AbelTesfai at334@kent.ac.uk

AbelTesfai BiochemicalSocietyStudentshipReport2011

A scientific report The aims of the project We wanted to use an alternative method of breaking up cells other than sonication as this was deemed too forceful and we want a method that allows micro compartments to be whole. Moreover, sonication leads to the presence of impurities, such as membrane proteins or cytoskeleton, thus we dont have micro-compartments isolated on their own. We want to explore two possible avenues that allow us to view micro-compartments and break down the cells: I. cefsulodin trying 100micrograms/ml. 2. sucrose -3 g of sucrose in 50ml flasks [175mM]. A description of the work carried out This project allowed me to be involved in making use of the following techniques for cefsulodin project: Transformation: For example we wanted pdu65 to be incorporated in to the vector p ET14b . Making lysogeny broth and taking OD readings using spectrometer. Techniques for sucrose project: Use of complement cells allow the introduction of vectors in to the bacterial cell and thus allow the process of transformation to occur. This involved incorporating Pet14B PDU65. LigationWheremyvectorandinsert[plysS]weredigestedwithrestrictionenzymes.Thisinvolved usingNEB2bufferwhichworkedequallyaswellfortworestrictionenzymestocutSacBgene. Assessment of the results and the outcomes of the studentship The effect of cefsulodin was not as significant as we hoped when adding to the samples:pet14b[control] and pet14 pdu65.Therefore we wanted to find out the right concentration of cefsulodin to add so we did a plate reader assay with different concentrations of the antibiotic cefsulodin. There was a marginal effect on the OD when adding 1mg/ml of cefsulodin: 100micrograms/ml of cefsulodin 1mg/ml of cefsulodin

In addition when observing the results under an electron microscope the pdu65 cells on average looked the same as the control. Moreover we could not conclude that the distinctive circular shapes in a few pdu65 cells were definitely micro compartments;

AbelTesfai at334@kent.ac.ukTuner pet14b pdu65-cef Tuner pet14b pdu65+cef

This meant we had to explore an alternative method such as adding sucrose with which we had better success and which seemed to be dependent on the expression of SacB gene. Therefore when SacB was present and the enzyme levansucrase was expressed then cells seemed to die much more readily and there was a sharp drop in OD of the cells with SacB contained in genome than those without. When adding 3g of sucrose, in powder form, to 50ml cultures that have reached an OD of 0.6 [exponential phase] there was an effect after one hour where the OD of the cells containing sacB fell sharply indicating death of cells.

This evidence coupled with running an SDS gel which showed SacB cultures had lighter protein bands lead us to conclude that sucrose is definitely breaking cells up. We then used 1litre flasks hoping to achieve the same effect as in 50ml culture. This experiment involved inducing protein production with IPTG and adding certain concentrations of sucrose. In the space of two days we tried three concentrations:6%,10% and 20% sucrose in 25ml of sonication buffer, used to resuspend the pellet formed from centrifugation of 1 litre flask. In these three experiments with 1litre cultures of plysS SacB pdu65 there was no sign of cell lysis .This is because there was no change in the cell density of the culture due to lysis as with 50ml cultures where you could see that colour of the SacB cultures was lighter in comparison to the control .We also took 1.5ml and centrifuged all of treated cells and found that pellets were formed indicating that cell lysis had not occurred. Changes made to our original proposal The aim of the project was never changed but the protocols and techniques we used did. Changes made include: using sucrose instead of cefsulodin towards the end of my placement. Using 1mg/ml of cefsulodin instead of 30micrograms/ml or 100micrograms/ml. the use of water instead of PBS when imaging with an electron microscope gave us cleaner images. we started to use NCE[minimal media] instead of LB[rich media] so that we get more distinct and isolated cells. Therefore the use of minimal media is better for imaging.

Future directions When adding 5g of sucrose powder in a litre flask we saw no evidence of lysis. This opposed the data we obtained from 50ml culture. The use of sucrose has not been thoroughly explored. For example we have not observed cells that have been lysed by sucrose with EM. This is because with 50ml flasks there is not enough biological material to view the cells in EM so have to scale up the volume to 1litre and add an appropriate amount of sucrose as we have not yet found the right concentration of sucrose to add in order to lyse the cells.

Studentship

AbelTesfai at334@kent.ac.ukThe studentship for me was about developing my own set of skills and confidence as well as helping to improve our chances of seeing micro-compartments. I have become increasingly more independent throughout the summer course in the work that I do. This was especially the case because I was able to revisit some techniques thus become ever more proficient at carrying out certain tasks such as making an SDS gel, running a gel electrophoresis and doing column chromatography. The value of my work was always appreciated by my supervisor and as a result I was able to go ahead and prove that cefsulodin was not a useful way of opening up E.coli cells.

BiochemicalSocietystudentshipreport2011

Adriana Guillermo Wiesinger Biochemical Society Studentship Report 2011

Dissectingtheroleofproteinkinasesininvasivecellmigration Supervisor: Dr Daimark Bennett, Institute of Integrative Biology, University of Liverpool Background: The acquisition of invasive cell behaviours, including motility and altered adhesion, underlies many important processes during normal development and is the first step towards metastasis, which is clinically the most important process in the progression of cancer. The goal of the project was to understand the mechanisms controlling invasive cell migration to help improve our knowledge of the molecular pathogenesis underlying cancer progression and to identify novel molecular targets for therapeutic intervention. Enzymes controlling reversible protein phosphorylation represent important classes of potential therapeutic targets, but, until recently, it had not been possible to systematically test the role of these enzymes in invasive cell migration in vivo. Taking advantage of advances in genome-wide in vivo screening technologies in Drosophila we have completed a genome-wide screen for protein kinases that regulate invasive cell migration in a Drosophila model of ovarian cancer cell metastasis revealing for the first time the involvement of these highly conserved enzymes in invasive cell migration in vivo.

Fig 1.Drosophila model of ovarian cancer. Drosophila egg chamber showing the migration of the GFP labelled border cell cluster in real time.

Aims: Our goal here was to characterise the role of the kinases that we have identified in more detail at the molecular and cellular level. The specific objectives of this study were to: 1) validate the role of protein kinases during invasive cell migration during Drosophila oogenesis by quantifying the effect of loss-of-function mutations and knockdown by RNAi; 2) establish the pattern of protein kinase gene expression during the invasive process; 3) phenotypically characterise these genes in more detail by live imaging. Description of work: To determine the role of reversible protein phosphorylation in Border cell migration, we targeted a subset of genes encoding protein kinases from our initial screen by RNAi. To achieve this we expressed selected lines from the publicly available genome-wide, transgenic RNAi libraries, specifically in GFP-labelled invasive Border cells using the GAL4-UAS expression system. The effect on migration was initially assessed by measuring the relative position of the border cell cluster by confocal microscopy and image analysis. We controlled for variability and potential nonspecificity of RNAi by using multiple independent lines for each gene. To obtain more insight into the role of these genes we investigated their expression pattern by RNA in situ hybridisation. Finally, we performed live imaging on one or two selected genotypes to characterise their effect in more detail. Results:

BiochemicalSocietystudentshipreport2011 In summary, this project identified 12 genes that putatively promote invasive cell migration in the Drosophila ovary, adding to the growing list of components that are required for this process. Our findings are of particular relevance to the understanding of egg chamber development and ovarian cancer, which is often not diagnosed in the clinic prior to the formation of metastatic forms and has a poor survival rate, but is also of relevance to other types of epithelial-derived cancers. Departures from the original proposal: To make best use of the time available, the order of experiments differed from that outlined in the proposal. The live imaging experiments were technically demanding and optimisation of the technique meant there was only time to perform a preliminary experiment. Future directions: Future experiments will include further validation of the genes using independent genetic or pharmaceutical tools. Ongoing live imaging experiments on selected genotypes will help to establish whether the genes we have identified are required for initial specification, cluster formation, detachment from adjacent follicle cells, final detachment of the cluster, directional protrusion or motility. Value of studentship to the student: I have thoroughly enjoyed doing this summer placement, as it has taught me a lot. Reading recommended papers helped me become more familiar with scientific writing, with terminology in cancer research as well as familiar with some techniques. It also made me realize that I am more than capable of understanding them now and I feel ready to extract information from the primary source rather than relying on books or the internet. Working as part of a team of PhD students as well as post-doctoral fellows I had the opportunity to ask questions about career pathways, which everyone kindly answered and now I know what steps to follow. Laboratory wise I just feel more confident in general, even with simple things such as dilutions and time management. I learnt many cell techniques and microscopy and even achieved to do an in situ hybridization on my own. I found live imaging incredibly exciting and I feel capable of using the technique on my own. My studentship was handson and I am glad I did it because now I feel more prepared for my third year project, I feel confident and I also feel capable of doing a PhD in the future. I would recommend a summer placement highly since for me it has been an invaluable experience. Value of studentship to the lab: Adriana was an excellent student who proved to be exceptionally diligent and hard working. Her enthusiasm for the work was especially pleasing and I think she benefited a great deal from the experience. Adriana generated a number of exciting results, which will be taken forward predominantly by a NWCRF-funded post-doctoral researcher in my lab. The many genetic and molecular biology reagents that Adriana has validated during the course of her studies will be invaluable for these further studies. For instance, labelled RNA probes that we generated will also be used to test the efficacy of gene knockdown in RNAi samples. Additional funding to further extend this work will be sought in due course. References: Morris, C.A., Benson, E. & White-Cooper, H. (2009) Determination of gene expression patterns using in situ hybridization to drosophila testes. Nature protocols 4(12), 1807-1019. Naora, H. & Montell. (2005) Ovarian Cancer metastasis: Integrating insights from disparate model organisms. Nature Reviews 5, 355-366.

BiochemicalSocietystudentshipreport2011 Prasad, M. & Montell, D.J. (2007) Cellular and Molecular Mechanisms of border cell migration analyzed using time-lapse live-cell imaging. Developmental Cell 12, 997-1005.

