Biomedical subjects

Martin von Bergen

Publications and source records attributed to Martin von Bergen.

15 recordsLinked to original sources

PubMed2026

Loss of the Mechanistic Target of Rapamycin Complex 1 Causes a Lethal Alpha-1 Antitrypsin Deficiency-Associated Liver Disease.

BACKGROUND & AIMS: SERPINA1 mutations cause retention of the otherwise secreted alpha-1 antitrypsin and lead to the proteotoxic alpha-1 antitrypsin deficiency-related liver disease. As mechanistic target of rapamycin is a key coordinator of proteostasis, we studied its role in alpha-1 antitrypsin deficiency-related liver disease. METHODS: PiZ mice overexpressing the characteristic SERPINA1 mutation were mated with rodents harboring a hepatocyte specific-ablation of the interaction partners regulatory-associated protein of mechanistic target of rapamycin or rapamycin-insensitive companion of mammalian target of rapamycin, corresponding to mechanistic target of rapamycin complexes 1 or 2, or with mice lacking mechanistic target of rapamycin. Serum proteomics, liver bulk proteomics, spatial proteomics, and metabolomics were applied to characterize molecular and metabolic alterations. RESULTS: At 2 months of age, PiZ-mTORΔhep and PiZ-RaptorΔhep but not PiZ-RictorΔhep mice showed signs of increased liver injury and mortality despite diminished hepatic alpha-1 antitrypsin accumulation. PiZ-RaptorΔhep animals displayed increased levels of the proapoptotic protein C/EBP homologous protein, but C/EBP homologous protein ablation did not rescue the phenotype. Serum proteomics revealed no signs of advanced synthetic liver failure but immature hepatocellular products. Liver bulk proteomics and small metabolite measurement demonstrated a metabolic reprogramming of PiZ-RaptorΔhep mice. Spatial proteomics revealed alterations in liver zonation with increased ammonia levels as the likely cause of death in PiZ-RaptorΔhep animals. CONCLUSIONS: In summary, in alpha-1 antitrypsin deficiency-related proteotoxic liver injury, regulatory-associated protein of mechanistic target of rapamycin preserves a liver zonation, thereby protecting from lethal metabolic dysregulation.

Animals↗

PubMed2026

Hypoosmolarity inhibits ammonia oxidation by terrestrial and freshwater Nitrosopumilaceae members.

Salinity strongly influences the physiology and distribution of nitrifying microorganisms, yet the effects of low salinity remain understudied. This study investigates the impact of hypoosmolarity on different groups of ammonia oxidizers in soil and freshwater reservoirs, as well as in pure culture isolates. In soil microcosms amended with ammonium, at low salinity levels (~120 μS/cm), comparable to values commonly found in pristine terrestrial and freshwater environments, the abundance of ammonia-oxidizing bacteria (AOB), dominated by Nitrosomonas oligotropha, significantly increased. In contrast, the growth of ammonia-oxidizing archaea (AOA), dominated by "Candidatus Nitrosotenuis" of the Nitrosopumilaceae family, was stimulated by high salinity (~760 μS/cm). In ammonium-fed freshwater microcosms, the abundance of AOB, dominated by N. oligotropha, significantly increased under both low (~170 μS/cm) and high salinity (~850 μS/cm) conditions. In the presence of allylthiourea (50 μM), used to inhibit bacterial ammonia oxidation, AOA were sensitive to low salinity in both soil and freshwater microcosms. Consistently, culture-dependent studies revealed marked growth inhibition of terrestrial AOA, especially members of Nitrosopumilaceae, under hypoosmolarity, unlike AOB and complete ammonia oxidizer (comammox) strains. Genomic analyses, along with transcriptomic studies, suggested that the sensitivity of AOA to hypoosmolarity stress was possibly due to a lack of osmoregulatory transport systems and their S-layer cell wall structure. Overall, this study indicates hypoosmolarity as an important factor shaping the ecological niches and distribution of ammonia oxidizers, as well as nitrification activities, in terrestrial and freshwater environments that are increasingly affected by intensified water cycles due to global change.

