Disruption of the zinc finger motifs in the Leishmania tarentolae LC-4 (=TbMP63) L-complex editing protein affects the stability of the L-complex

The uridine insertion/deletion editing complex, which we have termed the L-complex, is composed of at least 16 polypeptides stabilized entirely by protein-protein interactions. Three L-complex proteins contain zinc finger motifs that could be involved in these interactions. In Leishmania these proteins are labeled LC-1, LC-4, and LC-7b, and the orthologs in Trypanosoma brucei are labeled MP81, MP63, and MP42. Overexpression of TAP-tagged LC-4 in Leishmania tarentolae led to a partial localization of the protein in the L-complex together with the endogenous LC-4 protein, suggesting at least a dimeric organization. Disruption of zinc fingers 1 or 2 (ZnF-1 and ZnF-2) in the tagged LC-4 protein was performed by mutation of the two zinc-binding cysteines to glycines. Disruption of ZnF-1 led to a partial growth defect and a substantive breakdown of the L-complex, whereas disruption of ZnF-2 had no effect on cell growth and caused a partial breakdown of the L-complex. A close interaction of LC-4 with 2-4 proteins, including REL1 (RNA ligase) and LC-3, was suggested by chemical crosslinking and co-immunoprecipitation experiments. Our results suggest that both ZnF-1 and ZnF-2 in LC-4 play a role in protein-protein interactions and indicate that the LC-4 subcomplex may be required for formation or stability of the entire L-complex.     

Characterization of human UDP-glucose dehydrogenase. CYS-276 is required for the second of two successive oxidations

UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological overproduction of extracellular matrix components may be linked to the availability of UDP-glucuronic acid; therefore UGDH is an intriguing therapeutic target. Specific inhibition of human UGDH requires detailed knowledge of its catalytic mechanism, which has not been characterized. In this report, we have cloned, expressed, and affinity-purified the human enzyme and determined its steady state kinetic parameters. The human enzyme is active as a hexamer with values for Km and Vmax that agree well with those reported for a bovine homolog. We used crystal coordinates for Streptococcus pyogenes UGDH in complex with NAD+ cofactor and UDP-glucose substrate to generate a model of the enzyme active site. Based on this model, we selected Cys-276 and Lys-279 as likely catalytic residues and converted them to serine and alanine, respectively. Enzymatic activity of C276S and K279A point mutants was not measurable under normal assay conditions. Rate constants measured over several hours demonstrated that K279A continued to turn over, although 250-fold more slowly than wild type enzyme. C276S, however, performed only a single round of oxidation, indicating that it is essential for the second oxidation. This result is consistent with the postulated role of Cys-276 as a catalytic residue and supports its position in the reaction mechanism for the human enzyme. Lys-279 is likely to have a role in positioning active site residues and in maintaining the hexameric quaternary structure.     

Characterisation of a novel homodimeric N-acetyl-beta-D-glucosaminidase from Streptococcus gordonii

An N-acetyl-beta-D-glucosaminidase (GcnA) from Streptococcus gordonii FSS2 was cloned and sequenced. GcnA had a deduced molecular mass of 72,120 Da. The molecular weight after gel-filtration chromatography was 140,000 Da and by SDS-PAGE was 70,000 Da, indicating that the native protein was a homodimer. The deduced amino acid sequence had significant homology to a glycosyl hydrolase from Streptococcus pneumoniae and the conserved catalytic domain of the Family 20 glycosyl hydrolases. GcnA catalysed the hydrolysis of the synthetic substrates, 4-methylumbelliferyl (4MU)-N-acetyl-beta-D-glucosaminide, 4MU-N-acetyl-beta-D-galactosaminide, 4-MU-beta-D-N,N'-diacetylchitobioside, and 4-MU-beta-D-N,N',N'-chitotrioside as well as the respective chito-oligosaccharides. GcnA was optimally active at pH 6.6 and 42 degrees C. The Km for 4-MU-beta-D-N,N',N'-chitotrioside, 45 microM, was the lowest for all the substrates tested. Hg2+, Cu2+, Fe2+, and Zn2+ completely inhibited while Co2+, Mn2+, and Ni2+ partially inhibited activity. S. gordonii FSS2 and a GcnA negative mutant grew equally well on chito-oligosaccharides as substrates. The S. gordonii sequencing projects indicate two further N-acetyl-beta-D-glucosaminidase activities.     

Update on estrogen signaling

Our understanding of estrogen signaling has undergone a true paradigm shift over recent years, following the discovery in 1995 of a second estrogen receptor, estrogen receptor beta (ERbeta). In many contexts ERbeta appears to antagonize the actions of ERalpha (yin/yang relationship) although there also exist genes that are specifically regulated by one of the two receptors. Studies of ERbeta knockout mice have shown that ERbeta exerts important functions in the ovary, central nervous system, mammary gland, prostate gland, hematopoiesis, immune system, vessels and bone. The use of ERbeta-specific ligands against certain forms of cancer represents one of the many pharmaceutical possibilities that have been created thanks to the discovery of ERbeta.     

Developmental switch in brain nutrient transporter expression in the rat

Normal development of both human and rat brain is associated with a switch in metabolic fuel from a combination of glucose and ketone bodies in the immature brain to a nearly total reliance on glucose in the adult. The delivery of glucose, lactate, and ketone bodies from the blood to the brain requires specific transporter proteins, glucose and monocarboxylic acid transporter proteins (GLUTs and MCTs), respectively. Developmental expression of the GLUTs in rat brain, i.e., 55-kDa GLUT1 in the blood-brain barrier (BBB), 45-kDa GLUT1 and GLUT3 in vascular-free brain, corresponds to maturational increases in cerebral glucose uptake and utilization. It has been suggested that MCT expression peaks during suckling and sharply declines thereafter, although a comparable detailed study has not been done. This study investigated the temporal and regional expression of MCT1 and MCT2 mRNA and protein in the BBB and the nonvascular brain during postnatal development in the rat. The results confirmed maximal MCT1 mRNA and protein expression in the BBB during suckling and a decline with maturation, coincident with the switch to glucose as the predominant cerebral fuel. However, nonvascular MCT1 and MCT2 levels do not reflect changes in cerebral energy metabolism, suggesting a more complex regulation. Although MCT1 assumes a predominantly glial expression in postweanling brain, the concentration remains fairly constant, as does that of MCT2 in neurons. The maintenance of nonvascular MCT levels in the adult brain implies a major role for these proteins, in concert with the GLUTs in both neurons and astrocytes, to transfer glycolytic intermediates during cerebral energy metabolism.     

Evolution of flowering in response to day length: flipping the CONSTANS switch

Day length provides an important environmental cue by signalling conditions favourable for flowering. While Arabidopsis promotes flowering in response to long days, rice promotes flowering in response to short days. Despite this difference, a recent paper reveals that the network controlling this response is highly conserved in these distantly related plants, only the activity of one component is reversed. This reveals how an important developmental process can be diversified for adaptation by using the same set of genes, but regulating them differently.