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The COVID-19 disease caused by the SARS-CoV-2 coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL M due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL M , revealing the ligand-free structure of the active site and the conformation of the catalytic site cavity at near-physiological temperature. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.

The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium ‘Candidatus Liberibacter asiaticus’ ( C Las) and there is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all C Las isolates, as a molecular probe to understand C Las virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in C Las-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1 -expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants. Greening disease threatens the productivity of citrus crops worldwide yet the pathosystem is poorly understood. Here, Clark et al. show that an effector cloned from the associated bacteria can suppress host plant papain-like cysteine proteases' activity, suggesting its probable role in pathogenesis.

A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery. Modulating particular ubiquitin chains using binding molecules is challenging given the diversity of chain lengths and linkages found in vivo. Now, tight binding modulators that are specific to K48-linked ubiquitin chains have been found by combining protein synthesis and screening of macrocyclic peptide ligands.

Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the “lock and key” mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.

Fasciola hepatica is a global parasite of humans and their livestock. Regulation of parasite-secreted cathepsin L-like cysteine proteases associated with virulence is important to fine-tune parasite-host interaction. We uncovered a family of seven Kunitz-type (FhKT) inhibitors dispersed into five phylogenetic groups. The most highly expressed FhKT genes (group FhKT1) are secreted by the newly excysted juveniles (NEJs), the stage responsible for host infection. The FhKT1 inhibitors do not inhibit serine proteases but are potent inhibitors of parasite cathepsins L and host lysosomal cathepsin L, S and K cysteine proteases (inhibition constants < 10 nM). Their unusual inhibitory properties are due to (a) Leu 15 in the reactive site loop P1 position that sits at the water-exposed interface of the S1 and S1′ subsites of the cathepsin protease, and (b) Arg 19 which forms cation-π interactions with Trp 291 of the S1′ subsite and electrostatic interactions with Asp 125 of the S2′ subsite. FhKT1.3 is exceptional, however, as it also inhibits the serine protease trypsin due to replacement of the P1 Leu 15 in the reactive loop with Arg 15 . The atypical Kunitz-type inhibitor family likely regulate parasite cathepsin L proteases and/or impairs host immune cell activation by blocking lysosomal cathepsin proteases involved in antigen processing and presentation.

There is emerging evidence that the proteolytic machinery of plants plays important roles in defense against pathogens. The oomycete pathogen Phytophthora infestans, the agent of the devastating late blight disease of tomato (Lycopersicon esculentum) and potato (Solanum tuberosum), has evolved an arsenal of protease inhibitors to overcome the action of host proteases. Previously, we described a family of 14 Kazal-like extracellular serine protease inhibitors from P. infestans. Among these, EPI1 and EPI10 bind and inhibit the pathogenesis-related (PR) P69B subtilisin-like serine protease of tomato. Here, we describe EPIC1 to EPIC4, a new family of P. infestans secreted proteins with similarity to cystatin-like protease inhibitor domains. Among these, the epiC1 and epiC2 genes lacked orthologs in Phytophthora sojae and Phytophthora ramorum, were relatively fast-evolving within P. infestans, and were up-regulated during infection of tomato, suggesting a role during P. infestans-host interactions. Biochemical functional analyses revealed that EPIC2B interacts with and inhibits a novel papain-like extracellular cysteine protease, termed Phytophthora Inhibited Protease 1 (PIP1). Characterization of PIP1 revealed that it is a PR protein closely related to Rcr3, a tomato apoplastic cysteine protease that functions in fungal resistance. Altogether, this and earlier studies suggest that interplay between host proteases of diverse catalytic families and pathogen inhibitors is a general defense-counterdefense process in plant-pathogen interactions.

Background: Plants and insects have coexisted for million years and evolved a set of interactions which affect both organisms at different levels. Plants have developed various morphological and biochemical adaptations to cope with herbivores attacks. However, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) has become the major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100%. Results: The aim of this study was to investigate the in vivo effect of a serine proteinase inhibitor (BTI-CMe) and a cysteine proteinase inhibitor (Hv-CPI2) from barley on this insect and to examine the effect their expression has on tomato defensive responses. We found that larvae fed on tomato transgenic plants co-expressing both proteinase inhibitors showed a notable reduction in weight. Moreover, only 56% of these larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Transgenes expression had no harmful effect on Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae), a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We also found that barley cystatin expression promoted plant defense by inducing the expression of the tomato endogenous wound inducible Proteinase inhibitor 2 (Pin2) gene, increasing the production of glandular trichomes and altering the emission of volatile organic compounds. Conclusion: Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta.

Cruzain, a cysteine protease of Trypanosoma cruzi, is a validated target for the treatment of Chagas disease. Due to its high similarity in three-dimensional structure with human cathepsins and their sequence identity above 70% in the active site regions, identifying potent but selective cruzain inhibitors with low side effects on the host organism represents a significant challenge. Here a panel of nitrile ligands with varying potencies against cathepsin K, cathepsin L and cruzain, are studied by molecular dynamics simulations as both non-covalent and covalent complexes. Principal component analysis (PCA), identifies and quantifies patterns of ligand-induced conformational selection that enable the construction of a decision tree which can predict with high confidence a low-nanomolar inhibitor of each of three proteins, and determine the selectivity for one against others.

Pine wilt disease (PWD) is an infectious disease of pines that typically kills affected trees. The causal pathogen of PWD is the pine wood nematode (PWN), Bursaphelenchus xylophilus. Understanding of the disease has advanced in recent years through the use of a highly sensitive proteomics procedure and whole genome sequence analysis; in combination, these approaches have enabled identification of proteins secreted by PWNs. However, the roles of these proteins during the onset of parasitism have not yet been elucidated. In this study, we used a leaf-disk assay based on transient overexpression in Nicotiana benthamiana to allow functional screening of 10 candidate pathogenic proteins secreted by PWNs. These proteins were selected based on previous secretome and RNA-seq analyses. We found that five molecules induced significant cell death in tobacco plants relative to a GFP-only control. Three of these proteins (Bx-TH1, Bx-TH2, and Bx-CPI) may have a role in molecular mimicry and likely make important contributions to inducing hypersensitive responses in host plants.

Insects are constantly adapting to human-driven landscape changes; however, the roles of their gut microbiota in these processes remain largely unknown. The western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) (Coleoptera: Chrysomelidae) is a major corn pest that has been controlled via annual rotation between corn (Zea mays) and nonhost soybean (Glycine max) in the United States. This practice selected for a “rotation-resistant” variant (RR-WCR) with reduced ovipositional fidelity to cornfields. When in soybean fields, RR-WCRs also exhibit an elevated tolerance of antiherbivory defenses (i.e., cysteine protease inhibitors) expressed in soybean foliage. Here we show that gut bacterial microbiota is an important factor facilitating this corn specialist’s (WCR's) physiological adaptation to brief soybean herbivory. Comparisons of gut microbiota between RR- and wild-type WCR (WT-WCR) revealed concomitant shifts in bacterial community structure with host adaptation to soybean diets. Antibiotic suppression of gut bacteria significantly reduced RR-WCR tolerance of soybean herbivory to the level of WT-WCR, whereas WT-WCR were unaffected. Our findings demonstrate that gut bacteria help to facilitate rapid adaptation of insects in managed ecosystems.