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11. Chemometrics in Protein Formulation:Stability Governed by Repulsion and Protein Unfolding

Author

Kulakova, Alina, Augustijn, Dillen, El Bialy, Inas, Gentiluomo, Lorenzo, Greco, Maria Laura, Hervø-hansen, Stefan, Indrakumar, Sowmya, Mahapatra, Sujata, Martinez Morales, Marcello, Pohl, Christin, Polimeni, Marco, Roche, Aisling, Svilenov, Hristo L., Tosstorff, Andreas, Zalar, Matja, Curtis, Robin, Derrick, Jeremy P., Frieß, Wolfgang, Golovanov, Alexander P., Lund, Mikael, Nørgaard, Allan, Khan, Tarik A., Peters, Günther H. J., Pluen, Alain, Roessner, Dierk, Streicher, Werner W., Van Der Walle, Christopher F., Warwicker, Jim, Uddin, Shahid, Winter, Gerhard, Bukrinski, Jens Thostrup, Rinnan, Åsmund, Harris, Pernille, Kulakova, Alina, Augustijn, Dillen, El Bialy, Inas, Gentiluomo, Lorenzo, Greco, Maria Laura, Hervø-hansen, Stefan, Indrakumar, Sowmya, Mahapatra, Sujata, Martinez Morales, Marcello, Pohl, Christin, Polimeni, Marco, Roche, Aisling, Svilenov, Hristo L., Tosstorff, Andreas, Zalar, Matja, Curtis, Robin, Derrick, Jeremy P., Frieß, Wolfgang, Golovanov, Alexander P., Lund, Mikael, Nørgaard, Allan, Khan, Tarik A., Peters, Günther H. J., Pluen, Alain, Roessner, Dierk, Streicher, Werner W., Van Der Walle, Christopher F., Warwicker, Jim, Uddin, Shahid, Winter, Gerhard, Bukrinski, Jens Thostrup, Rinnan, Åsmund, and Harris, Pernille

Abstract

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein–protein repulsion and the initial monomer fraction are the most important properties to monitor.

Published
2023

12. Chemometrics in Protein Formulation: Stability Governed by Repulsion and Protein Unfolding

Author

Kulakova, Alina, primary, Augustijn, Dillen, additional, El Bialy, Inas, additional, Gentiluomo, Lorenzo, additional, Greco, Maria Laura, additional, Hervø-Hansen, Stefan, additional, Indrakumar, Sowmya, additional, Mahapatra, Sujata, additional, Martinez Morales, Marcello, additional, Pohl, Christin, additional, Polimeni, Marco, additional, Roche, Aisling, additional, Svilenov, Hristo L., additional, Tosstorff, Andreas, additional, Zalar, Matja, additional, Curtis, Robin, additional, Derrick, Jeremy P., additional, Frieß, Wolfgang, additional, Golovanov, Alexander P., additional, Lund, Mikael, additional, Nørgaard, Allan, additional, Khan, Tarik A., additional, Peters, Günther H. J., additional, Pluen, Alain, additional, Roessner, Dierk, additional, Streicher, Werner W., additional, van der Walle, Christopher F., additional, Warwicker, Jim, additional, Uddin, Shahid, additional, Winter, Gerhard, additional, Bukrinski, Jens Thostrup, additional, Rinnan, Åsmund, additional, and Harris, Pernille, additional

Published
2023
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13. Structure of Dimeric and Tetrameric Complexes of the BAR Domain Protein PICK1 Determined by Small-Angle X-Ray Scattering

Author

Karlsen, Morten L., Thorsen, Thor S., Johner, Niklaus, Ammendrup-Johnsen, Ina, Erlendsson, Simon, Tian, Xinsheng, Simonsen, Jens B., Høiberg-Nielsen, Rasmus, Christensen, Nikolaj M., Khelashvili, George, Streicher, Werner, Teilum, Kaare, Vestergaard, Bente, Weinstein, Harel, Gether, Ulrik, Arleth, Lise, and Madsen, Kenneth L.

Published
2015
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24. Reversible supramolecular assembly of the anti-microbial peptide plectasin into helical non-amyloid fibrils

Author

Pohl, Christin, primary, Effantin, Gregory, additional, Kandiah, Eaazhisai, additional, Meier, Sebastian, additional, Zeng, Guanghong, additional, Streicher, Werner, additional, Peters, Guenther H., additional, Schoehn, Guy, additional, Mueller-Dieckmann, Christoph, additional, Noergaard, Allan, additional, and Harris, Pernille, additional

Published
2021
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26. The Effect of Point Mutations on the Biophysical Properties of an Antimicrobial Peptide: Development of a Screening Protocol for Peptide Stability Screening

Author

Pohl, Christin, primary, Zalar, Matja, additional, Bialy, Inas El, additional, Indrakumar, Sowmya, additional, Peters, Günther H. J., additional, Friess, Wolfgang, additional, Golovanov, Alexander P., additional, Streicher, Werner W., additional, Noergaard, Allan, additional, and Harris, Pernille, additional

Published
2020
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27. Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization

Author

Gentiluomo, Lorenzo, Svilenov, Hristo L, Augustijn, Dillen, El Bialy, Inas, Greco, Maria Laura, Vitaliyivna Kulakova, Alina, Indrakumar, Sowmya, Mahapatra, Sujata, Morales, Marcello Martinez, Pohl, Christin, Roche, Aisling, Tosstorff, Andreas, Curtis, Robin, Derrick, Jeremy P, Nørgaard, Allan, Khan, Tarik A, Peters, Günther H.J., Pluen, Alain, Rinnan, Åsmund, Streicher, Werner W, van der Walle, Christopher F, Uddin, Shahid, Winter, Gerhard, Roessner, Dierk, Harris, Pernille, Frieß, Wolfgang, Gentiluomo, Lorenzo, Svilenov, Hristo L, Augustijn, Dillen, El Bialy, Inas, Greco, Maria Laura, Vitaliyivna Kulakova, Alina, Indrakumar, Sowmya, Mahapatra, Sujata, Morales, Marcello Martinez, Pohl, Christin, Roche, Aisling, Tosstorff, Andreas, Curtis, Robin, Derrick, Jeremy P, Nørgaard, Allan, Khan, Tarik A, Peters, Günther H.J., Pluen, Alain, Rinnan, Åsmund, Streicher, Werner W, van der Walle, Christopher F, Uddin, Shahid, Winter, Gerhard, Roessner, Dierk, Harris, Pernille, and Frieß, Wolfgang

Abstract

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.

