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RSC Advances, 2017

Colorimetric detection of cholic acid based on an aptamer adsorbed gold nanoprobe

Gu, Yueqing Lab, China Pharmaceutical University

Cholic acid (CA) has been proved as an effective clinical biomarker for disease diagnosis and treatment monitoring. However, traditional CA detection methods are tedious, time-consuming and expensive, and the results cannot be visualized. Herein, we present a facile, low-cost, fast and label-free biosensor platform based on a CA aptamer (48 nt) modified gold nanoprobe for CA detection. CA can bind withthe surface-adsorbed CA aptamers competitively and detach the aptamer from the gold nanoparticles (Au NPs). Au NPs modified with aptamers of different densities will grow into morphologically varied nanostructures. The result can be observed visibly and quantified easily with the localized surface plasmon resonance (LSPR) spectra. The detection limit (LOD) was 1 mM, which was lower than the existing colorimetric detection methods based on Au NPs. Furthermore, the affinity between CA and 48 nt aptamer was verified for the first time by MicroScale Thermophoresis (MST) technology.

Biochem Biophys Res Commun, 2017

Structural insights into the functional role of GMP in modulating the YfiBNR system

Jiang Tao Lab. Institute of Biophysics, CAS

YfiBNR, a tripartite cyclic-di-GMP (c-di-GMP) signalling system, plays an important role in biofilm formation of the gram-negative bacterium P. aeruginosa, which regulates the cellular processes strongly associated with chronic lung infections and drug resistance. The outer-membrane lipoprotein YfiB can release the inhibition of the inner membrane protein YfiN by sequestering the periplasmatic protein YfiR, resulting in the activation of diguanylate cyclase activity of YfiN and the production of c-di-GMP. In contrast to the extensive studies on c-di-GMP, little is known about how GMP acts in the YfiBNR system. Here, we report the crystal structures of YfiR complexed with GMP and YfiBL43P-YfiR complexed with GMP. In the YfiR-GMP complex, GMP is located in a hydrophilic pocket formed by R175/H177/R60, while in the YfiBL43P-YfiR-GMP complex, GMP is located in a slightly separated hydrophilic pocket, with GMP forming hydrogen bonds with both YfiB and YfiR. A binding affinity test and biofilm formation assay indicated that GMP may activate diguanylate cyclase activity to promote biofilm production by promoting the affinity of YfiB and YfiR. This finding thus provides a new perspective for preventing biofilm-related antibiotic resistance and chronic infections.

PLoS Pathog, 2017

Rice stripe virus NS3 protein regulates primary miRNA processing through association with the miRNA biogenesis factor OsDRB1 and facilitates virus infection in rice

Li Yi Lab. Peking University

MicroRNAs (miRNAs) are small regulatory RNAs processed from primary miRNA transcripts, and plant miRNAs play important roles in plant growth, development, and response to infection by microbes. Microbial infections broadly alter miRNA biogenesis, but the underlying mechanisms remain poorly understood. In this study, we report that the Rice stripe virus (RSV)-encoded nonstructural protein 3 (NS3) interacts with OsDRB1, an indispensable component of the rice (Oryza sativa) miRNA-processing complex. Moreover, the NS3-OsDRB1 interaction occurs at the sites required for OsDRB1 self-interaction, which is essential for miRNA biogenesis. Further analysis revealed that NS3 acts as a scaffold between OsDRB1 and pri-miRNAs to regulate their association and aids in vivo processing of pri-miRNAs. Genetic evidence in Arabidopsis showed that NS3 can partially substitute for the function of double-stranded RNA binding domain (dsRBD) of AtDRB1/AtHYL1 during miRNA biogenesis. As a result, NS3 induces the accumulation of several miRNAs, most of which target pivotal genes associated with development or pathogen resistance. In contrast, a mutant version of NS3 (mNS3), which still associated with OsDRB1 but has defects in pri-miRNA binding, reduces accumulation of these miRNAs. Transgenic rice lines expressing NS3 exhibited significantly higher susceptibility to RSV infection compared with non-transgenic wild-type plants, whereas the transgenic lines expressing mNS3 showed a less-sensitive response. Our findings revealed a previously unknown mechanism in which a viral protein hijacks OsDRB1, a key component of the processing complex, for miRNA biogenesis and enhances viral infection and pathogenesis in rice.

