Removal of acidic residues of the prodomain of PCSK9 increases its activity towards the LDL receptor

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and mediates intracellular degradation of the LDLR. The amino-terminus of mature PCSK9, residues 31–53 of the prodomain, has an inhibitory effect on this function of PCSK9, but the underlying mechanism is not fully understood. In this study, we have identified two highly conserved negatively charged segments (residues 32–40 and 48–50, respectively) within this part of the prodomain and performed deletions and substitutions to study their importance for degradation of the LDLRs.Deletion of the acidic residues of the longest negatively charged segment increased PCSK9’s ability to degrade the LDLR by 31%, whereas a modest 8% increase was observed when these residues were mutated to uncharged amino acids. Thus, both the length and the charge of this part of the prodomain were important for its inhibitory effect. Deletion of the residues of the shorter second negatively charged segment only increased PCSK9’s activity by 8%. Substitution of the amino acids of both charged segments to uncharged residues increased PCSK9’s activity by 36%. These findings indicate that the inhibitory effect of residues 31–53 of the prodomain is due to the negative charge of this segment. The underlying mechanism could involve the binding of this peptide segment to positively charged structures which are important for PCSK9’s activity. One possible candidate could be the histidine-rich C-terminal domain of PCSK9.     

Novel selective thiazoleacetic acids as CRTH2 antagonists developed from in silico derived hits. Part 1

Structure–activity relationships of three related series of 4-phenylthiazol-5-ylacetic acids, derived from two hits emanating from a focused library obtained by in silico screening, have been explored as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. Several compounds with double digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained in all subclasses. The most potent compound was [2-(4-chloro-benzyl)-4-(4-phenoxy-phenyl)-thiazol-5-yl]acetic acid having an binding affinity of 3.7 nM and functional antagonistic effect of 66 nM in a BRET and 12 nM in a cAMP assay with no functional activity for the other PGD2 DP receptor (27 μM in cAMP).     

Phosphorylation of eIF4E Confers Resistance to Cellular Stress and DNA-Damaging Agents through an Interaction with 4E-T: A Rationale for Novel Therapeutic Approaches

Phosphorylation of the eukaryotic translation initiation factor eIF4E is associated with malignant progression and poor cancer prognosis. Accordingly, here we have analyzed the association between eIF4E phosphorylation and cellular resistance to oxidative stress, starvation, and DNA-damaging agents in vitro. Using immortalized and cancer cell lines, retroviral expression of a phosphomimetic (S209D) form of eIF4E, but not phospho-dead (S209A) eIF4E or GFP control, significantly increased cellular resistance to stress induced by DNA-damaging agents (cisplatin), starvation (glucose+glutamine withdrawal), and oxidative stress (arsenite). De novo accumulation of eIF4E-containing cytoplasmic bodies colocalizing with the eIF4E-binding protein 4E-T was observed after expression of phosphomimetic S209D, but not S209A or wild-type eIF4E. Increased resistance to cellular stress induced by eIF4E-S209D was lost upon knockdown of endogenous 4E-T or use of an eIF4E-W73A-S209D mutant unable to bind 4E-T. Cancer cells treated with the Mnk1/2 inhibitor CGP57380 to prevent eIF4E phosphorylation and mouse embryonic fibroblasts derived from Mnk1/2 knockout mice were also more sensitive to arsenite and cisplatin treatment. Polysome analysis revealed an 80S peak 2 hours after arsenite treatment in cells overexpressing phosphomimetic eIF4E, indicating translational stalling. Nonetheless, a selective increase was observed in the synthesis of some proteins (cyclin D1, HuR, and Mcl-1). We conclude that phosphorylation of eIF4E confers resistance to various cell stressors and that a direct interaction or regulation of 4E-T by eIF4E is required. Further delineation of this process may identify novel therapeutic avenues for cancer treatment, and these results support the use of modern Mnk1/2 inhibitors in conjunction with standard therapy.     

