Biology subjects

Bieschke, J.

Publications and source records attributed to Bieschke, J..

9 recordsLinked to original sources

bioRxiv2025

Taf1 expression in mouse: novel transcripts and protein distribution

TATA-box binding protein associated factor 1 (TAF1) is the largest component of transcription factor IID (TFIID), a fundamental multiprotein complex for RNA polymerase II-mediated transcription. TAF1 is essential for promoter recognition, coactivator interaction, and normal development. Mutations in TAF1 cause developmental disorders and the lethal neurodegenerative disease X-linked dystonia-Parkinsonism (XDP). Our previous work suggested that this [~]170 kb gene has a more complex set of transcripts than currently catalogued. We therefore undertook a systematic assessment of Taf1 transcription in mouse, given its widespread use as a model organism and its high genetic homology with humans. Using targeted nanopore sequencing, we reveal extensive transcriptional diversity and differential abundance of Taf1 mRNAs across brain and body regions. We identify 19 novel transcript variants and multiple novel exons, and we predicted their protein domain architectures and modelled them using AlphaFold. Notably, we observed elevated RNA and protein expression in cerebellum compared to other brain regions. These findings substantially expand the transcriptional landscape of Taf1 and provide crucial insights to guide investigation of transcript-specific mechanisms in neurodevelopmental and neurodegenerative disorders, enabling the creation of more accurate disease models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/674157v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1c31231org.highwire.dtl.DTLVardef@1df8a57org.highwire.dtl.DTLVardef@f35bd8org.highwire.dtl.DTLVardef@112c264_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Alanine replacements in the structured C-terminal domain of the prion protein reveal conformationally variable regions as major determinants for prion propagation

Mutational analysis of the cellular prion protein (PrPC) has revealed various regions of the protein that modulate prion propagation. However, most approaches involve deletions, insertions, or replacements in the presence of the wild-type cellular protein, which may mask the true phenotype. Here, site-directed alanine mutagenesis of PrPC was conducted to identify sites particularly a surface patch of the protein pertinent to prion propagation in the absence of the wild-type prion protein. Mutations were targeted to the helical, sheet and loop regions of PrPC, or a combination thereof and the mutated proteins expressed in PK1 cells in which endogenous PrPC had been silenced. PK1 cells are a clone of mouse neuroblastoma cells that are highly susceptible to Rocky Mountain Laboratory mouse prions. Using the scrapie cell assay, a highly sensitive cell culture-based bioassay for quantifying infectious titres of mouse prions, we found that all mutations within the structured 121-230 domain, irrespective of secondary structure, severely reduced prion propagation. The reduction was most pronounced for mutations within conformationally variable regions of the protein (G123A.L124A.G125A and V188A.T191A.T192A) and those neighbouring or within helix 1 (S134A.R135A.M153A and H139A.G141A.D146A). While mutations G123A and G125A would likely disrupt the structure of the prion fibril, the other mutations are unlikely to cause disruption. Our data therefore suggests that conformationally variable regions within the structured domain of PrPC are the major determinants of prion propagation efficacy.

biochemistry↗

bioRxiv2022

Loss of the first β-strand of human prion protein generates an aggregation-competent partially "open" form

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). The pathway of PrP misfolding is still unclear, though previous data indicate the presence of a structural core in cellular PrP (PrPC), whose cooperative unfolding presents a substantial energy barrier on the path to prion formation. PrP is a GPI-anchored membrane protein, and a number of studies suggest that membrane interactions play an important role in the conversion of PrPC to its disease-associated form, including a transmembrane form of PrP in which a highly conserved region (residues 110 - 136) spans the ER membrane. Insertion of this region results in the detachment of the PrPC first {beta}-strand from the structural core. The effect of this removal on the structure, stability and self-association of the folded domain of PrPC is determined here through a biophysical characterisation of a truncated form of PrPC lacking this region. Whilst markedly destabilised, NMR chemical shifts show that the truncated protein exhibits tertiary structure characteristic of a fully folded protein and retains its native secondary structure elements, including the second strand of the PrP {beta}-sheet, but with altered conformational flexibility in the {beta}2-2 loop and first -helix. The latter is destabilised relative to the other helical regions of the protein, with markedly increased solvent exposure. This truncated form of PrP fibrilises more readily than the native form of the protein. These data suggest a stepwise mechanism, in which a destabilised "open" form of PrPC may be a key intermediate in the refolding to the fibrillar, pathogenic form of the protein.

biophysics↗

bioRxiv2022

Syntaxin 6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates

Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro, non-physiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening suggests that variants that increase syntaxin-6 expression in the brain (gene: STX6) are risk factors for sporadic Creutzfeldt-Jakob disease (CJD). Analysis of the protein in NAA revealed counterintuitively that syntaxin-6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, syntaxin-6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that, instead of highly ordered fibrils, in the presence of syntaxin-6 PrP formed less-ordered aggregates containing syntaxin-6. These data strongly suggest that the protein can directly alter the initial phase of PrP self-assembly and, uniquely, can act as an anti-chaperone, which promotes toxic aggregation intermediates by inhibiting fibril formation.

biochemistry↗

bioRxiv2022

Direct Observation of Competing Prion Protein Fibril Populations with Distinct Structures and Kinetics

