Cyclic Peptides from Graspetide Biosynthesis and Native Chemical Ligation.

The ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily of natural products includes many examples of cyclic peptides with diverse macrocyclization chemistries. The graspetides, one family of macrocyclized RiPPs, harbor side chain-side chain ester or amide linkages. We recently reported the structure and biosynthesis of the graspetide pre-fuscimiditide, a 22-amino-acid (aa) peptide with two ester cross-links forming a stem-loop structure. These cross-links are introduced by a single graspetide synthetase, the ATP-grasp enzyme ThfB. Here we show that ThfB can also catalyze the formation of amide or thioester cross-links in prefuscimiditide, with thioester formation being especially efficient. We further show that upon proteolysis to reveal an N-terminal cysteine residue, the thioester-linked peptide rapidly and quantitatively rearranges via native chemical ligation into an isopeptide-bonded head-to-tail cyclic peptide. The solution structure of this rearranged peptide was determined by using 2D NMR spectroscopy experiments. Our methodology offers a straightforward recombinant route to head-to-tail cyclic peptides.

The pearl jubilee of microcin J25: thirty years of research on an exceptional lasso peptide.

Covering: 1992 up to 2023Since their discovery, lasso peptides went from peculiarities to be recognized as a major family of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that were shown to be spread throughout the bacterial kingdom. Microcin J25 was first described in 1992, making it one of the earliest known lasso peptides. No other lasso peptide has since then been studied to such an extent as microcin J25, yet, previous review articles merely skimmed over all the research done on this exceptional lasso peptide. Therefore, to commemorate the 30th anniversary of its first report, we give a comprehensive overview of all literature related to microcin J25. This review article spans the early work towards the discovery of microcin J25, its biosynthetic gene cluster, and the elucidation of its three-dimensional, threaded lasso structure. Furthermore, the current knowledge about the biosynthesis of microcin J25 and lasso peptides in general is summarized and a detailed overview is given on the biological activities associated with microcin J25, including means of self-immunity, uptake into target bacteria, inhibition of the Gram-negative RNA polymerase, and the effects of microcin J25 on mitochondria. The in vitro and in vivo models used to study the potential utility of microcin J25 in a (veterinary) medicine context are discussed and the efforts that went into employing the microcin J25 scaffold in bioengineering contexts are summed up.

Classification of chemically modified red blood cells in microflow using machine learning video analysis.

We classify native and chemically modified red blood cells with an AI based video classifier. Using TensorFlow video analysis enables us to capture not only the morphology of the cell but also the trajectories of motion of individual red blood cells and their dynamics. We chemically modify cells in three different ways to model different pathological conditions and obtain classification accuracies for all three classification tasks of more than 90% between native and modified cells. Unlike standard cytometers that are based on immunophenotyping our microfluidic cytometer allows to rapidly categorize cells without any fluorescence labels simply by analysing the shape and flow of red blood cells.

Protein Engineering in Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs).

Ribosomally synthesized and post-translationally modified peptides (RiPPs) make up a rapidly growing superfamily of natural products. RiPPs exhibit an extraordinary range of structures, but they all begin as gene-encoded precursor peptides that are linear chains of amino acids produced by ribosomes. Given the gene-encoded nature of RiPP precursor peptides, the toolbox of protein engineering can be directly applied to these precursors. This Perspective will discuss examples of site-directed mutagenesis, noncanonical amino acid mutagenesis, and the construction and screening of combinatorial libraries as applied to RiPPs. These studies have led to important insights into the biosynthesis and bioactivity of RiPPs and the reengineering of RiPPs for entirely new functions.

Kinetics of Aspartimide Formation and Hydrolysis in Lasso Peptide Lihuanodin.

