Biology subjects

Wiener, R.

Publications and source records attributed to Wiener, R..

10 recordsLinked to original sources

bioRxiv2026

Dihydropyrimidines sustain aggressive cancer states by stabilizing DPYSL2

Cancer cell states are governed by coordinated signaling and metabolic networks, yet the mechanisms coupling metabolic activity to oncogenic signaling remain poorly understood. Here, we identify dihydropyrimidines (DHPs), metabolites generated during pyrimidine catabolism, as "signaling metabolites" that sustain aggressive cancer cell states. Depletion of DHPs, either by knockout of the pyrimidine catabolic enzyme DPYD or by expression of the DHP-degrading enzyme DPYS, suppressed STAT3 signaling and attenuated mesenchymal and inflammatory transcriptional programs. Mechanistically, DHPs stabilized DPYSL2, a JAK1-interacting adaptor protein required for efficient STAT3 activation. Structural modeling and thermal stability analyses further support a direct interaction between DHPs and DPYSL2. Accordingly, high DPYD expression was associated with EMT, inflammatory signaling, and poor-prognosis breast cancer subtypes across patient cohorts. Ultimately, our findings establish pyrimidine catabolism as a direct regulator of signaling competence and identify DHPs as signaling-active metabolites that couple cellular metabolic state to oncogenic transcriptional programs and mesenchymal identity.

cancer biology↗

bioRxiv2025

UFMylation orchestrates chromatin engagement of core NHEJ components to promote DNA double-strand break repair

DNA double-strand breaks (DSBs) are highly cytotoxic lesions whose misrepair can lead to genomic instability, cancer and developmental disorders. Through systematic screening of understudied ubiquitin-like modifiers (UBLs), we identify UFM1 as a previously unrecognised regulator of non-homologous end-joining (NHEJ). Using a structure-guided chemical biology strategy, we develop a photo-crosslinkable UFM1 probe and, together with high-resolution NMR, uncover non-canonical UFM1-binding regions in core NHEJ components, including XRCC4. Mechanistically, proximity-dependent proteomics reveals Ku70 as a key UFMylation substrate, establishing a functional axis in which XRCC4 engages UFMylated Ku70 to promote the chromatin assembly of NHEJ factors. Perturbation of UFM1 signalling, via UFSP2 depletion or a hypomorphic UBA5 allele in patient-derived fibroblasts, impairs these processes, linking UFMylation defects to altered regulation of DSB repair. Our findings define a complete UFM1 signalling module in genome maintenance and uncover a molecular connection between hereditary UFMylation disorders and dysregulated DSB repair pathways.

molecular biology↗

bioRxiv2024

Binding of SARS-CoV-2 nucleocapsid protein to uninfected epithelial cells induces antibody-mediated complement deposition

SARS-CoV-2 infection triggers strong antibody response toward Nucleocapsid-Protein (NP), suggesting extracellular presence beyond its intra-virion RNA binding. Interestingly, NP was found to decorate infected and proximal uninfected cell-surfaces. Here, we propose a new mechanism through which extracellular NP on uninfected cells contributes to COVID-19 pathogenicity. We show that NP binds to cell-surface sulfated linear-glycosaminoglycans by spatial rearrangement of its RNA-binding sites facilitated by the flexible, positively charged, linker. Coating of uninfected lung-derived cells with purified NP attracted anti-NP-IgG from lung fluids and sera collected from COVID-19 patients. The magnitude of this immune recognition was significantly elevated in moderate compared to mild COVID-19 cases. Importantly, binding of anti-NP-IgG present in sera generated clusters that triggered C3b deposition by the classical complement pathway. Heparin analog enoxaparin outcompeted NP-binding, rescuing cells from anti-NP IgG-mediated complement deposition. Our findings unveil how extracellular NP may exacerbate COVID-19 tissue damage, and suggest leads for preventative therapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/585388v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1084e20org.highwire.dtl.DTLVardef@4126caorg.highwire.dtl.DTLVardef@159c610org.highwire.dtl.DTLVardef@da7b7f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIIgG from patients sera target NP-bound cells resulting in complement activation C_LIO_LIThe flexible linker allows NP to both bind linear sulfated GAGs and wrap around RNA C_LIO_LIHeparin analogs prevent NP surface binding and alleviate complement activation C_LIO_LICell-ELISA anti-NP IgG levels differ between mild and moderate COVID-19 C_LI

immunology↗

bioRxiv2022

AlphaFold2-based fusion design deciphers crucial role of the E3 UFL1 N-terminal helix in E2 UFC1 binding and ufmylation

