Structural and functional studies of Arabidopsis thaliana legumain beta reveal isoform specific mechanisms of activation and substrate recognition.

The vacuolar cysteine protease legumain plays important functions in seed maturation and plant programmed cell death. Because of their dual protease and ligase activity, plant legumains have become of particular biotechnological interest, e.g. for the synthesis of cyclic peptides for drug design or for protein engineering. However, the molecular mechanisms behind their dual protease and ligase activities are still poorly understood, limiting their applications. Here, we present the crystal structure of Arabidopsis thaliana legumain isoform ß (AtLEGß) in its zymogen state. Combining structural and biochemical experiments, we show for the first time that plant legumains encode distinct, isoform-specific activation mechanisms. Whereas the autocatalytic activation of isoform γ (AtLEGγ) is controlled by the latency-conferring dimer state, the activation of the monomeric AtLEGß is concentration independent. Additionally, in AtLEGß the plant-characteristic two-chain intermediate state is stabilized by hydrophobic rather than ionic interactions, as in AtLEGγ, resulting in significantly different pH stability profiles. The crystal structure of AtLEGß revealed unrestricted nonprime substrate binding pockets, consistent with the broad substrate specificity, as determined by degradomic assays. Further to its protease activity, we show that AtLEGß exhibits a true peptide ligase activity. Whereas cleavage-dependent transpeptidase activity has been reported for other plant legumains, AtLEGß is the first example of a plant legumain capable of linking free termini. The discovery of these isoform-specific differences will allow us to identify and rationally design efficient ligases with application in biotechnology and drug development.

Development of a generic reversed-phase liquid chromatography method for protein quantification using analytical quality-by-design principles.

Biopharmaceutical drug substances are generally produced using fermentation technology and are subsequently purified in the following downstream process. For the determination of critical quality attributes (CQAs), such as target protein titer and purity, monitoring tools are required before quality control analysis. We herein present a novel reversed phase liquid chromatography method (RPLC), which enables facile and robust protein quantification during upstream and downstream processing of intracellularly produced proteins in E. coli. The overall goal was to develop a fast, robust and mass spectrometry compatible method which can baseline resolve and quantify each protein of interest. Method development consisted of three steps, oriented on an Analytical Quality by Design (AQbD) workflow: (i) the stationary phase as primary parameter was chosen based on state-of-the art technology thus minimizing protein on-column adsorption and providing high efficiency, (ii) secondary parameters (i.e. gradient conditions and column temperature) were optimized applying chromatographic modeling, and (iii) the established Method Operable Design Region (MODR) was challenged and confirmed during robustness testing, performed in-silico and experimentally by a Design of experiment (DoE) based approach. Finally, we validated the RPLC method for pivotal validation parameters (i.e. linearity, limit of quantification, and repeatability) and compared it for protein quantification against a well-established analytical methodology. The outcome of this study shows (i) a protocol for RPLC development using an AQbD principle for new method generation and (ii) a highly versatile RPLC method, suited for quick and straightforward recombinant protein titer measurement being applicable for the detection of a broad range of proteins.

Septal total atrial conduction time for prediction of atrial fibrillation in embolic stroke of unknown source: a pilot study.

BACKGROUND: Subclinical atrial fibrillation (AF) is the underlying cause in a relevant part of patients with embolic stroke of unknown source (ESUS). This pilot study aims to identify novel echocardiographic parameters predicting AF subsequently detected in patients originally hospitalized with ESUS. METHODS AND RESULTS: Patients with acute ischemic stroke [baseline diagnosis of ESUS (n = 69), stroke of macro- or microvascular cause (n = 16/25), stroke caused by AF (n = 5)] and controls with paroxysmal AF without acute ischemic stroke (n = 22) as well as healthy controls of young and old age (n = 21/17) in sinus rhythm were included (overall n = 175). Echocardiography was performed in all participants. Prolonged Holter-ECG-monitoring was performed in all stroke patients. In the overall cohort, septal total atrial conduction time (sPA-TDI), left atrial (LA) volume index to tissue Doppler velocity (LAVI/a`) and second negative peak strain rate during LA contraction (SRa), representing echocardiographic parameters of LA remodelling and function, were statistically significant different in patients with and without AF and predictive for subclinical AF (multivariate regression analysis: sPA-TDI: HR 1.06 [1.04-1.08], p < 0.001; LAVI/a`: HR 0.85, [0.74-0.97], p = 0.02; SRa: HR 2.35 [0.9-5.5], p = 0.05). Multivariate Cox regression analysis revealed sPA-TDI as an independent predictor of AF in ESUS patients (sPA-TDI: HR 1.10 [1.04-1.17], p = 0.001). A sPA-TDI of 126 ms strictly discriminated between presence and absence of subclinical AF within 48 h after initiation of Holter-ECG-monitoring in ESUS patients. CONCLUSIONS: sPA-TDI seems to be a strong independent predictor of subclinical AF in patients hospitalized for ESUS and might support risk-stratified clinical decision making in these patients. Septal Total Atrial Conduction Time (sPA-TDI) determined by echocardiography for prediction of Atrial Fibrillation in Embolic Stroke of Unknown Source (ESUS).

