Structure and properties of phospholipid-peptide monolayers containing monomeric SP-B(1-25) II. Peptide conformation by infrared spectroscopy

The conformation and orientation of synthetic monomeric human sequence SP-B(1-25) (mSP-B(1-25)) was studied in films with phospholipids at the air-water (A/W) interface by polarization modulation infrared reflectance absorption spectroscopy (PM-IRRAS). Modified two-dimensional infrared (2D IR) correlation analysis was applied to PM-IRRAS spectra to define changes in the secondary structure and rates of reorientation of mSP-B(1-25) in the monolayer during compression. PM-IRRAS spectra and 2D IR correlation analysis showed that, in pure films, mSP-B(1-25) had a major alpha-helical conformation plus regions of beta-sheet structure. These alpha-helical regions reoriented later during film compression than beta structural regions, and became oriented normal to the A/W interface as surface pressure increased. In mixed films with 4:1 mol:mol acyl chain perdeuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPC-d(62):DOPG), the IR spectra of mSP-B(1-25) showed that a significant, concentration-dependent conformational change occurred when mSP-B(1-25) was incorporated into a DPPC-d(62):DOPG monolayer. At an mSP-B(1-25) concentration of 10 wt.%, the peptide assumed a predominantly beta-sheet conformation with no contribution from alpha-helical structures. At lower, more physiological peptide concentrations, 2D IR correlation analysis showed that the propensity of mSP-B(1-25) to form alpha-helical structures was increased. In phospholipid films containing 5 wt.% mSP-B(1-25), a substantial alpha-helical peptide structural component was observed, but regions of alpha and beta structure reoriented together rather than independently during compression. In films containing 1 wt.% mSP-B(1-25), peptide conformation was predominantly alpha-helical and the helical regions reoriented later during compression than the remaining beta structural components. The increased alpha-helical structure of mSP-B(1-25) demonstrated here by PM-IRRAS and 2D IR correlation analysis in monolayers of 4:1 DPPC:DOPG containing 1 wt.% (and, to a lesser extent, 5 wt.%) peptide may be relevant for the formation of the intermediate order 'dendritic' surface phase observed in similar surface films by epi-fluorescence.     

Rat reduced-folate carrier-1 is localized basolaterally in MDCK kidney epithelial cells and contributes to the secretory transport of methotrexate and fluoresceinated methotrexate

The reduced-folate carrier (Rfc-1), previously also called methotrexate carrier-1 (MTX-1), was recently identified as accounting for approximately 30% of the methotrexate (Mtx) uptake into rat kidney slices. The localization of the carrier and its contribution to secretory or reabsorptive flux of the drug was therefore evaluated in polarized epithelial layers of Madin Darby canine kidney (MDCK) cells. Confocal laser scanning microscopy revealed that the HA-epitope-tagged protein was sorted to the basolateral side. In flux assays, the basolateral-to-apical transport of fluoresceinated methotrexate (FMTX) was two-fold higher than in the apical-to-basolateral direction across rat Rfc-1 transfected, but not mock-transfected, monolayers. The same observation was made for unlabeled Mtx. This secretory transport of FMTX was inhibited by an excess of 1 mM Mtx and was saturable and temperature-dependent. No differences in directional flux were observed for the pure fluorescein label. Removal of sodium resulted in a marked decrease of directional FMTX flux. The pH profile of the active transport component showed a trough around 6.5 and a maximum at acidic pH, as reported for uptake into Rfc-1-expressing cells. Thus, rat Rfc-1 is sorted to the basolateral side in polarized MDCK epithelial cells and mediates the secretion of Mtx, probably in co-operation with efflux proteins, such as multidrug resistance associated proteins, which are also expressed in these cells. This study was supported by the Deutsche Forschungsgemeinschaft (HO2103/1-2) and HO2512/1-1 (Kerstin U. Honscha).     

Activity of 3'-thioAMP derivatives as ribosomal P-site substrates

The ribosome is a large RNP complex but its main enzymatic activity, the peptidyl transferase, is a ribozyme. As many RNA enzymes use divalent metal ions in catalysis, one of the hypotheses put forward proposed that metal ions might aid peptide bond formation. To be able to test a possible coordination of a metal ion to the 3'-bridging oxygen of P-site substrates, a 3'-thioAMP was synthesized. Its chemical acylation with N-acetyl-L-leucine yielded both mono and diaminoacylated 3'-thioAMP. These thioated substrates were tested for peptide bond formation in an optimized fragment reaction in comparison with their unmodified counterparts. As the amino acid was predominantly linked to the unproductive 2'-OH in AcLeu-thioAMP (5), this substrate was barely active and not used for further analysis. In contrast, Di(AcLeu)-thioAMP (4) was more active than Di(AcLeu)-AMP (2) which is in line with the higher energy of thioesters. Both activities were slightly enhanced when Mn2+ containing buffers were employed in the assay. These data show that thioated P-site substrates are active in peptide bond formation and can in principle be used for metal-ion-rescue experiments in a full translation system.     

