Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is ...the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein-lipid interactions are critical during translocon-mediated membrane insertion.
All proteins of the intermembrane space of mitochondria are encoded by nuclear genes and synthesized in the cytosol. Many of these proteins lack presequences but are imported into mitochondria in an ...oxidation-driven process that relies on the activity of Mia40 and Erv1. Both factors form a disulfide relay system in which Mia40 functions as a receptor that transiently interacts with incoming polypeptides via disulfide bonds. Erv1 is a sulfhydryl oxidase that oxidizes and activates Mia40, but it has remained unclear how Erv1 itself is oxidized. Here, we show that Erv1 passes its electrons on to molecular oxygen via interaction with cytochrome c and cytochrome c oxidase. This connection to the respiratory chain increases the efficient oxidation of the relay system in mitochondria and prevents the formation of toxic hydrogen peroxide. Thus, analogous to the system in the bacterial periplasm, the disulfide relay in the intermembrane space is connected to the electron transport chain of the inner membrane.
A disulphide relay system mediates the import of cysteine‐containing proteins into the intermembrane space of mitochondria. This system consists of two essential proteins, Mia40 and Erv1, which bind ...to newly imported proteins by disulphide transfer. A third component, Hot13, was proposed to be important in the biogenesis of cysteine‐rich proteins of the intermembrane space, but the molecular function of Hot13 remained unclear. Here, we show that Hot13, a conserved zinc‐binding protein, interacts functionally and physically with the import receptor Mia40. It improves the Erv1‐dependent oxidation of Mia40 both in vivo and in vitro. As a consequence, in mutants lacking Hot13, the import of substrates of Mia40 is impaired, particularly in the presence of zinc ions. In mitochondria as well as in vitro, Hot13 can be functionally replaced by zinc‐binding chelators. We propose that Hot13 maintains Mia40 in a zinc‐free state, thereby facilitating its efficient oxidation by Erv1.
Puumala hantavirus (PUUV) infections usually show a mild or moderate clinical course, but may sometimes also lead to life-threatening disease. Here, we report on a 60-year-old female patient with ...common variable immunodeficiency (CVID) who developed a fatal PUUV infection with persistent renal failure, thrombocytopenia, and CNS infection with impaired consciousness and tetraparesis. Hantavirus-specific antibodies could not be detected due to the humoral immunodeficiency. Diagnosis and virological monitoring were based on the quantitative detection of PUUV RNA in blood, cerebrospinal fluid, bronchial lavage, and urine, where viral RNA was found over an unusually extended period of one month. Due to clinical deterioration and virus persistence, treatment with ribavirin was initiated. Additionally, fresh frozen plasma (FFP) from convalescent donors with a history of PUUV infection was administered. Despite viral clearance, the clinical condition of the patient did not improve and the patient died on day 81 of hospitalization. This case underlines the importance of the humoral immune response for the course of PUUV disease and illustrates the need for PCR-based virus diagnostics in those patients. Due to its potential antiviral activity, convalescent plasma should be considered in the therapy of severe hantavirus diseases.
The Mia40-Erv1 disulfide relay system is of high importance for mitochondrial biogenesis. Most so far identified substrates of this machinery contain either two cysteine-x(3)-cysteine (twin Cx(3)C) ...or two cysteine-x(9)-cysteine (twin Cx(9)C) motifs. While the first group is composed of well-characterized components of the mitochondrial import machinery, the molecular function of twin Cx(9)C proteins still remains unclear. To systematically characterize this protein family, we performed a database search to identify the full complement of Cx(9)C proteins in yeast. Thereby, we identified 14 potential family members, which, with one exception, are conserved among plants, fungi, and animals. Among these, three represent novel proteins, which we named Cmc2 to 4 (for Cx(9)C motif-containing protein) and which we demonstrated to be dependent for import on the Mia40-Erv1 disulfide relay. By testing deletion mutants of all 14 proteins for function of the respiratory chain, we found a critical function of most of these proteins for the assembly or stability of respiratory chain complexes. Our data suggest that already early during the evolution of eukaryotic cells, a multitude of twin Cx(9)C proteins developed, which exhibit largely nonredundant roles critical for the biogenesis of enzymes of the respiratory chain in mitochondria.
