Biology subjects

Kasperkiewicz, P.

Publications and source records attributed to Kasperkiewicz, P..

5 recordsLinked to original sources

bioRxiv2019

Noninvasive optical detection of Granzyme B from natural killer cells using enzyme-activated fluorogenic probes

Despite many studies on the cytotoxic protease granzyme B, key aspects of its function remain unexplored due to the lack of selective probes for its activity. In this study, we fully mapped the substrate preferences of GrB using a set of unnatural amino acids, demonstrating previously unknown GrB substrate preferences that we then used to design novel substrate-based inhibitors and a GrB-activatable activity-based probe. We showed that our GrB probes react poorly with caspases, making them ideal for the in-depth analysis of GrB localization and function in cells. With our quenched fluorescence substrate, we determined GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with the GrB attack MDA-MB-231 target cells, and active GrB influences its target cell killing efficiency.

biochemistry↗

bioRxiv2019

Detection of active Granzyme A in NK92 cells with fluorescent activity-based probe

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation; and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.

biochemistry↗

bioRxiv2019

Tissue-ABPP enables high-resolution confocal fluorescence imaging of serine hydrolase activity in cryosections - Application to glioma brain unveils activity hotspots originating from tumor-associated neutrophils

Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. The ABPP approach utilizes cell/tissue proteomes and features the FP warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Here, we advance the ABPP methodology to glioma brain cryosections, enabling high-resolution confocal fluorescence imaging of SH activity in different cell types of the tumor microenvironment, identified by using extensive immunohistochemistry on activity probe labeled sections. We name this technique tissue-ABPP to distinguish it from conventional gel-based ABPP. We show heightened SH activity in glioma vs. normal brain and unveil activity hotspots originating from tumor-associated neutrophils. Thorough optimization and validation is provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Tissue-ABPP enables a wide range of applications for confocal imaging of SH activity in any type of tissue or animal species.

cancer biology↗

bioRxiv2019

Leveraging peptide substrate libraries to design inhibitors of bacterial Lon protease

Lon is a widely-conserved housekeeping protease found in all domains of life. Bacterial Lon is involved in the recovery from various types of stress, including tolerance to fluoroquinolone antibiotics, and is linked to pathogenesis in a number of organisms. However, detailed functional studies of Lon have been limited by the lack of selective, cell-permeable inhibitors. Here we describe the use of positional scanning libraries of hybrid peptide substrates to profile the primary sequence specificity of bacterial Lon. In addition to identifying optimal natural amino acid binding preferences, we identified several non-natural residues that were leveraged to develop optimal peptide substrates as well as a potent peptidic boronic acid inhibitor of Lon. Treatment of E. coli with this inhibitor promotes UV-induced filamentation and reduces tolerance to ciprofloxacin, phenocopying established lon-deletion phenotypes. It is also non-toxic to mammalian cells due to its increased selectivity for Lon over the proteasome. Our results provide new insight into the primary substrate specificity of Lon and identify substrates and an inhibitor that will serve as useful tools for dissecting the diverse cellular functions of Lon.

microbiology↗

bioRxiv2019

The cytoprotective role of antioxidants in mammalian cells under rapidly varying temperature, pressure and UV conditions during stratospheric balloon campaign

1.Currently ongoing age of the dynamic development of the space industry brings the mankind closer to the routine manned space flights and space tourism. That progress leads to a demand for intensive astrobiological research aimed at improving strategies of the pharmacological protection of the human cells against extreme conditions. Although routine research in space remain out of our reach, it is worth noticing that unique severe environment of the Earths stratosphere have been found to mimic subcosmic conditions, giving rise to the opportunity for use of stratospheric surface as a research model for the astrobiological studies. Our study included launching balloon into the stratosphere containing the human normal and cancer cells treated with various compounds to examine whether these medicines are capable to protect the cells against the stress caused by rapidly varying temperature, pressure and radiation, especially UV. Due to oxidative stress caused by irradiation and temperature shock, we used natural compounds which display antioxidant properties, namely catechin isolated from green tea, honokiol derived from magnolia, curcumin from turmeric and cinnamon extract. "After-flight" laboratory tests displayed the most active antioxidants as potential agents which can minimize harmful impact of extreme conditions to the human cells.

cell biology↗