have measured their effects both on lens size and on refractive index. Our technique provides insights into the mechanisms involved in the development of this natural graded index micro-lens and its associated optical properties.
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http://dx.doi.org/10.1364/BOE.9.002176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946780PMC
May 2018

A rim-and-spoke hypothesis to explain the biomechanical roles for cytoplasmic intermediate filament networks.

J Cell Sci 2017 Oct;130(20):3437-3445

RWTH Aachen University, Institute of Molecular and Cellular Anatomy, Wendlingweg 2, 52074 Aachen, Germany

Textbook images of keratin intermediate filament (IF) networks in epithelial cells and the functional compromization of the epidermis by keratin mutations promulgate a mechanical role for this important cytoskeletal component. In stratified epithelia, keratin filaments form prominent radial spokes that are focused onto cell-cell contact sites, i.e. the desmosomes. In this Hypothesis, we draw attention to a subset of keratin filaments that are apposed to the plasma membrane. They form a rim of filaments interconnecting the desmosomes in a circumferential network. We hypothesize that they are part of a rim-and-spoke arrangement of IFs in epithelia. From our review of the literature, we extend this functional role for the subplasmalemmal rim of IFs to any cell, in which plasma membrane support is required, provided these filaments connect directly or indirectly to the plasma membrane. Furthermore, cytoplasmic IF networks physically link the outer nuclear and plasma membranes, but their participation in mechanotransduction processes remain largely unconsidered. Therefore, we also discuss the potential biomechanical and mechanosensory role(s) of the cytoplasmic IF network in terms of such a rim (i.e. subplasmalemmal)-and-spoke arrangement for cytoplasmic IF networks.
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http://dx.doi.org/10.1242/jcs.202168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518161PMC
October 2017

αB-crystallin is a sensor for assembly intermediates and for the subunit topology of desmin intermediate filaments.

Cell Stress Chaperones 2017 07 3;22(4):613-626. Epub 2017 May 3.

Department of Biosciences and the Biophysical Sciences Institute, University of Durham, Durham, UK.

Mutations in the small heat shock protein chaperone CRYAB (αB-crystallin/HSPB5) and the intermediate filament protein desmin, phenocopy each other causing cardiomyopathies. Whilst the binding sites for desmin on CRYAB have been determined, desmin epitopes responsible for CRYAB binding and also the parameters that determine CRYAB binding to desmin filaments are unknown. Using a combination of co-sedimentation centrifugation, viscometric assays and electron microscopy of negatively stained filaments to analyse the in vitro assembly of desmin filaments, we show that the binding of CRYAB to desmin is subject to its assembly status, to the subunit organization within filaments formed and to the integrity of the C-terminal tail domain of desmin. Our in vitro studies using a rapid assembly protocol, C-terminally truncated desmin and two disease-causing mutants (I451M and R454W) suggest that CRYAB is a sensor for the surface topology of the desmin filament. Our data also suggest that CRYAB performs an assembly chaperone role because the assembling filaments have different CRYAB-binding properties during the maturation process. We suggest that the capability of CRYAB to distinguish between filaments with different surface topologies due either to mutation (R454W) or assembly protocol is important to understanding the pathomechanism(s) of desmin-CRYAB myopathies.
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http://dx.doi.org/10.1007/s12192-017-0788-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465037PMC
July 2017

The functional roles of the unstructured N- and C-terminal regions in αB-crystallin and other mammalian small heat-shock proteins.

Cell Stress Chaperones 2017 07 8;22(4):627-638. Epub 2017 Apr 8.

Department of Biosciences, Durham University, Durham, DH1 3LE, UK.

Small heat-shock proteins (sHsps), such as αB-crystallin, are one of the major classes of molecular chaperone proteins. In vivo, under conditions of cellular stress, sHsps are the principal defence proteins that prevent large-scale protein aggregation. Progress in determining the structure of sHsps has been significant recently, particularly in relation to the conserved, central and β-sheet structured α-crystallin domain (ACD). However, an understanding of the structure and functional roles of the N- and C-terminal flanking regions has proved elusive mainly because of their unstructured and dynamic nature. In this paper, we propose functional roles for both flanking regions, based around three properties: (i) they act in a localised crowding manner to regulate interactions with target proteins during chaperone action, (ii) they protect the ACD from deleterious amyloid fibril formation and (iii) the flexibility of these regions, particularly at the extreme C-terminus in mammalian sHsps, provides solubility for sHsps under chaperone and non-chaperone conditions. In the eye lens, these properties are highly relevant as the crystallin proteins, in particular the two sHsps αA- and αB-crystallin, are present at very high concentrations.
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http://dx.doi.org/10.1007/s12192-017-0789-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465038PMC
July 2017

The eye lens - a paradigm for healthy living.

Authors:
Roy A Quinlan

Exp Eye Res 2017 03 7;156:1-2. Epub 2017 Feb 7.

School of Biological Sciences, Durham University, Science Site South Road, Durham, DH1 3LE, UK. Electronic address:

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http://dx.doi.org/10.1016/j.exer.2017.02.003DOI Listing
March 2017

Ionizing radiation induced cataracts: Recent biological and mechanistic developments and perspectives for future research.

Mutat Res 2016 Oct - Dec;770(Pt B):238-261. Epub 2016 Jul 29.

Radiation Safety Research Center, Nuclear Technology Research Laboratory, Central Research Institute of Electric Power Industry (CRIEPI), 2-11-1 Iwado-kita, Komae, Tokyo 201-8511, Japan. Electronic address:

The lens of the eye has long been considered as a radiosensitive tissue, but recent research has suggested that the radiosensitivity is even greater than previously thought. The 2012 recommendation of the International Commission on Radiological Protection (ICRP) to substantially reduce the annual occupational equivalent dose limit for the ocular lens has now been adopted in the European Union and is under consideration around the rest of the world. However, ICRP clearly states that the recommendations are chiefly based on epidemiological evidence because there are a very small number of studies that provide explicit biological, mechanistic evidence at doses <2Gy. This paper aims to present a review of recently published information on the biological and mechanistic aspects of cataracts induced by exposure to ionizing radiation (IR). The data were compiled by assessing the pertinent literature in several distinct areas which contribute to the understanding of IR induced cataracts, information regarding lens biology and general processes of cataractogenesis. Results from cellular and tissue level studies and animal models, and relevant human studies, were examined. The main focus was the biological effects of low linear energy transfer IR, but dosimetry issues and a number of other confounding factors were also considered. The results of this review clearly highlight a number of gaps in current knowledge. Overall, while there have been a number of recent advances in understanding, it remains unknown exactly how IR exposure contributes to opacification. A fuller understanding of how exposure to relatively low doses of IR promotes induction and/or progression of IR-induced cataracts will have important implications for prevention and treatment of this disease, as well as for the field of radiation protection.
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http://dx.doi.org/10.1016/j.mrrev.2016.07.010DOI Listing
May 2017

The lipidation profile of aquaporin-0 correlates with the acyl composition of phosphoethanolamine lipids in lens membranes.

Biochim Biophys Acta 2016 11 1;1858(11):2763-2768. Epub 2016 Jul 1.

Chemistry Department, Durham University, Durham DH1 3LE, UK; Biophysical Sciences Institute, Durham University, Durham DH1 3LE, UK. Electronic address:

The lens fiber major intrinsic protein (otherwise known as aquaporin-0 (AQP0), MIP26 and MP26) has been examined by mass spectrometry (MS) in order to determine the speciation of acyl modifications to the side chains of lysine residues and the N-terminal amino group. The speciation of acyl modifications to the side chain of one specific, highly conserved lysine residue (K238) and the N-terminal amino group of human and bovine AQP0 revealed, in decreasing order of abundance, oleoyl, palmitoyl, stearoyl, eicosenoyl, dihomo-γ-linolenoyl, palmitoleoyl and eicosadienoyl modifications. In the case of human AQP0, an arachidonoyl modification was also found at the N-terminus. The relative abundances of these modifications mirror the fatty acid composition of lens phosphatidylethanolamine lipids. This lipid class would be expected to be concentrated in the inner leaflet of the lens fiber membrane to which each of the potential AQP0 lipidation sites is proximal. Our data evidence a broad lipidation profile that is both species and site independent, suggesting a chemical-based ester aminolysis mechanism to explain such modifications.
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http://dx.doi.org/10.1016/j.bbamem.2016.06.026DOI Listing
November 2016

Sub-nanometre mapping of the aquaporin-water interface using multifrequency atomic force microscopy.

Soft Matter 2016 Dec;13(1):187-195

Department of Physics, Durham University, Durham, UK.

Aquaporins are integral membrane proteins that regulate the transport of water and small molecules in and out of the cell. In eye lens tissue, circulation of water, ions and metabolites is ensured by a microcirculation system in which aquaporin-0 (AQP0) plays a central role. AQP0 allows water to flow beyond the diffusion limit through lens membranes. AQP0 naturally arranges in a square lattice. The malfunction of AQP0 is related to numerous diseases such as cataracts. Despite considerable research into its structure, function and dynamics, the interface between the protein and the surrounding liquid and the effect of the lattice arrangement on the behaviour of water at the interface with the membrane are still not fully understood. Here we use a multifrequency atomic force microscopy (AFM) approach to map both the liquid at the interface with AQP0 and the protein itself with sub-nanometer resolution. Imaging using the fundamental eigenmode of the AFM cantilever probes mainly the interfacial water at the surface of the membrane. The results highlight a well-defined region that surrounds AQP0 tetramers and where water exhibits a higher affinity for the protein. Imaging in the second eigenmode is dominated by the mechanical response of the protein and provides sub-molecular details of the protein surface and the sub-surface structure. The relationship between modes and harmonics is also examined.
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http://dx.doi.org/10.1039/c6sm00751aDOI Listing
December 2016

Small molecules, both dietary and endogenous, influence the onset of lens cataracts.

Exp Eye Res 2017 03 31;156:87-94. Epub 2016 Mar 31.

Biophysical Sciences Institute, University of Durham, Durham DH1 3LE, UK; University of Durham, Durham DH1 3LE, UK. Electronic address:

How the lens ages successfully is a lesson in biological adaption and the emergent properties of its complement of cells and proteins. This living tissue contains some of the oldest proteins in our bodies and yet they remain functional for decades, despite exposure to UV light, to reactive oxygen species and all the other hazards to protein function. This remarkable feat is achieved by a shrewd investment in very stable proteins as lens crystallins, by providing a reservoir of ATP-independent protein chaperones unequalled by any other tissue and by an oxidation-resistant environment. In addition, glutathione, a free radical scavenger, is present in mM concentrations and the plasma membranes contain oxidation-resistant sphingolipids what compromises lens function as it ages? In this review, we examine the role of small molecules in the prevention or causation of cataracts, including those associated with diet, metabolic pathways and drug therapy (steroids).
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http://dx.doi.org/10.1016/j.exer.2016.03.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107344PMC
March 2017

Radiation protection of the eye lens in medical workers--basis and impact of the ICRP recommendations.

Br J Radiol 2016 1;89(1060):20151034. Epub 2016 Feb 1.

1 Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Chilton, Didcot, UK.

The aim of this article was to explore the evidence for the revised European Union basic safety standard (BSS) radiation dose limits to the lens of the eye, in the context of medical occupational radiation exposures. Publications in the open literature have been reviewed in order to draw conclusions on the exposure profiles and doses received by medical radiation workers and to bring together the limited evidence for cataract development in medical occupationally exposed populations. The current status of relevant radiation-protection and monitoring practices and procedures is also considered. In conclusion, medical radiation workers do receive high doses in some circumstances, and thus working practices will be impacted by the new BSS. However, there is strong evidence to suggest that compliance with the new lower dose limits will be possible, although education and training of staff alongside effective use of personal protective equipment will be paramount. A number of suggested actions are given with the aim of assisting medical and associated radiation-protection professionals in understanding the requirements.
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http://dx.doi.org/10.1259/bjr.20151034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846221PMC
July 2016

In vivo, Ex Vivo, and In Vitro Approaches to Study Intermediate Filaments in the Eye Lens.

Methods Enzymol 2016 3;568:581-611. Epub 2015 Nov 3.

Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, The University of Durham, Durham, United Kingdom. Electronic address:

The role of the eye lens is to focus light into the retina. To perform this unique function, the ocular lens must be transparent. Previous studies have demonstrated the expression of vimentin, BFSP1, and BFSP2 in the eye lens. These intermediate filament (IF) proteins are essential to the optical properties of the lens. They are also important to its biomechanical properties, to the shape of the lens fiber cells, and to the organization and function of the plasma membrane. The eye lens is an iconic model in developmental studies, as a result different vertebrate models, including zebrafish, have been developed to study lens formation. In the present chapter, we have summarized the new approaches and the more breakthrough models (e.g., iPSc) that can be used to study the function of IFs in the ocular lens. We have presented three different groups of models. The first group includes in vitro models, where IFs can be studied and manipulated in lens cell cultures. The second includes ex vivo models. These replicate better the complex lens cell differentiation processes and the role(s) played by IFs. The third class is the in vivo models, and here, we have focused on Zebrafish and new imaging approaches using selective plane illumination microscopy. Finally, we present protocols on how to use these lens models to study IFs.
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http://dx.doi.org/10.1016/bs.mie.2015.09.010DOI Listing
October 2016

Purification of Protein Chaperones and Their Functional Assays with Intermediate Filaments.

Methods Enzymol 2016 9;569:155-75. Epub 2015 Sep 9.

Biophysical Sciences Institute, University of Durham, Durham, United Kingdom.

Intermediate filament (IF) scaffolds facilitate small heat shock protein (sHSP) function, while IF function is sHSP dependent. sHSPs interact with IFs and the importance of this interaction is to maintain the individuality of the IFs and to modulate interfilament interactions both in networks and in assembly intermediates. Mutations in both sHSPs and their interacting IF proteins phenocopy each other in the human diseases they cause. This establishes a key functional relationship between these two very distinct protein families, and it also evidences the role of this cytoskeleton-chaperone complex in the cellular stress response. In this chapter, we describe the detailed experimental protocols for the preparation of purified IF proteins and sHSPs to facilitate the study in vitro of their functional interactions. In addition, we describe the detailed biochemical procedures to assess the effect of sHSP on the assembly of IFs, the binding to IFs, and the prevention of noncovalent filament-filament interactions using in vitro cosedimentation, electron microscopy, and viscosity assays. These assays are valuable research tools to study and manipulate sHSP-IF complexes in vitro and therefore to determine the structure-function detail of this complex, and how it contributes to cellular, tissue, and organismal homeostasis and the in vivo stress response.
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http://dx.doi.org/10.1016/bs.mie.2015.07.025DOI Listing
October 2016

DRUG DISCOVERY. A new dawn for cataracts.

Authors:
Roy A Quinlan

Science 2015 Nov;350(6261):636-7

Biophysical Sciences Institute, School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, UK.

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http://dx.doi.org/10.1126/science.aad6303DOI Listing
November 2015

A dimensionless ordered pull-through model of the mammalian lens epithelium evidences scaling across species and explains the age-dependent changes in cell density in the human lens.

J R Soc Interface 2015 Jul;12(108):20150391

We present a mathematical (ordered pull-through; OPT) model of the cell-density profile for the mammalian lens epithelium together with new experimental data. The model is based upon dimensionless parameters, an important criterion for inter-species comparisons where lens sizes can vary greatly (e.g. bovine (approx. 18 mm); mouse (approx. 2 mm)) and confirms that mammalian lenses scale with size. The validated model includes two parameters: β/α, which is the ratio of the proliferation rate in the peripheral and in the central region of the lens; and γ(GZ), a dimensionless pull-through parameter that accounts for the cell transition and exit from the epithelium into the lens body. Best-fit values were determined for mouse, rat, rabbit, bovine and human lens epithelia. The OPT model accounts for the peak in cell density at the periphery of the lens epithelium, a region where cell proliferation is concentrated and reaches a maximum coincident with the germinative zone. The β/α ratio correlates with the measured FGF-2 gradient, a morphogen critical to lens cell survival, proliferation and differentiation. As proliferation declines with age, the OPT model predicted age-dependent changes in cell-density profiles, which we observed in mouse and human lenses.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528606PMC
http://dx.doi.org/10.1098/rsif.2015.0391DOI Listing
July 2015

Nonlinear ionizing radiation-induced changes in eye lens cell proliferation, cyclin D1 expression and lens shape.

