bims-proned 2024-06-09 papers

bims-proned

Biomed News

on Proteostasis in neurodegeneration

Issue of 2024‒06‒09
nine papers selected by
Verena Kohler, Umeå University

Elucidating the mechanisms of α-Synuclein-lipid interactions using site-directed mutagenesis.
Amyloidogenic regions in beta-strands II and III modulate the aggregation and toxicity of SOD1 in living cells.
Liquid-liquid phase separation induced by crowding condition affects amyloid-β aggregation mechanism.
Cryo-EM structures of pathogenic fibrils and their impact on neurodegenerative disease research.
Alpha-synuclein aggregates are phosphatase resistant.
Distinctive contribution of two additional residues in protein aggregation of Aβ42 and Aβ40 isoforms.
A Potent Sybody Selectively Inhibits α-Synuclein Amyloid Formation by Binding to the P1 Region.
VCP regulates early tau seed amplification via specific cofactors.
Kinetic models reveal the interplay of protein production and aggregation.

Neurobiol Dis. 2024 Jun 03. pii: S0969-9961(24)00152-9. [Epub ahead of print]198 106553

Elucidating the mechanisms of α-Synuclein-lipid interactions using site-directed mutagenesis.

Abid Ali, Aidan P Holman, Axell Rodriguez, Luke Osborne, Dmitry Kurouski.

  α-Synuclein (α-syn) is a small protein that is involved in cell vesicle trafficking in neuronal synapses. A progressive aggregation of this protein is the expected molecular cause of Parkinson's disease, a disease that affects millions of people around the world. A growing body of evidence indicates that phospholipids can strongly accelerate α-syn aggregation and alter the toxicity of α-syn oligomers and fibrils formed in the presence of lipid vesicles. This effect is attributed to the presence of high copies of lysines in the N-terminus of the protein. In this study, we performed site-directed mutagenesis and replaced one out of two lysines at each of the five sites located in the α-syn N-terminus. Using several biophysical and cellular approaches, we investigated the extent to which six negatively charged fatty acids (FAs) could alter the aggregation properties of K10A, K23A, K32A, K43A, and K58A α-syn. We found that FAs uniquely modified the aggregation properties of K43A, K58A, and WT α-syn, as well as changed morphology of amyloid fibrils formed by these mutants. At the same time, FAs failed to cause substantial changes in the aggregation rates of K10A, K23A, and K32A α-syn, as well as alter the morphology and toxicity of the corresponding amyloid fibrils. Based on these results, we can conclude that K10, K23, and K32 amino acid residues play a critical role in protein-lipid interactions since their replacement on non-polar alanines strongly suppressed α-syn-lipid interactions.

Keywords:  AFM-IR; Fatty acids; Toxicity; a-Synuclein

DOI:  https://doi.org/10.1016/j.nbd.2024.106553
Open Biol. 2024 Jun;14(6): 230418

Amyloidogenic regions in beta-strands II and III modulate the aggregation and toxicity of SOD1 in living cells.

Luke McAlary, Jeremy R Nan, Clay Shyu, Mine Sher, Steven S Plotkin, Neil R Cashman.

  Mutations in the protein superoxide dismutase-1 (SOD1) promote its misfolding and aggregation, ultimately causing familial forms of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Currently, over 220 (mostly missense) ALS-causing mutations in the SOD1 protein have been identified, indicating that common structural features are responsible for aggregation and toxicity. Using in silico tools, we predicted amyloidogenic regions in the ALS-associated SOD1-G85R mutant, finding seven regions throughout the structure. Introduction of proline residues into β-strands II (I18P) or III (I35P) reduced the aggregation propensity and toxicity of SOD1-G85R in cells, significantly more so than proline mutations in other amyloidogenic regions. The I18P and I35P mutations also reduced the capability of SOD1-G85R to template onto previously formed non-proline mutant SOD1 aggregates as measured by fluorescence recovery after photobleaching. Finally, we found that, while the I18P and I35P mutants are less structurally stable than SOD1-G85R, the proline mutants are less aggregation-prone during proteasome inhibition, and less toxic to cells overall. Our research highlights the importance of a previously underappreciated SOD1 amyloidogenic region in β-strand II (15QGIINF20) to the aggregation and toxicity of SOD1 in ALS mutants, and suggests that β-strands II and III may be good targets for the development of SOD1-associated ALS therapies.

Keywords:  SOD1; amyloid; amyotrophic lateral sclerosis; protein aggregation; protein homeostasis

DOI:  https://doi.org/10.1098/rsob.230418
Soft Matter. 2024 Jun 07.

Liquid-liquid phase separation induced by crowding condition affects amyloid-β aggregation mechanism.

Ryuki Kobayashi, Hideki Nabika.

