Taisuke Nakayama

Regulation of Notch Signaling by Dynamic Changes in the Precision of S3 Cleavage of Notch-1†

ABSTRACT Intramembrane proteolysis by presenilin-dependent γ-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-β. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val→Gly and the Lys→Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.

γ-Secretase Modulators and Presenilin 1 Mutants Act Differently on Presenilin/γ-Secretase Function to Cleave Aβ42 and Aβ43

Deciphering the mechanism by which the relative Aβ42(43) to total Aβ ratio is regulated is central to understanding Alzheimer disease (AD) etiology; however, the mechanisms underlying changes in the Aβ42(43) ratio caused by familial mutations and γ-secretase modulators (GSMs) are unclear. Here, we show in vitro and in living cells that presenilin (PS)/γ-secretase cleaves Aβ42 into Aβ38, and Aβ43 into Aβ40 or Aβ38. Approximately 40% of Aβ38 is derived from Aβ43. Aβ42(43) cleavage is involved in the regulation of the Aβ42(43) ratio in living cells. GSMs increase the cleavage of PS/γ-secretase-bound Aβ42 (increase k(cat)) and slow its dissociation from the enzyme (decrease k(b)), whereas PS1 mutants and inverse GSMs show the opposite effects. Therefore, we suggest a concept to describe the Aβ42(43) production process and propose how GSMs act, and we suggest that a loss of PS/γ-secretase function to cleave Aβ42(43) may initiate AD and might represent a therapeutic target.

The 28‐amino acid form of an APLP1‐derived Aβ‐like peptide is a surrogate marker for Aβ42 production in the central nervous system

Surrogate markers for the Alzheimer disease (AD)‐associated 42‐amino acid form of amyloid‐β (Aβ42) have been sought because they may aid in the diagnosis of AD and for clarification of disease pathogenesis. Here, we demonstrate that human cerebrospinal fluid (CSF) contains three APLP1‐derived Aβ‐like peptides (APL1β) that are generated by β‐ and γ‐cleavages at a concentration of ∼4.5 nM. These novel peptides, APL1β25, APL1β27 and APL1β28, were not deposited in AD brains. Interestingly, most γ‐secretase modulators (GSMs) and familial AD‐associated presenilin1 mutants that up‐regulate the relative production of Aβ42 cause a parallel increase in the production of APL1β28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1β28 levels are higher than in non‐AD controls, while the relative Aβ42 levels are unchanged or lower. Most strikingly, the relative APL1β28 levels are higher in CSF from sporadic AD patients (regardless of whether they are at mild cognitive impairment or AD stage), than those of non‐AD controls. Based on these results, we propose the relative level of APL1β28 in the CSF as a candidate surrogate marker for the relative level of Aβ42 production in the brain.

Processes of β-Amyloid and Intracellular Cytoplasmic Domain Generation by Presenilin/γ-Secretase

Background/Aims: Following extracellular shedding, transmembrane domains (TMs) of β-amyloid precursor protein (βAPP) and Notch-1 undergo proteolysis by presenilin (PS)/γ-secretase at least at two sites, near the middle of the TM (γ-/S4 cleavage) and at the interface between cytosol and the TM (Ε-/S3 cleavage), releasing Alzheimer disease (AD)-associated β-amyloid (Aβ)/Notch-1β (Nβ) and βAPP intracellular cytoplasmic domain (AICD)/Notch-1 intracellular cytoplasmic domain (NICD). Inhibiting PS/γ-secretase activity is an essential approach to AD treatment, but it also decreases NICD production, which may cause severe side effects. Therefore, it is important to investigate the differences between the cleavages at the two sites. γ-/S4 and Ε-cleavages have diversity, and produce a number of Aβ/Nβ and AICD species. S3 cleavage diversity has been recently identified. It is significant that each cleavage occurs with strict precision, not randomly. Methods: Biochemical analysis of cultured cells was performed to explore the processing mechanisms. Results: Familial AD-associated PS1 mutations as well as a subset of nonsteroidal anti-inflammatory drugs cause similar changes in γ-/S4 cleavage precision, suggesting a common process for these cleavages near the middle of the TM. While the precision of the Ε-cleavage is drastically affected by physiological factors, that of Ε-/S3 cleavage is not. Conclusion: The processes of the two cleavages occurring in different portions of TMs may be diverse, thus representing possible targets for anti-AD therapeutics to selectively reduce Aβ.

The production ratios of AICDε51 and Aβ42 by intramembrane proteolysis of βAPP do not always change in parallel

Background:  During intramembrane proteolysis of β‐amyloid protein precursor (βAPP) by presenilin (PS)/γ‐secretase, ε‐cleavages at the membrane‐cytoplasmic border precede γ‐cleavages at the middle of the transmembrane domain. Generation ratios of Aβ42, a critical molecule for Alzheimers disease (AD) pathogenesis, and the major Aβ40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aβ42 production have been investigated by many pharmaceutical companies. Therefore, the ε‐cleavages, rather than the γ‐cleavage, might be more effective upstream targets for decreasing the relative generation of Aβ42. Thus, one might evaluate compounds by analyzing the generation ratio of the βAPP intracellular domain (AICD) species (ε‐cleavage‐derived), instead of that of Aβ42.

