The gateway to chloroplast: re-defining the function of chloroplast receptor proteins

Chloroplast biogenesis often requires a tight orchestration between gene expression (both plastidial and nuclear) and translocation of similar to 3000 nuclear-encoded proteins into the organelle. Protein translocation is achieved via two multimeric import machineries at the outer (TOC) and inner (TIC) envelope of chloroplast, respectively. Three components constitute the core element of the TOC complex: a beta-barrel protein translocation channel Toc75 and two receptor constituents, Toc159 and Toc34. A diverse set of distinct TOC complexes have recently been characterized and these diversified TOC complexes have evolved to coordinate the translocation of differentially expressed proteins. This review aims to describe the recent discoveries relating to the typical characteristics of these distinct TOC complexes, particularly the receptor constituents, which are the main contributors for TOC complex diversification

Characterization of the protein import pathway in pea chloroplast

In order to sustain their structure and metabolism, chloroplasts and other plastid types must import the majority of their proteins from the cytosol across the envelope membrane. Translocation of these precursor proteins across the double envelope membrane is achieved by two multimeric complexes - the so-called TOC and TIC complexes (Translocon at the Outer envelope of Chloroplast and Translocon at the Inner envelope of Chloroplast, respectively). N-terminal transit peptides essential for import of the precursor proteins are cleaved after their entry into the stroma. It was thus far believed that all of the different cytosolic precursor proteins would enter the chloroplast through the same, jointly acting TOC/TIC machineries. Recent evidence, however, suggests that multiple, regulated import pathways exist in plastids that involve different import machineries. Different combinations of TOC and TIC proteins were shown to establish different import sites in Arabidopsis thaliana with specificity for either photosynthetic proteins (the general import pathway) or non-photosynthetic „housekeeping“ proteins. Moreover, numerous non-canonical import pathways such as the import of Tic32 and AtQORH mediated by the yet unknown novel import pathway and the import via the secretory pathway were shown to exist. Proteomics studies have revealed the presence of a large number of plastid proteins lacking predictable N-terminal transit sequences for import. The import mechanism for the majority of these proteins has not been determined yet. Examples of the transit sequenceless precursor proteins are the chloroplast envelope quinone oxidoreductase homologue, AtQORH and the chloroplast inner envelope protein 32, Tic32. Both proteins are imported into the inner plastid envelope membrane by a non-canonical pathway (Toc159- and Toc75-independent) and without any proteolytic cleavage. In the present study not only the import characteristic of nine tentative ‘non-canonical’ chloroplast precursor proteins but also the new interactions between these precursor proteins and the proteins at the organellar surfaces were analyzed. Moreover, a non-canonical precursor protein without the classical transit peptide, the iron superoxide dismutase (FSD1) could be identified. Biochemical crosslinking experiments revealed that FSD1 interacts with new members of the Toc159 family in pea, namely PsToc132 and PsToc120. Using deletion mutants as well as a peptide scanning approach, regions of the precursor protein, which are involved in receptor binding could be defined. These are distributed across the entire sequence; surprisingly only the extreme N-terminus as well as a C-proximal domain turned out to be essential for targeting and import. En route into the plastid FSD1 engages components of the general import pathway, implying that in spite of the ‘non-canonical’ targeting information and recognition by a specific receptor, this precursor protein follows a similar way across the envelope as the majority of plastid precursor proteins.Um ihre Struktur und ihren Metabolismus aufrechtzuerhalten, müssen Plastiden den Hauptteil ihrer im Zytosol synthetisierten Proteine importieren, was deren Transfer über die Hüllmembranen erfordert. Importapparate in der äußeren und inneren Hüllmembran, genannt TOC (Translocon at the Outer envelope of Chloroplast) und TIC(Translocon at the Inner envelope of Chloroplast), wurden identifiziert, die den Import von diesen plastidären Proteinen vermitteln. N-terminale Transitpeptide, die für den Import dieser Präproteine/Vorstufenproteine unerlässlich sind, werden nach deren Import im Stroma abgespalten. Bisher wurde angenommen, dass alle verschiedenen im Cytosol gebildeten Vorstufenproteine über die gleiche TOC/TIC Maschinerie in den Chloroplasten transportiert werden. Neuere Analysen belegen jedoch die Existenz verschiedener, regulierter Importwege, die unterschiedlichen Importapparate involvieren. So konnte in der Modellpflanze Arabidopsis thaliana gezeigt werden, dass verschiedene Kombinationen von TOC und TIC Proteinen unterschiedliche Importwege bilden, die vorzugsweise entweder photosynthetisch aktive Proteine (der sogenannte ‚general import pathway‘) oder nicht-photosynthetisch aktive („housekeeping“) Proteine importieren. Weiterhin wurden zahlreiche nicht-klassische Importwege beschrieben, wie zum Beispiel der Import von Tic32 und AtQORH sowie der Import über das endoplasmatische Retikulum und den Golgi-Apparat. Proteom-Analysen ergaben, dass zahlreiche in Plastiden lokalisierte Proteine keine prognostizierbaren N-terminalen Transitpeptide besitzen. Die Art und Weise ihres Imports ist bisher noch relativ unbekannt. Zwei Beispiele solcher Proteine sind ein in der plastidären Hüllmembran lokalisiertes quinone-oxidoreduktase-homolog, genannt AtQORH und eins der TIC Komponenten,Tic32. Dessen Import in die innere Hüllmembran erfolgte unabhängig von Toc159 und Toc75; zwei Komponenten des Standardproteinimportapparates, sowie ohne jede proteolytische Spaltung. Die vorliegende Arbeit analysierte sowohl die molekulare Importeigenschaften der transitpeptidelosen plastidären Vorstufenproteine als auch deren Interaktion mit Proteinen an den Organellenoberflächen. Darüber hinaus wurde „iron superoxide dismutase“ (FSD1) als eins der transitpeptidlosen plastidären lokalisierten Proteine identifiziert. Biochemische Crosslinking-Analysen zeigten, dass FSD1 mit den neuen Toc159-Homologen in Erbsen, PsToc132 und PsToc120 interagiert. Diese Daten lassen stark vermuten, dass das Vorhandensein mehrerer Toc159-Homologe, welcher an den unterschiedlichen TOC-Komplexen in Arabidopsis thaliana beteiligt sind, in Erbsen als möglich erschien. Um die Beteiligung des PsToc120 Rezeptorproteins bei der Erkennung und Sortierung der Vorstufenproteine im Cytosol zu untersuchen, wurde eine Kombination aus Deletion und eines Peptid-Arrays des FSD1-Proteins angewendet. Die Bindedomänen zwischen dem PsToc120 Rezeptorprotein und dem Vorstufenprotein, FSD1, wurden bestimmt. Dies ist zufällig über die gesamte Sequenz verteilt. Erstaunlicherweise sind nur der extreme N-Terminus sowie die C-proximale Domäne von FSD1 nötig um die Zielsteuerung und den Import in den Chloroplasten zu gewährleisten. Außerdem zeigte eine systematische Charakterisierung der Importwege von FSD1, dass FSD1, während seines Transports in den Chloroplasten mit den Bestandteilen des Standardproteinimportapparates interagiert. Dies weist darauf hin, dass der Transport von FSD1 in den Chloroplasten, trotz seines ungewöhnlichen N-terminalen Transitpeptids und die Nutzung von speziellen Rezeptorkomponenten, auf die gleiche Weise wie die Mehrzahl der plastidären Proteine erfolgt

