This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions.

Perfect circle. We report the biosynthesis of a natively folded cyclotide, MCoTI‐II, in E. coli by intracellular backbone cyclization of a linear cyclotide–intein fusion precursor. The cyclized peptide then spontaneously folds into its native conformation. Biosynthetic access to correctly folded cyclotides allows the possibility of generating cell‐based combinatorial libraries that can be screened, inside living cells, for their ability to modulate or inhibit cellular processes.

Herein, we report for the first time the design and synthesis of a novel cyclotide able to efficiently inhibit HIV-1 viral replication by selectively targeting cytokine receptor CXCR4. This was accomplished by grafting a series of topologically modified CVX15 based peptides onto the loop 6 of cyclotide MCoTI-I. The most active compound produced in this study was a potent CXCR4 antagonist (EC50 ≈ 20 nM) and an efficient HIV-1 cell-entry blocker (EC50 ≈ 2 nM). This cyclotide also showed high stability in human serum thereby providing a promising lead compound for the design of a novel type of peptide-based anti-cancer and anti-HIV-1 therapeutics.

We have developed a multistep route to the fabrication of virus assembled nanostructures with chemoselective protein-to-surface linkers synthesized by an efficient solid-phase method. These linkers were used to create patterns of 30-to-50-nm-width-lines by scanning probe nanolithography. Genetically modified cow pea mosaic virus with unique cysteine residues at specific locations on their capsomers were assembled through covalent linkage on these patterns. The morphology of the assembled structures on these line patterns characterized by atomic force microscopy was found to be strongly influenced by the intervirion interactions.

Cell‐ing point: This study shows that MCoTI‐cyclotides can provide an ideal scaffold for the biosynthesis of large combinatorial libraries inside living E. coli cells. Coupled to an appropriate in vivo reporter system, this library may rapidly be screened, for example, by fluorescence‐activated cell sorting.

The present work describes a general method for the selective attachment of proteins to solid surfaces through their C-termini that can be used for the efficient creation of protein chips. Our method is based in the chemoselective reaction between a protein C-terminal alpha-thioester and a modified surface containing N-terminal Cys residues. alpha-Thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing engineered inteins. This new method was used to immobilize two fluorescent proteins and a functional SH3 domain using a protein microarrayer.

Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands.