Viral inosine triphosphatase: A mysterious enzyme with typical activity, but an atypical function.

Plant viruses typically have highly condensed genomes, yet the plant-pathogenic viruses Cassava brown streak virus, Ugandan cassava brown streak virus, and Euphorbia ringspot virus are unusual in encoding an enzyme not yet found in any other virus, the "house-cleaning" enzyme inosine triphosphatase. Inosine triphosphatases (ITPases) are highly conserved enzymes that occur in all kingdoms of life and perform a house-cleaning function by hydrolysing the noncanonical nucleotide inosine triphosphate to inosine monophosphate. The ITPases encoded by cassava brown streak virus and Ugandan cassava brown streak virus have been characterized biochemically and are shown to have typical ITPase activity. However, their biological role in virus infection has yet to be elucidated. Here we review what is known of viral-encoded ITPases and speculate on potential roles in infection with the aim of generating a greater understanding of cassava brown streak viruses, a group of the world's most devastating viruses.

The macrocyclizing protease butelase 1 remains autocatalytic and reveals the structural basis for ligase activity.

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform maturation of seed storage protein upon cleavage-dependent autoactivation in the low-pH environment of storage vacuoles. The AEPs have attracted attention for their macrocyclization reactions, and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show that butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide-binding region are the source of its efficient macrocyclization. All considered, it seems that either butelase 1 has not fully specialized or a requirement for autocatalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.

Structural basis of ribosomal peptide macrocyclization in plants.

Constrained, cyclic peptides encoded by plant genes represent a new generation of drug leads. Evolution has repeatedly recruited the Cys-protease asparaginyl endopeptidase (AEP) to perform their head-to-tail ligation. These macrocyclization reactions use the substrates amino terminus instead of water to deacylate, so a peptide bond is formed. How solvent-exposed plant AEPs macrocyclize is poorly understood. Here we present the crystal structure of an active plant AEP from the common sunflower, Helianthus annuus. The active site contained electron density for a tetrahedral intermediate with partial occupancy that predicted a binding mode for peptide macrocyclization. By substituting catalytic residues we could alter the ratio of cyclic to acyclic products. Moreover, we showed AEPs from other species lacking cyclic peptides can perform macrocyclization under favorable pH conditions. This structural characterization of AEP presents a logical framework for engineering superior enzymes that generate macrocyclic peptide drug leads.

Macrocyclization by asparaginyl endopeptidases.

Contents Summary 923 I. Introduction 923 II. Plant AEPs with macrocyclizing ability 924 III. Mechanism of macrocyclization by AEPs 925 IV. Conclusions 927 Acknowledgements 927 References 927 SUMMARY: Plant asparaginyl endopeptidases (AEPs) are important for the post-translational processing of seed storage proteins via cleavage of precursor proteins. Some AEPs also function as peptide bond-makers during the biosynthesis of several unrelated classes of cyclic peptides, namely the kalata-type cyclic peptides, PawS-Derived Peptides and cyclic knottins. These three families of gene-encoded peptides have different evolutionary origins, but all have recruited AEPs for their maturation. In the last few years, the field has advanced rapidly, with the biochemical characterization of three plant AEPs capable of peptide macrocyclization, and insights have been gained from the first AEP crystal structures, albeit mammalian ones. Although the biochemical studies have improved our understanding of the mechanism of action, the focus now is to understand what changes in AEP sequence and structure enable some plant AEPs to perform macrocyclization reactions.

Two proteins for the price of one: Structural studies of the dual-destiny protein preproalbumin with sunflower trypsin inhibitor-1.

Seed storage proteins are both an important source of nutrition for humans and essential for seedling establishment. Interestingly, unusual napin-type 2S seed storage albumin precursors in sunflowers contain a sequence that is released as a macrocyclic peptide during post-translational processing. The mechanism by which such peptides emerge from linear precursor proteins has received increased attention; however, the structural characterization of intact precursor proteins has been limited. Here, we report the 3D NMR structure of the Helianthus annuus PawS1 (preproalbumin with sunflower trypsin inhibitor-1) and provide new insights into the processing of this remarkable dual-destiny protein. In seeds, PawS1 is matured by asparaginyl endopeptidases (AEPs) into the cyclic peptide SFTI-1 (sunflower trypsin inhibitor-1) and a heterodimeric 2S albumin. The structure of PawS1 revealed that SFTI-1 and the albumin are independently folded into well-defined domains separated by a flexible linker. PawS1 was cleaved in vitro with recombinant sunflower HaAEP1 and in situ using a sunflower seed extract in a way that resembled the expected in vivo cleavages. Recombinant HaAEP1 cleaved PawS1 at multiple positions, and in situ, its flexible linker was removed, yielding fully mature heterodimeric albumin. Liberation and cyclization of SFTI-1, however, was inefficient, suggesting that specific seed conditions or components may be required for in vivo biosynthesis of SFTI-1. In summary, this study has revealed the 3D structure of a macrocyclic precursor protein and provided important mechanistic insights into the maturation of sunflower proalbumins into an albumin and a macrocyclic peptide.

Evidence for Ancient Origins of Bowman-Birk Inhibitors from Selaginella moellendorffii.

Bowman-Birk Inhibitors (BBIs) are a well-known family of plant protease inhibitors first described 70 years ago. BBIs are known only in the legume (Fabaceae) and cereal (Poaceae) families, but peptides that mimic their trypsin-inhibitory loops exist in sunflowers (Helianthus annuus) and frogs. The disparate biosynthetic origins and distant phylogenetic distribution implies these loops evolved independently, but their structural similarity suggests a common ancestor. Targeted bioinformatic searches for the BBI inhibitory loop discovered highly divergent BBI-like sequences in the seedless, vascular spikemoss Selaginella moellendorffii Using de novo transcriptomics, we confirmed expression of five transcripts in S. moellendorffii whose encoded proteins share homology with BBI inhibitory loops. The most highly expressed, BBI3, encodes a protein that inhibits trypsin. We needed to mutate two lysine residues to abolish trypsin inhibition, suggesting BBI3's mechanism of double-headed inhibition is shared with BBIs from angiosperms. As Selaginella belongs to the lycopod plant lineage, which diverged ∼200 to 230 million years before the common ancestor of angiosperms, its BBI-like proteins imply there was a common ancestor for legume and cereal BBIs. Indeed, we discovered BBI sequences in six angiosperm families outside the Fabaceae and Poaceae. These findings provide the evolutionary missing links between the well-known legume and cereal BBI gene families.

Treatment of chronic HCV with sofosbuvir and simeprevir in patients with cirrhosis and contraindications to interferon and/or ribavirin.

OBJECTIVES: Patients with chronic hepatitis C virus (HCV) and cirrhosis are in critical need of treatment that is both effective and tolerable. The combination of simeprevir (SMV), a protease inhibitor, and sofosbuvir (SOF), a polymerase inhibitor, without peginterferon and/or ribavirin (PEGINF/RBV) has been shown to achieve sustained virologic response (SVR) exceeding 90% in patients with HCV genotype 1 with prior nonresponse and/or cirrhosis. The present report describes the efficacy of SMV and SOF in patients with cirrhosis, prior or current hepatic decompensation, and other contraindications to PEGINF/RBV. METHODS: A total of 120 consecutive patients with cirrhosis and contraindications to PEGINF/RBV were treated with SMV and SOF for 12 weeks. The primary end point was SVR at 12 weeks after the completion of treatment. RESULTS: The mean age of the cohort was 60 years; 63% were male, 48% were Caucasian, 44% were African American, 69% were of genotype 1A, 49% were treatment naïve, 96% were interleukin-28B non-CC, 33% were of Child class B or C, and 25% had prior hepatic decompensation. The SVR by intention-to-treat was 81% with a relapse rate of 14%. The SVR by per-protocol analysis was 87% with a relapse rate of 13%. The only baseline factor associated with SVR by multifactor analysis was Child class. SVR in patients with Child class A, B, and C was 87, 77, and 67%, respectively. Eleven percent of the patients developed severe adverse events, which included sepsis (two), variceal bleeding (two), hepatocellular carcinoma (two), and hyperbilirubinemia (eight). One of the patients with sepsis died. Two patients developed relapse more than 12 weeks after stopping SMV and SOF. CONCLUSIONS: The combination of SMV and SOF achieves high rates of SVR in patients with advanced cirrhosis but is lower with worsening Child class.