Dr Victor Bolanos-Garcia
PhD. MSc, BSc, FHEA-UK
Senior Lecturer in Clinical Biochemistry
Department of Biological and Medical Sciences

Role
Senior Lecturer and Principal Investigator. Leader of the research group "Mechanisms of Regulation of Cell Division".
Teaching and supervision
Modules taught
Undergraduate Courses:
- Introduction to Biochemistry A and B;
- Biochemistry of Cell Function;
- Evidence-based Medicine;
- Clinical Biochemistry and Pharmacology;
- 3rd year Research Project supervisor.
Postgraduate courses:
- Advanced Molecular Techniques;
- Genome Sciences;
- Clinical Genetics and Diagnostics;
- Advances in Medical Genetics.
Since 2012, I have been the Module Lead of the undergraduate courses Introduction to Biochemistry A and Introduction to Biochemistry A and B. Module lead of the postgraduate course Genome Sciences.
Supervision
Since I joined Oxford Brookes University I have acted as the first supervisor of 3 PhD students:
- Natalie L. Curtis, 2017-2021;
- Maria Kapanidou, 2013-2017;
- Dave Gervais, 2014-2016.
As a second PhD supervisor:
- Samuel Connelly, 2016-2020;
- Teresa Minguez Vinas, 2016-2020;
- Brittany Almond, 2014-2018.
I also supervise MSc students that choose to do their research projects in my group.
I have provided day to day co-supervision to a number of PhD students of the University of Cambridge and elsewhere. To name a few:
- Qian Wu, 2009-2012, University of Cambridge;
- Takashi Ochi, 2008-2012, University of Cambridge;
- Deepti Gupta. 2008-2011, University of Cambridge;
- Ann Ling, 2008-2011, University of Cambridge;
- Lionel Chieze, 2007-2010, University of Rennes, France;
- Sheena D'Arcy, 2006-2010, University of Cambridge.
Research
I am a biochemist, structural biologist and cancer researcher who trained with Francisco Bolívar-Zapata (the co-creator of the plasmid pBR322, one of the first widely used E. coli cloning vectors, and a member of the pioneer team at UCSF that for the first time overproduced human proteins such as insulin and somatostatin in bacteria using genetic engineering techniques) at the National Autonomous University of Mexico (UNAM); Ermanno Gherardi at the Laboratory of Molecular Biology (LMB)-of the Medical Research Council (MRC) and Sir Tom Blundell at the University of Cambridge. As a Wellcome Trust International Research Fellow, I studied the mechanisms of regulation of cell proliferation and motility by the MET kinase receptor in collaboration with Gherardi and Blundell, which was followed by the study of cell signaling systems implicated in DNA damage and repair. The research experience acquired on the latter signalling systems prompted me to initiate work on the molecular mechanisms of regulation of cell division in higher organisms. Cell division is a critical biological event that is tightly regulated. In the human, defects in the process result in genome instability, birth and developmental defects, spontaneous miscarriage, cancer, and premature ageing. In essence, research in my group aims to understand how different signals in the cell work together to ensure accurate cell division. We are equally interested in the development of innovative therapeutics, ranging from small size compounds and peptides that bind to specific protein targets to nanomaterials than induce tumour cells sensitisation, in an attempt to tackle human malignancies associated with defects in cell division. To this aim we integrate protein biochemistry, protein biophysics and structure-guided drug development methods with medicinal chemistry, cellular biology and preclinical validation approaches.
Ultimately, understanding the dynamics and the structural features of the molecular interactions underpinning the control of cell division will open up new opportunities for improving the quality of life of an ever growing population and therefore contribute to reducing pressure on social and healthcare systems.
Research impact
Over the past two decades my group has accumulated an extensive body of work on the definition of the structure and function of key protein regulators of cell division. Also, using a structure-guided approach, we have identified and validated new drug targets to interfere with cell division in cancer cells. Highlights of recent contributions in this research area include:
- the identification and pre-clinical validation of new specific ligands of key mitosis regulators to interfere with aberrant cell division in tumour cells (unpublished data);
- development and validation of functionalised chitosan nanoparticles for the radiosensitisation of breast cancer cells (Biomolecules 2019);
- in collaboration with the Elowe lab, we discovered a novel mechanism of regulation of the central mitotic checkpoint kinase Mps1, which has opened up a new widow for therapeutic intervention of cancer (Curr Biol 2018);
- in collaboration with the Nilsson lab, we have identified and functionally characterised a new binding motif in the pseudokinase BubR1. The newly defined protein-protein interface has potential to be exploited to interfere with premature ageing (Trends Mol Medicine 2015 and Nature Comm 2014);
- first description of the function and structure of the conserved N-terminal domain of the mitotic checkpoint kinases Bub1, BubR1 and Mps1 (Biochem J 2012; J Biol Chem 2010; Structure 2009).
We also have generated materials, methods, protocols and data that are used by other researchers around the world. A fraction of our research output has been used as a model case and/or primary citation in manuals and catalogues of well-established commercial suppliers of reagents and biological materials including GE Healthcare, New England Biolabs, Hampton Research, Bruker AXS, Molecular Dimensions, and Jena Bioscience.
Research in our group continues to provide a valuable opportunity for high-quality training and transference of a wide range of high-level skills to the new generation of researchers thus contributing in the long term to the competitiveness of these important bioscience industries in the UK.
Groups
Projects as Principal Investigator, or Lead Academic if project is led by another Institution
- Inhibition of cell division to treat breast cancer of poor prognosis (27/09/2021 - 31/03/2022), funded by: Technology Strategy Board (Innovate UK), funding amount received by Brookes: £29,642
Publications
Journal articles
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Morales P, Curtis NL, Zárate SG, Bastida A, Bolanos-Garcia VM, 'Interfering with mRNA Methylation by the 2′O-Methyltransferase (NSP16) from SARS-CoV-2 to Tackle the COVID-19 Disease'
Catalysts 10 (9) (2020)
ISSN: 2073-4344AbstractPublished here Open Access on RADARThe pandemic associated to Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) has resulted in a huge number of deaths and infected people. Although several vaccine programmes are currently underway and have reached phase 3, and a few small size drugs repurposed to aid treatment of severe cases of COVID-19 infections, effective therapeutic options for this disease do not currently exist. NSP16 is a S-adenosyl-L-Methionine (SAM) dependent 2′O-Methyltransferase that converts mRNA cap-0 into cap-1 structure to prevent virus detection by cell innate immunity mechanisms. NSP16 methylates the ribose 2′O-position of the first nucleotide of the mRNA only in the presence of an interacting partner, the protein NSP10. This feature suggests that inhibition of the NSP16 may represent a therapeutic window to treat COVID-19. To test this idea, we performed comparative structural analyses of the NSP16 present in human coronaviruses and developed a sinefungin (SFG) similarity-based virtual screening campaign to assess the druggability of the SARS-CoV-2 NSP16 enzyme. Through these studies, we identified the SFG analogue 44601604 as a promising more potent inhibitor of NSP16 to limit viral replication in infected cells, favouring viral clearance.
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Curtis NL, Ruda GF, Brennan P, Bolanos-Garcia VM, 'Deregulation of Chromosome Segregation and Cancer'
Annual Review of Cancer Biology 4 (2020) pp.257-278
ISSN: 2472-3428AbstractPublished here Open Access on RADARThe mitotic spindle assembly checkpoint (SAC) is an intricate cell signaling system that ensures the high fidelity and timely segregation of chromosomes during cell division. Mistakes in this process can lead to the loss, gain, or rearrangement of the genetic material. Gross chromosomal aberrations are usually lethal but can cause birth and development defects as well as cancer. Despite advances in the identification of SAC protein components, important details of the interactions underpinning chromosome segregation regulation remain to be established. This review discusses the current understanding of the function, structure, mode of regulation, and dynamics of the assembly and disassembly of SAC subcomplexes, which ultimately safeguard the accurate transmission of a stable genome to descendants. We also discuss how diverse oncoviruses take control of human cell division by exploiting the SAC and the potential of this signaling circuitry as a pool of drug targets to develop effective cancer therapies.
