Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate

Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3C^pro) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3C^pro with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3C^pro, mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5-P5′ region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 Å resolution. Comparison with free enzyme reveals significant conformational changes in 3C^pro on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4-P2′ positions of the peptide. Strikingly, the deep S1′ specificity pocket needed to accommodate P1′-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5-P4′ region of synthetic substrates. The structure of the enzyme-peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P′ side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3C^pro to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1′ specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3C^pro.     

Expression and purification of BmrI restriction endonuclease and its N-terminal cleavage domain variants

BmrI (ACTGGG N5/N4) is one of the few metal-independent restriction endonucleases (REases) found in bacteria. The BmrI restriction-modification system was cloned by the methylase selection method, inverse PCR, and PCR. BmrI REase shows significant amino acid sequence identity to BfiI and a putative endonuclease MspBNCORF3798 from the sequenced Mesorhizobium sp. BNC1 genome. The EDTA-resistant BmrI REase was successfully over-expressed in a pre-modified E. coli strain from pET21a or pBAC-expIQ vectors. The recombinant BmrI REase shows strong promiscuous activity (star activity) in NEB buffers 1, 4, and an EDTA buffer. Star activity was diminished in buffers with 100-150 mM NaCl and 10 mM MgCl(2). His-tagged BmrI192, the N-terminal cleavage domain of BmrI, was expressed in E. coli and purified from inclusion bodies. The refolded BmrI192 protein possesses non-specific endonuclease activity. BmrI192 variants with a single Ser to Cys substitution (S76C or S90C) and BmrI200 (T200C) with a single Cys at the C-terminal end were also constructed and purified. BmrI200 digests both single-strand (ss) and double-strand (ds) DNA and the nuclease activity on ss DNA is at least 5-fold higher than that on ds DNA. The Cys-containing BmrI192 and BmrI200 nuclease variants may be useful for coupling to other DNA binding elements such as synthetic zinc fingers, thio-containing locked nucleic acids (LNA) or peptide nucleic acids (PNA).     

The genetic architecture of fitness in a seed beetle: assessing the potential for indirect genetic benefits of female choice

Background  

Quantifying the amount of standing genetic variation in fitness represents an empirical challenge. Unfortunately, the shortage of detailed studies of the genetic architecture of fitness has hampered progress in several domains of evolutionary biology. One such area is the study of sexual selection. In particular, the evolution of adaptive female choice by indirect genetic benefits relies on the presence of genetic variation for fitness. Female choice by genetic benefits fall broadly into good genes (additive) models and compatibility (non-additive) models where the strength of selection is dictated by the genetic architecture of fitness. To characterize the genetic architecture of fitness, we employed a quantitative genetic design (the diallel cross) in a population of the seed beetle Callosobruchus maculatus, which is known to exhibit post-copulatory female choice. From reciprocal crosses of inbred lines, we assayed egg production, egg-to-adult survival, and lifetime offspring production of the outbred F1 daughters (F1 productivity).     

The effects of seaweed extract inclusion on gut morphology, selected intestinal microbiota, nutrient digestibility, volatile fatty acid concentrations and the immune status of the weaned pig

An experiment (complete randomised design) was conducted to investigate the effects of Laminaria hyperborea and Laminaria digitata seaweed extract inclusion on gut morphology, selected intestinal microbiota populations, volatile fatty acid concentrations and the immune status of the weaned pig. Twenty-eight piglets (24 days of age, 6.5 ± 1.4 kg live weight) were assigned to one of four dietary treatments for 7 days and then sacrificed: (T1) basal diet (control); (T2) basal diet and 1.5 g/kg L. hyperborea seaweed extract; (T3) basal diet and 1.5 g/kg L. digitata seaweed extract; and (T4) basal diet and 1.5 g/kg of a combination of L. hyperborea and L. digitata seaweed extract. The seaweed extract contained both laminarin and fucoidan. Digesta samples were taken from the caecum and colon to measure the enterobacteria, bifidobacteria and lactobacilli populations and for volatile fatty acid analysis. Tissue samples were taken from the duodenum, jejunum and ileum for morphological examination. Blood samples were taken to determine the cytokine gene expression profile and to measure the phagocytotic capacity of the blood. Pigs offered diets containing L. hyperborea seaweed extract had less bifidobacteria in the colon (P < 0.05) and lactobacilli in the caecum (P < 0.05) and colon (P < 0.001). The inclusion of L. digitata seaweed extract resulted in lower populations of enterobacteria in the caecum and colon (P < 0.01), bifidobacteria in the caecum (P < 0.05), and lactobacilli in the caecum (P < 0.05) and colon (P < 0.001). Pigs offered the combination of L. hyperborea and L. digitata seaweed extracts had less enterobacteria (P < 0.05) and lactobacilli (P < 0.01) in the caecum and colon. Pigs offered the L. digitata-supplemented diet had a reduced villous height in the duodenum and jejunum (P < 0.05). The inclusion of the L. digitata seaweed extract increased the molar proportion of butyric acid in the colon (P < 0.05). There was a significant reduction in the ammonia concentration in the colon with the inclusion of L. hyperborea (P < 0.01) and L. digitata (P < 0.05) seaweed extracts. An increase in the expression of the Interleukin-8 mRNA was observed on day 6 with the supplementation of the combination of L. hyperborea and L. digitata seaweed extract (P < 0.05). The inclusion of L. hyperborea seaweed extract resulted in an increase in total monocyte number (P < 0.05). In conclusion, the supplementation of L. hyperborea and L. digitata seaweed extract alone and in combination reduced the enterobacteria, bifidobacteria and lactobacilli populations in the caecum and colon, while only marginal effects on the immune response was observed.     

