Hiroto Komano

Endoplasmic reticulum stress-inducible protein, Herp, enhances presenilin-mediated generation of amyloid beta-protein

Presenilin (PS) is essential for the γ-cleavage required for the generation of the C terminus of amyloid β-protein (Aβ). However, the mechanism underlying PS-mediated γ-cleavage remains unclear. We have identified Herp cDNA by our newly developed screening method for the isolation of cDNAs that increase the degree of γ-cleavage. Herp was originally identified as a homocysteine-responsive protein, and its expression is up-regulated by endoplasmic reticulum stress. Herp is an endoplasmic reticulum-localized membrane protein that has a ubiquitin-like domain. Here, we report that a high expression of Herp in cells increases the level of Aβ generation, although not in PS-deficient cells. We found that Herp interacts with both PS1 and PS2. Thus, Herp regulates PS-mediated Aβ generation, possibly through its binding to PS. Immunohistochemical analysis of a normal human brain section with an anti-Herp antibody revealed the exclusive staining of neurons and vascular smooth muscle cells. Moreover, the antibody strongly stained activated microglia in senile plaques in the brain of patients with Alzheimer disease. Taken together, Herp could be involved in Aβ accumulation, including the formation of senile plaques and vascular Aβ deposits.

1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, sapecin.

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4–12), one helix (residues 15–23), and two extended strands (residues 24–31 and 34–40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP‐5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three‐dimensional structure determination has revealed that a basic‐residue rich region and the hydrophobic surface face each other on the surface of sapecin.

Expression of the lectin gene in Sarcophaga peregrina during normal development and under conditions where the defence mechanism is activated

Abstract Expression of the Sarcophaga lectin gene was investigated using a cDNA clone as a probe. During normal development of Sarcophaga peregrina , the lectin gene was activated transiently in the embryos in 8-day old female flies and in the fat body of early pupae. The gene was also activated in the fat body of 3rd-instar larvae when sheep red blood cells were injected. Since, in Sarcophaga , this lectin has been shown to participate in scavenging sheep red blood cells introduced into 3rd-instar larvae, the same defence mechanism mediated by the lectin probably operates both in scavenging unnecessary tissues or cells produced during normal development and, in emergency, in eliminating invading foreign substances.

Participation of Sarcophagaperegrina humoral lectin in the lysis of sheep red blood cells injected into the abdominal cavity of larvae

Sarcophaga lectin which is induced in the hemolymph of Sarcophaga peregrina (flesh-fly) larvae when their body wall is injured with a hypodermic needle, was shown to participate in the lysis of sheep red blood cells introduced into the abdominal cavity of the larvae. This finding indicates that humoral lectin plays a role in the defence mechanism of invertebrates. The lysis of sheep red blood cells was enhanced greatly by preinjection of red blood cells, but less by preinjection of other cells. Thus the defence mechanism of this insect seems to be able to distinguish red blood cells from other cells.

Presenilin 1 Mutations Linked to Familial Alzheimer's Disease Increase the Intracellular Levels of Amyloid β-Protein 1–42 and Its N-Terminally Truncated Variant(s) Which Are Generated at Distinct Sites

Abstract: Mutations in the presenilin genes PS1 and PS2 cause the most common form of early‐onset familial Alzheimers disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid β‐protein (Aβ) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuro‐blastoma (Neuro 2a) cell lines expressing the human wild‐type (wt) or mutated PS1 (M146L or Δexon 10). The induction of mutated PS1 increased the intracellular levels of two distinct Aβ species ending at residue 42 that were likely to be Aβ1–42 and its N‐terminally truncated variant(s) Aβx‐42. The induction of mutated PS1 resulted in a higher level of intracellular Aβ1–42 than of intracellular Aβx‐42, whereas extracellular levels of Aβ1–42 and Aβx‐42 were increased proportionally. In addition, the intracellular generation of these Aβ42 species in wt and mutated PS1‐induced cells was completely blocked by brefeldin A, whereas it exhibited differential sensitivities to monensin: the increased accumulation of intracellular Aβx‐42 versus inhibition of intracellular Aβ1–42 generation. These data strongly suggest that Aβx‐42 is generated in a proximal Golgi, whereas Aβ1–42 is generated in a distal Golgi and/or a post‐Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42‐specific γ‐secretase cleavage that occurs in the normal β‐amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to β‐secretase cleavage or (b) in the distinct sites where Aβx‐42 and Aβ1–42 are generated.

Homocysteine-induced endoplasmic reticulum protein (Herp) is up-regulated in sporadic inclusion-body myositis and in endoplasmic reticulum stress-induced cultured human muscle fibers.

