Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a salient feature of amyotrophic lateral sclerosis (ALS), a debilitating neurodegenerative disorder affecting over 200 000 people worldwide. The protein undergoes both functional and pathogenic aggregation; the latter is irreversible and hypothesized to produce soluble oligomers that are toxic to neurons in addition to inclusions made of stable fibrous deposits. Despite progress made toward identifying disease-related proteins, the underlying pathogenic mechanism associated with these toxic oligomers remains elusive. Utilizing a multimodal approach that combines several measurement techniques (circular dichroism (CD), thioflavin T spectroscopy (ThT), Fourier transform infrared spectroscopy (FTIR)) and high spatial resolution imaging tools (electron microscopy (EM) and atomic force microscopy (AFM)), with soft ion mobility mass spectrometry (IM-MS) and atomistic molecular dynamics (MD) simulations, we explore the oligomerization mechanisms, structures, and assembly pathways of TDP-43307–319. This fragment is both amyloidogenic and toxic and is within the glycine-rich C-terminal domain essential for both toxicity and aggregation of the full-length protein. In addition to the wild-type peptide, two ALS-related mutants (A315T and A315E) and a non-axon-toxic mutant (G314V) were investigated to determine how mutations affect the oligomerization of TDP-43307–319 and structures of toxic oligomers. The results of our study provide new insights into how ALS-related mutants, A315T and A315E, accelerate or alter the pathogenic mechanism and highlight the role of an internal glycine, G314, in maintaining efficient packing known to be critical for functional oligomer assembly. More importantly, our data demonstrate that G314 plays a vital role in TDP-43 assembly and prevents cytotoxicity via its unique aversion to oligomers larger than trimer. Our observation is consistent with previous studies showing that G314V mutation of the full-length TDP-43 induced remediation of both axonotoxicity and neuronal apoptosis. Our findings reveal a distinct aggregation mechanism for each peptide and elucidate oligomeric species and possible structures that may be involved in the pathology of ALS.
TDP-43 aggregates are a salient feature of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and a variety of other neurodegenerative diseases, including Alzheimer's disease (AD). With an anticipated growth in the most susceptible demographic, projections predict neurodegenerative diseases will potentially affect 15 million people in the United States by 2050. Currently, there are no cures for ALS, FTD, or AD. Previous studies of the amyloidogenic core of TDP-43 have demonstrated that oligomers greater than a trimer are associated with toxicity. Utilizing a joint pharmacophore space (JPS) method, potential drugs have been designed specifically for amyloid-related diseases. These molecules were generated on the basis of key chemical features necessary for blood−brain barrier permeability, low adverse side effects, and target selectivity. Combining ion-mobility mass spectrometry and atomic force microscopy with the JPS computational method allows us to more efficiently evaluate a potential drug's efficacy in disrupting the development of putative toxic species. Our results demonstrate the dissociation of higher-order oligomers in the presence of these novel JPSgenerated inhibitors into smaller oligomer species. Additionally, drugs approved by the Food and Drug Administration for the treatment of ALS were also evaluated and demonstrated to maintain higher-order oligomeric assemblies. Possible mechanisms for the observed action of the JPS molecules are discussed.
Stress is ubiquitous to life and can irreparably damage essential biomolecules and organelles in cells. To survive, organisms must sense and adapt to stressful conditions. One highly conserved adaptive stress response is through the post-translational modification of proteins by the small ubiquitin-like modifier (SUMO). Here, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae . We found that cells exhibit a transient sumoylation response after acute exposure to ≤ 7.5% ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol exposure. Mass spectrometry analyses identified 18 proteins that are sumoylated after acute ethanol exposure, with 15 known to associate with chromatin. Upon further analysis, we found that the chromatin structural proteins Smc5 and Smc6 undergo ethanol-induced sumoylation that depends on the activity of the E3 SUMO ligase Mms21. Using cell-cycle arrest assays, we observed that Smc5 and Smc6 ethanol-induced sumoylation occurs during G1 and G2/M phases but not S phase. Acute ethanol exposure also resulted in the formation of Rad52 foci at levels comparable to Rad52 foci formation after exposure to the DNA alkylating agent methyl methanesulfonate (MMS). MMS exposure is known to induce the intra-S phase DNA damage checkpoint via Rad53 phosphorylation, but ethanol exposure did not induce Rad53 phosphorylation. Ethanol abrogated the effect of MMS on Rad53 phosphorylation when added simultaneously. From these studies, we propose that acute ethanol exposure induces a change in chromatin leading to sumoylation of specific chromatin-structural proteins.
Stress is a ubiquitous part of life that disrupts cellular function and, if unresolved, can irreparably damage essential biomolecules and organelles. All organisms can experience stress in the form of unfavorable environmental conditions including exposure to extreme temperatures, hypoxia, reactive oxygen species, alcohol, or shifts in osmolarity. To survive, organisms must sense these changes then react and adapt. One highly conserved adaptive response to stress is through protein sumoylation, which is a post-translational modification by the small ubiquitin-like modifier (SUMO) protein. In this study, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae. Although ethanol induces protein sumoylation, the targets and roles of sumoylation are largely unknown. Here, we found that cells exhibit a transient sumoylation response after exposure of cells to ≤ 7.5% volume/volume ethanol. The response peaks at 15 minutes and resolves by 60 minutes, indicating that cells have an adaptive response to low concentrations of ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol stress with no resolution by 60 minutes. To identify key targets of ethanol-induced sumoylation, we performed mass spectrometry analyses and identified 18 proteins with increased sumoylation after acute ethanol exposure, with 15 identified as known chromatin-associated proteins. Two of these proteins are the chromatin structural proteins Smc5 and Smc6, which are sumoylated by the activity of the SUMO ligase Mms21. Ethanol-induced Smc5/6 sumoylation occurs during G1 and G2M phases of the cell cycle but is abrogated during S phase despite the fact that other proteins are sumoylated during this phase. Acute ethanol exposure leads to formation of Rad52 foci indicating DNA damage similar to that observed with the addition of methyl methanesulfonate (MMS), which is an alkylating agent that damages DNA. Whereas MMS exposure induces the intra-S phase DNA damage checkpoint as observed by Rad53 phosphorylation, ethanol exposure does not induce the intra-S phase checkpoint and prevents Rad53 phosphorylation when added with MMS. From these results, we propose that ethanol induces a structural change in chromatin, possibly through DNA damage, and this causes sumoylation of conserved chromatin-associated proteins, including Smc5 and Smc6.
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