Biology subjects

Szymanski, W.

Publications and source records attributed to Szymanski, W..

7 recordsLinked to original sources

bioRxiv2026

Cell-specific FUS loss enhances ageing-associated transcriptional elongation in lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is an age-related, lethal lung disease affecting over 3 million people worldwide. During aging, impairment of diverse molecular processes compromises cellular homeostasis. Eukaryotic cells adapt to this ageing-associated stress by large-scale transcriptome changes affecting the expression of genes involved in signaling, protein homeostasis, cell plasticity and immune response. The molecular mechanisms regulating extensive transcriptome rearrangements leading to senescence in specific cell types of IPF lungs remain sparsely investigated. Here we uncover fundamental molecular mechanisms underlying cellular senescence by integrative analysis of multi-omics studies and functional experiments monitoring fibrotic and senescence hallmarks in primary lung fibroblasts from control and IPF patients. Loss-of-function of the DNA/RNA binding protein fused in sarcoma (FUS) in control fibroblasts enhanced fibrotic and senescence hallmarks resembling IPF fibroblasts. Dissecting the molecular mechanisms, reduced FUS levels in IPF fibroblasts abolished R-loop formation at transcription start sites (TSS), leading to increased RNA polymerase II (Pol II) recruitment and its phosphorylation at serine 2, resulting in enhanced translocation speed of elongating Pol II and increased expression of fibrosis- and senescence-related genes. Summarizing, FUS maintains low Pol II processivity in control fibroblasts by stabilizing R-loops at TSS, acting as a gatekeeper of fibrotic and age-associated transcriptional programs.

molecular biology↗

bioRxiv2026

Italia: A PARP-Directed Auger Electron-Emitting Agent for Targeted Radionuclide Therapy of Cancer

Poly(ADP-ribose) polymerase 1 (PARP1) is a central mediator of DNA damage repair and an established therapeutic target in homologous recombination-deficient cancers. Radiolabelled PARP inhibitors provide a strategy to deliver cytotoxic radiation directly to tumour DNA by exploiting PARP overexpression and trapping at sites of DNA damage. Here, we describe the design, radiosynthesis, and in vitro evaluation of [123I]Italia, a talazoparib-derived Auger electron-emitting agent for PARP-targeted radionuclide therapy. Stereochemically pure [123I]Italia, (8S,9R)-5-fluoro-8-(4-(iodo-123I)phenyl)-9-(1-methyl-1H-1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one was synthesised in one step via copper-mediated iodo-deboronation, achieving activity yields >80% and molar activities >6.2 {+/-} 3.1 GBq/{micro}mol (n=8). UPLC analysis confirmed radiochemical purity >97%. Italia exhibited potent PARP1 inhibition (IC50 0.48 nM) and in silico predicted binding affinity comparable to talazoparib. In a panel of PARP-expressing cancer cell lines, [123I]Italia demonstrated highest uptake at 60 min, PARP-selective uptake, predominant nuclear localisation (up to 60% of added activity) and chromatin association consistent with PARP trapping (up to 15% of total activity recorded). Uptake was reduced more than 50-fold by addition of an excess of any PARP inhibitor (e.g. olaparib, talazoparib, and rucaparib) and in PARP1 knockout cells, confirming target specificity. Clonogenic assays showed a marked, added activity-dependent reduction in survival of PARP-expressing cells following a brief one-hour exposure, whereas PARP1-deficient cells were resistant. Collectively, these findings identify [123I]Italia as a promising PARP-targeted Auger electron-emitting theranostic candidate that warrants further in vivo evaluation.

cancer biology↗

bioRxiv2025

A microprotein encoded by FERMT3 modulates endothelial cell protein catabolism and induces p53-mediated cell cycle arrest and senescence

BackgroundHuman endothelial cells express numerous microproteins (miPs) encoded by small open reading frames (smORFs) distributed throughout the genome, yet the biological functions of most remain unknown. This study set out to characterize a novel 69 amino acid miP encoded by a smORF located within the coding sequence of the FERM domain containing kindlin-3 transcript (miP-FERMT3). MethodsConfocal microscopy was used to determine the subcellular localization of miP-FERMT3 in endothelial cells and its interaction partners were determined by mass spectrometry and immunoblotting. RNA sequencing identified transcriptional alterations induced by miP-FERMT3 overexpression. Cell proliferation and cell cycle stages were assessed by live cell imaging, EdU incorporation and flow cytometry, while senescence was examined by senescence-associated {beta}-galactosidase staining, live cell imaging and RT-qPCR-based measurement of telomere length. ResultsIn endothelial cells miP-FERMT3 localized mainly to centriole subdistal appendages, where it interacted with proteins involved in ubiquitin- and proteasome-dependent protein catabolism, including PSMD9, CUL2 and TRIM8. Consistent with these interactions, cells expressing miP-FERMT3 exhibited increased global protein ubiquitination, enhanced centrosomal neddylation and elevated proteasomal activity. MiP-FERMT3 also promoted the nuclear accumulation of p53, which subsequently repressed FOXM1 expression, leading to the downregulation of genes required for cell-cycle progression and upregulation of genes involved in cell cycle inhibition, resulting in cell-cycle arrest. Cells expressing the miP also demonstrated multiple hallmarks of cellular senescence, including enlarged size, DNA damage, increased senescence-associated {beta}-galactosidase activity, telomere shortening and paracrine pro-inflammatory activation of naive endothelial cells. Analyses of independent murine and human transcriptomic and proteomic aging datasets further revealed that FERMT3 expression and protein abundance increase with age. ConclusionsThese findings identify miP-FERMT3 as a novel regulator of protein catabolism and p53-dependent cell cycle arrest and cellular senescence in endothelial cells. Given the aging-associated upregulation of FERMT3 in mouse and human endothelial cells, increased miP-FERMT3 expression may contribute to the onset of vascular senescence as a hallmark of aging.

