Privileged proteins with a second residence: dual targeting and conditional re-routing of mitochondrial proteins.

Almost all mitochondrial proteins are encoded by nuclear genes and synthesized in the cytosol as precursor proteins. Signals in the amino acid sequence of these precursors ensure their targeting and translocation into mitochondria. However, in many cases, only a certain fraction of a specific protein is transported into mitochondria, while the rest either remains in the cytosol or undergoes reverse translocation to the cytosol, and can populate other cellular compartments. This phenomenon is called dual localization which can be instigated by different mechanisms. These include alternative start or stop codons, differential transcripts, and ambiguous or competing targeting sequences. In many cases, dual localization might serve as an economic strategy to reduce the number of required genes; for example, when the same groups of enzymes are required both in mitochondria and chloroplasts or both in mitochondria and the nucleus/cytoplasm. Such cases frequently employ ambiguous targeting sequences to distribute proteins between both organelles. However, alternative localizations can also be used for signaling, for example when non-imported precursors serve as mitophagy signals or when they represent transcription factors in the nucleus to induce the mitochondrial unfolded stress response. This review provides an overview regarding the mechanisms and the physiological consequences of dual targeting.

Biliary Cast Syndrome and Secondary Sclerosing Cholangitis in Critically Ill Patient after Long-Term Treatment in the Intensive Care Unit.

Introduction: Secondary sclerosing cholangitis in critically ill patients (SSC-CIP) is a rare but underdiagnosed entity that occurs after life-threatening events and treatment in the intensive care unit (ICU). The etiology of SSC-CIP is not fully understood but may be caused by ischemic bile duct injury. SSC-CIP is a cholestatic liver disease that rapidly progresses to liver cirrhosis, with a high mortality rate in the first year of 50%. Endoscopic retrograde cholangiopancreatography (ERCP), which is the gold standard for diagnosing SSC-CIP, shows primary SC-like changes, usually in the intrahepatic bile ducts. Biliary cast formation is pathognomonic for SSC-CIP. No proven effective conservative treatment is available for SSC-CIP, and liver transplantation is the only curative therapy when liver cirrhosis or recurrent cholangitis occurs. Case Presentation: We report the case of a 47-year-old male patient who developed cholestasis after a long treatment in the ICU for severe pneumonia. ERCP showed characteristic findings with rarefication and multiple segmental stenosis in the intrahepatic bile ducts. We removed multiple biliary casts from the bile ducts. Conclusion: SSC-CIP should be considered for ICU patients with unclear cholestasis, especially when the cholestasis persists after recovery from the underlying disease. Early diagnosis is important to achieve better outcomes; without liver transplantation, the prognosis is generally poor.

Extracorporeal membrane oxygenation aggravates platelet glycoprotein V shedding and δ-granule deficiency in COVID-19-associated acute respiratory distress syndrome.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) is a lifesaving therapy in patients with acute respiratory distress syndrome (ARDS). Hemostatic complications are frequently observed in patients on ECMO and limit the success of this therapy. Platelets are key mediators of hemostasis enabling activation, aggregation, and thrombus formation by coming in contact with exposed matrix proteins via their surface receptors such as glycoprotein (GP) VI or GPIb/V/IX. Recent research has elucidated a regulatory role of the GPV subunit. The cleaved soluble GPV (sGPV) ectodomain was identified to spatiotemporally control fibrin formation through complex formation with thrombin. OBJECTIVES: We aimed to decipher the impact of ECMO on platelet phenotype and function, including the role of GPV and plasmatic sGPV. METHODS: We recruited 36 patients with ARDS in the wake of COVID-19 pneumonia and performed a longitudinal comparison of platelet phenotype and function in non-ECMO (n = 23) vs ECMO (n = 13) compared with those of healthy controls. Patients were assessed at up to 3 time points (t1 = days 1-3; t2 = days 4-6; and t3 = days 7-14 after cannulation/study inclusion). RESULTS: Agonist-induced platelet activation was assessed by flow cytometry and revealed decreased GPIIb/IIIa activation and α-granule release in all ARDS patients. During ECMO treatment, agonist-induced δ-granule release continuously decreased, which was independently confirmed by electron microscopy and was associated with a prolonged in vitro bleeding time. GPV expression on the platelet surface markedly decreased in ECMO patients compared with that in non-ECMO patients. Plasma sGPV levels were increased in ECMO patients and were associated with poor outcome. CONCLUSION: Our data demonstrate an ECMO-intrinsic platelet δ-granule deficiency and hemostatic dysfunction beyond the underlying ARDS.

