Ondřej Vaněk

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• PhD.
• Group Leader at Charles University in Prague

Natural killer (NK) cells are critical components of the innate immune system. Their primary role is to induce apoptosis in target cells, such as cancerous or virally infected cells. These targets are recognized through interactions between activating or inhibitory receptors on the NK cell surface. Among the activating receptors is the natural cyto...

Siglec-7, an immune checkpoint receptor, has emerged as a promising target for cancer immunotherapy due to its involvement in the regulation of immune and inflammatory responses. However, while its participation in immunoediting and immune evasion is well established, understanding its biological context, relevant ligands, and associated signalling...

Supplemental Data to "Engineered cytokine/antibody fusion proteins improve IL-2 delivery to pro-inflammatory cells and promote antitumor activity"

Progress in cytokine engineering is driving therapeutic translation by overcoming these proteins’ limitations as drugs. The IL-2 cytokine is a promising immune stimulant for cancer treatment but is limited by its concurrent activation of both pro-inflammatory immune effector cells and antiinflammatory regulatory T cells, toxicity at high doses, and...

We investigated two recently synthesized and characterized sialyl derivatives, bearing the Neu5Ac‐α‐(2‐6)‐Gal epitope, as promising binders for Siglec‐7, an inhibitory Siglec mainly found on natural killer cells. A variety of sialoglycan structures can be recognized by Siglec‐7 with implications in the modulation of immune responses. Notably, overe...

Progress in cytokine engineering is driving therapeutic translation by overcoming the inherent limitations of these proteins as drugs. The interleukin-2 (IL-2) cytokine harbors great promise as an immune stimulant for cancer treatment. However, the cytokine’s concurrent activation of both pro-inflammatory immune effector cells and anti-inflammatory...

Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the r...

Background Proteins are used as reagents in a broad range of scientific fields. The reliability and reproducibility of experimental data will largely depend on the quality of the (recombinant) proteins and, consequently, these should undergo thorough structural and functional controls. Depending on the downstream application and the biochemical cha...

Natural killer (NK) cells are a family of lymphocytes with a natural ability to kill infected, harmed, or malignantly transformed cells. As these cells are part of the innate immunity, the cytotoxic mechanisms are activated upon recognizing specific patterns without prior antigen sensitization. This recognition is crucial for NK cell function in th...

Targeted cancer immunotherapy is a promising tool for restoring immune surveillance and eradicating cancer cells. Hydrophilic polymers modified with coiled coil peptide tags can be used as universal carriers designed for cell-specific delivery of such biologically active proteins. Here, we describe the preparation of pHPMA-based copolymer conjugate...

The article “Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study” written by López-Méndez, B., Baron, B., Brautigam, C. A., Jowitt, T. A., Knauer, S. H., Uebel, S., Williams, M. A., Sedivy, A., Abian, O., Abreu, C., Adamczyk, M., Bal, W., Berger, S., Buell, A. K., Carolis, C., Dav...

NKp30 is an activating receptor on the surface of human natural killer (NK) cells. Its crystal structure has been published previously by Joyce et al. [1], PDB code 3NOI. B7-H6 is an activating immunoligand expressed by some tumor cells. Its structure in complex with NKp30 has been described by Li et al. [2], PDB code 3PV6. In this study, we presen...

Human NKR-P1 (CD161, KLRB1) and its ligand LLT1 (CLEC2D) are a prototypical inhibitory C-type lectin-like receptor:ligand pair of NK cells with a critical role in homing lymphocytes to immune-privileged sites, particularly in multiple sclerosis, rheumatoid arthritis, and Crohn's disease. Furthermore, NKR-P1:LLT1 inhibitory signaling is associated w...

Most of the structural proteins known today are composed of domains that carry their own functions while keeping their structural properties. It is supposed that such domains, when taken out of the context of the whole protein, can retain their original structure and function to a certain extent. Information on the specific functional and structura...

Vaccination is one of the greatest achievements in biomedical research preventing death and morbidity in many infectious diseases through the induction of pathogen‐specific humoral and cellular immune responses. Currently, no effective vaccines are available for pathogens with a highly variable antigenic load, such as the human immunodeficiency vir...

Glycan structures are common posttranslational modifications of proteins, which serve multiple important structural roles (for instance in protein folding), but also are crucial participants in cell–cell communications and in the regulation of immune responses. Through the interaction with glycan‐binding receptors, glycans are able to affect the ac...

