Biology subjects

Poreba, M.

Publications and source records attributed to Poreba, M..

8 recordsLinked to original sources

bioRxiv2025

Engineering cathepsin S selective chemical probes and antibody-drug conjugates through substrate profiling with unnatural amino acids

Cysteine cathepsins, particularly cathepsin S, are important regulators of proteolytic signaling in health and disease, including cancer progression and immune modulation. Despite their therapeutic relevance, selective chemical tools to study individual cathepsins remain limited due to overlapping substrate preferences. Here we present design of cathepsin S selective chemical probes and cathepsin S-cleavable antibody-drug conjugates (ADCs) through substrate profiling with unnatural amino acids. First, Hybrid Combinatorial Substrate Library (HyCoSuL) technology incorporating a broad spectrum of unnatural amino acids was applied to comprehensively investigate the substrate specificity of cathepsin S. This approach enabled the identification of highly selective tetrapeptide motifs that served as scaffolds for the design of optimized fluorogenic substrates, irreversible inhibitors, and fluorescent activity-based probes (ABPs). These tools demonstrated high selectivity toward cathepsin S over closely related family members in both biochemical and cellular settings. We then translated these findings to develop cathepsin S-activated ADCs, incorporating the optimized peptide motifs as protease-cleavable linkers for targeted payload release. Using these linkers, we further generated MMAE-based antibody-drug conjugates directed against HER-2 and the TROP-2 proteins, demonstrating cathepsin S-dependent cytotoxicity in HER-2-positive as well as HER-2-negative/TROP-2-positive breast cancer models. Finally, we employed anti-cathepsin S antibodies in combination with mass cytometry (CyTOF) to assess the spatial distribution of cathepsin S expression in breast cancer patient samples. By correlating cathepsin S levels with tumor-associated markers such as TROP-2 and HER-2, we identified potential co-expression patterns that support the rationale for personalized therapy using antibody-drug conjugates selectively activated by cathepsin S. This integrative approach provides a comprehensive platform for profiling cathepsin S activity at the molecular, cellular, and tissue levels, with broad implications for the development of precision therapeutics and diagnostic strategies in oncology.

cancer biology↗

bioRxiv2025

Evaluation of cytotoxic effects of diverse essential oils on human keratinocytes (HaCaT)

Essential oils (EOs) are widely used in cosmetics for their active and aromatic properties, yet their cytotoxicity remains a concern, especially regarding skin safety. This study aimed to evaluate the cytotoxicity of 32 essential oils on immortalized keratinocytes (HaCaT cells) using an MTS assay. Results demonstrated significant variability in cell viability, with 14 oils reducing viability below 50%. These findings underscore the need for further research into the specific chemical constituents responsible for the observed effects.

pharmacology and toxicology↗

bioRxiv2025

Single-cell profiling of CAR-T CD19 cell phenotypes and immune system dynamics in pediatric BCP-ALL