Elife, 2017

A viral protein promotes host SAMS1 activity and ethylene production for the benefit of virus infection

Li, Yi Lab. Peking University

Ethylene plays critical roles in plant development and biotic stress response, but the mechanism of ethylene in host antiviral response remains unclear. Here, we report that Rice dwarf virus (RDV) triggers ethylene production by stimulating the activity of S-adenosyl-L-methionine synthetase (SAMS), a key component of the ethylene synthesis pathway, resulting in elevated susceptibility to RDV. RDV-encoded Pns11 protein specifically interacted with OsSAMS1 to enhance its enzymatic activity, leading to higher ethylene levels in both RDV-infected and Pns11-overexpressing rice. Consistent with a counter-defense role for ethylene, Pns11-overexpressing rice, as well as those overexpressing OsSAMS1, were substantially more susceptible to RDV infection, and a similar effect was observed in rice plants treated with an ethylene precursor. Conversely, OsSAMS1-knockout mutants, as well as an osein2 mutant defective in ethylene signaling, resisted RDV infection more robustly. Our findings uncover a novel mechanism which RDV manipulates ethylene biosynthesis in the host plants to achieve efficient infection.

Plant Physiology, 2017

A novel chemical inhibitor of ABA signaling targets all ABA receptors

Zhu Jiankang Lab & Zhao Yang Lab, Institute of Plant Physiology and Ecology & Shanghai Center for Plant Stress Biology

Abscisic acid (ABA), the most important stress-induced phytohormone, regulates seed dormancy, germination, plant senescence and the abiotic stress response. ABA signaling is repressed by group-A protein phosphatases 2C (PP2Cs), and then ABA binds to its receptor of the PYR/PYL/RCAR family, which in turn inhibits PP2Cs and activates downstream ABA signaling. The agonist/antagonist of ABA receptors have the potential to unveil the ABA signaling machinery and to become lead compounds for agrochemicals, however, up to now, no broad-spectrum antagonists of ABA receptors blocking all PYR/PYL-PP2C interactions have been identified. Here, by using chemical genetics screenings, we identified ABA antagonist 1 (AA1), the first broad-spectrum antagonist of ABA receptors. Physiological analyses revealed that AA1 is sufficiently active to block ABA signaling. AA1 interfered with all the PYR/PYL-HAB1 interactions, and the diminished PYR/PYL-HAB1 interactions in turn restored the activity of HAB1. AA1 binds to all 13 members. Molecular dockings, the non-AA1 bound PYL2 variant and competitive binding assays demonstrated that AA1 enters into the ligand-binding pocket of PYL2. By using AA1, we tested the genetic relationships of ABA receptors with other core components of ABA signaling, demonstrating that AA1 is a powerful tool to sidestep this genetic redundancy of PYR/PYLs. Besides, the application of AA1 delays leaf senescence. Thus, our study developed an efficient broad-spectrum antagonist of ABA receptors, and demonstrated that plant senescence can be chemically controlled through AA1, with a simple and easy to synthesis structure, allowing its availability and utility as a chemical probe synthesized in large quantities, indicating its potential application in agriculture.

Proc Natl Acad Sci , 2017

Burkholderia cenocepacia integrates cis-2-dodecenoic acid and cyclic dimeric guanosine monophosphate signals to control virulence

Deng, Yinyue Lab. South China Agricultural University

Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In Burkholderia cenocepacia, the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal cis-2-dodecenoic acid, also called Burkholderia diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in rpfR and gtrR had overlapping effects on both the B. cenocepacia transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows B. cenocepacia to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria.