Open Biol 2015 Apr;5(4):150011

School of Biological and Biomedical Sciences, University of Durham, Durham DH1 3LE, UK Biophysical Sciences Institute, University of Durham, Durham DH1 3LE, UK

Elevated cataract risk after radiation exposure was established soon after the discovery of X-rays in 1895. Today, increased cataract incidence among medical imaging practitioners and after nuclear incidents has highlighted how little is still understood about the biological responses of the lens to low-dose ionizing radiation (IR). Here, we show for the first time that in mice, lens epithelial cells (LECs) in the peripheral region repair DNA double strand breaks (DSB) after exposure to 20 and 100 mGy more slowly compared with circulating blood lymphocytes, as demonstrated by counts of γH2AX foci in cell nuclei. LECs in the central region repaired DSBs faster than either LECs in the lens periphery or lymphocytes. Although DSB markers (γH2AX, 53BP1 and RAD51) in both lens regions showed linear dose responses at the 1 h timepoint, nonlinear responses were observed in lenses for EdU (5-ethynyl-2'-deoxy-uridine) incorporation, cyclin D1 staining and cell density after 24 h at 100 and 250 mGy. After 10 months, the lens aspect ratio was also altered, an indicator of the consequences of the altered cell proliferation and cell density changes. A best-fit model demonstrated a dose-response peak at 500 mGy. These data identify specific nonlinear biological responses to low (less than 1000 mGy) dose IR-induced DNA damage in the lens epithelium.
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http://dx.doi.org/10.1098/rsob.150011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4422125PMC
April 2015

A silk purse from a sow's ear-bioinspired materials based on α-helical coiled coils.

Curr Opin Cell Biol 2015 Feb 28;32:131-7. Epub 2015 Jan 28.

School of Biological and Biomedical Sciences, The University of Durham, Stockton Road, Durham DH1 3LE, UK; Biophysical Sciences Institute, The University of Durham, Stockton Road, Durham DH1 3LE, UK; Department of Chemistry, The University of Durham, Stockton Road, Durham DH1 3LE, UK.

This past few years have heralded remarkable times for intermediate filaments with new revelations of their structural properties that has included the first crystallographic-based model of vimentin to build on the experimental data of intra-filament interactions determined by chemical cross-linking. Now with these and other advances on their assembly, their biomechanical and their cell biological properties outlined in this review, the exploitation of the biomechanical and structural properties of intermediate filaments, their nanocomposites and biomimetic derivatives in the biomedical and private sectors has started.
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http://dx.doi.org/10.1016/j.ceb.2014.12.010DOI Listing
February 2015

A gradient of matrix-bound FGF-2 and perlecan is available to lens epithelial cells.

Exp Eye Res 2014 Mar 14;120:10-4. Epub 2013 Dec 14.

Biophysical Sciences Institute, School of Biological and Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, United Kingdom. Electronic address:

Fibroblast growth factors play a key role in regulating lens epithelial cell proliferation and differentiation via an anteroposterior gradient that exists between the aqueous and vitreous humours. FGF-2 is the most important for lens epithelial cell proliferation and differentiation. It has been proposed that the presentation of FGF-2 to the lens epithelial cells involves the lens capsule as a source of matrix-bound FGF-2. Here we used immunogold labelling to measure the matrix-bound FGF-2 gradient on the inner surface of the lens capsule in flat-mounted preparations to visualize the FGF-2 available to lens epithelial cells. We also correlated FGF-2 levels with levels of its matrix-binding partner perlecan, a heparan sulphate proteoglycan (HSPG) and found the levels of both to be highest at the lens equator. These also coincided with increased levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in lens epithelial cells that localised to condensed chromosomes of epithelial cells that were Ki-67 positive. The gradient of matrix-bound FGF-2 (anterior pole: 3.7 ± 1.3 particles/μm2; equator: 8.2 ± 1.9 particles/μm2; posterior pole: 4 ± 0.9 particles/μm2) and perlecan (anterior pole: 2.1 ± 0.4 particles/μm2; equator: 5 ± 2 particles/μm2; posterior pole: 1.9 ± 0.7 particles/μm2) available at the inner lens capsule surface was measured for the bovine lens. These data support the anteroposterior gradient hypothesis and provide the first measurement of the gradient for an important morphogen and its HSPG partner, perlecan, at the epithelial cell-lens capsule interface.
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http://dx.doi.org/10.1016/j.exer.2013.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988982PMC
March 2014

Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation.