Ammonia↗

PubMed2024

Revealing novel protein interaction partners of glyphosate in Escherichia coli.

Despite all debates about its safe use, glyphosate remains the most widely applied active ingredient in herbicide products, with renewed approval in the European Union until 2033. Non-target organisms are commonly exposed to glyphosate as a matter of its mode of application, with its broader environmental and biological impacts remaining under investigation. Glyphosate displays structural similarity to phosphoenolpyruvate (PEP), thereby competitively inhibiting the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), crucial for the synthesis of aromatic amino acids in plants, fungi, bacteria, and archaea. Most microbes, including the gut bacterium Escherichia coli (E. coli), possess a glyphosate-sensitive class I EPSPS, making them vulnerable to glyphosate's effects. Yet, little is known about glyphosate's interactions with other bacterial proteins or its broader modes of action at the proteome level. Here, we employed a quantitative proteomics and thermal proteome profiling (TPP) approach to identify novel protein binding partners of glyphosate in the E. coli proteome. Glyphosate exposure significantly altered amino acid synthesizing pathways. The abundance of shikimate pathway proteins was increased, suggesting a compensatory mechanism. Extracellular riboflavin concentrations were elevated upon glyphosate exposure, while intracellular levels remained stable. Beyond the target enzyme EPSPS, thermal proteome profiling indicated an effect of glyphosate on the thermal stability of certain proteins, including AroH and ProA, indicating interactions. Similar to the competitive binding between PEP and glyphosate at EPSPS, one reason for the interaction of AroH and ProA with the herbicide could be a high structural similarity between their substrates and glyphosate. Overall, glyphosate induced metabolic disturbances in E. coli, extending beyond its primary target, thereby providing new insights into glyphosate's broader impact on microbial systems.

Glyphosate↗

PubMed2024

Dysregulated cytokine and oxidative response in hyper-glycolytic monocytes in obesity.

INTRODUCTION: Obesity is associated with a plethora of health complications, including increased susceptibility to infections or decreased vaccine efficacy, partly due to dysregulated immune responses. Monocytes play a crucial role in innate immunity, yet their functional alterations in obesity remain poorly understood. METHODS: Here, we employed proteomic and metabolomic analyses to investigate monocyte characteristics in individuals with overweight, obesity, impaired glucose tolerance (IGT), and type 2 diabetes (T2D), compared to lean donors. RESULTS AND DISCUSSION: Our results revealed distinct molecular signatures in monocytes from individuals with obesity, with significant alterations in pathways related to metabolism, cellular migration, and phagocytosis. Moreover, LPS-induced activation of monocytes unveiled heightened metabolic reprogramming towards glycolysis in subjects with obesity accompanied by dysregulated cytokine responses and elevated oxidative stress. Additionally, monocytes from donors with obesity exhibited increased lipid droplet accumulation. These findings shed light on the immunometabolic dysregulation underlying obesity-associated immune dysfunction, highlighting potential targets for therapeutic intervention.

Humans↗

PubMed2006

Structural characterization of beta-sheeted oligomers formed on the pathway of oxidative prion protein aggregation in vitro.

The pathology of transmissible spongiform encephalopathies (TSEs) is strongly associated with the structural conversion of the cellular prion protein (PrPC) into a misfolded isoform (PrPSc) that assembles into amyloid fibrils. Since increased levels of oxidative stress have been linked to prion diseases, we investigated the metal-induced oxidation of human PrP (90-231). A novel in vitro conversion assay based on aerobic incubation of PrP in the presence of elemental copper pellets at pH 5 was established, resulting in aggregation of highly beta-sheeted prion proteins. We show for the first time that two discrete oligomeric species of elongated shape, approx. 25 mers and 100 mers, are formed on the pathway of oxidative PrP aggregation in vitro, which are well characterized regarding shape and size using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and electron microscopy (EM). Considering that small oligomers of highly similar size have recently been reported to show the highest specific infectivity within TSE-infected brain tissues of hamsters, the novel oligomers observed in this study are interesting candidates as agent causing neurodegenerative and/or self-propagating effects. Moreover, our results significantly strengthen the theory that oxidative stress might be an influence that leads to substantial structural conversions of PrP in vivo.