Cell Rep, 2017

A Bacterial Receptor PcrK Senses the Plant Hormone Cytokinin to Promote Adaptation to Oxidative Stress

Qian, Wei Lab. Institute of Microbiology, CAS

Recognition of the host plant is a prerequisite for infection by pathogenic bacteria. However, how bacterial cells sense plant-derived stimuli, especially chemicals that function in regulating plant development, remains completely unknown. Here, we have identified a membrane-bound histidine kinase of the phytopathogenic bacterium Xanthomonas campestris, PcrK, as a bacterial receptor that specifically detects the plant cytokinin 2-isopentenyladenine (2iP). 2iP binds to the extracytoplasmic region of PcrK to decrease its autokinase activity. Through a four-step phosphorelay, 2iP stimulation decreased the phosphorylation level of PcrR, the cognate response regulator of PcrK, to activate the phosphodiesterase activity of PcrR in degrading the second messenger 3',5'-cyclic diguanylic acid. 2iP perception by the PcrK-PcrR remarkably improves bacterial tolerance to oxidative stress by regulating the transcription of 56 genes, including the virulence-associated TonB-dependent receptor gene ctrA. Our results reveal an evolutionarily conserved, inter-kingdom signaling by which phytopathogenic bacteria intercept a plant hormone signal to promote adaptation to oxidative stress.

Sci Rep, 2017

Structure determination and activity manipulation of the turfgrass ABA receptor FePYR1

Zhu, Jiankang Lab. Shanghai Center for Plant Stress Biology, CAS

Turfgrass are widely cultivated ornamental plants that have important ecological, societal and economical values. However, many turfgrass species are susceptible to drought and demand frequent irrigation thus consuming large amounts of water. With the ultimate goal of improving drought resistance in turfgrass, we identified several ABA receptors in turfgrass that are important to mediate ABA signaling and drought stress response. The ABA receptor FePYR1 from turfgrass Festuca elata was demonstrated to bind ABA as a monomer. Crystal structure analysis revealed that FePYR1 recognizes and binds ABA by the common gate-latch-lock mechanism resembling the Arabidopsis ABA receptors, but the ABA binding pocket in FePYR1 shows discrepant residues resulting in different binding affinity to ABA. Structure-guided alterations of amino acid residues in FePYR1 generated ABA receptor variants with significantly increased ABA binding affinity. Expression of FePYR1 in Arabidopsis conferred enhanced drought resistance in the transgenic plants. These findings provided detailed information about FePYR1 and demonstrated that structure-assisted engineering could create superior ABA receptors for improving plant drought resistance. The detailed structural information of FePYR1 would also assist future rational design of small molecules targeting specific ABA receptors in economically important plant species.

Science, 2017

Arabidopsis pollen tube integrity and sperm release are regulated by RALF-mediated signaling

Qu, Lijia Lab. Peking University

In flowering plants, fertilization requires complex cell-to-cell communication events between the pollen tube and the female reproductive tissues, which are controlled by extracellular signaling molecules interacting with receptors at the pollen tube surface. We found that two such receptors in Arabidopsis, BUPS1 and BUPS2, and their peptide ligands, RALF4 and RALF19, are pollen tube-expressed and are required to maintain pollen tube integrity. BUPS1 and BUPS2 interact with receptors ANXUR1 and ANXUR2 via their ectodomains, and both sets of receptors bind RALF4 and RALF19. These receptor-ligand interactions are in competition with the female-derived ligand RALF34, which induces pollen tube bursting at nanomolar concentrations. We propose that RALF34 replaces RALF4 and RALF19 at the interface of pollen tube-female gametophyte contact, thereby deregulating BUPS-ANXUR signaling and in turn leading to pollen tube rupture and sperm release.

Nature, 2017

Structural basis of CRISPR-SpyCas9 inhibition by an anti-CRISPR protein

Huang, Zhiwei Lab, Harbin Institute of Technology

CRISPR-Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing 'off-switch' tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.

Nature, 2016

A receptor heteromer mediates the male perception of female attractants in plants

Yang Weicai Lab, Institute of Genetics and Developmental Biology, CAS

Sexual reproduction requires recognition between the male and female gametes. In flowering plants, the immobile sperms are delivered to the ovule-enclosed female gametophyte by guided pollen tube growth.  Although the female gametophyte-secreted peptides have been identified to be the chemotactic attractant to the pollen tube1–3, the male receptor(s) is still unknown. Here we identify a cell-surface receptor heteromer, MDIS1–MIK, on the pollen tube that perceives female attractant LURE1 in Arabidopsis thaliana. MDIS1, MIK1 and MIK2 are plasma-membrane-localized receptor-like kinases with extracellular leucine-rich repeats and an intracellular kinase domain. LURE1 specifically binds the extracellular domains of MDIS1, MIK1 and MIK2, whereas mdis1 and mik1 mik2 mutant pollen tubes respond less sensitively to LURE1. Furthermore, LURE1 triggers dimerization of the receptors and activates the kinase activity of MIK1. Importantly, transformation of AtMDIS1 to the sister species Capsella rubella can partially break down the reproductive isolation barrier. Our findings reveal a new  mechanism of the male perception of the female attracting signals.

