The spindle checkpoint proteins BUB1 and BUBR1: (SLiM)ming down to the basics.

Benzimidazole 1 (BUB1) and budding uninhibited by benzimidazole 1-related 1 (BUBR1) are multidomain paralogs with key roles in chromosome alignment during mitosis and the spindle assembly checkpoint (SAC), an evolutionarily conserved signaling pathway that monitors errors in chromosome segregation during cell division in eukaryotes. Although BUB1 and BUBR1 share a similar domain organization and short linear interaction motifs (SLiMs), they control distinct aspects of chromosome congression and the SAC. Here we discuss the roles of BUB1 and BUBR1 SLiMs in mitosis and complement this with additional insights gleamed from studying their evolution. We show that BUB1 and BUBR1 SLiMs form highly specific interactions that are carefully orchestrated in space and time and contend that they define BUB1 and BUBR1 as organizing hubs that drive SAC signaling and ensure genome stability.

On the Regulation of Mitosis by the Kinetochore, a Macromolecular Complex and Organising Hub of Eukaryotic Organisms.

The kinetochore is the multiprotein complex of eukaryotic organisms that is assembled on mitotic or meiotic centromeres to connect centromeric DNA with microtubules. Its function involves the coordinated action of more than 100 different proteins. The kinetochore acts as an organiser hub that establishes physical connections with microtubules and centromere-associated proteins and recruits central protein components of the spindle assembly checkpoint (SAC), an evolutionarily conserved surveillance mechanism of eukaryotic organisms that detects unattached kinetochores and destabilises incorrect kinetochore-microtubule attachments. The molecular communication between the kinetochore and the SAC is highly dynamic and tightly regulated to ensure that cells can progress towards anaphase until each chromosome is properly bi-oriented on the mitotic spindle. This is achieved through an interplay of highly cooperative interactions and concerted phosphorylation/dephosphorylation events that are organised in time and space.This contribution discusses our current understanding of the function, structure and regulation of the kinetochore, in particular, how its communication with the SAC results in the amplification of specific signals to exquisitely control the eukaryotic cell cycle. This contribution also addresses recent advances in machine learning approaches, cell imaging and proteomics techniques that have enhanced our understanding of the molecular mechanisms that ensure the high fidelity and timely segregation of the genetic material every time a cell divides as well as the current challenges in the study of this fascinating molecular machine.

Computational Biology Dynamics of Mps1 Kinase Molecular Interactions with Isoflavones Reveals a Chemical Scaffold with Potential to Develop New Therapeutics for the Treatment of Cancer.

The protein kinase Mps1 (monopolar spindle 1) is an important regulator of the Spindle Assembly Checkpoint (SAC), the evolutionary conserved checkpoint system of higher organisms that monitors the proper bipolar attachment of all chromosomes to the mitotic spindle during cell division. Defects in the catalytic activity and the transcription regulation of Mps1 are associated with genome instability, aneuploidy, and cancer. Moreover, multiple Mps1 missense and frameshift mutations have been reported in a wide range of types of cancer of different tissue origin. Due to these features, Mps1 arises as one promising drug target for cancer therapy. In this contribution, we developed a computational biology approach to study the dynamics of human Mps1 kinase interaction with isoflavones, a class of natural flavonoids, and compared their predicted mode of binding with that observed in the crystal structure of Mps1 in complex with reversine, a small-sized inhibitor of Mps1 and Aurora B kinases. We concluded that isoflavones define a chemical scaffold that can be used to develop new Mps1 inhibitors for the treatment of cancer associated with Mps1 amplification and aberrant chromosome segregation. In a broader context, the present report illustrates how modern chemoinformatics approaches can accelerate drug development in oncology.

Cdc20: At the Crossroads between Chromosome Segregation and Mitotic Exit.

Cell-division cycle protein 20 homologue (Cdc20) has important functions in chromosome segregation and mitotic exit. Cdc20 is the target of the spindle assembly checkpoint (SAC) and a key cofactor of the anaphase-promoting complex or cyclosome (APC/C) E3 ubiquitin ligase, thus regulating APC/C ubiquitin activity on specific substrates for their subsequent degradation by the proteasome. Here we discuss the roles of Cdc20 in SAC signalling and mitotic exit, describe how the integration of traditional approaches with emerging technologies has revealed new details of Cdc20 functions, comment about the potential of Cdc20 as a therapeutic target for the treatment of human malignancies, and discuss recent advances and controversies in the mechanistic understanding of the control of chromosome segregation during cell division.

The Anaphase Promoting Complex/Cyclosome (APC/C): A Versatile E3 Ubiquitin Ligase.

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.

Protein Complexes in the Nucleus: The Control of Chromosome Segregation.