spp. are frequently encountered in specimens from ICUs. However, most of these detections represent colonization. Nevertheless, clinical practice shows that a considerable proportion of these ...patients will receive antifungal therapy (AT). β-(1→3)-D-glucan (BDG) and mannan are fungal biomarkers with high negative predictive values. We aimed to examine whether biomarker-guided discontinuation of AT can reduce the antifungal consumption. Therefore, we conducted a prospective, randomized intervention study between 1 April 2019 and 31 March 2020. All adult ICU patients with a newly started systemic AT but without fungal infection were eligible for inclusion. Enrolled patients were randomized into an intervention and a control group. In both groups, serum BDG and mannan were determined on days 1 and 2 of AT. If all measurements were negative, AT was discontinued in the intervention group. The primary endpoint was antifungal use. The study was terminated after 12 months. Until this time-point, 41 patients had been included. In the intervention group (
= 19), AT was stopped in only two patients because all others showed either positive BDG and/or mannan levels. One of these two patients developed candidemia and AT had to be restarted. There was no significant difference in the primary and secondary endpoints. In summary, the strategy of using two negative BDG and mannan levels to stop AT failed to reduce antifungal consumption in our cohort. Indeed, there will inevitably be patients with invasive candidiasis in whom necessary AT is discontinued. The optimal patient population, biomarker set, and termination criteria are critical to the success of biomarker-based termination strategies.
Abstract
Background
Severe COVID-19 induced acute respiratory distress syndrome (ARDS) often requires extracorporeal membrane oxygenation (ECMO). Recent German health insurance data revealed low ICU ...survival rates. Patient characteristics and experience of the ECMO center may determine intensive care unit (ICU) survival. The current study aimed to identify factors affecting ICU survival of COVID-19 ECMO patients.
Methods
673 COVID-19 ARDS ECMO patients treated in 26 centers between January 1st 2020 and March 22nd 2021 were included. Data on clinical characteristics, adjunct therapies, complications, and outcome were documented. Block wise logistic regression analysis was applied to identify variables associated with ICU-survival.
Results
Most patients were between 50 and 70 years of age. PaO
2
/FiO
2
ratio prior to ECMO was 72 mmHg (IQR: 58–99). ICU survival was 31.4%. Survival was significantly lower during the 2nd wave of the COVID-19 pandemic. A subgroup of 284 (42%) patients fulfilling modified EOLIA criteria had a higher survival (38%) (
p
= 0.0014, OR 0.64 (CI 0.41–0.99)). Survival differed between low, intermediate, and high-volume centers with 20%, 30%, and 38%, respectively (
p
= 0.0024). Treatment in high volume centers resulted in an odds ratio of 0.55 (CI 0.28–1.02) compared to low volume centers. Additional factors associated with survival were younger age, shorter time between intubation and ECMO initiation, BMI > 35 (compared to < 25), absence of renal replacement therapy or major bleeding/thromboembolic events.
Conclusions
Structural and patient-related factors, including age, comorbidities and ECMO case volume, determined the survival of COVID-19 ECMO. These factors combined with a more liberal ECMO indication during the 2nd wave may explain the reasonably overall low survival rate. Careful selection of patients and treatment in high volume ECMO centers was associated with higher odds of ICU survival.
Trial registration
Registered in the German Clinical Trials Register (study ID: DRKS00022964, retrospectively registered, September 7th 2020,
https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00022964
.
Graphical abstract
To identify yeast mutants that show a strong redox dependence of the ability to respire, we systematically screened a yeast deletion library for mutants that require the presence of reductants for ...growth on nonfermentable carbon sources.
Respirative growth of 44 yeast mutants was significantly improved by the addition of dithiothreitol or glutathione. Two mutants that were strongly stimulated by reductants lacked the proteins Cmc1 and Coa4. Both proteins belong to the family of "twin Cx(9)C" proteins present in the intermembrane space of mitochondria. Deletion of CMC1 or COA4 leads to assembly defects of cytochrome c oxidase, in particular to the lack of Cox1 and rapid degradation of Cox2 and Cox3. Interestingly, the presence of the reductants does not suppress these assembly defects and the levels of cytochrome c oxidase remain reduced. Reductants and antioxidants such as ascorbic acid rather counteract the effects of hydrogen peroxide that is produced from partially assembled cytochrome c oxidase intermediates.
Here we show that oxidative stress generated by the accumulation of partially assembled respiratory chain complexes prevents growth on carbon sources that force cells to respire.
Defects in the assembly of cytochrome c oxidase can lead to increased production of hydrogen peroxide, which is sensed in cells and blocks their proliferation. We propose that this redox-regulated feedback regulation specifically slows down the propagation of cells carrying respiratory chain mutations in order to select for cells of high mitochondrial fitness.
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