Nature Strunctural & Molecular Biology

The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations

Suman Nag,Darshan V Trivedi,Saswata S Sarkar,Arjun S Adhikari,Margaret S Sunitha,Shirley Sutton,Kathleen M Ruppel&James A SpudichAuthor


Abstract: Hypertrophic cardiomyopathy (HCM) is primarily caused by mutations in β-cardiac myosin and myosin-binding protein-C (MyBP-C). Changes in the contractile parameters of myosin measured so far do not explain the clinical hypercontractility caused by such mutations. We propose that hypercontractility is due to an increase in the number of myosin heads (S1) that are accessible for force production. In support of this hypothesis, we demonstrate myosin tail (S2)-dependent functional regulation of actin-activated human β-cardiac myosin ATPase. In addition, we show that both S2 and MyBP-C bind to S1 and that phosphorylation of either S1 or MyBP-C weakens these interactions. Importantly, the S1-S2 interaction is also weakened by four myosin HCM-causing mutations but not by two other mutations. To explain these experimental results, we propose a working structural model involving multiple interactions, including those with myosin's own S2 and MyBP-C, that hold myosin in a sequestered state.

bioRxiv

Beyond the myosin mesa a potential unifying hypothesis on the underlying molecular basis of hyper contractility caused by a majority of hypertrophic cardiomyopathy mutations

Abstract:Hypertrophic cardiomyopathy (HCM), the most commonly occurring inherited cardiovascular disease, is primarily caused by mutations in human β-cardiac myosin and myosin binding protein-C. It has been thought that such mutations in myosin increase the intrinsic force of the motor, its velocity of contraction, or its ATPase activity, giving rise to hyper-contractility. We hypothesize that while these parameters are mildly affected by most myosin HCM-causing mutations, a major effect of a majority of myosin HCM mutations is likely to involve an increase in the number of myosin heads that are functionally accessible (Na) for interaction with actin in the sarcomere. We consider a model involving three types of interactions involving the myosin mesa and the converter domain of the myosin motor that hold myosin heads in a sequestered state, likely to be released in a graded manner as the demands on the heart increase: 1) the two myosin heads binding to one another, 2) one head binding to its own coiled-coil tail, and 3) the other head binding to myosin binding protein-C. In addition there is clear evidence of interaction between the coiled-coil tail of myosin and myosin binding protein-C. Experimentally, here we focus on myosin head binding to its own coiled-coil tail and to myosin binding protein-C. We show that phosphorylation of the myosin regulatory light chain and myosin binding protein-C weaken these respective associations, consistent with known enhancements of sarcomere function by these phosphorylations. We show that these interactions are weakened as a result of myosin HCM mutations, in a manner consistent with our structural model. Our data suggests a potential unifying hypothesis for the molecular basis of hyper-contractility caused by human hypertrophic cardiomyopathy myosin mutations, whereby the mutations give rise to an increase in the number of myosin heads that are functionally accessible for interaction with actin in the sarcomere, causing the hyper-contractility observed clinically.

Methods in Molecular Biology

Aptamer Binding Studies Using MicroScale Thermophoresis

Dennis Breitsprecher, Nina Schlinck, David Witte, Stefan Duhr, Philipp Baaske and Thomas Schubert

Abstract:The characterization and development of highly specific aptamers requires the analysis of the interaction strength between aptamer and target. MicroScale Thermophoresis (MST) is a rapid and precise method to quantify biomolecular interactions in solution at microliter scale. The basis of this technology is a physical effect referred to as thermophoresis, which describes the directed movement of molecules through temperature gradients. The thermophoretic properties of a molecule depend on its size, charge, and hydration shell. Since at least one of these parameters is altered upon binding of a ligand, this method can be used to analyze virtually any biomolecular interaction in any buffer or complex bioliquid. This section provides a detailed protocol describing how MST is used to obtain quantitative binding parameters for aptamer-target interactions. The two DNA-aptamers HD1 and HD22, which are targeted against human thrombin, are used as model systems to demonstrate a rapid and straightforward screening approach to determine optimal buffer conditions.


