Biomedical subjects

Stefanie M Hauck

Publications and source records attributed to Stefanie M Hauck.

11 recordsLinked to original sources

PubMed2026

Manipulation of protein translation and stem cell self-renewal by CRISPR activation of rRNA transcription.

Ribosomal RNA (rRNA) transcription rates vary during development, and their dysregulation is linked to diseases such as cancer and ribosomopathies. Owing to their high abundance and genomic redundancy, the functional significance of rRNA levels remains unclear. We developed TAPIR (Targeted Activation of Protein Translation), a CRISPR-based approach to elevate rRNA levels by inducing 47S ribosomal DNA transcription. TAPIR increased nucleolar size and enhanced protein synthesis, even in rapidly proliferating cells. In neural stem cells, elevated translation promoted self-renewal and proliferation in vitro and in vivo. Furthermore, TAPIR enabled the modeling and partial rescue of associated disease phenotypes. Our findings reveal that rRNA levels directly regulate translational output and that protein synthesis capacity can act as a key determinant of mammalian stem cell behavior.

Animals↗

PubMed2026

Unlocking the Circulating Proteome: Toward Clinical Translation.

Blood-based proteomics is approaching a translational inflection point. Driven by advances in measurement technologies, rapid expansion of analytical capabilities, and growing adoption across research and medical communities, there is increasing demand for clinically actionable biomarkers. As the field transitions away from purely large-scale discovery-oriented studies toward more informed, targeted, application-driven analyses, the generation of proteomic data is no longer the bottleneck. Instead, the central challenge is to translate these measurements into robust, reproducible, and clinically meaningful insights. In this Review, we assess recent technological and methodological developments, evaluate persistent preanalytical and interpretative limitations, and outline the key steps required for clinical translation. We focus on three deeply interconnected dimensions: the capabilities and constraints of current measurement platforms, the role of computational and machine learning approaches in extracting biological and clinical signals, and the emergence of large-scale population studies that create new opportunities for validation and generalization. Finally, we discuss a forward-looking vision in which proteomics plays a central role in dynamic, multilayered omics frameworks, where integration with genomics, temporal profiling, and imaging can deepen our understanding of health, disease, and therapeutic response.

Humans↗

PubMed2025

The CGG triplet repeat binding protein 1 counteracts R-loop induced transcription-replication stress.

The CGG triplet repeat binding protein 1 (CGGBP1) binds to CGG repeats and has several important cellular functions, but how this DNA sequence-specific binding factor affects transcription and replication processes is an open question. Here, we show that CGGBP1 binds human gene promoters containing short (< 5) CGG-repeat tracts prone to R-loop formation. Loss of CGGBP1 leads to deregulated transcription, transcription-replication-conflicts (TRCs) and accumulation of Serine-5 phosphorylated RNA polymerase II (RNAPII), indicative of promoter-proximal stalling and a defect in transcription elongation. Consistently, an episomal CGG-repeat-containing model locus as well as endogenous genes show deregulated transcription, R-loop accumulation and increased RNAPII chromatin occupancy in CGGBP1-depleted cells. We identify the DEAD-box RNA:DNA helicases DDX41 and DHX15 as interaction partners specifically recruited by CGGBP1. Co-depletion experiments show that DDX41 and CGGBP1 work in the same pathway to unwind R-loops and avoid TRCs. Together, our work shows that short trinucleotide repeats are a source of genome-destabilizing secondary structures, and cells rely on specific DNA-binding factors to maintain proper transcription and replication coordination at short CGG repeats.

Humans↗

PubMed2006

Differential analysis of Saccharomyces cerevisiae mitochondria by free flow electrophoresis.

One major problem concerning the electrophoresis of mitochondria is the heterogeneity of mitochondrial appearance especially under pathological conditions. We show here the use of zone electrophoresis in a free flow electrophoresis device (ZE-FFE) as an analytical sensor to discriminate between different yeast mitochondrial populations. Impairment of the structural properties of the organelles by hyperosmotic stress resulted in broad separation profiles. Conversely untreated mitochondria gave rise to homogeneous populations reflected by sharp separation profiles. Yeast mitochondria with altered respiratory activity accompanied by a different outer membrane proteome composition could be discriminated based on electrophoretic deflection. Proteolysis of the mitochondrial surface proteome and the deletion of a single major protein species of the mitochondrial outer membrane altered the ZE-FFE deflection of these organelles. To demonstrate the usefulness of ZE-FFE for the analysis of mitochondria associated with pathological processes, we analyzed mitochondrial fractions from an apoptotic yeast strain. The cdc48(S565G) strain carries a mutation in the CDC48 gene that is an essential participant in the endoplasmic reticulum-associated protein degradation pathway. Mutant cells accumulate polyubiquitinated proteins in microsomal and mitochondrial extracts. Subsequent ZE-FFE characterization could distinguish a mitochondrial subfraction specifically enriched with polyubiquitinated proteins from the majority of non-affected mitochondria. This result demonstrates that ZE-FFE may give important information on the specific properties of subpopulations of a mitochondrial preparation allowing a further detailed functional analysis.