Artilysation' of endolysin λSa2lys strongly improves its enzymatic and antibacterial activity against streptococci

Endolysins constitute a promising class of antibacterials against Gram-positive bacteria. Recently, endolysins have been engineered with selected peptides to obtain a new generation of lytic proteins, Artilysins, with specific activity against Gram-negative bacteria. Here, we demonstrate that artilysation can also be used to enhance the antibacterial activity of endolysins against Gram-positive bacteria and to reduce the dependence on external conditions. Art-240, a chimeric protein of the anti-streptococcal endolysin λSa2lys and the polycationic peptide PCNP, shows a similar species specificity as the parental endolysin, but the bactericidal activity against streptococci increases and is less affected by elevated NaCl concentrations and pH variations. Time-kill experiments and time-lapse microscopy demonstrate that the killing rate of Art-240 is approximately two-fold higher compared to wildtype endolysin λSa2lys, with a reduction in viable bacteria of 3 log units after 10min. In addition, lower doses of Art240 are required to achieve the same bactericidal effect

Association between the risk of seizure and COVID-19 vaccinations: A self-controlled case-series study

OBJECTIVE: The risk of seizure following BNT162b2 and CoronaVac vaccinations has been sparsely investigated. This study aimed to evaluate this association. METHOD: Patients who had their first seizure-related hospitalization between February 23, 2021 and January 31, 2022 were identified in Hong Kong. All seizure episodes happening on the day of vaccination (day 0) were excluded since clinicians validated that most of the cases on day 0 were syncopal episodes. Within-individual comparison using a modified self-controlled case series analysis was applied to estimate the incidence rate ratio (IRR) with 95% confidence intervals (CI) of seizure using conditional Poisson regression. RESULTS: We identified 1656 individuals who had their first seizure-related hospitalization (BNT162b2: 426; CoronaVac: 263; unvaccinated: 967) within the observation period. The incidence of seizure was 1.04 (95% CI: 0.80-1.33) and 1.11 (95% CI: 0.80-1.50) per 100,000 doses of BNT162b2 and CoronaVac administered respectively. 16 and 17 individuals received second dose after having first seizure within 28 days after first dose of BNT162b2 and CoronaVac vaccinations, respectively. None had recurrent seizures after the second dose. There was no increased risk during day 1-6 after the first (BNT162b2: IRR=1.39, 95% CI=0.75-2.58; CoronaVac: IRR=1.19, 95% CI=0.50-2.83) and second doses (BNT162b2: IRR=1.36, 95% CI 0.72-2.57; CoronaVac: IRR=0.71, 95% CI=0.22-2.30) of vaccinations. During 7-13, 14-20- and 21-27-days post-vaccination, no association was observed for both vaccines. SIGNIFICANCE: The findings demonstrated no increased risk of seizure following BNT162b2 and CoronaVac vaccinations. Future studies will be warranted to evaluate the risk of seizure following COVID-19 vaccinations in different populations with subsequent doses to ensure the generalizability

