Publications by authors named "Dieter A Wolf"

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 multiomic 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.

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∼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%. Although 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.

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Purpose: Whereas most human genes encode multiple mRNA isoforms with distinct function, clinical workflows for assessing this heterogeneity are not readily available. This is a substantial shortcoming, considering that up to 25% of disease-causing gene variants are suspected of disrupting mRNA splicing or mRNA abundance. Long-read sequencing can readily portray mRNA isoform diversity, but its sensitivity is relatively low due to insufficient transcriptome penetration.

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Article Synopsis
  • Simple DNA repeats are linked to hereditary ataxias, but traditional genetic diagnostics are often imprecise due to reliance on PCR-based methods.
  • The study developed a new method called clinical nanopore Cas9-targeted sequencing, which uses CRISPR-Cas9 and long-read sequencing for a more accurate analysis of 10 repeat loci associated with ataxias, simultaneously assessing various parameters like sequence and methylation.
  • In testing 100 undiagnosed ataxia patients, this method identified repeat expansions in 28 cases, highlighting biallelic expansions in RFC1 as the most common cause, and discovered a novel repeat motif, AGGGG.
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Purpose: Approximately 20% of patients with clinical familial adenomatous polyposis (FAP) remain unsolved after molecular genetic analysis of the and other polyposis genes, suggesting additional pathomechanisms.

Methods: We applied multidimensional genomic analysis employing chromosomal microarray profiling, optical mapping, long-read genome and RNA sequencing combined with FISH and standard PCR of genomic and complementary DNA to decode a patient with an attenuated FAP that had remained unsolved by Sanger sequencing and multigene panel next-generation sequencing for years.

Results: We identified a complex 3.

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SMIP004-7 is a small molecule inhibitor of mitochondrial respiration with selective in vivo anti-cancer activity through an as-yet unknown molecular target. We demonstrate here that SMIP004-7 targets drug-resistant cancer cells with stem-like features by inhibiting mitochondrial respiration complex I (NADH:ubiquinone oxidoreductase, complex I [CI]). Instead of affecting the quinone-binding site targeted by most CI inhibitors, SMIP004-7 and its cytochrome P450-dependent activated metabolite(s) have an uncompetitive mechanism of inhibition involving a distinct N-terminal region of catalytic subunit NDUFS2 that leads to rapid disassembly of CI.

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Exposure to heat stress triggers a well-defined acute response marked by HSF1-dependent transcriptional upregulation of heat shock proteins. Cells allowed to recover acquire thermotolerance, but this adaptation is poorly understood. By quantitative proteomics, we discovered selective upregulation of HSP70-family chaperone HSPA1 and its co-factors, HSPH1 and DNAJB1, in MCF7 breast cancer cells acquiring thermotolerance.

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Mammalian COP9 signalosome (CSN) exists as two variant complexes containing either CSN7A or CSN7B paralogs of unknown functional specialization. Constructing knockout cells, we found that CSN7A and CSN7B have overlapping functions in the deneddylation of cullin-RING ubiquitin ligases. Nevertheless, CSN has a unique function in DNA double-strand break (DSB) sensing, being selectively required for ataxia telangiectasia mutated (ATM)-dependent formation of NBS1 and γH2AX as well as DNA-damage-induced apoptosis triggered by mitomycin C and ionizing radiation.

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eIF3, a multi-subunit complex with numerous functions in canonical translation initiation, is known 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.

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Studies over the past three years have substantially expanded the involvements of eukaryotic initiation factor 3 (eIF3) in messenger RNA (mRNA) translation. It now appears that this multi-subunit complex is involved in every possible form of mRNA translation, controlling every step of protein synthesis from initiation to elongation, termination, and quality control in positive as well as negative fashion. Through the study of eIF3, we are beginning to appreciate protein synthesis as a highly integrated process coordinating protein production with protein folding, subcellular targeting, and degradation.

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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-2α (eIF2α). Using mouse embryonic fibroblasts (MEFs) and the fission yeast S. pombe, we report that rapid translational arrest and cell survival in response to hydrogen peroxide-induced oxidative stress do not rely on eIF2α kinases and eIF2α phosphorylation.

