Stability and Inter-domain Interactions Modulate Amyloid Binding Activity of a General Amyloid Interaction Motif.

The M13 tip protein, g3p, binds the C-terminal domain of the bacterial membrane protein TolA via ß-sheet augmentation, facilitating viral entry into Escherichia coli. G3p binding leads to rearrangement of the ß strands and partial unfolding of TolA. G3p also binds multiple amyloid assemblies with high affinity, and it can remodel them into amorphous aggregates. We previously showed that amyloid binding activity is defined by the two g3p N-terminal domains, which we call the general amyloid interaction motif (GAIM). GAIM-hIgG1Fc fusions, which add immune effector function to amyloid targeting of GAIM, mediate reduction of two CNS amyloid deposits, Aß plaques and tau tangles, in transgenic animal models of neurodegenerative disease. We carried out site-directed mutagenesis of GAIM to identify variants with altered amyloid binding and remodeling activity. A small set of residues along the inner strands of the two domains regulates both activities. The specificity of amyloid binding is governed by individual domain stability and inter-domain interactions. Our studies reveal several lines of similarity between GAIM binding to amyloids and g3p binding to its E. coli membrane target, TolA. Based on these studies, we designed new GAIM fusions that show enhanced binding potency towards multiple amyloid aggregates.

NPT088 reduces both amyloid-ß and tau pathologies in transgenic mice.

INTRODUCTION: Alzheimer's disease (AD) is characterized by appearance of both extracellular senile plaques and intracellular neurofibrillary tangles, comprised of aggregates of misfolded amyloid-ß (Aß) and hyper-phosphorylated tau, respectively. In a previous study, we demonstrated that g3p, a capsid protein from bacteriophage M13, binds to and remodels misfolded aggregates of proteins that assume an amyloid conformation. We engineered a fusion protein ("NPT088") consisting of the active fragment of g3p and human-IgG1-Fc. METHODS: Aged Tg2576 mice or rTg4510 mice received NPT088 weekly via IP injection. Cognitive and/or functional motor endpoints were monitored during dosing. Pathology was quantified biochemically and immunohistochemically. RESULTS: NPT088-lowered Aß plaque and improved cognitive performance of aged Tg2576 mice. Moreover, NPT088 reduced phospho-tau pathology, reduced brain atrophy, and improved cognition in rTg4510 mice. DISCUSSION: These observations establish NPT088 as a novel therapeutic approach and potential drug class that targets both Aß and tau, the hallmark pathologies of AD.

Remodeling Amyloid Fibers: Baker's Yeast Shows Us the Way.

Proteopathies are a large and diverse group of human diseases that are caused by protein misfolding. Well-known examples of proteopathies are Alzheimer's and Parkinson's disease, which are both linked to amyloid fibril formation. In this issue of Chemistry & Biology, Castellano et al. (2015) describe the way to harness the power of a protein from baker's yeast, Hsp104, to disaggregate the fibrils.

A bacteriophage capsid protein provides a general amyloid interaction motif (GAIM) that binds and remodels misfolded protein assemblies.

Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis-trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aß binding to g3p. NMR studies show that g3p binding to Aß fibers is predominantly through middle and C-terminal residues of the Aß subunit, indicating ß strand-g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aß fibers (fAß) (KD=9.4nM); (2); blocks fAß assembly (IC50~50nM) and (3) dissociates fAß (EC50=40-100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.

A new role for Hedgehogs in juxtacrine signaling.

The Hedgehog pathway plays important roles in embryonic development, adult stem cell maintenance and tumorigenesis. In mammals these effects are mediated by Sonic, Desert and Indian Hedgehog (Shh, Dhh and Ihh). Shh undergoes autocatalytic cleavage and dual lipidation prior to secretion and forming a response gradient. Post-translational processing and secretion of Dhh and Ihh ligands has not previously been investigated. This study reports on the synthesis, processing, secretion and signaling activities of SHH, IHH and DHH preproteins expressed in cultured cells, providing unexpected evidence that DHH does not undergo substantial autoprocessing or secretion, and does not function in paracrine signaling. Rather, DHH functions as a juxtacrine signaling ligand to activate a cell contact-mediated HH signaling response, consistent with its localised signaling in vivo. Further, the LnCAP prostate cancer cell, when induced to express endogenous DHH and SHH, is active only in juxtacrine signaling. Domain swap studies reveal that the C-terminal domain of HH regulates its processing and secretion. These findings establish a new regulatory role for HHs in cell-mediated juxtacrine signaling in development and cancer.

Fission yeast mitogen-activated protein kinase Sty1 interacts with translation factors.

Signaling by stress-activated mitogen-activated protein kinase (MAPK) pathways influences translation efficiency in mammalian cells and budding yeast. We have investigated the stress-activated MAPK from fission yeast, Sty1, and its downstream protein kinase, Mkp1/Srk1, for physically associated proteins using tandem affinity purification tagging. We find Sty1, but not Mkp1, to bind to the translation elongation factor eukaryotic elongation factor 2 (eEF2) and the translation initiation factor eukaryotic initiation factor 3a (eIF3a). The Sty1-eIF3a interaction is weakened under oxidative or hyperosmotic stress, whereas the Sty1-eEF2 interaction is stable. Nitrogen deprivation causes a transient strengthening of both the Sty1-eEF2 and the Sty1-Mkp1 interactions, overlapping with the time of maximal Sty1 activation. Analysis of polysome profiles from cells under oxidative stress, or after hyperosmotic shock or nitrogen deprivation, shows that translation in sty1 mutant cells recovers considerably less efficiently than that in the wild type. Cells lacking the Sty1-regulated transcription factor Atf1 are deficient in maintaining and recovering translational activity after hyperosmotic shock but not during oxidative stress or nitrogen starvation. In cells lacking Sty1, eIF3a levels are decreased, and phosphorylation of eIF3a is reduced. Taken together, our data point to a central role in translational adaptation for the stress-activated MAPK pathway in fission yeast similar to that in other investigated eukaryotes, with the exception that fission yeast MAPK-activated protein kinases seem not to be directly involved in this process.