Protein insertion into the inner membrane of mitochondria: routes and mechanisms.

The inner membrane of mitochondria contains hundreds of different integral membrane proteins. These proteins transport molecules into and out of the matrix, they carry out multifold catalytic reactions and they promote the biogenesis or degradation of mitochondrial constituents. Most inner membrane proteins are encoded by nuclear genes and synthesized in the cytosol from where they are imported into mitochondria by translocases in the outer and inner membrane. Three different import routes direct proteins into the inner membrane and allow them to acquire their appropriate membrane topology. First, mitochondrial import intermediates can be arrested at the level of the TIM23 inner membrane translocase by a stop-transfer sequence to reach the inner membrane by lateral insertion. Second, proteins can be fully translocated through the TIM23 complex into the matrix from where they insert into the inner membrane in an export-like reaction. Carriers and other polytopic membrane proteins embark on a third insertion pathway: these hydrophobic proteins employ the specialized TIM22 translocase to insert from the intermembrane space (IMS) into the inner membrane. This review article describes these three targeting routes and provides an overview of the machinery that promotes the topogenesis of mitochondrial inner membrane proteins.

GABA does not regulate stomatal CO2 signalling in Arabidopsis.

Optimal stomatal regulation is important for plant adaptation to changing environmental conditions and for maintaining crop yield. The guard-cell signal GABA is produced from glutamate by Glutamate Decarboxylase (GAD) during a reaction that generates carbon dioxide (CO2) as a by-product. Here, we investigated a putative connection between GABA signalling and the more clearly defined CO2 signalling pathway in guard cells. The GABA-deficient mutant lines gad2-1, gad2-2 and gad1/2/4/5 were examined for stomatal sensitivity to various CO2 concentrations. Our findings show a phenotypical discrepancy between the allelic mutant lines gad2-1 and gad2-2 - a weakened CO2 response in gad2-1 (GABI_474_E05) in contrast to a wild-type response in gad2-2 (SALK_028819) and gad1/2/4/5. Through transcriptomic and genomic investigation, we traced the response of gad2-1 to a deletion of full-length Mitogen-activated protein kinase 12 (MPK12) in the GABI-KAT line, thereafter as renamed gad2-1*. Guard cell-specific complementation of MPK12 restored the gad2-1* CO2 phenotype, which confirms the proposed importance of MPK12 to CO2 sensitivity. Additionally, we found that stomatal opening under low atmospheric CO2 occurs independently of the GABA-modulated opening-channel ALMT9. Our results confirm that GABA has a role in modulating the rate of stomatal opening and closing - but not in response to CO2  per se.

The ER-SURF pathway uses ER-mitochondria contact sites for protein targeting to mitochondria.

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER mitochondria encounter structure (ERMES) and Tom70, together with Djp1 and Lam6, are part of two parallel and partially redundant ER-to-mitochondria delivery routes. When ER-to-mitochondria transfer is prevented by loss of these two contact sites, many precursors of mitochondrial inner membrane proteins are left stranded on the ER membrane, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.

Distinct types of intramitochondrial protein aggregates protect mitochondria against proteotoxic stress.

Mitochondria consist of hundreds of proteins, most of which are inaccessible to the proteasomal quality control system of the cytosol. How cells stabilize the mitochondrial proteome during challenging conditions remains poorly understood. Here, we show that mitochondria form spatially defined protein aggregates as a stress-protecting mechanism. Two different types of intramitochondrial protein aggregates can be distinguished. The mitoribosomal protein Var1 (uS3m) undergoes a stress-induced transition from a soluble, chaperone-stabilized protein that is prevalent under benign conditions to an insoluble, aggregated form upon acute stress. The formation of Var1 bodies stabilizes mitochondrial proteostasis, presumably by sequestration of aggregation-prone proteins. The AAA chaperone Hsp78 is part of a second type of intramitochondrial aggregate that transiently sequesters proteins and promotes their folding or Pim1-mediated degradation. Thus, mitochondrial proteins actively control the formation of distinct types of intramitochondrial protein aggregates, which cooperate to stabilize the mitochondrial proteome during proteotoxic stress conditions.

Identifying a target group for selenium supplementation in high-risk cardiac surgery: a secondary analysis of the SUSTAIN CSX trial.

