Biology subjects

Sobczak, K.

Publications and source records attributed to Sobczak, K..

5 recordsLinked to original sources

bioRxiv2025

The unique molecular recognition features of Siglec-10: structural insights into sialoglycan and antibody interactions

Siglec-10 is a sialic acid-binding immunoglobulin-like lectin implicated in immune regulation, yet the molecular basis for ligand recognition and how this is functionally linked to immune modulation remains poorly defined. Herein, we present a multidisciplinary study encompassing structural, biochemical, and cellular approaches to elucidate Siglec-10-carbohydrate interactions and their functional consequences. The crystal structure of the extracellular domain of Siglec-10 in complex with 2-6 sialyllactose revealed the presence of two key arginine residues within the Siglec-10 binding site that interact with the carboxyl group of sialic acid, the canonical R119 and R127, suggesting potential dual contributions to ligand engagement. Saturation Transfer Difference (STD)-Nuclear Magnetic Resonance (NMR) confirmed that R119 is essential for sialoglycan binding in solution, whereas R127 appears dispensable for interactions with glycans under these conditions. In contrast, cell-based binding assays using primary human T cells and engineered monocytic lines demonstrated that both arginine residues (R119 and R127) are critical for cellular recognition, revealing a context-dependent interaction. By obtaining direct images at a molecular resolution of 6-7 nm, super-resolution microscopy further revealed glycan-independent dimerization of the Siglec-10 receptor on the surface of human monocytes. Ligand blockade mediated by anti-Siglec-10 mAb (clone S10A) restores CAR-T cell cytotoxicity in vitro, supporting its role as an immune checkpoint receptor. Finally, although CD24 was not identified as a Siglec-10 ligand on T cells, proximity labeling and mass spectrometry uncovered other sialylated glycoproteins that may mediate this interaction. Together, these results identify Siglec-10 as a modulatory receptor with structural and functional features distinct from other Siglec family members and highlight its potential for therapeutic targeting in cancer immunotherapy.

molecular biology↗

bioRxiv2025

Loss of UFMylation supports prostate cancer metastasis and rewires cell metabolism towards hexosamine biosynthesis

The acquisition of metastatic features in tumor cells encompasses genetic and non-genetic adaptation, including reprogramming of cellular metabolism. Here we show that loss of UFMylation reroutes glucose metabolism, promotes invasive capacity and supports prostate cancer metastasis. Through transcriptome-based bioinformatics analysis, we identified a reduction in the ubiquitin-like modifier UFM1 and its ligase UFL1 in metastatic prostate cancer. We demonstrate that loss of UFMylation results in enhanced cancer cell dissemination and a switch from cellular proliferation to invasion. Using biotin-based proteomics, we identified phosphofructokinase (PFKAP) as an unprecedented UFMylation substrate. Consistent with UFMylation playing a role in the regulation of phosphofructokinase activity, loss of UFMylation reduced glucose metabolism in favour of hexosamine biosynthesis, which resulted in elevated glycosylation of proteins relevant for cell invasion. These results reveal a role for UFMylation in the regulation of phosphofructokinase and glucose metabolism to support prostate cancer metastasis.

cancer biology↗

bioRxiv2024

Ribosomal composition affects the noncanonical translation and toxicity of polyglycine-containing proteins in fragile X-associated conditions

Expansion of CGG repeats (CGGexp) in the 5 untranslated region (5UTR) of the FMR1 gene underlies the fragile X premutation-associated conditions including tremor/ataxia syndrome, a late-onset neurodegenerative disease and fragile X-associated primary ovarian insufficiency. One common pathomechanism of these conditions is the repeat-associated non-AUG-initiated (RAN) translation of CGG repeats of mutant FMR1 mRNA, resulting in production of FMRpolyG, a toxic protein containing long polyglycine tract. To identify novel modifiers of RAN translation we used an RNA-tagging system and mass spectrometry-based screening. It revealed proteins enriched on CGGexp-containing FMR1 RNA in cellulo, including a ribosomal protein RPS26, a component of the 40S subunit. We demonstrated that depletion of RPS26 and its chaperone TSR2, modulates FMRpolyG production and its toxicity. We also found that the RPS26 insufficiency impacted translation of limited number of proteins, and 5UTRs of mRNAs encoding these proteins were short and guanosine and cytosine-rich. Moreover, the silencing of another component of the 40S subunit, the ribosomal protein RPS25, also induced repression of FMRpolyG biosynthesis. Results of this study suggest that the two 40S ribosomal proteins and chaperone TSR2 play an important role in noncanonical CGGexp-related RAN translation.

