A list of key parts of the work performed from Jan 01, 2016 to the Dec 31, 2018:
CASPASES chemical tools:
1. The development of small-molecule fluorogenic substrates and activity based probes for the investigation of apoptotic caspases (-3, -7, -8, -9, and -10).
2. Evaluation of the specificity of obtained activity based probes toward a panel of apoptotic caspases in in vitro experiments.
3. Using selected substrates and activity based probes we studied apoptosis mechanisms (extra- and intra-cellular) in various cancer cell lines.
4. We have developed new selective chemical tools (substrates and inhibitors) for investigation of human caspase 2. The selectivity of these probes were confirmed by performing kinetic studies and in vitro competitive labeling toward six human apoptotic caspases. We also demonstrated that one of the caspase-2 inhibitor can selectively block reversine-mediated caspase-2 activation in HCT-116 cells.
Related publications:
A) Poreba M et al Scientific Reports, 2017, doi: 10.1038/srep43135
B) Poreba M et al. Nature Protocols 2017, doi: 10.1038/nprot.2017.091
C) Ramirez MLG, Poreba M, et al. JBC 2018 doi: 10.1074/jbc.RA117.001329
D) Poreba M et al CDD 2019 doi: 10.1038/s41418-018-0110-y
E) Poreba M, et al CDD 2019 doi: 10.1038/s41418-019-0329-2
F) Groborz K et al CDD doi: 10.1038/s41418-019-0364-z
CATHEPSIN/LEGUMAIN chemical tools:
1. Using our HyCoSuL technology, we developed new very selective fluorescent-labeled small molecule inhibitors for studying human cysteine cathepsins (cathepsin L and cathepsin B) and legumain in cancer cells..
2. We tested the specificity of obtained activity based probes toward cathepsin L, cathepsin B and legumain in in vitro experiments and used them to visualize these enzymes in living cells (fluorescence microscopy).
Related publications:
G) Poreba M et al CCB 2016 doi: 10.1016/j.chembiol.2016.05.020
H) Kasperkiewicz P et al FEBS Journal 2017 doi: 10.1111/febs.14001
I) Poreba M et al Chemical Science 2018 doi: 10.1039/c7sc04303a
J) Poreba M et al Chemical Science 2019 doi: 10.1039/c9sc00997c
MASS CYTOMETRY:
1. We developed a first panel of metal-tagged, mass cytometry-suitable chemical probes for dissecting proteases activtiy. Next, we used these probes to visualize cathepsin L, cathepsin B, legumain, and caspase-3 in cancer cells and human Peripheral Blood Mononuclear Cells (PBMCs) using mass cytometry. This is the first time ever, when mass cytometry-suitable chemical probes are used to detect enzymes activity. Moreover, we developed an integrated platform where we used both metal-tagged probes and metal-tagged antibodies to dissect proteolytic landscape in various populations (T cells, B cells, NK cells) in human PBMCs.
2. We also used our metal-labeled probes for the visualization of proteases in mice-models (cancer cell lines and cells isolated from mouse organs - kidney, liver, brain, and spleen).
Related publication:
K) Poreba M et al 2019, under review
Important remark: Since our mass cytometry data were very promising in context of multiparametric enzymes analysis, the Rector of our University approved to purchase mass cytometer (Helios, 3rd generation) to support our further research projects. We received this instrument on October 2018, and we have set up the first mass cytometry facility in Poland. Currently, I am responsible for training of other researchers on this instrument. We also submitted several grant proposals in which mass cytometer is a key technology, making our research very competitive in our field.
