Toxicity and localization studies of a potential photodynamic therapy agent in Drosophila

Photodynamic therapy utilizes light, a photosensitizer, and molecular oxygen as a treatment modality for a variety of cancers. We have recently combined ruthenium(II) polypyridyl groups with a zinc(II) centered porphyrin as a new photosensitizer for the treatment of melanoma. In‐vitro studies have indicated that this photosensitizer is toxic to melanoma cells when irradiated with low energy light; however, it is nontoxic to normal cells under similar conditions. To determine the toxicity and cell viability of this compound in‐vivo we present, herein, a study using Drosophila melanogaster. In the absence of light, the new photosensitizer shows no discernible effects to fly larvae at various concentrations of compound and stages of larval development. When the larvae were fed the photosensitizer it was observed, by fluorescence microscopy, that the compound passes through the cell membrane and localizes in the cytosol at lower concentrations and the nucleus at slightly higher concentrations indicating that the compound is not immediately metabolized. genesis 52:309–314, 2014. © 2014 Wiley Periodicals, Inc.     

Novel Src homology 3 domain-binding motifs identified from proteomic screen of a Pro-rich region

The Src homology (SH) 3 domain has been shown recently to bind peptide sequences that lack the canonical PXXP motif. The diverse specificity in ligand recognition for a group of 15 SH3 domains has now been investigated using arrays of peptides derived from the proline-rich region of the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76). A screen of the peptide arrays using individual or mixed SH3 domains has allowed the identification of a number of candidate SH3-binding peptides. Although some peptides contain the conventional PXXP motif, most are devoid of such a motif and are instead enriched in basic residues. Fluorescent polarization measurements using soluble peptides and purified SH3 domains demonstrated that several SH3 domains, including those from growth factor receptor-bound protein 2 (Grb2), NCK, and phospholipase C (PLC)-gamma1, bound with moderate affinities (10-100 microm) to a group of non-conventional peptides. Of particular interest, the PLC-gamma1 SH3 domain was found to associate with SLP-76 through at least three distinct sites, two of which bore a novel KKPP motif and the other contained the classic PXXP sequence. Intriguingly mutation of critical residues for the three sites not only affected binding of SLP-76 to the PLC-gamma1 SH3 domain but also to the Grb2 C-terminal SH3 domain, indicating that the binding sites in SLP-76 for the two SH3 domains are overlapped. Our studies suggest that the SH3 domain is an inherently promiscuous interaction module capable of binding to peptides that may or may not contain a PXXP motif. Furthermore the identification of numerous non-conventional SH3-binding peptides in SLP-76 implies that the global ligand pool for SH3 domains in a mammalian proteome may be significantly greater than previously acknowledged.     

Optimization of the multiple enzymatic activities of the hepatitis C virus NS3 protein

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is known to possess multiple enzymatic activities. In addition to its well-characterized protease activity, HCV NS3 also has ATP hydrolase (ATPase) and nucleic acid unwinding (helicase) activities. We systematically studied the effect of common reagents on all three enzymatic activities with a view to improving assay sensitivity for compound screening and profiling. Inclusion of the detergent lauryl dimethylamine oxide (LDAO) improves protease and helicase activities significantly, allowing robust assays at much lower NS3 concentrations. These conditions enable a particularly sensitive protease assay that uses picomolar concentrations of NS3.     

Structure of the Plasmodium falciparum Circumsporozoite Protein, a Leading Malaria Vaccine Candidate

The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (R_h 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21–25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.Malaria caused by Plasmodium falciparum is a serious global health issue, resulting in an estimated 1.5 million deaths annually, primarily among infants and young children. Ongoing multifaceted global intervention strategies to control malaria include drug treatment, insecticide usage, bed-net use, and vaccine development. However, parasite and mosquito control measures have met with limited success resulting from an increased drug and insecticide resistance within the Plasmodia and mosquito populations, respectively. Vaccine development represents an encouraging approach given that previous animal and human studies using irradiated sporozoites demonstrated the feasibility of producing an efficacious vaccine (1–3). Although the exact immunologic correlates of protection remain elusive, an abundance of evidence indicates that protection against liver stage parasites is complex, involving multiple immune mechanisms (4–11).To date, the majority of the pre-erythrocytic stage vaccine development has focused on the circumsporozoite protein (CSP),² the predominant surface antigen on sporozoites. CSP can be segmented into three regions as follows: the N-terminal region containing region I; the central repeat region; and the C-terminal region containing the thrombospondin-like type I repeat (TSR). Initial CSP vaccine development focused on the central repeat region that contains the immunodominant B cell epitope (12). However, vaccine constructs quickly evolved to incorporate both the central repeat region containing the B cell epitopes and the C terminus containing the TSR domain, T cell epitopes, and B cell epitopes (13, 14). Currently, the most advanced and moderately effective malaria vaccine, RTS,S, is composed of a portion of the central repeat and the C-terminal regions linked to the hepatitis B surface antigen (15). However, recent studies have highlighted the physiological importance of the N-terminal region (16–19). Rathore et al. (19) not only demonstrated the role of the N-terminal region in liver cell attachment but also identified along with Ancsin and Kisilevsky (16) an epitope within the N-terminal region that interacted with liver cells through heparin sulfate (18). Moreover, this epitope was not only found to be immunogenic but the resulting antibodies were determined to be inhibitory in a sporozoite invasion assay (18). Peptides corresponding to the N-terminal region (PpCS-(22–110) and PpCS-(65–110)) were also recognized by sera obtained from individuals living in malaria-endemic regions (17).To better understand the structure of CSP and to produce good quality recombinant protein for human vaccine-directed studies, we generated full-length and near full-length recombinant CSP. We examined two expression systems, Escherichia coli and Pichia pastoris, to determine their feasibility to generate CSP. To assist the characterization of the rCSPs, we generated a panel of monoclonal antibodies (mAbs) that were characterized biologically prior to being used to examine the rCSPs. Additionally, each of the rCSP molecules was extensively biochemically and biophysically characterized. The results collated together have enabled the molecular modeling of CSP as a long flexible, rod-like protein.     

