Chaperone-like activity of tubulin. binding and reactivation of unfolded substrate enzymes

The eukaryotic cytoskeletal protein tubulin is a heterodimer of two subunits, alpha and beta, and is a building block unit of microtubules. In a previous communication we demonstrated that tubulin possesses chaperone-like activities by preventing the stress-induced aggregation of various proteins (Guha, S., Manna, T. K., Das, K. P., and Bhattacharyya, B. (1998) J. Biol. Chem. 273, 30077-30080). As an extension of this observation, we explored whether tubulin, like other known chaperones, also protected biological activity of proteins against thermal stress or increased the yields of active proteins during refolding from a denatured state. We show here that tubulin not only prevents the thermal aggregation of alcohol dehydrogenase and malic dehydrogenase but also protects them from loss of activity. We also show that tubulin prevents the aggregation of substrates during their refolding from a denatured state and forms a stable complex with denatured substrate. The activity of malic dehydrogenase, alpha-glucosidase, and lactate dehydrogenase during their refolding from urea or guanidium hydrochloride denatured states increased significantly in presence of tubulin compared with that without tubulin. These results suggest that tubulin, in addition to its role in mitosis, cell motility, and other cellular events, might be implicated in protein folding and protection from stress.     

A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome)

Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.     

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor

The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures.     

TNF-α induced altered signaling mechanism in human neutrophil

In the present study we investigated the TNF- induced signal transduction mechanism in human neutrophil. Exogenously added TNF- affects both PKC activity and its translocation from cytosol to the membrane. Endogenous protein phosphorylation pattern is inhibited in TNF- induced neutrophil in Ca-dependent and Ca-independent manner, including a major 47 and 66 kDa cytosolic proteins, which may be implicated in superoxide anion generation. However TNF- dose dependently enhances the expression of -PKC isotype but not the -PKC. Morphology and cell cytotoxicity are studied in TNF- treated neutrophil to understand the TNF- induced cell death or apoptosis and these experiment is further confirmed by DNA fragmentation analysis. These results clearly demonstrate that TNF- induces cellular death of human neutrophil at least in part by enhanced expression of Ca-independent -PKC. These observations provide an insight towards understanding the function of -PKC in apoptotic pathway.     

Cloning and sequencing of an apparently recombinant promoter for napin gene from Brassica campestris genomic library and its evolutionary significance

From a genomic library of Brassica campestris (brown sarson cv. B54), we have cloned and sequenced about 2 kb of upstream regulatory region from one of the 2S albumin-coding gene family. The sequence has several seed-specific promoter motifs. A sequence alignment of the 5' flanking regions of the available Brassica 2S storage protein genes showed that our sequence is a double crossover recombinant product of the two members of the napin gene family. A possible explanation of this fact is that Brassica species evolved through gene duplication and recombination from a common ancestor with fewer number of chromosomes and genes.     

Characterization of non-planar peptide groups in protein crystal structures

Peptide groups are generally assumed to be planar in protein structure, due to 'rigid' partial double bond character of peptide bonds, thus the value of peptide torsion angle omega should be restricted to 180 degrees for the usual trans form of peptide unit. However, on analyzing the ultra-high resolution protein crystal database, we find that in some cases, omega deviates >10 degrees from its usual value of 180 degrees, indicating significant non-planarity of peptide groups. Moreover, the non-planarity for most of the amino acids is found to be 'biased' towards values of omega smaller than 180 degrees. Similar trend for to is confirmed by the neutron diffraction data for proteins. The neutron diffraction database also reveals that non-planar peptide groups are generally correlated to 'pyramidal' structure of the peptide-nitrogen bonds. Consequently, the hydrogen atom of peptide group deviates from its planar position, as measured by the 'improper' torsion angle theta. Thus, we find that both the angles omega and theta point towards a significant amount of non-planarity of peptide groups, which cannot be ignored. The role of peptide nonplanarity in protein function is, however, not yet clear.     

