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Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture. The N-end rule pathway targets substrate proteins for proteasomal degradation. Here, White et al . show that Arabidopsis PLANT CYSTEINE OXIDASEs show dioxygenase activity producing Cys-sulfinic acid at the N-terminus of target proteins, which then act as direct substrates for arginyl transferase.

Aberrant activation of signaling by the Wnt pathway is strongly implicated in the onset and progression of numerous types of cancer. Owing to the persistent dependence of these tumors on Wnt signaling for growth and survival, inhibition of this pathway is considered an attractive mechanism-based therapeutic approach. Oncogenic activation of Wnt signaling can ensue from a variety of distinct aberrations in the signaling pathway, but most share the common feature of causing increased cellular levels of β-catenin by interfering with its constitutive degradation. β-Catenin serves as a central hub in Wnt signaling by engaging in crucial protein-protein interactions with both negative and positive effectors of the pathway. Direct interference with these protein-protein interactions is a biologically compelling approach toward suppression of β-catenin hyperactivity, but such interactions have proven intransigent with respect to small-molecule targeting. Hence β-catenin remains an elusive target for translational cancer therapy. Here we report the discovery of a hydrocarbon-stapled peptide that directly targets β-catenin and interferes with its ability to serve as a transcriptional coactivator for T-cell factor (TCF) proteins, the downstream transcriptional regulators of the Wnt pathway.

Bicyclic peptides are promising scaffolds for the development of inhibitors of biological targets that proved intractable by typical small molecules. So far, access to bioactive bicyclic peptide architectures is limited due to a lack of appropriate orthogonal ring-closing reactions. Here, we report chemically orthogonal ring-closing olefin (RCM) and alkyne metathesis (RCAM), which enable an efficient chemo- and regioselective synthesis of complex bicyclic peptide scaffolds with variable macrocycle geometries. We also demonstrate that the formed alkyne macrocycle can be functionalized subsequently. The orthogonal RCM/RCAM system was successfully used to evolve a monocyclic peptide inhibitor of the small GTPase Rab8 into a bicyclic ligand. This modified peptide shows the highest affinity for an activated Rab GTPase that has been reported so far. The RCM/RCAM-based formation of bicyclic peptides provides novel opportunities for the design of bioactive scaffolds suitable for the modulation of challenging protein targets. Bicyclic peptides can inhibit biological targets hard to address with small molecules. Here, the authors combine two orthogonal ring-closing reactions to produce bicyclic peptides with improved bioactivity thereby providing a strategy that can greatly improve the structural diversity of such peptides.

Transcription factors are key protein effectors in the regulation of gene transcription, and in many cases their activity is regulated via a complex network of protein–protein interactions (PPI). The chemical modulation of transcription factor activity is a long-standing goal in drug discovery but hampered by the difficulties associated with the targeting of PPIs, in particular when extended and flat protein interfaces are involved. Peptidomimetics have been applied to inhibit PPIs, however with variable success, as for certain interfaces the mimicry of a single secondary structure element is insufficient to obtain high binding affinities. Here, we describe the design and characterization of a stabilized protein tertiary structure that acts as an inhibitor of the interaction between the transcription factor TEAD and its co-repressor VGL4, both playing a central role in the Hippo signalling pathway. Modification of the inhibitor with a cell-penetrating entity yielded a cell-permeable proteomimetic that activates cell proliferation via regulation of the Hippo pathway, highlighting the potential of protein tertiary structure mimetics as an emerging class of PPI modulators. Targeting the interaction between transcription factor TEAD and its co-repressor VGL4 is an attractive strategy to chemically modulate Hippo signaling. Here, the authors develop a proteomimetic with stabilized tertiary structure that inhibits the TEAD:VGL4 interaction in vitro and in cells.

Proteins have evolved as a variable platform that provides access to molecules with diverse shapes, sizes and functions. These features have inspired chemists for decades to seek artificial mimetics of proteins with improved or novel properties. Such work has focused primarily on small protein fragments, often isolated secondary structures; however, there has lately been a growing interest in the design of artificial molecules that mimic larger, more complex tertiary folds. In this Perspective, we define these agents as ‘proteomimetics’ and discuss the recent advances in the field. Proteomimetics can be divided into three categories: protein domains with side-chain functionality that alters the native linear-chain topology; protein domains in which the chemical composition of the polypeptide backbone has been partially altered; and protein-like folded architectures that are composed entirely of non-natural monomer units. We give an overview of these proteomimetic approaches and outline remaining challenges facing the field.The complexity of proteins has inspired chemists to seek artificial mimetics of protein structure and function. Historically, most such work has focused on analogues of small, isolated segments; however, there is growing interest in mimicry of larger, intact tertiary folds. This Perspective surveys the emerging body of work on these agents, termed ‘proteomimetics’, discusses their construction and outlines some of the remaining challenges.

