Application of recombinant human leukemia inhibitory factor (LIF) produced in rice (Oryza sativa L.) for maintenance of mouse embryonic stem cells.

Embryonic and induced pluripotent stem cells have the ability to differentiate into any somatic cell type, and thus have potential to treat a number of diseases that are currently incurable. Application of these cells for clinical or industrial uses would require an increase in production to yield adequate numbers of viable cells. However, the relatively high costs of cytokines and growth factors required for maintenance of stem cells in the undifferentiated state have the potential to limit translational research. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is a key regulator in the maintenance of naïve states for both human and mouse stem cells. In this study, we describe a new recombinant human LIF (rhLIF) using a plant-based (rice) expression system. We found that rice-derived rhLIF possessed the same specific activity as commercial Escherichia coli-derived LIF and was capable of supporting mouse embryonic stem cell proliferation in the undifferentiated state as evidenced from pluripotency marker level analysis. Retention of the pluripotent state was found to be indistinguishable between rice-derived rhLIF and other recombinant LIF proteins currently on the market.

High-throughput screening AlphaScreen assay for identification of small-molecule inhibitors of ubiquitin E3 ligase SCFSkp2-Cks1.

Decreased levels of cell cycle inhibitor p27(Kip1) due to excessive degradation occur in a variety of aggressive human tumors. Since reduced p27(Kip1) expression has been associated with a poor prognosis in many human cancers and resistance to certain antitumor therapies, elevation of p27(Kip1) expression could improve prognosis and prevent excessive cell proliferation. SCF(Skp2) is one of the major ubiquitin E3 ligases responsible for degradation of p27(Kip1). Ubiquitination of p27(Kip1) also requires a small adaptor protein, Cks1, which facilitates substrate recruitment by bridging the interaction between Skp2 and p27(Kip1). It has been shown previously that a direct interaction between Cks1 and Skp2 is required for p27(Kip1) degradation. Accordingly, perturbation of the Skp2-Cks1 interaction may represent an attractive target for pharmacological intervention. Here we describe a high-throughput AlphaScreen assay for discovering small-molecule inhibitors of the Skp2-Cks1 protein-protein interaction in vitro. Two compounds (NSC689857 and NSC681152) were identified and validated through a structure-activity relationship analysis. Both compounds were also shown to inhibit p27(Kip1) ubiquitination in vitro. These studies demonstrate that disruption of the Skp2-Cks1 interaction provides a viable strategy to prevent p27(Kip1) ubiquitination and may potentially be useful for the control of excessive degradation of this cell cycle inhibitor in tumor cells.

Only one ATP-binding DnaX subunit is required for initiation complex formation by the Escherichia coli DNA polymerase III holoenzyme.

The DnaX complex (DnaX(3)δδ'χ psi) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, ß(2), onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. ß(2) binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for ß(2). DNA synthesis activity can be restored by increased concentrations of ß(2). In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances ß(2) loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.

Reconstitution of the B. subtilis replisome with 13 proteins including two distinct replicases.

We have expressed and purified 13 proteins predicted to be required for B. subtilis DNA replication. When combined with a circular DNA template with a 5' unpaired flap, these proteins reconstitute replication of both the leading and lagging strands at the physiological rate. Consistent with the in vivo requirement for two DNA polymerase III replicases for B. subtilis chromosomal replication, both PolC and DnaE are required for reconstitution of the replication fork in vitro. Leading strand synthesis requires PolC plus ten proteins; lagging strand synthesis additionally requires primase and DnaE. DnaE does not serve as the lagging strand replicase, like DNA polymerase delta in eukaryotes, but instead functions like eukaryotic DNA polymerase alpha, adding a stretch of deoxynucleotides to the RNA primer before handoff to PolC. Primase equilibrates with the fork prior to synthesis of each Okazaki fragment, and its concentration controls the frequency of initiation and Okazaki fragment size.

Parallel multiplicative target screening against divergent bacterial replicases: identification of specific inhibitors with broad spectrum potential.

Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction includes up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, in which inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.

Quinazolin-2-ylamino-quinazolin-4-ols as novel non-nucleoside inhibitors of bacterial DNA polymerase III.

High throughput screening led to the discovery of a novel series of quinazolin-2-ylamino-quinazolin-4-ols as a new class of DNA polymerase III inhibitors. The inhibition of chromosomal DNA replication results in bacterial cell death. The synthesis, structure-activity relationships and functional activity are described.

Discovery and characterization of the cryptic psi subunit of the pseudomonad DNA replicase.

We previously reconstituted a minimal DNA replicase from Pseudomonas aeruginosa consisting of alpha and epsilon (polymerase and editing nuclease), beta (processivity factor), and the essential tau, delta, and delta' components of the clamp loader complex (Jarvis, T., Beaudry, A., Bullard, J., Janjic, N., and McHenry, C. (2005) J. Biol. Chem. 280, 7890-7900). In Escherichia coli DNA polymerase III holoenzyme, chi and Psi are tightly associated clamp loader accessory subunits. The addition of E. coli chiPsi to the minimal P. aeruginosa replicase stimulated its activity, suggesting the existence of chi and Psi counterparts in P. aeruginosa. The P. aeruginosa chi subunit was recognizable from sequence similarity, but Psi was not. Here we report purification of an endogenous replication complex from P. aeruginosa. Identification of the components led to the discovery of the cryptic Psi subunit, encoded by holD. P. aeruginosa chi and Psi were co-expressed and purified as a 1:1 complex. P. aeruginosa chiPsi increased the specific activity of tau(3)deltadelta' 25-fold and enabled the holoenzyme to function under physiological salt conditions. A synergistic effect between chiPsi and single-stranded DNA binding protein was observed. Sequence similarity to P. aeruginosa Psi allowed us to identify Psi subunits from several other Pseudomonads and to predict probable translational start sites for this protein family. This represents the first identification of a highly divergent branch of the Psi family and confirms the existence of Psi in several organisms in which Psi was not identifiable based on sequence similarity alone.