Microscale thermophoresis (MST), and the closely related Temperature Related Intensity Change (TRIC), are synonyms for a recently developed measurement technique in the field of biophysics to quantify biomolecular interactions, using the (capillary-based) NanoTemper Monolith and (multiwell plate-based) Dianthus instruments. Although this technique...

Understanding materials with dimensions down to a few nanometers is of major importance for fundamental science as well as prospective applications. Structural transformation and phonon-confinement effects in the nanodiamonds (NDs) have been theoretically predicted below 3 nm in size. Here, we investigate the effect of size on the surface chemistry...

Recent years have seen a dramatic improvement in protein-design methodology. Nevertheless, most methods demand expert intervention, limiting their widespread adoption. By contrast, the PROSS algorithm for improving protein stability and heterologous expression levels has been successfully applied to a range of challenging enzymes and binding protei...

Constantly increasing attention to bioengineered proteins has led to the rapid development of new functional targets. Here we present the biophysical and functional characteristics of the newly designed CaM/AMBN-Ct fusion protein. The two-domain artificial target consists of calmodulin (CaM) and ameloblastin C-terminus (AMBN-Ct). CaM as a well-char...

Detonation nanodiamonds (DND), their properties and deaggregation remain an active field of research. Here we innovated the salt-assisted ultrasonic deaggregation (SAUD) by employing NaHCO3 (a milling agent with relatively low solubility) followed by dialysis (a purification step) and processed two DND and one laser-synthesized (LND) commercial pow...

Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel-the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues...

Phlebotomus perniciosus (Diptera: Phlebotominae) is a medically and veterinary important insect vector. It transmits the unicellular parasite Leishmania infantum that multiplies intracellularly in macrophages causing life-threatening visceral diseases. Leishmania establishment in the vertebrate host is substantially influenced by immunomodulatory p...

Supplementary Materials to Natural Killer Cell Activation Receptor NKp30 Oligomerization Depends on Its N-Glycosylation

NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The binding of plasma proteins to a drug carrier alters the circulation of nanoparticles (NPs) in the bloodstream, and, as a consequence, the anticancer efficiency of the entire nanoparticle drug delivery system. We investigate the possible interaction and the interaction mechanism of a polymeric drug delivery system based on N-(2-hydroxypropyl)met...

Working at the border between innate and adaptive immunity, natural killer (NK) cells play a key role in the immune system by protecting healthy cells and by eliminating malignantly transformed, stressed or virally infected cells. NK cell recognition of a target cell is mediated by a receptor “zipper” consisting of various activating and inhibitory...

Background Canine leishmaniasis (CanL) is a severe chronic disease caused by Leishmania infantum and transmitted by sand flies of which the main vector in the Western part of the Mediterranean basin is Phlebotomus perniciosus. Previously, an immunochromatographic test (ICT) was proposed to allow rapid evaluation of dog exposure to P. perniciosus. I...

The amine-binding properties of sand fly salivary yellow-related proteins (YRPs) were described only in Lutzomyia longipalpis sand flies. Here, we experimentally confirmed the kratagonist function of YRPs in the genus Phlebotomus. We utilized microscale thermophoresis technique to determine the amine-binding properties of YRPs in saliva of Phleboto...

Natural killer (NK) cells are a type of lymphocytes able to kill tumour and virally infected cells. Human NKR-P1 is one of the plenty of receptors anchored in the membrane of the cell and LLT1 is its ligand on a partner cell. Both NKR-P1 and LLT1 have extracellular part with C-type lectin like fold. The extracellular domains of NKR-P1 and LLT1 have...

Natural killer cells are white blood cells able to kill tumor, virus-infected or stressed cells. NKR-P1 is a C-type lectin like-receptor on surface of human natural killer cells and LLT1 is its binding partner belonging to the same structural family. Recently, we have expressed, purified and solved four crystal structures of the extracellular part...

β-N-acetylhexosaminidase from the fungus Aspergillus oryzae is a secreted extracellular enzyme that cleaves chitobiose into constituent monosaccharides. It belongs to the GH 20 glycoside hydrolase family and consists of two N-glycosylated catalytic cores noncovalently associated with two 10-kDa O-glycosylated propeptides. We used X-ray diffraction...

Color centers in diamond have shown excellent potential for applications in quantum information processing, photonics, and biology. Here we report chemical vapor deposition (CVD) growth of as thin as 5-6 nm nanocrystalline diamond (NCD) films with photoluminescence (PL) from silicon-vacancy (SiV) centers at 739 nm. Instead of conventional 4-6 nm de...

Human natural killer receptor protein 1 (NKR-P1, CD161, gene klrb1) is a C-type lectin-like receptor of natural killer (NK) cells responsible for recognition of its cognate protein ligand lectin-like transcript 1 (LLT1). NKR-P1 is the single human orthologue of the prototypical rodent NKR-P1 receptors. Naturally, human NKR-P1 is expressed on the su...

Myristoylation of the matrix (MA) domain mediates the transport and binding of Gag polyproteins to the plasma membrane (PM) and is required for the assembly of most retroviruses. In betaretroviruses, which assemble immature particles in the cytoplasm, myristoylation is dispensable for assembly but is crucial for particle transport to the PM. Oligom...

Natural killer (NK) cells are a type of lymphocytes which kill tumor, virally infected or stressed cells. Decision to kill a cell is made as a result of balance of signals from plenty of activating and inhibitory receptors on surface of the natural killer cells. LLT1 is a ligand expressed primarily on activated lymphocytes (including NK cells thems...

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 and then tr...

The efficacy of vaccine adjuvants such as Toll-like receptor agonists (TLRa) can be improved through formulation and delivery approaches. Here, we attached small molecule TLR-7/8a to polymer scaffolds (polymer-TLR-7/8a) and evaluated how different physicochemical properties of the TLR-7/8a and polymer carrier influenced the location, magnitude and...

Natural killer cells (NK cells) are large granular lymphocytes able to kill virally infected, stressed or tumor cells. Unlike T-cells, the activity of NK cells is innate. NKR-P1 (CD161) is a receptor on a surface of human NK cells. LLT1 is a ligand for NKR-Pl receptor, expressed primarily on activated lymphocytes and antigen presenting cells. The i...

Human embryonic kidney 293 cell line deficient in N-acetylglucosaminyltransferase I (HEK293S GnTI-) is well known tool for recombinant expression of proteins with homogeneous and deglycosylatable N-glycosylation, a feature crucial especially within protein crystallography [1]. So far production protocols using this cell line were based either on le...

Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at...

Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, gene KLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the first N-acetylglucosam...

Lectin-like transcript 1 (LLT1, gene clec2d) was identified to be a ligand for the single human NKR-P1 receptor present on NK and NK-T lymphocytes. Naturally, LLT1 is expressed on the surface of NK cells, stimulating IFN-γ production, and is up-regulated upon activation of other immune cells, e.g. TLR-stimulated dendritic cells and B cells or T cel...

Natural killer (NK) cells are large granular lymphocytes with innate immune reaction against tumor cells or cells affected by viral infection. They have a variety of receptors on their surface which mediate contact with cells tested by NK cells. Using X-ray crystallography, we determined a structure of an extracellular part of mouse C-type lectin r...

Coiled coils are a common structural motif in many natural proteins that can also be utilized in the design and preparation of drug delivery systems for the non-covalent connection of two macromolecules. In this work, two different pairs of peptides forming coiled coil hetero-oligomers were designed, synthesized and characterized. While the peptide...

Nitrilases are versatile enzymes in biocatalysis, mediating the hydrolysis of diverse nitriles under mild conditions. Some of their products, notably hydroxy acids, such as mandelic, 3-hydroxyvaleric, or glycolic acid, are of significant industrial importance [1]. Arylaliphatic nitrilases (arylacetonitrilases; EC 3.5.5.1, EC 3.5.5.5) can transform,...

Arylaliphatic nitrilases can under mild conditions and often in a stereo-selective manner transform α-hydroxy nitriles and α-aminonitriles - industrially important precursors in organic synthesis. Nitrilases are proteins with unique structural properties, being able to exist in a number of different homooligomeric species - dimers, short homo-oligo...

The detailed examination of enzymes molecules by mass spectrometry and other techniques continues to identify hundreds of distinct PTMs. Recently, global analyses of enzymes using methods of contemporary proteomics revealed widespread distribution of PTMs on many key enzymes distributed in all cellular compartments. Critically, patterns of multiple...

Interactions between C-type lectin-like NK cell receptors and their protein ligands form one of the key recognition mechanisms of the innate immune system that is involved in the elimination of cells that have been malignantly transformed, virally infected, or stressed by chemotherapy or other factors. We determined an x-ray structure for the extra...

Using a codon-optimized gene fragment, we report remarkable yields for extracellular domain of human NK cell receptor (NKp30ex) when produced on M9 minimal medium, even with low (2g/L) glucose concentration. The yields were identical using media containing (15)NH(4)Cl or (15)NH(4)Cl in combination with all-(13)C-d-glucose allowing to produce homoge...

