Cpn20: Siamese twins of the chaperonin world

The chloroplast cpn20 protein is a functional homolog of the cpn10 co-chaperonin, but its gene consists of two cpn10-like units joined head-to-tail by a short chain of amino acids. This double protein is unique to plastids and was shown to exist in plants as well plastid-containing parasites. In vitro assays showed that this cpn20 co-chaperonin is a functional homolog of cpn10. In terms of structure, existing data indicate that the oligomer is tetrameric, yet it interacts with a heptameric cpn60 partner. Thus, the functional oligomeric structure remains a mystery. In this review, we summarize what is known about this distinctive chaperonin and use a bioinformatics approach to examine the expression of cpn20 in Arabidopsis thaliana relative to other chaperonin genes in this species. In addition, we examine the primary structure of the two homologous domains for similarities and differences, in comparison with cpn10 from other species. Lastly, we hypothesize as to the oligomeric structure and raison d’être of this unusual co-chaperonin homolog. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

2 A prebiotic RNA apparatus functions within the contemporary ribosome

Ribosomes, the universal cellular machines, possess spectacular architecture accompanied by inherent mobility, allowing for their smooth performance as polymerases that translate the genetic code into proteins. The site for peptide bond formation is located within a universal internal semi-symmetrical region, which was identified within all contemporary ribosomes. The high conservation of this region implies its existence irrespective of environmental conditions and indicates that it may represent an ancient RNA molecular apparatus. Hence, we named it the “proto-ribosome”. This prebiotic pocket-like RNA entity is suggested to be capable to accommodate substrates whose stereochemistry enables the creation of chemical bonds. It could have evolved from an earlier catalytic RNA entity that we named the “pre-proto-ribosome”, presumed to be a molecular machine capable of performing various essential tasks in the RNA world, which was snatched by the amino acid invaders for producing proteins.     

Understanding differences between synthetic and natural antibodies can help improve antibody engineering

Synthetic libraries are a major source of human-like antibody (Ab) drug leads. To assess the similarity between natural Abs and the products of these libraries, we compared large sets of natural and synthetic Abs using “CDRs Analyzer,” a tool we introduce for structural analysis of Ab-antigen (Ag) interactions. Natural Abs, we found, recognize their Ags by combining multiple complementarity-determining regions (CDRs) to create an integrated interface. Synthetic Abs, however, rely dominantly, sometimes even exclusively on CDRH3. The increased contribution of CDRH3 to Ag binding in synthetic Abs comes with a substantial decrease in the involvement of CDRH2 and CDRH1. Furthermore, in natural Abs CDRs specialize in specific types of non-covalent interactions with the Ag. CDRH1 accounts for a significant portion of the cation-pi interactions; CDRH2 is the major source of salt-bridges and CDRH3 accounts for most hydrogen bonds. In synthetic Abs this specialization is lost, and CDRH3 becomes the main sources of all types of contacts. The reliance of synthetic Abs on CDRH3 reduces the complexity of their interaction with the Ag: More Ag residues contact only one CDR and fewer contact 3 CDRs or more. We suggest that the focus of engineering attempts on CDRH3 results in libraries enriched with variants that are not natural-like. This may affect not only Ag binding, but also Ab expression, stability and selectivity. Our findings can help guide library design, creating libraries that can bind more epitopes and Abs that better mimic the natural antigenic interactions.     

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.     

On the interaction of colicin E3 with the ribosome

Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone     

Unique properties of the camel erythrocyte membrane: II. Organization of membrane proteins

Camel erythrocyte membranes are distinguished by some unique properties of stability and composition. Notable is their abundance in proteins (protein: lipid ratio of 3 : 1). Membrane proteins of camel erythrocytes were compared with those of human erythrocytes, which have been intensively investigated. Proteins were extracted with various aqueous media (EDTA, alkaline or high ionic strength) and with ionic and non-ionic detergents and were analyzed by gel electrophoresis. In membranes of camel erythrocytes, the peripheral proteins constitute, proportionally, a much smaller fraction of total proteins than in the human erythrocyte, while their distribution is identical per unit of surface area. The camel erythrocyte membrane is particularly rich in integral proteins and in intramembranous particles. The proteins in this membrane are more closely organized than in the human system, as revealed by crosslinking and freeze-etching studies. It is proposed that protein-protein interaction of integral proteins, presumably constituting an “integral skeleton”, is a dominant structural feature stabilizing the camel erythrocyte membrane.     

Biofilm control in water by a UV-based advanced oxidation process

An ultraviolet (UV)-based advanced oxidation process (AOP), with hydrogen peroxide and medium-pressure (MP) UV light (H(2)O(2)/UV), was used as a pretreatment strategy for biofilm control in water. Suspended Pseudomonas aeruginosa cells were exposed to UV-based AOP treatment, and the adherent biofilm formed by the surviving cells was monitored. Control experiments using H(2)O(2) or MP UV irradiation alone could inhibit biofilm formation for only short periods of time (<24 h) post-treatment. In a H(2)O(2)/filtered-UV (>295 nm) system, an additive effect on biofilm control was shown vs filtered-UV irradiation alone, probably due to activity of the added hydroxyl radical (OH?). In a H(2)O(2)/full-UV (ie full UV spectrum, not filtered) system, this result was not obtained, possibly due to the germicidal UV photons overwhelming the AOP system. Generally, however, H(2)O(2)/UV prevented biofilm formation for longer periods (days) only when maintained with residual H(2)O(2). The ratio of surviving bacterial concentration post-treatment to residual H(2)O(2) concentration played an important role in biofilm prevention and bacterial regrowth. H(2)O(2) treatments alone resulted in poorer biofilm control compared to UV-based AOP treatments maintained with similar levels of residual H(2)O(2), indicating a possible advantage of AOP.     

Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

Amyloid aggregates of the calcium-binding EF-hand proteins, S100A8 and S100A9, have been found in the corpora amylacea of patients with prostate cancer and may play a role in carcinogenesis. Here we present a novel model system using the yeast Saccharomyces cerevisiae to study human S100A8 and S100A9 aggregation and toxicity. We found that S100A8, S100A9 and S100A8/9 cotransfomants form SDS-resistant non-toxic aggregates in yeast cells. Using fluorescently tagged proteins, we showed that S100A8 and S100A9 accumulate in foci. After prolonged induction, S100A8 foci localized to the cell vacuole, whereas the S100A9 foci remained in the cytoplasm when present alone, but entered the vacuole in cotransformants. Biochemical analysis of the proteins indicated that S100A8 and S100A9 alone or coexpressed together form amyloid-like aggregates in yeast. Expression of S100A8 and S100A9 in wild type yeast did not affect cell viability, but these proteins were toxic when expressed on a background of unrelated metastable temperature-sensitive mutant proteins, Cdc53-1p, Cdc34-2p, Srp1-31p and Sec27-1p. This finding suggests that the expression and aggregation of S100A8 and S100A9 may limit the capacity of the cellular proteostasis machinery. To test this hypothesis, we screened a set of chaperone deletion mutants and found that reducing the levels of the heat-shock proteins Hsp104p and Hsp70p was sufficient to induce S100A8 and S100A9 toxicity. This result indicates that the chaperone activity of the Hsp104/Hsp70 bi-chaperone system in wild type cells is sufficient to reduce S100A8 and S100A9 amyloid toxicity and preserve cellular proteostasis. Expression of human S100A8 and S100A9 in yeast thus provides a novel model system for the study of the interaction of amyloid deposits with the proteostasis machinery.