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Laboratory of Structural Biochemistry of Immune Recognition

Our research group is interested in processes of cell-cell recognition, i.e., how the immune cells, for example NK cells, recognize by receptors expressed on their surface the target proteins expressed on the surface of healthy cells or of tumour, virally infected or stressed cells. We are interested firstly in the structure-function relationship, using a wide range of modern methods of research of protein structure, and their properties and interactions, and secondly, how we could transfer the gained knowledge into a practical level, besides other things by designing artificial protein constructs which would help the immune cells to better recognize and eliminate the target cells.

Contact

Department of Biochemistry, Faculty of Science, Charles University

Hlavova 2030/8, 2nd floor, door No. 204

Tel: +420 221 951 272

E-mail: ondrej.vanek@natur.cuni.cz

 

People

Ondřej Vaněk – group leader

Jan Bláha – postdoctoral fellow (presently post-doc stay at EMBL Hamburg)

Barbora Kalousková - PhD. student

Ondřej Skořepa - PhD. student

Celeste de Sousa Santos Abreu - PhD. student

Shiva Nejadebrahim - PhD. student

Tomáš Otépka - MSc. student

Gabriela Chytrá - BSc. student

Maxmilián Hejl - BSc. student

Petra Kozojedová - BSc. student

Kristýna Pazderová - BSc. student

Matouš Tulpa - BSc. student

 

Aims

Structural research of complexes of NK cell receptora with their ligands

  - Used methods

          - Recombinant protein expression within the HEK293 human cell line

          - Biophysical characterization of proteins and their interactions (AUC, ITC, MST, DLS, DSF, SPR)

          - Protein crystallography, structural mass spectrometry, SAXS

Research of NK cell receptors arrangement on the surface of a living cell

  - Superresolution microscopy, fluorescence microscopy, flow cytometry, single-molecule localization microscopy (SMLM)

Production of fusion constructs of nanobodies with ligands of activation receptors of NK cells and testing their biological activity

  - Isolation of NK cells from the blood of healthy donors

  - Cytotoxicity tests of NK cells against tumour cell lines

Production of fusion constructs with IL-2 or growth factors

  - Expression, purification and study of properties of fusion proteins containing protein hormones or cytokines

 

Projects

Czech Science Foundation, 15-15181S (O. Vaněk)

Title: Harnessing soluble forms of NK cell receptors and their ligands for the generation of novel anticancer immunotherapeutics

Duration: 2015 - 2017

Grant Agency of the Charles University, 161216 (J. Bláha / B. Kalousková)

Title: Utilization of a structural description of the interaction of human lymphocyte complex NKR-P1 and LLT1 for development of immunoactive fusion therapeutics

Duration: 2016 - 2018

Grant Agency of the Charles University, 927916 (S. Pažický / O. Skořepa)

Title: Impact of NKp30 glycosylation and oligomerization on its complex formation - the key features for NK cells activation

Duration: 2016 - 2018

Ministry of Education, Youth and Sports of the Czech Republic, INTER-COST program, LTC17065 (O. Vaněk)

Title: Utilization of integrative molecular biophysical approaches for the functional studies of human natural killer cell receptors and formation of their complexes with tumour ligands

Duration: 2017 - 2020

Czech Science Foundation, 18-10687S (O. Vaněk)

Title: New ligands for old receptors of human natural killer cells: structure, assembly within the immune synapse and potential for therapy

Duration: 2018 - 2020

Grant Agency of the Charles University, 1740318 (D. Cmunt)

Title: Utilization of activation ligands and nanobodies for directed stimulation of NK cells cytotoxicity

Duration: 2018 - 2019

Grant Agency of the Charles University, 1378219 (B. Kalousková)

Title: Quaternary structure of selected human lymphocyte receptors through the looking glass of super-resolution microscopy: What is the biological reality behind the crystal structures?

Duration: 2019 - 2021

Czech Science Foundation, 20-13029S (O. Vaněk, spoluřešitel)

Title: Novel clinically relevant IL-2 immunotherapeutics for cancer treatment

Duration: 2020 - 2022

Grant Agency of the Charles University, 1436020 (J. Houserová)

Title: Structural biochemistry of NK cell complexes NKp44:PCNA and NKp46:Epa1, and utilization of Epa1 for stimulation of NK cells

Duration: 2020 - 2022

Grant Agency of the Charles University, 1436220 (C. Abreu)

Title: Activation of NK cell anti-tumour response using bispecific fusion proteins with nanobodies