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Mutations in the gene are associated with early onset Parkinsonism. The mouse, however, does not exhibit neurodegeneration or other Parkinson's disease (PD) phenotypes. Previously, we discovered that translation of Mcl-1, a pro-survival factor, is upregulated in the mouse, suggesting a compensatory mechanism during development.

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Di(1-indol-3-yl)(4-trifluoromethylphenyl)methane (DIM-Ph-4-CF) is an analog of orphan nuclear receptor 4A1 (NR4A1) ligand cytosporone B. We have synthesized several oxidation products of DIM-Ph-4-CF, focusing on analogs with electron-withdrawing or donating groups at their phenyl ring 4-positions, and examined their anti-cancer activity and mechanism-of-action. Mesylates (DIM-Ph-4-X OMss) having CF, COMe and Cl groups were more effective inhibitors of cancer cell viability than their precursors.

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Specificity of the ubiquitin proteasome system is controlled by ubiquitin E3 ligases, including their major representatives, the multisubunit cullin-RING ubiquitin (Ub) ligases (CRLs). More than 200 different CRLs are divided into seven families according to their cullin scaffolding proteins (CUL1-7) around which they are assembled. Research over two decades has revealed that different CRL families are specialized to fulfill specific cellular functions.

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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.

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Polysome profile analysis is widely used by investigators studying the mechanism and regulation of translation. The method described here uses high-velocity centrifugation of whole cell extracts on linear sucrose gradients to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes. Cycloheximide is included in the lysis buffer to "freeze" polysomes by blocking translation.

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Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac I potassium channel.

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Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry.

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The multi-subunit eukaryotic translation initiation factor eIF3 is thought to assist in the recruitment of ribosomes to mRNA. The expression of eIF3 subunits is frequently disrupted in human cancers, but the specific roles of individual subunits in mRNA translation and cancer remain elusive. Using global transcriptomic, proteomic, and metabolomic profiling, we found a striking failure of Schizosaccharomyces pombe cells lacking eIF3e and eIF3d to synthesize components of the mitochondrial electron transport chain, leading to a defect in respiration, endogenous oxidative stress, and premature aging.

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As a ubiquitin-like modifier, ISG15 is conjugated to many cellular proteins in a process termed protein ISGylation. However, the crosstalk between protein ISGylation and the ubiquitin proteasome system is not fully understood. Here, we report that cellular ubiquitin is a substrate of ISG15 and Lys 29 on ubiquitin is the major ISG15 acceptor site.

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SRC kinase is activated in castration resistant prostate cancer (CRPC), phosphorylates the androgen receptor (AR), and causes its ligand-independent activation as a transcription factor. However, activating SRC mutations are exceedingly rare in human tumors, and mechanisms of ectopic SRC activation therefore remain largely unknown. Performing a functional genomics screen, we found that downregulation of SRC inhibitory kinase CSK is sufficient to overcome growth arrest induced by depriving human prostate cancer cells of androgen.

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A series of recent reports has suggested PGC1α-driven upregulation of mitochondrial oxidative phosphorylation as a selective vulnerability of drug-resistant cancers. Accordingly, chemical inhibitors of respiration led to selective eradication of such cancer cells due to their preferential sensitivity to mitochondrial production of reactive oxygen species. These insights create a timely opportunity for a biomarker guided application of already existing and newly emerging mitochondrial inhibitors in recurrent drug-resistant cancer, including lymphomas, melanomas, and other malignant diseases marked by increased mitochondrial respiration.

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NKX3.1 is a homeobox transcription factor whose function as a prostate tumor suppressor remains insufficiently understood because neither the transcriptional program governed by NKX3.1, nor its interacting proteins have been fully revealed.

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We previously identified SMIP004 (N-(4-butyl-2-methyl-phenyl) acetamide) as a novel inducer of cancer-cell selective apoptosis of human prostate cancer cells. SMIP004 decreased the levels of positive cell cycle regulators, upregulated cyclin-dependent kinase inhibitors, and resulted in G1 arrest, inhibition of colony formation in soft agar, and cell death. However, the mechanism of SMIP004-induced cancer cell selective apoptosis remained unknown.

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