BACKGROUND: Recent data from the randomized SUSTAIN CSX trial could not confirm clinical benefits from perioperative selenium treatment in high-risk cardiac surgery patients. Underlying reasons may involve inadequate biosynthesis of glutathione peroxidase (GPx3), which is a key mediator of selenium's antioxidant effects. This secondary analysis aimed to identify patients with an increase in GPx3 activity following selenium treatment. We hypothesize that these responders might benefit from perioperative selenium treatment. METHODS: Patients were selected based on the availability of selenium biomarker information. Four subgroups were defined according to the patient's baseline status, including those with normal kidney function, reduced kidney function, selenium deficiency, and submaximal GPx3 activity. RESULTS: Two hundred and forty-four patients were included in this analysis. Overall, higher serum concentrations of selenium, selenoprotein P (SELENOP) and GPx3 were correlated with less organ injury. GPx3 activity at baseline was predictive of 6-month survival (AUC 0.73; p = 0.03). While selenium treatment elevated serum selenium and SELENOP concentrations but not GPx3 activity in the full patient cohort, subgroup analyses revealed that GPx3 activity increased in patients with reduced kidney function, selenium deficiency and low to moderate GPx3 activity. Clinical outcomes did not vary between selenium treatment and placebo in any of these subgroups, though the study was not powered to conclusively detect differences in outcomes. CONCLUSIONS: The identification of GPx3 responders encourages further refined investigations into the treatment effects of selenium in high-risk cardiac surgery patients.

Oxidation of Arabidopsis thaliana COX19 Using the Combined Action of ERV1 and Glutathione.

Protein import and oxidative folding within the intermembrane space (IMS) of mitochondria relies on the MIA40-ERV1 couple. The MIA40 oxidoreductase usually performs substrate recognition and oxidation and is then regenerated by the FAD-dependent oxidase ERV1. In most eukaryotes, both proteins are essential; however, MIA40 is dispensable in Arabidopsis thaliana. Previous complementation experiments have studied yeast mia40 mutants expressing a redox inactive, but import-competent versions of yeast Mia40 using A. thaliana ERV1 (AtERV1) suggest that AtERV1 catalyzes the oxidation of MIA40 substrates. We assessed the ability of both yeast and Arabidopsis MIA40 and ERV1 recombinant proteins to oxidize the apo-cytochrome reductase CCMH and the cytochrome c oxidase assembly protein COX19, a typical MIA40 substrate, in the presence or absence of glutathione, using in vitro cysteine alkylation and cytochrome c reduction assays. The presence of glutathione used at a physiological concentration and redox potential was sufficient to support the oxidation of COX19 by AtERV1, providing a likely explanation for why MIA40 is not essential for the import and oxidative folding of IMS-located proteins in Arabidopsis. The results point to fundamental biochemical differences between Arabidopsis and yeast ERV1 in catalyzing protein oxidation.

Factors Affecting the Duration of Surgery in the Management of Condylar Head Fractures.

Prolonged operation times should be avoided due to the associated complications and negative effects on the efficiency of the use of operating room resources. Surgical treatment of mandibular condylar head fractures is a well-established routine procedure at our department, nevertheless, we recognized fluctuating operating times. This study aims to pinpoint the influencing factors, in particular the hypothesis whether the efficiency of intraoperative muscle relaxation may decisively affect the duration of surgery. It analyses 168 mandibular condylar head fractures that were surgically treated in the period from 2007 to 2022 regarding the duration of the surgery and potential factors affecting it. The potential predictors' influence on the dependent variable operation time was mainly calculated as a bivariate analysis or linear regression. Efficiency of relaxation (p ≤ 0.001), fragmentation type (p = 0.031), and fracture age (p = 0.003) could be identified as decisive factors affecting the duration of surgery, as the first surgeon was a constant. In conclusion, surgical intervention should start as soon as possible after a traumatic incident. In addition, a dosage regimen to optimize the efficiency of relaxation should be established in future studies. Fragmentation type and concomitant fractures should also be considered for a more accurate estimation of the operating time.

High-dose methylprednisolone pulse therapy during refractory COVID-19 acute respiratory distress syndrome: a retrospective observational study.