Ufmylation, a protein modification by Ubiquitin-like (UBL) protein UFM1, plays a crucial role in several cellular processes including DNA damage response, protein translation and ER homeostasis. To date, little is known how the enzymes responsible for this modification coordinate their action. Here we have studied the details of UFL1 (E3) activity, its binding to UFC1 (E2), and its relation to UBA5 (E1), using a combination of structural modeling with Alphafold2, X-ray crystallography, NMR, and in vitro biochemical activity assays. Guided by an Alphafold2 model, we generated an active UFL1 fusion construct that includes its cofactor DDRGK1, and solved the first crystal structure of this critical interaction. This fusion construct also unveiled the importance of the N-terminal helix of UFL1 for its binding to UFC1, which was validated by ITC and NMR experiments. Importantly, the binding site suggested by our structural model of the UFL1-UFC1 interaction reveals a conserved interface, and suggests a competition for binding to UFC1 between UFL1 and UBA5, which we reconfirmed by NMR. Altogether, our study reveals a novel, terminal helix-mediated regulatory mechanism which coordinates the cascade of E1-E2-E3 mediated transfer of UFM1 to its substrate, and provides new leads to target this important modification. Significance statementUfmylation is an important post-translational modification, but little is known about the mechanistic details of its machinery, and in particular how the UFM1 E3 ligase (UFL1) binds and functions together with the E2 conjugating enzyme (UFC1). We combined AlphaFold2 modeling, X-ray crystallography, NMR and biochemical experiments to reveal crucial elements that govern UFL1 activity and ufmylation. We discover a crucial role for the UFL1 N-terminal helix in binding to UFC1 and productive ufmylation. This helix competes with the E1 (UBA5) C-terminal helix for binding to UFC1. Altogether, our findings uncover a new, helix-mediated regulatory mechanism in ufmylation.

biochemistry↗

bioRxiv2022

S6K1 phosphorylates Cdk1 and MSH6 to regulate DNA repair.

The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability.

cancer biology↗

bioRxiv2022

Intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2 induces robust neutralizing salivary IgA

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable sterilizing immunity at the mucosal level. Our study uncovers, strong neutralizing mucosal component (NT50 [≤] 50pM), emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the Receptor-Binding-Domain (RBD) of SARS-CoV-2 spike protein and demonstrate that these IgAs are key mediators of potent neutralization. RBD-targeting IgAs were found to associate with the Secretory Component, indicating their bona-fide transcytotic origin and their dimeric tetravalent nature. The mechanistic understanding of the exceptionally high neutralizing activity provided by mucosal IgA, acting at the first line of defence, will advance vaccination design and surveillance principles, pointing to novel treatment approaches, and to new routes of vaccine administration and boosting. Significance statementWe unveiled powerful mucosal neutralization upon BNT162b2 vaccination, mediated by temporary polymeric IgA and explored its longitudinal properties. We present a model, whereby the molecular architecture of polymeric mucosal IgA and its spatial properties are responsible for the outstanding SARS-CoV-2 neutralization potential. We established a methodology for quantitative comparison of immunoreactivity and neutralization for IgG and IgAs in serum and saliva in molar equivalents for standardization in diagnostics, surveillance of protection and for vaccine evaluations.

microbiology↗

bioRxiv2022

Regulation of major bacterial survival strategies by transcript sequestration in a membraneless organelle

Liquid-liquid phase separation (LLPS) of proteins was shown in recent years to regulate spatial organization of cell content without the need for membrane encapsulation in eukaryotes and prokaryotes. Yet evidence for the relevance of LLPS for bacterial cell functionality is largely missing. Here we show that the sugar metabolism-regulating clusters, recently shown by us to assemble in the E. coli cell poles by means of the novel protein TmaR, are formed via LLPS. A mutant screen uncovered residues and motifs in TmaR that are important for its condensation. Upon overexpression, TmaR undergoes irreversible liquid-to-solid transition, similar to the transition of disease-causing proteins in human, which impairs bacterial cell morphology and proliferation. Not only does RNA contribute to TmaR phase separation, but by ensuring polar localization and stability of flagella-related transcripts, TmaR enables cell motility and biofilm formation, thus providing a linkage between LLPS and major survival strategies in bacteria.