Adenosine Triphosphatases↗

PubMed2006

Identification and functional validation of novel autoantigens in equine uveitis.

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers.

Animals↗

PubMed2006

Calpain is activated in degenerating photoreceptors in the rd1 mouse.

The retinal degeneration (rd)1 mouse displays an inherited retinal degeneration and therefore allows studies of the molecular mechanisms behind the blinding disease retinitis pigmentosa. Activation of the calcium-dependent protease calpain has been suggested to play an important role in cell death in various tissues, but little is known about the expression and activity of calpain during inherited retinal degeneration. Using microarray techniques, transcript levels of cyclic AMP response element-binding protein (CREB)-1, calpastatin and of various calpain genes were analysed in the rd1 mouse compared with its wild-type control. Expression of distinct calpain isoforms and calpastatin was investigated using immunofluorescence and immunoblotting. Gene transcription and protein expression levels were compared with calpain activity using an enzymatic assay that allowed monitoring of calpain activity at the cellular level. We found that CREB-1 and calpastatin expression was reduced in rd1 retinas, whereas calpain activity was substantially increased in rd1 photoreceptors. Calpain activity peaked at postnatal day 13, together with rd1 photoreceptor cell death. Calpain-specific inhibitors decreased calpain activity in situ. These results indicate that activation of calpains correlates with rd1 photoreceptor cell death, which raises the possibility of using calpain inhibitors to prevent or delay photoreceptor degeneration.

Age Factors↗

PubMed2006

Differential protein profiling of primary versus immortalized human RPE cells identifies expression patterns associated with cytoskeletal remodeling and cell survival.

Functional research of retinal pigment epithelium (RPE) most often relies on utilization of RPE-derived cell lines in vitro. However, no studies about similarities and differences of the respective cell lines exist so far. Thus, we here analyze the proteome of the most popular RPE cell lines: ARPE-19 and hTERT and compare their constitutive and de novo synthesized protein expression profiles to human early passage retinal pigment epithelial cells (epRPE) by 2-D electrophoresis and MALDI-TOF peptide mass fingerprinting. In all three cell lines the baseline protein expression pattern corresponded well to the de novo synthesized cellular proteome. However, comparison of the protein profile of epRPE cells with that of hTERT-RPE cells revealed a higher abundance of proteins related to cell migration, adhesion, and extracellular matrix formation, paralleled by a down-regulation of proteins attributed to cell polarization, and showed an altered expression of detoxification enzymes in hTERT-RPE. ARPE-19 cells, however, exhibited a higher abundance of components of the microtubule cytoskeleton and differences in expression of proteins related to proliferation and cell death. epRPE cells, hTERT-RPE, and ARPE-19 therefore may respond differently with respect to certain functional properties, a finding that should prove valuable for future in vitro studies.

Cell Culture Techniques↗

PubMed2006

GDNF family ligands trigger indirect neuroprotective signaling in retinal glial cells.

Apoptotic cell death of photoreceptors is the final event leading to blindness in the heterogeneous group of inherited retinal degenerations. GDNF (glial cell-line-derived neurotrophic factor) was found to rescue photoreceptor function and survival very effectively in an animal model of retinal degeneration (M. Frasson, S. Picaud, T. Leveillard, M. Simonutti, S. Mohand-Said, H. Dreyfus, D. Hicks, and J. Sahel, Investig. Ophthalmol. Vis. Sci. 40:2724-2734, 1999). However, the cellular mechanism of GDNF action remained unresolved. We show here that in porcine retina, GDNF receptors GFRalpha-1 and RET are expressed on retinal Mueller glial cells (RMG) but not on photoreceptors. Additionally, RMG express the receptors for the GDNF family members artemin and neurturin (GFRalpha-2 and GFRalpha-3). We further investigated GDNF-, artemin-, and neurturin-induced signaling in isolated primary RMG and demonstrate three intracellular cascades, which are activated in vitro: MEK/ERK, stress-activated protein kinase (SAPK), and PKB/AKT pathways with different kinetics in dependence on stimulating GFL. We correlate the findings to intact porcine retina, where GDNF induces phosphorylation of ERK in the perinuclear region of RMG located in the inner nuclear layer. GDNF signaling resulted in transcriptional upregulation of FGF-2, which in turn was found to support photoreceptor survival in an in vitro assay. We provide here a detailed model of GDNF-induced signaling in mammalian retina and propose that the GDNF-induced rescue effect on mutated photoreceptors is an indirect effect mediated by retinal Mueller glial cells.

Animals↗

PubMed2005

Differential modification of phosducin protein in degenerating rd1 retina is associated with constitutively active Ca2+/calmodulin kinase II in rod outer segments.

