Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia, accounting for 60-70% of cases in subjects over 65 years of age. Several postulates have been put forward that relate AD neuropathology to intellectual and functional impairment. These range from free-radical-induced damage, through cholinergic dysfunction, to beta-amyloid-induced toxicity. However, therapeutic strategies aimed at improving the cognitive symptoms of patients via choline supplementation, cholinergic stimulation or beta-amyloid vaccination, have largely failed. A growing body of evidence suggests that perturbations in systems using the excitatory amino acid L-glutamate (L-Glu) may underlie the pathogenic mechanisms of (e.g.) hypoxia-ischemia, epilepsy, and chronic neurodegenerative disorders such as Huntington's disease and AD. Almost all neurons in the CNS carry the N-methyl-D-aspartate (NMDA) subtype of ionotropic L-glutamate receptors, which can mediate post-synaptic Ca2+ influx. Excitotoxicity resulting from excessive activation of NMDA receptors may enhance the localized vulnerability of neurons in a manner consistent with AD neuropathology, as a consequence of an altered regional distribution of NMDA receptor subtypes. This review discusses mechanisms for the involvement of the NMDA receptor complex and its interaction with polyamines in the pathogenesis of AD. NMDA receptor antagonists have potential for the therapeutic amelioration of AD. (C) 2004 Elsevier Ltd. All rights reserved.

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-Angstrom crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 Angstrom, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.

Crystallization of Arabidopsis thaliana acetohydroxyacid synthase in complex with the sulfonylurea herbicide chlorimuron ethyl

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) catalyses the formation of 2-acetolactate and 2-aceto-2-hydroxybutyrate as the first step in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine. The enzyme is inhibited by a wide range of substituted sulfonylureas and imidazolinones and many of these compounds are used as commercial herbicides. Here, the crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of Arabidopsis thaliana AHAS in complex with the sulfonylurea herbicide chlorimuron ethyl are reported. This is the first report of the structure of any plant protein in complex with a commercial herbicide. Crystals diffract to 3.0 Angstrom resolution, have unit-cell parameters a = b = 179.92, c = 185.82 Angstrom and belong to space group P6(4)22. Preliminary analysis indicates that there is one monomer in the asymmetric unit and that these are arranged as pairs of dimers in the crystal. The dimers form a very open hexagonal lattice, with a high solvent content of 81%.

5 '-Untranslated regions with multiple upstream AUG codons can support low-level translation via leaky scanning and reinitiation

Upstream AUGs (uAUGs) and upstream open reading frames (uORFs) are common features of mRNAs that encode regulatory proteins and have been shown to profoundly influence translation of the main ORF. In this study, we employed a series of artificial 5'-untranslated regions (5'-UTRs) containing one or more uAUGs/uORFs to systematically assess translation initiation at the main AUG by leaky scanning and reinitiation mechanisms. Constructs containing either one or two uAUGs in varying contexts but without an in-frame stop codon upstream of the main AUG were used to analyse the leaky scanning mechanism. This analysis largely confirmed the ranking of different AUG contextual sequences that was determined previously by Kozak. In addition, this ranking was the same for both the first and second uAUGs, although the magnitude of initiation efficiency differed. Moreover, similar to10% of ribosomes exhibited leaky scanning at uAUGs in the most favourable context and initiated at a downstream AUG. A second group of constructs containing different numbers of uORFs, each with optimal uAUGs, were used to measure the capacity for reinitiation. We found significant levels of initiation at the main ORF even in constructs containing four uORFs, with nearly 10% of ribosomes capable of reinitiating five times. This study shows that for mRNAs containing multiple uORFs/uAUGs, ribosome reinitiation and leaky scanning are efficient mechanisms for initiation at their main AUGs.

Anlaysis of complementary expression profiles following WT1 induction versus repression reveals the cholesterol/fatty acid synthetic pathways as a possible major target of WT1

The Wilms' tumour suppressor gene, WT1, encodes a zinc-finger protein that is mutated in Wilms' tumours and other malignancies. WT1 is one of the earliest genes expressed during kidney development. WT1 proteins can activate and repress putative target genes in vitro, although the in vivo relevance of such target genes often remains unverified. To better understand the role of WT1 in tumorigenesis and kidney development, we need to identify downstream target genes. In this study, we have expression pro. led human embryonic kidney 293 cells stably transfected to allow inducible WT1 expression and mouse mesonephric M15 cells transfected with a WT1 antisense construct to abolish endogenous expression of all WT1 isoforms to identify WT1-responsive genes. The complementary overlap between the two cell lines revealed a pronounced repression of genes involved in cholesterol biosynthesis by WT1. This pathway is transcriptionally regulated by the sterol responsive element-binding proteins (SREBPs). Here, we provide evidence that the C-terminal end of the WT1 protein can directly interact with SREBP, suggesting that WT1 may modify the transcriptional function of SREBPs via a direct protein-protein interaction. Therefore, the tumour suppressor activities of WT1 may be achieved by repressing the mevalonate pathway, thereby controlling cellular proliferation and promoting terminal differentiation.

