Functional and structural characterization of a prokaryotic peptide transporter with features similar to mammalian PEPT1

The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.     

Hepatitis C virus NS5A is a direct substrate of casein kinase I-alpha, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors

The hepatitis C virus encodes a single polyprotein that is processed by host and viral proteases to yield at least 10 mature viral proteins. The nonstructural (NS) protein 5A is a phosphoprotein, and experimental data indicate that the phosphorylation state of NS5A is important for the outcome of viral RNA replication. We were able to identify kinase inhibitors that specifically inhibit the formation of the hyperphosphorylated form of NS5A (p58) in cells. These kinase inhibitors were used for inhibitor affinity chromatography in order to identify the cellular targets of these compounds. The kinases casein kinase I (CKI), p38 MAPK, CIT (Citron Rho-interacting kinase), GAK, JNK2, PKA, RSK1/2, and RIPK2 were identified in the high affinity binding fractions of two NS5A hyperphosphorylation inhibitors (NS5A-p58-i). Even though these kinases are targets of the NS5A-p58-i, the only kinase showing an effect on NS5A hyperphosphorylation was confirmed to be CKI-alpha. Although this finding does not exclude the possibility that other kinase(s) might be involved in basal or regulatory phosphorylation of NS5A, we show here that NS5A is a direct substrate of CKI-alpha. Moreover, in vitro phosphorylation of NS5A by CKI-alpha resulted for the first time in the production of basal and hyperphosphorylated forms resembling those produced in cells. In vitro kinase reactions performed with NS5A peptides show that Ser-2204 is a preferred substrate residue for CKI-alpha after pre-phosphorylation of Ser-2201.     

Kunitz-type protease inhibitors group B from Solanum palustre

Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.     

Three-year weight change in successful weight losers who lost weight on a low-carbohydrate diet

Objective: The purpose of this study was to evaluate long‐term weight loss and eating and exercise behaviors of successful weight losers who lost weight using a low‐carbohydrate diet. Research Methods and Procedures: This study examined 3‐year changes in weight, diet, and physical activity in 891 subjects (96 low‐carbohydrate dieters and 795 others) who enrolled in the National Weight Control Registry between 1998 and 2001 and reported ≥30‐lb weight loss and ≥1 year weight loss maintenance. Results: Only 10.8% of participants reported losing weight after a low‐carbohydrate diet. At entry into the study, low‐carbohydrate diet users reported consuming more kcal/d (mean ± SD, 1895 ± 452 vs. 1398 ± 574); fewer calories in weekly physical activity (1595 ± 2499 vs. 2542 ± 2301); more calories from fat (64.0 ± 7.9% vs. 30.9 ± 13.1%), saturated fat (23.8 ± 4.1 vs. 10.5 ± 5.2), monounsaturated fat (24.4 ± 3.7 vs. 11.0 ± 5.1), and polyunsaturated fat (8.6 ± 2.7 vs. 5.5 ± 2.9); and less dietary restraint (10.8 ± 2.9 vs. 14.9 ± 3.9) compared with other Registry members. These differences persisted over time. No differences in 3‐year weight regain were observed between low‐carbohydrate dieters and other Registry members in intent‐to‐treat analyses (7.0 ± 7.1 vs. 5.7 ± 8.7 kg). Discussion: It is possible to achieve and maintain long‐term weight loss using a low‐carbohydrate diet. The long‐term health effects of weight loss associated with a high‐fat diet and low activity level merits further investigation.     

Proteoglycanomics: tools to unravel the biological function of glycosaminoglycans

Glycosylation is the most frequent PTM and contributes significantly to the function of proteins depending on the type of glycosylation. Especially glycan structures like the glycosaminoglycans are considered to constitute themselves the major function of the glycoconjugate which is therefore termed proteoglycan. Here we review recent views on and novel tools for analysing the proteoglycanome, which are directly related to the type of glycanation under investigation. We define the major function of the proteoglycanome to be its interaction with various proteins in many different (patho-)physiological conditions. This is exemplified by the differential glycosaminoglycan-interactome of healthy versus arthritic patient sera.     

Integrative Approach Uncovers New Patterns of Ecomorphological Convergence in Slow Arboreal Xenarthrans

Journal of Mammalian Evolution - Identifying ecomorphological convergence examples is a central focus in evolutionary biology. In xenarthrans, slow arboreality independently arose at least three...     

Grandisol as an attractant for the sugar beet pest Bothynoderes affinis and data on other Lixinae species

As a result of field tests in Bulgaria and Hungary, cis‐2‐isopropenyl‐l‐methylcyclobutane ethanol (racemic grandisol) is reported for the first time as an attractant for Bothynoderes affinis (Schrank) (Coleoptera: Curculionidae, Lixinae), a member of the pest weevil complex of sugar beet. Dose–response experiments in the field using Csalomon TAL (modified pitfall) traps (Plant Protection Institute, CAR HAS, Budapest, Hungary) showed that catches of B. affinis adults increased with increasing attractant dose. In a subsequent experiment studying the effect of trap color (white, blue, yellow, fluorescent yellow, and transparent) all traps with the lure caught more than non‐baited control traps, and the highest number of adults was recorded in transparent and yellow baited traps. Trap color had a significant effect on the number of B. affinis females captured. Transparent TAL traps baited with 1–10 mg grandisol applied on rubber dispensers are recommended for the detection and monitoring of B. affinis. In addition to the target species, 17 other Lixinae species were captured during the field experiments, demonstrating for the first time the possible role of grandisol in the chemical communication systems of some of these species. A second locality of Lixus punctiventris Boheman (Lixinae, Lixini) in Bulgaria is reported. TAL traps baited with grandisol might be a useful tool for surveying Lixinae diversity in different biotopes.