Duration: 2020 - 2022

Ministry of Education, Youth and Sports of the Czech Republic, INTER-COST program, LTC20078 (O. Vaněk)

Title: Glycan-dependent regulation of NK cell recognition: structural biology and novel methods of glycopeptide analysis

Duration: 2020 - 2023

 

Offered/Supervised/Defended theses

Ondřej Vaněk

 

Publications

2021

Stehlík Š., Henych J., Štenclová P., Král R., Zemenová P., Pangrác J., Vaněk O., Kromka A., Rezek B. (2021): Size and nitrogen inhomogeneity in detonation and laser synthesized primary nanodiamond particles revealed via salt-assisted deaggregation. Carbon 171, 230-239. IF 8,821 DOI: 10.1016/j.carbon.2020.09.026

2020

Janisova L., Gruzinov A., Zaborova O. V., Velychkivska N., Vaněk O., Chytil P., Etrych T., Janoušková O., Zhang X., Blanchet C., Papadakis C. M., Svergun D. I., Filippov S. K. (2020): Molecular mechanisms of the interactions of N-(2-hydroxypropyl)methacrylamide copolymers designed for cancer therapy with the blood plasma proteins. Pharmaceutics 12, 106. IF 4,421 DOI: 10.3390/pharmaceutics12020106

Skořepa O., Pažický S., Kalousková B., Bláha J., Abreu C., Ječmen T., Rosůlek M., Fish A., Sedivy A., Harlos K., Dohnálek J., Skálová T., Vaněk O.* (2020): Natural killer cell activation receptor NKp30 oligomerization depends on its N-glycosylation. Cancers 12, 1998. IF 6,126 DOI: 10.3390/cancers12071998

Sumová P., Polanská N., Leštinová T., Spitzová T., Kalousková B., Vaněk O., Volf P., Rohoušová I. (2020): Phlebotomus perniciosus recombinant salivary proteins polarize murine macrophages towards the anti-inflammatory phenotype. Front Cell Infect Microbiol 10, 427. IF 4,123 DOI: 10.3389/fcimb.2020.00427

2019

Sumová P., Šíma M., Kalousková B., Polanská N., Vaněk O., Oliveira F., Valenzuela J. G., Volf P. (2019): Amine-binding properties of salivary yellow-related proteins in phlebotomine sand flies. Insect Biochem Mol Biol 115, 103245. IF 3,827 DOI: 10.1016/j.ibmb.2019.103245

Willen L., Leštinová T., Kalousková B., Sumová P., Spitzová T., Velez R., Domenech E., Vaněk O., Gállego M., Mertens P., Volf P. (2019): Field study of the improved rapid sand fly exposure test in areas endemic for canine leishmaniasis. PLoS Negl Trop Dis 13, e0007832. IF 3,885 DOI: 10.1371/journal.pntd.0007832

Vaněk O.*, Celadová P., Skořepa O., Bláha J., Kalousková B., Dvorská A., Poláchová E., Pucholtová H., Kavan D., Pompach P., Hofbauerová K., Kopecký V. Jr., Mesci A., Voigt S., Carlyle J. R. (2019): Production of recombinant soluble dimeric C-type lectin-like receptors of rat natural killer cells. Sci Rep 9, 17836. IF 3,998 DOI: 10.1038/s41598-019-52114-8

2018

Škerlová J., Bláha J., Pachl P., Hofbauerová K., Kukačka Z., Man P., Pompach P., Novák P., Otwinowski Z., Brynda J., Vaněk O.*, Řezáčová P.* (2018): Crystal structure of native β-N-acetylhexosaminidase isolated from Aspergillus oryzae sheds light onto its substrate specificity, high stability, and regulation by propeptide. FEBS J 285, 580-598. IF 4,739 DOI: 10.1111/febs.14360

Malý M., Skálová T., Švecová L., Dohnálek J., Bláha J., Vaněk O., Harlos K., Kolenko P. (2018): Paired refinement: Impact of reintegration and rescaling. 8. Studentská vědecká konference fyziky pevných látek a materiálů ČVUT, 8-10. ISBN 978-80-01-06511-2

2017

Bláha J., Kalousková B., Skořepa O., Pažický S., Novák P., Vaněk O.* (2017): High-level expression and purification of soluble form of human natural killer cell receptor NKR-P1 in HEK293S GnTI- cells. Protein Expr Purif 140, 36-43. IF 1,338 DOI: 10.1016/j.pep.2017.07.016

