709 SummaryEthanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize ( Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-β -1,4-glucanase from Acidothermus cellulolyticus , was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei , also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall.With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T 2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.
Ras is a guanine nucleotide-binding protein that cycles between inactive GDP-bound and active GTP-bound states to regulate a diverse array of cellular processes, including cell growth, apoptosis, and differentiation. The guanine nucleotide-bound state of Ras is tightly maintained by regulatory factors to promote regulated growth control. A class of regulatory molecules that lead to Ras activation are guanine nucleotide exchange factors (GEFs). Ras GEFs bind to Ras and facilitate GDP release, followed by GTP incorporation and Ras activation. Nitric oxide (NO) has also been shown to promote guanine nucleotide exchange (GNE) on Ras and increase cellular Ras-GTP levels, but the process by which NOmediated GNE occurs is not clear. We initiated NMR structural and biochemical studies to elucidate how nitrosylation of Ras might lead to enhanced GNE. Surprisingly, our studies show that stable S-nitrosylation of Ras at Cys-118, does not affect the structure of Ras, its association with the Ras-binding domain of Raf (a downstream effector of Ras), or GNE rates relative to non-nitrosylated Ras. We have found, however, that the actual chemical process of nitrosylation, rather than the end-product of Ras S-nitrosylation, accounts for the enhanced GNE that we have observed and that has been previously observed by others.guanosine triphosphatase ͉ redox ͉ cancer ͉ reactive oxygen species ͉ reactive nitrogen species N early 20 years ago, nitric oxide (NO) was identified as a vaso-relaxation factor and was found to mediate its effects on smooth muscle by coordinating the heme moiety of guanylate cyclase, leading to enhanced production of cGMP (1). It was later shown that increased levels of cGMP modulate the function of other physiological targets, such as protein kinases, phosphodiesterases, and ion channels, causing vaso-relaxation (as reviewed in refs. 1-3). NO has also been shown to modulate cGMP-independent pathways by modifying a multitude of cellular biomolecules containing reactive metal centers, as well as cysteine residues (4-6). Although NO is able to modulate the activity of tyrosine kinases, serine͞threonine kinases, guanosine triphosphatases (GTPases), and phosphatases, the molecular basis of NO modification and the subsequent effect on enzyme activity are not completely understood. NO is generally believed to exert its effects on biomolecules by direct modification of a residue important for protein structure or enzymatic catalysis.The Ras GTPase is a protein product of the most commonly mutated oncogene in all of human cancer (ras), and has been identified as a target for S-nitrosylation by NO (7). Ras is a molecular switch that cycles between an inactive GDP-bound state and an active GTP-bound state, to regulate a number of cellular processes, including cell growth, differentiation and apoptosis (for reviews, see refs. 8-10). In its oncogenic form, Ras is populated in a GTP-bound state and is constitutively active. The chronic activation of Ras leads to persistent activation of downstream signaling cascades to pr...
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