Published
2020

28. The Effect of Point Mutations on the Biophysical Properties of an Antimicrobial Peptide:Development of a Screening Protocol for Peptide Stability Screening

Author

Pohl, Christin Alina, Zalar, Matja, Bialy, Inas El, Indrakumar, Sowmya, Peters, Günther H.J., Friess, Wolfgang, Golovanov, Alexander P., Streicher, Werner W., Noergaard, Allan, Harris, Pernille, Pohl, Christin Alina, Zalar, Matja, Bialy, Inas El, Indrakumar, Sowmya, Peters, Günther H.J., Friess, Wolfgang, Golovanov, Alexander P., Streicher, Werner W., Noergaard, Allan, and Harris, Pernille

Abstract

Therapeutic peptides and proteins show enormous potential in the pharmaceutical market, but high costs in discovery and development are limiting factors so far. Single or multiple point mutations are commonly introduced in protein drugs to increase their binding affinity or selectivity. They can also induce adverse properties, which might be overlooked in a functional screen, such as a decreased colloidal or thermal stability, leading to problems in later stages of the development. In this study, we address the effect of point mutations on the stability of the 4.4 kDa antimicrobial peptide plectasin, as a case study. We combined a systematic high-throughput biophysical screen of the peptide thermal and colloidal stability using dynamic light scattering and differential scanning calorimetry with structure-based methods including small-angle X-ray scattering, analytical ultracentrifugation, and nuclear magnetic resonance spectroscopy. Additionally, we applied molecular dynamics simulations to link obtained protein stability parameters to the protein's molecular structure. Despite their predicted structural similarities, all four plectasin variants showed substantially different behavior in solution. We observed an increasing propensity of plectasin to aggregate at a higher pH, and the introduced mutations influenced the type of aggregation. Our strategy for systematically assessing the stability and aggregation of protein drugs is generally applicable and is of particular relevance, given the increasing number of protein drugs in development.

Published
2020

29. Albumin-neprilysin fusion protein:understanding stability using small angle X-ray scattering and molecular dynamic simulations

Author

Kulakova, Alina, Indrakumar, Sowmya, Sønderby Tuelung, Pernille, Mahapatra, Sujata, Streicher, Werner W., Peters, Günther H.J., Harris, Pernille, Kulakova, Alina, Indrakumar, Sowmya, Sønderby Tuelung, Pernille, Mahapatra, Sujata, Streicher, Werner W., Peters, Günther H.J., and Harris, Pernille

Abstract

Fusion technology is widely used in protein-drug development to increase activity, stability, and bioavailability of protein therapeutics. Fusion proteins, like any other type of biopharmaceuticals, need to remain stable during production and storage. Due to the high complexity and additional intramolecular interactions, it is not possible to predict the behavior of fusion proteins based on the behavior the individual proteins. Therefore, understanding the stability of fusion proteins on the molecular level is crucial for the development of biopharmaceuticals. The current study on the albumin-neprilysin (HSA-NEP) fusion protein uses a combination of thermal and chemical unfolding with small angle X-ray scattering and molecular dynamics simulations to show a correlation between decreasing stability and increasing repulsive interactions, which is unusual for most biopharmaceuticals. It is also seen that HSA-NEP is not fully flexible: it is present in both compact and extended conformations. Additionally, the volume fraction of each conformation changes with pH. Finally, the presence of NaCl and arginine increases stability at pH 6.5, but decreases stability at pH 5.0.

Published
2020

31. Advancing Therapeutic Protein Discovery and Development through Comprehensive Computational and Biophysical Characterization

Author

Gentiluomo, Lorenzo, primary, Svilenov, Hristo L., additional, Augustijn, Dillen, additional, El Bialy, Inas, additional, Greco, Maria Laura, additional, Kulakova, Alina, additional, Indrakumar, Sowmya, additional, Mahapatra, Sujata, additional, Morales, Marcello Martinez, additional, Pohl, Christin, additional, Roche, Aisling, additional, Tosstorff, Andreas, additional, Curtis, Robin, additional, Derrick, Jeremy P., additional, Nørgaard, Allan, additional, Khan, Tarik A., additional, Peters, Günther H. J., additional, Pluen, Alain, additional, Rinnan, Åsmund, additional, Streicher, Werner W., additional, van der Walle, Christopher F., additional, Uddin, Shahid, additional, Winter, Gerhard, additional, Roessner, Dierk, additional, Harris, Pernille, additional, and Frieß, Wolfgang, additional

Published
2020
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33. Conformational Stability Study of a Therapeutic Peptide Plectasin Using Molecular Dynamics Simulations in Combination with NMR

Author

Indrakumar, Sowmya, Zalar, Matja, Pohl, Christin, Nørgaard, Allan, Streicher, Werner, Harris, Pernille, Golovanov, Alexander P., Peters, Günther H.J., Indrakumar, Sowmya, Zalar, Matja, Pohl, Christin, Nørgaard, Allan, Streicher, Werner, Harris, Pernille, Golovanov, Alexander P., and Peters, Günther H.J.

Abstract

Plectasin is a small, cysteine-rich peptide antibiotic which belongs to the class of antimicrobial peptides and has potential antibacterial activity against various Gram-positive bacteria. In the current study, the effect of pH and ionic strength (NaCl) on the conformational stability of plectasin variants has been investigated. At all physiochemical conditions, peptide secondary structures are intact throughout simulations. However, flexibility increases with pH because of the change in electrostatics around the distinct anionic tetrapeptide (9-12) stretch. Furthermore, plectasin interactions with NaCl were measured by determining the preferential interaction coefficients, Γ23. Generally, wild-type plectasin has higher preference for sodium ions as 9ASP is mutated in other variants. Overall, the Γ23 trend with pH for the two salt conditions remain the same for all variants predominately having accumulation of sodium ions around 10GLU and 12ASP. Insignificant changes in the overall peptide conformational stability are in agreement with the fact that plectasin has three cystines. Thermodynamic integration molecular dynamics simulations supplemented with nuclear magnetic resonance were employed to determine the degree of involvement of three different cystines to the overall structural integrity of the peptide. Both methods show the same order of cystine reduction and complete unfolding is observed only upon reduction of all cystines.