molecular biology↗

bioRxiv2022

Lactose-based surfactants complexable with oligonucleic acids as gene delivery agents

Advances in molecular medicine related to the treatment of genetic disorders and cancer require finding new pathways for gene transfer. Nonviral delivery methods rely on the application of polymers, liposomes and cationic lipid systems used as vehicle. Among these systems, there is increasing interest in surfactants, which, due to their ability to complex with nucleic acids can deliver into cell DNA or RNA molecules of almost any size, which is unattainable with viral gene delivery systems.The main aim of this study was to determine the effect of the concentration of lactose-based surfactants (zwitterionic derivatives of sulfobetaine with carbohydrate moieties) on the structure of DNA/RNA as well as to explore their abilities of nucleic acid complexation. Structural studies of DNA or RNA in complexes with surfactants of two types at various concentrations were conducted using circular dichroism (CD) spectroscopy, gel electrophoresis (GE) and synchrotron radiation small angle X-ray scattering (SR-SAXS). Our studies showed that the examined surfactants have excellent properties of forming complexes with DNA and RNA. Additionally, to determine the cytotoxicity and transfection abilities of the studied lipoplexes, preliminary tests were performed in HeLa and fibroblast cells. The obtained results suggest that these systems have relatively low toxicity; however, further research is needed in this area.

biophysics↗

bioRxiv2018

Global increase in circRNA levels in myotonic dystrophy

Splicing aberrations induced as a consequence of the sequestration of MBNL splicing factors on the DMPK transcript, which contains expanded CUG repeats, present a major pathomechanism of myotonic dystrophy type 1 (DM1). As MBNLs may also be important factors involved in the biogenesis of circular RNAs (circRNAs), we hypothesized that the level of circRNAs would be decreased in DM1. To test this hypothesis, we selected twenty well-validated circRNAs and analyzed their levels in several experimental systems (e.g., cell lines, DM muscle tissues, and a mouse model of DM1) using droplet digital PCR assays. We also explored the global level of circRNAs using two RNA-Seq datasets of DM1 muscle samples. Contrary to our original hypothesis, our results consistently showed a global increase in circRNA levels in DM1 and we identified numerous circRNAs that were increased in DM1. We also identified many genes (including muscle-specific genes) giving rise to numerous (>10) circRNAs. Thus, this study is the first to show an increase in global circRNA levels in DM1. We also provided preliminary results showing the association of circRNA level with muscle weakness and alternative splicing changes that are biomarkers of DM1 severity. Author SummaryRecently, a great deal of interest has been focused on a new class of RNA molecules called circular RNAs (circRNAs). To date, thousands of circRNAs have been found in different human cells/tissues. Although the function of circRNAs remains mostly unknown, circRNAs have emerged as an important component of the RNA-RNA and RNA-protein interactome. Thus, intensive efforts are being made to fully understand the biology and function of circRNAs, especially their role in human diseases. As an important role in the biogenesis of circRNA may be played by MBNL splicing factors, in this study we used DM1 (to a lesser extent, DM2) as a natural model in which the level of MBNLs is decreased. In contrast to the expected effect, our results consistently showed a global increase in circRNA levels in DM1. As a consequence, whole genome transcriptome analysis revealed dozens of circRNAs with significantly altered (mostly increased) levels in DM1. Furthermore, we observed that the circRNA levels were in many cases strongly associated with DM1 severity.

molecular biology↗