Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides

Nonribosomal peptide synthetases (NRPS) are large multienzyme complexes, which simultaneously represent template and biosynthetic machinery for the production of structurally diverse peptidic products that feature high pharmacological and biological activities. A key determinant of nonribosomal peptide product activity is the common macrocyclic structure of many compounds. Macrocyclization is catalyzed in the last step of nonribosomal synthesis by thioesterase (TE) domain activity. The herein presented work describes the first biochemical characterization of a TE domain of a streptomycete, the thioesterase of the S. coelicolor calcium-dependent antibiotic (CDA) synthetase. This recombinant cyclase catalyzes macrolactone formation of linear peptidyl-thioesters based on a sequence analogous to natural CDA. For substrate mimics, the phosphopantetheine cofactor was successfully substituted by various thioester leaving groups. The best rates for cyclization were determined for the thiophenol leaving group, revealing that chemical reactivity is more important for enzyme acylation than cofactor recognition. Interestingly, CDA cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr2 and Ser1 residues onto the C-terminal Trp11 of the acyl-enzyme intermediate. To further explore this relaxed regioselectivity of CDA TE, alterations to the peptide backbone and the fatty acyl chain were made. Substitution of either Thr2 or Ser1 by alanine led to selective formation of a decapeptide or undecapeptide lactone ring. However, the stereoselectivity of CDA cyclase was fully retained, thus accepting only L-configured Ser1 and Thr2 for cyclization. Elongation of the fatty acyl group by four methylene groups to the natural length (C6) of CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring, along with decreased hydrolysis of the peptidyl-thioester substrate. This provides evidence for the crucial role of the lipid chain in controlling the regio- and chemoselectivity of TE-mediated macrocyclization. CDA belongs to the group of acidic lipopeptides, which includes the clinically approved antibiotic daptomycin. To evaluate the capability of CDA cyclase for the chemoenzymatic generation of daptomycin, six daptomycin-specific residues were successively incorporated into linear CDA undecapeptidyl-thioesters. All these six substrates were efficiently cyclized by CDA TE. Simultaneous incorporation of all six of these residues into the peptide backbone and elongation of the N-terminus of CDA by two residues finally yielded a daptomycin derivative that lacked only the -methyl group of L-3-methylglutamate. In accordance with acidic lipopeptide antibiotics, the bioactivity of the chemoenzymatic assembled daptomycin analogue is dependent on the presence of calcium ions. To identify calcium-binding sites in the lipo-tridecapeptide chain of the daptomycin analogue, all four acidic residues were successively substituted by either Asn or Gln. Bioactivity studies revealed that only Asp7 and Asp9 are essential for antimicrobial potency. Moreover, these two residues are strictly conserved among all other nonribosomal acidic lipopeptides and the calcium-binding EF-motif of ribosomally assembled calmodulin. The final part of this work is dedicated to the selective detection of peptide cyclization by fluorescence resonance energy transfer (FRET). In this approach, peptide cyclization catalyzed by NRPS-derived TE domains brings the donor Trp and the acceptor Kyn (kynurenine) in sufficiently close proximity to enable efficient FRET. Theses fluorophores were readily incorporated into the peptide backbone by solid-phase peptide chemistry and show excellent spectral overlap between the donor emission and acceptor absorption. Application of this method provided a tool to track TE-mediated peptide cyclization in real-time. Furthermore, picomolar detection limits of cyclopeptides were realized, thereby facilitating kinetic studies of TE-mediated macrocyclization. The general utility of FRET-assisted detection of cyclopeptides was demonstrated for two cyclases, namely tyrocidine (Tyc) TE, and CDA TE. For the latter cyclase, this approach was combined with site-directed affinity labelling, opening the possibility for high-throughput enzymatic screening

Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides

Nonribosomal peptide synthetases (NRPS) are large multienzyme complexes, which simultaneously represent template and biosynthetic machinery for the production of structurally diverse peptidic products that feature high pharmacological and biological activities. A key determinant of nonribosomal peptide product activity is the common macrocyclic structure of many compounds. Macrocyclization is catalyzed in the last step of nonribosomal synthesis by thioesterase (TE) domain activity. The herein presented work describes the first biochemical characterization of a TE domain of a streptomycete, the thioesterase of the S. coelicolor calcium-dependent antibiotic (CDA) synthetase. This recombinant cyclase catalyzes macrolactone formation of linear peptidyl-thioesters based on a sequence analogous to natural CDA. For substrate mimics, the phosphopantetheine cofactor was successfully substituted by various thioester leaving groups. The best rates for cyclization were determined for the thiophenol leaving group, revealing that chemical reactivity is more important for enzyme acylation than cofactor recognition. Interestingly, CDA cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr2 and Ser1 residues onto the C-terminal Trp11 of the acyl-enzyme intermediate. To further explore this relaxed regioselectivity of CDA TE, alterations to the peptide backbone and the fatty acyl chain were made. Substitution of either Thr2 or Ser1 by alanine led to selective formation of a decapeptide or undecapeptide lactone ring. However, the stereoselectivity of CDA cyclase was fully retained, thus accepting only L-configured Ser1 and Thr2 for cyclization. Elongation of the fatty acyl group by four methylene groups to the natural length (C6) of CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring, along with decreased hydrolysis of the peptidyl-thioester substrate. This provides evidence for the crucial role of the lipid chain in controlling the regio- and chemoselectivity of TE-mediated macrocyclization. CDA belongs to the group of acidic lipopeptides, which includes the clinically approved antibiotic daptomycin. To evaluate the capability of CDA cyclase for the chemoenzymatic generation of daptomycin, six daptomycin-specific residues were successively incorporated into linear CDA undecapeptidyl-thioesters. All these six substrates were efficiently cyclized by CDA TE. Simultaneous incorporation of all six of these residues into the peptide backbone and elongation of the N-terminus of CDA by two residues finally yielded a daptomycin derivative that lacked only the -methyl group of L-3-methylglutamate. In accordance with acidic lipopeptide antibiotics, the bioactivity of the chemoenzymatic assembled daptomycin analogue is dependent on the presence of calcium ions. To identify calcium-binding sites in the lipo-tridecapeptide chain of the daptomycin analogue, all four acidic residues were successively substituted by either Asn or Gln. Bioactivity studies revealed that only Asp7 and Asp9 are essential for antimicrobial potency. Moreover, these two residues are strictly conserved among all other nonribosomal acidic lipopeptides and the calcium-binding EF-motif of ribosomally assembled calmodulin. The final part of this work is dedicated to the selective detection of peptide cyclization by fluorescence resonance energy transfer (FRET). In this approach, peptide cyclization catalyzed by NRPS-derived TE domains brings the donor Trp and the acceptor Kyn (kynurenine) in sufficiently close proximity to enable efficient FRET. Theses fluorophores were readily incorporated into the peptide backbone by solid-phase peptide chemistry and show excellent spectral overlap between the donor emission and acceptor absorption. Application of this method provided a tool to track TE-mediated peptide cyclization in real-time. Furthermore, picomolar detection limits of cyclopeptides were realized, thereby facilitating kinetic studies of TE-mediated macrocyclization. The general utility of FRET-assisted detection of cyclopeptides was demonstrated for two cyclases, namely tyrocidine (Tyc) TE, and CDA TE. For the latter cyclase, this approach was combined with site-directed affinity labelling, opening the possibility for high-throughput enzymatic screening

A comparison of third-generation semi-invasive arterial waveform analysis with thermodilution in patients undergoing coronary surgery

Uncalibrated semi-invasive continous monitoring of cardiac index (CI) has recently gained increasing interest. The aim of the present study was to compare the accuracy of CI determination based on arterial waveform analysis with transpulmonary thermodilution. Fifty patients scheduled for elective coronary surgery were studied after induction of anaesthesia and before and after cardiopulmonary bypass (CPB), respectively. Each patient was monitored with a central venous line, the PiCCO system, and the FloTrac/Vigileo-system. Measurements included CI derived by transpulmonary thermodilution and uncalibrated semi-invasive pulse contour analysis. Percentage changes of CI were calculated. There was a moderate, but significant correlation between pulse contour CI and thermodilution CI both before (r(2) = 0.72, P < 0.0001) and after (r(2) = 0.62, P < 0.0001) CPB, with a percentage error of 31% and 25%, respectively. Changes in pulse contour CI showed a significant correlation with changes in thermodilution CI both before (r(2) = 0.52, P < 0.0001) and after (r(2) = 0.67, P < 0.0001) CPB. Our findings demonstrated that uncalibrated semi-invasive monitoring system was able to reliably measure CI compared with transpulmonary thermodilution in patients undergoing elective coronary surgery. Furthermore, the semi-invasive monitoring device was able to track haemodynamic changes and trends

