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1. HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells

   https://doi.org/10.1126/science.abb4309  

   Yu, Haiyang; Lu, Shan; Gasior, Kelsey; Singh, Digvijay; Vazquez-Sanchez, Sonia; Tapia, Olga; Toprani, Divek; Beccari, Melinda S.; Yates, III, John R.; Da Cruz, Sandrine; et al (December 2020, Science)

   The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding–deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named “anisosomes”, have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)–dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43–containing anisosomes. These structures converted to aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel or solid phase. 

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2. A cellular model of TDP ‐43 induces phosphorylated TDP ‐43 aggregation with distinct changes in solubility and autophagy dysregulation

   https://doi.org/10.1111/febs.17413  

   Dopler, Matthew B; Abeer, Muhammad I; Arezoumandan, Sanaz; Cox, Keyshawn; Petersen, Tyler L; Daniel, Esther H; Cannon, Carlton L; Bautista, Angelica; Blancher, Kennedy D; Bland, Alysia M; et al (January 2025, The FEBS Journal)

   Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that affects neurons in the brain and spinal cord, causing loss of muscle control, and eventually leads to death. Phosphorylated transactive response DNA binding protein‐43 (TDP‐43) is the major pathological protein in both sporadic and familial ALS, forming cytoplasmic aggregates in over 95% of cases. Of the 10–15% of ALS cases that are familial, mutations in TDP‐43 represent about 5% of those with a family history. We have developed anin vitrooverexpression model by introducing three familial ALS mutations (A315T, M337V, and S379P) in the TDP‐43 (TARDBP) gene which we define as 3X‐TDP‐43. This overexpression model TDP‐43 shows deficits in autophagy flux and colocalization of TDP‐43 with stress granules. We also observe a progressive shift of TDP‐43 to the cytoplasm in this model. This overexpression model shows a reduction in solubility of phosphorylated TDP‐43 from RIPA to urea soluble. Four glycolytic enzymes, phosphoglycerate kinase one (PGK1), aldolase A (ALDOA), enolase 1 (ENO1), and pyruvate dehydrogenase kinase 1 (PDK1) show significant time‐dependent decreases in 3X‐TDP‐43 expressing cells. Shotgun proteomic analysis shows global changes in the importin subunit alpha‐1 (KPNA2), heat shock 70 kDa protein 1A (HSPA1A), and protein disulfide‐isomerase A3 (PDIA3) expression levels and coimmunoprecipitation reveals that these proteins complex with TDP‐43. Overall, these results suggest that the 3X‐TDP‐43 model may provide new insights into pathophysiology and an avenue for drug screeningin vitrofor those suffering from ALS and related TDP‐43 proteinopathies. 

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3. Role of the Cell Asymmetry Apparatus and Ribosome-Associated Chaperones in the Destabilization of a Saccharomyces cerevisiae Prion by Heat Shock

   https://doi.org/10.1534/genetics.119.302237  

   Howie, Rebecca L.; Jay-Garcia, Lina Manuela; Kiktev, Denis A.; Faber, Quincy L.; Murphy, Margaret; Rees, Katherine A.; Sachwani, Numera; Chernoff, Yury O. (July 2019, Genetics)

   Self-perpetuating transmissible protein aggregates, termed prions, are implicated in mammalian diseases and control phenotypically detectable traits in Saccharomyces cerevisiae . Yeast stress-inducible chaperone proteins, including Hsp104 and Hsp70-Ssa that counteract cytotoxic protein aggregation, also control prion propagation. Stress-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Short-term mild heat stress destabilizes [ PSI + ], a prion isoform of the yeast translation termination factor Sup35 . This destabilization is linked to the induction of the Hsp104 chaperone. Here, we show that the region of Hsp104 known to be required for curing by artificially overproduced Hsp104 is also required for heat-shock-mediated [ PSI + ] destabilization. Moreover, deletion of the SIR2 gene, coding for a deacetylase crucial for asymmetric segregation of heat-damaged proteins, also counteracts heat-shock-mediated destabilization of [ PSI + ], and Sup35 aggregates are colocalized with aggregates of heat-damaged proteins marked by Hsp104 -GFP. These results support the role of asymmetric segregation in prion destabilization. Finally, we show that depletion of the heat-shock noninducible ribosome-associated chaperone Hsp70-Ssb decreases heat-shock-mediated destabilization of [ PSI + ], while disruption of a cochaperone complex mediating the binding of Hsp70-Ssb to the ribosome increases prion loss. Our data indicate that Hsp70-Ssb relocates from the ribosome to the cytosol during heat stress. Cytosolic Hsp70-Ssb has been shown to antagonize the function of Hsp70-Ssa in prion propagation, which explains the Hsp70-Ssb effect on prion destabilization by heat shock. This result uncovers the stress-related role of a stress noninducible chaperone. 

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4. Phe-Gly motifs drive fibrillization of TDP-43’s prion-like domain condensates

   https://doi.org/10.1371/journal.pbio.3001198  

   Pantoja-Uceda, David; Stuani, Cristiana; Laurents, Douglas V.; McDermott, Ann E.; Buratti, Emanuele; Mompeán, Miguel (April 2021, PLOS Biology)

   Walters, Kylie J. (Ed.)

   Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms. 

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5. Zinc Alters the Supramolecular Organization of Nucleic Acid Complexes with Full-Length TIA1

   Yizhuo Yang, Keith J. (January 2023, bioRxiv)

   na (Ed.)

   T-Cell Intracellular Antigen-1 (TIA1) is a 43 kDa multi-domain RNA-binding protein involved in stress granule formation during eukaryotic stress response, and has been implicated in neurodegenerative diseases including Welander distal myopathy and amyotrophic lateral sclerosis. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids and a C-terminal Q/N-rich prion-related domain (PRD) which has been variously described as intrinsically disordered or prion inducing and is believed to play a role in promoting liquid-liquid phase separation connected with the assembly of stress granule formation. Motivated by the fact that our prior work shows RRMs 2 and 3 are well-ordered in an oligomeric full-length form, while RRM1 and the PRD appear to phase separate, the present work addresses whether the oligomeric form is functional and competent for binding, and probes the consequences of nucleic acid binding for oligomerization and protein conformation change. New SSNMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at the RRM2 domain, but also weakly at the RRM3 domain, and Zn2+ binds primarily to RRM3. Binding of Zn2+ and DNA was reversible for the full-length wild type oligomeric form, and did not lead to formation of amyloid fibrils, despite the presence of the C-terminal prion-related domain. While TIA1:DNA complexes appear as long “daisy chained” structures, the addition of Zn2+ caused the structures to collapse. We surmise that this points to a regulatory role for Zn2+. By occupying various “half” binding sites on RRM3 Zn2+ may shift the nucleic acid binding off RRM3 and onto RRM2. More importantly, the use of different half sites on different monomers may introduce a mesh of crosslinks in the supramolecular complex rendering it compact and markedly reducing the access to the nucleic acids (including transcripts) from solution. 

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