BackgroundChimeric Antigen Receptor T-cell (CAR-T) therapy targeting CD19 has transformed the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, the phenotypic heterogeneity of CAR+ T-cells and their interactions with the immune system remain poorly understood. Here, we characterize CAR+ T-cell subsets and persistence in patients receiving standard-of-care tisagenlecleucel in an effort to optimize therapeutic efficacy in pediatric BCP-ALL. MethodsNineteen pediatric patients with relapsed/refractory BCP-ALL treated with tisagenlecleucel in Poland were included in the study. CAR+ T-cell composition and the peripheral blood mononuclear cell (PBMC) immune system were assessed using mass cytometry, with qPCR validation of CAR+ T-cells. Machine learning algorithms in R classified phenotypes. Statistical analyses examined associations between CAR+ T-cell subsets, immune system changes, and CAR-T therapy outcomes, including cytokine release syndrome (CRS). ResultsInfusion product analysis showed a predominance of CD4+ Central Memory (16.6%), MAIT/NKT (7.04%), and Treg memory (63.3%) cells, with high consistency between paired products. Post-infusion, CAR+ Treg memory cells declined to <1%, while CD8+ subsets expanded. PBMC immune system analysis revealed increased monocyte and NK cell counts post-infusion. Patients who experienced CRS had fewer classical monocytes at day 7 and fewer transitional monocytes at day 28. Early and late NK cells increased post-infusion in all patients. However, pre-infusion levels were lower in patients experiencing CRS. CRS correlated with higher CAR+ CD8+ Terminal Effector and MAIT/NKT frequencies in infusion products. While CAR+ composition was not linked to disease burden, prior treatments, or therapy outcomes, changes in monocyte and NK cell dynamics were associated with disease burden, relapse, and CRS. ConclusionsThis study provides a detailed characterization of CAR-T CD19 cell composition and post-infusion dynamics in pediatric BCP-ALL patients treated with tisagenlecleucel. Infusion products were predominantly CAR+ CD4+ T cells with a Central Memory phenotype, including a notable Treg memory subset that declined post-infusion. CAR+ CD8+ subsets expanded, coinciding with immune system shifts. These changes correlated with clinical responses, including CRS, highlighting the role of the immune system in CAR-T therapy outcomes and informing strategies to optimize treatment. Summary boxO_ST_ABSWHAT IS ALREADY KNOWN ON THIS TOPICC_ST_ABSCD19-targeted CAR-T therapy has revolutionized the treatment of relapsed/refractory pediatric BCP-ALL. While studies have examined the diverse composition of CAR-T infusion products in adult large B-cell lymphoma (LBCL), there is limited data in the pediatric BCP-ALL setting. WHAT THIS STUDY ADDSThis study demonstrates that standard-of-care CAR-T infusion products (tisagenlecleucel) are primarily composed of CAR+ CD4+ Central Memory and regulatory T cells, which decline post-infusion as CAR+ CD8+ subsets expand. Additionally, monocyte and NK cell dynamics are closely linked to cytokine release syndrome (CRS) and treatment outcomes, emphasizing the critical role of immune system interactions in CAR-T therapy efficacy. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICYThese findings indicate that monitoring immune system shifts could enhance CRS risk assessment and improve patient stratification, leading to more personalized CAR-T therapy. Additionally, insights into CAR+ T-cell composition may guide the development of strategies to optimize CAR-T efficacy, potentially improving long-term treatment outcomes in pediatric BCP-ALL.

cancer biology↗

bioRxiv2025

Delivery of caspase inhibitors through GSDMD pores to inhibit pyroptosis

Caspase-1, -4, -5, and -11 activate Gasdermin D (GSDMD) to form pores in the plasma membrane. In addition to releasing interleukin (IL)-1{beta} and IL-18, GSDMD pores cause a lytic, proinflammatory form of cell death called pyroptosis. Blocking this pathway holds therapeutic promise for the treatment of inflammatory disorders, but clinical trials of cell permeable caspase inhibitors have been unsuccessful. Here, we describe covalent inhibitors of proinflammatory caspases that are impermeable to healthy cells but effectively block caspase-1 driven pyroptosis and IL-1{beta} secretion. Their failure to inhibit apoptosis implies inhibitor entry through GSDMD pores. Propidium iodide entered rescued cells, confirming transient membrane permeabilization via caspase-1 and GSDMD. Caspase-1 inhibition prevented rather than delayed cell death, likely due to the activation of membrane repair mechanisms neutralizing the initial GSDMD pores. Notably, inhibiting caspase-1 and -11 suppressed IL-1{beta} and IL-18 production in a mouse model of endotoxic shock. These findings underscore the therapeutic potential of exploiting GSDMD pores for the delivery of caspase inhibitors, offering a novel strategy for treating inflammatory diseases.