Structural features of interfacial tyrosine residue in ROBO1 fibronectin domain-antibody complex: Crystallographic, thermodynamic, and molecular dynamic analyses

ROBO1, fibronectin Type‐III domain (Fn)‐containing protein, is a novel immunotherapeutic target for hepatocellular carcinoma in humans. The crystal structure of the antigen‐binding fragment (Fab) of B2212A, the monoclonal antibody against the third Fn domain (Fn3) of ROBO1, was determined in pursuit of antibody drug for hepatocellular carcinoma. This effort was conducted in the presence or absence of the antigen, with the chemical features being investigated by determining the affinity of the antibody using molecular dynamics (MD) and thermodynamics. The structural comparison of B2212A Fab between the complex and the free form revealed that the interfacial TyrL50 (superscripts L, H, and F stand for the residues in the light chain, heavy chain, and Fn3, respectively) played important roles in Fn3 recognition. That is, the aromatic ring of TyrL50 pivoted toward PheF68, forming a CH/π interaction and a new hydrogen bond with the carbonyl O atom of PheF68. MD simulations predicted that the TyrL50‐PheF68 interaction almost entirely dominated Fab‐Fn3 binding, and Ala‐substitution of TyrL50 led to a reduced binding of the resultant complex. On the contrary, isothermal titration calorimetry experiments underscored that Ala‐substitution of TyrL50 caused an increase of the binding enthalpy between B2212A and Fn3, but importantly, it induced an increase of the binding entropy, resulting in a suppression of loss in the Gibbs free energy in total. These results suggest that mutation analysis considering the binding entropy as well as the binding enthalpy will aid in the development of novel antibody drugs for hepatocellular carcinoma.

Destruxin E Decreases Beta-Amyloid Generation by Reducing Colocalization of Beta-Amyloid-Cleaving Enzyme 1 and Beta-Amyloid Protein Precursor

Alzheimer-disease-associated β-amyloid (Aβ) is produced by sequential endoproteolysis of β-amyloid protein precursor (βAPP): the extracellular portion is shed by cleavage in the juxtamembrane region by β-amyloid-cleaving enzyme (BACE)/β-secretase, after which it is cleaved by presenilin (PS)/γ-secretase near the middle of the transmembrane domain. Thus, inhibition of either of the secretases reduces Aβ generation and is a fundamental strategy for the development of drugs to prevent Alzheimer disease. However, it is not clear how small compounds reduce Aβ production without inhibition of the secretases. Such compounds are expected to avoid some of the side effects of secretase inhibitors. Here, we report that destruxin E (Dx-E), a natural cyclic hexadepsipeptide, reduces Aβ generation without affecting BACE or PS/γ-secretase activity. In agreement with this, Dx-E did not inhibit Notch signaling. We found that Dx-E decreases colocalization of BACE1 and βAPP, which reduces β-cleavage of βAPP. Therefore, the data demonstrate that Dx-E represents a novel Aβ-reducing process which could have fewer side effects than secretase inhibitors.

Epiregulin Recognition Mechanisms by Anti-epiregulin Antibody 9E5 STRUCTURAL, FUNCTIONAL, AND MOLECULAR DYNAMICS SIMULATION ANALYSES

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1–3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro103. A molecular dynamics simulation and mutational analyses revealed that Arg40 in EPR is a key residue for the specific binding of 9E5 IgG. From isothermal titration calorimetry analysis, the dissociation constant was determined to be 6.5 nm. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)·EPR on the known complex structure of EGF·EGFR showed that the 9E5(Fab) paratope overlaps with Domains I and III on the EGFR, which reveals that the 9E5(Fab)·EPR complex could not bind to the EGFR. The 9E5 antibody will also be useful in medicine as a neutralizing antibody specific for colon cancer.

Macrophage colony stimulating factor is associated with excretion of amyloid‐β peptides from cerebrospinal fluid to peripheral blood

Background:  The process of aggregation of brain amyloid‐β peptides (Aβ) is thought to be associated with the pathogenesis of Alzheimers disease (AD). Amyloid‐β peptides are produced by sequential endoproteolysis by β‐site amyloid‐β protein precursor‐cleaving enzyme (BACE) followed by presenilin (PS)/γ‐secretase. There are several species of Aβ due to cleavage diversity of PS/γ‐secretase. The predominant species in human cerebrospinal fluid (CSF) or plasma is Aβ40, whereas Aβ42 is much more aggregatable and accumulated in senile plaques. The level of Aβ in the brain is determined by the balance between the generation and clearance of Aβ, including transport across the brain–blood barrier (BBB). Although the processes of Aβ generation and degradation have been studied in some detail, knowledge of the Aβ transport process across the BBB is limited. So far, low‐density lipoprotein receptor‐related protein (LRP1), P‐glycoprotein (P‐gp), and insulin‐like growth factor‐1 (IGF‐1) have been identified to modify the excretion of brain Aβ to the blood.

A trimeric structural fusion of an antagonistic tumor necrosis factor-α mutant enhances molecular stability and enables facile modification

Tumor necrosis factor-α (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein both in vitro and in vivo. Specifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retain in vitro molecular properties of receptor selectivity and antagonistic activity but displayed a marked increase in thermal stability. The residence time of scR1antTNF in vivo was also significantly prolonged. Furthermore, molecular modification using polyethylene glycol (PEG) was easily controlled by limiting the number of reactive sites. Taken together, our findings show that scR1antTNF displays enhanced molecular stability while maintaining biological activity compared with R1antTNF.