Natural killer (NK) cells are key components of innate immunity. The task of these cells is to kill virally infected cells or tumour cells. A NK cells have a variety of surface receptors to recognize target cells. Signals which the NK cell is getting from receptors could be activating or inhibitory (so they activate or inhibit cytotoxic response of the cell). Killing of an infected or otherwise damaged cell is mediated by so-called cytotoxic molecules (proteins) found in the NK cells. Recognition of the target cell induces immune synapse formation through which the cytotoxic molecules are introduced into the target cell, which leads to apoptosis.
One of the natural cytotoxicity receptors (NCRs) of NK cells is NKp44. NKp44 is a unique protein in the family of NCR. It has been observed that binding of a proliferating cell nuclear antigen (PCNA), a nuclear protein involved in DNA replication expressed on cell surface upon malignant transformation, to NKp44 leads to an inhibitory signal, even though it was assumed that NKp44 is an activating receptor.
The goal of this project is to characterise the interaction between NKp44 and PCNA and finally to try to crystallize the NKp44:PCNA complex. Both proteins are being prepared via recombinant expression in HEK293S GnTI- cell line. Purification is performed by affinity chromatography using TALON or protein A columns followed by gel filtration. PCNA has been already successfully prepared by intracellular expression in milligram amounts. Recombinant expression of extracellular domain of NKp44 failed so far, however, we are able to produce NKp44 in fusion with Fc fragment of human IgG by secreted expression to cell culture media. Although the yield of this NKp44 fusion construct is not sufficient for crystallization and optimisation of NKp44 production is still ongoing, it enabled us to perform preliminary biophysical characterization of NKp44:PCNA interaction using microscale thermophoresis and analytical ultracentrifugation.
This study was supported by Czech Science Foundation (18-10687S) and Ministry of Education, Youth and Sports of the Czech Republic (LTC17065 in frame of the COST Action CA15126).