cell biology↗

bioRxiv2024

Glycosaminoglycans act as activators of peptidylarginine deiminase 4

Peptidylarginine deiminase 4 (PAD4) is a citrullinating enzyme that is gathering increasing attention due to its possible involvement in physiological processes as well as in the pathogenesis of diseases like rheumatoid arthritis or thrombosis. PAD4 is activated by calcium ions, but the details of this mechanism are elusive, because in the human body, Ca2+ concentrations are too low for full activity. Given that glycosaminoglycans (GAGs) are also implicated in the development and progression of rheumatoid arthritis, we investigated the activation of PAD4 by GAGs using heparin as a model. We employed activity assays, chromatography techniques, molecular interaction measurements (MST and SPR), FACS, and immunocytochemistry to demonstrate the activation of PAD4 by GAGs. Our data show that PAD4 binds heparin with high affinity and forms high molecular weight complexes with heparin, consistent with heparin-bound tetramer formation. Heparin activates PAD4 by increasing the enzymes Ca2+ affinity threefold. We also show that the effectiveness of activation with heparin depends on the length of GAG used and its negative charge. Direct measurement of heparin binding to PAD4 confirmed tight interaction with nanomolar affinity. Mutagenesis of regions likely responsible for heparin binding showed that dimerization of PAD4 is necessary for efficient activation, but the distinct binding site was not determined as interaction with heparin likely occurs over larger surface of PAD4. Furthermore, we show that other GAG family members, including heparan and chondroitin sulphates, are also able to activate PAD4. We also found that disturbed production of GAGs by CHO cells results in reduced PAD4 binding efficiency. Finally, heparin induces NETosis in hPMNs in concentration-dependent manner, as measured by the release of DNA and citrullination of histone H3. In summary, we identify the first natural coactivator of PAD4, which is present in all individuals, potentially explaining the regulation of PAD4 activity in physiological conditions, and providing new insight into the development of rheumatoid arthritis and other PAD4-related diseases.

biochemistry↗

bioRxiv2023

Thermoprotection by a cell membrane-localized metacaspase in a green alga

Caspases are restricted to animals, while other organisms, including plants possess metacaspases (MCAs), a more ancient and broader class of structurally-related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis expressing nine MCAs with partly redundant activities. In contrast to streptophytes, most chlorophytes contain only one or two hitherto uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigate CrMCA-II, a single type II MCA from a model chlorophyte Chlamydomonas reinhardtii. Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlates with the dimerization. Most of CrMCA-II in the cell is present as a zymogen attached to the plasma membrane (PM). Deletion of CrMCA-II by CRISPR/Cas9 compromises thermotolerance leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restores thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we link the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

plant biology↗

bioRxiv2019

The Activome: multiplexed probing of activity of proteolytic enzymes using mass cytometry-compatible activity-based probes (TOF-probes)

The activome can be considered as a subset of the proteome that contains enzymes in their catalytically active form and can be interrogated by using probes targeted towards individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, simultaneous analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry. Using lanthanide-based tags instead of fluorophores gives us the ability to monitor the activity of multiple proteases in parallel. As proof of concept we developed a panel of cathepsin and legumain specific probes and showed that we were able to identify an activome of these proteases in two cell lines and peripheral blood mononuclear cells, providing a framework for the use of mass cytometry for multiplexed enzyme activity detection.

biochemistry↗

bioRxiv2018

Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies different amino acid preferences between peptide-based substrates and inhibitors.

Malarial dipeptidyl aminopeptidases (DPAPs) are cysteine proteases important for parasite development thus making them attractive drug targets. In order to develop inhibitors specific to the parasite enzymes it is necessary to map the determinants of substrate specificity of the parasite enzymes and its mammalian homologue cathepsin C (CatC). Here, we screened peptide-based libraries of substrates and covalent inhibitors to characterize the differences in specificity between parasite DPAPs and CatC, and used this information to develop highly selective DPAP1 and DPAP3 inhibitors. Interestingly, while the primary amino acid specificity of a protease is often used to develop potent inhibitors, we show that equally potent and highly specific inhibitors can be developed based on the sequences of non-optimal peptide substrates. Importantly, analysis of previously published data about the specificity of other proteases also unveiled significant discrepancies in the amino acid preference between substrates and inhibitors. In this article, we also discuss important structural and theoretical reasons that might account for these discrepancies. Overall, this study illustrates that focusing the development of protease inhibitors solely on substrate specificity might overlook important structural features that can be exploited to develop highly potent and selective compounds.

biochemistry↗