The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.     

A locus conferring tolerance to Theileria infection in African cattle

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Bos indicus (Boran) cattle demonstrating heritable tolerance to infection with T. parva (h² = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on bovine chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained variant in a paralogue of the FAF1 gene in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africa’s most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.     

Structural and biochemical analysis of Cellvibrio japonicus xylanase 10C: how variation in substrate-binding cleft influences the catalytic profile of family GH-10 xylanases

Microbial degradation of the plant cell wall is the primary mechanism by which carbon is utilized in the biosphere. The hydrolysis of xylan, by endo-beta-1,4-xylanases (xylanases), is one of the key reactions in this process. Although amino acid sequence variations are evident in the substrate binding cleft of "family GH10" xylanases (see afmb.cnrs-mrs.fr/CAZY/), their biochemical significance is unclear. The Cellvibrio japonicus GH10 xylanase CjXyn10C is a bi-modular enzyme comprising a GH10 catalytic module and a family 15 carbohydrate-binding module. The three-dimensional structure at 1.85 A, presented here, shows that the sequence joining the two modules is disordered, confirming that linker sequences in modular glycoside hydrolases are highly flexible. CjXyn10C hydrolyzes xylan at a rate similar to other previously described GH10 enzymes but displays very low activity against xylooligosaccharides. The poor activity on short substrates reflects weak binding at the -2 subsite of the enzyme. Comparison of CjXyn10C with other family GH10 enzymes reveals "polymorphisms" in the substrate binding cleft including a glutamate/glycine substitution at the -2 subsite and a tyrosine insertion in the -2/-3 glycone region of the substrate binding cleft, both of which contribute to the unusual properties of the enzyme. The CjXyn10C-substrate complex shows that Tyr-340 stacks against the xylose residue located at the -3 subsite, and the properties of Y340A support the view that this tyrosine plays a pivotal role in substrate binding at this location. The generic importance of using CjXyn10C as a template in predicting the biochemical properties of GH10 xylanases is discussed.     

Proteolytic enzyme activity in rat hindlimb muscles in fetus and during post-natal development

The enzymatic activity of two lysosomal enzymes, acid phosphatase and cathepsin D, was determined in fetus and during post-natal development of the rat gastrocnemius muscle in comparison to the histological differentiation of this muscle. The specific activity of cathepsin D and acid phosphatase was 7 and 2.5 fold higher in the muscle during development until 20 days after birth, than that of mature muscle, respectively. A trend of gradual decrease in the activity of these enzymes was observed concomitantly with the differentiation and maturation of the muscle from mononucleated cells in the fetus to myotubes formation at day 1 after birth, followed by the formation of "young" and then striated myofibers in 10- and 20-day old neonates, respectively. However, no correlation could be found between the lysosomal enzyme activity and the developmental stages of the muscle until 20 days after birth. It is suggested that the elevated activity of lysosomal acid hydrolases may be associated with late developmental processes from young to mature myofibers in normal skeletal muscle and not only in various pathological conditions.     

Associating Genes and Protein Complexes with Disease via Network Propagation

A fundamental challenge in human health is the identification of disease-causing genes. Recently, several studies have tackled this challenge via a network-based approach, motivated by the observation that genes causing the same or similar diseases tend to lie close to one another in a network of protein-protein or functional interactions. However, most of these approaches use only local network information in the inference process and are restricted to inferring single gene associations. Here, we provide a global, network-based method for prioritizing disease genes and inferring protein complex associations, which we call PRINCE. The method is based on formulating constraints on the prioritization function that relate to its smoothness over the network and usage of prior information. We exploit this function to predict not only genes but also protein complex associations with a disease of interest. We test our method on gene-disease association data, evaluating both the prioritization achieved and the protein complexes inferred. We show that our method outperforms extant approaches in both tasks. Using data on 1,369 diseases from the OMIM knowledgebase, our method is able (in a cross validation setting) to rank the true causal gene first for 34% of the diseases, and infer 139 disease-related complexes that are highly coherent in terms of the function, expression and conservation of their member proteins. Importantly, we apply our method to study three multi-factorial diseases for which some causal genes have been found already: prostate cancer, alzheimer and type 2 diabetes mellitus. PRINCE''s predictions for these diseases highly match the known literature, suggesting several novel causal genes and protein complexes for further investigation.     

Activity of single-stranded DNA endonucleases in mung bean is associated with cell division

A single-strand-specific endonuclease from mung bean sprouts is widely usedin molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.     

Mechanisms of initial endplate failure in the human vertebral body

Endplate failure occurs frequently in osteoporotic vertebral fractures and may be related to the development of high tensile strain. To determine whether the highest tensile strains in the vertebra occur in the endplates, and whether such high tensile strains are associated with the material behavior of the intervertebral disc, we used micro-CT-based finite element analysis to assess tissue-level strains in 22 elderly human vertebrae (81.5±9.6 years) that were compressed through simulated intervertebral discs. In each vertebra, we compared the highest tensile and compressive strains across the different compartments: endplates, cortical shell, and trabecular bone. The influence of Poisson-type expansion of the disc on the results was determined by compressing the vertebrae a second time in which we suppressed the Poisson expansion. We found that the highest tensile strains occurred within the endplates whereas the highest compressive strains occurred within the trabecular bone. The ratio of strain to assumed tissue-level yield strain was the highest for the endplates, indicating that the endplates had the greatest risk of initial failure. Suppressing the Poisson expansion of the disc decreased the amount of highly tensile-strained tissue in the endplates by 79.4±11.3%. These results indicate that the endplates are at the greatest risk of initial failure due to the development of high tensile strains, and that such high tensile strains are associated with the Poisson expansion of the disc. We conclude that initial failure of the vertebra is associated with high tensile strains in the endplates, which in turn are influenced by the material behavior of the disc.