Published
2019

34. Annexin I and annexin II N-terminal peptides binding to S100 protein family members: specificity and thermodynamic characterization

Author

Streicher, Werner W., Lopez, Maria M., and Makhatadze, George I.

Subjects
Annexins -- Chemical properties, Annexins -- Structure, Protein binding -- Analysis, Protein folding -- Analysis, Thermodynamics -- Analysis, Biological sciences, Chemistry
Abstract

The specificity of binding of representative members of the human S100 proteins to short N-terminal peptides of annexin I (AnI) and annexin II (AnII) was examined to understand exact biological functions of the S100 proteins in response to changing calcium levels. The thermodynamics of interactions indicated that both AnI and AnII undergo entropically driven coil-to-helix transition upon binding to their respective binding partners.

Published
2009

35. Unfolding thermodynamics of Trp-cage, a 20 residue miniprotein, studied by differential scanning calorimetry and circular dichroism spectroscopy

Author

Streicher, Werner W. and Makhatadze, George I.

Subjects
Bacterial proteins -- Structure, Bacterial proteins -- Properties, Calorimetry -- Analysis, Circular dichroism -- Analysis, Thermodynamics -- Analysis, Biological sciences, Chemistry
Abstract

The thermodynamics of unfolding of Trp-cage is studied by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy in order to analyze the unfolding mechanism. The thermodynamic parameters have estimated the stability of the Trp-cage miniprotein at 25 degree Celsius that is only 30% more than the thermal fluctuation energy.

Published
2007

36. Calorimetric evidence for a two-state unfolding of the beta-hairpin peptide trpzip4

Author

Streicher, Werner W. and Makhatadze, George I.

Subjects
Peptides -- Structure, Peptides -- Chemical properties, Proteins -- Structure, Proteins -- Research, Chemistry
Abstract

The effect of loop length, beta-sheet forming propensity, formation of cross-strand interactions, and types of interactions of stability is determined by using host-guest beta-hairpin models. The results have shown that stable beta-hairpins formed by the tryptophan zipper 4 (trpzip4), or beta-sheets, might be responsible for the two-state equilibrium and kinetic unfolding of all beta-proteins.

Published
2006

38. Investigating the function of Fc -specific binding of IgM to Plasmodium falciparum erythrocyte membrane protein 1 mediating erythrocyte rosetting

Author

Stevenson, Liz, Huda, Pie, Jeppesen, Anine, Laursen, Erik, Rowe, J Alexandra, Craig, Alister, Streicher, Werner, Barfod, Lea, and Hviid, Lars

Subjects
Erythrocytes, Plasmodium falciparum, Protozoan Proteins, wh_150, Antibodies, wc_750, Immunoglobulin Fc Fragments, qu_58.5, Immunoglobulin M, qx_135, Protozoan, embryonic structures, parasitic diseases, Humans, Antigens, Protein Binding
Abstract

Acquired protection from Plasmodium falciparum malaria takes years to develop, probably reflecting the ability of the parasites to evade immunity. A recent example of this is the binding of the Fc region of IgM to VAR2CSA-type PfEMP1. This interferes with specific IgG recognition and phagocytosis of opsonized infected erythrocytes (IEs) without compromising the placental IE adhesion mediated by this PfEMP1 type. IgM also binds via Fc to several other PfEMP1 proteins, where it has been proposed to facilitate rosetting (binding of uninfected erythrocytes to a central IE). To further dissect the functional role of Fc -mediated IgM binding to PfEMP1, we studied the PfEMP1 protein HB3VAR06, which mediates rosetting and binds IgM. Binding of IgM to this PfEMP1 involved the Fc domains Cμ3-Cμ4 in IgM and the penultimate DBL domain (DBLζ2) at the C-terminus of HB3VAR06. However, IgM binding did not inhibit specific IgG labelling of HB3VAR06 or shield IgG-opsonized IEs from phagocytosis. Instead, IgM was required for rosetting, and each pentameric IgM molecule could bind two HB3VAR06 molecules. Together, our data indicate that the primary function of Fc -mediated IgM binding in rosetting is not to shield IE from specific IgG recognition and phagocytosis as in VAR2CSA-type PfEMP1. Rather, the function appears to be strengthening of IE-erythrocyte interactions. In conclusion, our study provides new evidence on the molecular details and functional significance of rosetting, a long-recognized marker of parasites that cause severe P. falciparum malaria.

Published
2015
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39. A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Author