Discovery of 3,3-dimethyl-5-hydroxypipecolic hydroxamate-based inhibitors of aggrecanase and MMP-13

A series of pipecolic hydroxamate inhibitors of MMP-13 and aggrecanase was discovered based on screening known inhibitors of TNF-alpha converting enzyme (TACE). Potency versus aggrecanase was optimized by modification of the benzyloxyarylsulfonamide group. Incorporation of geminal alkyl substitution at the 3-position of the piperidine ring improved metabolic stability, presumably by increasing steric hindrance around the metabolically labile hydroxamic acid. This modification also resulted in dramatic improvement of aggrecanase activity with a slight reduction in selectivity versus MMP-1. Synthesis, structure activity relationships, and strategies to reduce metabolic clearance are described.     

Molecular extensibility of mini-dystrophins and a dystrophin rod construct

Muscular dystrophies arise with various mutations in dystrophin, implicating this protein in force transmission in normal muscle. With 24 three-helix, spectrin repeats interspersed with proline-rich hinges, dystrophin's large size is an impediment to gene therapy, prompting the construction of mini-dystrophins. Results thus far in dystrophic mice suggest that at least one hinge between repeats is necessary though not sufficient for palliative effect. One such mini-dystrophin is studied here in forced extension at the single molecule level. Delta2331 consists of repeats (R) and hinges (H) H1-R1-2 approximately H3 approximately R22-24-H4 linked by native (-) and non-native (approximately) sequence. This is compared to its core fragment R2 approximately H3 approximately R22 as well as an eight-repeat rod fragment middle (RFM: R8-15). We show by atomic force microscopy that all repeats extend and unfold at forces comparable to those that a few myosin molecules can generate. The hinge regions most often extend and transmit force while limiting tandem repeat unfolding. From 23-42 degrees C, the dystrophin constructs also appear less temperature-sensitive in unfolding compared to a well-studied betaI-spectrin construct. The results thus reveal new modes of dystrophin flexibility that may prove central to functions of both dystrophin and mini-dystrophins.     

MHC class II-associated invariant chain isoforms regulate pulmonary immune responses

Asthma, a chronic inflammatory disease of the lung, is characterized by reversible airway obstruction and airway hyperresponsiveness (AHR), and is associated with increased production of IgE and Th2-type cytokines (IL-4, IL-5, and IL-13). Development of inflammation within the asthmatic lung depends on MHC class II-restricted Ag presentation, leading to stimulation of CD4(+) T cells and cytokine generation. Conventional MHC class II pathways require both MHC-associated invariant chain (Ii) and HLA-DM (H2-M in mice) chaperone activities, but alternative modes of Ag presentation may also promote in vivo immunity. In this study, we demonstrate that Ii(-/-) and H2-M(-/-) mice fail to develop lung inflammation or AHR following sensitization and challenge with OVA in a mouse model of allergic inflammation. To assess potentially distinct contributions by Ii chain isoforms to lung immunity, we also compared allergen-induced lung inflammation, eosinophilia, IgE production, and AHR in mice genetically altered to express either p31 Ii or p41 Ii isoform alone. Sole expression of either Ii isoform alone facilitates development of allergen-induced lung inflammation and eosinophilia. However, animals expressing only the p31 Ii isoform exhibit abrogated IgE and AHR responses as compared with p41 Ii mice in this model of allergen-induced lung inflammation, suggesting that realization of complete immunity within the lung requires expression of p41 Ii. These findings reveal a crucial role of Ii and H2-M in controlling the immune response within the lung, and suggest that p31 Ii and p41 Ii manifest nonredundant roles in development of immunity.     

Drosophila paramyosin is important for myoblast fusion and essential for myofibril formation

Paramyosin is a major structural protein of thick filaments in invertebrate muscles. Coiled-coil dimers of paramyosin form a paracrystalline core of these filaments, and the motor protein myosin is arranged on the core surface. To investigate the function of paramyosin in myofibril assembly and muscle contraction, we functionally disrupted the Drosophila melanogaster paramyosin gene by mobilizing a P element located in its promoter region. Homozygous paramyosin mutants die at the late embryo stage. Mutants display defects in both myoblast fusion and in myofibril assembly in embryonic body wall muscles. Mutant embryos have an abnormal body wall muscle fiber pattern arising from defects in myoblast fusion. In addition, sarcomeric units do not assemble properly and muscle contractility is impaired. We confirmed that these defects are paramyosin-specific by rescuing the homozygous paramyosin mutant to adulthood with a paramyosin transgene. Antibody analysis of normal embryos demonstrated that paramyosin accumulates as a cytoplasmic protein in early embryo development before assembling into thick filaments. We conclude that paramyosin plays an unexpected role in myoblast fusion and is important for myofibril assembly and muscle contraction.     

Myosin IIb is unconventionally conventional

Members of the myosin II class of molecular motors have been referred to as "conventional," a term used to describe their ability to form thick filaments, their low duty ratio, the ability of individual motor-containing "heads" to operate independently of each other, and their rate-limiting phosphate release. These features ensure that those motors that have completed their power stroke dissociate rapidly enough to prevent them from interfering with those motors that are beginning theirs. However, in this study, we demonstrate that myosin IIB, a cytoplasmic myosin II particularly enriched in the central nervous system and cardiac tissue, has a number of features that it shares instead with "unconventional" myosin isoforms, including myosins V and VI. These include a high duty ratio, rate-limiting ADP release, and high ADP affinity. These features imply that myosin IIB serves a set of physiologic needs different from those served by its more conventional myosin II counterparts, and this work provides a plausible basis for explaining the physiologic role of this unconventionally conventional myosin.