Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.     

Evaluation of a series of anticonvulsant 1,2,3,4-tetrahydroisoquinolinyl-benzamides

SAR studies around a series of N-(tetrahydroisoquinolinyl)-2-methoxybenzamides, identified by high-throughput screening at the novel SB-204269 binding site, have provided compounds such as 13, 29-33 with high affinity and excellent anticonvulsant activity in animal models.     

Deciphering the unusual acylation pattern of Helicobacter pylori lipid A

The synthesis of “typical” hexa-acylated lipid A occurs via a nine-step enzymatic pathway, which is generally well conserved throughout all gram-negative bacteria. One exception to the rule is Helicobacter pylori, which has only eight homologs to the nine lipid A biosynthetic enzymes. The discrepancy occurs toward the end of the pathway, with H. pylori containing only a single putative secondary acyltransferase encoded by jhp0265. In Escherichia coli K-12, two late acyltransferases, termed LpxL and LpxM, are required for the biosynthesis of hexa-acylated lipid A. Detailed biochemical and genetic analyses reveal that H. pylori Jhp0265 (the protein encoded by jhp0265) is in fact an LpxL homolog, capable of transferring a stearoyl group to the hydroxyl group of the 2′ linked fatty acyl chain of lipid A. Despite the lack of a homolog to LpxM in the H. pylori genome, the organism synthesizes a hexa-acylated lipid A species, suggesting that an equivalent enzyme exists. Using radiolabeled lipid A substrates and acyl-acyl carrier protein as the fatty acyl donor, we were able to confirm the presence of a second H. pylori late acyl transferase by biochemical assays. After synthesis of the hexa-acylated lipid A species, several modification enzymes then function to produce the major lipid A species of H. pylori that is tetra-acylated. Jhp0634 was identified as an outer membrane deacylase that removes the 3′-linked acyl chains of H. pylori lipid A. Together, this work elucidates the biochemical machinery required for the acylation and deacylation of the lipid A domain of H. pylori lipopolysaccharide.     

Information quality in proteomics

Proteomics, the study of the protein complement of a biologicalsystem, is generating increasing quantities of data from rapidlydeveloping technologies employed in a variety of different experimentalworkflows. Experimental processes, e.g. for comparative 2D gelstudies or LC-MS/MS analyses of complex protein mixtures, involvea number of steps: from experimental design, through wet anddry lab operations, to publication of data in repositories andfinally to data annotation and maintenance. The presence ofinaccuracies throughout the processing pipeline, however, resultsin data that can be untrustworthy, thus offsetting the benefitsof high-throughput technology. While researchers and practitionersare generally aware of some of the information quality issuesassociated with public proteomics data, there are few acceptedcriteria and guidelines for dealing with them. In this article,we highlight factors that impact on the quality of experimentaldata and review current approaches to information quality managementin proteomics. Data quality issues are considered throughoutthe lifecycle of a proteomics experiment, from experiment designand technique selection, through data analysis, to archivingand sharing.      

Cysteine protease gene expression and proteolytic activity during senescence of Alstroemeria petals.

The functional life of the flower is terminated by senescence and/or abscission. Multiple processes contribute to produce the visible signs of petal wilting and inrolling that typify senescence, but one of the most important is that of protein degradation and remobilization. This is mediated in many species through protein ubiquitination and the action of specific protease enzymes. This paper reports the changes in protein and protease activity during development and senescence of Alstroemeria flowers, a Liliaceous species that shows very little sensitivity to ethylene during senescence and which shows perianth abscission 8-10 d after flower opening. Partial cDNAs of ubiquitin (ALSUQ1) and a putative cysteine protease (ALSCYP1) were cloned from Alstroemeria using degenerate PCR primers and the expression pattern of these genes was determined semi-quantitatively by RT-PCR. While the levels of ALSUQ1 only fluctuated slightly during floral development and senescence, there was a dramatic increase in the expression of ALSCYP1 indicating that this gene may encode an important enzyme for the proteolytic process in this species. Three papain class cysteine protease enzymes showing different patterns of activity during flower development were identified on zymograms, one of which showed a similar expression pattern to the cysteine protease cDNA.     

Acquisition of an Agrobacterium Ri Plasmid and Pathogenicity by Other α-Proteobacteria in Cucumber and Tomato Crops Affected by Root Mat

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring α-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium. An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring α-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).     

Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.