Zhao, Huaying, Ghirlando, Rodolfo, Alfonso, Carlos, Arisaka, Fumio, Attali, Ilan, Bain, David L., Bakhtina, Marina M., Becker, Donald F., Bedwell, Gregory J., Bekdemir, Ahmet, Besong, Tabot M.D., Birck, Catherine, Brautigam, Chad A., Brennerman, William, Byron, Olwyn, Bzowska, Agnieszka, Chaires, Jonathan B., Chaton, Catherine T., Connaghan, Keith D., Crowley, Kimberly A., Curth, Ute, Daviter, Tina, Dean, William L., Ebel, Christine, Eckert, Debra M., Eisele, Leslie E., Eisenstein, Edward, England, Patrick, Escalante, Carlos, Fagan, Jeffrey A., Fairman, Robert, Finn, Ron M., Fischle, Wolfgang, Gor, Jayesh, Gustafsson, Henning, Hall, Damien, Harding, Stephen E., Herr, Andrew B., Howell, Elizabeth E., Isaac, Richard S., Jao, Shu-Chuan, Jose, Davis, Kim, Soon-Jong, Kokona, Bashkim, Kornblatt, Jack A., Kosek, Dalibor, Krayukhina, Elena, Krzizike, Daniel, Kusznir, Eric A., Kwon, Hyewon, Larson, Adam, Laue, Thomas M., Le Roy, Aline, Leech, Andrew P., Lilie, Hauke, Luger, Karolin, Luque-Ortega, Juan R., Ma, Jia, May, Carrie A., Maynard, Ernest L., Modrak-Wojcik, Anna, Mok, Yee-Foong, Nagel-Steger, Luitgard, Narlikar, Geeta J., Noda, Masanori, Nourse, Amanda, Obsil, Tomas, Park, Chad K., Park, Jin-Ku, Pawelek, Peter D., Perdue, Erby E., Perkins, Stephen J., Perugini, Matthew A., Peterson, Craig L., Peverelli, Martin G., Piszczek, Grzegorz, Prag, Gali, Prevelige, Peter E., Raynal, Bertrand D.E., Rezabkova, Lenka, Richter, Klaus, Ringel, Alison E., Rosenberg, Rose, Rowe, Arthur J., Rufer, Arne C., Scott, David J., Seravalli, Javier G., Solovyova, Alexandra S., Song, Renjie, Staunton, David, Stoddard, Caitlin, Stott, Katherine, Strauss, Holger M., Streicher, Werner W., Sumida, John P., Swygert, Sarah G., Szczepanowski, Roman H., Tessmer, Ingrid, Toth, Ronald T., Tripathy, Ashutosh, Uchiyama, Susumu, Uebel, Stephan F.W., Unzai, Satoru, Gruber, Anna Vitlin, von Hippel, Peter H., Wandrey, Christine, Wang, Szu-Huan, Weitzel, Steven E., Wielgus-Kutrowska, Beata, Wolberger, Cynthia, Wolff, Martin, Wright, Edward, Wu, Yu-Sung, Wubben, Jacinta M., and Schuck, Peter

Abstract

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

Published
2015

40. A novel TPR-BEN domain interaction mediates PICH-BEND3 association

Author

Pitchai, Ganesha P, Kaulich, Manuel, Bizard, Anna H, Mesa, Pablo, Yao, Qi, Sarlos, Kata, Streicher, Werner W, Nigg, Erich A, Montoya, Guillermo, Hickson, Ian D, Pitchai, Ganesha P, Kaulich, Manuel, Bizard, Anna H, Mesa, Pablo, Yao, Qi, Sarlos, Kata, Streicher, Werner W, Nigg, Erich A, Montoya, Guillermo, and Hickson, Ian D

Abstract

PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain.

Published
2017

41. Characterization of the NTPR and BD1 interacting domains of the human PICH-BEND3 complex

Author

Pitchai, Ganesha P, Hickson, Ian D, Streicher, Werner, Montoya, Guillermo, Mesa, Pablo, Pitchai, Ganesha P, Hickson, Ian D, Streicher, Werner, Montoya, Guillermo, and Mesa, Pablo

Abstract

Chromosome integrity depends on DNA structure-specific processing complexes that resolve DNA entanglement between sister chromatids. If left unresolved, these entanglements can generate either chromatin bridging or ultrafine DNA bridging in the anaphase of mitosis. These bridge structures are defined by the presence of the PICH protein, which interacts with the BEND3 protein in mitosis. To obtain structural insights into PICH-BEND3 complex formation at the atomic level, their respective NTPR and BD1 domains were cloned, overexpressed and crystallized using 1.56 M ammonium sulfate as a precipitant at pH 7.0. The protein complex readily formed large hexagonal crystals belonging to space group P6122, with unit-cell parameters a = b = 47.28, c = 431.58 Å and with one heterodimer in the asymmetric unit. A complete multiwavelength anomalous dispersion (MAD) data set extending to 2.2 Å resolution was collected from a selenomethionine-labelled crystal at the Swiss Light Source.

Published
2016

42. Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

Author

Sedgwick, Garry G, Larsen, Marie Sofie Yoo, Lischetti, Tiziana, Streicher, Werner, Jersie-Christensen, Rosa Rakownikow, Olsen, Jesper V, Nilsson, Jakob, Sedgwick, Garry G, Larsen, Marie Sofie Yoo, Lischetti, Tiziana, Streicher, Werner, Jersie-Christensen, Rosa Rakownikow, Olsen, Jesper V, and Nilsson, Jakob

Abstract

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least two different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.

Published
2016

43. Structure and Interactions of a Dimeric Variant of sHIP, a Novel Virulence Determinant of Streptococcus pyogenes

Author

Diehl, Carl, Wisniewska, Magdalena, Frick, Inga-Maria, Streicher, Werner, Björck, Lars, Malmström, Johan, Wikström, Mats, Diehl, Carl, Wisniewska, Magdalena, Frick, Inga-Maria, Streicher, Werner, Björck, Lars, Malmström, Johan, and Wikström, Mats

Abstract

Streptococcus pyogenes is one of the most significant bacterial pathogens in the human population mostly causing superficial and uncomplicated infections (pharyngitis and impetigo) but also invasive and life-threatening disease. We have previously identified a virulence determinant, protein sHIP, which is secreted at higher levels by an invasive compared to a non-invasive strain of S. pyogenes. The present work presents a further characterization of the structural and functional properties of this bacterial protein. Biophysical and structural studies have shown that protein sHIP forms stable tetramers both in the crystal and in solution. The tetramers are composed of four helix-loop-helix motifs with the loop regions connecting the helices displaying a high degree of flexibility. Owing to interactions at the tetramer interface, the observed tetramer can be described as a dimer of dimers. We identified three residues at the tetramer interface (Leu84, Leu88, Tyr95), which due to largely non-polar side-chains, could be important determinants for protein oligomerization. Based on these observations, we produced a sHIP variant in which these residues were mutated to alanines. Biophysical experiments clearly indicated that the sHIP mutant appear only as dimers in solution confirming the importance of the interfacial residues for protein oligomerisation. Furthermore, we could show that the sHIP mutant interacts with intact histidine-rich glycoprotein (HRG) and the histidine-rich repeats in HRG, and inhibits their antibacterial activity to the same or even higher extent as compared to the wild type protein sHIP. We determined the crystal structure of the sHIP mutant, which, as a result of the high quality of the data, allowed us to improve the existing structural model of the protein. Finally, by employing NMR spectroscopy in solution, we generated a model for the complex between the sHIP mutant and an HRG-derived heparin-binding peptide, providing further molecular deta