Amino Acid Sequence↗

PubMed2006

The core of tau-paired helical filaments studied by scanning transmission electron microscopy and limited proteolysis.

In Alzheimer's disease and frontotemporal dementias the microtubule-associated protein tau forms intracellular paired helical filaments (PHFs). The filaments formed in vivo consist mainly of full-length molecules of the six different isoforms present in adult brain. The substructure of the PHF core is still elusive. Here we applied scanning transmission electron microscopy (STEM) and limited proteolysis to probe the mass distribution of PHFs and their surface exposure. Tau filaments assembled from the three repeat domain have a mass per length (MPL) of approximately 60 kDa/nm and filaments from full-length tau (htau40DeltaK280 mutant) have approximately 160 kDa/nm, compared with approximately 130 kDa/nm for PHFs from Alzheimer's brain. Polyanionic cofactors such as heparin accelerate assembly but are not incorporated into PHFs. Limited proteolysis combined with N-terminal sequencing and mass spectrometry of fragments reveals a protease-sensitive N-terminal half and semiresistant PHF core starting in the first repeat and reaching to the C-terminus of tau. Continued proteolysis leads to a fragment starting at the end of the first repeat and ending in the fourth repeat. PHFs from tau isoforms with four repeats revealed an additional cleavage site within the middle of the second repeat. Probing the PHFs with antibodies detecting epitopes either over longer stretches in the C-terminal half of tau or in the fourth repeat revealed that they grow in a polar manner. These data describe the physical parameters of the PHFs and enabled us to build a model of the molecular arrangement within the filamentous structures.

Alzheimer Disease↗

PubMed2006

Interactions of recombinant prions with compounds of therapeutical significance.

The transformation of the cellular prion protein (PrP(C)) into the infectious form (PrP(Sc)) is implicated in the invariably fatal transmissible spongiform encephalopathies. To identify a mechanism to prevent the undesired PrP(C)-->PrP(Sc) transformation, we investigated the interactions of recombinant prion proteins with a number of potential therapeutic agents which inhibit the PrP(Sc) formation, infectivity, and the accumulation of the misfolded form. We show that the prion aggregates formed in the presence of six compounds have no beta-structure, which is typical of the infectious form, and possess considerably higher alpha-helical content than the normal PrP(C). The investigated compounds stimulate the formation of alpha-helices and the destruction of beta-structure. They prevent the transformation of alpha-helical structure into beta-sheets. Probably, this is the reason for the resistance to PrP(C)-->PrP(Sc) transformation in the presence of these compounds. The results may be useful for the future therapy of neurodegenerative diseases.

Binding Sites↗

PubMed2006

Global hairpin folding of tau in solution.

The microtubule-associated protein tau stabilizes microtubules in its physiological role, whereas it forms insoluble aggregates (paired helical filaments) in Alzheimer's disease. Soluble tau is considered a natively unfolded protein whose residual folding and intramolecular interactions are largely undetermined. In this study, we have applied fluorescence resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) to examine the proximity and flexibility of tau domains and the global folding. FRET pairs spanning the tau molecule were created by inserting tryptophans (donor) and cysteines (labeled with IAEDANS as an acceptor) by site-directed mutagenesis. The observed FRET distances were significantly different from those expected for a random coil. Notably, the C-terminal end of tau folds over into the vicinity of the microtubule-binding repeat domain, the N-terminus remains outside the FRET distance of the repeat domain, yet both ends of the molecule approach one another. The interactions between the domains were obliterated by denaturation in GdnHCl. Paramagnetic spin-labels attached in various domains of tau were analyzed by EPR and exhibited a high mobility throughout. The data indicate that tau retains some global folding even in its "natively unfolded" state, combined with the high flexibility of the chain.

Circular Dichroism↗

PubMed2005

Inducible expression of Tau repeat domain in cell models of tauopathy: aggregation is toxic to cells but can be reversed by inhibitor drugs.

