Structural transitions in orb2 prion-like domain relevant for functional aggregation in memory consolidation

Grant BBM_TRA_0203 PD/BD/148028/2019 UIDB/04378/2020The recent structural elucidation of ex vivo Drosophila Orb2 fibrils revealed a novel amyloid formed by interdigitated Gln and His residue side chains belonging to the prion-like domain. However, atomic-level details on the conformational transitions associated with memory consolidation remain unknown. Here, we have characterized the nascent conformation and dynamics of the prion-like domain (PLD) of Orb2A using a nonconventional liquid-state NMR spectroscopy strategy based on 13C detection to afford an essentially complete set of 13Ca, 13Cb, 1Ha, and backbone 13CO and 15N assignments. At pH 4, where His residues are protonated, the PLD is disordered and flexible, except for a partially populated a-helix spanning residues 55–60, and binds RNA oligos, but not divalent cations. At pH 7, in contrast, His residues are predominantly neutral, and the Q/H segments adopt minor populations of helical structure, show decreased mobility and start to self-associate. At pH 7, the His residues do not bind RNA or Ca21, but do bind Zn21, which promotes further association. These findings represent a remarkable case of structural plasticity, based on which an updated model for Orb2A functional amyloidogenesis is suggested.publishersversionpublishe

Implications for phase separation

Funding Information: The authors would also like to acknowledge Prof. Dr. Jaime Mota, Dra. Irina Franco for the technical assistance with the microscopic experiments, Philip O'Toole for the aid in protein production and Dr. Aldino Viegas and Dr. David Pantoja-Uceda for the support and valuable discussions regarding NMR spectroscopy. This work was supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) for funding UCIBIO project (UIDP/04378/2020 and UIDB/04378/2020) and Associate Laboratory Institute for Health and Bioeconomy – i4HB Project (LA/P/0140/2020). The authors also thank FCT-Portugal for the PhD grant attributed to SF (PD/BD/148028/2019) under the PTNMRPhD Program. JO is a recipient of a Leonardo Grant from the Spanish BBVA Foundation (BBM_TRA_0203) and a Ramón y Cajal Grant (RYC2018-026042-I funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future.”) JO and DVL are supported by the Spanish Grants PID-2019-109276RA-I00 and PID-2019-109306RB-I00, respectively, both funded by MCIN/AEI/10.13039/501100011033. The NMR spectrometers are part of the National NMR Facility supported by FCT-Portugal (ROTEIRO/0031/2013–PINFRA/22161/2016, co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). The 800 MHz spectrometer present in the “Manuel Rico” NMR laboratory (LMR-CSIC) is a node of the Spanish Large-Scale National Facility (ICTS R-LRB-MR). Publisher Copyright: © 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.The mediation of liquid–liquid phase separation (LLPS) for fused in sarcoma (FUS) protein is generally attributed to the low-complexity, disordered domains and is enhanced at low temperature. The role of FUS folded domains on the LLPS process remains relatively unknown since most studies are mainly based on fragmented FUS domains. Here, we investigate the effect of metabolites on full-length (FL) FUS LLPS using turbidity assays and differential interference contrast (DIC) microscopy, and explore the behavior of the folded domains by nuclear magnetic resonance (NMR) spectroscopy. FL FUS LLPS is maximal at low concentrations of glucose and glutamate, moderate concentrations of NaCl, Zn2+, and Ca2+ and at the isoelectric pH. The FUS RNA recognition motif (RRM) and zinc-finger (ZnF) domains are found to undergo cold denaturation above 0°C at a temperature that is determined by the conformational stability of the ZnF domain. Cold unfolding exposes buried nonpolar residues that can participate in LLPS-promoting hydrophobic interactions. Therefore, these findings constitute the first evidence that FUS globular domains may have an active role in LLPS under cold stress conditions and in the assembly of stress granules, providing further insight into the environmental regulation of LLPS.publishersversionpublishe

NMR characterization of angiogenin variants and tRNAAla products impacting aberrant protein oligomerization

