Domain Organization, Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases

Cystathionine beta-synthase (CBS) is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS), fruit fly (dCBS) and yeast (yCBS) enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme’s catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet); however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme’s catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.This work was supported by Postdoctoral Fellowship 0920079G from the American Heart Association (to TM), by National Institutes of Health Grant HL065217, by American Heart Association Grant In-Aid 09GRNT2110159, by a grant from the Jerome Lejeune Foundation (all to JPK) and by a research contract RYC2009-04147 (to ALP). In addition, grant support (P11-CTS-07187, CSD2009-00088 and BIO2012-34937) to Dr. Jose M. Sanchez-Ruiz (University of Granada) and SGIker technical and human support (UPV/EHU, MICINN, GV/EJ, ESF) are gratefully acknowledged

Epidemiological characteristics, ventilator management, and clinical outcome in patients receiving invasive ventilation in intensive care units from 10 Asian middle-income countries (PRoVENT-iMiC): an international, multicenter, prospective study

Epidemiology, ventilator management, and outcome in patients receiving invasive ventilation in intensive care units (ICUs) in middle-income countries are largely unknown. PRactice of VENTilation in Middle-income Countries is an international multicenter 4-week observational study of invasively ventilated adult patients in 54 ICUs from 10 Asian countries conducted in 2017/18. Study outcomes included major ventilator settings (including tidal volume [VT] and positive end-expiratory pressure [PEEP]); the proportion of patients at risk for acute respiratory distress syndrome (ARDS), according to the lung injury prediction score (LIPS), or with ARDS; the incidence of pulmonary complications; and ICU mortality. In 1,315 patients included, median VT was similar in patients with LIPS < 4 and patients with LIPS ≥ 4, but lower in patients with ARDS (7.90 [6.8–8.9], 8.0 [6.8–9.2], and 7.0 [5.8–8.4] mL/kg Predicted body weight; P = 0.0001). Median PEEP was similar in patients with LIPS < 4 and LIPS ≥ 4, but higher in patients with ARDS (five [5–7], five [5–8], and 10 [5–12] cmH2O; P < 0.0001). The proportions of patients with LIPS ≥ 4 or with ARDS were 68% (95% CI: 66–71) and 7% (95% CI: 6–8), respectively. Pulmonary complications increased stepwise from patients with LIPS < 4 to patients with LIPS ≥ 4 and patients with ARDS (19%, 21%, and 38% respectively; P = 0.0002), with a similar trend in ICU mortality (17%, 34%, and 45% respectively; P < 0.0001). The capacity of the LIPS to predict development of ARDS was poor (receiver operating characteristic [ROC] area under the curve [AUC] of 0.62, 95% CI: 0.54–0.70). In Asian middle-income countries, where two-thirds of ventilated patients are at risk for ARDS according to the LIPS and pulmonary complications are frequent, setting of VT is globally in line with current recommendations