BiochemicalSocietystudentshipreport2011

AdrianaGuillermoWiesinger

Dissecting the role of protein kinases in invasive cell migration Student: Adriana Guillermo Wiesinger Supervisor: Dr Daimark Bennett, Institute of Integrative Biology, University of Liverpool Background: The acquisition of invasive cell behaviours, including motility and altered adhesion, underlies many important processes during normal development and is the first step towards metastasis, which is clinically the most important process in the progression of cancer. The goal of the project was to understand the mechanisms controlling invasive cell migration to help improve our knowledge of the molecular pathogenesis underlying cancer progression and to identify novel molecular targets for therapeutic intervention. Enzymes controlling reversible protein phosphorylation represent important classes of potential therapeutic targets, but, until recently, it had not been possible to systematically test the role of these enzymes in invasive cell migration in vivo. Taking advantage of advances in genome-wide in vivo screening technologies in Drosophila the Bennett lab has recently completed a genomewide screen for protein kinases that regulate invasive cell migration in a Drosophila model of ovarian cancer cell metastasis, revealing for the first time the involvement of these highly conserved enzymes in invasive cell migration in vivo. This work is of particular relevance to the understanding of egg chamber development and ovarian cancer, which is often not diagnosed in the clinic prior to the formation of metastatic forms and has a poor survival rate, but is also of relevance to other types of epithelial-derived cancers. Aims: Our goal here was to characterise the role of the kinases that have been identified in more detail at the molecular and cellular level. The specific objectives of this study were to: 1) validate the role of protein kinases during invasive cell migration during Drosophila oogenesis by quantifying the effect on migration of knockdown by RNAi; 2) establish the pattern of protein kinase gene expression during the invasive process; 3) phenotypically characterise these genes in more detail by live imaging. Description of work: To determine the role of reversible protein phosphorylation in Border cell migration, we targeted a subset of genes encoding protein kinases from the initial screen by RNAi. To achieve this we expressed selected lines from the publicly available genome-wide, transgenic RNAi libraries, specifically in GFP-labelled invasive Border cells using the GAL4UAS expression system. The effect on migration was initially assessed by measuring the relative position of the border cell cluster by confocal microscopy and image analysis. We controlled for variability and potential non-specificity of RNAi by using multiple independent lines for each gene. To obtain more insight into the role of these genes we investigated their expression pattern by RNA in situ hybridisation. Finally, we performed live imaging on two selected genotypes to characterise their effect in more detail. Departures from the original proposal: To make best use of the time available, the order of experiments differed slightly from that outlined in the proposal. The live imaging experiments were technically demanding and optimisation of the technique meant there was only time to perform one preliminary experiment. Results: Short hairpin RNA molecules designed against selected protein kinases were ectopically expressed specifically within border cells (BCs) in the Drosophila ovary, leading to depletion of a particular enzyme in BCs by RNA interference. Defective migration was confirmed for RNAi lines representing 12 protein kinase genes (e.g. see Figure 1A-D), providing evidence for a role for these genes in invasive cell migration in the Drosophila ovary. RNA in situ experiments to investigate the endogenous expression pattern of these genes was not conclusive and will need to be repeated, although staining with a positive control probe

BiochemicalSocietystudentshipreport2011

AdrianaGuillermoWiesinger

indicated that the approach was successful (Figure 1E-F). Live imaging confirmed the effect of one of the short hairpin RNA lines on border cell migration and provides a basis for ongoing experiments to investigate the precise function of the affected gene in this process.

AB E

CDF

Figure 1. Images of representative egg chambers showing normal (A-B) and aberrant (C-D) migration. (C,D) Defective migration was identified by retarded position of GFP-labelled BC clusters, relative to nurse cell/oocyte boundary (indicated with dotted line). GFP in green, DNA in red (A and C); transmitted light image (B and D). EF, representative images of early (E) and late (F) stage 9 egg chambers stained with a digoxygenin-labelled RNA probe for slbo, which is specifically expressed in the border cell cluster and served as a positive control for RNA in situ staining.

Future directions: Future experiments will include further validation of the genes using independent genetic or pharmaceutical tools. Ongoing live imaging experiments on selected genotypes will help to establish whether the genes we have identified are required for initial specification, cluster formation, detachment from adjacent follicle cells, final detachment of the cluster, directional protrusion or motility. Value of studentship to the student: I have thoroughly enjoyed doing this summer placement, as it has taught me a lot. Reading recommended papers helped me become more familiar with scientific writing, with terminology in cancer research as well as with the techniques. It also made me realise that I am more than capable of understanding original papers and am ready to extract information from the primary source rather than relying on books or the internet. Working as part of a team of PhD students as well as post-doctoral fellows I had the opportunity to ask questions about career pathways, which everyone kindly answered. I now feel more confident about which steps to follow. My confidence has also grown in the laboratory, even with simple things such as dilutions and time management. I learnt many cell biological techniques and was able to perform in situ hybridization experiments unaided. I found live imaging incredibly exciting and would relish the opportunity to do more confocal microscopy. My studentship was hands-on and I am glad I had this opportunity as I feel more prepared for my third year project, and better prepared and informed about doing a PhD in the future. I would recommend a summer placement highly, since for me it has been an invaluable experience. Value of studentship to the lab: Adriana was an excellent student who proved to be exceptionally diligent and hard working. Her enthusiasm for the work was especially pleasing and I think she benefited a great deal from the experience. Adriana generated a number of exciting results, which will be taken forward predominantly by a NWCRF-funded post-doctoral researcher in the lab. The many genetic and molecular biology reagents that Adriana has validated during the course of her studies will be invaluable for these further studies. For instance, labelled RNA probes that we generated will also be used to test the efficacy of gene knockdown in RNAi samples. Additional funding to further extend this work will be sought in due course.

Agnieszka Wieckowska

Biochemical Society Studentship Report 2011 Supervisor:DrDanMulvihill Institutionforplacement:UniversityofKent Usingpressureperturbationtochallengethedynamicsandintegrityoftheyeastactomyosin cytoskeleton Introduction:Pressureisaforceorinfluenceexertedonoragainstsomething.Usinghydrostatic pressuremayhaveinterestingeffectsonyeastcellsanditcouldhavesimilareffectsonothercellular organisms.Oneofthereasonsforusingpressureinsteadofotherkindsofstress(e.g.temperature) islackofdisruptionofintermolecularbondingthereforeproteinstructureisnotaffected.Pressure affectsthecellsystemsinanondestructiveandreversiblemanner.Anotheradvantageofusing pressurecanbeabilityofapplyingandreleasingpressurefromasampleextremelyrapidly,within milliseconds. Thecytoplasmofyeastcellscontainsanetworkofproteinfilamentscytoskeleton.Yeast cytoskeletonisbuildfromropelikepolymersoftubulinmictotubules.Thesearehighlydynamic andimportantformaintainingthecellstructure.Otherimportantfunctionsincludeproviding platformsforintracellulartransportandformingthespindleduringmitosis.Thesecondstructure formingyeastcytoskeletonareactinfilaments.Theyaredynamicstructuresandnormallyexistin bundlesornetworks.Theirfunctionistocontroltheshapeandsurfacemovements.Unlikeother eukaryoticcells,yeastsdonothavemostinsolubleandresistanttomechanicalstressintermediate filaments. Aimsoftheproject:Thisprojectaimstousepressureperturbationtochallengethedynamicsand integrityoftheyeastcytoskeletontoexploreitsroleinmaintainingpolarizedcellgrowth. Descriptionofworkcarriedout:Inorderto determinetheeffectsofpressureonyeast cellsanespeciallybuildpressurechamber (Fig.1)wasused.Waterfromthebeaker goesthroughthepipeintothepump.Water pushesdownonthemembranewhich exertshydrostaticpressureonthecells. Cellsareinsertedtothechamberusinga sterilesyringeandcanbeseenthroughand aneyepiece.Wholesysteminoperatedby computersoftware.

pump syringe pipes chamber

Figure1 Pressurechamber

eyepiece

Schizosaccharomycespombewildtypecellswereusedintheexperiments.Spombeweregrownin YES(30%glucose,5%yeastextract,4%peptone)at25Cwithaerationtoexponentialgrowth.Cells weresubjectedtoahydrostaticpressureof1,50,75,100,and200barforvariousperiodsoftimeat roomtemperature.Haemocytometerwasusedtodeterminethecelldensitybeforeandafterthe pressuretreatment.Imagesofthecells(controlcellsandpressurised)were takenbyafluorescentmicroscope tofollowchangesinthelengthofyeast cellswhicharebroughtaboutbychangesinpressure.

Figure2Viabilityplate

Viabilitytests(Fig.2)wereperformedbyinoculating200ofcontrolandpressurisedcellsonto3YES plateandincubatingthemin32C.After48hrsthenumberofformedcolonieswasrecorded. Results:Optimaleffectsofpressureoncellswereobtainedat200bar.After2hoursrapiddivisionof cellsandsmallersizeofthecellswereobserved.Thereasonforthatcouldbestressinducedoncells andfollowingquickermitosis.Cellskeptfor4hoursinthepressurechamberwerethesamesizeas controlcellshoweverafter6hourspressurisedcellsbegantostopdividingandgetlonger.After16 hourspressurisedcellswereconsiderablylonger,approximately23times.

1.Normal lengthcells

2.Elongated cells

3.Deadcells visible

Figure3Examplesofcellskeptunder0barfor14hrs,200barfor14hrsand200barfor18hrsrespectively.

Wild type S.pombe cells (Fig.3.1) divide when they reach a length of 14m when kept at atmospheric pressure. In contrast when kept at pressure (Fig3.2) cells have an elongated bent shape measuring up to 34m, with cytokinesis defects. The bent shape suggests a microtubule defect whereas lack of division may be due to an actin filament defects. Cells kept under 200 bar for longer than 14hrs (Fig3.3.) continue to elongate however some cells begin to die which was confirmed by viability tests (Fig.3). More extreme lengths are also visible since cells fail to divide most likely due to failing to form a Figure4RelativeViability.Cellsdieafterlonger spindle. Doubling time for wild type cells is 3 hours. Cells periodsat200barpressure.

which spent up to 6 hours in the pressure chamber divide just once whereas after 16 hours they divide just over two times. These two different results could be due to cells being in unknown stage of the cell cycle which means that cells could divide just after they were inserted into the chamber giving a higher cell density at the end of an experiment or a measuring error occurred. The control cells continue to grow and divide until nutrients are still available up to 8 generations. To conclude: Pressure reduces S. pombe viability at pressure of 200 bar, most likely due to defect in spindle formation. Cells are unable to divide because of microtubule defect and die from incomplete or incorrect chromosome segregation. Value of the studentship: This studentship was a great opportunity to help me develop my practical and personal skills as well as awareness of the workplace culture. The experience gained during 8 weeks in the laboratory will also be extremely valuable in my final year project and study. Having the opportunity to undertake such a placement also had a number of additional advantages; it helped me to decide to undertake further, postgraduate study upon completion of my degree programme and allowed me to develop more independence as a critical evaluator of experiments and data and to feed these thoughts back into future experimental approaches and design. Value of the studentship to the lab: During this project Agnieszka obtained a significant body ofdata on the effect pressure has upon cell growth and viability. These date indicate pressure has a strong but reversible effect upon the actin and microtubule cytoskeletons, research upon which are the main focus of this laboratory. This project defined the range of pressures to be used in future experiments. Using a novel pressure chamber for use with a live cell imaging system this will help us understand the impact pressure has upon cytoskeleton dynamics.

Alexandra Balueva Biochemical Society Studentship Report 2011

SUMMER VACATION STUDENTSHIP REPORT *RIBOSOMES HUNTING*University College London Department of Structural and Molecular Biology John Christodoulou group

The aims of the project The aim of this project was to study the process of protein folding on the ribosome using NMR spectroscopy. This project involved the production in E.coli, of selectively labeled ribosome nascent

complexes as well as their purification, so that folded properties of the nascent chains can be studied by NMR. For these studies, nascent chains ofFigure 1. Ribosome with attached nascent chain.

immunoglobulin-like domain (Dom5) were chosen.Dom5 of three different lengths were to be compared: Dom5 on

an extended linker (Dom5+110), Dom5 close to and within the ribosomal exit tunnel (Dom5+17) and a Dom5 truncation (Dom5745+17) which lacked 5 residues from its C-terminus. These constructs were selected in order to enhance the understanding of the early co-translational folding events on the ribosome (Fig.1). As nascent chains that are close or within the ribosomal exit tunnel (Fig.1) have limited motion, the methyl groups of Dom 5 were to be labeled with site-specific NMR probes, using selective13

CH3 isotopic labeling of the Ile, Leu and Val residues of the nascent chain against a perdeuterated

ribosome background, to enable the use of TROSY-based experiments. This strategy would permit snapshots representing structure of the Dom5 peptides emerging from the ribosomal exit tunnel to be obtained.

