The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

Potent inhibitors of the HIV-1 protease incorporating cyclic urea P1-P2 scaffold

We have developed synthetic approaches to novel analogues of 2-imidazolidinone scaffold 2, which was found to be an effective P1-P2 mimetic in HIV-1 protease inhibitor 4. This enabled a rapid synthesis of analogues of 4 and subsequently allowed us to evaluate and rationalize the SAR. Accordingly, trans relationship of P1 and P2 substituents in the P1-P2 mimetic, as found in a related 2-pyrrolidone-based scaffold 1, was found necessary for high potency against HIV-1 protease. Results of this study provided further rationale towards subsequent optimization of 2-pyrrolidone-based lead 3, which led us to potent and drug-like HIV-1 protease inhibitors described in a follow-on report (Bioorg. Med. Chem. Lett. 2004, 14, in press. ).     

Testing the quick meal hypothesis: The effect of pulp on hoarding and seed predation of Hymenaea courbaril by red‐rumped agoutis (Dasyprocta leporina)

Abstract: Red‐rumped agoutis (Dasyprocta leporina) are important seed dispersers/predators of Neotropical large‐seeded plants. Several species of seeds cached by agoutis have an edible reward, in contrast to temperate rodent‐dispersed diaspores. The quick meal hypothesis states that the presence of a reward such as edible pulp will enhance the efficiency of rodents as seed disperses by satiating the animal and, consequently, reducing seed predation and enhancing hoarding. In this study, this hypothesis was tested using as the reference system the pulp and seeds of Hymenaea courbaril. Seeds with and without pulp were offered to agoutis and the behaviour of each individual was recorded. Since the probability of predation and hoarding were complementary, we used the probability of predation. The proportion of agoutis that preyed on at least one seed was similar for seeds with (42.8% of individuals) and without (40.0% of individuals) pulp. In agoutis that preyed upon at least one seed, the probability that they killed a seed did not differ between seeds with (0.17 ± 0.03) and without (0.20 ± 0.08) pulp. Hence, these results do not support the ‘quick meal hypothesis’.     

Effectors of HIV-1 protease peptidolytic activity

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.     

Exploration of the P1 SAR of aldehyde cathepsin K inhibitors

The synthesis and biological activity of a series of aldehyde inhibitors of cathepsin K are reported. Exploration of the properties of the S(1) subsite with a series of alpha-amino aldehyde derivatives substituted at the P(1) position afforded compounds with cathepsin K IC(50)s between 52 microM and 15 nM.     

Variants in the interleukin-1 alpha and beta genes,and the risk for periodontal disease in dogs

Elevated levels of interleukin-1 (IL-1) have been shown to amplify the inflammatory response against periodontopathogenic bacteria. In humans, polymorphisms in the IL1A and IL1B genes are the most well-studied genetic polymorphisms associated with periodontal disease (PD). In contrast to human, there is a lack of knowledge on the genetic basis of canine PD. A case–control study was conducted in which a molecular analysis of dog IL1A and IL1B genes was performed. Of the eight genetic variants identified, seven in IL1A gene and one in IL1B gene, IL1A/1_g.388A >C and IL1A/1_g.521T >A showed statistically significant differences between groups (adjusted OR (95% CI): 0.15 (0.03–0.76), P= 0.022; 5.76 (1.03–32.1), P= 0.046, respectively). It suggests that in the studied population the IL1A/1_g.388C allele is associated with a decreased PD risk, whereas the IL1A/1_g.521A allele can confer an increased risk. Additionally, the IL1A/2_g.515G >T variation resulted in a change of amino acid, i.e. glycine to valine. In silico analysis suggests that this change can alter protein structure and function, predicting it to be deleterious or damaging. This work suggests that IL1 genetic variants may be important in PD susceptibility in canines.     

Prevalence of tick-borne encephalitis virus antibodies in dogs from Denmark

Background  

Large regions of central and eastern Europe are recognized as areas where tick-borne encephalitis virus (TBEV) is endemic, including countries neighbouring Denmark. It is therefore timely and relevant to determine if TBEV infections occur in Denmark. This study investigates the presence of antibodies against TBEV in a cross-section of the Danish canine population to assess the level of exposure to TBEV and possibly identify TBEV microfoci in Denmark.     

Extrafloral nectaries as a deterrent mechanism against seed predators in the chemically protected weed Crotalaria pallida (Leguminosae)

Abstract In several plants, extrafloral nectaries (EFN) are located close to the reproductive structures, suggesting that ants may act as a defence against specialized seed predators that overcome chemical defences. Alternatively, ants may also deter herbivores in a generalized manner, thereby protecting the whole plant. In this work, we examined the relationship between the chemically protected weed Crotalaria pallida Ait. (Leguminosae) that bears EFN, its specialized seed predator, the larvae of the arctiid moth Utetheisa ornatrix L. (Arctiidae) and ants. We tested two hypotheses related to the type of deterrence caused by ants. The Seed Predator Deterrence Hypothesis predicts that ant deterrence is directed primarily towards herbivores that destroy seeds and other reproductive structures, without attacking herbivores on vegetative structures. The General Deterrence Hypothesis states that ants are general in their effects, equally deterring herbivores in vegetative and reproductive structures. Our results supported the predictions of the Seed Predator Deterrence Hypothesis, namely, that (i) ant activity on EFN was related to the vulnerability of reproductive structures to attack by U. ornatrix; (ii) ant patrolling was restricted almost entirely to racemes; (iii) ants removed termites used as baits more frequently on racemes than on leaves; and (iv) U. ornatrix larvae were often expulsed from the racemes. These results indicate that EFN can act as another deterrent mechanism in chemically protected plants by promoting the expulsion of specialist seed predators.