In most bacteria and archaea, filaments of FtsZ protein organize cell division. FtsZ forms a ring structure at the division site and starts the recruitment of 10 to 20 downstream proteins that together form a multiprotein complex termed the divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time in detail how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division. Most bacteria and archaea use the tubulin homologue FtsZ as its central organizer of cell division. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic, and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. We show in biochemical assays that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the β domain of FtsQ shows that only residues 64 to 87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ-FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ-FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalizing view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches. IMPORTANCE In most bacteria and archaea, filaments of FtsZ protein organize cell division. FtsZ forms a ring structure at the division site and starts the recruitment of 10 to 20 downstream proteins that together form a multiprotein complex termed the divisome. The divisome is thought to facilitate many of the steps required to make two cells out of one. FtsQ and FtsB are part of the divisome, with FtsQ being a central hub, interacting with most of the other divisome components. Here we show for the first time in detail how FtsQ interacts with its downstream partner FtsB and show that mutations that disturb the interface between the two proteins effectively inhibit cell division.

Hyperactive Ras signalling is found in most cancers. Ras proteins are only active in membrane nanoclusters, which are therefore potential drug targets. We previously showed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering via direct interaction with the Ras binding domain (RBD) of Raf. Here, we establish that the B-Raf preference of Gal1 emerges from the divergence of the Raf RBDs at their proposed Gal1-binding interface. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B- and C-Raf-RBDs. Its 23-mer core fragment is sufficient to interfere with H-Ras nanoclustering, modulate Ras-signalling and moderately reduce cell viability. These latter two phenotypic effects may also emerge from the ability of L5UR to broadly engage with several RBD- and RA-domain containing Ras interactors. The L5UR-peptide core fragment is a starting point for the development of more specific reagents against Ras-nanoclustering and -interactors. The 23mer L5URcore peptide is identified, which binds with low micromolar affinity to Ras-binding domains, notably of Raf proteins, thus disrupting their galectin1-interaction, H-Ras-nanoclustering, -signalling and more broadly cell proliferation.

This protocol provides a detailed procedure for the preparation of stapled α-helical peptides, which have proven their potential as useful molecular probes and as next-generation therapeutics. Two crucial features of this protocol are (i) the construction of peptide substrates containing hindered α-methyl, α-alkenyl amino acids and (ii) the ring-closing olefin metathesis (RCM) of the resulting resin-bound peptide substrates. The stapling systems described in this protocol, namely bridging one or two turns of an α-helix, are highly adaptable to most peptide sequences, resulting in favorable RCM kinetics, helix stabilization and promotion of cellular uptake.

The rising incidence of multidrug resistance in Gram-negative bacteria underlines the urgency for novel treatment options. One promising new approach is the synergistic combination of antibiotics with antimicrobial peptides. However, the use of such peptides is not straightforward; they are often sensitive to proteolytic degradation, which greatly limits their clinical potential. One approach to increase stability is to apply a hydrocarbon staple to the antimicrobial peptide, thereby fixing them in an α-helical conformation, which renders them less exposed to proteolytic activity. In this work we applied several different hydrocarbon staples to two previously described peptides shown to act on the outer membrane, L6 and L8, and tested their activity in a zebrafish embryo infection model using a clinical isolate of Acinetobacter baumannii as a pathogen. We show that the introduction of such a hydrocarbon staple to the peptide L8 improves its in vivo potentiating activity on antibiotic treatment, without increasing its in vivo antimicrobial activity, toxicity or hemolytic activity.

There has been a strong effort to devise strategies to interfere with oncogenic Ras for cancer treatment. A review of recent advances in the development of small-molecule inhibitors that impair either Ras localization or protein interactions provides new optimism in this field. Despite intense efforts in pharmaceutical industry and academia, a therapeutic grip on oncogenic Ras proteins has remained elusive. Mutated Ras is associated with ∼20−30% of all human cancers often not responsive to established therapies. In particular, K-Ras, the most frequently mutated Ras isoform, is considered one of the most important but 'undruggable' targets in cancer research. Recently, new cavities on Ras for small-molecule ligands were identified, and selective direct targeting of mutated K-Ras G12C has become possible for what is to our knowledge the first time. In addition, impairment of Ras spatial organization, in particular via targeting the prenyl-binding Ras chaperone PDEδ, has opened a fresh perspective in anticancer research. These recent advances fuel hopes for the development of new drugs targeting Ras.