Stehlík Š., Varga M., Štenclová P., Ondič L., Ledinský M., Pangrác J., Vaněk O., Lipov J., Kromka A., Rezek B. (2017): Ultrathin Nanocrystalline Diamond Films with Silicon Vacancy Color Centers via Seeding by 2 nm Detonation Nanodiamonds. ACS Appl Mater Interfaces 9, 38842-38853. IF 8,097 DOI: 10.1021/acsami.7b14436

2016

Doležal M., Zábranský A., Dostál J., Vaněk O., Brynda J., Lepšík M., Hadravová R., Pichová I. (2016): Myristoylation drives dimerization of matrix protein from mouse mammary tumor virus. Retrovirology 13, 2. IF 3,867 DOI: 10.1186/s12977-015-0235-8

Stráňava M., Martínek V., Man P., Fojtíková V., Kavan D., Vaněk O., Shimizu T., Martínková M. (2016): Structural Characterization of the Heme-based Oxygen Sensor, AfGcHK, its Interactions with the Cognate Response Regulator, and their Combined Mechanism of Action in a Bacterial Two-component Signaling System. Proteins 84, 1375-1389. IF 2,289 DOI: 10.1002/prot.25083

2015

Bláha J., Pachl P., Novák P., Vaněk O.* (2015): Expression and purification of soluble and stable ectodomain of natural killer cell receptor LLT1 through high-density transfection of suspension adapted HEK293S GnTI- cells. Protein Expr Purif 109, 7-13. IF 1,407 DOI: 10.1016/j.pep.2015.01.006

Skálová T., Bláha J., Harlos K., Dušková J., Kovaľ T., Stránský J., Hašek J., Vaněk O., Dohnálek J. (2015): Four crystal structures of human LLT1, a ligand for human NKR-P1, in varied glycosylation and oligomerization states. Acta Crystallogr Sect D Biol Crystallogr 71, 578-591. IF 2,512 DOI: 10.1107/S1399004714027928

Machová I., Snášel J., Dostál J., Brynda J., Fanfrlík J., Singh M., Tarábek J., Vaněk O., Bednárová L., Pichová I. (2015): Structural and functional studies of phosphoenolpyruvate carboxykinase from Mycobacterium tuberculosis. PLoS ONE 10, e0120682. IF 3,057 DOI: 10.1371/journal.pone.0120682

Lynn G. M., Laga R., Darrah P. A., Ishizuka A. S., Balaci A. J., Dulcey A. E., Pechar M., Pola R., Gerner M. Y., Yamamoto A., Buechler C. R., Quinn K. M., Smelkinson M. G., Vaněk O., Cawood R., Hills T., Vasalatiy O., Kastenmüller K., Francica J. R., Stutts L., Tom J. K., Ryu K. A., Esser-Kahn A. P., Etrych T., Fisher K. D., Seymour L. W., Seder R. A. (2015): In vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity. Nat Biotechnol 33, 1201-1210. IF 43,113 DOI: 10.1038/nbt.3371

 

Collaboration

Academic partners

Dr. Mario Brameshuber, Technical University of Vienna, Austria

doc. Ario de Marco, University Nova Gorica, Slovenia

prof. James R. Carlyle, University Toronto, Canada

Dr. Jan Dohnálek, IBT AS CR, BIOCEV

Dr. Jakub Tomala, IBT AS CR, BIOCEV

Dr. Jana Dvořáková, IMCh AS CR

Industry and commercial partners

IOCB TECH s.r.o. (2017–2018)

Merck s.r.o. (2018)

 

Teaching (Department of Biochemistry, FoS CU)

MC250C17N - Advanced practical course in Biochemistry I

MC250C03 - Advanced practical course in biochemistry I for KATA

MC250C08 - Advanced practical course in biochemistry II

MC250P49 - Biology for biochemists

MC250P67 - Therapeutical proteins

MS750P02 - Introduction to knowledge and technology transfer

MC250P50 - Management of biochemistry

MC250P34 - Biochemistry as the theoretical basis of biomedicine

28 defended bachelor theses

18 defended master theses

1 defended PhD. thesis

 

Alumni

Supervision of bachelor theses

Hájková Alena (2007): Preparation of vector for the recombinant expression of human protein NKR-P1, receptor of natural killer cell

Němcová Petra (2007): Preparation of vector for the recombinant expression of protein NKR-P1A, receptor of rat natural killer cell

Novotná Lenka (2008): Preparation of vector for the recombinant expression of protein NKR-P1B, receptor of rat natural killer cell

Celadová Petra (2008): Molecular cloning and amplification of b and g isoforms of Clr gene, ligand for Nkrp1 receptors of mouse NK cells

