Agmatine transport into spinal nerve terminals is modulated by polyamine analogs

Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that Snca deletion had on whole brain lipid composition. We analysed masses of individual phospholipid (PL) classes and neutral lipid mass as well as PL acyl chain composition in brains from wild-type and Snca-/- mice. Although total brain PL mass was not altered, cardiolipin and phosphatidylglycerol mass decreased 16% and 27%, respectively, in Snca-/- mice. In addition, no changes were observed in plasmalogen or polyphosphoinositide mass. In ethanolamine glycerophospholipids and phosphatidylserine, docosahexaenoic acid (22 : 6n-3) was decreased 7%, while 16 : 0 was increased 1.1-fold and 1.4-fold, respectively. Surprisingly, brain cholesterol, cholesteryl ester, and triacylglycerol mass were increased 1.1-fold, 1.6-fold, and 1.4-fold, respectively in Snca-/- mice. In isolated myelin, cholesterol mass was also increased 1.3-fold, but because there was also a net increase in myelin PL mass, the cholesterol to PL ratio was unaltered. No changes in the expression of cholesterogenic enzymes were observed, suggesting these did not account for the observed changes in cholesterol. These data extend our previous results in astrocytes and kinetic studies in vivo demonstrating a role for Snca in brain lipid metabolism and demonstrate a clear impact on brain neutral lipid metabolism.     

The 1.4-A crystal structure of the S. pombe Pop2p deadenylase subunit unveils the configuration of an active enzyme

Deadenylation is the first and probably also rate-limiting step of controlled mRNA decay in eukaryotes and therefore central for the overall rate of gene expression. In yeast, the process is maintained by the mega-Dalton Ccr4-Not complex, of which both the Ccr4p and Pop2p subunits are 3′–5′ exonucleases potentially responsible for the deadenylation reaction. Here, we present the crystal structure of the Pop2p subunit from Schizosaccharomyces pombe determined to 1.4Å resolution and show that the enzyme is a competent ribonuclease with a tunable specificity towards poly-A. In contrast to S. cerevisiae Pop2p, the S. pombe enzyme contains a fully conserved DEDDh active site, and the high resolution allows for a detailed analysis of its configuration, including divalent metal ion binding. Functional data further indicates that the identity of the ions in the active site can modulate both activity and specificity of the enzyme, and finally structural superposition of single nucleotides and poly-A oligonucleotides provide insight into the catalytic cycle of the protein.     

Endocytic down-regulation of ErbB2 is stimulated by cleavage of its C-terminus

High ErbB2 levels are associated with cancer, and impaired endocytosis of ErbB2 could contribute to its overexpression. Therefore, knowledge about the mechanisms underlying endocytic down-regulation of ErbB2 is warranted. The C-terminus of ErbB2 can be cleaved after various stimuli, and after inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) to the N- and C-terminus of ErbB2, respectively (YFP-ErbB2-CFP). After geldanamycin stimulation YFP-ErbB2-CFP became cleaved in nonapoptotic cells in a proteasome-dependent manner, and a markedly larger relative amount of cleaved YFP-ErbB2-CFP was observed in early endosomes than in the plasma membrane. Furthermore, cleavage took place at the plasma membrane, and cleaved ErbB2 was internalized and degraded far more efficiently than full-length ErbB2. Concordantly, a C-terminally truncated ErbB2 was also readily endocytosed and degraded in lysosomes compared with full-length ErbB2. Altogether, we suggest that geldanamycin leads to C-terminal cleavage of ErbB2, which releases the receptor from a retention mechanism and causes endocytosis and lysosomal degradation of ErbB2.     

Crystal Structure of Barley Limit Dextrinase-Limit Dextrinase Inhibitor (LD-LDI) Complex Reveals Insights into Mechanism and Diversity of Cereal Type Inhibitors

Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.     

How many hydrogen-bonded α-turns are possible?

The formation of α-turns is a possibility to reverse the direction of peptide sequences via five amino acids. In this paper, a systematic conformational analysis was performed to find the possible isolated α-turns with a hydrogen bond between the first and fifth amino acid employing the methods of ab initio MO theory in vacuum (HF/6-31G*, B3LYP/6-311?+?G*) and in solution (CPCM/HF/6-31G*). Only few α-turn structures with glycine and alanine backbones fulfill the geometry criteria for the i←(i?+?4) hydrogen bond satisfactorily. The most stable representatives agree with structures found in the Protein Data Bank. There is a general tendency to form additional hydrogen bonds for smaller pseudocycles corresponding to β- and γ-turns with better hydrogen bond geometries. Sometimes, this competition weakens or even destroys the i←(i?+?4) hydrogen bond leading to very stable double β-turn structures. This is also the reason why an “ideal” α-turn with three central amino acids having the perfect backbone angle values of an α-helix could not be localized. There are numerous hints for stable α-turns with a distance between the \( {{\hbox{C}}_\alpha } \)-atoms of the first and fifth amino acid smaller than 6-7 Å, but without an i←(i?+?4) hydrogen bond.     

A crucial requirement for Hedgehog signaling in small cell lung cancer

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.     

Comparative genomics of <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> with emphasis on strains from meat

Lactobacillus sakei is a lactic acid bacterium important in food microbiology mainly due to its ability to ferment and preserve meat. The genome sequence of L. sakei strain 23K has revealed specialized metabolic capacities that reflect the bacterium’s adaption to meat products, and that differentiate it from other LAB. An extensive genomic diversity analysis was conducted to elucidate the core features of the species, and to provide a better comprehension of niche adaptation of the organism. Here, we describe the genomic comparison of 18 strains of L. sakei originating mainly from processed meat against the 23K strain by comparative genome hybridization. Pulsed field gel electrophoresis was used to estimate the genome sizes of the strains, which varied from 1.880 to 2.175 Mb, and the 23K genome was among the smallest. Consequently, a large part of the genome of this strain belongs to a common gene pool invariant in this species. The majority of genes important in adaption to meat products, the ability to flexibly use meat components, and robustness during meat processing and storage were conserved, such as genes involved in nucleoside scavenging, catabolism of arginine, and the ability to cope with changing redox and oxygen levels, which is indicative of the role these genes play in niche specialization within the L. sakei species. Moreover, an additional set of sequenced L. sakei genes beyond the 23K genome was present on the microarray used, and it was demonstrated that all the strains carry remnants of or complete bacteriocin operons. The genomic divergence corresponded mainly to five regions in the 23K genome, which showed features consistent with horizontal gene transfer. Carbohydrate-fermentation profiles of the strains were evaluated in light of the CGH data, and for most substrates, the genotypes were consistent with the phenotypes. We have demonstrated a highly conserved organization of the L. sakei genomes investigated, and the 23K strain is a suitable model organism to study core features of the L. sakei species.