Mistakes in the process of cell division can lead to the loss, gain or rearrangement of chromosomes. Significant chromosomal abnormalities are usually lethal to the cells and cause spontaneous miscarriages. However, in some cases, defects in the spindle assembly checkpoint lead to severe diseases, such as cancer and birth and development defects, including Down's syndrome. The timely and accurate control of chromosome segregation in mitosis relies on the spindle assembly checkpoint (SAC), an evolutionary conserved, self-regulated signalling system present in higher organisms. The spindle assembly checkpoint is orchestrated by dynamic interactions between spindle microtubules and the kinetochore , a multiprotein complex that constitutes the site for attachment of chromosomes to microtubule polymers to pull sister chromatids apart during cell division. This chapter discusses the current molecular understanding of the essential, highly dynamic molecular interactions underpinning spindle assembly checkpoint signalling and how the complex choreography of interactions can be coordinated in time and space to finely regulate the process. The potential of targeting this signalling pathway to interfere with the abnormal segregation of chromosomes, which occurs in diverse malignancies and the new opportunities that recent technological developments are opening up for a deeper understanding of the spindle assembly checkpoint are also discussed.

The dynamic protein Knl1 - a kinetochore rendezvous.

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.

BUB1 and BUBR1: multifaceted kinases of the cell cycle.

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.

Overexpression of Endoplasmic Reticulum Proteins from Arabidopsis thaliana in Baculovirus.

The overproduction of proteins of the endoplasmic reticulum (ER) of plant cells in prokaryotic heterologous gene expression system remains a technical challenge. Recent advances in genetically modified insect cell technology and virus engineering methods have paved the way to produce recombinant ER plant proteins, including those harboring posttranslational modifications, and therefore, to yield ER plant proteins that are natively folded and fully functional. The present contribution focuses on the baculovirus-expression system flashBAC, which overcomes certain technical hurdles found in other insect cell-based expression systems such as the generation of a bacmid and the negative selection of recombinant clones.

Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases.

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.

Structural and functional insights into the role of the N-terminal Mps1 TPR domain in the SAC (spindle assembly checkpoint).

The 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=0.1 nm) 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.

Crystal structure of human XLF/Cernunnos reveals unexpected differences from XRCC4 with implications for NHEJ.

The recently characterised 299-residue human XLF/Cernunnos protein plays a crucial role in DNA repair by non-homologous end joining (NHEJ) and interacts with the XRCC4-DNA Ligase IV complex. Here, we report the crystal structure of the XLF (1-233) homodimer at 2.3 A resolution, confirming the predicted structural similarity to XRCC4. The XLF coiled-coil, however, is shorter than that of XRCC4 and undergoes an unexpected reverse in direction giving rise to a short distorted four helical bundle and a C-terminal helical structure wedged between the coiled-coil and head domain. The existence of a dimer as the major species is confirmed by size-exclusion chromatography, analytical ultracentrifugation, small-angle X-ray scattering and other biophysical methods. We show that the XLF structure is not easily compatible with a proposed XRCC4:XLF heterodimer. However, we demonstrate interactions between dimers of XLF and XRCC4 by surface plasmon resonance and analyse these in terms of surface properties, amino-acid conservation and mutations in immunodeficient patients. Our data are most consistent with head-to-head interactions in a 2:2:1 XRCC4:XLF:Ligase IV complex.

The Biomolecules Journal Club: Highlights on Recent Papers-1.

We are glad to share with you our first Journal Club and to highlight some of the most interesting papers published recently [...].

Biochemical, biophysical, and functional characterisation of the E3 ubiquitin ligase APC/C regulator CDC20 from Arabidopsis thaliana.

The Anaphase Promoting Complex (APC/C), a large cullin-RING E3-type ubiquitin ligase, constitutes the ultimate target of the Spindle Assembly Checkpoint (SAC), an intricate regulatory circuit that ensures the high fidelity of chromosome segregation in eukaryotic organisms by delaying the onset of anaphase until each chromosome is properly bi-oriented on the mitotic spindle. Cell-division cycle protein 20 homologue (CDC20) is a key regulator of APC/C function in mitosis. The formation of the APC/CCDC20 complex is required for the ubiquitination and degradation of select substrates, which is necessary to maintain the mitotic state. In contrast to the roles of CDC20 in animal species, little is known about CDC20 roles in the regulation of chromosome segregation in plants. Here we address this gap in knowledge and report the expression in insect cells; the biochemical and biophysical characterisation of Arabidopsis thaliana (AtCDC20) WD40 domain; and the nuclear and cytoplasmic distribution of full-length AtCDC20 when transiently expressed in tobacco plants. We also show that most AtCDC20 degrons share a high sequence similarity to other eukaryotes, arguing in favour of conserved degron functions in AtCDC20. However, important exceptions were noted such as the lack of a canonical MAD1 binding motif; a fully conserved RRY-box in all six AtCDC20 isoforms instead of a CRY-box motif, and low conservation of key residues known to be phosphorylated by BUB1 and PLK1 in other species to ensure a robust SAC response. Taken together, our studies provide insights into AtCDC20 structure and function and the evolution of SAC signalling in plants.

Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition.

BubR1 is essential for the mitotic checkpoint that prevents aneuploidy in cellular progeny by triggering anaphase delay in response to kinetochores incorrectly/not attached to the mitotic spindle. Here, we define the molecular architecture of the functionally significant N-terminal region of human BubR1 and present the 1.8 A crystal structure of its tetratricopeptide repeat (TPR) domain. The structure reveals divergence from the classical TPR fold and is highly similar to the TPR domain of budding yeast Bub1. Shared distinctive features include a disordered loop insertion, a 3(10)-helix, a tight turn involving glycine positive Phi angles, and noncanonical packing of and between the TPR motifs. We also define the molecular determinants of the interaction between BubR1 and kinetochore protein Blinkin. We identify a shallow groove on the concave surface of the BubR1 TPR domain that forms multiple discrete and potentially cooperative interactions with Blinkin. Finally, we present evidence for a direct interaction between BubR1 and Bub1 mediated by regions C-terminal to their TPR domains. This interaction provides a mechanism for Bub1-dependent kinetochore recruitment of BubR1. We thus present novel molecular insights into the structure of BubR1 and its interactions at the kinetochore-microtubule interface. Our studies pave the way for future structure-directed engineering aimed at dissecting the roles of kinetochore-bound and other pools of BubR1 in vivo.

The crystal structure of the N-terminal region of BUB1 provides insight into the mechanism of BUB1 recruitment to kinetochores.

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 angstroms 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.

Interfacial Activity of Lipoprotein (a) Isoforms with a Variable Number of Kringle IV Type 2 Repeats: A New Indicator of Cardiovascular Risk?

OBJECTIVE: Lipoprotein (a) [Lp(a)] is an LDL-like particle constituted by lipids, apolipoprotein B100 and apolipoprotein (a) [apo(a)], a multidomain glycoprotein whose molecular mass is dependent on the genetically encoded number of Kringle IV type 2 (KIV-2) repeats. Because Lp(a) isoforms have been associated with cardiovascular risk (CVR), we have investigated if their interfacial properties can contribute to distinguish between low and high-risk groups and thus be used as a new CVR indicator. METHODS: Four Lp(a) variants, each carrying a different apo(a) isoform (K20, K24, K25, and K29), were purified from plasma of homozygous donors and their interfacial properties characterized using ellipsometry and surface pressure techniques. RESULTS: Ellipsometry measurements revealed that these isoforms had a similar propensity to form adsorbed layers at hydrophobic-hydrophilic interfaces, but surface pressure enabled to clearly separate them into two groups: K20 and K24 on one side, and K25 and K29 on the other side. CONCLUSION: Though K24 and K25 differ only by a single KIV-2 domain, their sharp difference in surface pressure suggests a critical threshold between the two Lp(a) forms, providing insights into the use of condensed matter approaches to monitor CVR. Our findings may represent a new laboratory window to assist medical decisions and to develop precision medicine treatments, practices, and products for CVR, which can be extended to other cardiovascular disease conditions.

A Molecular Modeling Approach to Identify Novel Inhibitors of the Major Facilitator Superfamily of Efflux Pump Transporters.

Multidrug efflux systems play a prominent role in medicine, as they are important contributors to bacterial antibiotic resistance. NorA is an efflux pump transporter from the major facilitator superfamily that expels numerous drug compounds across the inner membrane of Staphylococcus aureus (S. aureus). The design of novel inhibitors to combat drug efflux could offer new opportunities to avoid the problem of antibiotic resistance. In this study, we performed molecular modeling studies in an effort to discover novel NorA efflux pump inhibitors. A group of over 673 compounds from the PubChem database with a high (>80%) level of similarity to the chemical structure of capsaicin was used to study the binding affinity of small molecule compounds for the NorA efflux pump. Ten potential lead compounds displayed a good druggability profile, with one in particular (CID 44330438) providing new insight into the molecular mechanism of the inhibition of major facilitator superfamily (MFS) efflux pump transporters. It is our hope that the overall strategy described in this study, and the structural information of the potential novel inhibitors thus identified, will stimulate others to pursue the development of better drugs to tackle multidrug resistance in S. aureus.

Combinatorial Use of Chitosan Nanoparticles, Reversine, and Ionising Radiation on Breast Cancer Cells Associated with Mitosis Deregulation.

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.

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.

The tetratricopeptide motif repeat (TPR) is an alpha-helix-turn-alpha-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.