Holubová Martina (2008): Molecular cloning and amplification of B, D, and F isoforms of NKR-P1 mouse natural killer cell receptor

Bláha Jan (2010): Optimization of recombinant protein expression in HEK293 cell line

Čuperková Romana (2012): Optimization of Culture Medium for HEK293 Cell Line

Dvorská Anna (2012): Preparation and studies of complex of rat NK cell receptors NKR-P1B and Clrb

Leontovyč Adrian (2012): Preparation of expression vector for production of receptors CD69 and S1P1

Poláchová Edita (2012): Preparation of Expression Vectors That Regulate Cell Cycle and Apoptosis to Optimize Expression System of HEK293 Cell Line

Pucholtová Helena (2012): Preparation of dimeric form of mouse NK cell receptor NKR-P1C

Illéš David (2013): Optimization of purification of recombinant nitrilase from Arthroderma benhamiae

Mikulová Barbora (2013): Preparation of expression vectors for NKp65 and KACL, new members of human NK cell receptor family

Nový Jiří (2013): Preparation of the human NK cell receptor KACL

Petermannová Romana (2013): Production of auxin transporter PIN1 in HEK293 cell line

Slavata Lukáš (2013): Preparation of plasmids for expression of interleukin 2-fusion monoclonal antibody

Kalousková Barbora (2014): Preparation of glycosylated form of human immunoreceptor NKp30

Pažický Samuel (2014): Preparation of expression vectors for receptor NKp30 and its ligands B7-H6 and BAG-6

Skořepa Ondřej (2014): Preparation of lentiviral expression vector with reporter gene

Gáthy Lajos (2015): Recombinant expression and purification of C324S modified nitrilase from Arthroderma benhamiae

Prokopová Tereza (2015): Production and purification of recombinant receptor Clrb

Herynek Štěpán (2017): Recombinant preparation of proliferating cell nuclear antigen (PCNA)

Krejčová Kateřina (2017): Preparation of tumour ligand B7-H6 with coiled-coil tag and verifying of its binding to receptor NKp30

Hausleitner Filip (2018): The influence of S159A mutation on the oligomeric state of human NK cell receptor NKR-P1A

Houserová Jana (2018): Recombinant expression of protein NKp46 and its ligand Epa1

Nepokojová Tereza (2018): Preparation of fusion ligands and evaluation of their binding to NK cell receptors

Runová Alžběta (2018): Preparation of receptor AICL in fusion with Fc fragment of human IgG

Rožová Dominika (2020): Production of interleukin 2 in fusion with monoclonal antibody S4B6

 

Supervision of master theses

Hájková Alena (2009): Recombinant expression and studies on receptor hNKR-P1

Němcová Petra (2009): Recombinant expression and studies on rNKR-P1A receptor

Celadová Petra (2010): Preparation of rat NK cell receptors using HEK293T expression system

Bláha Jan (2012): Preparation and study of human lymphocyte receptor LLT1

Dvorská Anna (2014): Structural biology of complex of rat NK cell receptors NKR-P1B and Clrb

Hradilová Iveta (2014): Preparation and characterization of cyanide hydratase from Aspergillus niger and nitrilase from Arthroderma benhamiae

Poláchová Edita (2014): Optimization of HEK293 cell line expression system by regulation of cell cycle and apoptosis

Pucholtová Helena (2014): Production of mouse NK cell receptor NKR-P1C and seeking of its ligand

Zawadová Dorota (2014): Recombinant expression and purification of nitrilase from Neurospora crassa

Nový Jiří (2015): Preparation and study of human NK cell receptor AICL

Petermannová Romana (2015): Preparation of HEK293 cell line expressing auxin transporter PIN7 and testing of inhibitors of auxin transport

Kalousková Barbora (2016): Preparation of human NK cell activation receptor NKp80 and its ligand AICL

Pažický Samuel (2016): Study of NKp30 oligomerization and its interaction with B7-H6

Skořepa Ondřej (2016): Structural studies of rat NK cell receptor NKR-P1B and its ligand Clrb

Abreu de Sousa Santos Celeste (2019): Interaction of Galectin-1 with human NK cell receptors

Cmunt Denis (2019): Preparation of fusion domains of human immunoreceptors for their utilization in immunotherapy

Herynek Štěpán (2019): Structural biology of receptor NKp44 and its ligand PCNA

Houserová Jana (2020): Study of the interaction of receptor NKp46 with adhesin Epa1

 

Supervision of PhD. theses

Bláha Jan (2017): Study of structure and interaction of human lymphocyte receptors

 


 

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