MICA-G129R: A bifunctional fusion protein increases PRLR-positive breast cancer cell death in co-culture with natural killer cells.

Breast cancer cells were reported to up-regulate human prolactin receptor (PRLR) to assist their growth through the utilization of prolactin (PRL) as the growth factor, which makes PRLR a potential therapeutic target for breast cancer. On the other hand, advanced cancer cells tend to down-regulate or shed off stress signal proteins to evade immune surveillance and elimination. In this report, we created a fusion protein consisting of the extracellular domain of MHC class I chain-related protein (MICA), a stress signal protein and ligand of the activating receptor NKG2D of natural killer (NK) cells, and G129R, an antagonistic variant of PRL. We hypothesize that the MICA portion of the fusion protein binds to NKG2D to activate NK cells and the G129R portion binds to PRLR on breast cancer cells, so that the activated NK cells will kill the PRLR-positive breast cancer cells. We demonstrated that the MICA-G129R fusion protein not only binds to human natural killer NK-92 cells and PRLR-positive human breast cancer T-47D cells, but also promotes NK cells to release granzyme B and IFN-γ and enhances the cytotoxicity of NK cells specifically on PRLR-positive cells. The fusion protein, therefore, represents a new approach for the development of breast cancer specific immunotherapy.

In Vitro Assay for Plasmid Length DNA Strand Exchange by Human DMC1.

Meiosis is a specialized cell division that generates gametes. Meiotic recombination is essential not only to generate diversity in offspring, but also to hold homologous chromosomes together through chiasma allowing proper chromosome segregation. This process requires the meiosis-specific recombinase, DMC1. DMC1 facilitates the search for homology between the homologous chromosomes and is followed by DNA strand invasion and strand exchange to produce a linkage between the two homologous chromosomes. The development of biochemical in vitro assays and the purification of the requisite proteins factors has led to a better understanding of the molecular mechanisms of meiotic homologous recombination. In this chapter, a detailed in vitro assay to examine DNA strand exchange over 5000 bases of DNA catalyzed by human DMC1 is described. This method has proved to be valuable for examining the catalytic potential of hDMC1 and delineating the functional interaction with other HR factors.

In Vitro Assays for DNA Branch Migration.

Homologous recombination is a high-fidelity DNA double-strand break repair pathway that uses a homologous template to repair the break. Recombinases are the central enzymes that facilitate the strand invasion step of homologous recombination, which forms a DNA joint molecule. These DNA joint molecules can be moved through branch migration activity. In this chapter, we describe two assays to determine the branch migration activity and directionality of an enzyme. Monitoring the branch migration activity of an enzyme can provide insight into the roles of these factors in homologous recombination.

Stabilization of the Human DMC1 Nucleoprotein Filament.

The meiosis-specific recombinase, DMC1, is important for the generation of haploids during meiosis. DMC1 forms a helical nucleoprotein filament on ssDNA overhangs located at the processed double-stranded DNA break. The DMC1 filament performs a search for homology in homologous chromosome. Once homology is located, the DMC1 filament strand invades the homologous chromosome forming a displacement loop (D-loop). These connections are needed for accurate segregation to occur later in meiosis. Because DMC1 requires numerous accessory factors and specific ionic conditions to participate in this DNA repair process, in vitro assays were developed to understand how these accessory factors influence the biochemical properties of hDMC1. This chapter describes a method that can be used to investigate the stability of the human DMC1 nucleoprotein filament under various conditions and provides insight into an important early stage in DNA double-strand break repair by homologous recombination during meiosis.

Flow cytometric characterization of encystation in Entamoeba invadens.

Entamoeba histolytica causes dysentery and liver abscess mostly in countries that lack proper sanitation. Infection is acquired by ingestion of the cyst form in contaminated food or water. E. histolytica does not encyst in vitro; thus, E. invadens, a reptilian parasite that encysts in vitro, has been used as a surrogate. Cysts are small and possess chitin-rich walls. These are characteristics that may be exploited by flow cytometry. We stained encysting E. invadens cells with a fluorescent chitin stain, and analyzed fluorescence and forward scatter by flow cytometry. We demonstrate that flow cytometry can be used to track differentiation, reveal unique cell populations, and evaluate encystation inhibitors.

Homologous Recombination in Protozoan Parasites and Recombinase Inhibitors.