Liquid-liquid phase separation (LLPS) is common in the aggregation of proteins associated with neurodegenerative diseases. Many efforts have been made to reproduce crowded conditions with artificial polymeric materials to understand the effect of LLPS in physiological conditions with significantly highly concentrated proteins, such as intrinsically disordered proteins. Although the possibility that LLPS is involved in intracellular amyloid-β (Aβ) aggregation, a protein related to the pathogenesis of Alzheimer's disease, has been investigated, the relationship between LLPS and the aggregation of Aβ is poorly characterized. Thus, in this study, we mimicked the intracellular crowding environment using polyethylene glycol and dextran, used commonly as model polymers, to examine the relationship of Aβ with LLPS and aggregation dynamics in vitro. We confirmed that Aβ undergoes LLPS under specific polymer coexistence conditions. Moreover, the addition of different electrolytes modulated LLPS and fibril formation. These results suggest that hydrophobic and electrostatic interactions are the driving forces for the LLPS of Aβ. Similar to the role of the liposome interface, the interface of droplets induced by LLPS functioned as the site for heterogeneous nucleation. These findings offer valuable insights into the complex mechanisms of Aβ aggregation in vivo and may be useful in establishing therapeutic methods for Alzheimer's disease.

DOI: https://doi.org/10.1039/d4sm00470a
Neuron. 2024 May 29. pii: S0896-6273(24)00359-3. [Epub ahead of print]

Cryo-EM structures of pathogenic fibrils and their impact on neurodegenerative disease research.

Tiffany W Todd, Naeyma N Islam, Casey N Cook, Thomas R Caulfield, Leonard Petrucelli.

  Neurodegenerative diseases are commonly associated with the formation of aberrant protein aggregates within the brain, and ultrastructural analyses have revealed that the proteins within these inclusions often assemble into amyloid filaments. Cryoelectron microscopy (cryo-EM) has emerged as an effective method for determining the near-atomic structure of these disease-associated filamentous proteins, and the resulting structures have revolutionized the way we think about aberrant protein aggregation and propagation during disease progression. These structures have also revealed that individual fibril conformations may dictate different disease conditions, and this newfound knowledge has improved disease modeling in the lab and advanced the ongoing pursuit of clinical tools capable of distinguishing and targeting different pathogenic entities within living patients. In this review, we summarize some of the recently developed cryo-EM structures of ex vivo α-synuclein, tau, β-amyloid (Aβ), TAR DNA-binding protein 43 (TDP-43), and transmembrane protein 106B (TMEM106B) fibrils and discuss how these structures are being leveraged toward mechanistic research and therapeutic development.

Keywords:  Aβ; TDP-43; TMEM106B; amyloid; cryo-EM; tau; α-synuclein

DOI:  https://doi.org/10.1016/j.neuron.2024.05.012
Acta Neuropathol Commun. 2024 May 31. 12(1): 84

Alpha-synuclein aggregates are phosphatase resistant.

S G Choi, T Tittle, D Garcia-Prada, J H Kordower, R Melki, B A Killinger.

  Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.

Keywords:  Parkinson’s disease pathogenesis; Post-translational modifications; Postmortem interval; Protein aggregation

DOI:  https://doi.org/10.1186/s40478-024-01785-0
BMB Rep. 2024 Jun 05. pii: 6217. [Epub ahead of print]

Distinctive contribution of two additional residues in protein aggregation of Aβ42 and Aβ40 isoforms.

Dongjoon Im, Tae Su Choi.

Amyloid-β (Aβ) is one of the amyloidogenic intrinsically disordered proteins (IDPs) that self-assemble to protein aggregates, incurring cell malfunction and cytotoxicity. While Aβ has been known to regulate multiple physiological functions, such as enhancing synaptic functions, aiding in the recovery of the blood-brain barrier/brain injury, and exhibiting tumor suppression/antimicrobial activities, the hydrophobicity of the primary structure promotes pathological aggregations that are closely associated with the onset of Alzheimer's Disease (AD). Aβ proteins consist of multiple isoforms with 37-43 amino acid residues that are produced by the cleavage of amyloid-β precursor protein (APP). The hydrolytic products of APP are secreted to the extracellular regions of neuronal cells. Aβ 1-42 (Aβ42) and Aβ 1-40 (Aβ40) are dominant isoforms whose significance in AD pathogenesis has been highlighted in numerous studies to understand the molecular mechanism and develop AD diagnosis and therapeutic strategies. In this review, we focus on the differences between Aβ42 and Aβ40 in the molecular mechanism of amyloid aggregations mediated by the two additional residues (Ile41 and Ala42) of Aβ42. The current comprehension of Aβ42 and Aβ40 in AD progression is outlined, together with the structural features of Aβ42/Aβ40 amyloid fibrils, and the aggregation mechanisms of Aβ42/Aβ40. Furthermore, the impact of the heterogeneous distribution of Aβ isoforms during amyloid aggregations is discussed in the system mimicking the coexistence of Aβ42 and Aβ40 in human cerebrospinal fluid (CSF) and plasma.
J Med Chem. 2024 Jun 06.