Herp is a stress‐response protein localized in the endoplasmic reticulum (ER) membrane. Herp was proposed to improve ER‐folding, decrease ER protein load, and participate in ER‐associated degradation (ERAD). Intra‐muscle‐fiber ubiquitinated multiprotein‐aggregates containing, among other proteins, either amyloid‐β (Aβ) or phosphorylated tau are characteristic of sporadic inclusion‐body myositis (s‐IBM). ER stress and proteasome inhibition appear to play a role in s‐IBM pathogenesis. We have now studied Herp in s‐IBM muscle fibers and in ER‐stress‐induced or proteasome‐inhibited cultured human muscle fibers. In s‐IBM muscle fibers: (i) Herp was strongly immunoreactive in the form of aggregates, which co‐localized with Aβ, GRP78, and β2 proteasome subunit; (ii) Herp mRNA and protein were increased. In ER‐stress‐induced cultured human muscle fibers: (i) Herp immunoreactivity was diffusely increased; (ii) Herp mRNA and protein were increased. In proteasome‐inhibited cultured human muscle fibers: (i) Herp immunoreactivity was in the form of aggregates; (ii) Herp protein was increased, but its mRNA was not. Accordingly, in s‐IBM muscle fibers: (i) increase of Herp might be due to both ER‐stress and proteasome inhibition; (ii) co‐localization of Herp with Aβ, proteasome, and ER‐chaperone GRP78 could reflect its possible role in processing and degradation of cytotoxic proteins in ER.

Aβ42-to-Aβ40- and Angiotensin-converting Activities in Different Domains of Angiotensin-converting Enzyme

Amyloid β-protein 1–42 (Aβ42) is believed to play a causative role in the development of Alzheimer disease (AD), although it is a minor part of Aβ. In contrast, Aβ40 is the predominant secreted form of Aβ and recent studies have suggested that Aβ40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Aβ42 to Aβ40, and its inhibition enhances brain Aβ42 deposition (Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi, K., Gong, J. S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K., and Michikawa, M. (2007) J. Neurosci. 27, 8628–8635). ACE has two homologous domains, each having a functional active site. In the present study, we identified the domain of ACE, which is responsible for converting Aβ42 to Aβ40. Interestingly, Aβ42-to-Aβ40-converting activity is solely found in the N-domain of ACE and the angiotensin-converting activity is found predominantly in the C-domain of ACE. We also found that the N-linked glycosylation is essential for both Aβ42-to-Aβ40- and angiotensin-converting activities and that unglycosylated ACE rapidly degraded. The domain-specific converting activity of ACE suggests that ACE inhibitors could be designed to specifically target the angiotensin-converting C-domain, without inhibiting the Aβ42-to-Aβ40-converting activity of ACE or increasing neurotoxic Aβ42.

Involvement of Sarcophaga lectin in the development of imaginal discs of Sarcophaga peregrina in an autocrine manner.

The imaginal discs of Sarcophaga were found not to develop normally in the presence of galactose, a hapten sugar of Sarcophaga lectin, or anti-Sarcophaga lectin antibody. Wing and leg discs cultured with these substances became morphologically abnormal and no imaginal discs reached the stage of terminal differentiation, even in the presence of 20-hydroxyecdysone. The development of the imaginal discs was shown to be autonomously regulated in an autocrine manner by Sarcophaga lectin; namely Sarcophaga lectin was secreted by the imaginal discs in the presence of 20-hydroxyecdysone, and the stimulus of self-induced Sarcophaga lectin seemed to be indispensable for further development of the imaginal discs. Sarcophaga lectin was originally found as a defense protein, but these results show that it plays independent roles in both defense and development.

The ubiquitin-like domain of Herp is involved in Herp degradation, but not necessary for its enhancement of amyloid β-protein generation

Herp is an endoplasmic reticulum (ER)‐stress‐inducible membrane protein, which has a ubiquitin‐like domain (ULD). However, its biological function is as yet unknown. Previously, we reported that a high expression level of Herp in cells increases the generation of amyloid β‐protein (Aβ) and that Herp interacts with presenilin (PS). Here, we addressed the role of the ULD of Herp in Aβ generation and intracellular Herp stability. We found that the ULD is not essential for the enhancement of Aβ generation by Herp expression and the interaction of Herp with PS, but is involved in the rapid degradation of Herp, most likely via the ubiquitin/proteasome pathway. Thus, the ULD of Herp most likely plays a role in the regulation of the intracellular level of Herp under ER stress.

Random Mutagenesis of Presenilin-1 Identifies Novel Mutants Exclusively Generating Long Amyloid β-Peptides

Familial Alzheimer disease-causing mutations in the presenilins increase production of longer pathogenic amyloid β-peptides (Aβ42/43) by altering γ-secretase activity. The mechanism underlying this effect remains unknown, although it has been proposed that heteromeric macromolecular complexes containing presenilins mediate γ-secretase cleavage of the amyloid β-precursor protein. Using a random mutagenesis screen of presenilin-1 (PS1) for PS1 endoproteolysis-impairing mutations, we identified five unique mutants, including R278I-PS1 and L435H-PS1, that exclusively generated a high level of Aβ43, but did not support physiological PS1 endoproteolysis or Aβ40 generation. These mutants did not measurably alter the molecular size or subcellular localization of PS1 complexes. Pharmacological studies indicated that the up-regulation of activity for Aβ43 generation by these mutations was not further enhanced by the difluoroketone inhibitor DFK167 and was refractory to inhibition by sulindac sulfide. These results suggest that PS1 mutations can lead to a wide spectrum of changes in the activity and specificity of γ-secretase and that the effects of PS1 mutations and γ-secretase inhibitors on the specificity are mediated through a common mechanism.