ASN Neuro 2013 Nov 19;5(5):e00125. Epub 2013 Nov 19.

*Institute of Molecular Medicine, College of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan.

IF (intermediate filament) proteins can be cleaved by caspases to generate proapoptotic fragments as shown for desmin. These fragments can also cause filament aggregation. The hypothesis is that disease-causing mutations in IF proteins and their subsequent characteristic histopathological aggregates could involve caspases. GFAP (glial fibrillary acidic protein), a closely related IF protein expressed mainly in astrocytes, is also a putative caspase substrate. Mutations in GFAP cause AxD (Alexander disease). The overexpression of wild-type or mutant GFAP promotes cytoplasmic aggregate formation, with caspase activation and GFAP proteolysis. In this study, we report that GFAP is cleaved specifically by caspase 6 at VELD²²⁵ in its L12 linker domain in vitro. Caspase cleavage of GFAP at Asp²²⁵ produces two major cleavage products. While the C-GFAP (C-terminal GFAP) is unable to assemble into filaments, the N-GFAP (N-terminal GFAP) forms filamentous structures that are variable in width and prone to aggregation. The effect of N-GFAP is dominant, thus affecting normal filament assembly in a way that promotes filament aggregation. Transient transfection of N-GFAP into a human astrocytoma cell line induces the formation of cytoplasmic aggregates, which also disrupt the endogenous GFAP networks. In addition, we generated a neo-epitope antibody that recognizes caspase-cleaved but not the intact GFAP. Using this antibody, we demonstrate the presence of the caspase-generated GFAP fragment in transfected cells expressing a disease-causing mutant GFAP and in two mouse models of AxD. These findings suggest that caspase-mediated GFAP proteolysis may be a common event in the context of both the GFAP mutation and excess.
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http://dx.doi.org/10.1042/AN20130032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3833455PMC
November 2013

Chaperones: needed for both the good times and the bad times.

Philos Trans R Soc Lond B Biol Sci 2013 May 25;368(1617):20130091. Epub 2013 Mar 25.

School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UK.

In this issue, we explore the assembly roles of protein chaperones, mainly through the portal of their associated human diseases (e.g. cardiomyopathy, cataract, neurodegeneration, cancer and neuropathy). There is a diversity to chaperone function that goes beyond the current emphasis in the scientific literature on their undoubted roles in protein folding and refolding. The focus on chaperone-mediated protein folding needs to be broadened by the original Laskey discovery that a chaperone assists the assembly of an oligomeric structure, the nucleosome, and the subsequent suggestion by Ellis that other chaperones may function in assembly processes, as well as in folding. There have been a number of recent discoveries that extend this relatively neglected aspect of chaperone biology to include proteostasis, maintenance of the cellular redox potential, genome stability, transcriptional regulation and cytoskeletal dynamics. So central are these processes that we propose that chaperones stand at the crossroads of life and death because they mediate essential functions, not only during the bad times, but also in the good times. We suggest that chaperones facilitate the success of a species, and hence the evolution of individuals within populations, because of their contributions to so many key cellular processes, of which protein folding is only one.
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http://dx.doi.org/10.1098/rstb.2013.0091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638401PMC
May 2013

The specificity of the interaction between αB-crystallin and desmin filaments and its impact on filament aggregation and cell viability.

Philos Trans R Soc Lond B Biol Sci 2013 May 25;368(1617):20120375. Epub 2013 Mar 25.

School of Biological and Biomedical Sciences, The University of Durham, South Road, Durham DH1 3LE, UK.

