Biology subjects

Siemer, A. B.

Publications and source records attributed to Siemer, A. B..

4 recordsLinked to original sources

bioRxiv2024

Phosphorylation of α-synuclein fibrils at S129 changes DNAJB1 binding as probed by solid-state NMR

Amyloid fibrils composed of the protein -synuclein (aSyn) are implicated in the pathogenesis of synucleinopathies. These pathological fibrils, characterized by their rigid amyloid cores, also feature flexible intrinsically disordered regions (IDRs) that interact with various cellular components. Due to their solvent exposure and flexibility, the N- and C-termini, IDRs of aSyn fibrils, have been used as targets for immunotherapies and serve as binding sites for many chaperones. Chaperones are known to play a vital role in preventing and reversing amyloid formation in neurodegenerative diseases, but how they recognize misfolded proteins is often poorly understood. DNAJB1 is a co-chaperone that is part of a larger complex formed with Hsp70 and Apg2, which collectively disaggregate amyloid fibrils such as those formed by aSyn, tau, or huntingtin. Although the entire chaperone and co-chaperone complex are required for aggregate disassembly, DNAJB1 directly recognizes these amyloid fibrils. More specifically, DNAJB1 preferentially binds to the C-terminus of wild-type aSyn. However, over 90% of aSyn fibrils in Lewy bodies are phosphorylated at S129. Here, we determine the effect of S129 phosphorylation on DNAJB1-aSyn fibril binding using biochemical assays and solid-state NMR. We show that DNAJB1 preferentially binds aSyn pS129 fibrils and that the aSyn binding site of pS129 and wild-type fibrils is different. These results suggest that pS129 might influence chaperone-mediated degradation efficiency.

biophysics↗

bioRxiv2022

Solid-state NMR of paired helical filaments formed by the core tau fragment tau(297-391)

Aggregation of the tau protein into fibrillar cross-{beta} aggregates is a hallmark of Alzheimers diseases (AD) and many other neurodegenerative tauopathies. Recently, several core structures of patient-derived tau paired helical filaments (PHFs) have been solved revealing a structural variability that often correlates with a specific tauopathy. To further characterize the dynamics of these fibril cores, to screen for strain-specific small molecules as potential biomarkers and therapeutics, and to develop strain-specific antibodies, recombinant in-vitro models of tau filaments are needed. We recently showed that a 95-residue fragment of tau (from residue 297 to 391), termed dGAE, forms filaments in vitro in the absence of polyanionic co-factors often used for in vitro aggregation of full length tau. Tau(297-391) was identified as the proteolytic resistant core of tau PHFs and overlaps with the structures characterized by cryo-electron microscopy in ex-vivo PHFs, making it a promising model for the study of AD tau filaments in vitro. In the present study, we used solid-state NMR to characterize tau(297-391) filaments and show that such filaments assembled under non-reducing conditions are more dynamic and less ordered than those made in the presence of the reducing agent, DTT. We further report the resonance assignment of tau(297-392)+DTT filaments and compare it to existing core structures of tau.

biophysics↗

bioRxiv2021

Droplet and fibril formation of the functional amyloid Orb2

The functional amyloid Orb2 belongs to the cytoplasmic polyadenylation element binding (CPEB) protein family and plays an important role in long-term memory formation in Drosophila. The Orb2 domain structure combines RNA recognition motifs with low complexity sequences similar to many RNA binding proteins shown to form protein droplets via liquid-liquid phase separation (LLPS) in vivo and in vitro. This similarity suggests that Orb2 might also undergo LLPS. However, cellular Orb2 puncta have very little internal protein mobility and Orb2 forms fibrils in Drosophila brains that are functionally active indicating that LLPS might not play a role for Orb2. In the present work, we reconcile these two views on Orb2 droplet formation. We show that soluble Orb2 can indeed phase separate into protein droplets. However, these droplets have either no or only an extremely short-lived liquid phase and appear maturated right after formation. For Orb2 fragments that lack the C-terminal RNA binding domain (RBD), droplet formation is a prerequisite for fibril formation of an otherwise stable monomeric Orb2 solution. Solid-state NMR shows that these fibrils have additional well ordered static domains beside the Gln/His-rich fibril core. Further, we find that full-length Orb2B, which is by far the major component of Orb2 fibrils in vivo, does not transition into cross-{beta} fibrils but remains in the droplet phase. Together, our data suggest that phase separation might play a role in initiating the formation of functional Orb2 fibrils.

biophysics↗

bioRxiv2020

Structural model of the proline-rich domain of huntingtin exon-1 fibrils

Huntingtons disease (HD) is a heritable neurodegenerative disease that is caused by a CAG expansion in the first exon of the huntingtin gene. This expansion results in an elongated polyglutamine (polyQ) domain that increases the propensity of huntingtin exon-1 (HTTex1) to form cross-{beta} fibrils. While the polyQ domain is important for fibril formation, the dynamic, C-terminal proline-rich domain (PRD) of HTTex1 makes up a large fraction of the fibril surface. Because potential fibril toxicity has to be mediated by interactions of the fibril surface with its cellular environment, we wanted to model the conformational space adopted by the PRD. We ran 800 ns long molecular dynamics (MD) simulations of the PRD using an explicit water model optimized for intrinsically disordered proteins. These simulations accurately predicted our previous solid-state NMR data and newly acquired EPR DEER distances, lending confidence in their accuracy. The simulations show that the PRD generally forms an imperfect polyproline II (PPII) helical conformation. The two polyproline (polyP) regions within the PRD stay in a PPII helix for most of the simulation, whereas occasional kinks in the proline rich linker region cause an overall bend in the PRD structure. The dihedral angles of the glycine at the end of the second polyP region are very variable, effectively decoupling the highly dynamic 12 C-terminal residues from the rest of the PRD. Statement of SignificanceHD is caused by a polyQ expansion in the exon-1 of huntingtin, which results in the formation of fibrillar huntingtin aggregates. Although the polyQ domain is the site of the disease-causing mutation, the PRD domain of HTTex1 is important for fibril toxicity and contains many epitopes of fibril-specific HTTex1 antibodies. Here, we present a structural and dynamic model of the highly dynamic PRD domain using a combination of EPR, solid-state NMR, and MD simulations. This model paves the way for studying known HTTex1 fibril specific binders and designing new ones.

biophysics↗