Deletion of the copper transporter CaCCC2 reveals two distinct pathways for iron acquisition in Candida albicans

Efficient iron acquisition is an essential requirement for growth of pathogenic organisms in the iron-poor host environment. In Saccharomyces cerevisiae, high-affinity iron import depends on the multicopper ferroxidase ScFet3. ScFet3 biogenesis in the trans-Golgi compartment requires a copper-transporting P-type ATPase, ScCcc2. Here, we describe the isolation by functional complementation of a Ccc2 homologue from the pathogenic yeast Candida albicans. CaCcc2 is functionally distinct from a previously described C. albicans copper-transporting P-type ATPase, CaCrp1, which appears to be specifically involved in copper detoxification. Regulation of CaCCC2 and the phenotype of the homozygous CaCCC2 deletion indicate that it is required for high-affinity iron import, making it the bona fide CCC2 homologue of C. albicans. Remarkably, in a mouse model of systemic infection, the Caccc2Delta strain displayed robust proliferation and no significant reduction in pathogenicity, suggesting the existence of alternative mechanisms of iron uptake from host tissues. We identify haemin and haemoglobin as potential iron sources that can be used by C. albicans in a CaCcc2-independent manner.     

Monoclonal antibody specific to urochordate Botryllus schlosseri pyloric gland

We describe here the development of a new hybridoma cell line, CF12F6, that produces a specific antibody to Botryllus schlosseri (a colonial tunicate). The monoclonal antibody was isotyped as IgG1 (by enzyme-linked immunosorbent assay), and the cellular localization of the antigenic epitope that reacts specifically with CF12F6 was confined to the cells of the pyloric gland of the zooid (by immunohistochemistry). The pyloric gland participates in osmoregulation, digestion, glycogen storage, and various other secretion functions that will be studied further by the use of monoclonal antibody CF12F6, the first in botryllid ascidians that recognizes cells of a whole, single organ.     

Evolution of homo‐oligomerization of methionine S‐adenosyltransferases is replete with structure–function constrains

Homomers are prevalent in bacterial proteomes, particularly among core metabolic enzymes. Homomerization is often key to function and regulation, and interfaces that facilitate the formation of homomeric enzymes are subject to intense evolutionary change. However, our understanding of the molecular mechanisms that drive evolutionary variation in homomeric complexes is still lacking. How is the diversification of protein interfaces linked to variation in functional regulation and structural integrity of homomeric complexes? To address this question, we studied quaternary structure evolution of bacterial methionine S‐adenosyltransferases (MATs)—dihedral homotetramers formed along a large and conserved dimeric interface harboring two active sites, and a small, recently evolved, interdimeric interface. Here, we show that diversity in the physicochemical properties of small interfaces is directly linked to variability in the kinetic stability of MAT quaternary complexes and in modes of their functional regulation. Specifically, hydrophobic interactions within the small interface of Escherichia coli MAT render the functional homotetramer kinetically stable yet impose severe aggregation constraints on complex assembly. These constraints are alleviated by electrostatic interactions that accelerate dimer‐dimer assembly. In contrast, Neisseria gonorrhoeae MAT adopts a nonfunctional dimeric state due to the low hydrophobicity of its small interface and the high flexibility of its active site loops, which perturbs small interface integrity. Remarkably, in the presence of methionine and ATP, N. gonorrhoeae MAT undergoes substrate‐induced assembly into a functional tetrameric state. We suggest that evolution acts on the interdimeric interfaces of MATs to tailor the regulation of their activity and stability to unique organismal needs.     

A Relay Network of Extracellular Heme-Binding Proteins Drives C. albicans Iron Acquisition from Hemoglobin

Iron scavenging constitutes a crucial challenge for survival of pathogenic microorganisms in the iron-poor host environment. Candida albicans, like many microbial pathogens, is able to utilize iron from hemoglobin, the largest iron pool in the host''s body. Rbt5 is an extracellular glycosylphosphatidylinositol (GPI)-anchored heme-binding protein of the CFEM family that facilitates heme-iron uptake by an unknown mechanism. Here, we characterize an additional C. albicans CFEM protein gene, PGA7, deletion of which elicits a more severe heme-iron utilization phenotype than deletion of RBT5. The virulence of the pga7^−/− mutant is reduced in a mouse model of systemic infection, consistent with a requirement for heme-iron utilization for C. albicans pathogenicity. The Pga7 and Rbt5 proteins exhibit distinct cell wall attachment, and discrete localization within the cell envelope, with Rbt5 being more exposed than Pga7. Both proteins are shown here to efficiently extract heme from hemoglobin. Surprisingly, while Pga7 has a higher affinity for heme in vitro, we find that heme transfer can occur bi-directionally between Pga7 and Rbt5, supporting a model in which they cooperate in a heme-acquisition relay. Together, our data delineate the roles of Pga7 and Rbt5 in a cell surface protein network that transfers heme from extracellular hemoglobin to the endocytic pathway, and provide a paradigm for how receptors embedded in the cell wall matrix can mediate nutrient uptake across the fungal cell envelope.     

