Emily Bliss (Presenter)
UCL Institute of Child Health
Bio: My name is Emily Bliss, I’m currently in my third and final year of a PhD at University College London’s Institute of Child Health in their Biological Mass Spectrometry Centre under the supervision of Dr Kevin Mills. My main project is to investigate how mass spectrometry can be used to study the skin’s proteins, lipids and metabolites. In turn that leads on to the application of those techniques to study diseases such as atopic dermatitis, scarring and post-operation complications. My research project is funded by the Biomedical Research Council, National Institute of Health Research.
Authorship: Emily Bliss (1), Alex Virasami (2), Kate Bennett (1), Jeremy Pryce (2), Ryan O’Shaughnessy (1), Neil Sebire (2), Wei-Li Di (1), Wendy Heywood (1) and Kevin Mills (1)
(1) UCL Institute of Child Health, London, UK, (2) Great Ormond Street Hospital, London, UK
| Short Abstract Atopic eczema is a skin disease that affects approximately 25% of children and between 1 and 3% of the adult population in the UK. In atopic eczema the skin barrier is disturbed to the extent that it no longer functions to retain as much water as usual, resulting in an itchy, red skin rash. Label free proteomics was used to identify protein markers that may potentially be able to elucidate the mechanism behind this change. Fourteen proteins we identified as being significantly changed between control and atopic eczema elbow scrapings were selected for validation by immunohistochemical staining. Alpha-1 acid glycoprotein, bleomycin hydrolase, calmodulin-like protein 3, cathepsin D and dermcidin all showed changes, however calmodulin–like protein 5 was the most significant. |
Long Abstract
Eczema is a skin disease that affects approximately 25% of children and between 1 and 3% of the adult population in the UK(1). Eczema is not life-threatening, however it can be a very distressing condition to live with. There are seven different sub-types of eczema, the most common form is atopic eczema which affects 15% of children in the UK before the age of seven (2). It is a chronic inflammatory disease which presents in outbreaks of an itchy, dry, red rash, the underlying cause of which is unknown making it difficult to manage. In children the rash usually presents on the hands, face and skin folds. There is no cure to atopic eczema and due to its increasing prevalence it is becoming relatively costly to the National Health Service in the UK.
The skin is divided into 3 major structural layers: the epidermis, dermis and sub-cutaneous fat layer. The epidermis is the most superficial of those and is made up of a further 5 sub-layers. The “skin barrier” is found in the epidermis, in the sub-layer called the stratum corneum and acts among other things to protect the body from excessive water loss. For patients with atopic eczema however, this barrier has reduced function and therefore leads to excessive water loss from the skin, which in turn causes the skin manifestations.
The aim of this project was to use proteomics and mass spectrometry to investigate whether the skin barrier disturbances were arising as a result of protein changes in the skin. Initially skin scrapings were taken from volunteers with and without atopic eczema. Skin scrapings involved taking only the uppermost layers of cells that detach easily. Skin scrapings from the volunteers with atopic eczema were taken from both affected and unaffected sites of their body. The proteins from those samples were digested with trypsin and the respective peptides were then concentrated and desalted on a C18 trap column followed by chromatographic separation on a BEH C18 column using a nanoAcquity liquid chromatography system (Waters, Manchester) over a 1h gradient. Sixty minute mass spectrometry analyses were performed on a QToF Premier (Waters, Manchester) mass spectrometer in a label-free, data-independent acquisition mode, MSE whereby the peptides are simultaneously quantified and sequenced by using rapidly alternating high and low energy scans. Raw data was imported into ProteinLynx GlobalServer (Waters, Manchester) in order to identify the peptide masses corresponding to the fragmentation ion data. Proteins that were significantly different between the control and atopic dermatitis group were identified and their fold change was calculated.
Fourteen proteins were identified from the skin scrapings as being of particular interest in this study. Atopic eczema is not just a disease of the very surface of the skin where the skin scrapings were taken from, it originates in the skin barrier which is a few layers below the surface. In order to verify the roles that these proteins may play in the disease of atopic dermatitis throughout the whole thickness of the skin we decided to validate them using immunohistochemistry.
From the validation stage the protein that showed to be of particular interest was calmodulin-like protein 5 or calmodulin-like skin protein. It is a marker of late keratinocyte differentiation and in health it is expressed in a tight band, just one layer below the surface of the skin. However in the eczema samples it was expressed diffusely throughout the epidermis of the skin. Keratinocytes are the predominant cell type within the epidermis and they progress from basal layer progenitor cells to the surface of the skin. Once the keratinocytes have reached the surface of the skin or the stratum corneum of the epidermis to be specific they have fully differentiated into mature keratinocytes. It is shown that calmodulin-like protein 5 is secreted by keratinocytes in the epidermal layer as they differentiate and progress through the layers of the epidermis (3). The family of calmodulin proteins are responsible for regulating calcium transport and function within the epidermis (4). Keratinocyte maturation in the skin is calcium ion dependent (5). A gradient of calcium ion concentration increases as it progresses towards the surface of the skin, corresponding with the various stages of keratinocyte differentiation (6). In another skin disease psoriasis the calcium ion gradient within the epidermis has been shown to be disturbed (7).
To investigate further this link between differentiation of keratinocytes and eczema, we chose to stain the same tissue sections of skin for known markers of keratinocyte differentiation: involucrin and filaggrin. They are both markers of late keratinocyte differentiation in the skin. The results of this staining showed that both proteins demonstrated similar patterns of staining as calmodulin-like protein 5 itself had shown in the skin; dark concentrated staining in the uppermost layers of the epidermis in control tissue and diffuse staining throughout the whole epidermis for eczema tissues.
We concluded that calmodulin-like protein 5 has shown to be differentially expressed in eczema samples compared with controls in accordance with keratinocyte differentiation. It is therefore suggestive of the fact that differentiation of cells within the epidermis is disturbed in eczema alongside the link with the calcium ion gradient in the skin.
Further work is still required for this study to determine the exact cause of the differentiation disturbance, for which a greater number of samples would increase the power of the study.
References & Acknowledgements:
(1) GUPTA, R. et al. Time trends in allergic disorders in the UK. Thorax, v. 62, n. 1, p. 91-96, 2007. Disponível em: < http://thorax.bmj.com/content/62/1/91.abstract >.
(2) Types of Eczema: National Eczema Society. Disponível em: < http://www.eczema.org/types-of-eczema >. Acesso em: 08/09/2014.
(3) HASHIMOTO, Y. et al. Secreted calmodulin-like skin protein inhibits neuronal death in cell-based Alzheimer's disease models via the heterotrimeric Humanin receptor. Cell Death Dis, v. 4, p. e555, 2013. ISSN 2041-4889. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/23519124 >.
(4) MÉHUL, B. et al. Identification and cloning of a new calmodulin-like protein from human epidermis. J Biol Chem, v. 275, n. 17, p. 12841-7, Apr 2000. ISSN 0021-9258. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/10777582 >.
(5) HENNINGS, H. et al. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell, v. 19, n. 1, p. 245-54, Jan 1980. ISSN 0092-8674. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/6153576 >.
(6) BOYCE, S. T.; HAM, R. G. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol, v. 81, n. 1 Suppl, p. 33s-40s, Jul 1983. ISSN 0022-202X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/6345690 >.
(7) MENON, G. K.; ELIAS, P. M. Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arch Dermatol, v. 127, n. 1, p. 57-63, Jan 1991. ISSN 0003-987X. Disponível em: < http://www.ncbi.nlm.nih.gov/pubmed/1986708 >.
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