We generated several cell models of tauopathy in order to study the mechanisms of neurodegeneration in diseases involving abnormal changes of tau protein. N2a neuroblastoma cell lines were created that inducibly express different variants of the repeat domain of tau (tau(RD)) when exposed to doxycycline (Tet-On system). The following three constructs were chosen: (i) the repeat domain of tau that coincides with the core of Alzheimer paired helical filaments; (ii) the repeat domain with the deletion mutation DeltaK280 known from frontotemporal dementia and highly prone to spontaneous aggregation; and (iii) the repeat domain with DeltaK280 and two proline point mutations that inhibit aggregation. The comparison of wild-type, pro-aggregation, and anti-aggregation mutants shows the following. (a) Aggregation of tau(RD) is toxic to cells. (b) The degree of aggregation and toxicity depends on the propensity for beta-structure. (c) Soluble mutants of tau(RD) that cannot aggregate are not toxic. (d) Aggregation is preceded by fragmentation. (e) Fragmentation of tau(RD) in cells is initially due to a thrombin-like protease activity. (f) Phosphorylation of tau(RD) (at KXGS motifs) precedes aggregation but is not correlated with the degree of aggregation. (g) Aggregates of tau(RD) disappear when the expression is silenced, showing that aggregation is reversible. (h) Aggregation can be prevented by drugs and even pre-formed aggregates can be dissolved again by drugs. Thus, the cell models open up new insights into the relationship between the structure, expression, phosphorylation, aggregation, and toxicity of tau(RD) that can be used to test current hypotheses on tauopathy and to develop drugs that prevent the aggregation and degeneration of cells.

Alzheimer Disease↗

PubMed2005

Sites of tau important for aggregation populate {beta}-structure and bind to microtubules and polyanions.

The aggregation of the microtubule-associated tau protein and formation of "neurofibrillary tangles" is one of the hallmarks of Alzheimer disease. The mechanisms underlying the structural transition of innocuous, natively unfolded tau to neurotoxic forms and the detailed mechanisms of binding to microtubules are largely unknown. Here we report the high-resolution characterization of the repeat domain of soluble tau using multidimensional NMR spectroscopy. NMR secondary chemical shifts detect residual beta-structure for 8-10 residues at the beginning of repeats R2-R4. These regions correspond to sequence motifs known to form the core of the cross-beta-structure of tau-paired helical filaments. Chemical shift perturbation studies show that polyanions, which promote paired helical filament aggregation, as well as microtubules interact with tau through positive charges near the ends of the repeats and through the beta-forming motifs at the beginning of repeats 2 and 3. The high degree of similarity between the binding of polyanions and microtubules supports the hypothesis that stable microtubules prevent paired helical filament formation by blocking the tau-polyanion interaction sites, which are crucial for paired helical filament formation.

Alzheimer Disease↗

PubMed2005

Intrinsic fluorescent detection of tau conformation and aggregation.

The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Insights into the prerequisites and kinetics of the polymerization was obtained by the in vitro analysis of this process. In the past, fluorescent dyes were used to stain amyloidogenic material in histology and later on similar dyes were used in in vitro studies as well. To circumvent the flaws of extragenous dyes, namely the alteration of the polymerization kinetic or incompatibility with other chemical compounds needed for stability analysis, we applied tryptophan fluorescence to the in vitro analysis of PHF formation. Single tryptophans were introduced into the hexapeptide PHF6 within the third repeat, which was shown to be involved in beta sheet formation and scattered around the whole microtubule binding domain. Tryptophan fluorescence was then used to scan the microtubule binding domain for accessibility to quenching reagent in the soluble and the aggregated state and the fluorescence resonance energy transfer (FRET) between tryptophan and tyrosine 310. Furthermore, this approach enables the analysis of stability of PHFs in the presence of Guanidinium hydrochloride. The examples given here could be applied in modified ways to other amyloidogenic proteins.

Fluorescence Resonance Energy Transfer↗

PubMed2005

Screening for inhibitors of tau polymerization.

