Biology subjects

Majchrzak, M.

Publications and source records attributed to Majchrzak, M..

4 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↗

bioRxiv2023

Perilipin membrane association determines Lipid Droplet heterogeneity in differentiating adipocytes

The storage of fat within lipid droplets (LDs) of adipocytes is critical for whole-body health. Acute fatty acid (FA) uptake by differentiating adipocytes leads to the formation of at least two LD-classes marked by distinct perilipins (PLINs). How this LD-heterogeneity arises is an important yet unresolved cell biological problem. Here, we show that an unconventional integral membrane-segment (iMS) targets the adipocyte specific LD-surface factor PLIN1 to the endoplasmic reticulum (ER) and facilitates high affinity binding to the first LD-class. The other PLINs remain largely excluded from these LDs until FA-influx recruits them to a second LD-population. Preventing ER-targeting turns PLIN1 into a soluble, cytoplasmic LD-protein, reduces its LD-affinity and switches its LD-class specificity. Conversely, moving the iMS to PLIN2 leads to ER-insertion and formation of a separate LD-class. Our results shed light on how differences in organelle targeting and disparities in lipid-affinity of LD-surface factors contribute to formation of LD-heterogeneity. HighlightsO_LIPLIN1 is an integral membrane protein behaving as a class I LD protein. C_LIO_LIAn unconventional integral membrane segment (iMS) mediates ER insertion. C_LIO_LIThe iMS is required for LD-heterogeneity in differentiating adipocytes. C_LIO_LIHigh affinity LD-targeting from the ER is likely a gated process. C_LI eTOC blurbMajchrzak et al. use biochemistry, imaging and in vitro experiments to identify molecular features in PLIN1 that determine ER insertion and high lipid droplet affinity which are both important in the generation of LD-heterogeneity within differentiating adipocytes.

cell biology↗

bioRxiv2022

Functional brain-wide network mapping during acute stress exposure in rats: Interaction between the lateral habenula and cortical, amygdalar, hypothalamic and monoaminergic regions.

Upon stress exposure a broad network of structures comes into play in order to provide adequate responses and restore homeostasis. It has been known for decades that the main structures engaged during the stress response are the medial prefrontal cortex, the amygdala, the hippocampus, the hypothalamus, the monoaminergic systems (noradrenaline, dopamine, serotonin), and the periaqueductal gray. The lateral habenula (LHb) is an epithalamic structure directly connected to prefrontal cortical areas and to the amygdala, whereas it functionally interacts with the hippocampus. Also, it is a main modulator of monoaminergic systems. The LHb is activated upon exposure to basically all types of stressors, suggesting it is also involved in the stress response. However, it remains unknown if and how the LHb functionally interacts with the broad stress response network. In the current study we performed in rats a restraint stress procedure followed by immunohistochemical staining of the c-Fos protein throughout the brain. Using Graph Theory-based functional connectivity analyses, we confirm the principal hubs of the stress network (e.g. prefrontal cortex, amygdala, periventricular hypothalamus), and show that the LHb is engaged during stress exposure in close interaction with the medial prefrontal cortex, the lateral septum, and the medial habenula. In addition, we performed DREADD-induced LHb inactivation during the same restraint paradigm in order to explore its consequences on the stress response network. This last experiment gave contrasting results as the DREADD ligand alone, clozapine-N-oxide, was able to modify the network. GRAPHICAL ABSTRACT GRAPHICAL ABSTRACT TEXTIn this study, using immunohistochemical staining of the immediate early gene c-fos and graph theory-based functional correlational analyses, we aimed at unravelling the possible engagement of the lateral habenula (LHb) within the stress response network during acute stress exposure (10-min restraint) in rats. We found that the medial part of the LHb (LHbM) was preferentially engaged, and that this engagement was concomitant to this of structures such as the medial prefrontal cortex (mPFC), the insular cortex (Ins), hypothalamic (PVH) and thalamic (PVT) paraventricular nuclei, the extended amygdala, comprising the Bed nucleus of the stria terminalis (BNST) and the entire amygdala (AMG), as well as the dopaminergic ventral tegmental area (VTA) and the serotonergic dorsal raphe nucleus (RD). This suggests upon stressful situations the LHbM serves as a relay of cortical, thalamic, hypothalamic and temporal information, further transmitted to midbrain monoaminergic systems to probably initiate coping strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/491280v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a423eeorg.highwire.dtl.DTLVardef@32e1aaorg.highwire.dtl.DTLVardef@7d6106org.highwire.dtl.DTLVardef@16cff53_HPS_FORMAT_FIGEXP M_FIG C_FIG

animal behavior and cognition↗

bioRxiv2020

Correlation of pathogenic factors with antimicrobial resistance of clinical Proteus mirabilis strains

Proteus mirabilis is the third most common etiological factor of the urinary tract infection (UTI). It produces urease, which contributes to the formation of crystalline biofilm, considered to be one of the most important virulence factors of P. mirabilis strains, along with their ability to swarm on a solid surface. The aim of this study was to analyze the pathogenic properties of two selected groups of clinical P. mirabilis isolates, antimicrobial-susceptible and multidrug-resistant (MDR), collected from hospitals in different regions in Poland. The strains were examined based on virulence gene profiles, urease and hemolysin production, biofilm formation, and swarming properties. Additionally, the strains were differentiated based on the Dienes test and antibiotic susceptibility patterns. It turned out that the MDR strains exhibited kinship more often than susceptible ones. The strains which were able to form stronger biofilm had broader antimicrobial resistance profiles. It was also found that the strongest swarming motility correlated with susceptibility to most antibiotics. The correlations described in this work encourage further investigation of the mechanisms of pathogenicity of P. mirabilis. Author summaryProteus mirabilis is widely widespread in environment but also it is responsible for most Proteus infections, especially in human urinary tracts. They cause complicated, persistent infections especially due to the ability to form urinary stones. The clinical importance of P. mirabilis have been described in the literature many times. However, the role of pathogenic features with correlation to drug resistance require further investigation. In this research we analyzed thee virulence factors in relation to drug resistance of clinical P. mirabilis strains isolated from urine. The virulence genes, ureolytic and hemolytic activity, biofilm formation, swarming growth and strains kindship were analyzed. The most important observation was that the strains exhibited a stronger territorialism were kindred to a lower number of other strains, formed weaker biofilm and exhibited a lower resistance to antibiotics. Furthermore, we proved that the strains which were more likely to mutual growth, they were also less similar in the drug resistance profile but exhibited a higher resistance to antibiotics, which can be beneficial for different bacteria living together. We believe that P. mirabilis with strong territorialism can represent a wild group of strains with poor experience of antibiotic pressure. The environmental influence (toxins, antibiotics, bacterial neighbors) stimulates the development of a less dispersed community with stronger biofilm, exchange of genes and increase of resistance to antibiotics.

microbiology↗