Stability analysis for long-term storage of naked DNA: impact on nonviral in vivo gene transfer

The transfer of naked DNA is gaining growing acceptance for nonviral gene therapy. Integrity and stability of the DNA used in nonviral gene therapy is known to be decisive for efficacy of gene transfer and transgene expression. Thus, preclinical and clinical studies require the safe storage of DNA preparations to ensure defined quality and conformation. To evaluate the influence of potentially destructive processes on plasmid DNA associated with long-term storage, capillary gel electrophoresis (CGE) analysis of the LacZ-expressing pCMVbeta plasmid over a period of 13 months was performed. The CGE analysis revealed that stable storage conditions at -80 degrees C prevent an increase in open circular (oc) plasmid, preserving the covalently closed circular (ccc) form, which is sought for efficient gene transfer. By contrast, long-term storage of plasmid DNA at 4 degrees C leads to the rapid decline of the ccc form and the increase of oc and linear DNA molecules. The use of naked DNA stored for 1, 2, or 13 months at -80 degrees C showed similar in vivo transfer efficiencies by jet-injection. Therefore, analysis of plasmids by CGE allows the reliable determination of integrity and distribution of the topology of the DNA by quantitative means.     

The vesicular monoamine content regulates VMAT2 activity through Galphaq in mouse platelets. Evidence for autoregulation of vesicular transmitter uptake

Variations in the neurotransmitter content of secretory vesicles enable neurons to adapt to network changes. Vesicular content may be modulated by vesicle-associated Go(2), which down-regulates the activity of the vesicular monoamine transmitter transporters VMAT1 in neuroendocrine cells and VMAT2 in neurons. Blood platelets resemble serotonergic neurons with respect to transmitter storage and release. In streptolysin O-permeabilized platelets, VMAT2 activity is also down-regulated by the G protein activator guanosine 5'-(beta(i)gamma-imido)triphosphate (GMppNp). Using serotonin-depleted platelets from peripheral tryptophan hydroxylase knockout (Tph1-/-) mice, we show here that the vesicular filling initiates the G protein-mediated down-regulation of VMAT2 activity. GMppNp did not influence VMAT2 activity in naive platelets from Tph1-/- mice. GMppNp-mediated inhibition could be reconstituted, however, when preloading Tph1-/- platelets with serotonin or noradrenaline. Galpha(q) mediates the down-regulation of VMAT2 activity as revealed from uptake studies performed with platelets from Galpha(q) deletion mutants. Serotonergic, noradrenergic, as well as thromboxane A(2) receptors are not directly involved in the down-regulation of VMAT2 activity. It is concluded that in platelets the vesicle itself regulates transmitter transporter activity via its content and vesicle-associated Galpha(q).     

Targeted gene correction of hprt mutations by 45 base single-stranded oligonucleotides

Targeted correction of a single base in a gene of an eucaryotic cell by specific oligonucleotides is a yet controversial technique. Here, we introduce the correction of point mutations in the hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) gene as an additional model system to test targeted gene correction. In human, Hprt mutations cause Lesch-Nyhan syndrome. Using hamster V79 cells, we generated three cell lines with one hprt point mutation each. These cell lines were treated with specific single-stranded 45 base phosphothioate modified oligonucleotides and selected by HAT medium. The surviving clones were investigated for the correction of the respective hprt mutation. Treatment with the oligonucleotides was successful in repairing all three hprt mutations (hprt cDNA position 74, C --> T; position 151, C --> T; and position 400, G --> A). The correction efficiency was very low but reproducible. We suggest that this system allows one to investigate targeted gene correction in dependence on the target sequence and the oligonucleotides used.     

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.     

Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis

Chaperonins use ATPase cycling to promote conformational changes leading to protein folding. The prokaryotic chaperonin GroEL requires a cofactor, GroES, which serves as a "lid" enclosing substrates in the central cavity and confers an asymmetry on GroEL required for cooperative transitions driving the reaction. The eukaryotic chaperonin TRiC/CCT does not have such a cofactor but appears to have a "built-in" lid. Whether this seemingly symmetric chaperonin also operates through an asymmetric cycle is unclear. We show that unlike GroEL, TRiC does not close its lid upon nucleotide binding, but instead responds to the trigonal-bipyramidal transition state of ATP hydrolysis. Further, nucleotide analogs inducing this transition state confer an asymmetric conformation on TRiC. Similar to GroEL, lid closure in TRiC confines the substrates in the cavity and is essential for folding. Understanding the distinct mechanisms governing eukaryotic and bacterial chaperonin function may reveal how TRiC has evolved to fold specific eukaryotic proteins.     

Visualization of retroviral replication in living cells reveals budding into multivesicular bodies

Retroviral assembly and budding is driven by the Gag polyprotein and requires the host-derived vacuolar protein sorting (vps) machinery. With the exception of human immunodeficiency virus (HIV)-infected macrophages, current models predict that the vps machinery is recruited by Gag to viral budding sites at the cell surface. However, here we demonstrate that HIV Gag and murine leukemia virus (MLV) Gag also drive assembly intracellularly in cell types including 293 and HeLa cells, previously believed to exclusively support budding from the plasma membrane. Using live confocal microscopy in conjunction with electron microscopy of cells generating fluorescently labeled virions or virus-like particles, we observed that these retroviruses utilize late endosomal membranes/multivesicular bodies as assembly sites, implying an endosome-based pathway for viral egress. These data suggest that retroviruses can interact with the vps sorting machinery in a more traditional sense, directly linked to the mechanism by which cellular proteins are sorted into multivesicular endosomes.