A description of the work carried out During 8 weeks I was following a protocol for the production in E.coli, of ribosomes with attached nascent chains. The whole procedure took up to several days and included two

main steps. Firstly, cells BL21-GOLD(DE3) were transformed with plasmid containing Dom5 gene and culture of these cells was grown in an unlabelled medium which enabled high cell density growth. Afterwards cells were transferred to a medium containing13 15

C and

N

isotopes for labeling (these isotopes are routinely used in the laboratory and were chosen for the first experiments) and expression of Dom5-RNCs is induced by IPTG. The nascent chains were able to be stalled on the ribosome due to the presence of a 17 amino acid sequence at the C-terminus, which is from a protein called SecM. The second step involved purification of the ribosomes with attached Dom5 chains. After lysis of the E.coli, the ribosomes were separated from the lysate by sedimenting through a sucrose cushion. Afterwards ribosomes with attached nascent chains containing His-Tag were purified on a Ni affinity column. To further purify the RNCs, a sucroseFigure 2. Example of Western Blot for attached Dom5 chain quantification.

gradient was used. After the centrifugation of sucrose gradient, 70S fractions are collected and frozen for the further NMR analysis. To assess the extent to which the ribosome was

occupied by nascent chains, a western blot with a specific antibody against the His-tag is used (Fig. 2).

Results The expression and purification of ribosomes with attached nascent chains was performed several times. One significant challenging during the production of RNCs was to obtain a sample that was of a suitable concentration for NMR analysis. It was found that the concentrations of initial samples that were produced was not enough high for NMR analysis. One reason for this may have been due to the presence of proteases which is a common problem that needs to be overcome during RNC preparations. After gaining experience in handling RNCs, one preparation was very successful in which a stable sample of high concentration (>10M) and high occupancy (>90%) was produced for NMR. Unfortunately, additional time in the project was needed to continue with NMR analysis of this sample and the production of other labeled RNCs. These types of RNC together with different methods of labeling can be used to understand of the principles of protein folding as it occurs on the ribosome. Studies of this kind can help form the basis of important applications, for example, in drug design.

The value of the studentship This project was an excellent opportunity to discover the everyday life of the scientific laboratory and to see how the methods learned in the university are used in research. I found this internship very useful in terms of practical skills. I learned to use laboratory equipment for chromatography, to prepare buffers and media, and understood all the steps of ribosome purification. An internship in the laboratory is different from the university training, as there are real scientific problems to solve and all the members of the group have certain degree of independency in their work. It was also interesting to see how several fields of biology (cell culture, biochemistry, biophysics) are all interconnected in one scientific project. It was rewarding to be involved in a project where the samples prepared by me will be used for the NMR analysis by the members of the group and will contribute to the understanding of protein folding on the ribosome. I thank Dr. John Christodoulou, Dr. Lisa Cabrita and PhD student Xiaolin Wang for their support and useful explanations during the project.

Alice Johnson Biochemical Society Studentship Report 2011

Supervisor: Stefan Bagby, Bath University

Introduction And Aims Postsynaptic density (PSD) has a number of components involved in memory, a very important area of neuroscience. One such component is KIBRA, an essential PSD scaffolding protein, which has been linked to a variety of memory-related diseases such as Alzheimers. Furthermore, KIBRA is also involved in other pathways, including the kidney. However, the structure of this key protein is yet to be determined. The aims of the project were to express and purify the C2 domain of the KIBRA protein for use in nuclear magnetic resonance (NMR) and X-ray crystallography. This would allow us to clarify the binding properties of the C2 domain, which is a membrane-targeting module. We are looking specifically to see if the domain is calcium-modulated because other signalling proteins often contain a C2 domain that has shown affinity with calcium. The research group have previously found a variant KIBRA, with two linked single nucleotide polymorphisms (SNPs) that have occurred in the human KIBRA gene. These SNPs have lead to the substitution of two amino acids in the C2 domain. Therefore, the aim of the project is to compare the normal and variant domains to determine whether either of them binds calcium. Human KIBRA C2 domain C771A variant (658-785) sequence (minus pQE30 residues): GATRIQIALKYDEKNKQFAILIIQLSNLSALLQQQDQKVNIRVAVLPCSESTTCLFRTRPLDASDTLV FNEVFWVSMSYPALHQKTLRVDVCTTDRSHLEECLGGAQISLAEVCRSGERSTRWYNLLS Experimental methods Two different lengths of purified DNA coding for KIBRA C2 variants (C771A mutant) were provided by another lab in collaboration with ours, these were 658-785 and 648-795. We amplified these using PCR and then used the shorter length (658-785) as that was the only length to give us a positive result on the gel. This was then cut up using restriction enzymes and ligated into the PQE-30 vector (3461bp), which is Ampicillin resistant to help with selection. Once in the vectors, we transformed them into DH5 cells. We then purified the plasmids from the DH5 colonies that we grew on antibiotic-selection plates and transformed them into the BL21 (DE3) expression vectors. The bacteria were then grown up in a 1 in 10 culture at 35C and, once induced with imidazole after reaching a density of between 0.6-0.9, samples were taken at intervals of 1, 2, 3 and 17hours. These samples were run through a flow chromatography column, which bound our protein of interest via its His-tag. We then buffer-exchanged the purified protein to make sure we had no possibility of calcium contamination. NMR spectrometry was then used to help us to find out whether or not the C2 domain binds calcium. Results NMR Spectrums

Graph 3. Overlay of graph 1 and graph 2. Graph 1. 35C. 50 mM MES, pH 6.5, 50 mM NaCl

Graph 1 shows the Human KIBRA C2 domain, without the addition of calcium. We got 20 less peaks than expected, which could be explained by the flexible parts of the C2 domain, such as the calcium binding loops. However, on addition of 5mM CaCl2 there was no change, suggesting that either the C2 domain doesnt bind calcium or that calcium is already bound to the C2 domain. If it is calcium contamination then it could be because of calcium in the water or calcium as an impurity in any reagents used during protein preparation. Therefore, EGTA, a calcium chelator, was added to the sample and a few significant changes occurred (shown in graph 2). Graph 3 is an overlay of the first two graphs and clearly shows the interaction of the Human KIBRA C2 domain with Ca2+ and there are some clear shifts upon addition of EGTA. A sub-set of these peaks revert more quickly than others to pre-EGTA positions, which suggests an ordered occupancy of Ca2+ binding sites. Consequently, we can conclude that the Human KIBRA C2 domain C771A mutant does bind calcium. Future Directions More detailed studies of calcium and phospholipid binding are going to be carried out by sequence specific resonance assignment. Departures From Original Proposal As well as working on the Human KIBRA C2 domain I have also been helping with the research into the structures of HACE1 and TRAF2, both involved in human cancers. Value Of Studentship My summer placement has been a very beneficial experience, by increasing my confidence in the lab as well as teaching me new techniques and honing those I already knew. I met some incredibly influential people during my time at Bath University, who supported me when I needed help and provided detailed knowledge when I asked for it. They reminded me of why I love this subject so much and how exciting it can be. I have most definitely enjoyed

working in a research lab and this experience has helped me to expand my views on future career possibilities to include lab work. Furthermore, my placement has introduced me to the field of structural biology, which is not an area of science that would have particularly interested me before the summer. Consequently, the studentship has been an informative and invaluable experience. In addition, I feel I have contributed towards the research my lab was carrying out, and have helped them discover that the Human KIBRA C2 domain of the C771A mutant does in fact bind calcium.

Amanda Chaplin Biochemical Society Studentship Report 2011 TheBiochemicalSocietySummerVacationStudentshipReport,2011

Elucidatingtheregulonofacopperresponsivetranscriptionalregulatorin Streptomyceslividans Student:AmandaChaplin,Supervisor:DrJonathanWorrall

BackgroundStreptomycesareaclassofGrampositiveactinobacteriawhichareofhugeimportancetohumanwelfare as they produce an array of clinically useful antibiotics and serve as a model system for bacterial development.1 Copperisanessentialtransitionmetalinbiologyhoweverexcesscopperistoxictocellular systemsmeaninglevelsaretightlycontrolled.2Therehavebeenseveralcopperproteins/enzymesidentified in streptomyces which play key roles in development and antibiotic production.1 A new class of metalloregulatory repressor protein has recently been identified in several bacteria that sense elevated copper levels.1 In most bacteria the gene for this repressor protein is genetically coupled to a copper sensitiveoperon(cso)whichencodesforaCu(I)chaperoneproteinandaCu(I)effluxPtypeATPase.3When bufferedlevelsofcopperincreaseitisproposedthatthemetalloregulatorbindsCu(I)causinganallosteric changethatleadstoitsreleasefromtheoperatorDNAallowingexpressionoftheCu(I)effluxsystem.This newclassofmetalsensorproteinhasbeennamedcoppersensitiveoperonrepressor(CsoR).3 ACsoRlikeproteinhasbeenidentifiedinS.lividansencodedbytheSL4136gene.However,unlikeother CsoRproteinsidentifiedthesurroundinggenomicenvironmentdoesnotcontaincopperresistancegenes but genes encoding for proteins involved in inorganic phosphate transport.1 Using a bioinformatics approach three target operator sequences for 4136 have been identified (Fig. 1). These are found to be located next to two operons which encode for a Cu(I) chaperone and Ptype ATPases and also next to SL4136.1

Aims

Figure1Thethree4136operatortargetsequences.A)Genomicenvironmentof SL4136alsowiththetargetDNAsequencesidentifiedbybioinformatics,B)&C)Gene environmentfortwoothertargetsequencesofP4136.

1)ToexpressandpurifyHistaggedP4136. 2)Touse digestedS.lividansgenomicDNA to ascertainwhetherDNA targetsequences ofP4136canbe pulleddownusingHistaggedP4136. 3) To determine whether P4136 binds to the bioinformatic identified target sequences using electrophoreticmobilityshiftassays(EMSAs).

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ResultsandDiscussion IsolationandpurificationofHistaggedP4136:AplasmidharbouringthegeneforP4136wastransformedandoverexpressedinEscherichiacoli.Cells werelysedusinganemulsiflexandthelysatewasappliedtoaNi2+chromatographycolumnandG75gel filtrationtoproduceapureHistaggedP4136protein.

