Biology subjects

Wolf, D. A.

Publications and source records attributed to Wolf, D. A..

7 recordsLinked to original sources

bioRxiv2025

5' UTR-mediated retention of eIF3 on 80S ribosomes promotes co-translational folding of ER membrane proteins

Co-translational folding of nascent polypeptides is essential for protein function and cellular homeostasis. Ribosome-associated chaperones assist in this process, but coordination between their recruitment and translation initiation remains poorly understood. We report here that specific binding sites for eukaryotic translation initiation factor eIF3 within the 5 untranslated regions (5UTRs) of mRNAs promote its retention on 80S ribosomes during the synthesis of select endoplasmic reticulum (ER) membrane proteins. Disruption of these eIF3 binding sites leads to misfolding and sequestration of newly synthesized membrane proteins into ER whorls. Sequestration into ER whorls is exacerbated by HSP70 inhibition but can be rescued by overexpressing HSPA8 and HSPA1. Cross-linking assays reveal that 5UTR binding sites stabilize eIF3-80S interactions during early elongation, facilitating recruitment of HSPA8 to ribosomes. These findings indicate that genetic instructions within 5UTRs direct eIF3-mediated chaperone recruitment, ensuring proper co-translational folding of ER membrane proteins.

cell biology↗

bioRxiv2023

NDC80 status pinpoints mitotic kinase inhibitors as emerging therapeutic options in clear cell renal cell carcinoma

[~]30% of clear cell renal cell carcinoma (ccRCC) patients present with metastatic disease at the time of diagnosis, causing a dire 5-year survival rate of 13%. Whereas anti-PD-1 immunotherapy has improved survival, a strong need remains for new therapeutic options. Using integrated network analysis, we identified the mitotic regulator NDC80 as a predictor of ccRCC progression. Overexpression of NDC80 fosters the malignant phenotype by promoting cell cycle progression through S phase as well as boosting glycolysis and mitochondrial respiration. Despite high levels of immune infiltration, particularly derived from tumor resident CD8+ T cells with an exhausted phenotype, NDC80 defines a class of ccRCCs that poorly respond to immune checkpoint blockade. Instead, bioinformatics identified NDC80-high ccRCCs as sensitive to inhibitors of mitotic kinases, PLK1 and AURK, therapeutic approaches we validated in cell lines and mouse xenograft studies. Thus, NDC80 status pinpoints mitotic kinase inhibitors as promising therapeutic options in difficult-to-treat ccRCCs.

cancer biology↗

bioRxiv2022

eIF3 mRNA selectivity profiling reveals eIF3k as a cancer-relevant regulator of ribosome content

eIF3, whose subunits are frequently overexpressed in cancer, regulates mRNA translation from initiation to termination, but mRNA-selective functions of individual subunits remain poorly defined. Using multi-omic profiling upon acute depletion of eIF3 subunits, we observed that while eIF3a, b, e, and f markedly differed in their impact on eIF3 holo-complex formation and translation, they were each required for cancer cell proliferation and tumor growth. Remarkably, eIF3k showed the opposite pattern with depletion promoting global translation, cell proliferation, tumor growth, and stress resistance through repressing the synthesis of ribosomal proteins, especially RPS15A. Whereas ectopic expression of RPS15A mimicked the anabolic effects of eIF3k depletion, disruption of eIF3 binding to the 5-UTR of RSP15A mRNA negated them. Supported by mathematical modeling, our data uncovers eIF3k-l as a mRNA-specific module which, through controlling RPS15A translation, serves as a rheostat of ribosome content to secure spare translational capacity that can be mobilized during stress.

cell biology↗

bioRxiv2020

Convective forces increase rostral delivery of intrathecal antisense oligonucleotides in the cynomolgus monkey nervous system

BackgroundThe intrathecal (IT) dosing route introduces drugs directly into the CSF to bypass the blood-brain barrier and gain direct access to the CNS. We evaluated the use of convective forces acting on the cerebrospinal fluid as a means for increasing rostral delivery of IT dosed radioactive tracer molecules and antisense oligonucleotides (ASO) in the monkey CNS. We also measured the cerebral spinal fluid (CSF) volume in a group of cynomolgus monkeys. MethodsThere are three studies presented, in each of which cynomolgus monkeys were injected into the IT space with radioactive tracer molecules and/or ASO by lumbar puncture in either a low or high volume. The first study used the radioactive tracer 64Cu-DOTA and PET imaging to evaluate the effect of the convective forces. The second study combined the injection of the radioactive tracer 99mTc-DTPA and ASO, then used SPECT imaging and ex vivo tissue analysis of the effects of convective forces to bridge between the tracer and the ASO distributions. The third experiment evaluated the effects of different injection volumes on the distribution of an ASO. In the course of performing these studies we also measured the CSF volume in the subject monkeys by Magnetic Resonance Imaging. ResultsIt was consistently found that larger bolus dose volumes produced greater rostral distribution along the neuraxis. Thoracic percussive treatment also increased rostral distribution of low volume injections. There was little added benefit on distribution by combining the thoracic percussive treatment with the high-volume injection. The CSF volume of the monkeys was found to be 11.9 {+/-} 1.6 cm3. ConclusionsThese results indicate that increasing convective forces after IT injection increases distribution of molecules up the neuraxis. In particular, the use of high IT injection volumes will be useful to increase rostral CNS distribution of therapeutic ASOs for CNS diseases in the clinic.

