Conformational Dynamics of Transmembrane Domain 3 of Presenilin 1 Is Associated with the Trimming Activity of γ-Secretase.

γ-Secretase is an intramembrane-cleaving protease that generates the toxic species of the amyloid-ß peptide (Aß) that is responsible for the pathology of Alzheimer disease. The catalytic subunit of γ-secretase is presenilin 1 (PS1), which is a polytopic membrane protein with a hydrophilic catalytic pore. The length of the C terminus of Aß is proteolytically determined by its processive trimming by γ-secretase, although the precise mechanism still remains largely unknown. Here, we identified that transmembrane domain (TMD) 3 of human PS1 is involved in the formation of the intramembranous hydrophilic pore. Notably, the water accessibility of TMD3 was greatly altered by point mutations and compounds, which modify γ-secretase activity. The changes in the water accessibility of TMD3 was also correlated with Aß42 production. Moreover, crosslinking between TMD3 and TMD7 resulted in a loss of sensitivity to a γ-secretase modulator that reduces Aß42 production. Therefore, our findings indicate that the conformational dynamics of TMD3 is a prerequisite for regulation of the Aß trimming activity of γ-secretase.SIGNIFICANCE STATEMENT Modulation of γ-secretase activity to reduce the level of toxic amyloid-ß species is thought to be a therapeutic strategy for Alzheimer disease. However, the detailed mechanism of the regulation of amyloid-ß production, as well as the structure-and-activity relationship of γ-secretase remains unclear. Here we identified that the water accessibility around transmembrane domain 3 in presenilin 1 was increased along with a reduction in toxic amyloid-ß production. Our findings demonstrate how the structure of presenilin 1 dynamically changes during amyloid-ß production, and provides insights toward the development of treatments against Alzheimer disease.

Conformational Changes in Transmembrane Domain 4 of Presenilin 1 Are Associated with Altered Amyloid-ß 42 Production.

γ-Secretase is an intramembrane-cleaving protease that produces amyloid-ß peptide 42 (Aß42), which is the toxic and aggregation-prone species of Aß that causes Alzheimer's disease. Here, we used the substituted cysteine accessibility method to analyze the structure of transmembrane domains (TMDs) 4 and 5 of human presenilin 1 (PS1), a catalytic subunit of γ-secretase. We revealed that TMD4 and TMD5 face the intramembranous hydrophilic milieu together with TMD1, TMD6, TMD7, and TMD9 of PS1 to form the catalytic pore structure. Notably, we found a correlation in the distance between the cytosolic sides of TMD4/TMD7 and Aß42 production levels, suggesting that allosteric conformational changes of the cytosolic side of TMD4 affect Aß42-generating γ-secretase activity. Our results provide new insights into the relationship between the structure and activity of human PS1. SIGNIFICANCE STATEMENT: Modulation of γ-secretase activity to reduce toxic amyloid-ß peptide species is one plausible therapeutic approaches for Alzheimer's disease. However, precise mechanistic information of γ-secretase still remains unclear. Here we identified the conformational changes in transmembrane domains of presenilin 1 that affect the proteolytic activity of the γ-secretase. Our results highlight the importance of understanding the structural dynamics of presenilin 1 in drug development against Alzheimer's disease.

Allosteric regulation of γ-secretase activity by a phenylimidazole-type γ-secretase modulator.

γ-Secretase is an intramembrane-cleaving protease responsible for the generation of amyloid-ß (Aß) peptides. Recently, a series of compounds called γ-secretase modulators (GSMs) has been shown to decrease the levels of long toxic Aß species (i.e., Aß42), with a concomitant elevation of the production of shorter Aß species. In this study, we show that a phenylimidazole-type GSM allosterically induces conformational changes in the catalytic site of γ-secretase to augment the proteolytic activity. Analyses using the photoaffinity labeling technique and systematic mutational studies revealed that the phenylimidazole-type GSM targets a previously unidentified extracellular binding pocket within the N-terminal fragment of presenilin (PS). Collectively, we provide a model for the mechanism of action of the phenylimidazole-type GSM in which binding at the luminal side of PS induces a conformational change in the catalytic center of γ-secretase to modulate Aß production.

