Biology subjects

Vieweg, S.

Publications and source records attributed to Vieweg, S..

4 recordsLinked to original sources

bioRxiv2021

Towards deciphering the Nt17 code: How the sequence and conformation of the first 17 amino acids in Huntingtin regulate the aggregation, cellular properties, and neurotoxicity of mutant Httex1

Converging evidence points to the N-terminal domain comprising the first 17 amino acids of the Huntingtin protein (Nt17) as a key regulator of its aggregation, cellular properties and toxicity. In this study, we further investigated the interplay between Nt17 and the polyQ domain repeat length in regulating the aggregation and inclusion formation of exon 1 of the Huntingtin protein (Httex1). In addition, we investigated the effect of removing Nt17 or modulating its local structure on the membrane interactions, neuronal uptake, and toxicity of monomeric or fibrillar Httex1. Our results show that the polyQ and Nt17 domains synergistically modulate the aggregation propensity of Httex1 and that the Nt17 domain plays an important role in shaping the surface properties of mutant Httex1 fibrils and regulating their poly-Q-dependent growth, lateral association and neuronal uptake. Removal of Nt17 or disruption of its transient helical conformations slowed the aggregation of monomeric Httex1 in vitro, reduced inclusion formation in cells, enhanced the neuronal uptake and nuclear accumulation of monomeric Httex1 proteins, and was sufficient to prevent cell death induced by Httex1 72Q overexpression. Finally, we demonstrate that the uptake of Httex1 fibrils into primary neurons and the resulting toxicity are strongly influenced by mutations and phosphorylation events that influence the local helical propensity of Nt17. Altogether, our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation, internalization, and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, and neuronal toxicity. HighlightsO_LIThe Nt17 and polyQ domains synergistically promote Httex1 aggregation. C_LIO_LIThe Nt17 domain is a key determinant of the lateral association and morphology of fibrils. C_LIO_LIThe Nt17 domain and conformation regulate the nuclear/cytoplasmic distribution and toxicity of Httex1. C_LIO_LINt17 conformation is a key determinant of Httex1 fibril membrane interaction and cellular uptake. C_LIO_LINt17 serves as one of the master regulators of Httex1 aggregation, cellular uptake and toxicity. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/431207v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@7afd68org.highwire.dtl.DTLVardef@92c67aorg.highwire.dtl.DTLVardef@24823dorg.highwire.dtl.DTLVardef@e2a835_HPS_FORMAT_FIGEXP M_FIG The Nt17 domain: A master switch of Httex1 aggregation, uptake, subcellular localization and neurotoxicity. In this paper, we showed that 1) the Nt17 and polyQ domains synergistically promote Httex1 aggregation; 2) the Nt17 domain is a key determinant of the lateral association and morphology of fibrils in vitro, 3) Nt17 conformation is a key determinant of Httex1 fibril membrane interaction and cellular uptake in primary neurons; 4) the Nt17 domain and conformation regulate the nuclear/cytoplasmic distribution and toxicity of Httex1 in primary neurons. The figure was created with Biorender and https://www.vectorstock.com/royalty-free-vector/icon-on-and-off-toggle-switch-button-white-design-vector-30148026 C_FIG

neuroscience↗

bioRxiv2019

Unraveling the Complexity of Amyloid Polymorphism Using Gold Nanoparticles and Cryo-EM

The misfolding and self-assembly of proteins into {beta}-sheet-rich amyloid fibrils of various structures and morphologies is a hallmark of several neurodegenerative and systemic diseases. Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicity and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the use of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) to label the edges of synthetic, recombinant and native amyloid fibrils to assess amyloid morphological polymorphism using cryogenic transmission electron microscopy (cryo-EM). The fibrils studied were derived from amyloid proteins involved in disorders of the central nervous system (amyloid-{beta}, tau, -synuclein) and in systemic amyloidosis (a fragment of an immunoglobulin {lambda} light chain). The labeling efficiency enabled imaging and characterization of amyloid fibrils of different morphologies under hydrated conditions using cryo-EM. These NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including TEM with use of the negative stain or cryo-EM imaging. We also demonstrate the use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of an Alzheimers disease patient, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain (AL) amyloidosis. Analysis of the cryo-EM images of amyloids decorated with NPs shows exceptional homogeneity across the fibrils derived from human tissue in comparison to fibrils aggregated in vitro. The use of these NPs enabled us to gain novel insight into the structural features that distinguish amyloid fibrils formed in vivo from those formed in cell-free in vitro systems. Our findings demonstrate that these NPs represent a powerful tool for rapid imaging and profiling of amyloid morphological polymorphism in different types of samples, including those derived from complex biological aggregates found in human tissue and animal models of amyloid diseases. These advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM but also pave the way to elucidate the structural basis of amyloid strains and toxicity and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

neuroscience↗

bioRxiv2019

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72

Loss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111-phosphorylated Rab1B that does not demonstrate any major changes suggesting that the phosphorylated SF3 motif may disrupt effector-Switch II interactions. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A suggesting that small molecule activators of PINK1 may have therapeutic potential in patients harbouring LRRK2 mutations.

biochemistry↗

bioRxiv2019

Phosphorylation of Ser111 in Rab8a modulates Rabin8 dependent activation by perturbation of side chain interaction networks

GTPases are key-players in cellular signaling processes. Phosphorylation of Rab proteins, which belong to the Ras superfamily of small GTPases regulating intracellular transport, has recently been implicated in the pathogenesis of Parkinson Disease (PD). For Rab8a, it was shown that serine 111 phosphorylation (pS111) is dependent on the protein kinase PINK1, and that mimicking the phosphorylation at S111 by a serine/glutamate substitution (S111E) impaired Rab8a activation by its cognate nucleotide exchange factor (GEF) Rabin8. Here, we performed comparative Molecular Dynamics and free energy simulations on Rab8a and Rab8a:Rabin8 complexes to elucidate the molecular details on how pS111 and S111E may influence the interaction with Rabin8. The simulations indicate that S111E and pS111 establish an intramolecular interaction with arginine 79 (R79). In the complex, this interaction persists, and therefore perturbs a favorable intermolecular salt-bridge contact between R79 in Rab8a and the acidic aspartate 187 (D187) in Rabin8. Binding free analysis reveals that S111E and pS111, as well as the mutation R79A, in Rab8a drastically reduce the binding affinity to Rabin8. Combining the R79A mutation with S111E or pS111, respectively, nearly diminishes Rab8a-Rabin8 binding. In vitro experiments confirm our computational results showing that the nucleotide exchange rates of the respective Rab8a mutants are decreased by >80% in the presence of Rabin8 compared to wild type. In addition to specific insights into how S111 phosphorylation of Rab8a can influence GEF-mediated activation, the simulations demonstrate how side chain modifications in general can allosterically influence the network of surface side chain interactions between binding partners.

biophysics↗