Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.

In Alzheimer’s disease (AD), brain insulin and insulin-like growth factor (IGF) resistance and deficiency begin early, and worsen with severity of disease. The factors mediating progression of brain insulin/IGF resistance in AD are not well understood. We hypothesize that AD progression is mediated via negative cross-talk that promotes toxic ceramide generation and endoplasmic reticulum (ER) stress. The rationale is that insulin resistance dysregulates lipid metabolism and promotes ceramide accumulation, and thereby increases inflammation and stress. Consequences include disruption of cytoskeletal function and AβPP-Aβ secretion. The present study correlates AD stage with activation of pro-ceramide genes, ceramide levels, and molecular indices of ER stress in postmortem human brain tissue. The results demonstrated that in AD, brain insulin/IGF resistance was associated with constitutive activation of multiple pro-ceramidegenes, increased ceramide levels, and increased expression of pro-ER stress pathway genes and proteins. Expression of several pro-ceramide and pro-apoptotic ER stress pathway molecules increased with AD severity and brain insulin/IGF resistance. In contrast, ER stress molecules that help maintain homeostasis with respect to unfolded protein responses were mainly upregulated in the intermediate rather than late stage of AD. These findings support our hypothesis that in AD, a triangulated mal-signaling network initiated by brain insulin/IGF resistance is propagated by the dysregulation of ceramide and ER stress homeostasis, which themselves promote insulin resistance. Therefore, once established, this reverberating loop must be targeted using multi-pronged approaches to disrupt the AD neurodegeneration cascade.

In eukaryotes, all isoprenoid compounds share a common precursor, mevalonic acid, whose synthesis is catalyzed by the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. As one step towards a better understanding of the role that this enzyme plays in coordinating isoprenoid biosynthesis in plants, Arabidopsis thaliana HMG CoA reductase was ectopically expressed in transgenic Arabidopsis plants. By using this molecular genetic approach, several novel and fundamental observations have been made regarding isoprenoid biosynthesis in Arabidopsis. First, it was demonstrated that the overexpression of authentic Arabidopsis HMG CoA reductase is not sufficient to alter the bulk synthesis and accumulation of the abundant end products of the plant isoprenoid pathway. Second, active transcription of the transgene appears to co-activate and deregulate expression of the native gene, resulting in a striking elevation of HMG CoA reductase mRNA levels. Finally, although very high levels of HMG CoA reductase mRNA were expressed in these transgenic plants, only modest increases in enzyme activity could be detected. Taken together, these data suggest that HMG CoA reductase expression is regulated at multiple levels in plants as well as animals, and they provide a foundation for elucidating the molecular mechanisms for mevalonate regulation in A. thaliana.

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