Aspartimides are notorious as undesired side products in solid-phase peptide synthesis and in pharmaceutical formulations. However, we have discovered several ribosomally synthesized and post-translationally modified peptides (RiPPs) in which aspartimide is installed intentionally via enzymatic activity of protein l-isoaspartyl methyltransferase (PIMT) homologues. In the case of the lasso peptide lihuanodin, the methyltransferase LihM recognizes the lassoed substrate pre-lihuanodin, specifically methylating the side chain of an l-Asp residue in the ring portion of the lasso peptide. The subsequent nucleophilic attack from the adjacent amide leads to the formation of an aspartimide. The resulting aspartimide hydrolyzes regioselectively to l-Asp in buffers above pH 7. Here we report the first Michaelis-Menten kinetic measurements of such a RiPP-associated PIMT homologue, LihM, acting on its cognate substrate pre-lihuanodin. Additionally, we measured the rate of aspartimide hydrolysis, which allowed us to deduce the kinetics of the entire reaction network. The relative magnitudes of these rates explain the accumulation and relative stability of aspartimide-containing lihuanodin. We also demonstrate that the residue C-terminal to the aspartimide controls the regioselectivity of hydrolysis and thus the threadedness of the peptide.

Genome Mining and Discovery of Imiditides, a Family of RiPPs with a Class-Defining Aspartimide Modification.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large and diverse class of natural products of ribosomal origin. In the past decade, various sophisticated machine-learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Here, we show that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds, providing a complementary approach for novel RiPP discovery. Leveraging the fact that O-methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized a C-terminal motif unique to RiPP-associated O-methyltransferases as the search query to discover a novel family of RiPPs, the imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, distributed across Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaAM, encoded in the genome of Nonomuraea maritima. In contrast to other RiPP-associated PIMTs that recognize constrained peptides as substrates, the PIMT homologue in the mNmaAM BGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. Substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experiments and an AlphaFold model prediction. Our study shows that PIMT-mediated aspartimide formation is an emerging backbone modification strategy in the biosynthesis of multiple RiPP families.

LassoHTP: A High-Throughput Computational Tool for Lasso Peptide Structure Construction and Modeling.

Lasso peptides are a subclass of ribosomally synthesized and post-translationally modified peptides with a slipknot conformation. With superior thermal stability, protease resistance, and antimicrobial activity, lasso peptides are promising candidates for bioengineering and pharmaceutical applications. To enable high-throughput computational prediction and design of lasso peptides, we developed a software, LassoHTP, for automatic lasso peptide structure construction and modeling. LassoHTP consists of three modules, including the scaffold constructor, mutant generator, and molecular dynamics (MD) simulator. With a user-provided sequence and conformational annotation, LassoHTP can either generate the structure and conformational ensemble as is or conduct random mutagenesis. We used LassoHTP to construct eight known lasso peptide structures de novo and to simulate their conformational ensembles for 100 ns MD simulations. For benchmarking, we calculated the root mean square deviation (RMSD) of these ensembles with reference to their experimental crystal or NMR PDB structures; we also compared these RMSD values against those of the MD ensembles that are initiated from the PDB structures. Dihedral principal component analysis was also conducted. The results show that the LassoHTP-initiated ensembles are similar to those of the PDB-initiated ensembles. LassoHTP offers a computational platform to develop strategies for lasso peptide prediction and design.

Discovery, Characterization, and Bioactivity of the Achromonodins: Lasso Peptides Encoded by Achromobacter.

Through genome mining efforts, two lasso peptide biosynthetic gene clusters (BGCs) within two different species of Achromobacter, a genus that contains pathogenic organisms that can infect patients with cystic fibrosis, were discovered. Using gene-refactored BGCs in E. coli, these lasso peptides, which were named achromonodin-1 and achromonodin-2, were heterologously expressed. Achromonodin-1 is naturally encoded by certain isolates from the sputum of patients with cystic fibrosis. The NMR structure of achromonodin-1 was determined, demonstrating that it is a threaded lasso peptide with a large loop and short tail structure, reminiscent of previously characterized lasso peptides that inhibit RNA polymerase (RNAP). Achromonodin-1 inhibits RNAP in vitro and has potent, focused activity toward Achromobacter pulmonis, another isolate from the sputum of a cystic fibrosis patient. These efforts expand the repertoire of antimicrobial lasso peptides and provide insights into how Achromobacter isolates from certain ecological niches interact with each other.

Fecal microbiota composition is related to brown adipose tissue 18F-fluorodeoxyglucose uptake in young adults.