BACKGROUND: Current COVID-19 guidelines recommend the early use of systemic corticoids for COVID-19 acute respiratory distress syndrome (ARDS). It remains unknown if high-dose methylprednisolone pulse therapy (MPT) ameliorates refractory COVID-19 ARDS after many days of mechanical ventilation or rapid deterioration with or without extracorporeal membrane oxygenation (ECMO). METHODS: This is a retrospective observational study. Consecutive patients with COVID-19 ARDS treated with a parenteral high-dose methylprednisolone pulse therapy at the intensive care units (ICU) of two University Hospitals between January 1st 2021 and November 30st 2022 were included. Clinical data collection was at ICU admission, start of MPT, 3-, 10- and 14-days post MPT. RESULTS: Thirty-seven patients (mean age 55 ± 12 years) were included in the study. MPT started at a mean of 17 ± 12 days after mechanical ventilation. Nineteen patients (54%) received ECMO support when commencing MPT. Mean paO2/FiO2 significantly improved 3- (p = 0.034) and 10 days (p = 0.0313) post MPT. The same applied to the necessary FiO2 10 days after MPT (p = 0.0240). There were no serious infectious complications. Twenty-four patients (65%) survived to ICU discharge, including 13 out of 20 (65%) needing ECMO support. CONCLUSIONS: Late administration of high-dose MPT in a critical subset of refractory COVID-19 ARDS patients improved respiratory function and was associated with a higher-than-expected survival of 65%. These data suggest that high-dose MPT may be a viable salvage therapy in refractory COVID-19 ARDS.

The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.

Chasing the right tail: How the ER membrane complex recognizes its substrates.

Tail-anchored proteins are tethered to membranes of the ER, mitochondria, and peroxisomes. In this issue, Pleiner and colleagues (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202212007) show that the ER membrane complex (EMC) uses an inbuilt charge-dependent selectivity filter to specifically insert ER tail-anchored proteins according to their topology signals and to prevent the misincorporation of mitochondrial proteins.

Membrane insertases at a glance.

Protein translocases, such as the bacterial SecY complex, the Sec61 complex of the endoplasmic reticulum (ER) and the mitochondrial translocases, facilitate the transport of proteins across membranes. In addition, they catalyze the insertion of integral membrane proteins into the lipid bilayer. Several membrane insertases cooperate with these translocases, thereby promoting the topogenesis, folding and assembly of membrane proteins. Oxa1 and BamA family members serve as core components in the two major classes of membrane insertases. They facilitate the integration of proteins with α-helical transmembrane domains and of ß-barrel proteins into lipid bilayers, respectively. Members of the Oxa1 family were initially found in the internal membranes of bacteria, mitochondria and chloroplasts. Recent studies, however, also identified several Oxa1-type insertases in the ER, where they serve as catalytically active core subunits in the ER membrane protein complex (EMC), the guided entry of tail-anchored (GET) and the GET- and EMC-like (GEL) complex. The outer membrane of bacteria, mitochondria and chloroplasts contain ß-barrel proteins, which are inserted by members of the BamA family. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of these different types of membrane insertases and discuss their function.

The unfolded protein response of the endoplasmic reticulum supports mitochondrial biogenesis by buffering nonimported proteins.

Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of nonimported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitochondrial membrane proteins at the endoplasmic reticulum, thereby triggering the unfolded protein response (UPRER). Moreover, we find that mitochondrial membrane proteins are also routed to the ER under physiological conditions. The level of ER-resident mitochondrial precursors is enhanced by import defects as well as metabolic stimuli that increase the expression of mitochondrial proteins. Under such conditions, the UPRER is crucial to maintain protein homeostasis and cellular fitness. We propose the ER serves as a physiological buffer zone for those mitochondrial precursors that cannot be immediately imported into mitochondria while engaging the UPRER to adjust the ER proteostasis capacity to the extent of precursor accumulation.

The role of the proteasome in mitochondrial protein quality control.

The abundance of each cellular protein is dynamically adjusted to the prevailing metabolic and stress conditions by modulation of their synthesis and degradation rates. The proteasome represents the major machinery for the degradation of proteins in eukaryotic cells. How the ubiquitin-proteasome system (UPS) controls protein levels and removes superfluous and damaged proteins from the cytosol and the nucleus is well characterized. However, recent studies showed that the proteasome also plays a crucial role in mitochondrial protein quality control. This mitochondria-associated degradation (MAD) thereby acts on two layers: first, the proteasome removes mature, functionally compromised or mis-localized proteins from the mitochondrial surface; and second, the proteasome cleanses the mitochondrial import pore of import intermediates of nascent proteins that are stalled during translocation. In this review, we provide an overview about the components and their specific functions that facilitate proteasomal degradation of mitochondrial proteins in the yeast Saccharomyces cerevisiae. Thereby we explain how the proteasome, in conjunction with a set of intramitochondrial proteases, maintains mitochondrial protein homeostasis and dynamically adapts the levels of mitochondrial proteins to specific conditions.

Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast.

The biogenesis of mitoribosomes is an intricate process that relies on the coordinated synthesis of nuclear-encoded mitoribosomal proteins (MRPs) in the cytosol, their translocation across mitochondrial membranes, the transcription of rRNA molecules in the matrix as well as the assembly of the roughly 80 different constituents of the mitoribosome. Numerous chaperones, translocases, processing peptidases, and assembly factors of the cytosol and in mitochondria support this complex reaction. The budding yeast Saccharomyces cerevisiae served as a powerful model organism to unravel the different steps by which MRPs are imported into mitochondria, fold into their native structures, and assemble into functional ribosomes.In this chapter, we provide established protocols to study these different processes experimentally. In particular, we describe methods to purify mitochondria from yeast cells, to import radiolabeled MRPs into isolated mitochondria, and to elucidate the assembly reaction of MRPs by immunoprecipitation. These protocols and the list of dos and don'ts will enable beginners and experienced scientists to study the import and assembly of MRPs.

A modular cloning (MoClo) toolkit for reliable intracellular protein targeting in the yeast Saccharomyces cerevisiae.

Modular Cloning (MoClo) allows the combinatorial assembly of plasmids from standardized genetic parts without the need of error-prone PCR reactions. It is a very powerful strategy which enables highly flexible expression patterns without the need of repetitive cloning procedures. In this study, we describe an advanced MoClo toolkit that is designed for the baker's yeast Saccharomyces cerevisiae and optimized for the targeting of proteins of interest to specific cellular compartments. Comparing different targeting sequences, we developed signals to direct proteins with high specificity to the different mitochondrial subcompartments, such as the matrix and the intermembrane space (IMS). Furthermore, we optimized the subcellular targeting by controlling expression levels using a collection of different promoter cassettes; the MoClo strategy allows it to generate arrays of expression plasmids in parallel to optimize gene expression levels and reliable targeting for each given protein and cellular compartment. Thus, the MoClo strategy enables the generation of protein-expressing yeast plasmids that accurately target proteins of interest to various cellular compartments.

Prospective single-center study of health-related quality of life after COVID-19 in ICU and non-ICU patients.

Long-term sequelae in hospitalized Coronavirus Disease 2019 (COVID-19) patients may result in limited quality of life. The current study aimed to determine health-related quality of life (HRQoL) after COVID-19 hospitalization in non-intensive care unit (ICU) and ICU patients. This is a single-center study at the University Hospital of Wuerzburg, Germany. Patients eligible were hospitalized with COVID-19 between March 2020 and December 2020. Patients were interviewed 3 and 12 months after hospital discharge. Questionnaires included the European Quality of Life 5 Dimensions 5 Level (EQ-5D-5L), patient health questionnaire-9 (PHQ-9), the generalized anxiety disorder 7 scale (GAD-7), FACIT fatigue scale, perceived stress scale (PSS-10) and posttraumatic symptom scale 10 (PTSS-10). 85 patients were included in the study. The EQ5D-5L-Index significantly differed between non-ICU (0.78 ± 0.33 and 0.84 ± 0.23) and ICU (0.71 ± 0.27; 0.74 ± 0.2) patients after 3- and 12-months. Of non-ICU 87% and 80% of ICU survivors lived at home without support after 12 months. One-third of ICU and half of the non-ICU patients returned to work. A higher percentage of ICU patients was limited in their activities of daily living compared to non-ICU patients. Depression and fatigue were present in one fifth of the ICU patients. Stress levels remained high with only 24% of non-ICU and 3% of ICU patients (p = 0.0186) having low perceived stress. Posttraumatic symptoms were present in 5% of non-ICU and 10% of ICU patients. HRQoL is limited in COVID-19 ICU patients 3- and 12-months post COVID-19 hospitalization, with significantly less improvement at 12-months compared to non-ICU patients. Mental disorders were common highlighting the complexity of post-COVID-19 symptoms as well as the necessity to educate patients and primary care providers about monitoring mental well-being post COVID-19.