IdentifyingP4136DNAtargetsequences:The genomic DNA of S.lividans was digested using the restriction enzyme Sau3AI to produce digested fragments(lane3Fig.2).Histagged4136proteinwasthenincubatedwiththefragmentsandloadedtoa Ni2+column.Thiswaswashedextensivelywithahighsalt buffer followed by elution of Histagged P4136. The eluantwasconcentratedandtheDNAextractedusinga phenolchloroform and DNA precipitation procedure. The extracted DNA was then incubated with a BamHI digested pUC19 plasmid and transformed into JM109 cells and plated on IPTG/Xgal LB/Agar plates. This producedblueandwhitecoloniesandthewhitecolonies were picked and cultured overnight for DNA isolation Figure 2 The using a fermentas GeneJET Plasmid Miniprep Kit. The Figure 3 Agarose gel Genomic DNA of miniprepped DNA was then digested using the showing the pUC19 S.lividans and the following restrictionenzymesEcoR1andHindIIItotestwhetheran plasmid DNA once digested insert had been cloned. These were observed on an digestions with EcoR1 and with the restriction HindIII enzymeSau3AI. agarose gel, with Fig. 3 showing that four colonies Lane1&9showstheDNA contained an insert. Clones containing an insert were markersthenlanes 4, 5, 7 sentforsequencingandtheresultsshowedthattheinsertcontainedS.lividans & 9 were sent for DNA butthesequencedid notmatchthe targetDNA sequences identifiedby sequencing. bioinformatics.*

AmplifyingIntergenicRegionsandEMSAs:The three target sequences for P4136 were amplified from S. lividans genomic DNA. The annealing temperaturesweremodifiedtoobtainthedesiredproduct.ThePCRproductwasranonanagarosegeland extractedusingaQIAquickGelExtractionKit.SeveralEMSAswerethenconductedwithoneshowninFig.4. ThethreeDNAtargets(1045,2730and4136)wereincubatedwithP4136andCu4136.Intheabsenceof copperaclearlowmobilitybandisobservedcorrespondingtoaP4136:DNAcomplex.Uponbindingcopper the 2730 and 4136 targets show a highmobility band that corresponds to the free DNA target. This suggeststhatonbindingcoppertheaffinityforDNAbyP4136issignificantlyreduced.

Summary Histagged 4136 protein was successfully overexpressed and purified. The identification of 4136 target sites using the approach of digested genomic DNA proved difficult. Some S.lividans DNA wasfoundinseveralclonesoncesequencedbutdidnotmatch thetargetDNAsequencesof4136identifiedbybioinformatics. P4136 target sequences were successfully amplified from genomicDNAandEMSAsrevealedthatallthreetargetsbound toP4136.

ValueofthestudentshipFigure4EMSAofP4136targetsequences;1045, 2730&4137incubatedwithapo4136andCu 4136.Lanes1,4,7targetDNAsequenceinthe absenceofP4136.Lanes2,5,8DNAtargetinthe presenceofP4136.Lanes3,6,9,DNAtargetinthe presenceofCuP4136.

Workinginthelabhasbeenabrilliantexperience;Ihavegaineda greatdealofknowledgeandunderstandingofmanyscientific techniquesandbeenabletoputmuchofthetheoryIhavelearnt

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intopractise.IwasabletocarryoutahugerangeoflaboratorytechniquesincludingEMSAs,agarosegels, PCRsandmanyothers.Ihavealsodevelopedmyabilitytoovercomeanexperimentaldifficultywhichwere oftenfacedandwiththehelpofmysupervisorandPhDstudentswasabletoadapttheexperimentto obtainthedesiredoutcome.Inowfeelmoreconfidentandexcitedaboutcarryingoutmyfinalyearlab basedresearchdissertation.Overallthestudentshiphasbeenstimulating,educational,andatruly insightfulexperienceoftheworkcarriedoutwithinaresearchlaboratory.

References:1. 2. Worrall,J.A.R.andVijenboom,E.(2010)CoppermininginStreptomyces:enzymes,naturalproductsanddevelopment, NaturalProductReports,27,742756. Baker,J.Sengupta,M.Jayaswal,R.K.andMorrissey,J.A.(2011)TheStaphylococcusaureusCsoRregulatesboth chromosomalandplasmidencodedcopperresistancemechanisms,EnvironmentalMicrobiology,13,24952507. Liu,T.Ramesh,A.Ma,Z.Ward,S.Zhang,L.George,G.Talaat,A.Sacchettini,J.andGiedroc,D.P.(2007)CsoRisanovel Mycobacteriumturberculosiscoppersensingtranscriptionalregulator,Naturechemicalbiology,3,6068.

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AndrewPountain

AndrewPountain BiochemicalSocietyStudentshipReport2011 MySummerStudentshipwiththeRudenkoLab,ImperialCollege Aimsoftheproject:Workwiththeeukaryoticparasite,Trypanosomabrucei,servestwofunctions:firstly,understandingitsbiology hasdirectmedicalimplications,T.bruceibeingthecausativepathogeninAfricansleepingsicknessandnagana, arelateddiseaseinlivestock;however,asanearlybranchingeukaryote,itservesasahighlyinformative modelforunderstandingtheregulationofeukaryoticgeneexpression,inbothfunctionalandevolutionary terms.Indiscussingtheproject,itisworthfirstnotingthattheaimshadchangedsomewhatonstartingit, fromthatdescribedintheapplication.TheoriginalaimwastoinvestigateRCC1domaincontainingproteinsby RNAiknockdown.However,subsequentlyitwasdeemedafarmoreinterestingandusefulproject,forthelab andformyself,tocreateT.bruceicelllinescontainingconstructsforDamID.Thistechniqueactsasan alternativetoChromatinImmunoprecipitation(ChIP),inthatitcanbeusedtomapthebindingofspecific chromatinassociatedproteinswithinthegenome.However,itisfelttobemoresensitivetotransient interactionsand,unlikeChIP,hasneverbeforebeenperformedforaT.bruceiprotein.Thetechniquerelieson theconstructionofafusionproteinbetweenthetrypanosomechromatinassociatedproteinandabacterial DNAmethyltransferase,DamMethylase,whichcatalysestheSAMdependentmethylationofadeninesinthe sequenceGATC.AdeninemethylationisnotfoundotherwiseinT.brucei,allowingtheanalysisofthe methylationpatternasameansofdirectlymappingthebindingofthefusionprotein.Thisisalarge,and potentiallysignificantproject,andthereforeIwasunabletoseeitthroughfromstarttofinish.However,Iwas abletotransfectthetrypanosomeswiththefusionconstructsanddobasicphenotypeanalysisononecellline.

DescriptionoftheworkIdid:Initialworkontheprojectinvolvedtheinsertionofthedammethylasegeneintoashuttlevector.Iinitially clonedthegenebyPCRfromEscherichiacoligDNAandinserteditintothevectorbydigestionofbothvector anddamgenewithHindIIIandBamHI,followedbyligationandtransformationofE.colicells,platingthemout ontoampicillintoselectfortransformants.IpickedcoloniesandcheckedforcorrectinsertionbycolonyPCR andvisualisationonanagarosegel.Miniprepswerethendonetoextracttheplasmidfromindividualclones, beforesendingoffsamplesforsequencing,confirmingcorrectcloningofthedamgene. Next,IinsertedtheT.bruceichromatinassociatedproteingenesintothevectorbackbone,sothatthe proteinswouldbeCterminallytaggedwiththedammethylase.5proteingeneswereclonedfromT.brucei gDNA:tbISWI,achromatinremodellingfactor;NLP(nucleoplasminlikeprotein),atranscriptionalregulator; 50kDaand70kDa,twohypotheticalproteinsshownbyChIPtobindspecificsitesontheT.bruceigenome; BDF3,abromodomainfactoronwhichpreviouschromatinbindingdatahasbeenpublished.RNAimediated knockdownofthefirstfourproteinshasbeenshowntoleadtoderepressionofRNApolItranscribedVSG expressionsites.VSG(variablesurfaceglycoprotein)istheantigeniccoatproteininbloodstreamformT. brucei,whosemanyvariantscanbeswitchedinordertoevadethehostimmunesystem.Hencenormallythe monoallelicexpressionofVSGgenesistightlycontrolled.BDF3istoactasacontrol,totestthattheDamID systemisworkingproperlyintrypanosomes.InordertoinserttheT.bruceigenesintothevectorbackbone, ClonetechsligationfreeInFusionsystemwasused.ThisinvolvesthecloningofT.bruceigenesbyPCRusing primersthatcomplementtheregionsofthevectorborderingthegeneinthefinalconstruct.Ilinearisedthe vectorbydigestionwithHindIIIand,usingthemanufacturersuppliedmix(theexactcontentsofwhicharefar fromclear!),insertedthegenesintothevectorbackbone,beforetransformingE.colicellswiththeconstructs. Theadvantagesofthissystemareitshightransformationefficiencyandthelackofrequirementforrestriction enzymes.GiventhatsomeofthegenescontainHindIIIsites,stickyendligationwouldhavebeenahighly difficultprocess.Asbefore,cloneswereselectedbycolonyPCRandcheckedbysequencing.

AndrewPountain The

Figure1:titrationofnuclearlocaliseddam expressionwithdoxycycline

Figure2:DICimage(left);DAPIstaining(middle);IFlabellingof BDF3_damfusion(right)

nextstagewasthetransfectionofbloodstreamformT.BruceicellsusingtheelectroporationbasedAmaxa Nucleofectortechnique.Cellswereplatedoutintowellsatdifferentconcentrations,allowingtheselectionfor monoclonalpopulationswithphleomycin.Cellswerelefttogrowbeforecloneswereselectedandglycerol stabilatesmadeforpreservationinliquidnitrogen. OtherworkconsistedofphenotypicanalysisofclonescontainingtheBDF3_damconstruct.Thisincluded growthcurves,WesternBlotanalysisandimmunofluorescencemicroscopy(IF).Thefusionproteinswere undercontrolofatetracycline(tet)inducedexpressionsystem.Totestfortoxicityoftheconstructorof overexpressionoftheprotein,Iobtainedgrowthcurvesfortheclones.Intheabsenceoftetracycline,these showednodifferenceingrowthratefromtheparentalcellline.However,after24hourstetinduction,growth slowedandcellsstartedtodie.Thisimpliesthatoverexpressionistoxic.However,analysisdonebyaprevious studentonacelllinecontaininganucleartargeteddammethylaseproteinshowedthattheleakyexpression ofthepromoterwithouttetinductionisenoughforsignificantlevelsofmethylation.Overexpressionleadsto veryhighbackgroundmethylation.IdidWesternBlotanalysisonthiscelllineafterinductionwithdifferent concentrationsofdoxycycline(amorewatersolubletetracyclinederivative)toshowthatexpressioncanalso becontrolledfromthispromoterifrequired(seefig.1).FurtherWesternBlotanalysisshowedcorrect expressionoftheBDF3_damfusionproteininmytransfectedcelllines.IalsodidIFmicroscopytocheckfor correctsubcellularlocalisationofthefusionprotein(seefig.2),showingthattheproteincolocalisedwithDAPI stainingofthenucleus(notethatthesecondspotwithDAPIstainingiskinetoplastDNA).

ProjectassessmentandfuturedirectionsBasedonresultsfromanalysisoftheBDF3_damcelllines,theprojectappearstohavebeenhighlysuccessful tothispoint.Problemsencounteredincludedthesizeofgenesthathadtobecloned(thetbISWIgeneis3480 bp),requiringtheuseofahighfidelity(andhence,expensive)PhusionTMpolymerase,andthecleartoxicityof fusionproteinoverexpression.ItislikelythatthistoxicityisduetooverexpressionoftheBDF3activity(RNAi hasshownthistobeanessentialprotein),sincethistoxicitywasnotobservedinpreviousexperimentson nuclearlocaliseddammethylase,implyingadeninemethylationitselfisnotharmful.Asthisisanongoing project,futuredirectionsareclear:similarphenotypicanalysismustbemadeoftheothertransfectedcell lines,beforeanalysisofthemethylationpattern.DamIDexperimentsinotherorganismshaveoftenused microarrayorsequencingbasedtechniquestomapthewholegenome.However,sincemygroupislookingat specificsites,itislikelythattheywilluseaqPCRbasedmethod(asdoneforthecelllinetransfectedwith nuclearlocaliseddammethylase),toanalysethepercentageofDNAcutbymethylationstatespecific restrictionenzymes.Thenuclearlocaliseddammethylasecelllineswillbeanecessarycontrol,allowingusto accountforbackgroundmethylation(whichisnonuniform:ithasbeenshownthatmethylationisreducedin chromatinbelievedtobemorehighlycompacted).Ifitworks,thiswillbeafirstforT.bruceiandmayserveto complementworkbyChIPanalysisandothertechniques.Hopefully,itwillalsogoontoprovidenew information,highlightinginteractionspickedupbythehighactivityofdammethylasewhichhavebeenmissed byChIP. TheValueofthisStudentshiptoMyselfandtotheLab:

AndrewPountain Duringmyeightweeksworkingonthisproject,Ihavegainedahugeamountofexperienceofavarietyofcore molecularbiologytechniques,asdescribedabove.Ihavealsolearntahugeamountwhichwillbeofgreathelp formydegree.Whatisoffargreatersignificance,however,istheexperienceIhavegainedsimplythroughmy submersioninaresearchenvironment.Ithasgivenmeachancetoseetherealitiesofresearch(although perhapstherelativelackofthingsgoingwronginthisprojectmayhavedistortedmyview!)andtogainbetter judgementofpotentialcareeroptionsinthefuture.Therequirementwhenworkingwitheukaryoticcelllines forregulartissueculture,forexample,whichwouldnotoccurwhenworkingwithbacteria,viruses,orprotein, isapotentialconsiderationwhichwouldneverotherwisehaveoccurredtome.Nonetheless,thisis counterbalancedbythedeeplyinteresting,complex,andunusualsystemsemployedbytheseorganisms,both ingeneregulationandimmuneevasion.Intermsofvaluetothelab,onlytimewilltell.Whilstmytimeworking hereisover,theprojectcontinuesanditwillbeinterestingtohearhowitgoes.Theoutcomemaybethe demonstrationofausefulnewtechniqueintrypanosomeresearch(previousstudieshavebeeninorganisms suchasDrosophila,Arabidopsis,andC.elegans)yieldingnewinsightsintogeneregulation.Alternatively,the projectmaysimplynotworkout,butanegativeresultisaresultnevertheless!Personally,Iamhopingforthe former,butwewillsimplyhavetosee.PerhapsthemostprofoundthingIhavelearnthereaboutbiological researchisthatcellswillsimplydotheirownthing,irregardlessoftheresearchersbestintentions.

The Biochemical Society

AnitaMurphy Biochemical Society Studentship Report2011

AnitaMurphy UniversityCollegeCork, Ireland. Bsc.Biochemistry PrincipalInvestigator:Dr.PaulYoung. BioSciencesInstitute, UniversityCollegeCork.

[INVESTIGATIONOF POTENTIALFUNCTIONSOF MYOZENININTHECENTRAL NERVOUSSYSTEMAND PURIFICATIONOFACTININ HETERODIMERS]

[INVESTIGATIONOFPOTENTIALFUNCTIONSOFMYOZENININ THECENTRALNERVOUSSYSTEMANDPURIFICATIONOF ACTININHETERODIMERS] TheBiochemicalSociety BriefBackground InmyoriginalproposalsubmittedbackinFebruarythetitleofmyresearchprojectwasThe InvestigationofPotentialFunctionsofMyozeninintheNervousSystem.However,duetothefact thatmyprojectworkinvolvedtheuseofbraintissueofmiceofembryonicandpostnatal developmentalstagesmeantthatIhadtowaitforeachmousetoreachitsrequiredstageof development,andasaconsequenceofthisIcouldntgetactivelyinvolvedinthisprojectuntilthe laterweeksofmyscholarship.InthemeantimeIworkedwithtwoproteins;actinin1andactinin 4. actinin actininisanactincrosslinkingproteinthatformsanantiparalleldimer.ItsNterminusconsistsof twocalponinhomologydomains;thisistheactinbindinghead.ItsCterminusisacalmodulinlike domainconsistingoftwoEFhands,whileitscentralregionconsistsoffourhelicalspectrinlike repeats.Therearefourisoformsofactinin,1,2,3and4,twomuscleisoforms,2and3,and2non muscleisoforms1and4.Themuscleisoformsarefoundinskeletalmuscle(OteyandCarpen, 2004).actinin1,thenonmuscleisoform,isfoundinfocaladhesions(Hondaetal.,1998).actinin 4,theothernonmuscleisoform,isconcentratedattheleadingedgeofmotilecells,suggestingit playsaroleincellmotilityandalsocancermetastasis(Yamamotoetal.,2007).Asalready mentionedactininmonomerscometogethertoformdimers,atfirsttheywerethoughttoonly formhomodimers,butthenheterodimerswerenotedtoformbetweenthemuscleisoforms(Gache etal.,1984).ThePhDstudentthatIwasworkingwithformostoftheeightweeks,KateFoley,has discoveredthatheterodimersformbetweenthenonmuscleisoforms.Thisiswheremylabwork camein.Inacellexpressinggenesforactinin1andactinin4homodimersbetweentheactinin 1swillform,likewiseforactinin4andheterodimerswillform,containingonemonomerofeach. AspartofherPhD,Katewouldliketostudytheseheterodimersinmoredetailandtheonlywayto dothiswouldbetoseparatethehomodimersfromtheheterodimers.

Actinin1Homodimer

Actinin4Homodimer

ActininHeterodimer

Asamethodfordoingthismyprojectwastoattachanaffinitytaggenesequencetoeachofthe actiningenesequences(ACTN)sothatthehetrodimerscouldbepurifiedfromthehomodimers usingaffinitychromatography.actinin1wastobetaggedwithaGlutathioneSTransferasetag (GST)andactinin4wastobetaggedwithaMaltoseBindingProteintag(MBP).Thelogicbehind thisisasfollows:ifamixtureofhomoandheterodimersofactinin,witheachmonomercontaining itsmentionedtag,ispassedthroughachromatographycolumncontainingglutathioneagarose beads,thosemonomersofeachdimercontainingtheGSTtagwillbindtothebeadsandthosethat dontwillpassthroughasGlutathionehasahighaffinityforGST.Thismeansthattheactinin4

[INVESTIGATIONOFPOTENTIALFUNCTIONSOFMYOZENININ THECENTRALNERVOUSSYSTEMANDPURIFICATIONOF TheBiochemicalSociety ACTININHETERODIMERS] homodimerswillpassthroughandbeseparatedfromtheactinin1homodimersand heterodimers.Toelutethisactinin1homodimerandheterodimermixtureglutathioneisaddedto thecolumntodisplacethetaggedproteins.Thiselutionisthenpassedthroughanothercolumn housingamylosecoatedbeads;amylosehasahighaffinityfortheMBP.Theheterodimers, containingactinin4withaMBPtagwillbindtotheamyloseandremaininthecolumnwhilethe actinin1homodimers,containingtheGSTtagwillpassthrough.Asaresulttheheterodimersare purifiedfromthehomodimersandcanbeelutedbypouringfreeamylosethroughthecolumn. Whilethesetagsfacilitatepurificationoftherecombinantproteintheyhavethepotentialto interferewiththebiologicalactivityoftheprotein(Waugh,2005)weaddedaTEVprotease recognitionsequencetotheNterminal,rightbeforetheactualtagthatwillberecognisedbyaTEV proteaseandcleavethetagfromtheprotein.BoththeGSTandMBPaswellastheTEVprotease haveanadditional6histidinetagtofacilitatetheirremovalfromtheuntaggedheterodimerusinga Niaffinitycolumn.

Passactinin1dimers, actinin4dimersand actininheterodimers throughthecolumn.

Passactinin1 homodimersand theheterodimers throughthe column.

Containsglutathione agarosebeadswhichwill bindtotheglutathiones transferase(gst)tag.

Containsamylosecoatedbeads whichbindtomaltosebinding protein(mbp)tag.

Heterodimersandactinin1willremainin columntobeelutedlaterwhileactinin4will passthroughandbeseparatedfromthe mixture.

Actinin1homodimerswillpassthrough,but heterodimers,containingMBPtag,willbind, henceseparatinghomofromheterodimers.

Asastartingpointforthisprojectbothactinin1isoformswereavailableinpET24basedvectorsthat encodeaGSTtag.Forcoexpressionofbothproteinswithdifferenttags,actinin4hadtofirstlybe taggedwithaMBPtag.ThenGSTtaggedactinin1andMBPtaggedactinin4hadtobesubcloned intovectorsthathaddifferentantibioticresistancesanddifferentoriginsofreplicationsothatthey couldbecotransformedintoandmaintainedinE.coliBL21cellsforproteinexpression.Toachieve thisactinin4wascutfromthestartingvectorusingXhoIandEcoR1restrictionenzymes,andligated