Protein oligomerization is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Human Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads to abnormal protein oligomerization, resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson's disease. While a score of disease linked h-ANG mutants has been studied by X-ray diffraction, some elude crystallization. There is also a debate regarding the structure that RNA fragments adopt after cleavage by h-ANG. Here, to better understand the beginning of the process that leads to aberrant protein oligomerization, the solution secondary structure and residue-level dynamics of WT h-ANG and two mutants i.e., H13A and R121C, are characterized by multidimensional heteronuclear NMR spectroscopy under near-physiological conditions. All three variants are found to adopt well folded and highly rigid structures in the solution, although the elements of secondary structure are somewhat shorter than those observed in crystallography studies. R121C alters the environment of nearby residues only. By contrast, the mutation H13A affects local residues as well as nearby active site residues K40 and H114. The conformation characterization by CD and 1D 1H NMR spectroscopies of tRNAAla before and after h-ANG cleavage reveals a retention of the duplex structure and little or no G-quadruplex formation

Dimerization of human angiogenin and of variants involved in neurodegenerative diseases

Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies

Molecular mechanism for the synchronized electrostatic coacervation and co-aggregation of a-synuclein and tau

Amyloid aggregation of α-synuclein (αS) is the hallmark of Parkinson’s disease and other synucleinopathies. Recently, Tau protein, generally associated with Alzheimer’s disease, has been linked to αS pathology and observed to co- localize in αS-rich disease inclusions, although the molecular mechanisms for the co-aggregation of both proteins remain elusive. We report here that αS phase-separates into liquid condensates by electrostatic complex coacerva- tion with positively charged polypeptides such as Tau. Condensates undergo either fast gelation or coalescence followed by slow amyloid aggregation depending on the affinity of αS for the poly-cation and the rate of valence exhaustion of the condensate network. By combining a set of advanced bio- physical techniques, we have been able to characterize αS/Tau liquid-liquid phase separation and identified key factors that lead to the formation of hetero-aggregates containing both proteins in the interior of the liquid protein condensates

Metamorphism in TDP-43 prion-like domain determines chaperone recognition

The RNA binding protein TDP-43 forms cytoplasmic inclusions via its C-terminal prion-like domain in several neurodegenerative diseases. Aberrant TDP-43 aggregation arises upon phase de-mixing and transitions from liquid to solid states, following still unknown structural conversions which are primed by oxidative stress and chaperone inhibition. Despite the well-established protective roles for molecular chaperones against protein aggregation pathologies, knowledge on the determinants of chaperone recognition in disease-related prions is scarce. Here we show that chaperones and cochaperones primarily recognize the structured elements in TDP-43´s prionlike domain. Significantly, while HSP70 and HSP90 chaperones promote TDP43 phase separation, co-chaperones from the three classes of the large human HSP40 family (namely DNAJA2, DNAJB1, DNAJB4 and DNAJC7) show strikingly different effects on TDP-43 de-mixing. Dismantling of the second helical element in TDP-43 prion-like domain by methionine sulfoxidation impacts phase separation and amyloid formation, abrogates chaperone recognition and alters phosphorylation by casein kinase-1δ. Our results show that metamorphism in the post-translationally modified TDP-43 prion-like domain encodes determinants that command mechanisms with major relevance in diseas

Common Features at the Start of the Neurodegeneration Cascade

A single-molecule study reveals that neurotoxic proteins share common structural features that may trigger neurodegeneration, thus identifying new targets for therapy and diagnosis

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

The Preparation and Characterization of Reduced Methylated Ribonuclease T1

Program year: 1989/1990Digitized from print original stored in HDRTo study the contribution of disulfide bonds to protein stablity, a derivative of ribonuclease T1 (RNase T1) with both cystine residues reduced and methylated has been prepared using an improved procedure adapted from that of Heinrikson Heinrikson, R.L. (1971) J. Biol. Chern. 246, 4090. Characterization using urea and thermal denaturation show that the reduced, methylated ribonuclease T1 is about 8 kcal/mol less stable than RNase T1, but is .5 and 1 kcal/mol more stable than reduced carboxyamidomethylated and reduced carboxymethylated RNase T1, respectively. It is suggested that this difference in stability arises from the increase in conformational entropy of the unfolded state and the size and hydrophobicity of the blocking group