Engineering a Dimeric Caspase-9: A Re-evaluation of the Induced Proximity Model for Caspase Activation

Caspases are responsible for the execution of programmed cell death (apoptosis) and must undergo proteolytic activation, in response to apoptotic stimuli, to function. The mechanism of initiator caspase activation has been generalized by the induced proximity model, which is thought to drive dimerization-mediated activation of caspases. The initiator caspase, caspase-9, exists predominantly as a monomer in solution. To examine the induced proximity model, we engineered a constitutively dimeric caspase-9 by relieving steric hindrance at the dimer interface. Crystal structure of the engineered caspase-9 closely resembles that of the wild-type (WT) caspase-9, including all relevant structural details and the asymmetric nature of two monomers. Compared to the WT caspase-9, this engineered dimer exhibits a higher level of catalytic activity in vitro and induces more efficient cell death when expressed. However, the catalytic activity of the dimeric caspase-9 is only a small fraction of that for the Apaf-1-activated caspase-9. Furthermore, in contrast to the WT caspase-9, the activity of the dimeric caspase-9 can no longer be significantly enhanced in an Apaf-1-dependent manner. These findings suggest that dimerization of caspase-9 may be qualitatively different from its activation by Apaf-1, and in conjunction with other evidence, posit an induced conformation model for the activation of initiator caspases

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

The Dimeric Caspase-9 Closely Resembles the WT Caspase-9

<div><p>(A) Overall structure of the dimeric caspase-9 (C287S). The structural core is shown in blue; the solvent-exposed active site loops are shown in magenta. The β6 and β6′ strands are highlighted in red. Note the asymmetry of the dimer.</p> <p>(B) Structural superposition of the dimeric caspase-9 (blue and magenta) and the WT caspase-9 (grey and green). The only significant structural difference is in the solvent-exposed active site loops, which is due to the inactive nature of the dimeric caspase-9 (C287S).</p> <p>(C) A stereo comparison of the region surrounding strands β6 and β6′ in WT and dimeric (engineered) caspase-9. The side chains of the WT and dimeric caspase-9 are shown in orange and yellow, respectively. To avoid congestion in the graphic, residues from only one caspase-9 monomer are labeled. The Cys²⁸⁷ in the upper left corner is the catalytic residue in the active site of the asymmetric caspase-9.</p> <p>(D) A stereo comparison of the region surrounding strands β6 and β6′ in caspase-3 and dimeric (engineered) caspase-9. The side chains of the caspase-3 and dimeric caspase-9 are shown in green and yellow, respectively. The labels refer to amino acids in caspase-3.</p></div

The Dimeric Caspase-9 Exhibits Higher Catalytic Activity and Stronger Cell-Killing Activity than the WT Protein

<div><p>(A) A time course experiment of caspase-9 activity using procaspase-3 (C163A) as the substrate revealed that the dimeric caspase-9 (residues 1–416) exhibited an approximately 5-fold higher level of catalytic activity than the monomeric WT caspase-9. The concentrations were: WT and engineered caspase-9, 0.5 μM; procaspase-3 (C163A), 33 μM.</p> <p>(B) Comparison of catalytic activity for the WT and dimeric caspase-9 using fluorescent substrate LEHD-AFC. Both the full-length and Δ139 dimeric caspase-9 displayed higher activities than their WT counterparts.</p> <p>(C) The dimeric caspase-9 induced apoptosis more effectively than the WT protein. Mammalian expression vector pcDNA3 constructs encoding WT or dimeric caspase-9 were transfected into HeLa or 293 cells. The extent of cell death induced by each construct was examined.</p> <p>(D) The dimeric but not the WT caspase-9 promoted the activation of caspase-3. The expression of caspase-9 variants (upper panel) and the processing of caspase-3 (lower panel) from the cell extracts were detected by antibodies against caspase-9 and -3, respectively. No endogenous caspase-9 band was visible in the vector-transfected cells at lower exposure.</p></div

The Dimeric Caspase-9 Exhibits a Much Lower Activity Than the Apaf-1-Activated WT Caspase-9

<div><p>(A) Using fluorescent substrate LEHD-AFC, the catalytic activity of the Apaf-1-activated WT caspase-9 was approximately 35-fold greater than that of the dimeric caspase-9.</p> <p>(B) A time course comparison of the catalytic activities between the Apaf-1-activated WT caspase-9 and the dimeric caspase-9. WT and dimeric caspase-9, 0.5 μM; Apaf-1 (residues 1–570), 2 μM; LEHD-AFC, 200 μM.</p></div

The β6 Strand Is the Major Determinant for the Dimerization of Caspases

<div><p>(A) Comparison of the dimerization interfaces of caspase-3 and caspase-9. Caspases-3 and -9 exist primarily as a dimer and a monomer, respectively, in solution. However, inhibitor-bound caspase-9 was crystallized in its dimeric form [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030183#pbio-0030183-b19" target="_blank">19</a>]. The overall structures of caspases-3 and -9 are similar. A close-up view of the dimerization interfaces reveals sharp variation of residues on the β6 strand between caspase-3 and -9, which likely contributes to their different propensity for dimer formation. For caspase-9, Phe⁴⁰⁴ on strand β6 appears to impede dimerization as it sterically clashes with Phe⁴⁰⁴′ of the adjacent monomer [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030183#pbio-0030183-b19" target="_blank">19</a>].</p> <p>(B) A sequence alignment of the residues on and surrounding the strand β6 in four representative caspases. Similar to caspase-3, caspases-6 and -7 also exist as homodimers in solution. Residues on β6 are highlighted in blue and orange for the monomeric and dimeric caspases, respectively. Note that the variation of residues on β6 strand appears to correlate with the propensity for dimerization.</p></div

The Dimeric Caspase-9 Exists as a Monomer in Solution

<p>The apparent molecular masses for the WT and dimeric caspase-9 (full-length, residues 1–416) were analyzed by gel filtration. Relevant peak fractions were visualized by SDS-PAGE followed by Coomassie staining. The dimeric caspase-9 was eluted from the column with a molecular mass about twice that of the WT protein.</p