New roles for ubiquitin in the assembly and function of neuronal circuits

A series of recent papers highlight a prominent role for ubiquitin in the formation and function of neural circuits. These new results focus attention on the molecular remodeling that occurs at various decision points in the life of growth cones and synapses.     

Design,activity, and structure of a highly specific artificial endonuclease

We have generated an artificial highly specific endonuclease by fusing domains of homing endonucleases I-DmoI and I-CreI and creating a new 1400 A(2) protein interface between these domains. Protein engineering was accomplished by combining computational redesign and an in vivo protein-folding screen. The resulting enzyme, E-DreI (Engineered I-DmoI/I-CreI), binds a long chimeric DNA target site with nanomolar affinity, cleaving it precisely at a rate equivalent to its natural parents. The structure of an E-DreI/DNA complex demonstrates the accuracy of the protein interface redesign algorithm and reveals how catalytic function is maintained during the creation of the new endonuclease. These results indicate that it may be possible to generate novel highly specific DNA binding proteins from homing endonucleases.     

ZK 156718, a low calcemic, antiproliferative, and prodifferentiating vitamin D analog.

The physiologically active form of vitamin D, 1,25-dihydroxyvitamin D(3), plays an important role not only in the establishment and maintenance of calcium metabolism, but also in regulating cell growth and differentiation. Because the clinical usefulness of 1,25-dihydroxyvitamin D(3) is limited by its tendency to cause hypercalcemia, new analogs with a better therapeutic profile have been synthesized, including ZK 156718. We compared the effects of 1,25-dihydroxyvitamin D(3) and ZK 156718 on growth, differentiation, and on p21(Waf1/Cip1) and p27(Kip1) expression in human colon cancer cells (Caco-2). Whereas ZK 156718 at the concentration [10(-8) M] was as potent as 10(-6) M 1,25-dihydroxyvitamin D(3) in inducing differentiation and p21(Waf1/Cip1) expression, it was even more effective in inhibiting cell growth and stimulating p27(Kip1) expression than 1,25-dihydroxyvitamin D(3) itself. In summary, our study presents a new and potent vitamin D analog with a decreased metabolic stability, making it useful for the treatment of a diversity of clinical disorders.     

Interleukin 1B gene polymorphism is associated with baseline C-reactive protein levels in healthy individuals

C-reactive protein (CRP) is a sensitive marker of inflammation induced by both IL-6 and IL-1. Thus, genetic variation in these genes could be associated with the variety in C-reactive protein levels, and therefore with the severity of the entire inflammatory response. Even a subtle elevation in baseline CRP levels in healthy individuals has been found to significantly increase the risk for cardiovascular diseases. Therefore, to find out the possible role of pro-inflammatory cytokines in CRP baseline regulation we conducted a study of 338 healthy blood donors whose CRP levels were determined and whose single nucleotide polymorphisms of IL1A(C/T)-889, IL1B(C/T)-511, IL1B(C/T) + 3954, IL6(G/C)-174 and ILRN (a VNTR) both genotyped and haplotyped. The data revealed an association between CRP levels and the IL1B + 3954 genotype. Also, the bilocus haplotype IL1B-511*1/IL1B + 3954*2 was more frequent in subjects with below median CRP levels (< 0.72 mg/l), and composite genotype analysis of IL1B-511/IL1B + 3954 supported this finding. Our findings suggest that in healthy people, basal CRP levels are regulated by IL1B but not by IL6 genetics.     

Gene expression profiling of resting and activated vascular smooth muscle cells by serial analysis of gene expression and clustering analysis

Migration and proliferation of vascular smooth muscle cells (SMCs) are key events in atherosclerosis. However, little is known about alterations in gene expression upon transition of the quiescent, contractile SMC to the proliferative SMC. We performed serial analysis of gene expression (SAGE) of cultured, human SMCs, either grown under resting circumstances or activated with an atherogenic stimulus. Analysis of tags, representing 47,209 and 47,259 mRNAs from a library of resting and activated SMCs, respectively, identified 105 tags induced and 52 tags repressed greater than fivefold. To evaluate the relevance in SMC biology of unmatched, regulated tags, we performed hierarchical clustering analysis, based on their expression profiles in public SAGE databases, and clustered these novel genes in distinct groups. The regulation in SMCs was confirmed by Northern blotting for representative genes of these groups. Plasminogen activator inhibitor-2 has not been associated with atherosclerosis before and was localized to atherosclerotic lesions.     