Mortality in patients with cardiogenic shock treated with the Impella CP microaxial pump for isolated left ventricular failure.

AIMS: Cardiogenic shock is still associated with high mortality rates of around 50%. Intra-aortic counterpulsation had been frequently used in cardiogenic shock, but was previously found to provide no mortality benefit. We investigated the effect of an interdisciplinary and multiprofessional routine strategy of early invasive haemodynamic support in combination with complete revascularization in patients with cardiogenic shock before admission to our intensive care unit. METHODS AND RESULTS: We analysed all cardiogenic shock patients (mean age 62±13 years) presenting at our institution between 2013 and mid 2016, who received an Impella CP microaxial pump for isolated left ventricle support (n=61). Sixty-one per cent (n=37) had been resuscitated before Impella insertion. Overall mortality was 48% (n=29/61) at 30 days. Thirty-day mortality was higher in resuscitated patients (resuscitated: 65% (n=24/37); non-resuscitated: 21% (n=5/24)). When applying the inclusion/exclusion criteria of the SHOCK-II trial, eligible patients (n=25) had a markedly lower mortality (24% (n=6/25) at 30 days) compared with the published trial (~40% in both arms). The observed mortality of SHOCK-II-like patients in the registry was also lower compared with their predicted mortality using IABP-Shock II score (49%) and CardShock score (36%). CONCLUSION: The results of this registry suggest that using a standardized protocol including early active haemodynamic support with Impella CP in cardiogenic shock in patients with isolated left ventricle failure may be associated with improved outcomes and lower than previously reported or predicted mortality rates. Pre-implantation cardiac arrest critically influences observed mortality. The results support the case for a randomized trial.

Mortality in Patients With Out-of-Hospital Cardiac Arrest Undergoing a Standardized Protocol Including Therapeutic Hypothermia and Routine Coronary Angiography: Experience From the HACORE Registry.

OBJECTIVES: This study sought to analyze the impact of mandatory therapeutic hypothermia and cardiac catheterization in the absence of overt noncardiac cause of arrest as part of the Hannover Cardiac Resuscitation Algorithm before intensive care admission. BACKGROUND: Despite advanced therapies, out-of-hospital cardiac arrest (OHCA) is still associated with high mortality rates. Recently, the TTM (Target Temperature Management 33°C Versus 36°C After Out-of-Hospital Cardiac Arrest)-trial caused severe uncertainty about the efficacy of and need for therapeutic hypothermia. Furthermore, the role of early coronary angiography in OHCA survivors without ST-segment elevation remains undetermined. METHODS: In the HACORE (HAnnover Cooling REgistry) we investigated 233 consecutive patients (median age 64 [interquartile range: 53 to 74] years) with OHCA admitted to our institution between January 2011 and December 2015 who were treated according to the algorithm. RESULTS: A total of 73% had ventricular fibrillation as primary rhythm. Return of spontaneous circulation was achieved after 20 (interquartile range: 10 to 30) min. Immediate percutaneous coronary angiography was performed in 96% and coronary angioplasty in 59% of all cases. ST-segment elevation was present in 47%. Critical coronary stenosis requiring percutaneous coronary intervention was present in 67% of patients with and 52% of patients without ST-segment elevation. Overall 30-day intrahospital mortality in this real-world registry was 37%. Patients in our local registry who matched the inclusion/exclusion criteria of the TTM-trial (n = 145) had a markedly lower 30-day mortality (27%) compared with the published trial (44%). CONCLUSIONS: Standardized treatment of patients with OHCA following a strict protocol incorporating computed tomography, cardiac catheterization and revascularization, liberal use of active hemodynamic support in presence of shock, and mandatory therapeutic hypothermia results in mortality rates lower than previously reported.