Published
2016

44. Proceedings of the Frontiers of Retrovirology Conference 2016

Author

Zurnic, Irena, primary, Hütter, Sylvia, additional, Lehmann, Ute, additional, Stanke, Nicole, additional, Reh, Juliane, additional, Kern, Tobias, additional, Lindel, Fabian, additional, Gerresheim, Gesche, additional, Hamann, Martin, additional, Müllers, Erik, additional, Lesbats, Paul, additional, Cherepanov, Peter, additional, Serrao, Erik, additional, Engelman, Alan, additional, Lindemann, Dirk, additional, Da Silva Santos, Claire, additional, Tartour, Kevin, additional, Cimarelli, Andrea, additional, Burdick, Rya, additional, Chen, Jianbo, additional, Sastri, Jaya, additional, Hu, Wei-Shau, additional, Pathak, Vinay, additional, Keppler, Oliver T., additional, Pradeau, Karine, additional, Eiler, Sylvia, additional, Levy, Nicolas, additional, Lennon, Sarah, additional, Cianferani, Sarah, additional, Emiliani, Stéphane, additional, Ruff, Marc, additional, Parissi, Vincent, additional, Rato, Sylvie, additional, Rausell, Antonio, additional, Munoz, Miguel, additional, Telenti, Amalio, additional, Ciuffi, Angela, additional, Zhyvoloup, Alexander, additional, Melamed, Anat, additional, Anderson, Ian, additional, Planas, Delphine, additional, Kriston-Vizi, Janos, additional, Ketteler, Robin, additional, Lee, Chen- Hsuin, additional, Merritt, Andy, additional, Ancuta, Petronela, additional, Bangham, Charles, additional, Fassati, Ariberto, additional, Rodari, Anthony, additional, Van Driessche, Benoit, additional, Galais, Mathilde, additional, Delacourt, Nadége, additional, Fauquenoy, Sylvain, additional, Vanhulle, Caroline, additional, Kula, Anna, additional, Burny, Arsène, additional, Rohr, Olivier, additional, Van Lint, Carine, additional, van Montfort, Thijs, additional, van der Sluis, Renee, additional, Speijer, Dave, additional, Berkhout, Ben, additional, Meng, Bo, additional, Rutkowski, Andrzej, additional, Berry, Neil, additional, Dölken, Lars, additional, Lever, Andrew, additional, Schuster, Thomas, additional, Asbach, Benedikt, additional, Wagner, Ralf, additional, Gross, Christine, additional, Wiesmann, Veit, additional, Kalmer, Martina, additional, Wittenberg, Thomas, additional, Gettemans, Jan, additional, Thoma-Kress, Andrea K., additional, Li, Minghua, additional, Freed, Eric O., additional, Liu, Shan-Lu, additional, Müller, Janis, additional, Münch, Jan, additional, Sewald, Xaver, additional, Uchil, Pradeep, additional, Ladinsky, Mark, additional, Beloor, Jagadish, additional, Pi, Ruoxi, additional, Herrmann, Christin, additional, Motamedi, Nasim, additional, Murooka, Thomas, additional, Brehm, Michael, additional, Greiner, Dale, additional, Mempel, Thorsten, additional, Bjorkman, Pamela, additional, Kumar, Priti, additional, Mothes, Walther, additional, Joas, Simone, additional, Parrish, Erica, additional, Gnanadurai, Clement Wesley, additional, Lump, Edina, additional, Stürzel, Christina M., additional, Parrish, Nicholas F., additional, Sauermann, Ulrike, additional, Töpfer, Katharina, additional, Schultheiss, Tina, additional, Bosinger, Steven, additional, Silvestri, Guido, additional, Apetrei, Cristian, additional, Huot, Nicholas, additional, Müller-Trutwin, Michaela, additional, Sauter, Daniel, additional, Hahn, Beatrice H., additional, Stahl-Hennig, Christiane, additional, Kirchhoff, Frank, additional, Schumann, Gerald, additional, Jung-Klawitter, Sabine, additional, Fuchs, Nina V., additional, Upton, Kyle R., additional, Muñoz-Lopez, Martin, additional, Shukla, Ruchi, additional, Wang, Jichang, additional, Garcia-Canadas, Marta, additional, Lopez-Ruiz, Cesar, additional, Gerhardt, Daniel J., additional, Sebe, Attila, additional, Grabundzija, Ivana, additional, Gerdes, Patricia, additional, Merkert, Sylvia, additional, Pulgarin, Andres, additional, Bock, Anja, additional, Held, Ulrike, additional, Witthuhn, Anett, additional, Haase, Alexandra, additional, Wolvetang, Ernst J., additional, Martin, Ulrich, additional, Ivics, Zoltán, additional, Izsvák, Zsuzsanna, additional, Garcia-Perez, J., additional, Faulkner, Geoffrey J., additional, Hurst, Tara, additional, Katzourakis, Aris, additional, Magiorkinis, Gkikas, additional, Schott, Kerstin, additional, Derua, Rita, additional, Seifried, Janna, additional, Reuter, Andreas, additional, Schmitz, Heike, additional, Tondera, Christiane, additional, Brandariz-Nuñez, Alberto, additional, Diaz-Griffero, Felipe, additional, Janssens, Veerle, additional, König, Renate, additional, Baldauf, Hanna-Mari, additional, Stegmann, Lena, additional, Schwarz, Sarah-Marie, additional, Trotard, Maud, additional, Martin, Margarethe, additional, Lenzi, Gina, additional, Burggraf, Manja, additional, Pan, Xiaoyu, additional, Fregoso, Oliver I., additional, Lim, Efrem S., additional, Abraham, Libin, additional, Erikson, Elina, additional, Nguyen, Laura, additional, Ambiel, Ina, additional, Rutsch, Frank, additional, Kim, Baek, additional, Emerman, Michael, additional, Fackler, Oliver T., additional, Wittmann, Sabine, additional, Behrendt, Rayk, additional, Volkmann, Bianca, additional, Eissmann, Kristin, additional, Gramberg, Thomas, additional, Bolduan, Sebastian, additional, Koppensteiner, Herwig, additional, Regensburg, Stefanie, additional, Brack-Werner, Ruth, additional, Draenert, Rika, additional, Schindler, Michael, additional, Ducroux, Aurélie, additional, Xu, Shuting, additional, Ponnurangam, Aparna, additional, Franz, Sergej, additional, Malassa, Angelina, additional, Ewald, Ellen, additional, Goffinet, Christine, additional, Fung, Sin-Yee, additional, Chan, Ching-Ping, additional, Yuen, Chun-Kit, additional, Kok, Kin-Hang, additional, Chan, Chin-Ping, additional, Jin, Dong-Yan, additional, Dittmer, Ulf, additional, Kmiec, Dorota, additional, Iyer, Shilpa, additional, Stürzel, Christina, additional, Hahn, Beatrice, additional, Ariumi, Yasuo, additional, Yasuda-Inoue, Mariko, additional, Kawano, Koudai, additional, Tateishi, Satoshi, additional, Turelli, Priscilla, additional, Compton, Alex, additional, Roy, Nicolas, additional, Porrot, Françoise, additional, Billet, Anne, additional, Casartelli, Nicoletta, additional, Yount, Jacob, additional, Liang, Chen, additional, Schwartz, Oliver, additional, Magnus, Carsten, additional, Reh, Lucia, additional, Moore, Penny, additional, Uhr, Therese, additional, Weber, Jacqueline, additional, Morris, Lynn, additional, Trkola, Alexandra, additional, Grindberg, Rashel