OBJECTIVE: Human brown adipose tissue (BAT) has gained considerable attention as a potential therapeutic target for obesity and its related cardiometabolic diseases; however, whether the gut microbiota might be an efficient stimulus to activate BAT metabolism remains to be ascertained. We aimed to investigate the association of fecal microbiota composition with BAT volume and activity and mean radiodensity in young adults. METHODS: 82 young adults (58 women, 21.8 ± 2.2 years old) participated in this cross-sectional study. DNA was extracted from fecal samples and 16S rRNA sequencing was performed to analyse the fecal microbiota composition. BAT was determined via a static 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography-computed tomography scan (PET/CT) after a 2 h personalized cooling protocol. 18F-FDG uptake was also quantified in white adipose tissue (WAT) and skeletal muscles. RESULTS: The relative abundance of Akkermansia, Lachnospiraceae sp. and Ruminococcus genera was negatively correlated with BAT volume, BAT SUVmean and BAT SUVpeak (all rho ≤ - 0.232, P ≤ 0.027), whereas the relative abundance of Bifidobacterium genus was positively correlated with BAT SUVmean and BAT SUVpeak (all rho ≥ 0.262, P ≤ 0.012). On the other hand, the relative abundance of Sutterellaceae and Bifidobacteriaceae families was positively correlated with 18F-FDG uptake by WAT and skeletal muscles (all rho ≥ 0.213, P ≤ 0.042). All the analyses were adjusted for the PET/CT scan date as a proxy of seasonality. CONCLUSION: Our results suggest that fecal microbiota composition is involved in the regulation of BAT and glucose uptake by other tissues in young adults. Further studies are needed to confirm these findings. CLINICAL TRIAL INFORMATION: ClinicalTrials.gov no. NCT02365129 (registered 18 February 2015).

Mechanistic Analysis of the Biosynthesis of the Aspartimidylated Graspetide Amycolimiditide.

Several classes of ribosomally synthesized and post-translationally modified peptides (RiPPs) are composed of multiple macrocycles. The enzymes that assemble these macrocycles must surmount the challenge of installing a single specific set of linkages out of dozens of distinct possibilities. One class of RiPPs that includes multiple macrocycles are the graspetides, named after the ATP-grasp enzymes that install ester or amide linkages between pairs of nucleophilic and electrophilic side chains. Here, using heterologous expression and NMR spectroscopy, we characterize the connectivity and structure of amycolimiditide, a 29 aa graspetide with a stem-loop structure. The stem includes four esters and extends over 20 Å. The loop of amycolimiditide is distinguished by the presence of an aspartimide moiety, installed by a dedicated O-methyltransferase enzyme. We further characterize the biosynthesis of amycolimiditide in vitro, showing that the amycolimiditide ATP-grasp enzyme AmdB operates in a strict vectorial manner, installing esters starting at the loop and proceeding down the stem. Surprisingly, the O-methyltransferase AmdM that aspartimidylates amycolimiditide prefers a substrate with all four esters installed, despite the fact that the most distal ester is ∼30 Å away from the site of aspartimidylation. This study provides insights into the structure and diversity of aspartimidylated graspetides and also provides fresh insights into how RiPP biosynthetic enzymes engage their peptide substrates.

The Shuttling Cascade in Lasso Peptide Benenodin-1 is Controlled by Non-Covalent Interactions.

The lasso peptide benenodin-1, a naturally occurring and bacterially produced [1]rotaxane, undergoes a reversible zip tie-like motion under heat activation, in which a peptidic wheel stepwise translates along a molecular thread in a cascade of "tail/loop pulling" equilibria. Conformational and structural analyses of four translational isomers, in solution and in the gas phase, reveal that the equilibrium distribution is controlled by mechanical and non-covalent forces within the lasso peptide. Furthermore, each dynamic pulling step is accompanied by a major restructuring of the intramolecular hydrogen bonding network between wheel and thread, which affects the peptide's physico-chemical properties.

ParST is a widespread toxin-antitoxin module that targets nucleotide metabolism.

Toxin-antitoxin (TA) systems interfere with essential cellular processes and are implicated in bacterial lifestyle adaptations such as persistence and the biofilm formation. Here, we present structural, biochemical, and functional data on an uncharacterized TA system, the COG5654-COG5642 pair. Bioinformatic analysis showed that this TA pair is found in 2,942 of the 16,286 distinct bacterial species in the RefSeq database. We solved a structure of the toxin bound to a fragment of the antitoxin to 1.50 Å. This structure suggested that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin specifically modifies phosphoribosyl pyrophosphate synthetase (Prs), an essential enzyme in nucleotide biosynthesis conserved in all organisms. We propose renaming the toxin ParT for Prs ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT is a unique example of an intracellular protein mART in bacteria and is the smallest known mART. This work demonstrates that TA systems can induce bacteriostasis through interference with nucleotide biosynthesis.

Structural mechanism of transcription inhibition by lasso peptides microcin J25 and capistruin.

We report crystal structures of the antibacterial lasso peptides microcin J25 (MccJ25) and capistruin (Cap) bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). Both peptides bind within the RNAP secondary channel, through which NTP substrates enter the RNAP active site, and sterically block trigger-loop folding, which is essential for efficient catalysis by the RNAP. MccJ25 binds deep within the secondary channel in a manner expected to interfere with NTP substrate binding, explaining the partial competitive mechanism of inhibition with respect to NTPs found previously [Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004) Mol Cell 14:739-751]. The Cap binding determinant on RNAP overlaps, but is not identical to, that of MccJ25. Cap binds further from the RNAP active site and does not sterically interfere with NTP binding, and we show that Cap inhibition is partially noncompetitive with respect to NTPs. This work lays the groundwork for structure determination of other lasso peptides that target the bacterial RNAP and provides a structural foundation to guide lasso peptide antimicrobial engineering approaches.

Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification.

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with genes encoding protein l-isoaspartyl methyltransferase (PIMT) homologues. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an l-aspartate side chain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium Thermobifida cellulosilytica, while lihuanodin is encoded in the genome of firmicute Lihuaxuella thermophila. Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.

New developments in RiPP discovery, enzymology and engineering.

Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.

Real-life assessment of standardized contrast-enhanced ultrasound (CEUS) and CEUS algorithms (CEUS LI-RADS®/ESCULAP) in hepatic nodules in cirrhotic patients-a prospective multicenter study.

OBJECTIVES: Hepatocellular carcinoma (HCC) can be diagnosed non-invasively with contrast-enhanced ultrasound (CEUS) in cirrhosis if the characteristic pattern of arterial phase hyperenhancement followed by hypoenhancement is present. Recent studies suggest that diagnosis based on this "hyper-hypo" pattern needs further refinement. This study compares the diagnostic accuracies of standardized CEUS for HCC according to the current guideline definition and following the newly developed CEUS algorithms (CEUS LI-RADS®, ESCULAP) in a prospective multicenter real-life setting. METHODS: Cirrhotic patients with liver lesions on B-mode ultrasound were recruited prospectively from 04/2018 to 04/2019, and clinical and imaging data were collected. The CEUS standard included an additional examination point after 4-6 min in case of no washout after 3 min. The diagnostic accuracies of CEUS following the guidelines ("hyper-hypo" pattern), based on the examiner's subjective interpretation ("CEUS subjective"), and based on the CEUS algorithms ESCULAP and CEUS LI-RADS® were compared. RESULTS: In total, 470 cirrhotic patients were recruited in 43 centers. The final diagnosis was HCC in 378 cases (80.4%) according to the reference standard (histology 77.4%, MRI 16.4%, CT 6.2%). The "hyper-hypo" pattern yielded 74.3% sensitivity and 63% specificity. "CEUS subjective" showed a higher diagnostic accuracy (sensitivity, 91.5%; specificity, 67.4%; positive predictive value, 92%; negative predictive value, 66%). Sensitivity was higher for ESCULAP (95%) and "CEUS subjective" (91.5%) versus CEUS LI-RADS® (65.2%; p < 0.001). Specificity was highest for CEUS LI-RADS® (78.6%; p < 0.001). CONCLUSIONS: CEUS has an excellent diagnostic accuracy for the non-invasive diagnosis of HCC in cirrhosis. CEUS algorithms may be a helpful refinement of the "hyper-hypo" pattern defined by current HCC guidelines. KEY POINTS: • Contrast-enhanced ultrasound (CEUS) has a high diagnostic accuracy for the non-invasive diagnosis of hepatocellular carcinoma (HCC) in cirrhosis. • The CEUS algorithm ESCULAP (Erlanger Synopsis for Contrast-enhanced Ultrasound for Liver lesion Assessment in Patients at risk) showed the highest sensitivity, whereas the CEUS LI-RADS® (Contrast-Enhanced UltraSound Liver Imaging Reporting and Data System) algorithm yielded the highest specificity. • A standardized CEUS examination procedure with an additional examination point in the late phase, after 4-6 min in lesions with no washout after 3 min, is vital.