Metagenomes of complex microbial consortia derived from different soils as sources for novel genes conferring formation of carbonyls from short-chain polyols on Escherichia coli

Metagenomic DNA libraries from three different soil samples (meadow, sugar beet field, cropland) were constructed. The three unamplified libraries comprised approximately 1267000 independent clones and harbored approximately 4.05 Gbp of environmental DNA. Approximately 300000 recombinant Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from short-chain (C2 to C4) polyols such as 1,2-ethanediol, 2,3-butanediol, and a mixture of glycerol and 1,2-propanediol on indicator agar. Twenty-four positive E. COLI clones were obtained during the initial screen. Fifteen of them contained recombinant plasmids, designated pAK201-215, which conferred a stable carbonyl-forming phenotype on E. coli Sequencing revealed that the inserts of pAK201-215 encoded 26 complete and 14 incomplete predicted protein-encoding genes. Most of these genes were similar to genes with unknown functions from other microorganisms or unrelated to any other known gene. The further analysis was focused on the 7 plasmids (pAK204, pAK206, pAK208, and pAK210-213) recovered from the positive clones, which exhibited an NAD(H)-dependent alcohol oxidoreductase activity with polyols or the correlating carbonyls as substrates in crude extracts. Three genes (ORF6, ORF24, and ORF25) conferring this activity were identified during subcloning of the inserts of pAK204, pAK211, and pAK212. The sequences of the three deduced gene products revealed no significant similarities to known alcohol oxidoreductases, but contained putative glycine-rich regions, which are characteristic for binding of nicotinamide cofactors.     

Extensive MHC class I-restricted CD8 T lymphocyte responses against various yeast genera in humans

The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeast-specific CD8 T cells expressed interferon-gamma but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses.     

Intraspecific Variation of Unusual Phospholipids from Corynebacterium spp. Containing a Novel Fatty Acid

The novel fatty acid trans-9-methyl-10-octadecenoic acid was isolated from the coryneform bacterial strain LMG 3820 (previously misidentified as Arthrobacter globiformis) and identified by spectroscopic methods and chemical derivatization. This fatty acid is attached to the unusual lipid acyl phosphatidylglycerol. Five different species of this lipid type were identified; their structures were elucidated by tandem mass spectrometry and are reported here for the first time. Additionally, we identified three different cardiolipins, two bearing the novel fatty acid. The characteristic 10-methyl-octadecanoic acid was present only in phosphatidylinositol. Because of the unusual fatty acid pattern of strain LMG 3820, the 16S rDNA sequence was determined and showed regions of identity to sequences of Corynebacterium variabilis DSM 20132^T and DSM 20536. All three strains possessed the novel fatty acid, identifying trans-9-methyl-10-octadecenoic acid as a potential biomarker characteristic for this taxon. Surprisingly, the fatty acid and relative abundances of phospholipids of Corynebacterium sp. strain LMG 3820 were similar to those of the type strain but different from those of Corynebacterium variabilis DSM 20536, although all three strains possessed identical 16S rDNA sequences and strains DSM 20132^T and DSM 20536 have 90.5% DNA-DNA homology. This is one of the rare cases wherein different organisms with identical 16S rDNA sequences have been observed to present recognizably different fatty acid and lipid compositions. Since methylation of a fatty acid considerably lowers the transition temperature of the corresponding lipid resulting in a more flexible cell membrane, the intraspecific variation in the lipid composition, coinciding with the morphological and Gram stain reaction variability of this species, probably offers an advantage for this species to inhabit different environmental niches.     

Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin

Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington’s disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on “gutless” adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.