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Piña Olmos S, Díaz Torres R, Elbakrawy E, Hughes L, Mckenna J, Hill MA, Kadhim M, Ramírez Noguera P, Bolanos-Garcia VM, 'Combinatorial Use of Chitosan Nanoparticles, Reversine, and Ionising Radiation on Breast Cancer Cells Associated with Mitosis Deregulation.'
Biomolecules 9 (5) (2019)
ISSN: ISSN 2218-273XAbstractPublished here Open Access on RADARBreast cancer is the most commonly occurring cancer in women worldwide and the second most common cancer overall. The development of new therapies to treat this devastating malignancy is needed urgently. Nanoparticles are one class of nanomaterial with multiple applications in medicine, ranging from their use as drug delivery systems and the promotion of changes in cell morphology to the control of gene transcription. Nanoparticles made of the natural polymer chitosan are easy to produce, have a very low immunogenic profile, and diffuse easily into cells. One hallmark feature of cancer, including breast tumours, is the genome instability caused by defects in the spindle-assembly checkpoint (SAC), the molecular signalling mechanism that ensures the timely and high-fidelity transmission of the genetic material to an offspring. In recent years, the use of nanoparticles to treat cancer cells has gained momentum. This is in part because nanoparticles made of different materials can sensitise cancer cells to chemotherapy and radiotherapy. These advances prompted us to study the potential sensitising effect of chitosan-based nanoparticles on breast cancer cells treated with reversine, which is a small molecule inhibitor of Mps1 and Aurora B that induces premature exit from mitosis, aneuploidy, and cell death, before and after exposure of the cancer cells to X-ray irradiation. Our measurements of metabolic activity as an indicator of cell viability, DNA damage by alkaline comet assay, and immunofluorescence using anti-P-H3 as a mitotic biomarker indicate that chitosan nanoparticles elicit cellular responses that affect mitosis and cell viability and can sensitise breast cancer cells to X-ray radiation (2Gy). We also show that such a sensitisation effect is not caused by direct damage to the DNA by the nanoparticles. Taken together, our data indicates that chitosan nanoparticles have potential application for the treatment of breast cancer as adjunct to radiotherapy.
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Fernando Rodríguez,Carla Vallejos,Víctor M. Bolanos-Garcia,Diana Ponce, Nancy Unanue, Francisco Garay, Fernando Cassorla,Mariana Aracena., 'Co-occurrence of Noonan and Cardio-facio-cutaneous syndrome features in a patient with KRAS variant'
Journal of Pediatric Genetics 7 (4) (2018) pp.158-163
ISSN: 2146-4596 eISSN: 2146-460XAbstractWe report the case of a 3-year-old girl, who is the third child of nonconsanguineous parents with short stature, hypertrophic cardiomyopathy and mild dysmorphic features; all suggestive of Noonan syndrome. In addition, the patient presents with feeding difficulties, deep palmar and plantar creases, sparse hair, and delayed psychomotor and language development, all characteristics frequently observed in Cardio-facio-cutaneous syndrome. Molecular analysis of the Ras/MAPK pathway genes using high resolution melting curve analysis and gene sequencing revealed a de novo KRAS amino acid substitution of leucine to tryptophan at codon 53 (p.L53W). This substitution was recently described in an Iranian patient with Noonan syndrome. The findings described in the present report expand the phenotypic heterogeneity observed in RASopathy patients harbouring a KRAS substitution, and advocate for the inclusion of genes with low mutational frequency in genetic screening protocols for Noonan syndrome and other RASophaties.Published here Open Access on RADAR -
Guillaume Combes, Helena Barysz, Chantal Garand, Luciano Gama Braga,Ibrahim Alharbi,Philippe Thebault, Luc Murakami, Dominic P. Bryne, Stasa Stankovic, Patrick A. Eyers, Victor M. Bolanos-Garcia, William C. Earnshaw, John Maciejowski, Prasad V. Jallepalli, and Sabine Elowe, 'Mps1 Phosphorylates Its N-Terminal Extension to Relieve Autoinhibition and Activate the Spindle Assembly Checkpoint'
Current Biology 28 (6) (2018) pp.872-883.e5
ISSN: 0960-9822AbstractMonopolar spindle 1 (Mps1) is a conserved apical kinase in the spindle assembly checkpoint (SAC) that ensures accurate segregation of chromosomes during mitosis. Mps1 undergoes extensive auto- and transphosphorylation, but the regulatory and functional consequences of these modifications remain unclear. Recent findings highlight the importance of intermolecular interactions between the N-terminal extension (NTE) of Mps1 and the Hec1 subunit of the NDC80 complex, which control Mps1 localization at kinetochores and activation of the SAC. Whether the NTE regulates other mitotic functions of Mps1 remains unknown. Here, we report that phosphorylation within the NTE contributes to Mps1 activation through relief of catalytic autoinhibition that is mediated by the NTE itself. Moreover, we find that this regulatory NTE function is independent of its role in Mps1 kinetochore recruitment. We demonstrate that the NTE autoinhibitory mechanism impinges most strongly on Mps1- dependent SAC functions and propose thatMps1 activation likely occurs sequentially through dimerization of a ‘‘prone-to-autophosphorylate’’ Mps1 conformer followed by autophosphorylation of the NTE prior to maximal kinase activation segment trans-autophosphorylation. Our observations underline the importance of autoregulated Mps1 activity in generation and maintenance of a robust SAC in human cells.Published here Open Access on RADAR -
Kapadinou M, Curtis NL, Bolanos-Garcia V, 'Cdc20: At the Crossroads between Chromosome Segregation and Mitotic Exit'
Trends in Biochemical Sciences 42 (3) (2017) pp.193-205
ISSN: 0968-0004 eISSN: 0167-7640AbstractReviewPublished here Open Access on RADAR -
Kapanidou M, Lee S, Bolanos-Garcia VM, 'BubR1 kinase: protection against aneuploidy and premature aging'
Trends in Molecular Medicine 21 (6) (2015) pp.364-372
ISSN: 1471-4914 eISSN: 1471-499XAbstractPublished here Open Access on RADARThe multidomain protein kinase BubR1 is a central component of the mitotic spindle assembly checkpoint (SAC), an essential self-monitoring system of the eukaryotic cell cycle that ensures the high fidelity of chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bi-oriented on the mitotic spindle. We discuss the roles of BubR1 in the SAC and the implications of BubR1-mediated interactions that protect against aneuploidy. We also describe the emerging roles of BubR1 in cellular processes that extend beyond the SAC, discuss how mice models have revealed unanticipated functions for BubR1 in the regulation of normal aging, and the potential role of BubR1 as therapeutic target for the development of innovative anticancer therapies
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Lischetti T, Zhang G, Sedgwick GG, Bolanos-Garcia VM, Nilsson J, 'The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing.'
Nature Communications 5 (2014)
ISSN: 2041-1723AbstractPublished here Open Access on RADARImproperly attached kinetochores activate the spindle assembly checkpoint (SAC) and by an unknown mechanism catalyse the binding of two checkpoint proteins, Mad2 and BubR1, to Cdc20 forming the mitotic checkpoint complex (MCC). Here, to address the functional role of Cdc20 kinetochore localization in the SAC, we delineate the molecular details of its interaction with kinetochores. We find that BubR1 recruits the bulk of Cdc20 to kinetochores through its internal Cdc20 binding domain (IC20BD). We show that preventing Cdc20 kinetochore localization by removal of the IC20BD has a limited effect on the SAC because the IC20BD is also required for efficient SAC silencing. Indeed, the IC20BD can disrupt the MCC providing a mechanism for its role in SAC silencing. We thus uncover an unexpected dual function of the second Cdc20 binding site in BubR1 in promoting both efficient SAC signalling and SAC silencing.
