Artur Krężel

Protein–drug conjugates (PDCs) constitute a rapidly growing group of precise anticancer agents. Receptor-mediated endocytosis is a critical step in PDC action, which ensures the delivery of PDCs into the interior of cancer cells. Here, we report TriFHS-MMAE, the first multivalent PDC specifically targeting heparan sulfate proteoglycans (HSPGs) overexpressed by pancreatic cancer cells, which induces ultrafast and highly efficient aggregation-dependent endocytosis (ADE) of HSPGs. Using high-content screening with a near-kinome-wide library of inhibitors, we identified signaling pathways that govern ADE of HSPGs and discovered cascades that selectively operate in pancreatic cancer cells versus healthy cells. We show that priming cells with identified endocytic chemical modulators improves the targeting of the TriFHS-MMAE conjugate in vitro and in vivo. Overall, these findings provide insights into the interplay among signaling, endocytosis, and PDCs and support the development of specific therapies for pancreatic cancer.

Complementary mass spectrometry-based approaches and molecular dynamics simulations were integrated to map Zn( ii ) distribution in partially metalated Zn _4–6 -metallothionein-3 species relevant to zinc metabolism.

introduction to the 22nd international Biometals Webinars The spotlights of these iBWs will be firstly on zinc binding comparisons to metallothioneins from various organisms. Metallothioneins aremetal-binding proteins essential in metallic stress conditions. The second spotlight will be on iron homeostasis in the opportunistic pathogen Pseudomonas aeruginosa.The focus will be on this homeostasis in bacterial biofims more resistant to antibiotherapy Polymorphism and metamorphism of metallothioneins in zinc homeostasis Artur Krężel is Professor of Biological Sciences and Department Chair of Chemical Biology at the University of Wrocław (Poland). He received his diploma in chemistry (master of science, 2000) and a Ph.D. degree in bioinorganic chemistry (2004) from the University of Wroclaw, Poland, under the supervision of Prof. Wojciech Bal. During his postdoctoral training (2004-2007) at the University of Texas Medical Branch in Galveston, he worked with Prof. Wolfgang Maret on mechanisms of cellular zinc homeostasis, which culminated in the discovery of metallothioneins as physiological zinc ion buffers. He then accepted a position as an assistant professor at the Faculty of Biotechnology of his alma mater. In 2011, he received a D.Sc. degree from his university. Currently, he is a full professor and head of the Department of Chemical Biology. His research concentrates on several areas at the interface of inorganic biochemistry, biophysics, and chemical biology, particularly understanding the molecular bases of zinc and copper metabolism. Examples of this endeavor are structure-function relationships, the stability of metalloproteins, protein folding and thermodynamics, and the development of new analytical methods, such as fluorescent probes for selective protein modifications and metal sensing, mass spectrometry approaches for studying metalloproteins (un)folding and structures. Iron homeostasis in Pseudomonas aeruginosa biofilms Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes both acute and chronic infections in vulnerable populations. Treatment of P. aeruginosa infections is increasingly challenging due to multi-drug resistance, and biofilm formation during infection further increases antibiotic tolerance. Iron, which is sequestered by the host innate immune system, is also a key nutrient that is required for P. aeruginosa biofilm formation. The iron-responsive PrrF small regulatory RNAs (sRNAs) are key to P. aeruginosa’s iron starvation response, promote the production of the PQS quorum sensing molecule, and are required for virulence in murine lung infection. Prior work showed that the PrrF sRNAs are dispensable for biofilm formation; however, these studies were performed using flow-cell biofilms grown at room temperature. Here we demonstrate a temperature dependency for PrrF in P. aeruginosa biofilm formation: the genes for these sRNAs are required for optimal biofilm formation at 37°C but not 25°C. We further show that a ∆pqsA mutant, which lacks production of PQS and related metabolites, phenocopies the ∆prrF mutant. These studies demonstrate the importance of the PrrF sRNAs in P. aeruginosa biofilm formation at body temperature and reveal a previously under-appreciated role of temperature in iron homeostasis and P. aeruginosa biofilm physiology.

Garlic (Allium sativum) is a key culinary ingredient and agricultural product that releases its pungent aroma when cut or crushed due to the presence of volatile organosulfur compounds. These compounds, specifically allicin and ajoene, rapidly modify human metallothioneins (MTs), leading to the dissociation of Zn(II) ions. This study employed a top-down protein MS analysis to identify the most reactive cysteine sites in MT2, confirming that both compounds induce S-thioallylation and alter Zn(II) binding. Spectrophotometric studies indicated that both compounds mobilized nearly six molar equivalents of Zn(II) from MT2 after one hour, with ajoene demonstrating slightly higher dissociation efficiency than allicin. Moreover, allicin mobilized Zn(II) from all human isoforms of MT (MT1-MT4), with the lowest efficiency observed for MT4. Mass spectrometry analysis revealed a diverse array of modified complexes, with ajoene resulting in more complex formations than allicin. Reduced glutathione interacts with both untreated and allicin- or ajoene-treated MTs, forming mixed and partially oxidized complexes, highlighting the relationship between these compounds depending on the cell’s redox state. The use of reducing agents demonstrated that MTs’ S-thioallylation is reversible, allowing the protein to rebind Zn(II) after reduction, thereby maintaining its role in zinc homeostasis and rendering the effects of allicin temporary. The health implications of the modifications are significant, given the established role of Zn(II) in inhibiting pro-inflammatory pathways and enhancing antioxidant responses. Zn(II) ions were shown to inhibit the activation of the NF-κB pathway, which regulates pro-inflammatory cytokines. Our findings indicate that the modification of metallothioneins (MTs) by garlic-derived organosulfur compounds may play a crucial role in zinc homeostasis, with implications for immune regulation in humans, and warrant further investigation into their potential therapeutic applications. Graphical Abstract

