TAR DNA-binding protein 43 (TDP-43) has been identified as the major ubiquitinated aggregates in the inclusion bodies in the patients of amyotrophic lateral sclerosis (ALS) since 2006 and become a crucial culprit for ALS and related motor neuron diseases. Recent literature has further indicated that the major components of these aggregates are hyper-phosphorylated TDP-43 C-terminus. In an effort to clarify the conformational and physical properties of its disordered C-terminal domain, we have synthesized several peptide fragments and shown that only D1 within D1-4 can form twisted fibrils with a cross section of approximately 11 nm in width under the incubation of phosphate buffer. In contrast, the D2-4 peptides all formed amorphous aggregates, showing different aggregation propensities. In addition to D1, two pathological mutant peptides, A315T and G294A, can also form fibrils that share similar shape and morphology with neuronal cytoplasmic inclusions. We propose that the residues with this region (287-322), which contains myriads of glycine repeats, may contribute significantly to the fiber formation as well as aggregation propensity. Moreover, from the conformational characterizations of D1, A315T, and G294A with EM, CD, fluorescence, and Raman spectroscopy, we found that all three peptides formed an amyloid structure, providing insights into the nature of its aggregation vis a vis the other fragments in the C-terminus of TDP-43.

The formation of the N-terminal b-hairpin of ubiquitin is thought to be an early event in the folding of this small protein.Previously, we have shown that a peptide corresponding to residues 1-17 of ubiquitin folds autonomously and is likely to have a native-like hairpin register. To investigate the causes of the stability of this fold, we have made mutations in the amino acids at the apex of the turn. We find that in a peptide where Thr9 is replaced by Asp, U~1-17!T9D, the native conformation is stabilized with respect to the wild-type sequence, so much so that we are able to characterize the structure of the mutant peptide fully by NMR spectroscopy. The data indicate that U~1-17!T9D peptide does indeed form a hairpin with a native-like register and a type I turn with a G1 b-bulge, as in the full-length protein. The reason for the greater stability of the U~1-17!T9D mutant remains uncertain, but there are nuclear Overhauser effects between the side chains of Asp9 and Lys11, which may indicate that a charge-charge interaction between these residues is responsible.Keywords: b-hairpin; b-sheet; NMR; peptide; peptide conformations; structure; ubiquitin; X-PLOR The b-hairpin is one of the most commonly occurring structural motifs in globular protein folds: most antiparallel b-sheets contain at least some strand pairs that are contiguous in sequence and connected by a relatively tight turn. There has also been much speculation and some experimental evidence to support the idea that hairpins may play a key role in initiating the assembly of b-structures in folding~Ptitsyn, 1991; Muñoz et al., 1997!. In recent years, there has been considerable interest in using b-hairpins as model systems for developing our understanding of b-structure and several examples both of protein fragments and of de novo designed peptides capable of folding autonomously as hairpins have now been identified~Blanco et al., 1994;Searle et al., 1995;Ramirez-Alvarado et al., 1996!. The lessons learned from exploring the effects of sequence variations in these systems have been reviewed recently~Griffiths- Jones et al., 1998;Ramirez-Alvarado et al., 1999!. Sequence variations cannot only alter the stability of hairpin structures, but also the register of the b-strands. In the arms of the hairpin, a high b-propensity of individual residues is important for stability. However, specific pairwise contacts, especially between side chains interacting across the hairpin, are also important and optimization of these is hypothesized to be an important contribution in fixing the strand register~Smith & Regan, 1995;Wouters & Curmi, 1995;Hutchinson et al., 1998!. The turn sequence is also important: it is clear from analysis of hairpins within intact protein structures that some turn types are prevalent in b-hairpins. Sequence compatibility with these preferred structures has been shown in peptide studies to be an important determinant of structural specificity and stability~Ramirez- Alvarado et al., 1997;De Alba et al., 1999;Griffiths-Jones et al., 19...

More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various diseases, such as Alzheimer's disease, prion disease, and systematic amyloidosis. Amyloid fibrils are an ordered protein aggregate with a lamellar cross-beta structure. Enhancing amyloid clearance is one of the targets of the therapy of these amyloid-related diseases. Although there is debate on whether the toxicity is due to amyloids or their precursors, research on the degradation of amyloids may help prevent or alleviate these diseases. In this study, we explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin.

Proteolytic processing of amyloid precursor protein (APP) C-terminal fragments (CTFs) by γ-secretase underlies the pathogenesis of Alzheimer's disease (AD). An RNA interference screen using APP-CTF [99-residue CTF (C99)]-and Notch-specific γ-secretase interaction assays identified a unique ErbB2-centered signaling network that was predicted to preferentially govern the proteostasis of APP-C99. Consistently, significantly elevated levels of ErbB2 were confirmed in the hippocampus of human AD brains. We then found that ErbB2 effectively suppressed autophagic flux by physically dissociating Beclin-1 from the Vps34-Vps15 complex independent of its kinase activity. Down-regulation of ErbB2 by CL-387,785 decreased the levels of C99 and secreted amyloid-β in cellular, zebrafish, and mouse models of AD, through the activation of autophagy. Oral administration of an ErbB2-targeted CL-387,785 for 3 wk significantly improves the cognitive functions of APP/presenilin-1 (PS1) transgenic mice. This work unveils a noncanonical function of ErbB2 in modulating autophagy and establishes ErbB2 as a therapeutic target for AD.ErbB2 | Alzheimer's disease | Aβ | C99 | autophagy A myloid plaques are the primary cause of neurodegeneration in the brains of patients with Alzheimer's disease (AD) (1). Amyloid plaques are composed of amyloid-β (Aβ) peptides that are produced by stepwise cleavages of amyloid precursor protein (APP) by β-and γ-secretase (2). Therapeutic approaches toward treatment of AD developed in the past decade have centered on the prevention of Aβ production (3). The majority of these studies focused on either the augmentation of α-secretase activity, which can reduce the production of Aβ, or the inhibition of β-/γ-secretase activities (4). Unfortunately, the nonselective inhibition of β-secretase and γ-secretase results in unavoidable side effects due to the interference of other physiological substrates of β-secretase and γ-secretase (5, 6).ErbB2 is a member of the epidermal growth factor receptor (EGFR)/ErbB family [which consists of four closely related receptor tyrosine kinases (ErbB1-4, also known as HER1-4)] and is tightly associated with neuritic plaques in AD (7). The correlation between EGFR/ErbB signaling and AD pathogenesis has been well documented in various studies (8-10). Ras GTPase activation mediates EGF-induced stimulation of γ-secretase to increase the nuclear function of the APP intracellular domain (AICD) (11). Consistent with the role of EGF signaling in AD, the intracellular mediators downstream of EGF signaling (which include Grb2, ShcA, and Abl) directly or indirectly interact with APP (12); these findings support the correlation between EGFR/ ErbB-dependent signaling and AD susceptibility.Autophagy controls the clearance of misfolded proteins and damaged organelles, and plays an essential role in maintaining neuronal functions (13,14). Previous studies have demonstrated that autophagy is instrumental to the clearance of proteins related to neurodegenerative diseases; these proteins include polyg...