CRYAB (αB-crystallin) is expressed in many tissues and yet the R120G mutation in CRYAB causes tissue-specific pathologies, namely cardiomyopathy and cataract. Here, we present evidence to demonstrate that there is a specific functional interaction of CRYAB with desmin intermediate filaments that predisposes myocytes to disease caused by the R120G mutation. We use a variety of biochemical and biophysical techniques to show that plant, animal and ascidian small heat-shock proteins (sHSPs) can interact with intermediate filaments. Nevertheless, the mutation R120G in CRYAB does specifically change that interaction when compared with equivalent substitutions in HSP27 (R140G) and into the Caenorhabditis elegans HSP16.2 (R95G). By transient transfection, we show that R120G CRYAB specifically promotes intermediate filament aggregation in MCF7 cells. The transient transfection of R120G CRYAB alone has no significant effect upon cell viability, although bundling of the endogenous intermediate filament network occurs and the mitochondria are concentrated into the perinuclear region. The combination of R120G CRYAB co-transfected with wild-type desmin, however, causes a significant reduction in cell viability. Therefore, we suggest that while there is an innate ability of sHSPs to interact with and to bind to intermediate filaments, it is the specific combination of desmin and CRYAB that compromises cell viability and this is potentially the key to the muscle pathology caused by the R120G CRYAB.
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http://dx.doi.org/10.1098/rstb.2012.0375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638400PMC
May 2013

Changes in the quaternary structure and function of MjHSP16.5 attributable to deletion of the IXI motif and introduction of the substitution, R107G, in the α-crystallin domain.

Philos Trans R Soc Lond B Biol Sci 2013 May 25;368(1617):20120327. Epub 2013 Mar 25.

Biophysical Sciences Institute, University of Durham, , South Road, Durham DH1 LE, UK.

The archael small heat-shock protein (sHSP), MjHSP16.5, forms a 24-subunit oligomer with octahedral symmetry. Here, we demonstrate that the IXI motif present in the C-terminal domain is necessary for the oligomerization of MjHSP16.5. Removal increased the in vitro chaperone activity with citrate synthase as the client protein. Less predictable were the effects of the R107G substitution in MjHSP16.5 because of the differences in the oligomerization of metazoan and non-metazoan sHSPs. We present the crystal structure for MjHSP16.5 R107G and compare this with an improved (2.5 Å) crystal structure for wild-type (WT) MjHSP16.5. Although no significant structural differences were found in the crystal, using cryo-electron microscopy, we identified two 24mer species with octahedral symmetry for the WT MjHSP16.5 both at room temperature and at 60°C, all showing two major species with the same diameter of 12.4 nm. Similarly, at room temperature, there are also two kinds of 12.4 nm oligomers for R107G MjHSP16.5, but in the 60°C sample, a larger 24mer species with a diameter of 13.6 nm was observed with significant changes in the fourfold symmetry axis and dimer-dimer interface. This highly conserved arginine, therefore, contributes to the quaternary organization of non-metazoan sHSP oligomers. Potentially, the R107G substitution has functional consequences as R107G MjHSP16.5 was far superior to the WT protein in protecting βL-crystallin against heat-induced aggregation.
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http://dx.doi.org/10.1098/rstb.2012.0327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3638399PMC
May 2013

Evolution of the vertebrate beaded filament protein, Bfsp2; comparing the in vitro assembly properties of a "tailed" zebrafish Bfsp2 to its "tailless" human orthologue.

Exp Eye Res 2012 Jan 11;94(1):192-202. Epub 2011 Dec 11.

In bony fishes, Bfsp2 orthologues are predicted to possess a C-terminal tail domain, which is absent from avian, amphibian and mammalian Bfsp2 sequences. These sequences, are however, not conserved between fish species and therefore questions whether they have a functional role. For other intermediate filament proteins, the C-terminal tail domain is important for both filament assembly and regulating interactions between filaments. We confirm that zebrafish has a single Bfsp2 gene by radiation mapping. Two transcripts (bfsp2α and bfsp2β) are produced by alternative splicing of the last exon. Using a polyclonal antibody specific to a tridecameric peptide in the C-terminal tail domain common to both zebrafish Bfsp2 splice variants, we have confirmed its expression in zebrafish lens fibre cells. We have also determined the in vitro assembly properties of zebrafish Bfsp2α and conclude that the C-terminal sequences are required to regulate not only the diameter and uniformity of the in vitro assembly filaments, but also their filament-filament associations in vitro. Therefore we conclude zebrafish Bfsp2α is a functional orthologue conforming more closely to the conventional domain structure of intermediate filament proteins. Data mining of the genome databases suggest that the loss of this tail domain could occur in several stages leading eventually to completely tailless orthologues, such as human BFSP2.
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http://dx.doi.org/10.1016/j.exer.2011.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593068PMC
January 2012