Characterization and Identification of Hepatic mRNA Related to Copper Metabolism and Homeostasis in Cattle

Copper is an essential trace mineral required for growth and development. Copper homeostasis within the cell is mediated by the expression of the Cu transporter protein (CTR1), ATPase7A (ATP7A), ATPase7B (ATP7B), Cox17, and Cu chaperone for Cu–Zn superoxide dismutase (CCS) which helps to regulate Cu uptake, export, and intracellular compartmentalization in non-ruminants. Copper also serves as a cofactor of antioxidant, superoxide dismutase1 (SOD1). Liver tissue from eighteen Holstein bull calves (average BW 201?±?58.5 kg, 7.3?±?1.9 months) from a previous experiment were utilized to characterize and identify hepatic mRNA related to Cu metabolism and homeostasis in cattle. Hepatic Cu concentration was determined via flame atomic absorption, and total RNA was extracted using TRI reagent and purified using RNeasy. Hepatic Cu concentrations ranged from 86 to 801 mg of Cu/kg DM. Real-time polymerase chain reaction analysis revealed that CTR1, ATP7A, and ATP7B mRNA expressions were negatively correlated with hepatic Cu concentration, while CCS (P?=?0.0887) and SOD1 had a tendency (P?=?0.0733) to be negatively correlated to hepatic Cu concentration. These data indicate that higher than normal hepatic Cu concentration downregulates gene expression of CTR1, ATP7A, ATP7B, and Cox17, which are involved in bovine liver copper homeostasis.     

The Apaf‐1•procaspase‐9 apoptosome complex functions as a proteolytic‐based molecular timer

During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that apoptosome‐mediated cleavage of procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector procaspase‐3, procaspase‐9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase‐9 possessed higher affinity for the apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase‐9 per se contributed little to the activation of procaspase‐3. Thus, the Apaf‐1 apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of procaspase‐9 sets the overall duration of the timer, procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate procaspase‐3) dictates how fast the timer ‘ticks’ over.     

Paclitaxel reduces regulatory T cell numbers and inhibitory function and enhances the anti-tumor effects of the TLR9 agonist PF-3512676 in the mouse

The anti-tumor properties of Toll-like receptor (TLR) 9 agonist CpG oligodeoxynucleotides (ODN) are enhanced by combinations with several cytotoxic chemotherapy regimens. The mechanisms of this added benefit, however, remain unclear. We now report that, similar to the depletion of regulatory T cells (Treg) using anti-CD25, paclitaxel increased the anti-tumor effect of the TLR9 agonist PF-3512676 in a CD8⁺ T cell-dependent fashion. Paclitaxel treatment decreased Treg numbers in a TLR4-independent fashion, and preferentially affected cycling Treg expressing high levels of FoxP3. The paclitaxel-induced reduction in Treg FoxP3 expression was associated with reduced inhibitory function. Adoptively transferred tumor-antigen specific CD8⁺ T cells proliferated better in mice treated with paclitaxel and their recruitment in the tumor was increased. However, the systemic frequency of PF-3512676-induced tumor-antigen specific effector CD8⁺ T cells decreased with paclitaxel, suggesting opposite effects of paclitaxel on the anti-tumor response. Finally, gene expression profiling and studies of tumor-associated immune cells revealed a complex modulation of the PF-3512676-induced immune response by paclitaxel, including a decrease of IL-10 expression and an increase in IL-17-secreting CD4⁺ T cells. Collectively, these data suggest that paclitaxel combined with PF-3512676 may not only promote a better anti-tumor CD8⁺ response though increased recruitment in the tumor, possibly through Treg depletion and suppression, but also exerts more complex immune modulatory effects.