V., additional, Schlaepfer, Erika, additional, Schreiber, Gideon, additional, Simon, Viviana, additional, Speck, Roberto F., additional, Debyser, Zeger, additional, Vranckx, Lenard, additional, Demeulemeester, Jonas, additional, Saleh, Suha, additional, Verdin, Eric, additional, Cereseto, Anna, additional, Christ, Frauke, additional, Gijsbers, Rik, additional, Wang, Gang, additional, Zhao, Na, additional, Das, Atze T., additional, Köstler, Josef, additional, Perdiguero, Beatriz, additional, Esteban, Mariano, additional, Jacobs, Bertram L., additional, Montefiori, David C., additional, LaBranche, Celia C., additional, Yates, Nicole L., additional, Tomaras, Georgia D., additional, Ferrari, Guido, additional, Foulds, Kathryn E., additional, Roederer, Mario, additional, Landucci, Gary, additional, Forthal, Donald N., additional, Seaman, Michael S., additional, Hawkins, Natalie, additional, Self, Steven G., additional, Phogat, Sanjay, additional, Tartaglia, James, additional, Barnett, Susan W., additional, Burke, Brian, additional, Cristillo, Anthony D., additional, Ding, Song, additional, Heeney, Jonathan L., additional, Pantaleo, Giuseppe, additional, Stab, Viktoria, additional, Ensser, Armin, additional, Tippler, Bettina, additional, Burton, Dennis, additional, Tenbusch, Matthias, additional, Überla, Klaus, additional, Alter, Galit, additional, Lofano, Giuseppe, additional, Dugast, Anne-Sophie, additional, Kulkarni, Viraj, additional, Suscovich, Todd, additional, Opazo, Tatiana, additional, Barraza, Felipe, additional, Herrera, Diego, additional, Garces, Andrea, additional, Schwenke, Tomas, additional, Tapia, Diego, additional, Cancino, Jorge, additional, Arriagada, Gloria, additional, Haußner, Christina, additional, Damm, Dominik, additional, Rohrhofer, Anette, additional, Schmidt, Barbara, additional, Eichler, Jutta, additional, Midgley, Rebecca, additional, Wheeldon, James, additional, Piguet, Vincent, additional, Khopkar, Priyanka, additional, Rohamare, Megha, additional, Kulkarni, Smita, additional, Godinho-Santos, Ana, additional, Hance, Allan, additional, Goncalves, Joao, additional, Mammano, Fabrizio, additional, Gasser, Romain, additional, Hamoudi, Meriem, additional, Pellicciotta, Martina, additional, Zhou, Zhicheng, additional, Visdeloup, Clara, additional, Colin, Philippe, additional, Braibant, Martine, additional, Lagane, Bernard, additional, Negroni, Matteo, additional, Wamara, Jula, additional, Bannert, Norbert, additional, Mesplede, Thibault, additional, Osman, Nathan, additional, Anstett, Kaitlin, additional, Liang, Jiaming Calvin, additional, Pham, Hanh Thi, additional, Wainberg, Mark, additional, Shao, Wei, additional, Shan, Jigui, additional, Kearney, Mary, additional, Wu, Xiaolin, additional, Maldarelli, Frank, additional, Mellors, John, additional, Luke, Brian, additional, Coffin, John, additional, Hughes, Stephen, additional, Fricke, Thomas, additional, Opp, Silvana, additional, Shepard, Caitlin, additional, Ivanov, Dmitri, additional, Valle-Casuso, Jose, additional, Kanja, Marine, additional, Cappy, Pierre, additional, Lener, Daniela, additional, Knyazhanskaya, Ekaterina, additional, Anisenko, Andrey, additional, Zatsepin, Timofey, additional, Gottikh, Marina, additional, Komkov, Alexander, additional, Minervina, Anastasia, additional, Nugmanov, Gaiaz, additional, Nazarov, Vadim, additional, Khodosevich, Konstantin, additional, Mamedov, Ilgar, additional, Lebedev, Yuri, additional, Colomer-Lluch, Marta, additional, Serra-Moreno, Ruth, additional, Sarracino, Ambra, additional, Gharu, Lavina, additional, Pasternak, Alexander, additional, Marcello, Alessandro, additional, McCartin, Ann Marie, additional, Kulkarni, Anurag, additional, Le Douce, Valentin, additional, Gautier, Virginie, additional, Baeyens, Ann, additional, Naessens, Evelien, additional, Van Nuffel, Anouk, additional, Weening, Karin, additional, Reilly, Anne- Marie, additional, Claeys, Eva, additional, Trypsteen, Wim, additional, Vandekerckhove, Linos, additional, Eyckerman, Sven, additional, Gevaert, Kris, additional, Verhasselt, Bruno, additional, Mok, Hoi Ping, additional, Norton, Nicholas, additional, Fun, Axel, additional, Hirst, Jack, additional, Wills, Mark, additional, Miklik, Dalibor, additional, Senigl, Filip, additional, Hejnar, Jiri, additional, Sakuragi, Jun-ichi, additional, Sakuragi, Sayuri, additional, Yokoyama, Masaru, additional, Shioda, Tatsuo, additional, Sato, Hironori, additional, Bodem, Jochen, additional, Moschall, Rebecca, additional, Denk, Sarah, additional, Erkelenz, Steffen, additional, Schenk, Christian, additional, Schaal, Heiner, additional, Donhauser, Norbert, additional, Socher, Ellen, additional, Millen, Sebastian, additional, Sticht, Heinrich, additional, Mann, Melanie, additional, Wei, Guochao, additional, Betts, Matthew J., additional, Liu, Yang, additional, Kehl, Timo, additional, Russell, Robert B., additional, Löchelt, Martin, additional, Hohn, Oliver, additional, Mostafa, Saeed, additional, Hanke, Kirsten, additional, Norley, Stephen, additional, Chen, Chia-Yen, additional, Shingai, Masashi, additional, Borrego, Pedro, additional, Taveira, Nuno, additional, Strebel, Klaus, additional, Hellmund, Chris, additional, Friedrich, Melanie, additional, Hahn, Friedrich, additional, Setz, Christian, additional, Rauch, Pia, additional, Fraedrich, Kirsten, additional, Matthaei, Alina, additional, Henklein, Petra, additional, Traxdorf, Maximilian, additional, Fossen, Torgils, additional, Schubert, Ulrich, additional, Khwaja, Aya, additional, Galilee, Meytal, additional, Alian, Akram, additional, Schwalbe, Birco, additional, Hauser, Heiko, additional, Schreiber, Michael, additional, Scherpenisse, Mirte, additional, Cho, Young-Keol, additional, Kim, Jungeun, additional, Jeong, Daeun, additional, Trejbalova, Katerina, additional, Benesova, Martina, additional, Kucerova, Dana, additional, Vernerova, Zdenka, additional, Amouroux, Rachel, additional, Hajkova, Petra, additional, Elleder, Daniel, additional, Hron, Tomas, additional, Farkasova, Helena, additional, Padhi, Abinash, additional, Paces, Jan, additional, Zhu, Henan, additional, Gifford, Robert, additional, Murcia, Pablo, additional, Carrozza, Maria