Simultaneous production and partitioning of heterologous polyketide and isoprenoid natural products in an <Emphasis Type="Italic">Escherichia coli</Emphasis> two-phase bioprocess

Natural products have long served as rich sources of drugs possessing a wide range of pharmacological activities. The discovery and development of natural product drug candidates is often hampered by the inability to efficiently scale and produce a molecule of interest, due to inherent qualities of the native producer. Heterologous biosynthesis in an engineering and process-friendly host emerged as an option to produce complex natural products. Escherichia coli has previously been utilized to produce complex precursors to two popular natural product drugs, erythromycin and paclitaxel. These two molecules represent two of the largest classes of natural products, polyketides and isoprenoids, respectively. In this study, we have developed a platform E. coli strain capable of simultaneous production of both product precursors at titers greater than 15 mg l⁻¹. The utilization of a two-phase batch bioreactor allowed for very strong in situ separation (having a partitioning coefficient of greater than 5,000), which would facilitate downstream purification processes. The system developed here could also be used in metagenomic studies to screen environmental DNA for natural product discovery and preliminary production experiments.     

Role of PDZK1 protein in apical membrane expression of renal sodium-coupled phosphate transporters

The sodium-dependent phosphate (Na/P(i)) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of P(i). The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in P(i) reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/P(i) transporters. Pdzk1(-/-) mice adapted to chronic low P(i) diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/P(i) transporters with NHERF-1 and PDZK1 by FRET. FRET measurements showed a much stronger interaction of NHERF-1 with NaPi-2a than with NaPi-2c. However, both Na/P(i) transporters showed similar FRET efficiencies with PDZK1. Interestingly, in cells adapted to low P(i) concentrations, there were increases in NaPi-2c/PDZK1 and NaPi-2a/NHERF-1 interactions. The differential affinity of the Na/P(i) transporters for NHERF-1 and PDZK1 proteins could partially explain their differential regulation and/or stability in the apical membrane. In this regard, direct interaction between NaPi-2c and PDZK1 seems to play an important role in the physiological regulation of NaPi-2c.     

Tumor-associated a2 vacuolar ATPase acts as a key mediator of cancer-related inflammation by inducing pro-tumorigenic properties in monocytes

Cancer-related inflammation profoundly affects tumor progression. Tumor-associated macrophages (TAMs) are known regulators of that inflammation, but the factors that initiate cancer-related inflammation are poorly understood. Tumor invasiveness and poor clinical outcome are linked to increased expression of cell surface-associated vacuolar adenosine triphosphatases. The a2 isoform vacuolar adenosine triphosphatase is found on the surface on many solid tumors, and we have identified a peptide cleaved from a2 isoform vacuolar adenosine triphosphatase called a2NTD. a2NTD has properties necessary to induce monocytes into a pro-oncogenic TAM phenotype. The peptide upregulated both pro- and anti-inflammatory mediators. These included IL-1β and IL-10, which are important in promoting inflammation and immune escape by tumor cells. The secretion of inflammatory cytokine IL-1β was dependent on ATP, K(+) efflux, and reactive oxygen species, all mediators that activate the inflammasome. These findings describe a mechanism by which tumor cells affect the maturation of TAMs via a nontraditional cytokine-like signal, the a2NTD peptide.     

Diversity of operation in ATP-dependent chromatin remodelers

Chromatin is actively restructured by a group of proteins that belong to the family of ATP-dependent DNA translocases. These chromatin remodelers can assemble, relocate or remove nucleosomes, the fundamental building blocks of chromatin. The family of ATP-dependent chromatin remodelers has many properties in common, but there are also important differences that may account for their varying roles in the cell. Some of the important characteristics of these complexes have begun to be revealed such as their interactions with chromatin and their mechanism of operation. The different domains of chromatin remodelers are discussed in terms of their targets and functional roles in mobilizing nucleosomes. The techniques that have driven these findings are discussed and how these have helped develop the current models for how nucleosomes are remodeled. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function.     

Placental ATPase expression is a link between multiple causes of spontaneous abortion in mice

The a2 isoform of vacuolar ATPase (ATP6V0A2 referred to as a2V) plays a pivotal role in successful pregnancy and provides a microenvironment to maintain the delicate immunological balance at the feto-maternal interaction. We studied the expression of a2V mRNA in embryos and placenta of abortion-prone (female CBA × male DBA) murine matings or LPS (lipopolysaccharide)-treated mice. The expression of a2V was significantly higher in the placentas of nonabortion-prone (female BALB/c × male BALB/c and female CBA × male BALB/c) matings compared with the abortion-prone (female CBA × male DBA) mating. The expression of a2V was significantly decreased in the placentas treated with LPS in both female CBA × male DBA and female BALB/c × male BALB/c mating combinations with increased Lif, Il1b, and Tnf expression in the placenta. Decreased expression of a2V in the placenta is directly correlated with high percentages of pregnancy loss in abortion-prone mating (female CBA × male DBA) as well as in LPS-treated animals. The normal expression of placental a2V on Day 16 in the nonabortion-prone matings correlated with higher Mcp1 (monocyte chemotactic protein 1) gene expression, markedly higher infiltration of M1 and M2 macrophages, and no significant polarization patterns (M1/M2 = 1.2-1.6). However, in the abortion-prone mating, decreased placental a2V expression correlated with significantly lower Mcp1 gene expression with less infiltration of M1 and M2 macrophages and with polarization patterns skewed to M1 phenotypes (M1/M2 = 3.9-4.2). These data indicate that the higher expression of placental a2V is associated with dynamic infiltration of M1 and M2 macrophages through the induction of Mcp1 expression. This strengthens our hypothesis that a2V regulates the delicate cytokine and chemokine networks that coordinate the recruitment of macrophages for successful placental development and growth at the feto-maternal interface.