Keywords
Monolith NT.115Pico, Protein-Nucleic acids interaction, high affinity interaction, Biomolecular Interaction

Journal Homepage

Orthogonal Methods for Characterizing the Unfolding of Therapeutic

Orthogonal Methods for Characterizing the Unfolding of Therapeutic Monoclonal Antibodies: Differential Scanning Calorimetry, Isothermal Chemical Denaturation, and Intrinsic Fluorescence with Concomitant Static Light Scattering

Methods in Enzymology


Deniz B. Temel, Pavel Landsman, Mark L. Brader

Abstract
Evaluating prospective protein pharmaceutical stability from accelerated screening is a critical challenge in biotherapeutic discovery and development. Measurements of protein unfolding transitions are widely employed for comparing candidate molecules and formulations; however, the interrelationships between intrinsic protein conformational stability and pharmaceutical robustness are complex and thermal unfolding measurements can be misleading. Beyond the discovery phase of drug development, astute formulation design is one of the most crucial factors enabling the protein to resist damage to its higher order structure-initially from bioprocessing stresses, then from stresses encountered during its journey from the product manufacturing site to the bloodstream of the patient. Therapeutic monoclonal antibodies are multidomain proteins that represent a large and growing segment of the biotechnology pipeline. In this chapter, we describe how differential scanning calorimetry may be leveraged synergistically with isothermal chemical denaturation and intrinsic fluorescence with concomitant static light scattering to elucidate characteristics of mAb unfolding and aggregation that are helpful toward understanding and designing optimal pharmaceutical compositions for these molecules.


An Automated Microscale Thermophoresis Screening Approach for...

An Automated Microscale Thermophoresis Screening Approach for Fragment-Based Lead Discovery
Journal of Biomolecular Screening

Pawel Linke, Kwame Amaning, Melanie Maschberger, Francois Vallee, Valerie Steier, Philipp Baaske, Stefan Duhr, Dennis Breitsprecher, Alexey Rak


Abstract
Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns.

Keywords
Monolith NT.Automated, Protein-Fragment Interaction, Screening, Mulitple Component Interactions, Biomolecular Interaction

DNA-binding proteins from marine bacteria expand the known sequence...

DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats

Nucleic Acids Research


Orlando de Lange, Christina Wolf, Philipp Thiel, Jens Krüger, Christian Kleusch,Oliver Kohlbacher, Thomas Lahaye


Abstract
Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats.


Keywords
Monolith NT.115, Protein-Nucleic Acids, Biomolecular Interaction, Prometheus NT.48, Protein Stability





Competition between MPS1 and microtubules at kinetochores regulates...

Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling

Science


Abstract
Cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules through kinetochores and the spindle assembly checkpoint (SAC) is satisfied. We show that the amino-terminal localization module of the SAC protein kinase MPS1 (monopolar spindle 1) directly interacts with the HEC1 (highly expressed in cancer 1) calponin homology domain in the NDC80 (nuclear division cycle 80) kinetochore complex in vitro, in a phosphorylation-dependent manner. Microtubule polymers disrupted this interaction. In cells, MPS1 binding to kinetochores or to ectopic NDC80 complexes was prevented by end-on microtubule attachment, independent of known kinetochore protein-removal mechanisms. Competition for kinetochore binding between SAC proteins and microtubules provides a direct and perhaps evolutionarily conserved way to detect a properly organized spindle ready for cell division.


Keywords: 
Monolith NT.115, Protein-Protein, Multiple Component Interactions, Biomolecular Interaction

CELL DIVISION CYCLE. Kinetochore attachment sensed by competitive...