neuroscience↗

bioRxiv2019

Crosstalk between eIF2α and eEF2 phosphorylation pathways optimizes translational arrest in response to oxidative stress

The cellular stress response triggers a cascade of events leading to transcriptional reprogramming and a transient inhibition of global protein synthesis, which is thought to be mediated by phosphorylation of eukaryotic initiation factor-2 (eIF2). Using mouse embryonic fibroblasts (MEFs) and the fission yeast S. pombe, we report here that rapid translational arrest and cell survival in response to hydrogen peroxide-induced oxidative stress do not rely on eIF2 kinases and eIF2 phosphorylation. Rather H2O2 induces a block in elongation through phosphorylation of eukaryotic elongation factor 2 (eEF2). Kinetic and dose-response analyses uncovered crosstalk between the eIF2 and eEF2 phosphorylation pathways, indicating that, in MEFs, eEF2 phosphorylation initiates the acute shutdown in translation, which is then maintained by eIF2 phosphorylation. Our results challenge the common conception that eIF2 phosphorylation is the primary trigger of translational arrest in response to oxidative stress and point to integrated control that may facilitate the survival of cancer cells.\n\nHIGHLIGHTSO_LIOxidative stress-induced translation arrest is independent of eIF2 phosphorylation\nC_LIO_LIOxidative stress blocks translation elongation\nC_LIO_LIOxidative stress triggers eEF2 kinase activation\nC_LIO_LIeEF2K KO cells are hypersensitive to oxidative stress\nC_LI

cell biology↗

bioRxiv2019

eIF3 promotes early translation elongation to ensure mitochondrial homeostasis and skeletal muscle health

eIF3 is a multi-subunit complex with numerous functions in canonical translation initiation. eIF3 was also found to interact with 40S and 60S ribosomal proteins and translation elongation factors, but a direct involvement in translation elongation has never been demonstrated. We found that eIF3 deficiency reduced early ribosomal elongation speed between codons 25 and 75 on a set of [~]2,700 mRNAs encoding proteins associated with mitochondrial and membrane functions, resulting in defective synthesis of their encoded proteins. To promote elongation, eIF3 interacts with 80S ribosomes translating the first [~]60 codons and serves to recruit protein quality control factors, functions required for normal mitochondrial physiology. Accordingly, eIF3e+/- mice accumulate defective mitochondria in skeletal muscle and show a progressive decline in muscle strength. Hence, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for mitochondrial physiology and muscle health.

cell biology↗

bioRxiv2017

Trade-off and flexibility in the dynamic regulation of the cullin-RING ubiquitin ligase repertoire

Cullin-RING ubiquitin ligases (CRLs) catalyze the ubiquitylation of substrates many of which are degraded by the 26S proteasome. Their modular architecture enables recognition of numerous substrates via exchangeable substrate receptors that competitively bind to a cullin scaffold with high affinity. Due to the plasticity of these interactions there is ongoing uncertainty how cells maintain a flexible CRL repertoire in view of changing substrate loads. Based on a series of in vivo and in vitro studies, different groups proposed that the exchange of substrate receptors is mediated by a protein exchange factor named cullin-associated and neddylation-dissociated 1 (Cand1). Here, we have performed quantitative mathematical modeling to support this hypothesis. To this end we first show that the exchange activity of Cand1 necessarily leads to a trade-off between high ligase activity and fast receptor exchange. Supported by previous in vivo studies we argue that this trade-off yields an optimal Cand1 concentration where the time scale for substrate degradation becomes minimal. In a second step we show through simulations that (i) substrates bias the CRL repertoire leading to preferential assembly of ligases for which substrates are available and (ii) differences in binding affinities create a temporal hierarchy for the degradation of substrates. Together, our results provide general constraints for the operating regimes of molecular exchange systems and suggest that Cand1 endows the CRL network with the properties of an \"on demand\" system allowing cells to dynamically adjust their CRL repertoire to fluctuating substrate abundances.\n\nAuthor summaryCullin-RING ubiquitin ligases (CRLs) are multisubunit protein complexes where exchangeable substrate receptors (SRs) assemble on a cullin scaffold to mediate ubiquitylation and subsequent degradation of a large variety of substrates. In humans there are hundreds of different CRLs having potentially thousands of substrates. Due to the high affinity of cullin-SR interactions, it has long been a mystery how cells would maintain flexibility to sample the entire SR repertoire in order to match fluctuating substrate loads. Recent experiments indicate that the exchange of different SRs is mediated by a novel protein exchange factor (Cand1). However, the proposed biochemical function of Cand1 as a promoter of CRL activity remained difficult to reconcile with previous reports of Cand1 acting as an inhibitor of CRL activity in vitro. Here we show that these two findings are not contradictory, but that the exchange activity of Cand1 necessarily leads to a trade-off between high ligase activity and fast receptor exchange which leads us to predict an optimal Cand1 concentration and a temporal hierarchy for substrate degradation. Our results support the view that Cand1 endows the CRL network with the flexibility of an \"on demand\" system where relative CRL abundances are dictated by substrate availability.

systems biology↗