Roslyn M Bill | Aston University (original) (raw)

Papers by Roslyn M Bill

Biochimica Et Biophysica Acta - Biomembranes, May 1, 2024

Water is essential for all life because it is required for the proper functioning of the cells an... more Water is essential for all life because it is required for the proper functioning of the cells and tissues of all organisms. It crosses biological membranes down osmotic gradients through the pores of aquaporin membrane channels at rates of up to 3 billion molecules per second. In the twenty years since Peter Agre was awarded the 2003 Nobel Prize in Chemistry for the discovery of the aquaporin family, aquaporin structure and function have become established in the literature. As a consequence, we understand in fine detail the mechanism by which aquaporins facilitate membrane water flow while excluding protons. We also know that some aquaporins facilitate the permeation of other small neutral solutes, ions or even unexpected substrates across biological membranes. The thirteen aquaporins in the human body have been implicated in pathologies including oedema, epilepsy, cancer cell migration, tumour angiogenesis, metabolic disorders and inflammation. Surprisingly, however, there is no aquaporin-targeted drug in the clinic. Some scientists have therefore concluded that aquaporins are intrinsically non-druggable targets. Discovering medicines to treat disorders of water homeostasis is thus an enduring challenge for the aquaporin field. Success in this endeavour will meet the urgent clinical need of millions of patients suffering from a range of life-threatening conditions and for whom no pharmacological interventions are currently available.

To study the function and structure of membrane proteins, high quantities of pure and stable prot... more To study the function and structure of membrane proteins, high quantities of pure and stable protein are needed. One of the first hurdles in accomplishing this is expression of the membrane protein at high levels and in a functional state. Membrane proteins are naturally expressed at low levels, so finding a suitable host for overexpression is imperative. Multidrug resistance protein 4 (MRP4) or ATP-binding cassette subfamily C member 4 (ABCC4) is a multi-transmembrane protein that is able to transport a range of organic anionic compounds (both endogenous and xenobiotic) out of the cell. This versatile transporter has been linked with extracellular signaling pathways and cellular protection, along with conferring drug resistance in cancers. Here we report the use of MRP4 as a case study to be expressed in three different expression systems: mammalian, insect, and yeast cells, to gain the highest yield possible. Interestingly, using the baculovirus expression system with Sf9 insect cells produced the highest protein yields. Vesicular transport assays were used to confirm that MRP4 expressed in Sf9 was functional using a fluorescent cAMP analogue (fluo-cAMP) instead of the traditional radiolabeled substrates. MRP4 transported fluo-cAMP in an ATP-dependent manner. The specificity of functional expression of MRP4 was validated by the use of nonhydrolyzable ATP analogues and MRP4 inhibitor MK571. Functionally expressed MRP4 in Sf9 cells can now be used in downstream processes such as solubilization and purification in order to better understand its function and structure.

HAL (Le Centre pour la Communication Scientifique Directe), Aug 31, 2015

We have shown previously that astrocyte adhesion is induced by the interaction of the neuronal pr... more We have shown previously that astrocyte adhesion is induced by the interaction of the neuronal protein Thy-1 with avb3 integrin and Syndecan-4 in astrocytes. However, the signaling mechanisms triggered downstream of these astrocyte receptors remain unknown. PAR-3 is an important adaptor protein that participates in the polarization of many cell types by forming a complex with PAR-6/aPKC; however, PAR-3 is not part of this complex during astrocyte polarization. Therefore, PAR-3 function in astrocyte adhesion remains to be determined. Here, we evaluated the participation of both Syndecan-4 and PAR-3 in astrocyte adhesion induced by Thy-1. Rat DITNC-1 astrocytes were transfected with siRNA against PAR-3, Syndecan-4 or siRNA control, treated with Thy-1-Fc or TRAIL-R2-Fc as a control and then assayed for wound-healing and focal adhesion formation. For focal adhesion analysis the cells were stained with anti-vinculin. Focal adhesion number and morphology were evaluated by confocal imaging. Our results show in DITNC-1 cells transfected with siRNA for PAR-3 or Syndecan-4 and stimulated with Thy-1 that wound-closure was decreased compared with siRNA control-transfected cells. For cells transfected with PAR-3 or Syndecan-4 siRNA larger focal adhesions than control cells were observed, implicating PAR-3 and Syndecan-4 in astrocyte adhesion induced by Thy-1. Whether these proteins regulate focal adhesion disassembly will be addressed in future studies.

Methods in molecular biology, 2012

The use of general descriptive names, registered names, trademarks, service marks, etc. in this p... more The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

Methods in molecular biology, 2019

New mass spectrometry approaches enable antibody-independent tracking of protein production. Here... more New mass spectrometry approaches enable antibody-independent tracking of protein production. Herein, we outline an antibody-independent mass spectrometry method for tracking recombinant protein production in the methylotrophic yeast Pichia pastoris system.

Journal of Biomolecular Structure and Dynamics

Lipases are enzymes of industrial importance responsible for the hydrolysis of ester bonds of tri... more Lipases are enzymes of industrial importance responsible for the hydrolysis of ester bonds of triglycerides. A lipolytic fungus was isolated and subsequently identified based on the ITS sequence analysis as putative Aspergillus flavus with accession number LC424503. The gene coding for extracellular triacylglycerol lipase was isolated from Aspergillus flavus species, sequenced, and characterised using bioinformatics tools. An open reading frame of 420 amino acid sequence was obtained and designated as Aspergillus flavus lipase (AFL) sequence. Alignment of the amino acid sequence with other lipases revealed the presence GHSLG sequence which is the lipase consensus sequence Gly-X1-Ser-X2-Gly indicating that it a classical lipase. A catalytic active site lid domain composed of TYITDTIIDLS amino acids sequence was also revealed. This lid protects the active site, control the catalytic activity and substrate selectivity in lipases. The 3-Dimensional structural model shared 34.08% sequence identity with a lipase from Yarrowia lipolytica covering 272 amino acid residues of the template model. A search of the lipase engineering database using AFL sequence revealed that it belongs to the class GX-lipase, superfamily abH23 and homologous family abH23.02, molecular weight and isoelectric point values of 46.95 KDa and 5.7, respectively. N-glycosylation sites were predicted at residues 164, 236 and 333, with potentials of 0.7250, 0.7037 and 0.7048, respectively. O-glycosylation sites were predicted at residues 355, 358, 360 and 366. A signal sequence of 37 amino acids was revealed at the N-terminal of the polypeptide. This is a short peptide sequence that marks a protein for transport across the cell membrane and indicates that AFL is an extracellular lipase. The findings on the structural and molecular properties of Aspergillus flavus lipase in this work will be crucial in future studies aiming at engineering the enzyme for biotechnology applications.Communicated by Ramaswamy H. Sarma.

Methods in molecular biology, 2022

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2021

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Contact Lens and Anterior Eye, 2018