The crystal structure of a natural DNA polymerase complexed with mirror DNA.

The intrinsic l-DNA binding properties of a natural DNA polymerase was discovered. The binding affinity of Dpo4 polymerase for l-DNA was comparable to that for d-DNA. The crystal structure of Dpo4/l-DNA complex revealed a dimer formed by the little finger domain that provides a binding site for l-DNA.

Extracellular pyruvate kinase M2 facilitates cell migration by upregulating claudin-1 expression in colon cancer cells.

Extensive studies have been reported the non-canonical functions of pyruvate kinase M2 (PKM2) as a kinase, transcriptional regulator, and even cell-to-cell communicator, emphasizing its importance in various signaling pathways. However, the role of secreted PKM2 in cancer progression and its signaling pathway is yet to be elucidated. In this study, we found that extracellular PKM2 enhanced the migration of low-metastatic, benign colon cancer cells by upregulating claudin-1 expression and internalizing it to the cytoplasm and nucleus. Knock-down of claudin-1 significantly reduced extracellular PKM2-induced cell migration. Inhibition of either protein kinase C (PKC) or epidermal growth factor receptor (EGFR) resulted in a reduction of extracellular PKM2-mediated claudin-1 expression, suggesting EGFR-PKC-claudin-1 as a signaling pathway in the extracellular PKM2-mediated tumorigenesis of colon cancer cells.

Metabolic engineering of Escherichia coli to produce a monophosphoryl lipid A adjuvant.

Monophosphoryl lipid A (MPLA) species, including MPL (a trade name of GlaxoSmithKline) and GLA (a trade name of Immune Design, a subsidiary of Merck), are widely used as an adjuvant in vaccines, allergy drugs, and immunotherapy to boost the immune response. Even though MPLA is a derivative of lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, bacterial strains producing MPLA have not been found in nature nor engineered. In fact, MPLA generation involves expensive and laborious procedures based on synthetic routes or chemical transformation of precursors isolated from Gram-negative bacteria. Here, we report the engineering of an Escherichia coli strain for in situ production and accumulation of MPLA. Furthermore, we establish a succinct method for purifying MPLA from the engineered E. coli strain. We show that the purified MPLA (named EcML) stimulates the mouse immune system to generate antigen-specific IgG antibodies similarly to commercially available MPLA, but with a dramatically reduced manufacturing time and cost. Our system, employing the first engineered E. coli strain that directly produces the adjuvant EcML, could transform the current standard of industrial MPLA production.

The Lipid A 1-Phosphatase, LpxE, Functionally Connects Multiple Layers of Bacterial Envelope Biogenesis.

Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C55-PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that Aquifex aeolicus LpxE structurally resembles YodM in Bacillus subtilis, a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak in vitro activity on C55-PP, and rescues Escherichia coli deficient in PGP and C55-PP phosphatase activities; deletion of lpxE in Francisella novicida reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C55-PP phosphatase activity. Suppression of plasmid-borne lpxE in F. novicida deficient in chromosomally encoded C55-PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death. These discoveries implicate LpxE as the first example of a multifunctional regulatory enzyme that orchestrates lipid A modification, O-antigen production, and peptidoglycan biogenesis to remodel multiple layers of the Gram-negative bacterial envelope.IMPORTANCE Dephosphorylation of the lipid A 1-phosphate by LpxE in Gram-negative bacteria plays important roles in antibiotic resistance, bacterial virulence, and modulation of the host immune system. Our results demonstrate that in addition to removing the 1-phosphate from lipid A, LpxEs also dephosphorylate undecaprenyl pyrophosphate, an important metabolite for the synthesis of the essential envelope components, peptidoglycan and O-antigen. Therefore, LpxEs participate in multiple layers of biogenesis of the Gram-negative bacterial envelope and increase antibiotic resistance. This discovery marks an important step toward understanding the regulation and biogenesis of the Gram-negative bacterial envelope.

Caspase-4 disaggregates lipopolysaccharide micelles via LPS-CARD interaction.

Lipopolysaccharides (LPS) are a major component of the outer membrane of Gram-negative bacteria and are pathogen-associated molecular patterns recognized by the TLR4/MD2 complex that induces an inflammatory response. Recently, the cytosolic receptors caspase-4/-5/-11 that bind LPS inside the cell and trigger inflammasome activation or pyroptosis, have been identified. Despite the important roles of caspase-4 in human immune responses, few studies have investigated its biochemical characteristics and interactions with LPS. Since caspase-4 (C258A) purified from an Escherichia coli host forms aggregates, monomeric proteins including full-length caspase-4, caspase-4 (C258A), and the CARD domain of caspase-4 have been purified from the insect cell system. Here, we report the overexpression and purification of monomeric caspase-4 (C258A) and CARD domain from E. coli and demonstrate that purified caspase-4 (C258A) and CARD domain bind large LPS micelles and disaggregate them to small complexes. As the molar ratio of caspase-4 to LPS increases, the size of the caspase-4/LPS complex decreases. Our results present a new function of caspase-4 and set the stage for structural and biochemical studies, and drug discovery targeting LPS/caspase-4 interactions by establishing a facile purification method to obtain large quantities of purified caspase-4 (C258A) and the CARD domain.