Luisa, additional, Niewiadomska, Anna-Maria, additional, Mazzei, Maurizio, additional, Abi-Said, Mounir, additional, Hughes, Joseph, additional, Hué, Stéphane, additional, Obasa, Adetayo, additional, Jacobs, Graeme, additional, Engelbrecht, Susan, additional, Mack, Katharina, additional, Starz, Kathrin, additional, Geyer, Matthias, additional, Bibollet-Ruche, Frederic, additional, Leoz, Marie, additional, Plantier, Jean Christophe, additional, Argaw-Denboba, Ayele, additional, Balestrieri, Emanuela, additional, Serafino, Annalucia, additional, Bucci, Ilaria, additional, Cipriani, Chiara, additional, Spadafora, Corrado, additional, Sinibaldi-Vallebona, Paolo, additional, Matteucci, Claudia, additional, Jayashree, S. Nandi, additional, Neogi, Ujjwal, additional, Chhangani, Anil K., additional, Rathore, Shravan Sing, additional, Mathur, Bajrang R. J., additional, Abati, Adeyemi, additional, Koç, B. Taylan, additional, Oğuzoğlu, Tuba Çiğdem, additional, Shimauchi, Takatoshi, additional, Caucheteux, Stephan, additional, Turpin, Jocelyn, additional, Finsterbusch, Katja, additional, Tokura, Yoshiki, additional, Souriant, Shanti, additional, Balboa, Luciana, additional, Pingris, Karine, additional, Kviatcowsky, Denise, additional, Raynaud-Messina, Brigitte, additional, Cougoule, Céline, additional, Mercier, Ingrid, additional, Kuroda, Marcelo, additional, González-Montaner, Pablo, additional, Inwentarz, Sandra, additional, Moraña, Eduardo Jose, additional, del Carmen Sasiain, Maria, additional, Neyrolles, Olivier, additional, Maridonneau-Parini, Isabelle, additional, Lugo-Villarino, Geanncarlo, additional, Vérollet, Christel, additional, Herrmann, Alexandra, additional, Thomas, Dominique, additional, Bouzas, Nerea Ferreirós, additional, Lahaye, Xavier, additional, Bhargava, Anvita, additional, Satoh, Takeshi, additional, Gentili, Matteo, additional, Cerboni, Silvia, additional, Silvin, Aymeric, additional, Conrad, Cécile, additional, Ahmed-Belkacem, Hakim, additional, Rodriguez, Elisa C., additional, Guichou, Jean-François, additional, Bosquet, Nathalie, additional, Piel, Matthieu, additional, Le Grand, Roger, additional, King, Megan, additional, Pawlotsky, Jean-Michel, additional, Manel, Nicolas, additional, Hofmann, Henning, additional, Vanwalscappel, Benedicte, additional, Bloch, Nicolin, additional, Landau, Nathaniel, additional, Indik, Stanislav, additional, Hagen, Benedikt, additional, Valle-Casuso, José Carlos, additional, Allouch, Awatef, additional, David, Annie, additional, Barré-Sinoussi, Françoise, additional, Benkirane, Monsef, additional, Pancino, Gianfranco, additional, Saez-Cirion, Asier, additional, Lee, Wing-Yiu, additional, Sloan, Richard, additional, Schulte, Bianca, additional, Blomberg, Jonas, additional, Vargiu, Luana, additional, Rodriguez-Tomé, Patricia, additional, Tramontano, Enzo, additional, Sperber, Göran, additional, Kumari, Namita, additional, Ammosova, Tatiana, additional, Diaz, Sharmeen, additional, Oneal, Patricia, additional, Nekhai, Sergei, additional, Fahrny, Audrey, additional, Gers-Huber, Gustavo, additional, Audigé, Annette, additional, Jayaprakash, Anitha, additional, Sachidanandam, Ravi, additional, Hernandez, Matt, additional, Dillon-White, Marsha, additional, Maze, Emmanuel, additional, Ham, Claire, additional, Almond, Neil, additional, Towers, Greg, additional, Belshaw, Robert, additional, de Sousa-Pereira, Patrícia, additional, Abrantes, Joana, additional, Pizzato, Massimo, additional, Esteves, Pedro J., additional, Kahle, Tanja, additional, Schmitt, Sven, additional, Merkel, Laura, additional, Reuter, Nina, additional, Stamminger, Thomas, additional, Rosa, Ilaria Dalla, additional, Bishop, Kate, additional, Spinazzola, Antonella, additional, Groom, Harriet, additional, Vieyres, Gabrielle, additional, Müsken, Mathias, additional, Zillinger, Thomas, additional, Hornung, Veit, additional, Barchet, Winfried, additional, Häussler, Susanne, additional, Pietschmann, Thomas, additional, Javed, Aneela, additional, Leuchte, Nicole, additional, Salinas, Gabriela, additional, Opitz, Lennart, additional, Sopper, Sieghart, additional, Mummert, Christiane, additional, Hofmann, Christian, additional, Hückelhoven, Angela G., additional, Bergmann, Silke, additional, Müller-Schmucker, Sandra M., additional, Harrer, Ellen G., additional, Dörrie, Jan, additional, Schaft, Niels, additional, Harrer, Thomas, additional, Cardinaux, Laure, additional, Zahno, M.- L., additional, Vogt, H.- R., additional, Zanoni, R., additional, Bertoni, G., additional, Muenchhoff, Maximilian, additional, Goulder, Philip, additional, Keppler, Oliver, additional, Rebensburg, Stephanie, additional, Helfer, Markus, additional, Zhang, Yuwei, additional, Chen, Huicheng, additional, Bernier, Annie, additional, Gosselin, Annie, additional, Routy, Jean- Pierre, additional, Wöhrl, Birgitta, additional, Schneider, Anna, additional, Corona, Angela, additional, Spöring, Imke, additional, Jordan, Mareike, additional, Buchholz, Bernd, additional, Maccioni, Elias, additional, Di Santo, Roberto, additional, Schweimer, Kristian, additional, Schölz, Christian, additional, Weinert, Brian, additional, Wagner, Sebastian, additional, Beli, Petra, additional, Miyake, Yasuyuki, additional, Qi, Jun, additional, Jensen, Lars, additional, Streicher, Werner, additional, McCarthy, Anna, additional, Westwood, Nicholas, additional, Lain, Sonia, additional, Cox, Jürgen, additional, Matthias, Patrick, additional, Mann, Matthias, additional, Bradner, James, additional, Choudhary, Chunaram, additional, Stern, Marcel, additional, Valletta, Elena, additional, Frezza, Caterina, additional, Marino-Merlo, Francesca, additional, Grelli, Sandro, additional, Serafino, Anna Lucia, additional, Mastino, Antonio, additional, Macchi, Beatrice, additional, Kaulfuß, Meike, additional, Windmann, Sonja, additional, Bayer, Wibke, additional, Mikasi, Sello, additional, Heß, Rebecca, additional, Bonsmann, Michael Storcksdieck gen., additional, Kirschning, Carsten, additional, Lepenies, Bernd, additional, Kolenbrander, Anne, additional, Temchura, Vladimir, additional, Iijima, Kenta, additional, Kobayashi, Junya, additional, and Ishizaka, Yukihito, additional