microbiology↗

bioRxiv2021

Structural basis for long-chain isoprenoids synthesis by cis-prenyltransferases

Isoprenoids are the largest group of natural products, found in all living organisms and play an essential role in numerous cellular processes. These compounds are synthesized by prenyltransferases, catalyzing the condensation reaction between an allylic diphosphate primer and a variable number of isopentenyl diphosphate (C5) units. This superfamily of enzymes can be subdivided into trans- or cis-prenyltransferases according to the stereoisomerism of the product. The cis branch can be further classified according to product length. While the active site volume was suggested to determine the final length in enzymes synthesizing short- and medium-chain products (up to C60), long-chain enzymes (up to C120) and rubber synthases (>C10,000) fail to conform to this paradigm. Here, to resolve the structural basis for long-chain isoprenoid synthesis, we focused on the human cis-prenyltransferase complex (hcis-PT). This enzyme, peripheral to the endoplasmic reticulum membrane, produces the precursor for dolichol phosphate, a membrane residing glycosyl carrier. In line with its crucial role in the cellular protein glycosylation machinery, disease-causing mutations in hcis-PT were shown to result in a wide spectrum of clinical phenotypes. The crystallographic structures of hcis-PT in four different substrate/product-bound conformations revealed an outlet enabling product elongation into the bulk solvent. Moreover, hydrogen-deuterium exchange mass spectrometry analysis in solution showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Finally, using a fluorescent substrate analog and a fluorescently-labeled lipid nanodiscs, we show that product elongation and membrane association are closely correlated. Together, our results support directional product synthesis in long-chain enzymes and rubber synthases, with a distinct substrate inlet and product outlet, allowing direct membrane insertion of the elongating isoprenoid during catalysis. This mechanism uncouples active site volume from product length and circumvents the need to expulse hydrophobic product into a polar environment prior to membrane insertion.

biochemistry↗

bioRxiv2020

RNA binding of Hfq monomers promotes RelA-mediated hexamerization in a limiting Hfq environment

The RNA chaperone Hfq acting as a hexamer, is a known mediator of post-transcriptional regulation expediting basepairing between small RNAs (sRNAs) and their target mRNAs. However, the intricate details associated with Hfq-RNA biogenesis are still unclear. Previously, we reported that the stringent response regulator, RelA is a functional partner of Hfq that facilitates Hfq-mediated sRNA-mRNA regulation in vivo and induces Hfq hexamerization in vitro. Here, for the first time we show that RelA-mediated Hfq hexamerization requires an initial binding of RNA, preferably sRNA to Hfq monomers. By interacting with a Shine-Dalgarno-like sequence (GGAG) in the sRNA, RelA stabilizes the initially unstable complex of RNA bound-Hfq monomer, enabling the attachment of more Hfq subunits to form a functional hexamer. Overall, our study showing that RNA binding to Hfq monomers is at the heart of RelA-mediated Hfq hexamerization, challenges the previous concept that only Hfq hexamers can bind RNA.

microbiology↗

bioRxiv2017

Conformational locking of Ufm1 upon binding to the Ufm1-interacting sequence of Uba5

Ubiquitin fold modifier 1 (Ufm1) is a ubiquitin-like protein (UBL) found in eukaryotic organisms which plays a crucial role in ER stress management and signal transduction. The crystal structure of UFM1 and its E1 (Uba5) in complex shows that Ufm1 binds to the adenylation domain of UBA5 and interacts with a separate Ufm1-interacting sequence (UIS) in the C-terminus of UBA5. The UIS interacts with Ufm1 on the opposite side of Ufm1 protein from the adenylation domain of Uba5 and the reason for this second interaction site is unclear. We analyzed Ufm1 bound to the UIS sequence through molecular dynamics simulations in order to identify additional functions for this interaction. We found that the residues in the adenylation interaction site of Ufm1 have less movement when the UIS peptide was bound to Ufm1 and formed a structure that aligns well with Ufm1 bound to the Uba5 adenylation domain. We further identified an amino acid that connects the UIS to the adenylation domain interacting site. Mutation of this amino acid decreases charging activity and shifts the Ufm1 conformation population toward the unlocked configuration even in the presence of the UIS peptide. These data suggest a role for the Uba5 UIS in stimulating activation of Ufm1.

biochemistry↗