Structural analyses of Arabidopsis thaliana legumain γ reveal differential recognition and processing of proteolysis and ligation substrates.

Legumain is a dual-function protease-peptide ligase whose activities are of great interest to researchers studying plant physiology and to biotechnological applications. However, the molecular mechanisms determining the specificities for proteolysis and ligation are unclear because structural information on the substrate recognition by a fully activated plant legumain is unavailable. Here, we present the X-ray structure of Arabidopsis thaliana legumain isoform γ (AtLEGγ) in complex with the covalent peptidic Ac-YVAD chloromethyl ketone (CMK) inhibitor targeting the catalytic cysteine. Mapping of the specificity pockets preceding the substrate-cleavage site explained the known substrate preference. The comparison of inhibited and free AtLEGγ structures disclosed a substrate-induced disorder-order transition with synergistic rearrangements in the substrate-recognition sites. Docking and in vitro studies with an AtLEGγ ligase substrate, sunflower trypsin inhibitor (SFTI), revealed a canonical, protease substrate-like binding to the active site-binding pockets preceding and following the cleavage site. We found the interaction of the second residue after the scissile bond, P2'-S2', to be critical for deciding on proteolysis versus cyclization. cis-trans-Isomerization of the cyclic peptide product triggered its release from the AtLEGγ active site and prevented inadvertent cleavage. The presented integrative mechanisms of proteolysis and ligation (transpeptidation) explain the interdependence of legumain and its preferred substrates and provide a rational framework for engineering optimized proteases, ligases, and substrates.

Crystal Structure of Plant Legumain Reveals a Unique Two-Chain State with pH-Dependent Activity Regulation.

The vacuolar cysteine protease legumain can cleave and selectively rebuild peptide bonds, thereby vastly expanding the sequential repertoire of biomolecules. In this context, plant legumains have recently attracted particular interest. Furthermore, legumains have important roles in many physiological processes, including programmed cell death. Their efficient peptide bond ligase activity has gained tremendous interest in the design of cyclic peptides for drug design. However, the mechanistic understanding of these dual activities is incomplete and partly conflicting. Here, we present the crystal structure of a plant legumain, Arabidopsis thaliana isoform-γ (AtLEGγ). Employing a conserved legumain fold, the plant legumain AtLEGγ revealed unique mechanisms of autoactivation, including a plant-specific two-chain activation state, which remains conformationally stable at neutral pH, which is a prerequisite for full ligase activity and survival in different cell compartments. The charge distribution around the α6-helix mediates the pH-dependent dimerization and serves as a gatekeeper for the active site, thus regulating its protease and ligase activity.

Distinct Roles of Catalytic Cysteine and Histidine in the Protease and Ligase Mechanisms of Human Legumain As Revealed by DFT-Based QM/MM Simulations.

The cysteine protease enzyme legumain hydrolyzes peptide bonds with high specificity after asparagine and under more acidic conditions after aspartic acid [Baker E. N.J. Mol. Biol.1980, 141, 441-484; Baker E. N.; J. Mol. Biol.1977, 111, 207-210; Drenth J.; Biochemistry1976, 15, 3731-3738; Menard R.; J. Cell. Biochem.1994, 137; Polgar L.Eur. J. Biochem.1978, 88, 513-521; Storer A. C.; Methods Enzymol.1994, 244, 486-500. Remarkably, legumain additionally exhibits ligase activity that prevails at pH > 5.5. The atomic reaction mechanisms including their pH dependence are only partly understood. Here we present a density functional theory (DFT)-based quantum mechanics/molecular mechanics (QM/MM) study of the detailed reaction mechanism of both activities for human legumain in solution. Contrasting the situation in other papain-like proteases, our calculations reveal that the active site Cys189 must be present in the protonated state for a productive nucleophilic attack and simultaneous rupture of the scissile peptide bond, consistent with the experimental pH profile of legumain-catalyzed cleavages. The resulting thioester intermediate (INT1) is converted by water attack on the thioester into a second intermediate, a diol (INT2), which is released by proton abstraction by Cys189. Surprisingly, we found that ligation is not the exact reverse of the proteolysis but can proceed via two distinct routes. Whereas the transpeptidation route involves aminolysis of the thioester (INT1), at pH 6 a cysteine-independent, histidine-assisted ligation route was found. Given legumain's important roles in immunity, cancer, and neurodegenerative diseases, our findings open up possibilities for targeted drug design in these fields.