Breast cancer is the most common malignancy in women, with approximately 20-30% of all diagnosed cases characterized by HER2 overexpression. Several HER2-targeted cytotoxic conjugates have been developed, but their efficacy is limited. One of the main obstacles restraining the effectiveness of HER2-specific cytotoxic conjugates is their low internalization, as HER2 is immobile mainly on the cell surface. Therefore, there is a need to develop novel HER2-selective cytotoxic conjugates that will overcome HER2 immovability and, by this, ensure efficient drug delivery into HER2-overexpressing cancer cells. Here, we present a novel system for generating high affinity, self-assembling, inherently fluorescent, and multivalent HER2 ligands. The developed HER2-specific ligands largely overcome the innate stability of HER2 in the plasma membrane by triggering clathrin-independent aggregation-dependent endocytosis of the receptor. To exploit the pro-endocytic potential of developed proteins, we constructed the tetravalent fluorescent cytotoxic conjugate TetraF_HER2-vcMMAE and demonstrated its high potency and selectivity against HER2+ breast cancer cells.

Zinc (Zn²⁺) plays a pivotal role in T-cell activation by modulating the interactions between the co-receptors CD4 and CD8α and the Src-family kinase Lck. A central structural feature in this regulation is the zinc clasp, a Zn²⁺-mediated CD4/CD8α-Lck receptor interface that stabilizes these complexes during T cell receptor signaling. Recent findings reveal that the stability of CD4-Lck and CD8α-Lck complexes is differentially regulated by Zn²⁺, which acts as a dynamic signaling molecule during T-cell activation. Here, we discuss the structural dynamics of these interactions and the impact of Zn²⁺ on CD4 dimerization, palmitoylation, and membrane interactions, which are crucial for effective T-cell responses. These mechanisms underscore a broader framework in which zinc biology intersects with co-receptor-Lck coupling to guide T-cell development, lineage fidelity, and functional specialization. Beyond immunobiology, zinc-dependent protein-protein interactions offer promising opportunities for biotechnological innovation, particularly in the design of molecular systems that exploit zinc-mediated structural control.

The transcription factor p53 is exquisitely sensitive and selective to a broad variety of cellular environments. Several studies have reported that oxidative stress weakens the p53-DNA binding affinity for certain promoters depending on the oxidation mechanism. Despite this body of work, the precise mechanisms by which the physiologically relevant DNA-p53 tetramer complex senses cellular stresses caused by H₂O₂ are still unknown. Here, we employed native mass spectrometry (MS) and ion mobility (IM)-MS coupled to chemical labelling and H₂O₂-induced oxidation to examine the mechanism of redox regulation of the p53-p21 complex. Our approach has found that two reactive cysteines in p53 protect against H₂O₂-induced oxidation by forming reversible sulfenates.

The expanding field of site-specific ligation of proteins and peptides has catalyzed the development of novel methods that enhance molecular modification. Among these methods, enzymatic strategies have emerged as dominant due to their specificity and efficiency in modifying proteins under mild conditions. Asparaginyl endopeptidase is a group of cyclotide-producing cysteine proteases from plants. These plant cysteine proteases, known for their specificity, effectively recognize the tripeptide motif (Asx-Xaa-Yaa) and cleave at the C-terminal side of Asx residues, forming acyl-enzyme intermediates that facilitate transpeptidation. Butelase 1 stands out as the most efficient AEP for protein engineering, yet challenges in its expression and purification limit its accessibility for widespread research and industrial use. To address these challenges, we engineered a new, catalytically efficient variant of Butelase 1, Butelase AY, by mutating the gatekeeping residues Val237Ala and Thr238Tyr within the LAD-1 region. These modifications significantly enhanced the stability and yield of Butelase AY, allowing for successful application in various peptide and protein engineering tasks. Butelase AY was tested on the peptide GLGKY, the globular protein GFP, and the intrinsically disordered protein α-synuclein, effectively labeling them with a fluorescent probe. Notably, Butelase AY maintained its efficiency with substrates containing unnatural amino acids, making it a promising candidate for biorthogonal applications. Importantly, the mutations did not compromise the enzyme’s specificity, as it continued to process model peptides and native protein substrates with N-term NHV recognition motifs effectively. In conclusion, Butelase AY presents a novel recombinant tool for diverse protein labeling and modifications, particularly in biorthogonal strategies. This innovation has the potential to expand applications in biotechnology and therapeutic development, ultimately revolutionizing protein engineering and its utility in synthetic biology.

To understand how particular post-translational modifications (PTMs) affect the function of a target protein, it is essential to first prepare and investigate the target with the modification at the desired position. This drives the continuous development of site-specific protein modification technologies. Here, we present the chemical synthesis and application of the biarsenical linker SrtCrAsH-EDT2, which has a dual labeling functionality. This linker, containing a sortase A recognition motif, can be conjugated with any protein containing the LPXTG motif at the C terminus, such as ubiquitin and the SUMO tag, and then attached to a protein of interest (POI) containing a terminal or bipartite (intramolecularly placed) tetracysteine motif. This modification of the POI facilitates the straightforward and rapid incorporation of PTMs, which are further highlighted by the fluorescent biarsenical probe. Consequently, this directly correlates proteins' physical properties and cellular roles under various physiological conditions or in disease states. The proposed one-pot labeling methodology can be utilized to explore the effects of PTMs on proteins, affecting their structure, function, localization, and interactions within the cellular environment. Understanding these effects is crucial for uncovering the complex mechanisms that regulate cellular function and dysfunction.