Predatory efficiency of the water bug Sphaerodema annulatum on mosquito larvae (Culex quinquefasciatus) and its effect on the adult emergence

The daily number of IV instar larva of Culex quinquefasciatus killed, rate of pupation and adult emergence was noted in presence of the predatory water bug Sphaerodema annulatum for a period of seven consecutive days, experimentally, in the laboratory. The rate of IV instar larva killed by the water bugs on an average was 65.17 per day. The rate of pupation ranged between 7.6 and 48 in control while in presence of water bugs it ranged between 6 and 35. The rate of adult emergence in control experiments varied between 1.4 and 4.8 per day, which was reduced to only 0.4-28.8 per day in case of the water bugs. The results clearly indicate that the water bugs on its way of predation reduces the rate of pupation and adult emergence of Cx. quinquefasciatus significantly which calls for an extensive field trials.     

Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway

Scaffold proteins mediate efficient and specific signaling in several mitogen-activated protein (MAP) kinase cascades. In the yeast high osmolarity response pathway, the MAP kinase kinase Pbs2 is thought to function as a scaffold, since it binds the osmosensor Sho1, the upstream MAP kinase kinase kinase Ste11, and the downstream MAP kinase Hog1. Nonetheless, previous work has shown that Ste11 can be activated even when Pbs2 is deleted, resulting in inappropriate crosstalk to the mating pathway. We have found a region in the C terminus of Sho1 that binds Ste11 independently of Pbs2 and is required for crosstalk. These data support a model in which Sho1 has at least two separable interaction regions: one that binds Ste11 and mediates its activation, and one that binds Pbs2, directing Ste11 to act on Pbs2. Thus, a network of interactions provided by both Sho1 and Pbs2 appears to direct pathway information flow.     

An empirical bayes adjustment to increase the sensitivity of detecting differentially expressed genes in microarray experiments

MOTIVATION: Detection of differentially expressed genes is one of the major goals of microarray experiments. Pairwise comparison for each gene is not appropriate without controlling the overall (experimentwise) type 1 error rate. Dudoit et al. have advocated use of permutation-based step-down P-value adjustments to correct the observed significance levels for the individual (i.e. for each gene) two sample t-tests. RESULTS: In this paper, we consider an ANOVA formulation of the gene expression levels corresponding to multiple tissue types. We provide resampling-based step-down adjustments to correct the observed significance levels for the individual ANOVA t-tests for each gene and for each pair of tissue type comparisons. More importantly, we introduce a novel empirical Bayes adjustment to the t-test statistics that can be incorporated into the step-down procedure. Using simulated data, we show that the empirical Bayes adjustment improved the sensitivity of detecting differentially expressed genes up to 16%, while maintaining a high level of specificity. This adjustment also reduces the false non-discovery rate to some degree at the cost of a modest increase in the false discovery rate. We illustrate our approach using a human colon cancer dataset consisting of oligonucleotide arrays of normal, adenoma and carcinoma cells. The number of genes with differential expression level declared statistically significant was about 50 when comparing normal to adenoma cells and about five when comparing adenoma to carcinoma cells. This list includes genes previously known to be associated with colon cancer as well as some novel genes. AVAILABILITY: R code for the empirical Bayes adjustment and step-down P-value calculation via resampling are available from the supplementary web-site. Supplementary information: http://www.mathstat.gsu.edu/~matsnd/EB/supp.htm     

The transitional ER localization mechanism of Pichia pastoris Sec12

COPII vesicles assemble at ER subdomains called transitional ER (tER) sites, but the mechanism that generates tER sites is unknown. To study tER biogenesis, we analyzed the transmembrane protein Sec12, which initiates COPII vesicle formation. Sec12 is concentrated at discrete tER sites in the budding yeast Pichia pastoris. We find that P. pastoris Sec12 exchanges rapidly between tER sites and the general ER. The tER localization of Sec12 is saturable and is mediated by interaction of the Sec12 cytosolic domain with a partner component. This interaction apparently requires oligomerization of the Sec12 lumenal domain. Redistribution of P. pastoris Sec12 to the general ER does not perturb the localization of downstream tER components, suggesting that Sec12 and other COPII proteins associate with a tER scaffold. These results provide evidence that tER sites form by a network of dynamic associations at the cytosolic face of the ER.