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Processing of amyloid-β (Aβ) precursor protein (APP) by γ-secretase produces multiple species of Aβ: Aβ40, short Aβ peptides (Aβ37-39), and longer Aβ peptides (Aβ42-43). γ-Secretase modulators, a class of Alzheimer's disease therapeutics, reduce production of the pathogenic Aβ42 but increase the relative abundance of short Aβ peptides. To evaluate the pathological relevance of these peptides, we expressed Aβ36-40 and Aβ42-43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on Aβ42 toxicity. In contrast to Aβ42, the short Aβ peptides were not toxic and, when coexpressed with Aβ42, were protective in a dose-dependent fashion. In parallel, we explored the effects of recombinant adeno-associated virus-mediated expression of Aβ38 and Aβ40 in mice. When expressed in nontransgenic mice at levels sufficient to drive Aβ42 deposition, Aβ38 and Aβ40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower Aβ42 by raising the levels of short Aβ peptides could attenuate the toxic effects of Aβ42., (© 2018 Moore et al.)

Granulins (GRNs) are a family of small (∼6 kDa) proteins generated by the proteolytic processing of their precursor, progranulin (PGRN), in many cell types. Both PGRN and GRNs are implicated in a plethora of biological functions, often in opposing roles to each other. Lately, GRNs have generated significant attention due to their implicated roles in neurodegenerative disorders. Despite their physiological and pathological significance, the structure-function relationships of GRNs are poorly defined. GRNs contain 12 conserved cysteines forming six intramolecular disulfide bonds, making them rather exceptional, even among a few proteins with high disulfide bond density. Solution NMR investigations in the past have revealed a unique structure containing putative interdigitated disulfide bonds for several GRNs, but GRN-3 was unsolvable due to its heterogeneity and disorder. In our previous report, we showed that abrogation of disulfide bonds in GRN-3 renders the protein completely disordered (Ghag et al., Prot Eng Des Sel 2016). In this study, we report the cellular expression and biophysical analysis of fully oxidized, native GRN-3. Our results indicate that both E. coli and human embryonic kidney (HEK) cells do not exclusively make GRN-3 with homogenous disulfide bonds, likely due to the high cysteine density within the protein. Biophysical analysis suggests that GRN-3 structure is dominated by irregular loops held together only by disulfide bonds, which induced remarkable thermal stability to the protein despite the lack of regular secondary structure. This unusual handshake between disulfide bonds and disorder within GRN-3 could suggest a unique adaptation of intrinsically disordered proteins towards structural stability., (© 2017 The Protein Society.)

γ-Secretase catalyzes the final cleavage of the amyloid precursor protein (APP), resulting in the production of amyloid-β (Aβ) peptides with different carboxyl termini. Presenilin (PSEN) and amyloid precursor protein (APP) mutations linked to early onset familial Alzheimer's disease modify the profile of Aβ isoforms generated, by altering both the initial γ-secretase cleavage site and subsequent processivity in a manner that leads to increased levels of the more amyloidogenic Aβ42 and in some circumstances Aβ43. Compounds termed γ-secretase modulators (GSMs) and inverse GSMs (iGSMs) can decrease and increase levels of Aβ42, respectively. As GSMs lower the level of production of pathogenic forms of long Aβ isoforms, they are of great interest as potential Alzheimer's disease therapeutics. The factors that regulate GSM modulation are not fully understood; however, there is a growing body of evidence that supports the hypothesis that GSM activity is influenced by the amino acid sequence of the γ-secretase substrate. We have evaluated whether mutations near the luminal border of the transmembrane domain (TMD) of APP alter the ability of both acidic, nonsteroidal anti-inflammatory drug-derived carboxylate and nonacidic, phenylimidazole-derived classes of GSMs and iGSMs to modulate γ-secretase cleavage. Our data show that point mutations can dramatically reduce the sensitivity to modulation of cleavage by GSMs but have weaker effects on iGSM activity. These studies support the concept that the effect of GSMs may be substrate selective; for APP, it is dependent on the amino acid sequence of the substrate near the junction of the extracellular domain and luminal segment of the TMD.