cell biology↗

bioRxiv2025

Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

BackgroundA large number of microproteins (miPs) encoded by small open reading frames (smORFs) are expressed in endothelial cells, yet their function remains largely unknown. In this study, we characterized a novel 46-amino-acid miP encoded by a smORF within the proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2) transcript that was upregulated under inflammatory conditions and we refer to as miP-PSTPIP2. MethodsImmunoprecipitation coupled with mass spectrometry-based proteomics, immunoblotting, immunofluorescence and proximity ligation assays were used to identify and validate miP-PSTPIP2 interacting proteins in human endothelial cells. The impact of adenovirus-mediated overexpression of miP-PSTPIP2 on endocytosis, cytoskeleton dynamics and abundance of proteins involved in these processes was investigated by confocal microscopy and immunoblotting. Live cell imaging was used to assess endothelial cell migration and vascular permeability. ResultsmiP-PSTPIP2 physically associated with caveolar proteins, proteins involved in the regulation of cytoskeleton dynamics, intracellular transport, clathrin adaptor activity, as well as nuclear proteins. Human endothelial cells overexpressing miP-PSTPIP2 demonstrated enhanced endocytosis and transcytosis of transferrin as well as low-density lipoprotein. Mechanistically, miP-PSTPIP2 modulated Arp2/3-mediated actin nucleation and branching, which are required for dynamic cytoskeleton rearrangements. Moreover, altered cytoskeleton dynamics in miP-PSTPIP2-expressing endothelial cells resulted in impaired cell migration as well as increased permeability and monocyte trans-endothelial migration. ConclusionsmiP-PSTPIP2 is an inflammation-induced endothelial miP that regulates Arp2/3-dependent actin dynamics, thereby enhancing lipid uptake and leukocyte permeability. Its upregulation under inflammatory conditions suggests a contributory role in endothelial dysfunction and vascular inflammation.

cell biology↗

bioRxiv2022

Patient-derived parathyroid organoids as tracer and drug-screening application model

Parathyroid diseases are characterized by dysregulation of calcium homeostasis and alterations in parathyroid hormone (PTH) excretion. The understanding of parathyroid hyperplastic growth and the development of parathyroid-targeted treatment and imaging tracers could benefit from in vitro models. Therefore, we aim to establish stem cell-derived, three-dimensional organoids representing human parathyroid tissue in vitro. Patient-derived hyperplastic parathyroid tissue was dispersed and parathyroid organoids (PTO) were cultured and characterized. PTO-derived cells were shown to exhibit in vitro self-renewal over several passages, indicative of the presence of putative stem cells. Immunofluorescence and RNA-sequencing confirm that PTO phenocopy hyperplastic parathyroid tissue. Exposure of PTO to increasing calcium concentrations and to PTH-lowering drugs resulted in a significantly reduced PTH excretion. Next to this, the PTO showed specific binding of 11C-methionine to the targeted receptor. Additionally, when organoids were incubated with 99mTc-sestamibi, we observed a higher uptake in PTOs from patients with a 99mTc-sestamibi positive scan compared to patients with a negative scan. These data show functionality of PTOs resembling the parathyroid. In conclusion, we present a patient-derived PTO culture, that recapitulates the originating tissue on gene and protein expression and functionality. This PTO model paves the way for future physiology studies and therapeutic target and tracer discovery.

cell biology↗

bioRxiv2022

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system

In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics, and live-cell microscopy, we determined the contribution of the pilotin to the assembly, function, and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. In line, most export apparatus subcomplexes are mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, while absence of the pilotin all but prevents export of early T3SS substrates, such as the needle subunits, it has little effect on secretion of late T3SS substrates, including the virulence effectors. We found that pilotins transiently interact with other injectisome components such as the secretin in the outer membrane, but mostly form transient mobile clusters in the bacterial membrane, which do not colocalize with assembled injectisomes. Together, these findings provide a new view on the role of pilotins during and after assembly of type III secretion injectisomes.

microbiology↗

bioRxiv2020

Correction of frameshift mutations in the atpB gene by translational recoding in chloroplasts of Oenothera and tobacco.

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding, is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch of atpB (encoding a {beta}-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, non-functional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, transplastomic tobacco lines were generated, in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We found that insertion of two adenines was more efficiently compensated than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding. Plants carrying a disrupted oligoA stretch have an albino-phenotype, indicating absence of indel correction. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in mutants that otherwise would be lethal.

plant biology↗