Laureates of FNP programmes from the Wrocław University of Science and Technology (WUST), in partnership with American researchers, have developed a new method to simultaneously scan the activity of proteolytic enzymes in cells. Their research results have been published in the prestigious Journal of the American Chemical Society (JACS). The first author of the paper is Marcin Poręba, PhD Eng., a two-time laureate of START; and the second Polish author is Professor Marcin Drąg, a laureate of multiple FNP programmes and the FNP Award 2019. Part of the project was completed under the TEAM-NET programme by the Foundation for Polish Science.
In the published paper, its authors describe how they had developed a set of chemical markers labelled with stable isotopes of transition metals (mainly lanthanides) to scan specific proteolytic enzymes using mass cytometry. Mass cytometry is an advanced technology that uses stable metal isotopes (usually lanthanides) as markers for antibodies or DNA probes. Due to the fact that each of these metals (markers) has a specific molecular weight, many such metals can be used during a single experiment, because their weight spectrums do not overlap. As a result, more than 40 cell parameters can be measured at the same time, which is significantly more than for flow cytometry which uses fluorescent markers. Over the past few years, there have been rapid advancements in mass cytometry, both in technical terms and in relation to its application in the study of various diseases, such as autoimmune diseases, infections, and cancer. Mass cytometry made it possible to accurately determine the immunophenotype of specific (sub)types of autoimmune diseases and cancer, which helped categorise them better and ultimately recommend optimum treatment. Moreover, mass cytometry is now gaining recognition in clinical research as a useful tool to monitor treatment progress and forecast therapy effectiveness.
The authors of the study were the first to create chemical markers for proteolythic enzymes that can be used in mass cytometry. And this discovery facilitates simultaneous examination of multiple enzymes, which can provide better insights into many cellular processes, including those leading to disease development. This set of enzyme-specific chemical markers will support the exploration of the activome, a concept developed by WUST scientists and defined as the active part of the proteome. The paper focuses on the analysis of the activity of four medically significant proteolythic enzymes, which are involved in the development of many diseases, including cancer. These enzymes are legumain, cathepsin L, cathepsin B, and neutrophil elastase. The researchers have determined the expression and activity levels for these enzymes in specific populations of peripheral blood mononuclear cells (PBMCs). This will help better understand the mechanisms behind the emergence and development of diseases in which proteolythic enzymes play a crucial role.
One of such diseases is neutropenia – an abnormally low concentration of neutrophils (neutrophil granulocytes) in peripheral blood, that can be either permanent or temporary. It is believed that one of its causes is abnormal catalytic activity of neutrophil elastase. With a selective chemical marker marked with a specific lanthanide for elastase, combined with an antibody panel for peripheral blood cells (also marked with lanthanides), the researchers were able to accurately identify in which peripheral blood cell populations from healthy donors the enzyme showed catalytic activity. This part of the study directly contributes to the Foundation’s TEAM-NET grant entitled “FIXNET: We Will Cure Neutropenia”. Now the authors are using their technology to study the activity of not only elastase but also other neutrophil serine proteases in samples from neutropenia patients. The study is implemented in partnership with the Medical University of Lodz.
The lead author of the published work is Marcin Poręba, PhD Eng., who had worked on the study in the laboratory of Professor Guy Salvesen (Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA) as part of his Marie Skłodowska-Curie Global Fellowship grant. Later the study was continued in the laboratory of Professor Marcin Drąg at the Wrocław University of Science and Technology, in cooperation with Katarzyna Groborz, MSc Eng., the first co-author. Currently, Marcin Poręba, Phd Eng., is contributing to TEAM-NET, where he is in charge of using chemical markers marked with metals to study neutropenia. At the WUST, the project is being coordinated by Professor Marcin Drąg. It is also important to note that WUST’s Department of Chemical Biology and Bioimaging, where the researchers work, is the only facility in Poland to have Helios by Fluidigm, a unique mass cytrometry system for imaging cells in suspension, and Hyperion, a system for imaging cells and tissues on slides.
- You can find the publication here
- For more information about the WUST team and their TEAM-NET project
- For more information about TEAM-NET
- Learn about FIXNET: We Will Cure Neutropenia ( a project supported from TEAM-NET)