[INVESTIGATIONOFPOTENTIALFUNCTIONSOFMYOZENININ THECENTRALNERVOUSSYSTEMANDPURIFICATIONOF ACTININHETERODIMERS] TheBiochemicalSociety intothepET24dMBPTevvector,thatthatencodesanMBPtagandTEVcleavagesite.MBPTEV actinin4wasthencutfromthisvectorusingrestrictionenzymesAsc1andapartialdigestusing NcoIandclonedintoapACYCvectorthathadbeencutwithNcoIandMluI.pACYChasKanomycin resistanceandap15Abasedoriginofreplicationandthuswouldbecompatibleforcoexpression withanAmpicillinresistantpETvectorthathasapBR322basedorigin.Thiscloningwassuccessful andtheresultantpACYCMBPactinin4vectorwastransformedintoBL21cellsandexpressionof MBPactinin4verifiedbyproteingelelectrophoresis(seeimageattached).Thesecondgoalofthis partoftheprojectwastosubclonetheGSTTevactinin1intoavectorpET8cavectorwithAmpicillin resistance.TheinserthadtobecutwithrestrictionenzymeEcoR1,thenbluntedwithKlenow polymeraseandthencutwithXbaI,thevectorhadtobealsocutwithrestrictionenzymeMluI, bluntedasbeforeandthencutwithXbaIsothatthetwocouldligatetogether,creatinga recombinantDNAmolecule.Thiswasnotachievedinthetimeframeoftheprojectduetodifficulties withtheefficienciesofthebluntendligation. Ididsomedifficultycarryingoutthisproject,notintheactuallabworkbutmoresoduethefactthat scienceitselfisverypernicketyandfastidious.Thelabworkwasquitesimpleinprincipaland involvedseveralprocessesofrestrictiondigestion,ligation,transformationintolivingcompetent cellstoproduceacolonyofcellsthatallexpresstheplasmidofinterest,growingofbacterial culturesofoneofthesecoloniestoincreasetheyieldoftherecombinantDNAmolecule,purification oftheplasmidandthentransformingtheplasmidintoBL21cellstoexpressthetaggedprotein.I managedtotagtheactinin4withMBPeventuallygotittoexpressinthepACYCvector.Thisstep tookalongtimetoachievebecauseinordertoinsertitintosaidvector,itfirsthadtobecutfrom thepET24dvector,asmentionedabove,andthisrequiredapartialdigest,whichareknowntobe verysensitiveandrequirepinpointprecisionandtiming.Thecloningofthetaggedactinin1intothe pET8cvectorrequiredboththeendsofthevectorandtheinserttobeblunted,andligationbetween bluntendsisknowntobefarlessefficientthanligationbetweenoverhangs.Irepeatedeachligation aspectoftheprotocolseveraltimesbuttonoavail.IfeelhoweverthatifgivenmoretimeIwould mostdefinitelyhavebeenabletocompletethispartoftheprojectalso,perhapsusingastrategy thatdidnotinvolveligatingbluntends. Myozenin Myozeninisaproteinthathasonlybeevernotedtobepresentintheskeletalmuscle,locatedinthe zdiscofthesacromerewhereitinteractswithactincrosslinkingproteinsactininand filamin(Faulkneretal.,2000).Myozeninbindstothe23rdrepeatofthe24Iglikemoleculesinthe filaminproteinanditbindstothespectrinlikerepeatsoftheactinin,bothofwhichare responsibleforthedimerizationofthesetwoactincrosslinkingproteins.Thissuggeststhat myozeninplaysaroleinmodulatingthespacingbetweenthemonomersofeachdimerandhence thespacingbetweentheactinfilaments(Takadaetal.,2001).Myozeninalsointeractswith calcineurin,aserine/threoninephosphatase.Thisphosphataseactivatesanotherproteincalled nuclearfactorofactivatedTcells(NFAT),whichwhenactivatedinducesaslowtwitchmusclefiber type.Myozeninisfoundonlyinfasttypemusclefibers,suggestingthatitnegativelyregulates calcineurin(Freyetal.,2008).

[INVESTIGATIONOFPOTENTIALFUNCTIONSOFMYOZENININ THECENTRALNERVOUSSYSTEMANDPURIFICATIONOF TheBiochemicalSociety ACTININHETERODIMERS] Calcineurinandactininarealsopresentinthebrain.CalcineurindephosphorylatesaGTPasecalled dynamin,whichtriggersendocytosisofvesiclesandreceptors(SmillieandCousin,2005),ergoplaying aroleinsynapticplasticitywhileactininmodulatesNMDAreceptorsandcalcium/calmodulin dependentproteinkinaseII(Walikonisetal.,2001).Actinalsoplaysanimportantroleinthe formationofsynapsesandmorphologicalchangesofthesynapseaswellasaxonanddendrite formation(DillonandGoda,2005).Forthepurposeofmyprojectwethoughtthatmaybemyozenin, viaitsinteractionswithactinin,maytethercalcineurintothesynapticregionandhenceplayarole insynapticplasticity. MymainprojectinmystudyofmyozeninwastoexaminetheexpressionofmyozeninmRNAinthe brain.Becausemyozeninistransientlyexpressedintheheartduringembryogenesisandlater becomesrestrictedtoskeletalmuscle(Freyetal.,2000)wehypothesizedthatmyozeninmightonly bepresentinthebrainatacertaindevelopmentaltimepointorinaparticularareaofthebrain.In ordertostudythetemporalprofileofmyozeninexpressionwecarriedoutaReverseTranscriptase (RT)PCR.Complementary(c)DNAwaspreparedfrommousebraintissueatembryonicandpost natalstagesandmyozeninspecificprimerswereusedtoperformthePCR.cDNAisDNAthatis synthesizedfrommRNAthatwaspurifiedfrombraintissueinareactioncatalyzedbyreverse transcriptase.cDNAwaspreparedfrombraintissuefromanembryonicmouse,P1,P8,P14,P28and P180mice.Ifmyozeninwaspresentinanyofthestages,theprimerswouldannealtoitscDNA sequencethesequencewouldbeelongated,viaTaqPolymerase.Whenallsampleswererunona gel,thoselaneswiththebrightestbandsrepresentedthosestageswiththebestexpressionof myozenin.Lane1:Embryo Lane2:P1 Lane3:P8 Lane4:P14 Lane5:P28 Lane6:P180 Lane7:NegativeControl(NoReverse Transcriptase) Lane8:NegativeNegativeControl(No Template)

Fromthisweconcludedthatmyozeninwasrelativelyuniformlyexpressedthroughoutdevelopment inthebrain.Alsoitsexpressioninthebrainwasrelativelylow,muchlowerthaninmuscle(datanot shown).Toexaminethespatialpatternofmyozeninexpressionwedecidedtocarryouttheinsitu hybridizationattheP1andP28developmentalstages. InthisexperimentmymaintaskwastoclonethemyozeningenesequenceinapBluescriptvector, growuptwobacterialculturesofthisandpurifytwotubestheplasmidofinterest.Inthemakingof theriboprobefortheinsituIplayedaveryminorroleduetothefactthatRNAworkwasinvolved andRNAisverysusceptibletodegradationbyRNases.Iwashoweververyhappytoobserveand learnnewtechniquesthatImayneedinthefuture.ThepBluescriptvectorwaschosenbecauseit containstwoRNApolymerasebindingsites,T3andT7Terminal.Inordertomaketheprobethe vectorhadtobelinearized.IcutoneportionofthepurifiedplasmidwithXhoIsothatits transcriptionwasunderthecontroloftheT3terminalandinthe5to3directionandwouldmakea

[INVESTIGATIONOFPOTENTIALFUNCTIONSOFMYOZENININ THECENTRALNERVOUSSYSTEMANDPURIFICATIONOF ACTININHETERODIMERS] TheBiochemicalSociety senseprobe,thecontrol,andIcuttheotherportionofthepurifiedplasmidwithEcoR1sothatthe transcriptionoftheinsertwasunderthecontroloftheT7terminalinthe3to5directionand wouldmaketheantisenseprobe,theactualprobe.Theantisenseprobeistheexactreplicaofthe antisensestandofthedoublestranded(ds)DNAmolecule.Thisantisensestrandisacomplimentof thesensestrand,thegenesequence,ofthedsDNAmolecule;hencetheantisenseriboprobeisa complimentofthegenesequenceofmyozenin,ifthisgenesequenceispresentinthebraintissue theriboprobewillannealtoit.Inordertodetectthiscomplementationtheriboprobethatwemade wastaggedwithDigoxigenin(DIG)andthenduringthehybridizationprocessanantiDIGantibody conjugatedtoalkalinephosphatasewasadded.ThisantiDIGantibodybindstoDIGandthealkaline phosphatasecatalyzesareactionallowingforprobedetection. Unfortunately,onviewingoftheslides,signalwaspresentforboththesenseandantisenseprobes, suggestingperhapsthatfurtheroptimizationofthehybridizationconditionsmightberequired.IfI hadhadmoretimeIwouldhaverepeatedthisinsitutoachievemoresatisfactoryresults.Overall thoughourrtPCRresultsindicatethatmyozeninisexpressedinthebrain,albeitatlowlevels.Thus theinvestigationofmyozeninfunctioninthenervoussystemprobablywarrantsfurther investigation. Conclusions IwouldliketotakethisopportunitytothankTheBiochemicalSocietyforthistremendous,valuable andsometimeschallengingexperience.IsetoutnottowinTheNobelPrize,(yet!),buttolearn aboutanduseimportantandpopularlabtoolsandtechniquesandthatisexactlywhatIachieved. Molecularcloning,rtPCR,proteinexpressionandgelelectrophoresisaresomeofthemostusefullab techniquesthatIwilldefinitelyneedtouseinthefutureandaftermyeightweeksthatIwill certainlybeabletouse.DespitethefactthatIcouldntsuccessivelytagbothactinins,Ilearneda valuablelessonintheprocess;thatalifeinresearchhasitsupsanddowns,youhavetolearntobe patient,tryalternativeapproachesandjustkeepgoing.IknowthatifIhadhadmoretimeIwould havetaggedbothactininsandIwouldhavepurifiedtheheterodimers.Thisscholarshiphasnotbeen detrimentalinmyinterestofresearchbuthaswhetmyappetiteforit.Ilookforwardtopursuinga careerinscience. References DILLON,C.&GODA,Y.(2005)Theactincytoskeleton:integratingformandfunctionatthesynapse. AnnuRevNeurosci,28,2555. FAULKNER,G.,PALLAVICINI,A.,COMELLI,A.,SALAMON,M.,BORTOLETTO,G.,IEVOLELLA,C., TREVISAN,S.,KOJIC,S.,DALLAVECCHIA,F.,LAVEDER,P.,VALLE,G.&LANFRANCHI,G.(2000) FATZ,afilamin,actinin,andtelethoninbindingproteinoftheZdiscofskeletalmuscle.J BiolChem,275,4123442. FREY,N.,FRANK,D.,LIPPL,S.,KUHN,C.,KOGLER,H.,BARRIENTOS,T.,ROHR,C.,WILL,R.,MULLER,O. J.,WEILER,H.,BASSELDUBY,R.,KATUS,H.A.&OLSON,E.N.(2008)Calsarcin2deficiency increasesexercisecapacityinmicethroughcalcineurin/NFATactivation.JClinInvest,118, 3598608. FREY,N.,RICHARDSON,J.A.&OLSON,E.N.(2000)Calsarcins,anovelfamilyofsarcomeric calcineurinbindingproteins.ProcNatlAcadSciUSA,97,146327.

[INVESTIGATIONOFPOTENTIALFUNCTIONSOFMYOZENININ THECENTRALNERVOUSSYSTEMANDPURIFICATIONOF TheBiochemicalSociety ACTININHETERODIMERS] GACHE,Y.,LANDON,F.&OLOMUCKI,A.(1984)Polymorphismofalphaactininfromhumanblood platelets.Homodimericandheterodimericforms.EurJBiochem,141,5761. HONDA,K.,YAMADA,T.,ENDO,R.,INO,Y.,GOTOH,M.,TSUDA,H.,YAMADA,Y.,CHIBA,H.& HIROHASHI,S.(1998)Actinin4,anovelactinbundlingproteinassociatedwithcellmotility andcancerinvasion.JCellBiol,140,138393. OTEY,C.A.&CARPEN,O.(2004)Alphaactininrevisited:afreshlookatanoldplayer.CellMotil Cytoskeleton,58,10411. SMILLIE,K.J.&COUSIN,M.A.(2005)DynaminIphosphorylationandthecontrolofsynapticvesicle endocytosis.BiochemSocSymp,8797. TAKADA,F.,VANDERWOUDE,D.L.,TONG,H.Q.,THOMPSON,T.G.,WATKINS,S.C.,KUNKEL,L.M.& BEGGS,A.H.(2001)Myozenin:analphaactininandgammafilaminbindingproteinof skeletalmuscleZlines.ProcNatlAcadSciUSA,98,1595600. WALIKONIS,R.S.,OGUNI,A.,KHOROSHEVA,E.M.,JENG,C.J.,ASUNCION,F.J.&KENNEDY,M.B. (2001)Densin180formsaternarycomplexwiththe(alpha)subunitofCa2+/calmodulin dependentproteinkinaseIIand(alpha)actinin.JNeurosci,21,42333. WAUGH,D.S.(2005)Makingthemostofaffinitytags.TrendsBiotechnol,23,31620. YAMAMOTO,S.,TSUDA,H.,HONDA,K.,KITA,T.,TAKANO,M.,TAMAI,S.,INAZAWA,J.,YAMADA,T. &MATSUBARA,O.(2007)Actinin4expressioninovariancancer:anovelprognostic indicatorindependentofclinicalstageandhistologicaltype.ModPathol,20,127885.

Anna Woroniuk Biochemical Society Studentship Report 2011Supervisor: Dr Simon Cook, The Babraham Institute THE EFFECT OF AZD6244 REMOVAL ON THE PROLIFERATION AND SURVIVAL OF AZD6244-RESISTANTCOLORECTAL CANCER CELL LINES

BACKGROUND AND PROJECT AIMS: AZD6244 is a novel MEK1/2 inhibitor currently in phase I/II clinical trials. AZD6244 is ATP uncompetitive, binding to an allosteric pocket close to the kinase active site and thereby inhibiting signalling through the RAF-MEK-ERK pathway. Tumour cell lines with activating BRAF or KRAS mutations are highly dependent on the RAF-MEK-ERK pathway for proliferation and survival; therefore the application of AZD6244 can trigger cell cycle arrest and/or apoptosis. However prolonged exposure of tumour cells to AZD6244 results in the emergence of cells with acquired resistance. Little et al (2011) determined that the mechanism through which acquired resistance develops is the intrachromosomal amplification of the driving oncogene; BRAF or KRAS. Higher expression levels of BRAF or KRAS can activate more MEK1/2 to overcome inhibition and promote increased ERK activation, cell proliferation and survival. However, despite BRAF/KRAS amplification, AZD6244 resistant tumour cells re-model the ERK1/2 pathway to maintain parental levels of active P-ERK1/2 even in the presence of drug. Long-term drug removal studies have shown the AZD6244-resistant COLO205 cells (C6244-R cells) lose the BRAF amplification and become re-sensitized to the anti-proliferative effects of the drug. This begs the question: why should BRAF600E be selected against following drug withdrawal? This project focused on examining the short-term effects of AZD6244 removal on resistant tumour cell lines, since it is known that too much ERK1/2 signalling can cause cell cycle arrest. EXPERIMENTAL PROTOCOL: AZD6244-resistant versions of three tumour cell lines (COLO205, HT29 and HCT116) were used (Little et al 2011). Results are shown for the COLO205/C6244-R cell pair. 72 hour AZD6244 removal time courses were implemented; medium was replaced at time 0 with drug-free medium and cells were harvested at 0,1,2,4,8,16,24,48 and 72 hour time points. In order to control for any stress induced by replacing medium, a plus-AZD6244 control was conducted in which the medium was replaced with fresh AZD6244-containing medium. Flow cytometric analysis was used to quantitate the sub-G1, G1, S and G2/M fractions of the cell population to indicate the effect of drug removal on cell cycle profile. Cell lysates were analysed using SDS-polyacrylamide gel electrophoresis and western blotting to determine the expression levels of cell cycle marker proteins and markers of apoptosis and autophagy. RESULTS: Cell cycle analysis - C6244-R cells Shortly after AZD6244 withdrawal (time 0) the G2/M fraction increased (2-8 hrs) as those cells in cycle progressed through G2 and M (Fig 1). This may have been facilitated by the increase in ERK1/2 activation as MEK1/2 inhibition was relieved (see below Fig 3). In addition, a pronounced and sustained G1 arrest was observed from 16 hours onwards to 72 hours after drug removal. The plus-AZD6244 control cells exhibited little significant trend over the time course (Fig 2), which indicated that any changes in cell cycle profile observed were due to the removal of AZD6244 not the simple change of medium. A similar trend in cell cycle profile (a pronounced G1 arrest after 16 hours) was observed when AZD6244 was removed from AZD6244-resistant HT29 cells. No trend was observed in the cell cycle analysis of AZD6244- resistant HCT116 cells.

Figure 1

Western Blot analysis - C6244-R cells: Western blotting was used to evaluate the effects of AZD6244 removal on the expression patterns of cell cycle markers and constituents of the RAF-MEK-ERK pathway (Fig 3). Levels of p-MEK1/2 are much higher in the resistant C6244-R cells compared to the parental cells due to the amplification of the driving oncogene, BRAF600E. ERK1/2 was rapidly and very strongly activated within 1 hour of drug removal and this was sustained for the full 72 hours of the time-course. +AZD6244 media had no effect. An increase in the expression of the CDK inhibitors p21 and p16 was observed from 2 hours after AZD6244 removal onwards, correlating with the timings of the increase in G2/M and G1 arrest. This suggests that increased CDK inhibitor expression may have a role in mediating the cell cycle arrest observed following AZD6244 removal. BIMEL and p62 were examined as markers of apoptosis and autophagy respectively. Removal of AZD6244 produced a characteristic shift in the BIMEL band, and a decrease in its abundance, consistent with the ability of ERK1/2 to promote the phosphorylation of BIM and its proteasomal degradation, which may act to prevent apoptosis. The blots also showed that resistant cell lines can tolerate higher basal levels of BIM than the parental cell line. p62 levels gradually increased which may indicate an induction of autophagy.

Figure 2

FUTURE WORK: Figure 3 Further extension of these initial experiments could include: Evaluation of the role of p21 in the G1 cell cycle arrest through RNAi knockdown of p21 in AZD6244-resistant cell populations. The ability of the RNAi knockdown to rescue the arrest observed might implicate p21 in mediating the cell cycle arrest. Thymidine incorporation experiments to measure the total proliferation of cells over the time course and extended time courses. Proliferation rates may indicate whether the increased G2/M fraction represents a burst of proliferation or G2/M arrest. Markers of senescence and autophagy could also be examined. Further reversion experiments with AZD6244-resistant HCT116 and HT29 cell lines could be carried out to determine the capacity of these cell lines for re-sensitization to the anti-proliferative effects of the drug. Comparison of the results of both the long-term and short-term drug removal time courses could indicate any correlation between the cell cycle arrest and the reversibility of AZD6244 resistance. DEVIATIONS FROM ORIGINAL PROPOSAL: Time did not permit the validation of dual-specificity phosphatise RNAi knock-downs in AZD6244-resistant cell lines. OUTCOMES AND VALUES OF THE STUDENTSHIP: I am very grateful to both the Cook laboratory and the Biochemical Society for providing me with the opportunity to gain invaluable practical laboratory experience and skills and to develop my knowledge of the theoretical concepts associated with cell signalling in tumour cells. Undertaking the studentship has further cemented my intention to apply for a PhD after the completion of my degree within the cancer research field. The studentship has also enabled me to generate preliminary data which will contribute to an on-going project within the Cook laboratory. Comments by Dr Simon Cook: Anna worked hard and with great care, read widely around her subject and was popular with other lab members. Her placement has proved to be very successful for us. The data she has generated has been of a very high quality, has pump-primed a new project (as I hoped) and I expect it to form a figure in a future paper; if this proves to be the case she will receive co-authorship and the Biochemical Society will receive an acknowledgement. Many thanks to Anna and the Biochem Soc.

Ben Chappell Biochemical Society Studentship Report 2011

Orthogonally cleavable linkers in Chemical ProteomicsAims and objectivesChemical proteomics involves the study of the structures and functions of proteins via chemical techniques.1 Typical chemical proteomic approaches involve the selective introduction of a chemical handle, for instant an activity probe, to proteins of interest which then facilitates their separation from the rest of the proteome.2 Chemical proteomic techniques are extensively utilised within the Tate group to investigate the Nmyristoylation of proteins, with the ultimate aim of producing well understood therapeutic agents for opportunistic infections of humans by fungi and 3 parasitic protozoa. This post translational modification involves the attachment of the fatty acid, myristate, to the N-terminal glycine residue of the targeted proteins. of endogenously biotinylated proteins and hydrophobic proteins that bind non-specifically to the beads.6 An alternative to these forcing conditions invokes the use of an orthogonally cleavable linker and a proposed protocol is illustrated on the right.

Analogues of myristic acid bearing biologically inert alkyne tags are also tolerated by Nmyristoyltransferase (NMT), which provides a method of introducing a chemical handle to proteins of interest.3 After the cells fed by this myristic acid analogue have been lysed, the alkyne tag can be used to install a biotin moiety via a Cu(I) catalysed alkyneazide [3+2] cycloaddition or click capture reaction.4 Biotins strong affinity for streptavidin resin beads provides a method of separation of these proteins of 5 interest from the remainder of the lysate. In order to isolate these N-myristoylated proteins for further analysis, removal from the beads is required. Breaking the biotin-streptavidin interactions requires extremely forcing conditions, which often results in the co-elution

J. Cox and M. Mann, Cell 2007,130, 395 - 398 N. Jessani and B. Cravatt, Current Opinion in Chemical Biology 2004, 8, 54 - 59 3 M. Wright, W. Heal, D. Mann and E. Tate, J. Chem. Biol. 2010, 3, 19 - 35 4 N. Agard, J. Prescher, and C. Bertozzi, J. Am. Chem. Soc.2004, 126, 1504615047 5 M. Wilchek and E. Bayer, Methods in Enzymology 1990, 184, 5-13 2 1

6

R. Lue, G. Chen, Y. Hu, Q. Zhu and S. Yao, J. Am. Chem. Soc., 2004, 126, 10551062

O OH11

YNC12 YNC12 introduced to cell culture O11

Incorporation of tagged myristate into proteins of interest by PTM

N H N3

O

Cell lysis followed by capture of alkyne tags via 'click reaction'

Cat. Cu(I)

N N N

O11

Lysate incubated with streptavidin beads

O Captured proteins stick to resin

N H

N N N Cleavage of linker

O11

N H

O

N N N

O11

N H

O

KEY Biotin Streptavidin beads Cleavable linker

Design and synthesis of the linkerA Levulinoyl ester based linker has recently been employed in activity-based protein profiling.7 It was

believed that this linker could be incorporated within a capture reagent for the proposed protocol.