Cardiac expression of the cystic fibrosis transmembrane conductance regulator involves novel Exon 1 usage to produce a unique amino-terminal protein

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel present in many cells. In cardiomyocytes, we report that multiple exon 1 usage and alternative splicing produces four CFTR transcripts, with different 5'-untranslated regions, CFTRTRAD-139, CFTR-1C/-1A, CFTR-1C, and CFTR-1B. CFTR transcripts containing the novel upstream exons (exons -1C, -1B, and -1A) represent more than 90% of cardiac expressed CFTR mRNA. Regulation of cardiac CFTR expression, in response to developmental and pathological stimuli, is exclusively due to the modulation of CFTR-1C and CFTR-1C/-1A expression. Upstream open reading frames have been identified in the 5'-untranslated regions of all CFTR transcripts that, in conjunction with adjacent stem-loop structures, modulate the efficiency of translation initiation at the AUG codon of the main CFTR coding region in CFTRTRAD-139 and CFTR-1C/-1A transcripts. Exon(-1A), only present in CFTR-1C/-1A transcripts, encodes an AUG codon that is in-frame with the main CFTR open reading frame, the efficient translation of which produces a novel CFTR protein isoform with a curtailed amino terminus. As the expression of this CFTR transcript parallels the spatial and temporal distribution of the cAMP-activated whole-cell current density in normal and diseased hearts, we suggest that CFTR-1C/-1A provides the molecular basis for the cardiac cAMP-activated chloride channel. Our findings provide further insight into the complex nature of in vivo CFTR expression, to which multiple mRNA transcripts, protein isoforms, and post-transcriptional regulatory mechanisms are now added.

Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells

Direct continuity between the membranes of cisternae in the Golgi complex in mammalian cells rarely has been observed; when seen, its documentation has been equivocal. Here we have used dual-axis electron microscope tomography to examine the architecture of the Golgi in three dimensions at approximate to6-nm resolution in rapidly frozen, freeze-substituted murine cells that make and secrete insulin in response to glucose challenge. Our data show three types of direct connections between Golgi cisternae that are normally distinct from one another. These connections all bypass interceding cisternae. We propose that when pancreatic beta cells are stimulated to synthesize and secrete insulin rapidly in vivo, such connections provide a continuous lumen that facilitates the rapid transit of large amounts of newly made protein for secretion. The heterotypic fusion of cisternae, even transiently, raises important questions about the molecular mechanisms that (i) facilitate the fusion/fission of cisternal membranes and control the directionality and specificity of such events, and (it) retain Golgi processing enzymes at specific places within individual cisternae when two cisternae at different levels in the Golgi have fused, maintaining the sequential processing hierarchy that is a hallmark of Golgi organization.

Suicide inhibition of acetohydroxyacid synthase by hydroxypyruvate

Acetohydroxyacid synthase (Ec 2.2.1.6) catalyses the thiamine diphosphate-dependent reaction between two molecules of pyruvate yielding 2-acetolactacte and CO2. The enzyme will also utilise hydroxypyruvate with a k(cat) value that is 12% of that observed with pyruvate. When hydroxypyruvate is the substrate, the enzyme undergoes progressive inactivation with kinetics that are characteristic of suicide inhibition. It is proposed that the dihydroxyethyl-thiamine diphosphate intermediate can expel a hydroxide ion forming an enol that rearranges to a bound acetyl group.

Multi-centre Phase II trial of the polyamine synthesis inhibitor SAM486A (CGP48664) in patients with metastatic melanoma

Purpose: To determine the activity and tolerability of SAM496A, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), in patients with metastatic melanoma who had not received prior chemotherapy. Selected patients were offered participation in two sub-studies examining early changes in tumor metabolism with FDG-PET and changes in tumor polyamine content. Patients and methods: Fifteen patients with measurable metastatic melanoma, normal cardiac function, and no known CNS metastases were eligible and received SAM486A by 1-hour IV infusion daily for 5 days every 3 weeks. Response was assessed by SWOG criteria. Results: No patient had a confirmed partial response. Fatigue/lethargy, myalgia and neutropenia were the main toxicities but no febrile neutropenia or grade 4 non-hematological toxicity occurred. Five patients had PET scans pre-treatment and on days 8-12 of cycle 1. No patient had reduction of tumor metabolism. Serial biopsy in one patient showed alterations in polyamines consistent with SAMDC inhibition. Conclusions: Using the present dose and schedule of administration, SAM486A does not have significant therapeutic potential in patients with metastatic melanoma.

Domain relationships in thiamine diphosphate-dependent enzymes

Three-dimensional structures have been determined for 13 different enzymes that use thiamine diphosphate (ThDP) as a cofactor. These enzymes fall into five families, where members within a family have similar structures. In different families, there are similarities between some domains that clearly point to a common ancestor for all of these enzymes. Where the enzyme structures differ, evolutionary relationships between families can be discerned. Here, I present an analysis of these families and propose an evolutionary pathway to explain the diversity of structures that are now known.

Thiamin nutrition and catalysis-induced instability of thiamin diphosphate

Thiamin (vitamin B1) is required in animal diets because it is the precursor of the enzyme cofactor, thiamin diphosphate. Unlike other B vitamins, the dietary thiamin requirement is proportional to non-fat energy intake but there is no obvious biochemical reason for this relationship. In the present communication we show for two enzymes that the cofactor undergoes a slow destruction during catalysis, which may explain the interdependence of thiamin and energy intakes.