Different Incubation Times of Cells After Gene Electrotransfer in Fetal Bovine Serum Affect Cell Viability,but Not Transfection Efficiency

Electroporation as a delivery method is increasingly important in gene therapy, not only in vivo but also in in vitro experimental systems. Different applications of gene electrotransfer require high viability of cells and high transfection efficiency of gene electrotransfer. It was already demonstrated that the addition of fetal bovine serum (FBS) immediately after gene electrotransfer leads to improved cell survival and transfection efficiency. Therefore, the aim of the study was to determine whether prolonged incubation of cells in FBS, for more than standard 5 min, can lead to increased transfection efficiency and improved cell survival. Different murine melanoma and murine and human endothelial cell lines were transfected with plasmid encoding green fluorescent protein and then incubated for different periods of time in FBS (5–30 min). Transfection efficiency was determined by flow cytometry and fluorescence microscopy and cell survival by cell viability assay. Prolonged incubation of cells in FBS after gene electrotransfer had varying effect on cell survival, which was decreased in melanoma cell lines B16F1 and B16F10, minimally affected in SVEC4-10 and HUVEC cells and increased in 2H11 cell at 30 min of incubation time in FBS. On the other hand, transfection efficiency of gene electrotransfer was not affected by long incubation of cell in FBS, regardless of the cell line used. The results of our study emphasize the importance of optimization of gene electrotransfer protocol for particular cells and specific purposes of gene electrotransfer, taking into account the importance of transfection efficiency and cell survival.     

Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase C betaII in the diabetic fat tissue

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is an important negative modulator of insulin signaling. Previously, we showed that glycogen synthase kinase-3 (GSK-3) phosphorylates IRS-1 at Ser(332). However, the fact that GSK-3 requires prephosphorylation of its substrates suggested that Ser(336) on IRS-1 was the "priming" site phosphorylated by an as yet unknown protein kinase. Here, we sought to identify this "priming kinase" and to examine the phosphorylation of IRS-1 at Ser(336) and Ser(332) in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser(336). Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKCalpha or PKCbetaII isoforms in cells enhanced IRS-1 phosphorylation at Ser(336) and Ser(332), and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser(336). The expression level and activation state of PKCbetaII, but not PKCalpha, were remarkably elevated in the fat tissues of diabetic ob/ob mice and in high-fat diet-fed mice compared with that from lean animals. Elevated levels of PKCbetaII were also associated with enhanced phosphorylation of IRS-1 at Ser(336/332) and elevated activity of GSK-3beta. Finally, adenoviral mediated expression of PKCbetaII in adipocytes enhancedphosphorylation of IRS-1 at Ser(336). Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCbetaII and GSK-3 at Ser(336) and Ser(332). Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.     

Cannabis reinforcement and dependence: role of the cannabinoid CB1 receptor.

Awareness of cannabis dependence as a clinically relevant issue has grown in recent years. Clinical and laboratory studies demonstrate that chronic marijuana smokers can experience withdrawal symptoms upon cessation of marijuana smoking and have difficulty abstaining from marijuana use. This paper will review data implicating the cannabinoid CB1 receptor in regulating the behavioral effects of Delta(9)-tetrahydrocannobinol (THC), the primary psychoactive component of cannabis, across a range of species. The behavioral effects that will be discussed include those that directly contribute to the maintenance of chronic marijuana smoking, such as reward, subjective effects, and the positive and negative reinforcing effects of marijuana, THC and synthetic cannabinoids. The role of the CB1 receptor in the development of marijuana dependence and expression of withdrawal will also be discussed. Lastly, treatment options that may alleviate withdrawal symptoms and promote marijuana abstinence will be considered.