A Potent Sybody Selectively Inhibits α-Synuclein Amyloid Formation by Binding to the P1 Region.

Dimitra Gialama, Devkee M Vadukul, Rebecca J Thrush, Sheena E Radford, Francesco A Aprile.

Increasing research efforts focus on exploiting antibodies to inhibit the amyloid formation of neurodegenerative proteins. Nevertheless, it is challenging to discover antibodies that inhibit this process in a specific manner. Using ribosome display, we screened for synthetic single-domain antibodies, i.e., sybodies, of the P1 region of α-synuclein (residues 36-42), a protein that forms amyloid in Parkinson's disease and multiple-system atrophy. Hits were assessed for direct binding to a P1 peptide and the inhibition of amyloid formation. We discovered a sybody, named αSP1, that inhibits amyloid formation of α-synuclein at substoichiometric concentrations in a specific manner, even within highly crowded heterogeneous mixtures. Fluorescence resonance energy transfer-based binding assays and seeding experiments with and without αSP1 further demonstrate the importance of the P1 region for both primary and secondary nucleation mechanisms of amyloid assembly.

DOI: https://doi.org/10.1021/acs.jmedchem.3c02408
Res Sq. 2024 May 22. pii: rs.3.rs-4307848. [Epub ahead of print]

VCP regulates early tau seed amplification via specific cofactors.

Sushobhna Batra, Jaime Iii Vaquer-Alicea, Clarissa Valdez, Skyler P Taylor, Victor A Manon, Anthony R Vega, Omar M Kashmer, Sourav Kolay, Andrew Lemoff, Nigel J Cairns, Charles L White, Marc I Diamond.

Background Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. In vitro seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu happens within hours, implicating a machinery with unknown players that controls this process in the acute phase. Methods We used proximity labeling to identify factors that control seed amplification within 5h of seed exposure. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity 5h after seeded intracellular tau aggregation. Valosin containing protein (VCP/p97) was the top hit. VCP harbors dominant mutations that underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We used immortalized cells and human neurons to study the effects of VCP on tau seeding. We exposed cells to fibrils or brain homogenates in cell culture media and measured effects on uptake and induction of intracellular tau aggregation following various genetic and chemical manipulations of VCP. Results VCP knockdown reduced tau seeding. Chemical inhibitors had opposing effects on aggregation in HEK293T tau biosensor cells and human neurons alike: ML-240 increased seeding efficiency, whereas NMS-873 decreased it. The inhibitors were effective only when administered within 8h of seed exposure, indicating a role for VCP early in seed processing. We screened 30 VCP co-factors in HEK293T biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. By contrast, reduction of FAF2 increased tau seeding. Conclusions Divergent effects on tau seeding of chemical inhibitors and cofactor reduction indicate that VCP regulates this process. This is consistent with a dedicated cytoplasmic processing complex based on VCP that directs seeds acutely towards degradation vs. amplification.

DOI: https://doi.org/10.21203/rs.3.rs-4307848/v1
Chem Sci. 2024 Jun 05. 15(22): 8430-8442

Kinetic models reveal the interplay of protein production and aggregation.

Jiapeng Wei, Georg Meisl, Alexander Dear, Matthijs Oosterhuis, Ronald Melki, Cecilia Emanuelsson, Sara Linse, Tuomas P J Knowles.

Protein aggregation is a key process in the development of many neurodegenerative disorders, including dementias such as Alzheimer's disease. Significant progress has been made in understanding the molecular mechanisms of aggregate formation in pure buffer systems, much of which was enabled by the development of integrated rate laws that allowed for mechanistic analysis of aggregation kinetics. However, in order to translate these findings into disease-relevant conclusions and to make predictions about the effect of potential alterations to the aggregation reactions by the addition of putative inhibitors, the current models need to be extended to account for the altered situation encountered in living systems. In particular, in vivo, the total protein concentrations typically do not remain constant and aggregation-prone monomers are constantly being produced but also degraded by cells. Here, we build a theoretical model that explicitly takes into account monomer production, derive integrated rate laws and discuss the resulting scaling laws and limiting behaviours. We demonstrate that our models are suited for the aggregation-prone Huntington's disease-associated peptide HttQ45 utilizing a system for continuous in situ monomer production and the aggregation of the tumour suppressor protein P53. The aggregation-prone HttQ45 monomer was produced through enzymatic cleavage of a larger construct in which a fused protein domain served as an internal inhibitor. For P53, only the unfolded monomers form aggregates, making the unfolding a rate-limiting step which constitutes a source of aggregation-prone monomers. The new model opens up possibilities for a quantitative description of aggregation in living systems, allowing for example the modelling of inhibitors of aggregation in a dynamic environment of continuous protein synthesis.

DOI: https://doi.org/10.1039/d4sc00088a