The histopathological diagnosis of Alzheimer's disease relies on two kinds of proteinaceous aggregates: the extracellular plaques built from filaments of the Abeta-peptide and the intracellular tangles consisting of tau polymerized into Paired Helical Filaments (PHFs). The order of aggregation events is still under debate, but it is well accepted that tau-related changes have an important impact on the viability of neurons. In neurons, early morphological changes are seen in axons which begin to loose and retract synapses. This process is accompanied by an increase of aggregated tau protein. Thus the prevention of tau aggregation seems to be a valuable target for therapy of Alzheimer's disease. Here we present a screening procedure by which we identified inhibitors of tau polymerization. In the primary screen we used a thioflavin-S based assay which detects PHF formation in solution. These initial hits were further analyzed for their capacity to depolymerize preformed PHFs. These results were confirmed by several secondary assays (tryptophan fluorescence, pelleting, filter trapping and electron microscopy). By this approach it is possible to identify small molecule compounds which prevent or reverse the aggregation of tau and thereby might improve the viability of neurons in a therapeutic approach.

Alzheimer Disease↗

PubMed2004

Tau aggregation is driven by a transition from random coil to beta sheet structure.

The abnormal aggregation of the microtubule associated protein tau into paired helical filaments (PHFs) is one the hallmarks of Alzheimer's disease. The soluble protein is one of the longest natively unfolded proteins, lacking significant amounts of secondary structure over a sequence of 441 amino acids in the longest isoform. Furthermore, the unfolded character is consistent with some notable features of the protein like stability towards heat and acid treatment. It is still unclear how these characteristics support the physiological function of binding to and stabilization of microtubules. We review here some recent studies on how an unfolded protein such as tau can adopt beta-structure, which then leads to the highly ordered morphology of the PHFs. The core sequence for both microtubule binding and PHF formation is the microtubule binding domain containing three or four repeats. This region alone is sufficient for PHF formation and mostly unfolded in the soluble state. A search for sequence motifs within this region crucial for PHF building revealed two hexapeptides in the second and the third repeat. Some of the genetically linked cases of FTDP-17 show missense mutations in or adjacent to these hexapeptide motifs. Proteins containing the P301L and the DeltaK280 mutations exhibit accelerated aggregation. The importance of the two hexapeptides stems from their capacity to undergo a conformational change from a random coil to a beta sheet structure. The increase of beta sheet structure is a typical feature of an amyloidogenic protein and is the basis of other characteristics like a decreased sensitivity towards proteolytic degradation and Congo red binding. PHFs aggregated in vitro and in vivo contain beta-sheet structure, as judged by circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction.

Alzheimer Disease↗

PubMed2004

Anthraquinones inhibit tau aggregation and dissolve Alzheimer's paired helical filaments in vitro and in cells.

The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC50 values of 1-5 microm and to disassemble preformed PHFs at DC50 values of 2-4 microm. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.

Alzheimer Disease↗

PubMed2002

Structure, stability, and aggregation of paired helical filaments from tau protein and FTDP-17 mutants probed by tryptophan scanning mutagenesis.

By using tryptophan scanning mutagenesis, we observed the kinetics and structure of the polymerization of tau into paired helical filaments (PHFs) independently of exogenous reporter dyes. The fluorescence exhibits pronounced blue shifts due to burial of the residue inside PHFs, depending on Trp position. The effect is greatest near the center of the repeat domain, showing that the packing is tightest near the beta-structure inducing hexapeptide motifs. The tryptophan response allows measurement of PHF stability made by different tau isoforms and mutants. Unexpectedly, the stability of PHFs is quite low (denaturation half-points approximately 1.0 m GdnHCl), implying that incipient aggregation should be reversible and that the observed high stability of Alzheimer PHFs is due to other factors. The stability increases with the number of repeats and with tau mutants promoting beta-structure, arguing for a gain of toxic function in frontotemporal dementias. Fluorescence resonance energy transfer (FRET) was used to analyze the distances of Tyr(310) to tryptophans in different positions. The degree of FRET in the soluble protein was position-dependent, with highest signals within the second and third repeats but low or no signals further away. In PHFs most mutants showed FRET, indicating that tight packing results from assembly of tau into PHFs.

Alzheimer Disease↗