One symptom, two arrhythmias: the rare and the even rarer.

BACKGROUND: Wolff-Parkinson-White (WPW) syndrome and idiopathic left ventricular tachycardia (ILVT) are rare and up to now the coexistence of both entities has rarely been reported. In patients with ventricular preexcitation the underlying mechanism of paroxysmal tachycardia most likely is atrioventricular reentrant tachycardia (AVRT). However, without ECG documentation of the tachycardia diagnosis of the underlying mechanism cannot be made due to similar clinical presentation of AVRT and ILVT. CASE PRESENTATION: We report a case of a two-staged occurrence of two rare arrhythmias in a young adult, who was admitted to our hospital twice within 6 months because of paroxysmal tachycardia. WPW syndrome and ILVT as underlying arrhythmias have been diagnosed and were ablated successfully. CONCLUSIONS: This case highlights the diagnostic defiance of rare tachycardia entities and the paramount importance of ECG documentation and analysis of all available tachycardia ECGs.

Acquired von Willebrand syndrome in cardiogenic shock patients on mechanical circulatory microaxial pump support.

Early use of mechanical circulatory support, e.g. veno-arterial extracorporeal membrane oxygenation (ECMO) or left ventricular unloading by microaxial pump in refractory cardiogenic shock is recommended in current guidelines. Development of acquired von Willebrand Syndrome (AVWS) in patients with left ventricular assist devices (LVADs) and ECMO has been reported. There is an increasing number of patients treated with the Impella® CP microaxial pump for left ventricular unloading. However, the prevalence of AVWS in these high risk patients is unknown and needs to be determined. We therefore screened 21 patients (68 ± 11years) treated with Impella® (17 for cardiogenic shock, 4 for protected PCI) for the presence of AVWS by determining von Willebrand factor multimers, VWF collagen binding capacity and VWF antigen. During the time course of Impella® support, 20/21 patients (95%) developed AVWS (mean duration of support: 135 ± 114 hours, mean time from device implantation to first diagnosis of AVWS: 10.6 ± 10.8 hours). Our data indicate that AVWS is a common phenomenon during left ventricular unloading via microaxial pump support. Thus, AVWS has to be considered as contributing factor for potential bleeding complications in this high risk patient population, especially in the context of dual antiplatelet therapy.

Leukocytoclastic vasculitis associated with endocarditis in a patient with transposition of the great arteries and mechanical valve replacement.

Immunological vascular phenomena can be the initial manifestation of bacterial infection and endocarditis. Here, we report a rare case of leukocytoclastic vasculitis without immune complexes or cryoglobulinemia in a patient with infective endocarditis, congenital heart disease, and a prior mechanical valve replacement. The patient completely recovered following antibiotic therapy, and skin lesions disappeared without immune suppression, which suggested infection-mediated vasculitis. While the treatment of leukocytoclastic vasculitis typically involves immunosuppressive therapy, the treatment for infection-mediated vasculitis is eradication of the infection.

Efficacy of prasugrel administration immediately after percutaneous coronary intervention in ST-elevation myocardial infarction.