CELL DIVISION CYCLE. Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80C

Science


Abstract
The spindle checkpoint of the cell division cycle senses kinetochores that are not attached to microtubules and prevents precocious onset of anaphase, which can lead to aneuploidy. The nuclear division cycle 80 complex (Ndc80C) is a major microtubule receptor at the kinetochore. Ndc80C also mediates the kinetochore recruitment of checkpoint proteins. We found that the checkpoint protein kinase monopolar spindle 1 (Mps1) directly bound to Ndc80C through two independent interactions. Both interactions involved the microtubule-binding surfaces of Ndc80C and were directly inhibited in the presence of microtubules. Elimination of one such interaction in human cells caused checkpoint defects expected from a failure to detect unattached kinetochores. Competition between Mps1 and microtubules for Ndc80C binding thus constitutes a direct mechanism for the detection of unattached kinetochores.


Keywords: Monolith NT.115, Protein-Protein, Biomolecular Interaction

VCAM-1 directed target-sensitive liposomes carrying CCR2...

VCAM-1 directed target-sensitive liposomes carrying CCR2 antagonists bind to activated endothelium and reduce adhesion and transmigration of monocytes

European Journal of Pharmaceutics and Biopharmaceutics


Manuela Calin, Daniela Stan, Martin Schlesinger, Viorel Simion, Mariana Deleanu, Cristina Ana Constantinescu, Ana-Maria Gan, Monica Madalina Pirvulescu, Elena Butoi, Ileana Manduteanu, Marian Bota, Marius Enachescu, Lubor Borsig, Gerd Bendas, Maya Simionescu


Abstract

Chemokines are critically involved in the development of chronic inflammatory-associated diseases such as atherosclerosis. We hypothesized that targeted delivery of compounds to the surface of activated endothelial cells (EC) interferes with chemokine/receptor interaction and thereby efficiently blocks inflammation. We developed PEGylated target-sensitive liposomes (TSL) encapsulating a CCR2 antagonist (Teijin compound 1) coupled with a specific peptide recognized by endothelial VCAM-1 (Vp-TSL-Tj). TSL were characterized for size (by dynamic light scattering), the amount of peptide coupled at the liposomal surface and Teijin release (by HPLC). We report that Vp-TSL-Tj binds specifically to activated EC in vitro and in situ, release the entrapped Teijin and prevent the transmigration of monocytes through activated EC. This is the first evidence that nanocarriers which transport and release chemokine inhibitors at specific pathological sites can reduce chemokine-dependent inflammatory processes.


Keywords: CCR2 antagonist; Endothelium; Monocyte; Target-sensitive liposomes; Targeted drug delivery; VCAM-1

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Surface acoustic wave biosensor as a functional quality method in...

Surface acoustic wave biosensor as a functional quality method in pharmaceutics

Sensors and Actuators B: Chemical


Nathalie Bracke, Sophia Barhdadi, Evelien Wynendaele, Bert Gevaert, Matthias D’Hondt, Bart De Spiegeleer


Abstract

With the recent successes of biopharmaceuticals, new bioanalytical tools are needed for the functional quality characterization of new biological entities and biosimilars towards marketing authorization approval and during the product life. Biosensors are emerging technologies in the pharmaceutical field in drug discovery, development and Quality Control (QC) stages; however, they are not yet included in any pharmacopoeia. Surface acoustic wave (SAW) biosensors allow the user to detect and quantify binding events, giving information not only on affinity (KD) and kinetics (kon and koff), but also on viscoelastic effects. We present here an analytical quality by design (aQbD) based approach using somatropin (rhGH, recombinant human growth hormone) and derivatives as model biotechnological drugs and an antibody interaction partner to evaluate the functional quality. Using techniques described in ICH Q2, Q5 and Q8-11, we evaluated the performance of this SAW biosensor technique from a pharmaceutical quality point of view. CMAs (Critical Method Attributes) such as sensitivity, selectivity and variability are evaluated as a function of different experimental variables using DoEs (design of experiments). We suggest qualification tests as well as SSTs (system suitability tests), as required by the pharmaceutical competent authorities and recommend the inclusion of biosensor techniques into the different pharmacopoeia and pharmaceutical guidelines.


Keywords: Surface acoustic wave biosensor; Analytical quality by design; (Modified) somatropin; Pharmaceutical GLP/GMP; Biosensor method development