Mechanisms of action of ribosomally synthesized and posttranslationally modified peptides (RiPPs).

Natural products remain a critical source of medicines and drug leads. One of the most rapidly growing superclasses of natural products is RiPPs: ribosomally synthesized and posttranslationally modified peptides. RiPPs have rich and diverse bioactivities. This review highlights examples of the molecular mechanisms of action that underly those bioactivities. Particular emphasis is placed on RiPP/target interactions for which there is structural information. This detailed mechanism of action work is critical toward the development of RiPPs as therapeutics and can also be used to prioritize hits in RiPP genome mining studies.

Discovery and structure of the antimicrobial lasso peptide citrocin.

We report the identification of citrocin, a 19-amino acid-long antimicrobial lasso peptide from the bacteria Citrobacter pasteurii and Citrobacter braakii We refactored the citrocin gene cluster and heterologously expressed it in Escherichia coli We determined citrocin's NMR structure in water and found that is reminiscent of that of microcin J25 (MccJ25), an RNA polymerase-inhibiting lasso peptide that hijacks the TonB-dependent transporter FhuA to gain entry into cells. Citrocin has moderate antimicrobial activity against E. coli and Citrobacter strains. We then performed an in vitro RNA polymerase (RNAP) inhibition assay using citrocin and microcin J25 against E. coli RNAP. Citrocin has a higher minimal inhibition concentration than microcin J25 does against E. coli but surprisingly is ∼100-fold more potent as an RNAP inhibitor. This suggests that citrocin uptake by E. coli is limited. We found that unlike MccJ25, citrocin's activity against E. coli relied on neither of the two proton motive force-linked systems, Ton and Tol-Pal, for transport across the outer membrane. The structure of citrocin contains a patch of positive charge consisting of Lys-5 and Arg-17. We performed mutagenesis on these residues and found that the R17Y construct was matured into a lasso peptide but no longer had activity, showing the importance of this side chain for antimicrobial activity. In summary, we heterologously expressed and structurally and biochemically characterized an antimicrobial lasso peptide, citrocin. Despite being similar to MccJ25 in sequence, citrocin has an altered activity profile and does not use the same outer-membrane transporter to enter susceptible cells.

Discovery of Ubonodin, an Antimicrobial Lasso Peptide Active against Members of the Burkholderia cepacia Complex.

We report the heterologous expression, structure, and antimicrobial activity of a lasso peptide, ubonodin, encoded in the genome of Burkholderia ubonensis. The topology of ubonodin is unprecedented amongst lasso peptides, with 18 of its 28 amino acids found in the mechanically bonded loop segment. Ubonodin inhibits RNA polymerase in vitro and has potent antimicrobial activity against several pathogenic members of the Burkholderia genus, most notably B. cepacia and B. multivorans, causative agents of lung infections in cystic fibrosis patients.

Heterologous and in Vitro Reconstitution of Fuscanodin, a Lasso Peptide from Thermobifida fusca.

Lasso peptides are a class of ribosomally derived natural products typified by their threaded rotaxane structure. The conversion of a linear precursor peptide into a lasso peptide structure requires two enzymatic activities: cleavage of the precursor via a cysteine protease and cyclization via isopeptide bond formation. In vitro studies of lasso peptide enzymology have been hampered by difficulties in obtaining pure, soluble enzymes. We reasoned that thermophilic bacteria would be a good source for well-behaved lasso peptide biosynthetic enzymes. The genome of the thermophilic actinobacterium Thermobifida fusca encodes for a lasso peptide with an unprecedented Trp residue at its N-terminus, a peptide we have named fuscanodin. Here we reconstitute fuscanodin biosynthesis in vitro with purified components, establishing a minimal fuscanodin synthetase. These experiments have allowed us to probe the kinetics of lasso peptide biosynthesis for the first time, and we report initial rates of fuscanodin biosynthesis. The fuscanodin biosynthetic enzymes are insensitive to substrate concentration and operate in a near single-turnover regime in vitro. While lasso peptides are often touted for their stability to both chaotropic and thermal challenges, fuscanodin is found to undergo a conformational change consistent with lasso peptide unthreading in organic solvents at room temperature.