The Influence of Pathological Mutations and Proline Substitutions in TDP-43 Glycine-Rich Peptides on Its Amyloid Properties and Cellular Toxicity

Abstract

TAR DNA-binding protein (TDP-43) was identified as the major ubiquitinated component deposited in the inclusion bodies in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) in 2006. Later on, numerous ALS-related mutations were found in either the glycine or glutamine/asparagine-rich region on the TDP-43 C-terminus, which hinted on the importance of mutations on the disease pathogenesis. However, how the structural conversion was influenced by the mutations and the biological significance of these peptides remains unclear. In this work, various peptides bearing pathogenic or de novo designed mutations were synthesized and displayed their ability to form twisted amyloid fibers, cause liposome leakage, and mediate cellular toxicity as confirmed by transmission electron microscopy (TEM), circular dichroism (CD), Thioflavin T (ThT) assay, Raman spectroscopy, calcein leakage assay, and cell viability assay. We have also shown that replacing glycines with prolines, known to obstruct β-sheet formation, at the different positions in these peptides may influence the amyloidogenesis process and neurotoxicity. In these cases, GGG308PPP mutant was not able to form beta-amyloid, cause liposome leakage, nor jeopardized cell survival, which hinted on the importance of the glycines (308–310) during amyloidogenesis.

Templated Aggregation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Seeding with TDP-43 Peptide Fibrils

“…10. Our sequence 274 -353 overlaps well with those reported by Huang and co-workers (28,29), although other sequences were not identified in this study. The discrepancy may be due to the difference in the length of peptides used in in vitro aggregate formation assay (we prepared 40-mers in contrast with their short 8 -12-mer peptides).…”

“…Huang and co-workers (28,29) showed that peptides in the sequence (287-322) have a propensity for aggregation, and this tendency was altered by substitution of glycines with prolines. The sequences reported in previous studies and identified in this study are summarized in Fig.…”

Mechanisms of Membrane Pore Formation by Amyloidogenic Peptides in Amyotrophic Lateral Sclerosis

“…While electron microscopy studies have revealed the formation of amorphous aggregates, the low fluorescence intensity after incubation with Thioflavin T (ThT) confirmed that GGG308PPP could not form fibrils. [11] These experiments suggest that the glycines 308-310 are required for both membrane disruption and fibril formation of D1 (Supporting Information, Table S1). …”

“…Huang et al have shown that D1 peptides form pores in lipid membranes that are sufficiently large to allow leakage of calcein (MW % 620 Da) from large unilamellar vesicles. [11] In contrast, the GGG308PPP mutant does not form membrane pores. While electron microscopy studies have revealed the formation of amorphous aggregates, the low fluorescence intensity after incubation with Thioflavin T (ThT) confirmed that GGG308PPP could not form fibrils.…”

“…[8] Previous studies have shown that the glycine-rich D1 domain, a fragment of C-terminal domain of TDP-43, is sufficient to induce fibril formation in phosphate buffer and forms pores in lipid bilayers. [9][10][11] Some studies proposed that early stage fibrils and/or oligomers of neurodegenerative proteins are more toxic than mature fibrils of the misfolded proteins; [12] however, the mechanism of pore formation, conduction, and the structure of the pore remain unknown.Herein, we study domain 1 (D1, residues 287-322) from the C-terminal moiety of TDP-43, a glycine-rich domain that is essential for protein aggregation and fibril formation.[9] We also study the D1 triple mutant GGG308PPP, where three C-terminal glycine residues (308-310) are substituted by three proline residues to severely reduce the flexibility and conformational plasticity of the peptide (Table TS1). Huang et al have shown that D1 peptides form pores in lipid membranes that are sufficiently large to allow leakage of calcein (MW % 620 Da) from large unilamellar vesicles.…”