Biology subjects

Gurzeler, L.-A.

Publications and source records attributed to Gurzeler, L.-A..

6 recordsLinked to original sources

bioRxiv2026

USP9X promotes the degradation of trapped translation factors on collided ribosomes

Various ribosome-associated quality control pathways safeguard translation fidelity by detecting and resolving aberrant translation events. Recently, a pathway that senses stalled ribosomes with an occluded A-site has been described. The small molecules NVS1.1 and Ternatin-4 induce ribosome stalling by trapping eRF1 and eEF1A1, respectively, within the ribosomal A-site, thereby promoting their ubiquitination and proteasomal degradation. Additionally to the previously identified factors GCN1, RNF14, and RNF25, we identify here the deubiquitinase USP9X as a regulator of this pathway. In the absence of USP9X deubiquitinase activity, eRF1 is still ubiquitinated but fails to undergo efficient degradation. However, compared to wildtype cells, K6-linked ubiquitin chains accumulate on eRF1 in cells lacking USP9X activity, suggesting that USP9X trims K6-linked ubiquitin chains during a late step of substrate processing. Furthermore, clearance of trapped eRF1 engages a feedback mechanism involving 4EHP and the integrated stress response (ISR) to suppress translation, whereas impaired degradation prevents translational shutdown.

molecular biology↗

bioRxiv2024

A Fibronectin (FN)-Silk 3D Cell Culture Model as a Screening Tool for Repurposed Antifibrotic Drug Candidates for Endometriosis

This study advances sustainable pharmaceutical research for endometriosis by aligning with the UN Sustainable Development Goals on health, gender equality, and responsible consumption in developing in vitro 3D cell culture models of endometriotic pathophysiology. Fibrosis is a key aspect of endometriosis, yet current models to study it remain limited, especially in 3D. This work aims to bridge the translational gap between in vitro fibrosis research and preclinical testing of non-hormonal drug candidates. When grown in a 3D matrix of sustainably produced silk protein functionalized with a fibronectin-derived cell adhesion motif (FN-silk), endometrial stromal and epithelial cells respond to transforming growth factor beta-1 (TGF-{beta}1) in a physiological manner as probed at the mRNA level. For stromal cells, this response to TGF-{beta}1 is not observed in spheroids, while epithelial cell spheroids behave similarly to epithelial cell FN-silk networks. Pirfenidone, an antifibrotic drug approved for the treatment of idiopathic pulmonary fibrosis, reverses TGF-{beta}1-induced upregulation of mRNA transcripts involved in fibroblast-to-myofibroblast transdifferentiation of endometrial stromal cells in FN-silk networks, supporting the drugs potential as a repurposed non-hormonal therapy for endometriosis. This study demonstrates how a sustainable approach - from project conceptualization to material selection - can be integrated into pharmaceutical research for womens health. Table of contentsThis paper presents in vitro 3D cell culture models of fibrosis in endometriosis. Endometrial stromal and epithelial cells cultured in networks of silk protein functionalized with a fibronectin-derived cell adhesion motif showed physiological-like fibrotic behavior. Pirfenidone was able to reverse fibrosis of endometrial stromal cells in vitro, demonstrating this models suitability as a screening tool for antifibrotic drugs for endometriosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/616776v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@12727c3org.highwire.dtl.DTLVardef@1a39292org.highwire.dtl.DTLVardef@1ebdb2aorg.highwire.dtl.DTLVardef@181d6ac_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control

Suppression of premature termination codons (PTC) by translational readthrough is a promising strategy to treat a wide variety of severe genetic diseases caused by nonsense mutations. Here, we present two novel and potent readthrough promoters - NVS1.1 and NVS2.1 - that restore substantial levels of functional full-length CFTR and IDUA proteins in disease models for cystic fibrosis and Hurler syndrome, respectively. In contrast to other readthrough promoters that affect stop codon decoding, the NVS compounds stimulate PTC suppression by triggering rapid proteasomal degradation of the translation termination factor eRF1. Our results show that this occurs by trapping eRF1 in the terminating ribosome, causing ribosome stalls and subsequent ribosome collisions, activating a novel branch of the ribosome-associated quality control (RQC) network that involves the translational stress sensor GCN1 and the catalytic activity of the E3 ubiquitin ligases RNF14 and RNF25.

molecular biology↗

bioRxiv2021

Production of human translation-competent lysates using dual centrifugation

Protein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. We optimized the lysate preparation to yield cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well as in an IRES-mediated way. Reduction of the phosphorylation state of eIF2 using recombinant GADD34 and 2-aminopurine considerably boosts the protein output, reinforcing the potential of this method to produce recombinant proteins from human lysates.

molecular biology↗

bioRxiv2020

SARS-CoV-2 Nsp1 binds ribosomal mRNA channel to inhibit translation

The non-structural protein 1 (Nsp1), also referred to as the host shutoff factor, is the first viral protein that is synthesized in SARS-CoV-2 infected human cells to suppress host innate immune functions1,2. By combining cryo-electron microscopy and biochemical experiments, we show that SARS-CoV-2 Nsp1 binds to the human 40S subunit in ribosomal complexes including the 43S pre-initiation complex. The protein inserts its C-terminal domain at the entrance to the mRNA channel where it interferes with mRNA binding. We observe potent translation inhibition in the presence of Nsp1 in lysates from human cells. Based on the high-resolution structure of the 40S-Nsp1 complex, we identify residues of Nsp1 crucial for mediating translation inhibition. We further show that the full-length 5’ untranslated region of the genomic viral mRNA stimulates translation in vitro, suggesting that SARS-CoV-2 combines inhibition of translation by Nsp1 with efficient translation of the viral mRNA to achieve expression of viral genes3.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Human NMD ensues independently of stable ribosome stalling

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty and the regulation of normal mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system revealed similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling revealed a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.

molecular biology↗