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Silva PM, Reis RM, Bolanos-Garcia VM, Florindo C, Tavares ÁA, Bousbaa H, 'Dynein-dependent transport of spindle assembly checkpoint proteins off kinetochores toward spindle poles'
FEBS Letters 588 (17) (2014) pp.3265-3275
ISSN: 0014-5793 eISSN: 1873-3468AbstractPublished hereA predominant mechanism of spindle assembly checkpoint (SAC) silencing is dynein-mediatedtransport of certain kinetochore proteins along microtubules. There are still conflicting data as towhich SAC proteins are dynein cargoes. Using two ATP reduction assays, we found that the coreSAC proteins Mad1, Mad2, Bub1, BubR1, and Bub3 redistributed from attached kinetochores to spin-dle poles, in a dynein-dependent manner. This redistribution still occurred in metaphase-arreste dcells, at a time when the SAC should be satisfie d and silenced. Unexpectedly, we found that a poolof Hec1 and Mis12 also relocalizes to spindle poles, suggesting KMN components as additionaldynein cargoes. The potential significance of these results for SAC silencing is discussed.
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Ghongane P, Kapanidou M, Asghar A, Elowe S, Bolanos-Garcia VM, 'The dynamic protein Knl1 - a kinetochore rendezvous'
Journal of Cell Science 127 (2014) pp.3415-3423
ISSN: 0021-9533 eISSN: 1477-9137AbstractPublished hereKnl1 (also known as CASC5, UniProt Q8NG31) is an evolutionarily conserved scaffolding protein that is required for proper kinetochore assembly, spindle assembly checkpoint (SAC) function and chromosome congression. A number of recent reports have confirmed the prominence of Knl1 in these processes and provided molecular details and structural features that dictate Knl1 functions in higher organisms. Knl1 recruits SAC components to the kinetochore and is the substrate of certain protein kinases and phosphatases, the interplay of which ensures the exquisite regulation of the aforementioned processes. In this Commentary, we discuss the overall domain organization of Knl1 and the roles of this protein as a versatile docking platform. We present emerging roles of the protein interaction motifs present in Knl1, including the RVSF, SILK, MELT and KI motifs, and their role in the recruitment and regulation of the SAC proteins Bub1, BubR1, Bub3 and Aurora B. Finally, we explore how the regions of low structural complexity that characterize Knl1 are implicated in the cooperative interactions that mediate binding partner recognition and scaffolding activity by Knl1.
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Lee S, Bolanos-Garcia VM, 'The dynamics of signal amplification by macromolecular assemblies for the control of chromosome segregation.'
Frontiers in Physiology 29 (5) (2014) pp.1-11
ISSN: 1664-042XAbstractPublished hereThe control of chromosome segregation relies on the spindle assembly checkpoint (SAC), a complex regulatory system that ensures the high fidelity of chromosome segregation in higher organisms by delaying the onset of anaphase until each chromosome is properly bi-oriented on the mitotic spindle. Central to this process is the establishment of multiple yet specific protein-protein interactions in a narrow time-space window. Here we discuss the highly dynamic nature of multi-protein complexes that control chromosome segregation in which an intricate network of weak but cooperative interactions modulate signal amplification to ensure a proper SAC response. We also discuss the current structural understanding of the communication between the SAC and the kinetochore; how transient interactions can regulate the assembly and disassembly of the SAC as well as the challenges and opportunities for the definition and the manipulation of the flow of information in SAC signaling.
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Tófoli de Araújo F, Bolanos-Garcia VM, Pereira CT, Sanches M, Oshiro EE, Ferreira RC, Chigardze DY, Barbosa JA, de Souza Ferreira LC, Benedetti CE, Blundell TL, Balan A, 'Structural and physiological analyses of the alkanesulphonate-binding protein (SsuA) of the citrus pathogen Xanthomonas citri'
PLoS ONE 8 (11) (2013)
ISSN: 1932-6203AbstractPublished hereThe uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. Until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the Xanthomonas genus, which encompasses several pathogenic species. In the present study, we characterised the alkanesulphonate uptake system (Ssu) of Xanthomonas axonopodis pv. citri 306 strain (X. citri), the etiological agent of citrus canker.
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Bolanos-Garcia VM, Nilsson J, Blundell TL, 'The architecture of the BubR1 tetratricopeptide tandem repeat defines a protein motif underlying mitotic checkpoint-kinetochore communication.'
BioArchitecture 2 (1) (2012) pp.23-27
ISSN: 1949-0992 eISSN: 1949-100XAbstractPublished here Open Access on RADARThe accurate and timely transmission of the genetic material to progeny during successive rounds of cell division is sine qua non for the maintenance of genome stability. Eukaryotic cells have evolved a surveillance mechanism, the mitotic spindle assembly checkpoint (SAC), to prevent premature advance to anaphase before every chromosome is properly attached to microtubules of the mitotic spindle. The architecture of the KNL1-BubR1 complex reveals important features of the molecular recognition between SAC components and the kinetochore. The interaction is important for a functional SAC as substitution of BubR1 residues engaged in KNL1 binding impaired the SAC and BubR1 recruitment into checkpoint complexes in stable cell lines. Here we discuss the implications of the disorder-to-order transition of KNL1 upon BubR1 binding for SAC signaling and propose a mechanistic model of how BUBs binding may affect the recognition of KNL1 by its other interacting partners.
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Scotti C, Sommi P, Pasquetto MV, Cappelletti D, Stivala S, Mignosi P, Savio M, Chiarelli LR, Valentini G, Bolanos-Garcia VM, Merrell DS, Franchini S, Verona ML, Bolis C, Solcia E, Manca R, Franciotta D, Casasco A, Filipazzi P, Zardini E, Vannini V, 'Cell-cycle inhibition by Helicobacter pylori L-asparaginase'
PLoS ONE 5 (11) (2012)
ISSN: 1932-6203AbstractPublished hereHelicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application.
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Lee S, Thebault P, Freschi L, Beaufils S, Blundell TL, Landry CR, Bolanos-Garcia VM, Elowe S, 'Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases'
Journal of Biological Chemistry 287 (8) (2012) pp.5988-6001
ISSN: 0021-9258AbstractPublished hereKinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.
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Chièze L, Bolanos-Garcia VM, Le Caër G, Renault A, Vié V, Beaufils S, 'Difference in lipid packing sensitivity of exchangeable apolipoproteins apoA-I and apoA-II: an important determinant for their distinctive role in lipid metabolism.'
BBA - Biochimica et Biophysica Acta 1818 (11) (2012) pp.2732-2741
ISSN: 0006-3002AbstractExchangeable apolipoproteins A-I and A-II play distinct roles in reverse cholesterol transport. ApoA-I interacts with phospholipids and cholesterol of the cell membrane to make high density lipoprotein particles whereas apolipoprotein A-II interacts with high density lipoprotein particles to release apolipoprotein A-I. The two proteins show a high activity at the aqueous solution/lipid interface and are characterized by a high content of amphipathic α-helices built upon repetition of the same structural motif. We set out to investigate to what extent the number of α-helix repeats of this structural motif modulates the affinity of the protein for lipids and the sensitivity to lipid packing. To this aim we have compared the insertion of apolipoproteins A-I and A-II in phospholipid monolayers formed on a Langmuir trough in conditions where lipid packing, surface pressure and charge were controlled. We also used atomic force microscopy to obtain high resolution topographic images of the surface at a resolution of several nanometers and performed statistical image analysis to calculate the spatial distribution and geometrical shape of apolipoproteins A-I and A-II clusters. Our data indicate that apolipoprotein A-I is sensitive to packing of zwitterionic lipids but insensitive to the packing of negatively charged lipids. Interestingly, apolipoprotein A-II proved to be insensitive to the packing of zwitterionic lipids. The different sensitivity to lipid packing provides clues as to why apolipoprotein A-II barely forms nascent high density lipoprotein particles while apolipoprotein A-I promotes their formation. We conclude that the different interfacial behaviors of apolipoprotein A-I and apolipoprotein A-II in lipidic monolayers are important determinants of their distinctive roles in lipid metabolism.
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Bolanos-Garcia VM, Wu Q, Ochi T, Chirgadze DY, Sibanda BL, Blundell TL, 'Spatial and temporal organization of multi-protein assemblies: achieving sensitive control in information-rich cell-regulatory systems'
Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 370 (1969) (2012) pp.3023-3039
ISSN: 1364-503X eISSN: 1471-2962AbstractPublished hereThe regulation of cellular processes in living organisms requires signalling systems that have a high signal-to-noise ratio. This is usually achieved by transient, multi-protein complexes that assemble cooperatively. Even in the crowded environment of the cell, such assemblies are unlikely to form by chance, thereby providing a sensitive regulation of cellular processes. Furthermore, selectivity and sensitivity may be achieved by the requirement for concerted folding and binding of previously unfolded components. We illustrate these features by focusing on two essential signalling pathways of eukaryotic cells: first, the monitoring and repair of DNA damage by non-homologous end joining, and second, the mitotic spindle assembly checkpoint, which detects and corrects defective attachments of chromosomes to the kinetochore. We show that multi-protein assemblies moderate the full range of functional complexity and diversity in the two signalling systems. Deciphering the nature of the interactions is central to understanding the mechanisms that control the flow of information in cell signalling and regulation.
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Thebault P, Chirgadze D, Dou Z, Blundell T, Elowes S, Bolanos-Garcia V, 'Structural and functional insights into the role of the N-terminal Mps1 TPR domain in the SAC (spindle assembly checkpoint)'
Biochemical Journal 448 (3) (2012) pp.321-328
ISSN: 0264-6021AbstractThe SAC (spindle assembly checkpoint) is a surveillance system that ensures the timely and accurate transmission of the genetic material to offspring. The process implies kinetochore targeting of the mitotic kinases Bub1 (budding uninhibited by benzamidine 1), BubR1 (Bub1 related) and Mps1 (monopolar spindle 1), which is mediated by the N-terminus of each kinase. In the present study we report the 1.8 Å (1 Å=0.1nm) crystal structure of the TPR (tetratricopeptide repeat) domain in the N-terminal region of human Mps1. The structure reveals an overall high similarity to the TPR motif of the mitotic checkpoint kinases Bub1 and BubR1, and a number of unique features that include the absence of the binding site for the kinetochore structural component KNL1 (kinetochore-null 1; blinkin), and determinants of dimerization. Moreover, we show that a stretch of amino acids at the very N-terminus of Mps1 is required for dimer formation, and that interfering with dimerization results in mislocalization and misregulation of kinase activity. The results of the present study provide an important insight into the molecular details of the mitotic functions of Mps1 including features that dictate substrate selectivity and kinetochore docking.Published here -
Ochi T, Wu Q, Chirgadze D Y, Günter Grossmann J, Bolanos-Garcia V M, Blundell T L, 'Structural insights into the role of domain flexibility in human DNA ligase IV'
Structure 20 (7) (2012) pp.1212-1222
ISSN: 0969-2126AbstractKnowledge of the architecture of DNA ligase IV (LigIV) and interactions with XRCC4 and XLF-Cernunnos is necessary for understanding its role in the ligation of double-strand breaks during nonhomologous end joining. Here we report the structure of a subdomain of the nucleotidyltrasferase domain of human LigIV and provide insights into the residues associated with LIG4 syndrome. We use this structural information together with the known structures of the BRCT/XRCC4 complex and those of LigIV orthologs to interpret small-angle X-ray scattering of LigIV in complex with XRCC4 and size exclusion chromatography of LigIV, XRCC4, and XLF-Cernunnos. Our results suggest that the flexibility of the catalytic region is limited in a manner that affects the formation of the LigIV/XRCC4/XLF-Cernunnos complex.Published here -
Simpson PJ, Cota E, Bolanos-Garcia VM, '¹H, ¹³C and ¹⁵N resonance assignments of the kinetochore localisation domain of BUBR1, a central component of the spindle assembly checkpoint.'
Biomolecular NMR Assignments 6 (1) (2011) pp.115-118
ISSN: 1874-2718AbstractPublished hereHuman BUBR1 is a 120 kDa protein that plays a central role in the spindle assembly checkpoint (SAC), the evolutionary conserved and self-regulatory system of higher organisms that monitors and repairs defects in chromosome segregation in mitotic cells. BUBR1 is organised into several domains, with an N-terminal region responsible for its localisation into the kinetochore, the multi-component proteinaceous network that assembles onto chromosomes upon mitotic entry. We have expressed and purified uniformly-15N/13C N-terminal BUBR1 and assigned backbone and side-chain resonances bound to an unlabelled peptide from the protein Blinkin, an element essential for recruitment of BUBR1 to the kinetochore. These assignments provide insights on the Blinkin interaction interface and form the basis of the three-dimensional structure determination of a BUBR1-Blinkin complex.
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Bolanos-Garcia VM, Blundell TL, 'BUB1 and BUBR1: multifaceted kinases of the cell cycle'
Trends in Biochemical Sciences 36 (3) (2011) pp.141-150
ISSN: 0968-0004 eISSN: 0167-7640AbstractPublished hereThe multidomain protein kinases BUB1 and BUBR1 (Mad3 in yeast, worms and plants) are central components of the mitotic checkpoint for spindle assembly (SAC). This evolutionarily conserved and essential self-monitoring system of the eukaryotic cell cycle ensures the high fidelity of chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bi-oriented on the mitotic spindle. Despite their amino acid sequence conservation and similar domain organization, BUB1 and BUBR1 perform different functions in the SAC. Recent structural information provides crucial molecular insights into the regulation and recognition of BUB1 and BUBR1, and a solid foundation to dissect the roles of these proteins in the control of chromosome segregation in normal and oncogenic cells.
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Przewloka M R, Venkei Z, Bolanos-Garcia V M, Debski J, Dadlez M, Glove D M, 'CENP-C is a structural platform for kinetochore assembly'
Current Biology 21 (5) (2011) pp.399-405
ISSN: 0960-9822Published here -
Chièze L, Bolanos-Garcia VM, Pinot M, Desbat B, Renault A, Beaufils S, Vié V, 'Fluid and condensed ApoA-I/phospholipid monolayers provide insights into ApoA-I membrane insertion'
Journal of Molecular Biology 410 (1) (2011) pp.60-76
ISSN: 0022-2836AbstractPublished hereApolipoprotein A-I (ApoA-I) is a protein implicated in the solubilization of lipids and cholesterol from cellular membranes. The study of ApoA-I in phospholipid (PL) monolayers brings relevant information about ApoA-I/PL interactions. We investigated the influence of PL charge and acyl chain organization on the interaction with ApoA-I using dipalmitoyl-phosphatidylcholine, dioleoyl-phosphatidylcholine and dipalmitoyl-phosphatidylglycerol monolayers coupled to ellipsometric, surface pressure, atomic force microscopy and infrared (polarization modulation infrared reflection–absorption spectroscopy) measurements. We show that monolayer compressibility is the major factor controlling protein insertion into PL monolayers and show evidence of the requirement of a minimal distance between lipid headgroups for insertion to occur, Moreover, we demonstrate that ApoA-I inserts deepest at the highest compressibility of the protein monolayer and that the presence of an anionic headgroup increases the amount of protein inserted in the PL monolayer and prevents the steric constrains imposed by the spacing of the headgroup. We also defined the geometry of protein clusters into the lipid monolayer by atomic force microscopy and show evidence of the geometry dependence upon the lipid charge and the distance between headgroups. Finally, we show that ApoA-I helices have a specific orientation when associated to form clusters and that this is influenced by the character of PL charges. Taken together, our results suggest that the interaction of ApoA-I with the cellular membrane may be driven by a mechanism that resembles that of antimicrobial peptide/lipid interaction.
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Bolanos-Garcia V M, Lischetti T, Matak-Vinković D, Cota E, Simpson P J, Chirgadze D Y, Spring D R, Robinson C V, Nilsson J, Blundell T L, 'Structure of a Blinkin-BUBR1 complex reveals an interaction crucial for kinetochore-mitotic checkpoint regulation via unanticipated binding site'
Structure 19 (11) (2011) pp.1691-1700
ISSN: 0969-2126AbstractPublished hereThe maintenance of genomic stability relies on the spindle assembly checkpoint (SAC), which ensures accurate chromosome segregation by delaying the onset of anaphase until all chromosomes are properly bioriented and attached to the mitotic spindle. BUB1 and BUBR1 kinases are central for this process and by interacting with Blinkin, link the SAC with the kinetochore, the macromolecular assembly that connects microtubules with centromeric DNA. Here, we identify the Blinkin motif critical for interaction with BUBR1, define the stoichiometry and affinity of the interaction, and present a 2.2Å resolution crystal structure of the complex. The structure defines an unanticipated BUBR1 region responsible for the interaction and reveals a novel Blinkin motif that undergoes a disorder-to-order transition upon ligand binding. We also show that substitution of several BUBR1 residues engaged in binding Blinkin leads to defects in the SAC, thus providing the first molecular details of the recognition mechanism underlying kinetochore-SAC signaling.
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D'Arcy S, Davies O R, Blundell T L, Bolanos-Garcia V M, 'Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition'
Journal of Biological Chemistry 285 (19) (2010) pp.14764-14776
ISSN: 0021-9258Published here -
Ochi T, Sibanda BL, Wu Q, Chirgadze DY, Bolanos-Garcia VM, Blundell TL, 'Structural biology of DNA repair: spatial organisation of the multicomponent complexes of nonhomologous end joining'
Journal of Nucleic Acids (2010)
ISSN: 2090-0201AbstractPublished hereNonhomologous end joining (NHEJ) plays a major role in double-strand break DNA repair, which involves a series of steps mediated by multiprotein complexes. A ring-shaped Ku70/Ku80 heterodimer forms first at broken DNA ends, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) binds to mediate synapsis and nucleases process DNA overhangs. DNA ligase IV (LigIV) is recruited as a complex with XRCC4 for ligation, with XLF/Cernunnos, playing a role in enhancing activity of LigIV. We describe how a combination of methods—X-ray crystallography, electron microscopy and small angle X-ray scattering—can give insights into the transient multicomponent complexes that mediate NHEJ. We first consider the organisation of DNA-PKcs/Ku70/Ku80/DNA complex (DNA-PK) and then discuss emerging evidence concerning LigIV/XRCC4/XLF/DNA and higher-order complexes. We conclude by discussing roles of multiprotein systems in maintaining high signal-to-noise and the value of structural studies in developing new therapies in oncology and elsewhere.
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Bolanos-Garcia VM, 'Assessment of the mitotic spindle assembly checkpoint (SAC) as the target of anticancer therapies'
Current Cancer Drug Targets 8 (22) (2009) pp.3631-3635
ISSN: 1568-0096AbstractPublished hereThe mitotic spindle assembly checkpoint (SAC) is an essential control system of the eukaryotic cell cycle. This surveillance mechanism monitors the kinetochore, the multi-component complex that assembles on the centromeric DNA and attaches chromosomes to the microtubules of the spindle. The recruitment of mitotic checkpoint proteins to kinetochores that are not correctly attached to microtubules initiates a signalling cascade that results in the CDC20-dependent inhibition of the anaphase-promoting complex/cyclosome (APC/C). Mutations in the genes encoding for diverse SAC proteins have been identified in human tumour cells and associated with chromosome segregation and cancer progression. This work describes the current understanding on the organisation, function and structure of SAC components and shows this knowledge assists the identification of those that may constitute suitable targets for the clinical treatment of cancer.
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Bolanos-Garcia VM, Chayen NE, 'New directions in conventional methods of protein crystallization'
Progress in Biophysics and Molecular Biology 2009 (101) (2009) pp.3-12
ISSN: 0079-6107 eISSN: 1873-1732AbstractPublished hereNovel strategies and techniques that are based on conventional crystallization methods for crystallizing proteins are described and discussed. New directions for rendering proteins and protein complexes to become more amenable to crystallization are also presented.
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Ochi T, Bolanos-Garcia VM, Stojanoff V, Moreno A, 'Perspectives on protein crystallisation'
Progress in Biophysics and Molecular Biology 101 (1-3) (2009) pp.56-63
ISSN: 0079-6107 eISSN: 1873-1732AbstractPublished hereThis final part on ‘perspectives’ is focused on new strategies that can be used to crystallise proteins and improve the crystal quality of macromolecular complexes using any of the methods reviewed in this focused issue. Some advantages and disadvantages, limitations, and plausible applications to high-resolution X-ray crystallography are discussed.
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Blundell TL, Bolanos-Garcia VM, 'Targeting protein interactions of p53 for therapeutic intervention: success in a frustrated landscape'
Cell Cycle 8 (22) (2009) pp.3631-3632
ISSN: 1538-4101AbstractPublished hereComment on: Design of a novel MDM2 binding peptide based on the p53 family. Madhumalar A, et al. Cell Cycle 2009; 8:2828-36
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Bolanos-Garcia VM, Kiyomitsu T, D'Arcy S, Chirgadze DY, Grossmann JG, Matak-Vinkovic D, Venkitaraman AR, Yanagida M, Robinson CV, Blundell TL., 'The crystal structure of the N-terminal region of BUB1 provides insight into the mechanism of BUB1 recruitment to kinetochores'
Structure 17 (1) (2009) pp.105-116
ISSN: 0969-2126AbstractPublished hereThe interaction of the central mitotic checkpoint component BUB1 with the mitotic kinetochore protein Blinkin is required for the kinetochore localization and function of BUB1 in the mitotic spindle assembly checkpoint, the regulatory mechanism of the cell cycle that ensures the even distribution of chromosomes during the transition from metaphase to anaphase. Here, we report the 1.74 Å resolution crystal structure of the N-terminal region of BUB1. The structure is organized as a tandem arrangement of three divergent units of the tetratricopeptide motif. Functional assays in vivo of native and site-specific mutants identify the residues of human BUB1 important for the interaction with Blinkin and define one region of potential therapeutic interest. The structure provides insight into the molecular basis of Blinkin-specific recognition by BUB1 and, on a broader perspective, of the mechanism that mediates kinetochore localization of BUB1 in checkpoint-activated cells.
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Rodriguez F A, Contreras C, Bolanos-Garcia V, Allende J E, 'Protein kinase CK2 as an ectokinase: the role of the regulatory CK2B subunit'
Proceedings of the National Academy of Sciences 105 (15) (2008) pp.5693-5698
ISSN: 0027-8424 eISSN: 1091-6490AbstractProtein kinase CK2 (also known as casein kinase 2) is present in the cytoplasm, nuclei, and several other organelles. In addition, this enzyme has been found bound to the external side of the cell membrane where it acts as an ectokinase phosphorylating several extracellular proteins. Previous experiments with transfection of HEK-293T cells demonstrated that expression of both subunits, CK2α (catalytic) and CK2β (regulatory), was necessary for the appearance of the ectopic enzyme as an ectokinase. In this work, using deletion and point mutations of CK2β, it was possible to demonstrate that the region between amino acids 20 and 33 was necessary for the export of the enzyme as an ectokinase. Phenylalanines 21 and 22 and acidic residues in positions 26-28 are involved in the structural aspects that are required for export. However, the region encompassing amino acids 20-33 of CK2β is not sufficient to make the carboxyl half of this subunit functional in bringing CK2 to the ectokinase locus. In cells transfected with only CK2β, it was demonstrated that 3-4% of the subunit is exported to the cell medium, but the subunit is not bound to the external membrane.Published here -
Beaufils S, Grossmann JG, Renault A, Bolanos-Garcia VM, 'Characterization of the tetratricopeptide-containing domain of BUB1, BUBR1, and PP5 proves that domain amphiphilicity over amino acid sequence specificity governs protein adsorption and interfacial activity.'
Journal of Physical Chemistry B (Soft Condensed Matter and Biophysical Chemistry) 112 (27) (2008) pp.7984-7991
ISSN: 1520-6106 eISSN: 1520-5207AbstractPublished hereThe tetratricopeptide motif repeat (TPR) is an α-helix-turn-α-helix motif that typically mediates protein−protein and, in some cases, protein−lipid interactions. Because of its success, this motif has been preserved through evolution and can be identified in proteins of a wide range of functions in lower and higher organisms. The N-terminal region of BUB1, BUBR1, and protein phosphatase 5 (PP5) contains tandem arrangements of the TPR motif. BUB1 and BUBR1 are conserved multidomain protein kinases that play a key role in the mitotic checkpoint, the mechanism that ensures the synchrony of chromosome segregation. PP5 is an enzyme that targets a wide range of protein substrates including single transmembrane receptors and mammalian cryptochromes. The N-terminal TPR domain of PP5 regulates the activity of the C-terminal catalytic domain through direct interaction with protein and lipid molecules. We portray the biophysical and biochemical properties of the tandem arrangements of the TPR motif of BUB1, BUBR1, and PP5 using far-UV spectroscopy, solution X-ray scattering, null ellipsometry, surface rheology measurements, and Brewster angle microscopy (BAM) observations. We show that, despite the low amino acid sequence conservation and different function, the TPR motif repeats of the three proteins exhibit similar interfacial properties including adsorption kinetics, high surface activity, and the formation of stable, rigid films at the air/water interface. Our studies demonstrate that domain amphiphilicity is of higher importance than amino acid sequence specificity in the determination of protein adsorption and interfacial activity.
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Li Y, Chirgadze DY, Bolanos-Garcia VM, Sibanda BL, Davies OR, Ahnesorg P, Jackson SP, Blundell TL, 'Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ'
EMBO Journal 27 (1) (2008) pp.290-300
ISSN: 0261-4189 eISSN: 1460-2075Published here -
Bolanos-Garcia VM, Renault A, Beaufils S, 'Surface rheology and adsorption kinetics reveal the relative amphiphilicity, interfacial activity, and stability of human exchangeable apolipoproteins'
Biophysical Journal 94 (5) (2008) pp.1735-1745
ISSN: 0006-3495 eISSN: 1542-0086AbstractPublished hereExchangeable apolipoproteins are located in the surface of lipoprotein particles and regulate lipid metabolism through direct protein-protein and protein-lipid interactions. These proteins are characterized by the presence of tandem repeats of amphiphatic α-helix segments and a high surface activity in monolayers and lipoprotein surfaces. A noteworthy aspect in the description of the function of exchangeable apolipoproteins is the requirement of a quantitative account of the relation between their physicochemical and structural characteristics and changes in the mesoscopic system parameters such as the maximum surface pressure and relative stability at interfaces. To comply with this demand, we set out to establish the relations among α-helix amphiphilicity, surface concentration, and surface rheology of apolipoproteins ApoA-I, ApoA-II, ApoC-I, ApoC-II, and ApoC-III adsorbed at the air-water interface. Our studies render further insights into the interfacial properties of exchangeable apolipoproteins, including the kinetics of their adsorption and the physical properties of the interfacial layer.
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Bolanos-Garcia VM, 'The N-terminal, TPR-containing domain of the mitotic checkpoint protein BUBR1 does not bind fatty acids'
Computational Biology and Chemistry 32 (2) (2008) pp.139-140
ISSN: 1476-9271Published here -
Harvey BM, Hong H, Jones MA, Hughes-Thomas ZA, Goss RM, Heathcote ML, Bolanos-Garcia VM, Kroutil W, Staunton J, Leadlay PF, Spencer JB, 'Evidence that a novel thioesterase is responsible for polyketide chain release during biosynthesis of the polyether ionophore monensin'
ChemBioChem 7 (9) (2006) pp.1435-1442
ISSN: 1439-4227 eISSN: 1439-7633Published here -
Bolanos-Garcia VM, Fernandez-Recio J, Allende JE, Blundell TL, 'Identifying interaction motifs in CK2beta--a ubiquitous kinase regulatory subunit'
Trends in Biochemical Sciences 31 (12) (2006) pp.654-661
ISSN: 0968-0004 eISSN: 0167-7640AbstractCasein kinase 2 (CK2) is probably the most ubiquitous serine/threonine kinase found in eukaryotes: it phosphorylates >300 cellular proteins, ranging from transcription factors to proteins involved in chromatin structure and cell division. CK2 is a heterotetrameric enzyme that induces neoplastic growth when overexpressed. The beta subunit of CK2 (CK2beta) functions as the regulator of the catalytic CK2alpha and CK2alpha' subunits, enhancing their stability, activity and specificity. However, CK2beta also functions as a multisubstrate docking platform for several other binding partners. Here, we discuss the organization and roles of interaction motifs of CK2beta, postulate new protein-interaction sites and map these to the known interaction motifs, and show how the resulting complexity of interactions mediated by CK2 gives rise to the versatile functions of this pleiotropic protein kinase.
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Bolanos-Garcia VM, Davies OR, 'Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography'
BBA - Biochimica et Biophysica Acta 1760 (9) (2006) pp.1304-1313
ISSN: 0006-3002AbstractPublished hereImmobilised metal affinity chromatography (IMAC) is the most widely used technique for single-step purification of recombinant proteins. However, despite its use in the purification of heterologue proteins in the eubacteria Escherichia coli for decades, the presence of native E. coli proteins that exhibit a high affinity for divalent cations such as nickel, cobalt or copper has remained problematic. This is of particular relevance when recombinant molecules are not expressed at high levels or when their overexpression induces that of native bacterial proteins due to pleiotropism and/or in response to stress conditions. Identification of such contaminating proteins is clearly relevant to those involved in the purification of histidine-tagged proteins either at small/medium scale or in high-throughput processes. The work presented here reviews the native proteins from E. coli most commonly co-purified by IMAC, including Fur, Crp, ArgE, SlyD, GlmS, GlgA, ODO1, ODO2, YadF and YfbG. The binding of these proteins to metal-chelating resins can mostly be explained by their native metal-binding functions or their possession of surface clusters of histidine residues. However, some proteins fall outside these categories, implying that a further class of interactions may account for their ability to co-purify with histidine-tagged proteins. We propose a classification of these E. coli native proteins based on their physicochemical, structural and functional properties.
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Bolanos-Garcia VM, 'Aurora kinases'
International Journal of Biochemistry and Cell Biology 37 (8) (2005) pp.1572-1577
ISSN: 1357-2725AbstractPublished hereAurora kinases A (also known as Aurora, Aurora-2, AIK, AIR-1, AIRK1, AYK1, BTAK, Eg2, MmIAK1 and STK15), Aurora B (also known as Aurora-1, AIM-1, AIK2, AIR-2, AIRK-2, ARK2, IAL-1 and STK12) and Aurora C (also known as AIK3) participate in several biological processes, including cytokinesis and dysregulated chromosome segregation. These important regulators of mitosis are over-expressed in diverse solid tumors. One member of this family of serine–threonine kinases, human Aurora A, has been proposed as a drugable target in pancreatic cancer. The recent determination of the three-dimensional structure of Aurora A has shown that Aurora kinases exhibit unique conformations around the activation loop region. This property has boosted the search and development of inhibitors of Aurora kinases, which might also function as novel antioncogenic agents.
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Bolanos-Garcia VM, 'MET meet adaptors: functional and structural implications in downstream signalling mediated by the Met receptor'
Molecular and Cellular Biochemistry 276 (2005) pp.149-157
ISSN: 0300-8177 eISSN: 1573-4919AbstractThe tyrosin kinase Met receptor regulates multiple cellular events, ranging from cell motility and angiogenesis to morphological differentiation and tissue regeneration. To conduce these activities, the cytoplasmic C-terminal region of this receptor acts as a docking site for multiple protein substrates, including Grb2, Gab1, STAT3, Shc, SHIP-1 and Src. These substrates are characterised by the presence of multiple domains, including the PH, PTB, SH2 and SH3 domains, which directly interact with the multisubstrate C-terminal region of Met. How this receptor recognises and binds a specific substrate in a space-temporal mode is a central question in cell signalling. The recently solved crystal structure of the tyrosine kinase domain of the Met receptor and that of domains of diverse Met substrates provides the molecular framework to understand Met substrate specificity. This structural information also gives new insights on the plasticity of Met signalling and the implications of Met deregulation in tumorigenic processes. In the light of these advances, the present work discusses the molecular basis of Met-substrate recognition and its functional implications in signalling events mediated by this pleiotropic receptor
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Bolanos-Garcia VM, Beaufils S, Renault A, Grossmann JG, Brewerton S, Lee M, Venkitaraman A, Blundell TL, 'The conserved N-terminal region of the mitotic checkpoint protein BUBR1: a putative TPR motif of high surface activity'
Biophysical Journal 89 (4) (2005) pp.2640-2649
ISSN: 0006-3495 eISSN: 1542-0086AbstractPublished hereBUBR1, a key component of the mitotic spindle checkpoint, is a multidomain protein kinase that is activated in response to kinetochore tension. Although BUB1 and BUBR1 play an important role in cell division, very little is known about their structural characteristics. We show that the conserved N-terminal region of BUBR1, comprising residues 1–204, is a globular domain of high α-helical content (≈60%), stable in the pH range 4–9 and probably organized as a tetratricopeptide motif repeat (TPR), most closely resembling residues 16–181 of protein phosphatase 5. Because the latter presents a continuous amphipathic groove and is regulated by binding certain fatty acids, we compared the properties of BUBR1(1–204) and TPR-PP5(16–181) at air/water interfaces and found that both proteins exhibited a similar surface activity and formed stable, rigid monolayers. The deletion of a region that probably comprises several α-helices of BUBR1 indicates that long-range interactions are essential for the stability of the N-terminal domain. The presence of the putative TPR motif strongly suggests that the N-terminal domain of BUBR1 is involved in direct protein-protein interactions and/or protein-lipid interactions
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Tapia JC, Bolanos-Garcia VM, Sayed M, Allende CC, Allende JE, 'Cell cycle regulatory protein p27KIP1 is a substrate and interacts with the protein kinase CK2'
International Journal of Biochemistry and Cell Biology 91 (5) (2004) pp.865-879
ISSN: 1357-2725AbstractPublished hereThe protein kinase CK2 is constituted by two catalytic (α and/or α′) and two regulatory (β) subunits. CK2 phosphorylates more than 300 proteins with important functions in the cell cycle. This study has looked at the relation between CK2 and p27KIP1, which is a regulator of the cell cycle and a known inhibitor of cyclin-dependent kinases (Cdk). We demonstrated that in vitro recombinant Xenopus laevis CK2 can phosphorylate recombinant human p27KIP1, but this phosphorylation occurs only in the presence of the regulatory β subunit. The principal site of phosphorylation is serine-83. Analysis using pull down and surface plasmon resonance (SPR) techniques showed that p27KIP1 interacts with the β subunit through two domains present in the amino and carboxyl ends, while CD spectra showed that p27KIP1 phosphorylation by CK2 affects its secondary structure. Altogether, these results suggest that p27KIP1 phosphorylation by CK2 probably involves a docking event mediated by the CK2β subunit. The phosphorylation of p27KIP1 by CK2 may affect its biological activity.
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Bertrand L, Sayed MF, Pei XY, Parisini E, Dhanaraj V, Bolanos-Garcia VM, Allende JE, Blundell TL, 'Structure of the regulatory subunit of CK2 in the presence of a p21WAF1 peptide demonstrates flexibility of the acidic loop'
Acta Crystallographica Section D: Biological Crystallography 60 (2004) pp.1698-1704
ISSN: 0907-4449AbstractPublished hereA truncated form of the regulatory subunit of the protein kinase CK2[beta] (residues 1-178) has been crystallized in the presence of a fragment of the cyclin-dependent kinase inhibitor p21WAF1 (residues 46-65) and the structure solved at 2.9 Å resolution by molecular replacement. The core of the CK2[beta] dimer shows a high structural similarity with that identified in previous structural analyses of the dimer and the holoenzyme. However, the electron density corresponding to the substrate-binding acidic loop (residues 55-64) indicates two conformations that differ from that of the holoenzyme structure [Niefind et al. (2001), EMBO J. 20, 5320-5331]. Difference electron density near the dimerization region in each of the eight protomers in the asymmetric unit is attributed to between one and eight amino-acid residues of a complexed fragment of p21WAF1. This binding site corresponds to the solvent-accessible part of the conserved zinc-finger motif.
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del Carmen Ramírez-Medeles M, Aguilar MB, Miguel RN, Bolanos-García VM, García-Hernández E, Soriano-García M, 'Amino acid sequence, biochemical characterization, and comparative modeling of a nonspecific lipid transfer protein from Amaranthus hypochondriacus'
Archives of Biochemistry and Biophysics 415 (1) (2003) pp.24-33
ISSN: 0003-9861AbstractPlant nonspecific lipid transfer proteins (nsLTPs) are characterized by their ability to bind a broad range of hydrophobic ligands in vitro. Their biological function has not yet been elucidated, but they could play a major role in plant defense to physical and biological stress. An nsLTP was isolated from Amaranthus hypochondriacus seeds and purified by gel filtration and reversed-phase high-performance liquid chromatography techniques. The molecular mass of the protein as determined by mass spectrometry is 9747.29 Da. Data from amino acid sequence, circular dichroism and binding/displacement of a fluorescent lipid revealed that it belongs to the nsLTP1 family. The protein shows the α-helical secondary structure typical for plant nsLTPs 1 and shares 40 to 57% sequence identity with nsLTPs 1 from other plant species and 100% identity with an nsLTP1 from Amaranthus caudatus. A model structure of the protein in complex with stearate based on known structures of maize and rice nsLTPs 1 suggests a protein fold complexed with lipids closely related to that of maize nsLTP1.
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Bolanos-Garcia VM, Miguel RN, 'On the structure and function of apolipoproteins: more than a family of lipid-binding proteins'
Progress in Biophysics and Molecular Biology 83 (1) (2003) pp.47-68
ISSN: 0079-6107 eISSN: 1873-1732AbstractPublished hereExchangeable apolipoproteins have been the subject of intense biomedical investigation for decades. However, only in recent years the elucidation of the three-dimensional structure reported for several members of the apolipoprotein family has provided insights into their functions at a molecular level for the first time. Moreover, the role of exchangeable apolipoproteins in several cellular events distinct from lipid metabolism has recently been described. This review summarizes these contributions, which have not only allowed the identification of the apolipoprotein domains that determine substrate binding specificity and/or affinity but also the plausible molecular mechanism(s) involved.
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Bolanos-Garcia V, 'The use of oil in a counter-diffusive system allows to control nucleation and coarsening during protein crystallization'
Journal of Crystal Growth 253 (1-4) (2003) pp.517-523
ISSN: 0022-0248AbstractA method is described to control nucleation and coarsening in the process of protein crystallization. The control of these events is achieved by combining a biphasic oil/water system with a multi-compartment crystallization device. The addition of oil resulted in a significant decrease of the diffusion rate of a precipitant agent solution. When compared with crystal growth in the absence of oil, significantly smaller amounts of initially precipitated protein were observed. Thus, this method shifts crystal growth from a nucleation-coarsening (Ostwald ripening) equilibrium to a new equilibrium where Ostwald ripening is drastically diminished. Moreover, larger, rod-shape, high quality single crystals can also be obtained as a result of this equilibrium shift.Published here -
Blundell T, Bolanos-Garcia V, Chirgadze D, Harmer N, Lo T, Pellegrini L, Sibanda B, 'Asymmetry in the multiprotein systems of molecular biology'
Structural Chemistry 13 (3-4) (2002) pp.405-412
ISSN: 1040-0400Published here -
Reyes-Grajeda JP, Jáuregui-Zúñiga D, Rodríguez-Romero A, Hernández-Santoyo A, Bolanos-Garcia VM, Moreno A, 'Crystallization and preliminary x-ray analysis of ovocleidin-17 a major protein of the gallus gallus eggshell calcified layer'
Protein and Peptide Letters 9 (3) (2002) pp.253-257
ISSN: 0929-8665 eISSN: 1875-5305AbstractPublished hereIn this work, we report the crystallization of ovocleidin-17, the major protein of the avian eggshell calcified layer and the preliminary X-ray characterization of this soluble protein which is implied into the CaCO3 formation of the eggshell in avians. Crystals belong to one of the trigonal space group P3 with cell dimensions a= b= 59.53 Å and c = 83.33 Å, and α=β= 90 and γ =120 . Crystals diffract up to 3.0 Å. -
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Bolaños-García V, Ramos S, Castillo R, Xicohtencatl-Cortes J, Mas-Oliva J, 'Monolayers of apolipoproteins at the air/water interface'
Journal of Physical Chemistry B (Soft Condensed Matter and Biophysical Chemistry) 105 (24) (2001) pp.5757-5765
ISSN: 1520-6106 eISSN: 1520-5207Published here -
Bolanos-García V, Màs-Oliva J, Ramos S, Castillo R, 'Phase transitions in monolayers of human apolipoprotein C-I'
Journal of Physical Chemistry B (Soft Condensed Matter and Biophysical Chemistry) 103 (30) (1999) pp.6236-6242
ISSN: 1520-6106 eISSN: 1520-5207Published here -
Bolaños-García V, Mas-Oliva J, Soriano-García M, Moreno A, 'Precrystallization of human apoprotein A-I based on its aggregation behavior in solution studied by dynamic light scattering'
Journal of Molecular Structure 440 (1-3) (1998) pp.1-8
ISSN: 0022-2860Published here -
Bolanos-García VM, Soriano-García M, Mas-Oliva J, bolanos, 'Stability of the C-terminal peptide of CETP mediated through an (i, i + 4) array'
BBA - Biochimica et Biophysica Acta 1384 (1) (1998) pp.7-15
ISSN: 0006-3002AbstractBased on circular dichroism (CD), we have found an essential (i, i + 4) alpha-helix stabilizing array in the C-terminus region for the cholesteryl ester transfer protein (CETP) between histidine 466 and aspartic acid 470. This region apparently corresponds to an amphipathic alpha-helix. The behavior of this peptide in solution in comparison with a mutant peptide (D470N) was also analyzed by dynamic light scattering (DLS). The results showed that alpha-helix stabilization is not due to peptide aggregation. The thermodynamic estimation of stability supports the idea that the phenomenon is carried out through an (i, i + 4) array. The representation of the C-terminal region as an amphipathic alpha-helical peptide shows that lipid-binding activity might be in part due to both the asymmetric polar/non-polar residue distribution and to the presence of an (i, i + 4) array important for helix stability.
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Bolanos-García VM, Soriano-García M, Mas-Oliva J, 'CETP and exchangeable apoproteins: common features in lipid binding activity'
Molecular and Cellular Biochemistry 175 (1-2) (1997) pp.1-10
ISSN: 0300-8177 eISSN: 1573-4919AbstractIn order to define the active domain for lipid binding in CETP (cholesteryl ester transfer protein), our study discusses some fundamental physicochemical properties of this molecule such as hydrophobic moment, protein active surface and helix amphipathicity, in comparison to the properties reported for a series of apoproteins including apoAI, apoAII, apoCI, CII, CIII and apoE. Our study suggests that CETP corresponds to a protein with an active surface slightly lower than the one calculated for the exchangeable apoproteins AI, AII, CI, CII, CIII and E. Arrays type (i, i + 3) and (i, i + 4) were found in the region associated to lipid binding in these apoproteins. Seven such arrays located in the amphipathic alpha-helices of CETP are also suggested to contribute to the overall lipid binding activity as a consequence of alpha-helix stability. It is proposed that for lipid binding to occur in both types of molecules, the possibility of a conformational specificity given by a redundant stereochemical code can be actively operating.
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Bolanos-García V, Juárez-Martínez G, Panneerselvam K, Soriano-García M, '17β-estradiol 3-benzoate'
Acta Crystallographica Section C: Crystal Structure Communications 52 (8) (1996) pp.1997-2000
ISSN: 0108-2701AbstractPublished hereIn the title compound, 17[beta]-hydroxyestra-1,3,5(10)-trien-3-yl benzoate, C25H28O3, the B, C and D rings adopt envelope, chair and envelope conformations, respectively. Both phenyl rings are planar. The structure is internally stabilized by C-H...O hydrogen-bond interactions and is also externally stabilized by one O-H...O and one C-H...O hydrogen bond.
Book chapters
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Curtis NL, Bolanos-Garcia VM, 'The Anaphase Promoting Complex/Cyclosome (APC/C): A Versatile E3 Ubiquitin Ligase' in Prof. Dr. Dr. J. Robin Harris and Dr. Jon Marles-Wright (ed.), Subcellular Biochemistry, Springer (2019)
ISBN: 9783030281502 eISBN: 9783030281519AbstractPublished here Open Access on RADARIn the present chapter we discuss the essential roles of the human E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C) in mitosis as well as the emerging evidence of important APC/C roles in cellular processes beyond cell division control such as regulation of genomic integrity and cell differentiation of the nervous system. We consider the potential incipient role of APC/C dysregulation in the pathophysiology of the neurological disorder Alzheimer’s disease (AD). We also discuss how certain Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) viruses take control of the host’s cell division regulatory system through harnessing APC/C ubiquitin ligase activity and hypothesise the plausible molecular mechanisms underpinning virus manipulation of the APC/C. We also examine how defects in the function of this multisubunit protein assembly drive abnormal cell proliferation and lastly argue the potential of APC/C as a promising therapeutic target for the development of innovative therapies for the treatment of chronic malignancies such as cancer.
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Bolanos-Garcia V, 'Protein Complexes in the Nucleus: the control of chromosome segregation' in Robin Harris J, Marles-Wright JM (ed.), Macromolecular Protein Complexes, Springer International Publishing (2017)
ISBN: 978-3-319-46501-2AbstractChapter 16Published here
Professional information
Memberships of professional bodies
- 2015-to date. Member of the Cancer Research UK Oxford Centre.
- 2015-to date. Member of the Oxford Ageing Network (OxAgeN).
- 2013-to date. Fellow of the Higher Education Academy (HEA) of the United Kingdom.
- 2012-to date. Member of the Biochemical Society.
- 2009-to date. Member of the Cambridge Cancer Centre.
- 2000-to date. Member of the British Crystallographic Association (BCA), United Kingdom.
Conferences
I regularly attend and present research advances in ad hoc conferences on cell division control; cancer; drug discovery; protein biophysics and protein structural biology.
Consultancy
- 2020 to date. Advisory Editorial Board Member. Subcellular Biochemistry (SCBI). Springer Nature. Heidelberg, Germany.
- 2020-to date. International Evaluator. National Council of Science and Technology (CONACyT). Government of Mexico. Mexico.
- 2019-to date. Editorial Board Member. Biomolecules. Basel, Switzerland.
- 2019-to date. International Advisory Board Member. Imagine IF accelerator. Republic of Serbia. In this role I provide advise on R&D activities to new SMEs in Serbia, which then pitch for funding at an annual international event organised by The Innovation Forum and IF Imagine.
Further details
Mentoring Experience
I have provided management and mentoring support to many colleagues with less experience, which ultimately has contributed to their academic and personal development. One example of my strong commitment to support junior colleagues beyond my current organisation of adscription is my formal role as Postdoctoral Mentor for the Postdoc Academy, University of Cambridge.
Funding
Research funding from external sources. As a PI, Co-investigator, Named Investigator or Collaborator, I have been involved in rising over £15M from the UK Research Councils (MRC, BBSRC), charities (The Wellcome Trust, CRUK) and other organisations (Innovate UK; CNRS, France; CONACyT, Mexico; Fondazione Cariplo, Italy; Instituts de Recherche en Santé, Canada; The Spanish National Research Council, CSIC, Spain).
Further information
- ORCiD file: https://orcid.org/0000-0003-2700-1479
- Twitter: twitter.com/VictorBolanosGa
- Linkedin: linkedin.com/in/victor-bolanos-garcia-92470a12