Retinitis pigmentosa comprises a heterogeneous group of incurable progressive blinding diseases with unknown pathogenic mechanisms. The retinal degeneration 1 (rd1) mouse is a retinitis pigmentosa model that carries a mutation in a rod photoreceptor-specific phosphodiesterase gene, leading to rapid degeneration of these cells. Elucidation of the molecular differences between rd1 and healthy retinae is crucial for explaining this degeneration and could assist in suggesting novel therapies. Here we used high resolution proteomics to compare the proteomes of the rd1 mouse retina and its congenic, wild-type counterpart at postnatal day 11 when photoreceptor death is profound. Over 3000 protein spots were consistently resolved by two-dimensional gel electrophoresis and subjected to a rigorous filtering procedure involving computer-based spot analyses. Five proteins were accepted as being differentially expressed in the rd1 model and subsequently identified by mass spectrometry. The difference in one such protein, phosducin, related to an altered modification pattern in the rd1 retina rather than to changed expression levels. Additional experiments showed phosducin in healthy retinae to be highly phosphorylated in the dark- but not in the light-adapted phase. In contrast, rd1 phosducin was highly phosphorylated irrespective of light status, indicating a dysfunctional rd1 light/dark response. The increased rd1 phosducin phosphorylation coincided with increased activation of calcium/calmodulin-activated protein kinase II, which is known to utilize phosducin as a substrate. Given the increased rod calcium levels present in the rd1 mutation, calcium-evoked overactivation of this kinase may be an early and long sought for step in events leading to photoreceptor degeneration in the rd1 mouse.

Animals↗

PubMed2005

Proteomic analysis of the porcine interphotoreceptor matrix.

The interphotoreceptor matrix (IPM) is located between photoreceptors and pigment epithelium in the retina and is involved in fundamental functions of the visual cycle. These include visual pigment chromophore exchange, retinal adhesion, metabolite trafficking, and growth factor presentation. In general, IPM preparations are contaminated with intracellular proteins, as has also been described for other body fluids. This study aimed at identifying new components of the IPM by discriminating between truly secreted proteins and proteins that are part of the IPM for secondary reasons. "Soluble" porcine IPM was extracted from retina and pigment epithelium with PBS by two different procedures, followed by extraction with water alone that released "insoluble" IPM matrix sheets. Samples from all preparations were separated by 2-DE and a total of 140 protein spots were identified by MALDI-TOF and/or CapLC Q-TOF MS. Although identified proteins included several already known in the IPM, the majority had not been previously described in this structure. Gene ontology classifications allocated the identified proteins into nine different functional networks. The IPM preparations also included intracellular proteins from cells adjacent to the IPM, which may have resulted from cell disruption. This underlines the experimental difficulties of a biochemical analysis of the IPM as an intact compartment. We show here a strategy for predicting the probability of identified IPM proteins occurring in vivo by combined high-resolution protein separation methods with computational prediction methods. Thus, a set of potentially neuroprotective proteins could be extracted, including PEA-15, peroxiredoxin 5, alpha-B-crystallin, macrophage migration inhibitory factor, 78 kDa glucose-regulated protein (GRP78), protein disulfide-isomerase, and PEP-19, which have not been previously associated with the IPM. Furthermore, with immunohistochemical staining we could confirm the localization of GRP78 in the IPM on porcine eye sections, thus validating the proposed prediction method.

Animals↗

PubMed2003

Proteomic profiling of primary retinal Müller glia cells reveals a shift in expression patterns upon adaptation to in vitro conditions.

Cultured primary retinal Müller glia cells (RMG), a glia cell spanning the entire neuroretina, have recently gained increased attention, especially with respect to their presumed in vivo role in supporting photoreceptor function and survival. Cultured RMG cells, however, are at risk to lose much of their in vivo features. To determine the conditions of isolated primary RMG cells best corresponding with their physiological role in the intact retina, we profiled the respective proteomes of RMG freshly isolated from intact pig eye, as well as from cultured material at different timepoints. Protein samples were separated by high-resolution two-dimensional electrophoresis (2-DE), and isolated proteins were identified by matrix-assisted laser desorption ionization time-of- flight (MALDI-TOF) peptide mass fingerprint. Compared with freshly isolated RMG, the in vitro protein expression patterns remain relatively stable for the first 3 days in culture but change dramatically thereafter. Proteins involved in specific RMG physiological functions, such as glycolysis, transmitter recycling, CO2 siphoning, visual pigment cycle, and detoxification, are either downregulated or absent. In contrast, cytoskeletal proteins, as well as proteins involved in motility and in proliferation, are upregulated during culture. In the present report, we show for the first time, on a systematic level, that profound changes in the RMG proteome reflect transdifferentiation from a multifunctional, highly differentiated glial cell to a dedifferentiated fibroblast-like phenotype in culture.

Animals↗