Drug Discovery Unit Annual report 2021

Website: https://drugdiscovery.dundee.ac.uk

Drug Discovery Unit Annual report 2021

Website: https://drugdiscovery.dundee.ac.uk

Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.Peer reviewe

Drug Discovery Unit Annual report 2021

Website: https://drugdiscovery.dundee.ac.uk

Crystal structure of a tripartite complex between C3dg, C-terminal domains of factor H and OspE of Borrelia burgdorferi

Complement is an important part of innate immunity. The alternative pathway of complement is activated when the main opsonin, C3b coats non-protected surfaces leading to opsonisation, phagocytosis and cell lysis. The alternative pathway is tightly controlled to prevent autoactivation towards host cells. The main regulator of the alternative pathway is factor H (FH), a soluble glycoprotein that terminates complement activation in multiple ways. FH recognizes host cell surfaces via domains 19–20 (FH19-20). All microbes including Borrelia burgdorferi, the causative agent of Lyme borreliosis, must evade complement activation to allow the infectious agent to survive in its host. One major mechanism that Borrelia uses is to recruit FH from host. Several outer surface proteins (Osp) have been described to bind FH via the C-terminus, and OspE is one of them. Here we report the structure of the tripartite complex formed by OspE, FH19-20 and C3dg at 3.18 Å, showing that OspE and C3dg can bind simultaneously to FH19-20. This verifies that FH19-20 interacts via the “common microbial binding site” on domain 20 with OspE and simultaneously and independently via domain 19 with C3dg. The spatial organization of the tripartite complex explains how OspE on the bacterial surface binds FH19-20, leaving FH fully available to protect the bacteria against complement. Additionally, formation of tripartite complex between FH, microbial protein and C3dg might enable enhanced protection, particularly on those regions on the bacteria where previous complement activation led to deposition of C3d. This might be especially important for slow-growing bacteria that cause chronic disease like Borrelia burgdorferi.Peer reviewe

Biophysical analysis of an oligomerization-attenuated variant of the Leishmania donovani dynamin-1-like protein

Chemotherapy is a cornerstone in the battle against leishmaniasis, a neglected tropical disease caused by Leishmania parasites that affects millions worldwide. An alarming number of reports are describing treatment failure with currently available drugs, thereby explaining the dire need for the discovery of novel compounds, preferably with yet unexplored modes of action. In this respect L. donovani dynamin-1 like protein (LdoDLP1) is of interest as mutations in LdoDLP1 were recently shown to confer resistance to a new antileishmanial compound, suggesting it to be a potential drug target. Through a combination of biochemical, structural, and biophysical methods, we were able to show that wild-type LdoDLP1 has a strong inherent propensity to self-assemble into higher-order oligomers. Guided by structural modeling, a selection of nine point mutations (including resistance markers) were screened for oligomerization behavior to generate self-assembly impaired LdoDLP1 mutants that would occur in solution as dimers and/or tetramers. This led to the identification of a double mutant (G354D/R357S) that exhibits significantly altered and reduced, yet not completely abolished, oligomerization behavior. Further characterization of the LdoDLP1 G354D/R357S double mutant using small-angle X-ray scattering (SAXS) revealed that a fraction of the protein population occurs as a dimer in solution. Additionally, SAXS analysis experimentally confirmed that LdoDLP1, like other dynamin-like proteins, lacks the structurally defined pleckstrin homology (PH) domain of classical dynamins but instead possesses an intrinsically disordered B insert, grouping it among the dynamin-like proteins that play key roles in processes such as mitochondrial fission

A new yeast strain for valorisation of vinasse, a rum distillery waste product

Background: Waste valorisation refers to processes of reusing or recycling waste materials to create valuable products. In the Rum distillery industry, the primary waste byproducts include bagasse, a solid waste made up of sugar cane residue and vinasse, a thick and acidic liquid. Although vinasse has been repurposed in agricultural fields, it has also contributed to both soil and ocean pollution. Despite several potential solutions having been suggested, an effective and environmentally safe use for vinasse has yet to be found. Results: The valorisation of vinasse for biofuel production was explored by assessing its potential as a growth medium for lipid production by non-conventional yeasts. The oleaginous yeast strain Yarrowia lipolytica, known for its lipid production capabilities, was initially tested on vinasse but required further adaptation and optimization. To circumvent this, we isolated a novel yeast strain from old vinasse waste, named V1, which demonstrated strong growth potential. The growth conditions of V1, including temperature and acidity, were characterized, and its potential for bioengineering was evaluated. This strain exhibited resistance to highly acidic pH levels and higher temperatures when cultivated on YPV, an artificial laboratory medium designed to mimic the acidity and glycerol content of vinasse. Whole genome sequencing (WGS) identified V1 as Pichia kudriavzevii. We demonstrated that V1 could be transformed with Yarrowia lipolytica vectors using the classical yeast heat shock protocol, thus enabling potential genetic engineering. Finally, lipid content in V1 was analysed in different conditions, confirming the strain's potential for biofuel production. Conclusions: Pichia kudriavzevii is not a traditional yeast, but its ability to adapt and grow under extreme pH and higher temperature conditions makes it a promising candidate for rum industry waste management applications. This strain could potentially be utilised to convert vinasse and other food waste products into valuable biofuels. Although further research is required to engineer and optimize this novel strain for vinasse cultivation, our findings highlight its great potential as a micro-factory in rum-producing regions and high locations, where agricultural waste is in need of valorisation solutions

Artificial membranes for membrane protein purification, functionality and structure studies.

Membrane proteins represent one of the most important targets for pharmaceutical companies. Unfortunately, technical limitations have long been a major hindrance in our understanding of the function and structure of such proteins. Recent years have seen the refinement of classical approaches and the emergence of new technologies that have resulted in a significant step forward in the field of membrane protein research. This review summarizes some of the current techniques used for studying membrane proteins, with overall advantages and drawbacks for each method

Intrinsic acyl-CoA thioesterase activity of a peroxisomal ATP binding cassette transporter is required for transport and metabolism of fatty acids.

Peroxisomes are organelles that perform diverse metabolic functions in different organisms, but a common function is β-oxidation of a variety of long chain aliphatic, branched, and aromatic carboxylic acids. Import of substrates into peroxisomes for β-oxidation is mediated by ATP binding cassette (ABC) transporter proteins of subfamily D, which includes the human adrenoleukodystropy protein (ALDP) defective in X-linked adrenoleukodystrophy (X-ALD). Whether substrates are transported as CoA esters or free acids has been a matter of debate. Using COMATOSE (CTS), a plant representative of the ABCD family, we demonstrate that there is a functional and physical interaction between the ABC transporter and the peroxisomal long chain acyl-CoA synthetases (LACS)6 and -7. We expressed recombinant CTS in insect cells and showed that membranes from infected cells possess fatty acyl-CoA thioesterase activity, which is stimulated by ATP. A mutant, in which Serine 810 is replaced by asparagine (S810N) is defective in fatty acid degradation in vivo, retains ATPase activity but has strongly reduced thioesterase activity, providing strong evidence for the biological relevance of this activity. Thus, CTS, and most likely the other ABCD family members, represent rare examples of polytopic membrane proteins with an intrinsic additional enzymatic function that may regulate the entry of substrates into the β-oxidation pathway. The cleavage of CoA raises questions about the side of the membrane where this occurs and this is discussed in the context of the peroxisomal coenzyme A (CoA) budget