Prasugrel, a potent thienopyridine, achieves stronger inhibition of platelet activation than clopidogrel. However, onset of inhibition is significantly delayed in patients with acute ST-elevation myocardial infarction (STEMI), as haemodynamic instability and morphine application seem to exhibit significant influence. Since rapid onset of effect was demonstrated in non-STEMI patients when prasugrel was administered only after percutaneous coronary intervention (PCI) without increasing cardiovascular event rates we assessed the efficacy of prasugrel loading immediately after PCI for STEMI instead of pre-loading before revascularisation. We investigated 50 consecutive patients with acute STEMI (mean age 56 ± 10 years) admitted for primary PCI. Prasugrel efficacy was assessed by platelet reactivity index (PRI; VASP assay) before, 1, 2, 4, 6, 12, and 24 hours following an oral loading dose of 60 mg immediately after PCI. High on-treatment platelet reactivity (HTPR) was defined as PRI>50 %. Prasugrel significantly and rapidly reduced platelet reactivity in acute STEMI patients (p<0.0001 at each time point vs control). Morphine application resulted in a significantly higher HTPR rate among patients having received morphine less than 1 hour before prasugrel loading (p<0.001) while concomitant metoclopramide (MCP) treatment did not significantly affect prasugrel efficacy. In conclusion, in contrast to previous reports describing a significant delay in onset of prasugrel-mediated P2Y12 inhibition in acute STEMI, we observed a rapid onset with low HTPR rates comparable to those observed in stable non-STEMI patients. Prasugrel administered directly after primary PCI might therefore be a useful therapeutic strategy in patients with STEMI to provide strong and effective P2Y12 inhibition.

Platelet inhibition with prasugrel in patients with acute myocardial infarction undergoing therapeutic hypothermia after cardiopulmonary resuscitation.

Acute myocardial infarction (AMI) is the leading cause for out-of-hospital cardiac arrest. Therapeutic hypothermia improves neurological outcome in combination with early revascularisation, but seems to affect clopidogrel responsiveness. The more potent thienopyridine prasugrel has not yet been sufficiently evaluated during therapeutic hypothermia. We investigated 23 consecutive AMI patients (61 ± 11 years) following out-of-hospital resuscitation undergoing revascularisation and therapeutic hypothermia. Prasugrel efficacy was assessed by the platelet-reactivity-index (PRI) before and 2, 4, 6, 12, 24, 48, and 72 hours (h) following a loading dose of 60 mg via a gastric tube. Mean PRI (± SD) was 70 ± 12 % prior to loading and 60 ± 16 % (2 h, ns), 52 ± 21 % (4 h, p< 0.01), 42 ± 26 % (6 h, p< 0.01), 37 ± 21 % (12 h, p< 0.01), 27 ± 23 % (24 h, p< 0.01), 18 ± 14 % (48 h, p< 0.01), and 13 ± 10 % (72 h, p< 0.01) after loading. Sufficient platelet inhibition occurred later compared to stable AMI patients (6 h vs 2 h); however, high on-treatment platelet reactivity significantly decreased over time and was non-existent after 72 h (PRI> 50 %: 2 h: 72 %, 4 h: 52 %, 6 h: 43 %, 12 h: 29 %, 24 h: 17 %, 48 h: 5 %, 72 h: 0 %). There was no relation between 30-day mortality rate (26 %) and PRI values. Prasugrel significantly reduced platelet reactivity even during vasopressor use, analgosedation and therapeutic hypothermia. Despite a significant delay compared to stable AMI patients, sufficient platelet inhibition was reached in 83 % of patients within 24 h. Therefore, prasugrel administration via gastric tube might be a useful therapeutic strategy in these patients at high risk, providing potent and effective P2Y12 inhibition.

Covalent Protein Labeling by Enzymatic Phosphocholination.

We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP-choline derivatives to N-termini, C-termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG-phosphocholine) is introduced to attach the conjugated cargo.

Novel biomechanical quantification methodology for lumbar intraforaminal spinal nerve adhesion in a laminectomy and disc injury rat model.

Spinal nerve fibrosis following injury or surgical intervention may play an important role in the pathophysiology of chronic back pain. In this current study, we demonstrate the role of biomechanical quantification of lumbar intraforaminal spinal nerve adhesion and tethering in the analysis of the post-laminectomy condition and describe a direct methodology to make this measurement. Twenty age-matched Sprague-Dawley male rats were divided into operative and non-operative (control) groups. Operative animals underwent a bilateral L5-L6 laminectomy with right-side L5-6 disc injury, a post-laminectomy pain model previously published by this lab. At eight weeks, animals were sacrificed and the strength of adhesion of the L5 intraforaminal spinal nerve to surrounding structures was quantified using a novel biomechanical methodology. Operative animals were found to have a significantly greater load to displace the intact right L5 spinal nerve through the intervertebral foramen when compared to control animals. The findings show that the post-laminectomy condition creates quantifiable fibrosis of the spinal nerve to surrounding structures and supports the conclusion that this fibrosis may play a role in the post-laminectomy pain syndrome.