Highly tumor-specific DNA nanostructures discovered by in vivo screening of a nucleic acid cage library and their applications in tumor-targeted drug delivery.

Enormous efforts have been made to harness nanoparticles showing extravasation around tumors for tumor-targeted drug carriers. Owing to the complexity of in vivo environments, however, it is very difficult to rationally design a nanoconstruct showing high tumor specificity. Here, we show an approach to develop tumor-specific drug carriers by screening a library of self-assembled nucleic acid cages in vivo. After preparation of a library of 16 nucleic acid cages by combining the sugar backbone and the shape of cages, we screened the biodistribution of the cages intravenously injected into tumor-bearing mice, to discover the cages with high tumor-specificity. This tumor specificity was found to be closely related with serum stability, cancer cell uptake efficiency, and macrophage evasion rate. We further utilized the cages showing high tumor specificity as carriers for the delivery of not only a cytotoxic small molecule drug but also a macromolecular apoptotic protein exclusively into the tumor tissue to induce tumor-specific damage. The results demonstrate that our library-based strategy to discover tumor-targeted carriers can be an efficient way to develop anti-cancer nanomedicines with tumor specificity and enhanced potency.

Streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular and tumor delivery of enzymes.

Despite the extremely high substrate specificity and catalytically amplified activity of enzymes, the lack of efficient cellular internalization limits their application as therapeutics. To overcome this limitation and to harness enzymes as practical biologics for targeting intracellular functions, we developed the streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular delivery of various enzymes. The hybrid consists of streptavidin, which provides a stoichiometrically controlled loading site for the enzyme cargo and an L-DNA (mirror DNA) tetrahedron, which provides the intracellular delivery potential. Due to the cell-penetrating ability of the mirror DNA tetrahedron of this hybrid, enzymes loaded on streptavidin can be efficiently delivered into the cells, intracellularly expressing their activity. In addition, we demonstrate tumor delivery of enzymes in an animal model by utilizing the potential of the hybrid to accumulate in tumors. Strikingly, the hybrid is able to transfer the apoptotic enzyme specifically into tumor cells, leading to strong suppression of tumor growth without causing significant damage to other tissues. These results suggest that the hybrid may allow anti-proliferative enzymes and proteins to be utilized as anticancer drugs.

A facile method to prepare large quantities of active caspase-3 overexpressed by auto-induction in the C41(DE3) strain.

Since human Caspase-3, a member of the cysteine protease family, plays important roles not only in the apoptosis pathway as an executioner protein, but also in neurological disorders as a critical factor, biomedical researchers have been interested in the development of modulators of caspase-3 activity. Such studies require large quantities of purified active caspase-3. So far, purification of soluble caspase-3 from full-length human caspase-3 in Escherichia coli (E. coli) yields only several mg from a liter of culture media. Therefore, a number of alternative strategies to purify active caspase-3 have been described in the literature, including refolding and protein engineering. In this study, we systematically study the effects of host E. coli strains and growth conditions on purifications of active caspase-3 from full-length human caspase-3. Using a combination of conditions that include use of the C41(DE3) strain, low-temperature expression, and auto-induction that induces caspase-3 expression depending on metabolic state of the individual host cell, we are able to obtain 14-17 mg caspase-3 per liter of culture, an amount that is about 7 times larger than published results. This optimized expression and purification method for caspase-3 can be easily scaled up to facilitate the demand for active enzyme.

The action of HIF-3α variants on HIF-2α-HIF-1ß heterodimer formation is directly probed in live cells.

Hypoxia-inducible factors (HIFs), consisting of α and ß subunits, activate various genes to adapt to low oxygen environments through their heterodimeric complex formation in the nucleus. While most of the studies have been extensively focused on the HIF-1α isoform, the effect of HIF-α isoforms on the complex formation between HIF-2α and HIF-1ß in live cells has not been reported in detail. To probe these interactions in a physiological condition, we established a fluorescence resonance energy transfer (FRET) assay by introducing fluorescent reporter proteins onto the N-termini of HIF-2α and HIF-1ß in live PC3 cells. After thorough validations of our FRET assay system, we showed that both HIF-1α and HIF-3α variants likely function as negative regulators on the heterodimer formation of HIF-2α with HIF-1ß in cells. We also characterized the localization and stabilization of HIF-3α variants and measured the interaction between HIF-3α variants and other HIF isoforms in live cells. In contrast to the previous results showing HIF-3α-mediated blockage of HIF-1α translocation, the presence of HIF-3α did not affect the localization of HIF-2α, suggesting distinct roles of HIF-3α in regulation of two HIF-α isoforms.

Kdo hydroxylase is an inner core assembly enzyme in the Ko-containing lipopolysaccharide biosynthesis.

The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains d-glycero-d-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe(2+)/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVAin vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis.