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PhD; MSc, BSc. FHEA-UK
Department of Biological and Medical Sciences
Faculty of Health and Life Sciences
+44 (0)1865 484146
Senior Lecturer and Principal Investigator. Leader of the research group "Mechanisms of Regulation of Cell Division".
My years of experience in teaching in Higher Education institutions in the UK spans different areas and settings and include lecturing, supervision and teaching to small groups, preparation of teaching material and examination papers, marking of coursework and exams, development of new postgraduate teaching programmes, mentoring, examination of PhD and MSc dissertations and the day-to-day supervision of undergraduate and postgraduate students and overseas research visitors. The majority of my former students from Cambridge, Oxford, and overseas have stayed in touch long after the termination of their courses/research projects. They often said they have found the experience of working under my supervision as inspiring, challenging and intellectually stimulating. They usually contact me for advice as they progress in their careers and/or ask me to write letters of reference to support scholarship and/or admission applications to postgraduate programmes at Biomedical Schools in the UK and abroad.
2012 to date. Leader of the Module “Genomic Sciences” (MSc Programme) and Introduction to Biochemistry A and B Modules (Undergraduate programme); Lecturing in a range of postgraduate and undergraduate courses underpinning Chemistry, Biochemistry, Pharmacology, Bioinformatics, Evidence-based Medicine, Cancer and key topics on Molecular and Cellular Biology.
2005-2011. Small group teaching. Supervisor of the papers MVST Par1A (Molecules in Medical Science) and the Natural Sciences Tripos (Part IA and B and Part II Biochemistry, Molecular and Cellular Biology and Physiology of Organisms) to undergraduate students from Queen’s, St Edmund’s and Homerton College, University of Cambridge, England.
Taught undergraduate courses.Module BIOS4006: Introduction to Biochemistry AModule BIOS4007: Introduction to Biochemistry B
Module BIOS5003: Biochemistry of Cell Function Module BIOS6008: Evidence Based Medicine and Diagnostics
Module BIOS6010: Project
Module BIOS6011: Clinical Biochemistry
Taught Postgraduate courses
Module BIOS7001: Advanced Molecular Techniques
Module BIOS7002: Genome Sciences
Module BIOS7006: Clinical Genetics and Diagnostics
Module BIOS7007: Advances in Medical Genetics
Miss Natalie L. Curtis. (PhD student) Oxford Brookes University. 2017-2021.
Miss Maria Kapanidou. (PhD student) Oxford Brookes University. 2013-2017.
Dr Dave Gervais. PhD by published work. Oxford Brookes University. 2014-2016.
Mr Samuel Connelly. 2016-2020, PhD. Oxford Brookes University, England.
Miss Teresa Minguez Vinas. 2016-2020, PhD. Oxford Brookes University, England.
Miss Brittany Almond. 2014-2018, PhD. Oxford Brookes University, England.
Miss Qian Wu. 2009-2012, PhD. University of Cambridge, England.
Mr. Takashi Ochi. 2008-2012, PhD. University of Cambridge, England.
Miss Deepti Gupta. 2008-2011, PhD. University of Cambridge, England.
Miss Ann Ling. 2008-2011, PhD. University of Cambridge, England.
Mr. Lionel Chieze. 2007-2010, PhD. University of Rennes, France.
Miss Sheena D'Arcy. 2006-2010, PhD. University of Cambridge, England.
The mechanistic understanding of cell division constitutes an important problem in the Life Sciences because anomalies in the processes are associated with birth and development defects, neuropathies, premature ageing and cancer. In essence, I am trying to understand how proteins can associate to form larger macromolecular assemblies to ensure timely and accurate cell division. I am equally interested in exploring ways in which the structural and mechanistic understanding of the process can be translated into the clinic. To this aim, my group integrates a multidisciplinary approach to obtain a molecular understanding of the function and structure of the macromolecular complexes that regulate the SAC. This is a matter of great interest in Biochemistry, Human and Animal Biology and Medicine. The research area has clear potential for application in diagnostic and treatment of birth and developmental defects, cancer and aging-associated disorders, which have a great impact for the wellbeing and the Health and Biotechnology sectors. Our studies combine Structural Biology and chemoinformatics methods with protein biochemistry, protein biophysics and molecular and cell biology approaches. In this way my group has accumulated an extensive body of work on the 3D structure and function of protein complexes that ensure genome stability and provided new insights for the molecular understanding of signal transduction mechanisms underpinning cell division in eukaryotic organisms. It also has led to the generation of novel molecules with drug-like properties to interfere with cell division in cancer cells.
2015-to date. Member of the Cancer Research UK Oxford Centre.
2015-to date. Member of the Oxford Ageing Network (OxAgeN).
2015-to date. Member of the Oxford UBIQUITIN Club, University of Oxford, UK.
2013-to date. Fellow of the Higher Education Academy (HEA) of the United Kingdom.
2013 - 2014. Editor of Advances in Enzyme Research.
2012-to date. Member of the Biochemical Society.
2009-to date. Member of the Cambridge Cancer Centre.
2003-to date. Member of the European Crystallographic Association (ECA).
2000-to date. Member of the British Crystallographic Association (BCA), United Kingdom.
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, Cancer Research UK) and other organisations (CNRS, France; CONACyT, Mexico; Fondazione Cariplo, Italy; Instituts de Recherche en Santé, Canada).
Research Grants Awarded (Selected examples only)
Structural and Functional Characteristation of the SAC
I am happy to be contacted at any time to discuss Postdoctoral openings, PhD and research training opportunities in my group.
I have developed an original, sustainable and international competitive research programme and generated materials, methods, protocols and data that are used by other researchers around the world. A fraction of my 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 such as GE Healthcare, Hampton Research, Bruker AXS, and New England Biolabs.
My research on cell signalling span basic and translational bioscience that underpins the major strategic objectives of the UK research councils (BBSRC, MRC, EPSRC), charities (The Wellcome Trust, CRUK) and the EU (Horizon 2020 Programme): understanding healthy aging, the molecular basis of tumour formation and growth and of birth and development disorders; developing industrial biotechnology and the control of infectious diseases through the targeting of cell division regulation.
Research Excellence Framework (REF) 2021.
I am a member of the Oxford Brookes University “Biomedicine” group, REF2021.
The 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.
The 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.
Breast 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.
The 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
Improperly 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.
The 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.
A 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.
Knl1 (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.
The 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.
The 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.
Helicobacter 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.
The 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.
Exchangeable 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.
Kinetochore 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.
The 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.
The 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.
Human 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.
Apolipoprotein 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.
Nonhomologous 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.
Novel 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.
Comment on: Design of a novel MDM2 binding peptide based on the p53 family. Madhumalar A, et al. Cell Cycle 2009; 8:2828-36
This 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.
The 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.
The 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.
The 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.
Exchangeable 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.
Immobilised 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.
Casein 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.
BUBR1, 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
The 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
Aurora 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.
The 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.
A 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.
Exchangeable 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.
Plant 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.
In 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. -
Based 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.
In 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.
In 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.
Cell Division Regulation: Structural Biology; Protein Biochemistry; Protein Bioinformatics; Protein Biophysics; Early Drug Discovery.
2020-to date. Postdoctoral Mentor. The Postdoc Academy, University of Cambridge, England, UK.
2020-to date. Advisory Editorial Board. Subcellular Biochemistry (SCBI), Springer. Heidelberg, Germany.
2019-to date. Editorial Board Member. Biomolecules. Basel, Switzerland.
2019-to date. International Advisory Board Member. Imagine IF accelerator. Republic of Serbia.
2017-2020. R&D Innovation Committee Member. Faculty of Health and Life Sciences, Oxford Brookes University (OBU).
2016-to 2018. Advisory Board Member. The Innovation Forum, Oxford branch.
2016-to 2018. iCASE Industrial Panel Committee Member, BBSRC Doctoral training Programme (DTP). Univ. of Oxford-Oxford Brookes University-Hartwell Research Campus-Pirbright Institute-Diamond Ligh Source, ISIS Neutron and Muon Source-STFC Rutherford Appleton Laboratory.
2016-2019. Oxford Brookes University representative, Oxford Academic Health Science Centre (AHSC) Theme 2 Strategy Group.
2014-to 2019. Knowledge Exchange & Impact Faculty Lead, Oxford Brookes University.
2009-2010. Acting Manager of the Baculovirus Expression Facility. Dept. Biochemistry, University of Cambridge, UK.
2015-to date. Member of the Oxford UBIQUITIN Club, University of Oxford
2009-to date. Member of the Cambridge Cancer Centre (http://www.cancer.cam.ac.uk).
Invited speaker (past six years only)
2011-2012. Appointed as a Post-doctoral Teaching Associate after a competitive application process. Department of Biochemistry, University of Cambridge, England. 2006-2012. Research Associate, University of Cambridge. (Department of Biochemistry, Group of Prof. Sir Tom L. Blundell). Cambridge, England.
2003-2006. Research Associate, University of Cambridge. (Department of Biochemistry, Group of Prof. Sir Tom L. Blundell in collaboration with Prof. Ashok Venkitaraman, Cambridge). 2000-2003. Wellcome Trust International Research Fellow, Laboratory of Molecular Biology (LMB) of the Medical Research Council (MRC) and the University of Cambridge. Cambridge, United Kingdom.
My research programme bridges together a quantitative understanding of the interactions underpinning chromosome segregation with biochemical, biophysical, molecular, cellular, and structural biology methods. The interdisciplinary nature and substantial societal impact of the research concerns central areas of bioscience: Cell and Developmental Biology, Cell Cycle regulation, Cancer, Drug Discovery, Pharmacology, Radiation Biology, Functional Genomics, Systems Biology, Clinical Biochemistry, Medicine, Healthy Ageing, Structural Biology and the human and animal health industries, where an understanding of the cell regulatory systems, the structure-guided design of vaccines and new drugs and the causes and treatment of genome instability and age-associated malignancies remains important. The research represents 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. Understanding the dynamics and the structural features of the molecular interactions underpinning mitotic checkpoint signalling will ultimately 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.
I am a biochemist and structural biologist 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 like 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)-MRC and Sir Tom L 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. The research experience acquired on this signalling system prompted me to initiate work on the spindle assembly checkpoint (SAC), the regulatory mechanism of higher organisms that ensures the proper segregation of chromosomes upon cell division. The initial studies were conducted in collaboration with Ashok Venkitaraman (MRC Cancer Unit, Cambridge) and Tom L. Blundell and supported by Cancer Research UK.