Characterization of encapsulated graphene layers using extreme ultraviolet coherence tomography

Many applications of two-dimensional materials such as graphene require the encapsulation in bulk material. While a variety of methods exist for the structural and functional characterization of uncovered 2D materials, there is a need for methods that image encapsulated 2D materials as well as the surrounding matter. In this work, we use extreme ultraviolet coherence tomography to image graphene flakes buried beneath 200 nm of silicon. We show that we can identify mono-, bi-, and trilayers of graphene and quantify the thickness of the silicon bulk on top by measuring the depth-resolved reflectivity. Furthermore, we estimate the quality of the graphene interface by incorporating a model that includes the interface roughness. These results are verified by atomic force microscopy and prove that extreme ultraviolet coherence tomography is a suitable tool for imaging 2D materials embedded in bulk materials

Dynamics of chromosome organization in a minimal bacterial cell

Computational models of cells cannot be considered complete unless they include the most fundamental process of life, the replication and inheritance of genetic material. By creating a computational framework to model systems of replicating bacterial chromosomes as polymers at 10 bp resolution with Brownian dynamics, we investigate changes in chromosome organization during replication and extend the applicability of an existing whole-cell model (WCM) for a genetically minimal bacterium, JCVI-syn3A, to the entire cell-cycle. To achieve cell-scale chromosome structures that are realistic, we model the chromosome as a self-avoiding homopolymer with bending and torsional stiffnesses that capture the essential mechanical properties of dsDNA in Syn3A. In addition, the conformations of the circular DNA must avoid overlapping with ribosomes identitied in cryo-electron tomograms. While Syn3A lacks the complex regulatory systems known to orchestrate chromosome segregation in other bacteria, its minimized genome retains essential loop-extruding structural maintenance of chromosomes (SMC) protein complexes (SMC-scpAB) and topoisomerases. Through implementing the effects of these proteins in our simulations of replicating chromosomes, we find that they alone are sufficient for simultaneous chromosome segregation across all generations within nested theta structures. This supports previous studies suggesting loop-extrusion serves as a near-universal mechanism for chromosome organization within bacterial and eukaryotic cells. Furthermore, we analyze ribosome diffusion under the influence of the chromosome and calculate in silico chromosome contact maps that capture inter-daughter interactions. Finally, we present a methodology to map the polymer model of the chromosome to a Martini coarse-grained representation to prepare molecular dynamics models of entire Syn3A cells, which serves as an ultimate means of validation for cell states predicted by the WCM. </p

Mutations in PINK1 and Parkin Impair Ubiquitination of Mitofusins in Human Fibroblasts

PINK1 and Parkin mutations cause recessive Parkinson's disease (PD). In Drosophila and SH-SY5Y cells, Parkin is recruited by PINK1 to damaged mitochondria, where it ubiquitinates Mitofusins and consequently promotes mitochondrial fission and mitophagy

Sind alle Denker traurig? – Fallstudien zum melancholischen Grund des Schöpferischen in Asien und Europa

»Warum erweisen sich alle außergewöhnlichen Männer in Philosophie oder Politik oder Dichtung oder in den Künsten als Melancholiker?« So beginnen die pseudo­ aristotelischen Problemata Physica XXX,1. Die Frage­ form enthält schon die Behauptung, die seit mehr als zwei Jahrtausenden in der abendländischen Tradition scheinbar ungebrochen wiederholt wird und deren vermeintlich kontinuierliche Wirkmacht als bestens er­ forscht gilt. Analysiert man die Genealogie, Medialität und Dynamik dieses Denkbilds vom melancholischen Grund des Schöpferischen jedoch aus morphomatischer Sicht, so ergeben sich bisher kaum beantwortete Fra­ gen: Warum verblasste diese so eindrücklich gestaltete Vorstellung von der Schöpferkraft der Melancholie sofort wieder, und was bewirkte ihre triumphale Wiederkehr in der Renaissance? Wie verhielt es sich mit der tatsäch­ lichen Wirkmacht von Dürers Melencolia I, in Literatur, Bildender Kunst und Philosophie, bevor dieser Kupfer­ stich von den Kunsthistorikern Fritz Saxl und Erwin Panofsky besprochen wurde? Im Abendland wird die Melancholie seit der Renaissance als Genieausweis gehandelt – wie jedoch sieht es in anderen Kulturtradi­ tionen aus? Hat die Melancholie des Schöpferischen ›Schwestern‹ in anderen Weltkulturen? Welche Artefakte, Theorien und Praktiken bezeugen dies? Die Autoren des vorliegenden Bandes gehen diesen und weiteren Fragen in ihren Beiträgen nach, die zu einem Großteil aus Fallstudien bestehen. Sie verhandeln zum einen Hauptmanifestationen der Vorstellung melancho­ lischen Schöpfertums in Europa und setzen zum anderen einen ebenso wichtigen Akzent auf den asiatischen Raum