Published
2016
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49. Interaction of RECQ4 and MCM10 is important for efficient DNA replication origin firing in human cells

Author

Kliszczak, Maciej, Sedlackova, Hana, Pitchai, Ganesha P, Streicher, Werner W, Krejci, Lumir, Hickson, Ian D, Kliszczak, Maciej, Sedlackova, Hana, Pitchai, Ganesha P, Streicher, Werner W, Krejci, Lumir, and Hickson, Ian D

Abstract

DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants are able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing.

Published
2015

50. Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose)

Author

Altmeyer, Matthias, Neelsen, Kai J, Teloni, Federico, Pozdnyakova, Irina, Pellegrino, Stefania, Grøfte, Merete, Rask, Maj-Britt Druedahl, Streicher, Werner, Jungmichel, Stephanie, Nielsen, Michael Lund, Lukas, Jiri, Altmeyer, Matthias, Neelsen, Kai J, Teloni, Federico, Pozdnyakova, Irina, Pellegrino, Stefania, Grøfte, Merete, Rask, Maj-Britt Druedahl, Streicher, Werner, Jungmichel, Stephanie, Nielsen, Michael Lund, and Lukas, Jiri

Abstract

Intrinsically disordered proteins can phase separate from the soluble intracellular space, and tend to aggregate under pathological conditions. The physiological functions and molecular triggers of liquid demixing by phase separation are not well understood. Here we show in vitro and in vivo that the nucleic acid-mimicking biopolymer poly(ADP-ribose) (PAR) nucleates intracellular liquid demixing. PAR levels are markedly induced at sites of DNA damage, and we provide evidence that PAR-seeded liquid demixing results in rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites. Demixing, which relies on electrostatic interactions between positively charged RGG repeats and negatively charged PAR, is amplified by aggregation-prone prion-like domains, and orchestrates the earliest cellular responses to DNA breakage. We propose that PAR-seeded liquid demixing is a general mechanism to dynamically reorganize the soluble nuclear space with implications for pathological protein aggregation caused by derailed phase separation.

Published
2015

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