Natural killer cells (NK cells) belong to lymphocytes, besides more familiar B and T-lymphocytes. They comprise 5-10% of lymphocytes in blood and their role in the immune system is to discover and kill cells with cancer and cells infected by viruses. NK cells have a number of receptors on their surface, which are used for contact with other cells a...

The glycan code of glycoproteins can be conceptually defined at molecular level by the sequence of well characterized glycans attached to evolutionarily predetermined amino acids along the polypeptide chain. Functional consequences of protein glycosylation are numerous, and include a hierarchy of properties from general physicochemical characterist...

α-N-Acetylgalactosaminidase (α-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal α-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the α-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The α-GalNAc-ase gene contains...

The structure of the H107R variant of the extracellular domain of the mouse natural killer cell receptor NKR-P1A has been determined by X-ray diffraction at 2.3 Å resolution from a merohedrally twinned crystal. Unlike the structure of the wild-type receptor in space group I4(1)22 with a single chain per asymmetric unit, the crystals of the variant...

We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected t...

Receptor proteins at the cell surface regulate the ability of natural killer cells to recognize and kill a variety of aberrant target cells. The structural features determining the function of natural killer receptor proteins 1 (NKR-P1s) are largely unknown. In the present work, refined homology models are generated for the C-type lectin-like extra...

Fungal β-N-acetylhexosaminidases are inducible extracellular enzymes with many biotechnological applications. The enzyme from Penicillium oxalicum has unique enzymatic properties despite its close evolutionary relationship with other fungal hexosaminidases. It has high GalNAcase activity, tolerates substrates with the modified N-acyl group better a...

Receptors belonging to NKR-P1 family and their specific Clr ligands form an alternative missing self recognition system critical in immunity against tumors and viruses, elimination of tumor cells subjected to genotoxic stress, activation of T cell dependent immune response, and hypertension. The three-dimensional structure of the extracellular doma...

Fungal β-N-acetylhexosaminidases are enzymes that are used in the chemoenzymatic synthesis of biologically interesting oligosaccharides. The enzyme from Aspergillus oryzae was produced and purified from its natural source and crystallized using the hanging-drop vapour-diffusion method. Diffraction data from two crystal forms (primitive monoclinic a...

Mouse NKR-P1C(B6) receptor corresponding to NK1.1 alloantigen is one of the most widespread surface markers of mouse NK and NKT cells in C57BL/6 mice detected by monoclonal antibody PK136. Although functional studies revealed the ability of this receptor to activate both natural killing and production of cytokines upon antibody crosslinking, the li...

Alignment of DNA sequences of fungal nitrilase and cyanide hydratase genes (pdf file).

Electron micrograph of Nit-ANigRec after re-folding (tif file).

PDB-file of the refined homology model of nitrilase subunit, prepared in YASARA.

Electron micrograph of Nit-ANigRec before re-folding (tif file).

Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases a...

Sedimentation analysis of macromolecules carried out with analytical ultracentrifuge is a powerful method for the study of proteins, nucleic acids and other polymers and their various complexes. Monitoring sedimentation of macromolecules in the centrifugal field allows their hydro-dynamic and thermodynamic characterization in solution, i.e. in nati...

Mouse NKR-P1F:Clr-g receptor:ligand pair is important com-ponent of the receptor ‘‘zipper’’ that occurs at the contact between natural killer cell and its target cell, and represent a recently discovered example of lectin-lectin interactions important for recognition of immune cell subsets. Previously, immunologists have brought evidence for the in...

Natural killer cells play a significant role in the immune response against tumor and infected cells. NK cells express a wide variety of surface receptors, including NKRP1, a C-type lectin-like family of both activating and inhibitory receptors. Recently, ligands have been found for some of these previously orphan molecules, some of them lying with...

Mouse NKR-P1B/D:Clrb receptor pair represents a recently dis-covered example of lectin – lectin interactions. In order to study this interaction by biochemical techniques, we have amplified the individual cDNA clones for the receptors by RT-PCR from B6/BL mice spleens and transferred DNA fragments coding for the extracellular ligand binding domains...

CD69 is a widespread receptor of the immune system cells. Although a physiological ligand for this receptor is unknown, we have previously proved its affinity towards a range of ligands including calcium, carbohydrates, and charged compounds such as carboxylated calixarenes. Moreover, a pentapeptide sequence LELTE derived from a mycobacterial heat s...

Natural killer cells are an intensively studied part of immune system due to their ability to directly kill cancer cells. Recent research in their C-type lectin-like receptors repertoire has shown that ligands of some of these previously orphan receptors are lying within their own family, describing a lectin-lectin interaction. This is also the cas...