Stepwise prediction of conformational discontinuous B-cell epitopes using the Mapitope algorithm

Mapping the epitope of an antibody is of great interest, since it contributes much to our understanding of the mechanisms of molecular recognition and provides the basis for rational vaccine design. Here we present Mapitope, a computer algorithm for epitope mapping. The algorithm input is a set of affinity isolated peptides obtained by screening phage display peptide-libraries with the antibody of interest. The output is usually 1-3 epitope candidates on the surface of the atomic structure of the antigen. We have systematically tested the performance of Mapitope by assessing the effect of the algorithm parameters on the final prediction. Thus, we have examined the effect of the statistical threshold (ST) parameter, relating to the frequency distribution and enrichment of amino acid pairs from the isolated peptides and the D (distance) and E (exposure) parameters which relate to the physical parameters of the antigen. Two model systems were analyzed in which the antibody of interest had previously been co-crystallized with the antigen and thus the epitope is a given. The Mapitope algorithm successfully predicted the epitopes in both models. Accordingly, we formulated a stepwise paradigm for the prediction of discontinuous conformational epitopes using peptides obtained from screening phage display libraries. We applied this paradigm to successfully predict the epitope of the Trastuzumab antibody on the surface of the Her-2/neu receptor in a third model system.     

COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER–ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins’ role in ER-to-Golgi transport.     

Promoters maintain their relative activity levels under different growth conditions

Most genes change expression levels across conditions, but it is unclear which of these changes represents specific regulation and what determines their quantitative degree. Here, we accurately measured activities of ～900 S. cerevisiae and ～1800 E. coli promoters using fluorescent reporters. We show that in both organisms 60–90% of promoters change their expression between conditions by a constant global scaling factor that depends only on the conditions and not on the promoter's identity. Quantifying such global effects allows precise characterization of specific regulation—promoters deviating from the global scale line. These are organized into few functionally related groups that also adhere to scale lines and preserve their relative activities across conditions. Thus, only several scaling factors suffice to accurately describe genome‐wide expression profiles across conditions. We present a parameter‐free passive resource allocation model that quantitatively accounts for the global scaling factors. It suggests that many changes in expression across conditions result from global effects and not specific regulation, and provides means for quantitative interpretation of expression profiles.     

Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin

Tiamulin, a prominent member of the pleuromutilin class of antibiotics, is a potent inhibitor of protein synthesis in bacteria. Up to now the effect of pleuromutilins on the ribosome has not been determined on a molecular level. The 3.5 A structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin provides for the first time a detailed picture of its interactions with the 23S rRNA, thus explaining the molecular mechanism of the antimicrobial activity of the pleuromutilin class of antibiotics. Our results show that tiamulin is located within the peptidyl transferase center (PTC) of the 50S ribosomal subunit with its tricyclic mutilin core positioned in a tight pocket at the A-tRNA binding site. Also, the extension, which protrudes from its mutilin core, partially overlaps with the P-tRNA binding site. Thereby, tiamulin directly inhibits peptide bond formation. Comparison of the tiamulin binding site with other PTC targeting drugs, like chloramphenicol, clindamycin and streptogramins, may facilitate the design of modified or hybridized drugs that extend the applicability of this class of antibiotics.     

Guidance of primordial germ cell migration by the chemokine SDF-1

The signals directing primordial germ cell (PGC) migration in vertebrates are largely unknown. We demonstrate that sdf-1 mRNA is expressed in locations where PGCs are found and toward which they migrate in wild-type as well as in mutant embryos in which PGC migration is abnormal. Knocking down SDF-1 or its receptor CXCR4 results in severe defects in PGC migration. Specifically, PGCs that do not receive the SDF-1 signal exhibit lack of directional movement toward their target and arrive at ectopic positions within the embryo. Finally, we show that the PGCs can be attracted toward an ectopic source of the chemokine, strongly suggesting that this molecule provides a key directional cue for the PGCs.     

Oxygen-mediated cold-acclimation in cucumber (Cucumis sativus) seedlings

Cold acclimation of etiolated cucumber seedlings, consisting of cooling at 12°C for 48 h followed by a warming period at 25°C, led to tolerance to subsequent chilling at 2°C. Tolerance, as evidenced by freedom from chilling injury and continued growth, developed during the warming period in a time-course manner for 12 h but decreased with prolonged warming. A similar increase and subsequent decrease was also observed in the content of palmitic, linoleic and linolenic acids in total lipid fraction from cucumber hypocotyl tissue. During the warming period supra-ambient oxygen stimulated, whereas subambient oxygen inhibited, the increase in fatty acid content as well as development of chilling tolerance. A strong correlation between oxygen-mediated changes in fatty acid content and associated development of cold tolerance suggests that both these processes are interrelated. Cold acclimation, but not cold stress, led to an increase followed by a decrease in CO₂ evolution suggesting that a respiratory upsurge is yet another feature of cold acclimation in cucumbers.     

Cold-induced enzyme inactivation: how does cooling lead to pyridoxal phosphate-aldimine bond cleavage in tryptophanase?

The phenomenon of cold scission or cold lability, which entails a widespread variety of oligomeric enzymes, is still enigmatic. The effect of cooling on the activity and the quaternary structure of the pyridoxal 5'-phosphate (PLP)-dependent enzyme, tryptophanase (Tnase), was studied utilizing single photon counting time-resolved spectrofluorometry. Upon cooling of holo-wild-type (wt) Tnase and its W330F mutant from 25 degrees C to 2 degrees C, a reduction in PLP fluorescence lifetime and rotational correlation time as well as inactivation and dissociation from tetramers to dimers were observed for both enzymes. Fluorescence anisotropy invariably decreased as a consequence of cooling, whether it was accompanied by a slight decrease in activity without significant dissociation, or by a substantial decrease in activity that was associated with either a partial or major dissociation. These results support the suggested conformational change that precedes the PLP-aldimine bond scission. It is proposed that cold inactivation is initiated by the weakening of hydrophobic interactions, leading to conformational changes which are the driving force for the aldimine bond cleavage.     

The function and regulation of vasa-like genes in germ-cell development

The vasa gene, essential for germ-cell development, was originally identified in Drosophila, and has since been found in other invertebrates and vertebrates. Analysis of these vasa homologs has revealed a highly conserved role for Vasa protein among different organisms, as well as some important differences in its regulation.