Regulation of Golgi turnover by CALCOCO1-mediated selective autophagy

The Golgi complex is essential for the processing, sorting, and trafficking of newly synthesized proteins and lipids. Golgi turnover is regulated to meet different cellular physiological demands. The role of autophagy in the turnover of Golgi, however, has not been clarified. Here we show that CALCOCO1 binds the Golgi-resident palmitoyltransferase ZDHHC17 to facilitate Golgi degradation by autophagy during starvation. Depletion of CALCOCO1 in cells causes expansion of the Golgi and accumulation of its structural and membrane proteins. ZDHHC17 itself is degraded by autophagy together with other Golgi membrane proteins such as TMEM165. Taken together, our data suggest a model in which CALCOCO1 mediates selective Golgiphagy to control Golgi size and morphology in eukaryotic cells via its interaction with ZDHHC17.     

Extrapair paternity and genetic diversity: the white‐throated dipper Cinclus cinclus

Passerine bird species vary considerably in the frequency of extrapair paternity, but the factors causing this variation are not well understood. There is some comparative evidence that extrapair paternity is associated with the population level of genetic diversity, but there is no consensus of how genetic diversity should be measured and compared across species or populations. Here we report a low frequency of extrapair paternity (2% extrapair offspring) in a Norwegian population of the white‐throated dipper Cinclus cinclus, which shows strong signs of reduced genetic diversity. We encountered difficulties in constructing a robust parentage analysis system for the species, largely due to consistently low polymorphism levels in 100 heterologous microsatellite markers. Furthermore, single‐nucleotide polymorphisms (SNPs) were almost absent in intron sequences in 10 nuclear genes (>5 kb) that are much more polymorphic in other species. Hence, our results seem consistent with the genetic diversity hypothesis that predicts a low frequency of extrapair paternity in species with low genetic diversity. Heterologous microsatellite markers are generally unsuitable for interspecific comparisons of genetic diversity as they show strong phylogenetic dependency in polymorphism levels. We suggest that SNP rates at homologous nuclear introns, like those presented here, can provide a useful method for obtaining unbiased estimates of genome‐wide genetic diversity across populations and species.     

Analysis of the mycosamine biosynthesis and attachment genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455

The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the beta-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed. The recombinant NysDIII protein was expressed in Escherichia coli and purified, and its in vitro GDP-mannose dehydratase activity was demonstrated. The nysDI and nysDII genes were inactivated individually in S. noursei, and analyses of the resulting mutants showed that both genes produced nystatinolide and 10-deoxynystatinolide as major products. Expression of the nysDI and nysDII genes in trans in the respective mutants partially restored nystatin biosynthesis in both cases, supporting the predicted roles of these two genes in mycosamine biosynthesis and attachment. Both antifungal and hemolytic activities of the purified nystatinolides were shown to be strongly reduced compared to those of nystatin, confirming the importance of the mycosamine moiety for the biological activity of nystatin.     

mRNA expression patterns of the BPI/LBP molecule in the Atlantic cod (Gadus morhua L.)

Bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP) are important components of the mammalian innate defence system against Gram-negative infections. cDNA encoding a protein related to mammalian BPI and LBP have been cloned from several teleosts including the Atlantic cod (Gadus morhua L.). Using real-time PCR an increase in cod BPI/LBP expression in whole blood and peritoneal cells was demonstrated one, two and four days after intraperitoneal injection of inactivated Vibrio anguillarum. Although constitutively produced in the head kidney, a moderate rise of BPI/LBP mRNA production was seen on day two in this organ. After seven days the BPI/LBP mRNA levels at the three locations examined were almost back to normal. In situ hybridisation demonstrated a leucocytic localisation and morphology of the BPI/LBP expressing cells in various tissues. A combination of in situ hybridisation and peroxidase staining of head kidney leucocytes showed that the BPI/LBP producing cells are peroxidase positive and possible neutrophil like cells. The results suggest that the cod BPI/LBP is important in the first-line defence against bacterial infections and has a function more related to the mammalian BPI molecule than the LBP counterpart.