Mitochondria-Endoplasmic Reticulum Contact Sites Dynamics and Calcium Homeostasis Are Differentially Disrupted in PINK1-PD or PRKN-PD Neurons

Background: It is generally believed that the pathogenesis of PINK1/parkin-related Parkinson's disease (PD) is due to a disturbance in mitochondrial quality control. However, recent studies have found that PINK1 and Parkin play a significant role in mitochondrial calcium homeostasis and are involved in the regulation of mitochondria-endoplasmic reticulum contact sites (MERCSs). Objective: The aim of our study was to perform an in-depth analysis of the role of MERCSs and impaired calcium homeostasis in PINK1/Parkin-linked PD.MethodsIn our study, we used induced pluripotent stem cell-derived dopaminergic neurons from patients with PD with loss-of-function mutations in PINK1 or PRKN. We employed a split-GFP-based contact site sensor in combination with the calcium-sensitive dye Rhod-2 AM and applied Airyscan live-cell super-resolution microscopy to determine how MERCSs are involved in the regulation of mitochondrial calcium homeostasis. Results: Our results showed that thapsigargin-induced calcium stress leads to an increase of the abundance of narrow MERCSs in wild-type neurons. Intriguingly, calcium levels at the MERCSs remained stable, whereas the increased net calcium influx resulted in elevated mitochondrial calcium levels. However, PINK1-PD or PRKN-PD neurons showed an increased abundance of MERCSs at baseline, accompanied by an inability to further increase MERCSs upon thapsigargin-induced calcium stress. Consequently, calcium distribution at MERCSs and within mitochondria was disrupted. Conclusions: Our results demonstrated how the endoplasmic reticulum and mitochondria work together to cope with calcium stress in wild-type neurons. In addition, our results suggests that PRKN deficiency affects the dynamics and composition of MERCSs differently from PINK1 deficiency, resulting in differentially affected calcium homeostasis. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Molecular dynamics simulation of an entire cell

The ultimate microscope, directed at a cell, would reveal the dynamics of all the cell’s components with atomic resolution. In contrast to their real-world counterparts, computational microscopes are currently on the brink of meeting this challenge. In this perspective, we show how an integrative approach can be employed to model an entire cell, the minimal cell, JCVI-syn3A, at full complexity. This step opens the way to interrogate the cell’s spatio-temporal evolution with molecular dynamics simulations, an approach that can be extended to other cell types in the near future

Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation

Ewing sarcoma (EwS) is characterized by EWSR1-ETS fusion transcription factors converting polymorphic GGAA microsatellites (mSats) into potent neo-enhancers. Although the paucity of additional mutations makes EwS a genuine model to study principles of cooperation between dominant fusion oncogenes and neo-enhancers, this is impeded by the limited number of well-characterized models. Here we present the Ewing Sarcoma Cell Line Atlas (ESCLA), comprising whole-genome, DNA methylation, transcriptome, proteome, and chromatin immunoprecipitation sequencing (ChIP-seq) data of 18 cell lines with inducible EWSR1-ETS knockdown. The ESCLA shows hundreds of EWSR1-ETS-targets, the nature of EWSR1-ETS-preferred GGAA mSats, and putative indirect modes of EWSR1-ETS-mediated gene regulation, converging in the duality of a specific but plastic EwS signature. We identify heterogeneously regulated EWSR1-ETS-targets as potential prognostic EwS biomarkers. Our freely available ESCLA (http://r2platform.com/escla/) is a rich resource for EwS research and highlights the power of comprehensive datasets to unravel principles of heterogeneous gene regulation by chimeric transcription factors