Use of endobronchial one-way valves reveals questions on etiology of spontaneous pneumothorax: report of three cases

Spontaneous pneumothoraces are believed to arise when air from the supplying airway exit via a ruptured visceral pleural bleb into the pleural cavity. Endobronchial one-way valves (EBVs) allow air exit (but not entry) from individual segmental airways. Systematic deployment of EBVs was applied to three patients with secondary spontaneous pneumothoraces and persistent airleak. In all cases, balloon-catheter occlusion of the upper lobe bronchus stopped the airleak. EBVs applied to individual upper lobe segmental airways failed to terminate the airleak, which only stopped after placements of multiple EBVs to occlude all upper lobe segments. The observation questions the traditional belief of 'one-airway-one-bleb-one-leak' in spontaneous pneumothorax

Simple non-invasive scoring systems and histological scores in predicting mortality in patients with non-alcoholic fatty liver disease: A systematic review and meta-analysis

[Background and Aim] There is debate among the hepatology community regarding the simple non-invasive scoring systems and histological scores (even it was developed for histological classification) in predicting clinical outcomes in patients with non-alcoholic fatty liver disease (NAFLD). This study aimed to determine whether the presence of simple non-invasive scoring systems and histological scores could predict all-cause mortality, liver-related mortality, and liver disease decompensation (liver failure, cirrhosis, hepatocellular carcinoma, or decompensated liver disease).[Methods] The pooled hazard ratio of prognostic factors and incidence rate per 1000 person-years in patients with NAFLD was calculated and further adjusted by two different models of handling the duplicated data.[Results] A total of 19 longitudinal studies were included. Most simple non-invasive scoring systems (Fibrosis-4 Score, BARD, and aspartate aminotransferase-to-platelet ratio index ) and histological scores (NAFLD activity score, Brunt, and "steatosis, activity, and fibrosis" ) failed in predicting mortality, and only the NAFLD fibrosis score > 0.676 showed prognostic ability to all-cause mortality (four studies, 7564 patients, 118 352 person-years followed up, pooled hazard ratio 1.44, 95% confidence interval [CI] 1.05–1.96). The incidence rate per 1000 person-years of all-cause mortality, liver-related mortality, cardiovascular-related mortality, and liver disease decompensation resulted in a pooled incidence rate per 1000 person-years of 22.65 (14 studies, 95% CI 9.62–53.31), 3.19 (7 studies, 95% CI 1.14–8.93), 6.02 (6 studies, 95% CI 4.69–7.74), and 11.46 (4 studies, 95% CI 5.33–24.63), respectively.[Conclusion] Non-alcoholic fatty liver disease fibrosis score showed promising prognostic value to all-cause mortality. Most present simple non-invasive scoring systems and histological scores failed to predict clinical outcomes.Peer reviewe

Linear B-cell epitopes in the spike and nucleocapsid proteins as markers of SARS-CoV-2 exposure and disease severity

BACKGROUND Given the unceasing worldwide surge in COVID-19 cases, there is an imperative need to develop highly specific and sensitive serology assays to define exposure to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). METHODS Pooled plasma samples from PCR positive COVID-19 patients were used to identify linear B-cell epitopes from a SARS-CoV-2 peptide library of spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins by peptide-based ELISA. Hit epitopes were further validated with 79 COVID-19 patients with different disease severity status, 13 seasonal human CoV, 20 recovered SARS patients and 22 healthy donors. FINDINGS Four immunodominant epitopes, S14P5, S20P2, S21P2 and N4P5, were identified on the S and N viral proteins. IgG responses to all identified epitopes displayed a strong detection profile, with N4P5 achieving the highest level of specificity (100%) and sensitivity (>96%) against SARS-CoV-2. Furthermore, the magnitude of IgG responses to S14P5, S21P2 and N4P5 were strongly associated with disease severity. INTERPRETATION IgG responses to the peptide epitopes can serve as useful indicators for the degree of immunopathology in COVID-19 patients, and function as higly specific and sensitive sero-immunosurveillance tools for recent or past SARS-CoV-2 infections. The flexibility of these epitopes to be used alone or in combination will allow for the development of improved point-of-care-tests (POCTs)