[Conservative treatment improved corrosive esophagitis and pneumomediastinum in a patient who ingested bleaching agent containing sodium hypochlorite and sodium hydroxide].

A 69-year-old man was admitted to the emergency department 3 hours after ingestion of a bleaching agent containing hypochlorous acid and sodium hydroxide in a suicide attempt. Enhanced chest computed tomography scans taken on admission indicated an edematous esophagus and air bubbles in the mediastinum. He underwent endotracheal intubation and mechanical ventilation until day 9 because of laryngeal edema. On day 10, his endoscopy indicated diffuse reddish mucosal hyperemia, erosions, and lacerated mucosal lesions in the esophagus that were indicative of grade 2b corrosive esophagitis. Treatment with a proton pump inhibitor was initiated, with which the condition of the esophagus improved, and on day 44, a slight stricture of the upper part of the esophagus was observed. He was discharged on day 64 without any complaints. The ingestion of sodium hypochlorite induces corrosive esophagitis and acute phase of gastritis. Ingestion of any corrosive agent is known as a risk factor for esophagus cancer in the long-term. In such cases with esophageal stricture, esophagectomy is recommended for preventing esophagus cancer. Considering the age of the patient, however, he did not undergo esophagectomy.

Binding of longer Aß to transmembrane domain 1 of presenilin 1 impacts on Aß42 generation.

BACKGROUND: Amyloid-ß peptide ending at 42nd residue (Aß42) is believed as a pathogenic peptide for Alzheimer disease. Although γ-secretase is a responsible protease to generate Aß through a processive cleavage, the proteolytic mechanism of γ-secretase at molecular level is poorly understood. RESULTS: We found that the transmembrane domain (TMD) 1 of presenilin (PS) 1, a catalytic subunit for the γ-secretase, as a key modulatory domain for Aß42 production. Aß42-lowering and -raising γ-secretase modulators (GSMs) directly targeted TMD1 of PS1 and affected its structure. A point mutation in TMD1 caused an aberrant secretion of longer Aß species including Aß45 that are the precursor of Aß42. We further found that the helical surface of TMD1 is involved in the binding of Aß45/48 and that the binding was altered by GSMs as well as TMD1 mutation. CONCLUSIONS: Binding between PS1 TMD1 and longer Aß is critical for Aß42 production.

[Survey of analytical works for drugs at emergency and critical care centers with high-performance instruments provided by the Ministry of Health and Welfare (at present: Ministry of Health, Labour, and Welfare) in fiscal 1998--continuation of survey with 2008 survey results as point of reference].

In a 2008 survey of the 73 emergency and critical care centers around the nation that were equipped with the drug and chemical analytical instrument provided by the Ministry of Welfare (currently the Ministry of Health, Labour, and Welfare) in 1998, 36 of those facilities were using the analytical instruments. Of these 36 facilities, a follow-up survey of the 17 facilities that recorded 50 or analyses per year. Responses were gained from 16 of the facilities and we learned that of those, 14 facilities (87.5%) were conducting analyses using the instrument. There was a positive mutual correlation between the annual number of cases of the 14 facilities conducting analyses with the instrument and the number of work hours. Depending on the instrument in use, average analytical instrument parts and maintenance expenses were roughly three million yen and consumables required a maximum three million yen for analysis of 51-200 cases per year. From this, we calculate that such expenses can be covered under the allowed budget for advanced emergency and critical care centers of 5,000 NHI points (1 point = 10 yen). We found there were few facilities using the instrument for all 15 of the toxic substances recommended for testing by the Japanese Society for Clinical Toxicology. There tended to be no use of the analytical instrument for compounds with no toxicology cases. However, flexible responses were noted at each facility in relation to frequently analyzed compounds. It is thought that a reevaluation of compounds subject to analysis is required.

[Study of the serum concentrations of acetaminophen overdose].