P. Geurink, B. Florea, N. Li, M. Witte, J. Verasdonck, C. Kuo, G. van der Marel, H Overkleeft, Angew. Chemie 2010, 122, 6954 - 6957 7

1

2

3

4

The linker is cleaved by hydrazine as shown below.iPr O N3 O O iPr NH2NH2 iPr N3 N N H O HO iPr

1)MyristicacidfedHelalysate,Tamracapturereagent.2)YNC12fedHela lysate,Tamracapturereagent.3)MyristicacidfedHelalysate,Levulinoyl estercapturereagent.4)YNC12fedHelalysate,Levulinoylestercapture reagent.

The work carried out regarding the synthesis of the capture reagent is described at the end of this report in the Appendix.

The next task was to establish that the linker could be cleaved and N-myristoylated proteins could be released from the streptavidin beads. Unfortunately when Western Blotting against MARCKS (a protein known to be N-myristoylated)3 was performed no captured MARCKS was detected with either capture reagent. This observation was attributed to a low concentration of tagged MARCKS within the lysate, which was below the detection limit of the experiment. However, blotting against histadine tags in a lysate where the protein pARF was overexpressed gave a much more encouraging result.

Chemical proteomics using the Levulinoyl ester capture reagentWith the new capture reagent in hand, its effectiveness could now be tested. It was imperative to first ensure that alkyne tagged lysates could still be captured with the new reagent in the click reaction. Hela cells were cultured and split. One culture was fed with myristic acid containing media, whereas the other was fed with YNC12 acid containing media. The cells were then lysed. The click capture reaction was then performed on both myristic acid and YNC12 fed lysates using both the new capture reagent and the established TAMRA capture reagent used internally within the Tate group.8 Anti-biotin Western blotting confirmed that the new capture reagent could still capture alkyne tagged proteins as reflected by the lower molecular weight signals in lane 4.

1 2

3

4

5

6 7

8 9 10 11

1) All blue protein ladder. 2) MA fed, after click. 3) YNC12 fed, after click. 4) MA fed, supernatant from pull down onto beads. 5) YNC12 fed, supernatant from pull down onto beads. 6) MA fed, hydrazine treatment. 7) YNC12 fed, hydrazine treatment. 8) MA fed, 0.2% SDS in PBS wash. 9) YNC12 fed, 0.2% SDS in PBS wash. 10) MA fed, beads boiled in 2% SDS in PBS for 10 min. 11) YNC12 fed, beads boiled in 2% SDS in PBS for 10 min.

Lane 7 indicated that cleavage was successful and that the cleavage conditions were leaving proteins that either bound non-specifically or were endogenously biotinylated unaffected as shown by the presence of signals in lanes 10 and 11.

8 W. Heal,B. Jovanovic, S. Bessin, M. Wright, A. Mageebc and E. Tate, Chem. Commun. 2011, 47, 40814083

Further work

Further experiments regarding this capture reagent are required before it can be definitively proven that the reagent is working as envisaged. However the results gained thus far are certainly very promising. Work is also planned to modify the linker to make it compatible with SPPS techniques.O O O N H iPr O O O iPr OH

protein side of the linker, allowing proteins of interest to be visualised by in gel fluorescence.

Value of the studentshipFrom the placement, the group has been given an encouraging result upon which to base future work in this area. A number of intermediates have also been synthesised to support this endeavour. As for myself, this placement has introduced me to a new, varied and exciting area of chemistry. Initially, I was a little daunted at the prospect of needing to learn the new practical techniques and language required to work within Chemical Biology. However, after completing this studentship, learning some of the techniques and the language, Ive realised that a limited Biology background is not necessarily an insurmountable problem. Making my own tools using chemical synthesis and then going on to use them to attempt to understand Biology was very rewarding and something I would be very interested in doing for both my final year Masters project and a PhD afterwards.

This material can be made in a few steps from acid 9, of which 1.8 g was made available as a result of this placement. The successful synthesis of this material would allow the development of a 2nd generation Leuvinolic ester based capture reagent that contains a fluorophore. Upon cleavage of the 2nd generation capture reagent, the fluorophore would remain on the

Appendix: Synthetic SchemeO EtO P EtO BnO CHO THF rt 72% 1 2 O OtBu BnO O OtBu MeOH rt 99% 3 H2 Pd/C BnO O OtBu

NaH

EtO2C

CO2Et Ethylene Glycol EtO2C PPTS O 4 Toluene Dean Stark Reflux 91% O 5 O

i) KOH, EtOH, 50 C CO2Et ii) EtOCOCl, Et3N, THF, rt iii) NaBH4, THF, H2O, rt 31%

OH CO2Et

O 6 CH2Cl2 quant.

O

MsCl, Et3N

N3 CO2H HCl O 10 3 DIC DMAP CH2Cl2 rt THF rt quant.

N3 CO2H NaOH O 9 O MeOH rt quant.

N3 CO2Et NaN3 O 8 O DMF 75 C 93%

OMs CO2Et

O 7

O

O iPr O OtBu TFA CH2Cl2 rt 26% (Over 2 steps) N3 O 12 iPr O O iPr

O OH

N3 O 11

O iPr

Acid 12 was to be coupled to amino-PEG-biotin under PyBOP/DiPEA conditions to provide the final capture reagent, unfortunately this could not be performed within the timeframe of the placement. However, a small quantity (~ 10 mg) of the succinimide ester of acid 12 was made available at the start of the project. Amino-PEG-biotin was directly coupled with this succinimide ester to provide 1.2 mg of the final capture reagent. This material was used in the chemical proteomics experiments.

Page |1

BenWades BiochemicalSocietyStudentshipReport2011

Understanding the role of lipids in the regulation of glycsyltransferase localisation and its implications in muscular dystrophyBenjamin Owen WadeIntroduction Muscular dystrophy (MD) is a complex disease with many facets, however they all exhibit a breakdown of the interaction between the extracellular matrix (ECM) and muscle fibres, typically due to a lack of integrity in the dystroglycan complex. The dystroglycanopathies are one such family of muscular dystrophys where aberrant glycosylation has been shown to disrupt the integrity of the dystroglycan complex. One member of this family, Fukuyama muscular dystrophy, has been linked to the miss-localisation of a protein Fukutin-1 (FK1) which is normally found in the Golgi Apparatus (GA) and is vital for the O-linked glycosylation of the dystroglycan complex1. Recent studies have shown that the localisation of this protein to the GA is mediated by its transmembrane domain and its interactions with the surrounding lipid2. However a molecular understanding of this process is far from clear. Currently there are two main models that have been proposed; the lipid sorting model and the oligomerization model3-6. To understand how the protein responds to changes in lipid bilayer composition we are using solid-state NMR to investigate the orientation of the protein and how it responds to changes in lipid composition. A pre-requisite for these studies is the introduction of 15N labels into the peptide backbone, reconstitution into the lipid bilayer and the subsequent preparation of oriented bilayer samples. Material and methods Protein purification and expression The expression and purification of the peptide FK1TMD was carried out by the procedure present in P.Marius et al.7. Briefly 10ml starter cultures were grown at 37C for 8 hours in LB broth and ampicillin (100g/mL). The cultures were then expanded to 100ml in M9 minimal media containing ampicillin (100g/mL) and 15N labelled ammonium chloride (1g/L). These were left to grow for 16 hours. They were then expanded to 1 litre cultures and left until an OD660 of 0.6 was reached. Protein expression was then induced by the addition of isopropyl--D-thiogalactopyranoside (IPTG) to a final concentration of 1mM. Bacteria were cultured for a further 4 hours. Bacteria were then harvested by centrifugation (5000rpm for 20min, 4C). Purification was carried out by re-suspending the pellet in 70mls of PBS and 1mM PMSF. The samples were sonicated to break open the cells, whilst cooling the samples on ice. After sonication we obtained the membrane fraction by ultracentrifugation (35,000rpm for 35min 4C). This pellet was solubilised in PBS, 50mM imidazole and 15mM DDM for 1 hour while stirring. The sample was clarified by centrifugation (13,000rpm for 15 min, 4C). The supernatant was then loaded onto pre-equilibrated Ni-NTA column (GE Healthcare, UK). The column was then washed with 200mM imdazole and the protein eluted with 600mM imdazole. Protein purification was monitored by SDS page. Oriented bilayers Oriented bilayers of deuterated Palmitoyl-2-oleoyl-sn-glycerol3-phosphocholine (POPC-d31) were prepared from both a vesicle suspension and a methanolic solution. In both cases glass slides were cleaned using the standard procedure of washing in strong acid and then methanol. Methanol alignment was carried out by the following method. A solution of the concentration of 10mg/ml of POPC-d31 dissolved in methanol was made up. This solution was then added to the glass plates which were then left to dry until the methanol had evaporated. The glass plates were then placed in a vacuum overnight to remove any methanol. They were then re-hydrated and 10 glass plates stacked on top of each other and loaded into a 5mm NMR tube. To incorporate the protein into the membrane, the lipids were co-solubilised with the peptide in methanol at a ratio of 100:1 and aligned on the glass plates as described above. Solid state NMR All solid-state NMR measurements were made on a 300 MHz Chemagnetics Infinity+ using a 5mm double resonance static probe at 25C. The data was acquired using a standard quadrupole echo sequence with 5 s pulses and an interpulse delay of 50 s. Typically 64000 acquisitions were accumulated and prior to Fourier transform, 200 Hz line broadening was applied. Results Protein expression and purification

Benjamin Owen Wade

Protein localisation

01/09/2011

Page |2 Analysis of the supernatant following sonication (SN1) and solubilisation (SN2) reveal a ladder, as expected for the release of soluble and membrane proteins respectively. No distinct band is visible for the FK1TMD due to its low expression levels. Fractions eluted from the Ni-affinity column (Lanes 1 to 10) contain little protein whilst in Lanes 7-10 a diffuse band appears at 4 kDa which corresponds to molecular weight of the FK1TMD Solid state NMR The structure and orientation of the FK1TMD in its native environment, the lipid bilayer, can be determined through solidstate NMR studies of aligned biomembranes. We measured the 0 and 90 deuterium spectrum of orientated POPC-d31 with and without the protein reconstituted into the membrane, Figure 2. This enabled us to ascertain the optimal conditions for the preparation of aligned bilayer and ensure that the incorporation of the protein did not disrupt the bilayer integrity and orientation. The spectra of the aligned samples are shown in Figure 2 and demonstrate that the samples are well-oriented. The spectra are symmetrical with each pair of peaks representing one of the CD2 groups on the fatty acid chain. The quadrupole splitting is smaller for the groups which are at the end of the chain as they are more disordered. The orientation dependence of the quadrupolar coupling is8. Figure 1 Shows the SDS-PAGE of the purification of labelled FK1TMD (Gel stained with Sypro Orange).M 1 2 3 4 5 6 7 8 9 10 Sn1 SN2 SN3

Analysis to the spectra of lipid bilayer with FK1TMD reconstituted into the bilayer reveals (Figure 2C/D) also show a similar high degree of orientation. This means that at a lipid to protein r