Cell-free protein production of a gamma secretase homolog.

Cleavage of the transmembrane domain (TMD) of amyloid-ß precursor protein (APP) by γ-secretase, an intramembrane aspartyl protease, generates Aß peptides of various lengths that form plaques in the brains of Alzheimer's disease patients. Although the debate has not been finally resolved whether these plaques trigger the onset of Alzheimer's or are side products, disease-related mutations suggest their implication in the etiology of the dementia. These occur both in presenilin, the catalytic subunit of γ-secretase, and in the TMD of APP. Despite two seminal cryo-electron microscopy structures that show the complex of γ-secretase with its substrates APP and Notch, the mechanism of γ-secretase is not yet fully understood. Especially on which basis it selects its substrates is still an enigma. The presenilin homolog PSH from the archaeon Methanoculleus marisnigri JR1 (MCMJR1) is catalytically active without accessory proteins in contrast to γ-secretase making it an excellent model for studies of the basic cleavage process. We here focused on the cell-free expression of PSH screening a range of conditions. Cleavage assays to verify the activity show that not only the yield, but mainly the activity of the protease depends on the careful selection of expression conditions. Optimal results were found for a cell-free expression at relatively low temperature, 20 °C, employing cell lysates prepared from E. coli Rosetta cells. To speed up protein preparation for immediate functional assays, a crude purification protocol was developed. This allows to produce ready-made PSH in a fast and efficient manner in less than two days.

Substrate Selection Criteria in Regulated Intramembrane Proteolysis.

Alzheimer's disease is the most common form of dementia encountered in an aging population. Characteristic amyloid deposits of Aß peptides in the brain are generated through cleavage of amyloid precursor protein (APP) by γ-secretase, an intramembrane protease. Cryo-EM structures of substrate γ-secretase complexes revealed details of the process, but how substrates are recognized and enter the catalytic site is still largely ignored. γ-Secretase cleaves a diverse range of substrate sequences without a common consensus sequence, but strikingly, single point mutations within the transmembrane domain (TMD) of specific substrates may greatly affect cleavage efficiencies. Previously, conformational flexibility was hypothesized to be the main criterion for substrate selection. Here we review the 3D structure and dynamics of several γ-secretase substrate TMDs and compare them with mutants shown to affect the cleavage efficiency. In addition, we present structural and dynamic data on ITGB1, a known nonsubstrate of γ-secretase. A comparison of biophysical details between these TMDs and changes generated by introducing crucial mutations allowed us to unravel common principles that differ between substrates and nonsubstrates. We identified three motifs in the investigated substrates: a highly flexible transmembrane domain, a destabilization of the cleavage region, and a basic signature at the end of the transmembrane helix. None of these appears to be exclusive. While conformational flexibility on its own may increase cleavage efficiency in well-known substrates like APP or Notch1, our data suggest that the three motifs seem to be rather variably combined to determine whether a transmembrane helix is efficiently recognized as a γ-secretase substrate.

The association of vaccination status with perceived discrimination in patients with COVID-19: results from a cross-sectional study.

STUDY AIMS: During the COVID-19 pandemic, there was increasing pressure to be vaccinated to prevent further spread of the virus and improve outcomes. At the same time, part of the population expressed reluctance to vaccination, for various reasons. Only a few studies have compared the perceptions of vaccinated and non-vaccinated patients being treated in hospitals for COVID-19. Our aim was to investigate the association between vaccination status and perceived healthcare-associated discrimination in patients with COVID-19 receiving hospital treatment. METHODS: Adult patients presenting to the emergency department or hospitalised for inpatient care due to or with COVID-19 from 1 June to 31 December 2021 in two Swiss hospitals were eligible. The primary endpoint was patients' perceived healthcare-associated discrimination, measured with the Discrimination in Medical Settings (DMS) scale. Secondary endpoints included different aspects of perceived quality of care and symptoms of psychological distress measured with the Hospital Anxiety and Depression Scale. RESULTS: Non-vaccinated patients (n = 113) had significantly higher DMS scores compared to vaccinated patients (n = 80) (mean: 9.54 points [SD: 4.84] vs 7.79 points [SD: 1.85]; adjusted difference: 1.18 [95% CI: 0.04-2.33 points]) and 21 of 80 vaccinated patients felt discriminated against vs 54 of 113 non-vaccinated patients (adjusted OR: 2.09 [95% CI: 1.10-3.99 ]). Non-vaccinated patients reported lower scores regarding respectful treatment by the nursing team (mean: 8.39 points [SD: 2.39] vs 9.30 points [SD: 1.09]; adjusted difference: -0.6 [95% CI: -1.18 - -0.02 points]). CONCLUSION: We found an association between vaccination status and perceived healthcare-associated discrimination. Healthcare workers should act in a professional manner regardless of a patient's vaccination status; in doing so, they might prevent the creation of negative perceptions in patients.