Acetaminophen (APAP) is a commonly used nonsteroidal analgesic because it is considered safe. However, APAP is a major cause of acute poisoning because of its easy availability. APAP overdose causes hepatic failure. A previous study reported a case of death occurring 3-4 days after APAP overdose. Serum APAP level is an index for administration of N-acetyl-L-cysteine (NAC). We investigated cases of APAP overdose to determine the correlation between serum APAP level and estimated APAP dosage, NAC medication, hepatic failure, etc. In one case, we found that the use of estimated APAP dosage alone led to inappropriate NAC medication. Moreover, there were cases in which serum APAP level increased 4 hr after APAP overdose. Repeated cases of APAP overdose suggested that the presence of NAC medication caused a difference in liver function test values.

Participation of transmembrane domain 1 of presenilin 1 in the catalytic pore structure of the γ-secretase.

γ-Secretase is an intramembrane-cleaving protease that is responsible for the generation of amyloid-ß peptides linked to the pathogenesis of Alzheimer's disease. Using a substituted cysteine accessibility method, we have previously shown that the hydrophilic "catalytic pore" structure of γ-secretase is formed by the transmembrane domains (TMDs) 6, 7, and 9 of presenilin 1 (PS1), the catalytic subunit of γ-secretase, within the membrane. Here, we analyzed the structure in and around the first hydrophobic region, the putative TMD1, of PS1, of which the precise function as well as three-dimensional location within γ-secretase remained unknown. We found that TMD1 is located in proximity to the catalytic GxGD and PAL motifs within the C-terminal fragment of PS1, facing directly the catalytic pore. Competition experiments using known γ-secretase inhibitors suggested that the N-terminal region of TMD1 functions as a subsite during proteolytic action of the γ-secretase. Intriguingly, binding of inhibitors affected water accessibility of residues at the membrane border of TMD1, suggesting the possibility of a dynamic motion of TMD1 during the catalytic process. Our results provide mechanistic insights into the functional role of TMD1 of PS1 in the intramembrane-cleaving activity of the γ-secretase.

Functional analysis of the transmembrane domains of presenilin 1: participation of transmembrane domains 2 and 6 in the formation of initial substrate-binding site of gamma-secretase.

gamma-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2, which mediates intramembrane proteolysis of a range of type I transmembrane proteins. We previously analyzed the functional roles of the N-terminal transmembrane domains (TMDs) 1-6 of PS1 in the assembly and proteolytic activity of the gamma-secretase using a series of TMD-swap PS1 mutants. Here we applied the TMD-swap method to all the TMDs of PS1 for the structure-function analysis of the proteolytic mechanism of gamma-secretase. We found that TMD2- or -6-swapped mutant PS1 failed to bind the helical peptide-based, substrate-mimic gamma-secretase inhibitor. Cross-linking experiments revealed that both TMD2 and TMD6 of PS1 locate in proximity to the TMD9, the latter being implicated in the initial substrate binding. Taken together, our data suggest that TMD2 and the luminal side of TMD6 are involved in the formation of the initial substrate-binding site of the gamma-secretase complex.

HSP70 inducers from Chinese herbs and their effect on melanin production.

Skin hyperpigmentation disorders as a result of abnormal melanin production induced by ultraviolet (UV) irradiation are both a clinical and a cosmetic problem. This melanin production is mediated by tyrosinase whose expression is positively regulated by microphthalmia-associated transcription factor (MITF). We recently found that expression of heat shock protein 70 (HSP70) inhibits melanin production. In this study, we searched for HSP70 inducers from Chinese herbs and selected an ethanol extract of Eupatorium lindleyanum (E. lindleyanum). Not only melanin production but also the activity and expression of tyrosinase were significantly suppressed in cells treated with E. lindleyanum extract as well as in HSP70-overexpressing cells. The expression of MITF was clearly suppressed in cells treated with E. lindleyanum extract but not in HSP70-overexpressing cells. These results suggest that E. lindleyanum extract suppresses the expression of tyrosinase and melanin production through both HSP70-dependent and HSP70-independent mechanisms.

Activation of lactoperoxidase by heme-linked protonation and heme-independent iodide binding.

Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red-shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe-His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme-linked protonation site.