Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.glutamine metabolism | Warburg effect

The latest version of CATH (class, architecture, topology, homology) (version 3.2), released in July 2008 (http://www.cathdb.info), contains 1 14 215 domains, 2178 Homologous superfamilies and 1110 fold groups. We have assigned 20 330 new domains, 87 new homologous superfamilies and 26 new folds since CATH release version 3.1. A total of 28 064 new domains have been assigned since our NAR 2007 database publication (CATH version 3.0). The CATH website has been completely redesigned and includes more comprehensive documentation. We have revisited the CATH architecture level as part of the development of a ‘Protein Chart’ and present information on the population of each architecture. The CATHEDRAL structure comparison algorithm has been improved and used to characterize structural diversity in CATH superfamilies and structural overlaps between superfamilies. Although the majority of superfamilies in CATH are not structurally diverse and do not overlap significantly with other superfamilies, ∼4% of superfamilies are very diverse and these are the superfamilies that are most highly populated in both the PDB and in the genomes. Information on the degree of structural diversity in each superfamily and structural overlaps between superfamilies can now be downloaded from the CATH website.

Molecular cloning of components of protective antigenic preparations has suggested that related parasite fatty acid-binding proteins could form the basis of the protective immune crossreactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. Molecular models of the two parasite proteins showed that both molecules adopt the same basic three-dimensional structure, consisting of a barrel-shaped molecule formed by 10 antiparallel (8-pleated strands joined by short loops, and revealed the likely presence of crossreactive, discontinuous epitopes principally derived from amino acids in the C-terminal portions of the molecules. A recombinant form of the S. mansoni antigen, rSml4, protected outbred Swiss mice by up to 67% against challenge with S. mansoni cercariae in the absence of adjuvant and without provoking any observable autoimmune response. The same antigen also provided complete protection against challenge with F. hepatica metacercariae in the same animal model. The results suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni, of veterinary and human importance, respectively. Schistosomiasis, caused principally by Schistosoma mansoni, S. haematobium, and S. japonicum, afflicts some 200 million individuals in tropical regions of the world. Fascioliasis caused by Fasciola hepatica is an economically important disease of cattle and sheep in Europe, the Americas, Australia, and New Zealand. There are no vaccines against Schistosoma or Fasciola species; however, there is evidence for protective immune crossreactivity between S. mansoni and F. hepatica. Hillyer and coworkers (1-3) have isolated a low molecular weight F. hepatica fraction that protects against both S. mansoni and F. hepatica infections. A component of this fraction is an antigen with homology to mammalian fatty acid-binding proteins that is termed Fhl5 (4). A similar antigen, Sm14, was cloned from S. mansoni following studies of a protective saline extract of adult worms, SE (5). These results suggested that the pair of similar parasite proteins could mediate immune crossreaction and represent the basis of a subunit vaccine effective against both species. We have investigated the molecular relationship of Fhl5, Sml4, and mammalian fatty acid-binding proteins (FABPs) (6) (9), and rat intestine (10) were obtained directly from the Brookhaven Protein Data Bank (accession codes 2HMB, 1ALB, and 2IFB, respectively). Sequences of known crystal structure were aligned by leastsquares superposition of the molecules using C' coordinates alone, and the remaining sequences were subsequently incorporated into the alignment by the method of Barton and Sternberg (11) as implemented in the AMPS package.For the construction of the Sm14 model, the backbone of the 10 (3-strands and three a-helices was based on that of 1ALB. (3-Turn types were determined on the basis of the position of glycine and/or asparagine and aspartic residues within ...

Septins are able to polymerize into long apolar filaments and have long been considered to be a component of the cytoskeleton alongside intermediate filaments (which are also apolar in nature), microtubules and actin filaments (which are not). Their central guanosine triphosphate (GTP)-binding domain, which is essential for stabilizing the filament itself, is flanked by N- and C-terminal domains for which no direct structural information is yet available. In most cases, physiological filaments are built from a number of different septin monomers, and in the case of mammalian septins this is most commonly either three or four. Comprehending the structural basis for the spontaneous assembly of such filaments requires a deeper understanding of the interfaces between individual GTP-binding domains than is currently available. Nevertheless, in this review we will summarize the considerable progress which has been made over the course of the last 10 years. We will provide a brief description of each structure determined to date and comment on how it has added to the body of knowledge which is rapidly growing. Rather than simply repeat data which have already been described in the literature, as far as is possible we will try to take advantage of the full set of information now available (mostly derived from human septins) and draw the reader's attention to some of the details of the structures themselves and the filaments they form which have not be commented on previously. An additional aim is to clarify some misconceptions.

The structure of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from glycosomes of the parasite Trypanosoma cruzi, causative agent of Chagas' disease, is reported. The final model at 2.8 A î includes the bound cofactor NAD + and 90 water molecules per monomer and resulted in an R ftor of 20.1%, R free = 22.3%, with good geometry indicators. The structure has no ions bound at the active site resulting in a large change in the side chain conformation of Arg PRW which as a consequence forms a salt bridge to Asp PIH in the present structure. We propose that this conformational change could be important for the reaction mechanism and possibly a common feature of many GAPDH structures. Comparison with the human enzyme indicates that interfering with this salt bridge could be a new approach to specific inhibitor design, as the equivalent to Asp PIH is a leucine in the mammalian enzymes. z 1998 Federation of European Biochemical Societies.

From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Septins are GTP binding proteins considered to be novel components of the cytoskeleton. They polymerize into filaments based on hexameric or octameric core particles in which two copies of either three or four different septins, respectively, assemble into a specific sequence. Viable combinations of the 13 human septins are believed to obey substitution rules in which the different septins involved must come from distinct subgroups. The hexameric assembly, for example, has been reported to be SEPT7–SEPT6–SEPT2–SEPT2–SEPT6–SEPT7. Here, we have replaced SEPT2 by SEPT5 according to the substitution rules and used transmission electron microscopy to demonstrate that the resulting recombinant complex assembles into hexameric particles which are inverted with respect that predicted previously. MBP‐SEPT5 constructs and immunostaining show that SEPT5 occupies the terminal positions of the hexamer. We further show that this is also true for the assembly including SEPT2, in direct contradiction with that reported previously. Consequently, both complexes expose an NC interface, as reported for yeast, which we show to be more susceptible to high salt concentrations. The correct assembly for the canonical combination of septins 2‐6‐7 is therefore established to be SEPT2–SEPT6–SEPT7–SEPT7–SEPT6–SEPT2, implying the need for revision of the mechanisms involved in filament assembly.

Asp49 plays a fundamental role in supporting catalysis by phospholipases A2 by coordinating the calcium ion which aids in the stabilization of the tetrahedral intermediate. In several myotoxins from the venoms of Viperidae snakes, this aspartic acid is substituted by lysine. The loss of calcium binding capacity by these mutants has become regarded as the standard explanation for their greatly reduced or nonexistent phospholipolytic activity. Here we describe the crystal structure of one such Lys49 PLA2, piratoxin-II, in which a fatty acid molecule is observed within the substrate channel. This suggests that such toxins may be active enzymes in which catalysis is interrupted at the stage of substrate release. Comparison of the present structure with other PLA2s, both active and inactive, identifies Lys122 as one of the likely causes of the increased affinity for fatty acid in Lys49 enzymes. Its interaction with the mainchain carbonyl of Cys29 is expected to lead to hyperpolarization of the peptide bond between residues 29 and 30 leading to an increased affinity for the fatty acid headgroup. This strongly bound fatty acid may serve as an anchor to secure the toxin within the membrane thus facilitating its pathological effects.