Effect of sterol composition on the activity of the yeast G-protein-coupled receptor Ste2

The main sterol of the human cell membrane is cholesterol, whereas in yeast it is ergosterol. In this study, we constructed a cholesterol-producing yeast strain by disrupting the genes related to ergosterol synthesis and inserting the genes related to cholesterol synthesis. The total sterols of the mutant yeast were extracted and the sterol composition was analyzed by GC-MS. We confirmed that cholesterol was produced instead of ergosterol in yeast and subsequently examined the activity of the yeast G-protein-coupled receptor (GPCR) Ste2p. Ste2p signaling was assessed in wild type (WT) with ergosterol and the cholesterol-producing yeast instead of ergosterol to determine whether sterol composition affects the activity of the yeast GPCR. Our results demonstrated that Ste2p could transduce a signal even in the cholesterol-rich membrane, but the maximum signal intensity was weaker than that transduced in the ergosterol-rich original (WT) membrane. This result indicates that sterol composition affects the activity of yeast GPCRs, and thus, this provides new insight into GPCR-mediated transduction using yeast for future fundamental and applied studies on GPCRs from yeast to other organisms.     

A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein

Expression and purification of aggregation‐prone and disulfide‐containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high‐value target proteins. Here, we present a novel gene‐fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag‐Acidic‐Target Tag) results in near‐complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one‐step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization.     

Reduced expression of the NADPH oxidase NOX4 is a hallmark of adipocyte differentiation

Adipocyte differentiation is a complex process regulated among other factors by insulin and the production of reactive oxygen species (ROS). NOX4 is a ROS generating NADPH oxidase enzyme mediating insulin's action in 3T3L1 adipocytes. In the present paper we show that NOX4 is expressed at high levels both in white and brown preadipocytes and that differentiation into adipocytes results in a decrease in their NOX4 mRNA content. These in vitro results were confirmed in vivo by demonstrating that in intact adipose tissue the majority of NOX4 expressing cells are localized within the preadipocyte containing stromal/vascular fraction, rather than in the portion consisting of mature adipocytes. In line with these observations, quantification of NOX4 mRNA in fat derived from different rodent models of insulin resistance indicated that alteration in NOX4 expression reflects changes in the ratio of adipocyte/interstitial fractions. In conclusion, we reveal that decreased NOX4 mRNA content is a hallmark of adipocyte differentiation and that NOX4 expression measured in whole adipose tissue is not an unequivocal indicator of intact or impaired insulin action.     

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops

Background

Cytosine- and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34-base long cytosine-rich sequence and its complementary guanine-rich strand corresponding to the first intron of the n-myc gene were studied. Both sequences, not yet studied, contain a 12-base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively.

Methods

Spectroscopic, mass spectrometry and separation techniques, as well as multivariate data analysis methods, were used to unravel the species and conformations present.

Results

The cytosine-rich sequence forms two i-motifs that differ in the protonation of bases located in the loops. A stable Watson–Crick hairpin is formed by the bases in the first loop, stabilizing the i-motif structure. The guanine-rich sequence adopts a parallel G-quadruplex structure that is stable throughout the pH range 3–7, despite the protonation of cytosine and adenine bases at lower pH values. The presence of G-quadruplex aggregates was confirmed using separation techniques. When mixed, G-quadruplex and i-motif coexist with the Watson–Crick duplex across a pH range from approximately 3.0 to 6.5.

Conclusions

Two cytosine- and guanine-rich sequences in n-myc gene may form stable i-motif and G-quadruplex structures even in the presence of long loops. pH modulates the equilibria involving the intramolecular structures and the intermolecular Watson–Crick duplex.

General significance

Watson–Crick hairpins located in the intramolecular G-quadruplexes and i-motifs in the promoter regions of oncogenes could play a role in stabilizing these structures.     

An essential enzyme for phospholipid synthesis associates with the Bacillus subtilis divisome

The mechanism by which the membrane synthetic machinery might be co‐organized with the cell‐division architecture during the bacterial cell cycle remains to be investigated. We characterized a key enzyme of phospholipid and fatty acid synthesis in Bacillus subtilis, the acyl–acyl carrier protein phosphate acyltransferase (PlsX), and identified it as a component of the cell‐division machinery. Comprehensive interaction analysis revealed that PlsX interacts with FtsA, the FtsZ‐anchoring protein. PlsX mainly localized at the potential division site independent of FtsA and FtsZ and then colocalized with FtsA. By multidirectional approaches, we revealed that the Z‐ring stabilizes the association of PlsX at the septum and pole. The localization of PlsX is also affected by the progression of DNA replication. PlsX is needed for cell division and its inactivation leads to aberrant Z‐ring formation. We propose that PlsX localization is prior to Z‐ring formation in the hierarchy of septum formation events and that PlsX is important for co‐ordinating membrane synthesis with cell division in order to properly complete septum formation.