Introduction: A hallmark of Alzheimer's disease (AD) is the presence of senile plaques composed of aggregated amyloid β (Aβ) peptides. Pathological aging (PA) is a postmortem classification that has been used to describe brains with plaque pathology similar in extent to AD, minimal cortical tau pathology, and no accompanying history of cognitive decline in the brain donor prior to death. PA may represent either a prodromal phase of AD, a benign form of Aβ accumulation, or inherent individual resistance to the toxic effects of Aβ accumulation. To attempt to distinguish between these possibilities we have systematically characterized Aβ peptides in a postmortem series of PA, AD and non-demented control (NDC) brains., Methods: Aβ was sequentially extracted with tris buffered saline (TBS), radioimmunoprecipitation buffer (RIPA), 2% sodium dodecyl sulfate (SDS) and 70% formic acid (FA) from the pre-frontal cortex of 16 AD, eight PA, and six NDC patients. These extracts were analyzed by 1) a panel of Aβ sandwich ELISAs, 2) immunoprecipitation followed by mass spectrometry (IP/MS) and 3) western blotting. These studies enabled us to asses Aβ levels and solubility, peptide profiles and oligomeric assemblies., Results: In almost all extracts (TBS, RIPA, 2% SDS and 70% FA) the average levels of Aβ1-40, Aβ1-42, Aβ total, and Aβx-42 were greatest in AD. On average, levels were slightly lower in PA, and there was extensive overlap between Aβ levels in individual PA and AD cases. The profiles of Aβ peptides detected using IP/MS techniques also showed extensive similarity between the PA and AD brain extracts. In select AD brain extracts, we detected more amino-terminally truncated Aβ peptides compared to PA patients, but these peptides represented a minor portion of the Aβ observed. No consistent differences in the Aβ assemblies were observed by western blotting in the PA and AD groups., Conclusions: We found extensive overlap with only subtle quantitative differences between Aβ levels, peptide profiles, solubility, and SDS-stable oligomeric assemblies in the PA and AD brains. These cross-sectional data indicate that Aβ accumulation in PA and AD is remarkably similar. Such data would be consistent with PA representing a prodromal stage of AD or a resistance to the toxic effects of Aβ.

A subset of non-steroidal anti-inflammatory drugs modulates the γ cleavage site in the amyloid precursor protein (APP) to selectively reduce production of Aβ42. It is unclear precisely how these γ-secretase modulators (GSMs) act to preferentially spare Aβ40 production as well as Notch processing and signaling. In an effort to determine the substrate requirements in NSAID/GSM activity, we determined the effects of sulindac sulfide and flurbiprofen on γ-cleavage of artificial constructs containing several γ-secretase substrates. Using FLAG-tagged constructs that expressed extracellularly truncated APP, Notch-1, or CD44, we found that these substrates have different sensitivities to sulindac sulfide. γ-Secretase cleavage of APP was altered by sulindac sulfide, but CD44 and Notch-1 were either insensitive or only minimally altered by this compound. Using chimeric APP constructs, we observed that the transmembrane domain (TMD) of APP played a pivotal role in determining drug sensitivity. Substituting the APP TMD with that of APLP2 retained the sensitivity to γ-cleavage modulation, but replacing TMDs from Notch-1 or ErbB4 rendered the resultant molecules insensitive to drug treatment. Specifically, the GXXXG motif within APP appeared to be critical to GSM activity. Consequently, the modulatory effects on γ-cleavage appears to be substrate-dependent. We hypothesize that the substrate present in the γ-secretase complex influences the conformation of the complex so that the binding site of GSMs is either stabilized or less favorable to influence the cleavage of the respective substrates.

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.

Inhibition of gamma-secretase cleavage of the amyloid precursor protein (APP) is a prime target for the development of therapeutics for treating Alzheimer's disease; however, complete inhibition of this activity would also impair the processing of many other proteins, including the APP homologues, amyloid precursor-like protein (APLP) 1 and 2. To prevent unwanted side effects, therapeutically useful gamma-secretase inhibitors should specifically target APP processing while sparing cleavage of other gamma-substrates. Thus, since APLP1 and APLP2 are more similar to APP than any of the other known gamma-secretase substrates and have important physiological roles in their own right, we reasoned that comparison of the effect of gamma-secretase inhibitors on APLP processing should provide a sensitive indicator of the selectivity of putative inhibitors. To address this issue, we have optimized microsome and cell culture assays to monitor the gamma-secretase proteolysis of APP and APLPs. Production of the gamma-secretase-generated intracellular domain (ICD) occurs more rapidly from APLP1 than from either APLP2 or APP, suggesting that APLP1 is a better gamma-substrate and that substrate recognition is not restricted to the highly conserved amino acid sequences surrounding the epsilon-site. As expected, the well-characterized gamma-secretase modulator, fenofibrate, did not inhibit ICD release, whereas a related compound, FT-9, inhibited gamma-secretase both in microsomes and in whole cells. Importantly, FT-9 displayed a preferential effect, inhibiting cleavage of APP much more effectively than cleavage of APLP1. These findings suggest that selective inhibitors can be developed and that screening of compounds against APP and APLPs should assist in this process.

Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.