In prion diseases, fibrillar assemblies of misfolded prion protein (PrP) self-propagate by incorporating PrP monomers. Using total internal reflection and transient amyloid binding super-resolution microscopy, our study analyses elongation of single PrP fibrils to reveal polymorphic populations, featuring structural and dynamic heterogeneity similar to prion strains, which were previously hidden in ensemble measurements. PrP fibrils elongated along a preferred direction by an intermittent stop- and-go mechanism. Fibrils fell into three main populations, which each displayed distinct elongation mechanisms incorporating different monomer structures and which maintained their properties even under elongation conditions favouring a different fibril type. Elongation of RML and ME7 prion rods likewise exhibited unique kinetic features. The discovery of polymorphic fibril populations of amyloid and prions growing in competition suggests that prions may present as quasispecies of structural isomorphs and that the replication environment may tilt the balance between prion isomorphs and amyloid species. HighlightsO_LISynthetic prion fibril populations contain structurally distinct fibril types C_LIO_LIFibril types faithfully elongate by different mechanisms C_LIO_LIFibril types compete for substrate depending on environment C_LIO_LIFibril populations model quasi-species behavior of prion strains C_LI eTOCReplication of different prion strains causes distinct disease phenotypes. Sun et al. analyzed the growth of individual synthetic prion protein fibrils by super-resolution microscopy and found populations of structurally distinct fibril types, which grew in competition to each other as a quasi-species, recapitulating basic prion strain characteristics in vitro.

biophysics↗

bioRxiv2021

VCP protects neurons from proteopathic seeding

Uptake and spread of proteopathic seeds, such as S, Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery necessary for this process is poorly understood. Using a genome-wide CRISPR-Cas9 screen, we identified Valosin Containing Protein (VCP) as a suppressor of S seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) with muscle and neuronal degeneration. VCP inhibition or disease mutations increase S seeding in cells and neurons. This is not associated with an increase in seed uptake and is similar to treatment with the lysosomal damaging agent, LLoME. Intrastriatal injection of S seeds into VCP disease mice enhances seeding efficiency compared with controls. This is not specific to S since VCP inhibition or disease mutations increased TDP-43 seeding in neurons. These data support that VCP protects against proteopathic spread of pathogenic aggregates. The spread of distinct aggregate species may dictate pleiotropic phenotypes and pathologies in VCP associated MSP.

neuroscience↗

bioRxiv2020

Brazilin Removes Toxic alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium

Alpha-synuclein (-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinsons disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (A{beta}) and -syn, prompting our inquiry in its mechanism of action. Here we test the effects of Brazilin on both seeded and unseeded -syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of -syn by a unique mechanism that is distinct from other polyphenols and is also distinct from its effect on A{beta}. Brazilin preserves the natively unfolded state of -syn by stabilizing the compact conformation of the -syn monomer over the aggregation-competent extended conformation. Molecular docking of Brazilin shows the molecule to interact both with unfolded -syn monomers and with the cross-{beta} sheet structure of -syn fibrils. Brazilin eliminates seeding competence of -syn assemblies from Parkinsons disease patient brain tissue, and treatment of pre-formed fibril assemblies with Brazilin significantly reduces their toxicity in primary neurons. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinsons Disease. Highlights- The natural polyphenol Brazilin binds to monomeric, oligomeric and fibrillar -syn - Brazilin shifts the equilibrium away from aggregation-competent monomer conformations - Brazilin inactivates seeding-competent -syn isolated from Parkinson patients brains - Brazilin detoxifies -syn aggregation intermediates and stabilizes mature amyloid fibrils Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=117 HEIGHT=200 SRC="FIGDIR/small/318220v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@447629org.highwire.dtl.DTLVardef@a9871dorg.highwire.dtl.DTLVardef@1e5a83dorg.highwire.dtl.DTLVardef@8843eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Megadalton-sized dityrosine aggregates of α-synuclein retain high degrees of structural disorder and internal dynamics.

Heterogeneous aggregates of the human protein -synuclein (Syn) are abundantly found in Lewy body inclusions of Parkinsons disease patients. While structural information on classical Syn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of Syn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-{beta} amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict Syn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species rather than cytochrome c are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining Syn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative Syn aggregation scavenges cytochrome cs activity into the formation of amorphous, high molecular-weight structures that may contribute to aggregate diversity in Lewy body deposits.

biophysics↗

bioRxiv2018

TDP-43 oligomers detected as initial intermediate species during aggregate formation

Aggregates of the RNA binding protein TDP-43 are a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which are neurodegenerative disorders with overlapping clinical, genetic and pathological features. Mutations in the TDP-43 gene are causative of ALS, supporting its central role in pathogenesis. The process of TDP-43 aggregation remains poorly understood and whether this includes formation of intermediate complexes is unknown. We characterized aggregates derived from purified TDP-43 as a function of time and analyzed them under semi-denaturing conditions. Our assays identified oligomeric complexes at the initial time points prior to the formation of large aggregates, suggesting that ordered oligomerization is an intermediate step of TDP-43 aggregation. In addition, we analyzed liquid-liquid phase separation of TDP-43 and detected similar oligomeric assembly upon the maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, ALS-linked mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that the aggregates generated from purified protein seed intracellular aggregation, which is detected by established markers of TDP-43 pathology. Remarkably, cytoplasmic aggregate propagation is detected earlier with A315T and M337V and is 50% more widespread than with wild-type aggregates. Our findings provide evidence for a controlled process of TDP-43 self-assembly into intermediate structures that provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.

biochemistry↗