Homologous recombination (HR) is a DNA double-strand break (DSB) repair pathway that utilizes a homologous template to fully repair the damaged DNA. HR is critical to maintain genome stability and to ensure genetic diversity during meiosis. A specialized class of enzymes known as recombinases facilitate the exchange of genetic information between sister chromatids or homologous chromosomes with the help of numerous protein accessory factors. The majority of the HR machinery is highly conserved among eukaryotes. In many protozoan parasites, HR is an essential DSB repair pathway that allows these organisms to adapt to environmental conditions and evade host immune systems through genetic recombination. Therefore, small molecule inhibitors, capable of disrupting HR in protozoan parasites, represent potential therapeutic options. A number of small molecule inhibitors were identified that disrupt the activities of the human recombinase RAD51. Recent studies have examined the effect of two of these molecules on the Entamoeba recombinases. Here, we discuss the current understandings of HR in the protozoan parasites Trypanosoma, Leishmania, Plasmodium, and Entamoeba, and we review the small molecule inhibitors known to disrupt human RAD51 activity.

Data on Rad51 amino acid sequences from higher and lower eukaryotic model organisms and parasites.

This paper contains data related to the research article titled "Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase" (Kelso et al., in press) [1]. The known and putative amino acid sequence of Rad51, the central enzyme of homologous recombination, from nineteen different higher and lower eukaryotic organisms was analyzed. Here, we show amino acid conservation using a multiple sequence alignment, overall sequence identities using a percent identity matrix, and the evolutionary relationship between organisms using a neighbor-joining tree.

The ß-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing.

Homologous recombination (HR) is a template-driven repair pathway that mends DNA double-stranded breaks (DSBs), and thus helps to maintain genome stability. The RAD51 recombinase facilitates DNA joint formation during HR, but to accomplish this task, RAD51 must be loaded onto the single-stranded DNA. DSS1, a candidate gene for split hand/split foot syndrome, provides the ability to recognize RPA-coated ssDNA to the tumor suppressor BRCA2, which is complexed with RAD51. Together BRCA2-DSS1 displace RPA and load RAD51 onto the ssDNA. In addition, the BRCA2 interacting protein BCCIP normally colocalizes with chromatin bound BRCA2, and upon DSB induction, RAD51 colocalizes with BRCA2-BCCIP foci. Down-regulation of BCCIP reduces DSB repair and disrupts BRCA2 and RAD51 foci formation. While BCCIP is known to interact with BRCA2, the relationship between BCCIP and RAD51 is not known. In this study, we investigated the biochemical role of the ß-isoform of BCCIP in relation to the RAD51 recombinase. We demonstrate that BCCIPß binds DNA and physically and functionally interacts with RAD51 to stimulate its homologous DNA pairing activity. Notably, this stimulatory effect is not the result of RAD51 nucleoprotein filament stabilization; rather, we demonstrate that BCCIPß induces a conformational change within the RAD51 filament that promotes release of ADP to help maintain an active presynaptic filament. Our findings reveal a functional role for BCCIPß as a RAD51 accessory factor in HR.

Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase.

The protozoan parasite responsible for human amoebiasis is Entamoeba histolytica. An important facet of the life cycle of E. histolytica involves the conversion of the mature trophozoite to a cyst. This transition is thought to involve homologous recombination (HR), which is dependent upon the Rad51 recombinase. Here, a biochemical characterization of highly purified ehRad51 protein is presented. The ehRad51 protein preferentially binds ssDNA, forms a presynaptic filament and possesses ATP hydrolysis activity that is stimulated by the presence of DNA. Evidence is provided that ehRad51 catalyzes robust DNA strand exchange over at least 5.4 kilobase pairs. Although the homologous DNA pairing activity of ehRad51 is weak, it is strongly enhanced by the presence of two HR accessory cofactors, calcium and Hop2-Mnd1. The biochemical system described herein was used to demonstrate the potential for targeting ehRad51 with two small molecule inhibitors of human RAD51. We show that 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited ehRad51 by interfering with DNA binding and attenuated encystation in Entamoeba invadens, while B02 had no effect on ehRad51 strand exchange activity. These results provide insight into the underlying mechanism of homology-directed DNA repair in E. histolytica.

Sequestering survivin to functionalized nanoparticles: a strategy to enhance apoptosis in cancer cells.

Survivin belongs to the family of inhibitor of apoptosis proteins (IAP) and is present in most cancers while being below detection limits in most terminally differentiated adult tissues, making it an attractive protein to target for diagnostic and, potentially, therapeutic roles. Sub-100 nm poly(propargyl acrylate) (PA) particles were surface modified through the copper-catalyzed azide/alkyne cycloaddition of an azide-terminated survivin ligand derivative (azTM) originally proposed by Abbott Laboratories and speculated to bind directly to survivin (protein) at its dimer interface. Using affinity pull-down studies, it was determined that the PA/azTM nanoparticles selectively bind survivin and the particles can enhance apoptotic cell death in glioblastoma cell lines and other survivin over-expressing cell lines such as A549 and MCF7 relative to cells incubated with the original Abbott-derived small molecule inhibitor.

Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1.

Meiosis depends on homologous recombination (HR) in most sexually reproducing organisms. Efficient meiotic HR requires the activity of the meiosis-specific recombinase, Dmc1. Previous work shows Dmc1 is expressed in Entamoeba histolytica, a eukaryotic parasite responsible for amoebiasis throughout the world, suggesting this organism undergoes meiosis. Here, we demonstrate Dmc1 protein is expressed in E. histolytica. We show that purified ehDmc1 forms presynaptic filaments and catalyzes ATP-dependent homologous DNA pairing and DNA strand exchange over at least several thousand base pairs. The DNA pairing and strand exchange activities are enhanced by the presence of calcium and the meiosis-specific recombination accessory factor, Hop2-Mnd1. In combination, calcium and Hop2-Mnd1 dramatically increase the rate of DNA strand exchange activity of ehDmc1. The biochemical system described herein provides a basis on which to better understand the role of ehDmc1 and other HR proteins in E. histolytica.

Role of the conserved lysine within the Walker A motif of human DMC1.

During meiosis, the RAD51 recombinase and its meiosis-specific homolog DMC1 mediate DNA strand exchange between homologous chromosomes. The proteins form a right-handed nucleoprotein complex on ssDNA called the presynaptic filament. In an ATP-dependent manner, the presynaptic filament searches for homology to form a physical connection with the homologous chromosome. We constructed two variants of hDMC1 altering the conserved lysine residue of the Walker A motif to arginine (hDMC1(K132R)) or alanine (hDMC1(K132A)). The hDMC1 variants were expressed in Escherichia coli and purified to near homogeneity. Both hDMC1(K132R) and hDMC1(K132A) variants were devoid of ATP hydrolysis. The hDMC1(K132R) variant was attenuated for ATP binding that was partially restored by the addition of either ssDNA or calcium. The hDMC1(K132R) variant was partially capable of homologous DNA pairing and strand exchange in the presence of calcium and protecting DNA from a nuclease, while the hDMC1(K132A) variant was inactive. These results suggest that the conserved lysine of the Walker A motif in hDMC1 plays a key role in ATP binding. Furthermore, the binding of calcium and ssDNA promotes a conformational change in the ATP binding pocket of hDMC1 that promotes ATP binding. Our results provide evidence that the conserved lysine in the Walker A motif of hDMC1 is critical for ATP binding which is required for presynaptic filament formation.

Substrate-baited nanoparticles: a catch and release strategy for enzyme recognition and harvesting.

The isolation of a single type of protein from a complex mixture is vital for the characterization of the function, structure, and interactions of the protein of interest and is typically the most laborious aspect of the protein purification process. In this work, a model system is utilized to show the efficacy of synthesizing a "baited" nanoparticle to capture and recycle enzymes (proteins that catalyze chemical reactions) from crude cell lysate. Enzyme trapping and recycling is illustrated with the carbazole 1,9a-dioxygenase (CARDO) system, an enzyme important in bioremediation and natural product synthesis. The enzymes are baited with azide-modified carbazolyl moieties attached to poly(propargyl acrylate) nanoparticles through a click transformation. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicates the single-step procedure to immobilize the enzymes on the particles is capable of significantly concentrating the protein from raw lysate and sequestering all required components of the protein to maintain bioactivity. These results establish a universal model applicable to concentrating and extracting known substrate-protein pairs, but it can be an invaluable tool in recognizing unknown protein-ligand affinities.

Novel attributes of Hed1 affect dynamics and activity of the Rad51 presynaptic filament during meiotic recombination.

During meiosis, recombination events that occur between homologous chromosomes help prepare the chromosome pairs for proper disjunction in meiosis I. The concurrent action of the Rad51 and Dmc1 recombinases is necessary for an interhomolog bias. Notably, the activity of Rad51 is tightly controlled, so as to minimize the use of the sister chromatid as recombination partner. We demonstrated recently that Hed1, a meiosis-specific protein in Saccharomyces cerevisiae, restricts the access of the recombinase accessory factor Rad54 to presynaptic filaments of Rad51. We now show that Hed1 undergoes self-association in a Rad51-dependent manner and binds ssDNA. We also find a strong stabilizing effect of Hed1 on the Rad51 presynaptic filament. Biochemical and genetic analyses of mutants indicate that these Hed1 attributes are germane for its recombination regulatory and Rad51 presynaptic filament stabilization functions. Our results shed light on the mechanism of action of Hed1 in meiotic recombination control.

Visualization of human Dmc1 presynaptic filaments.

Meiosis is initiated by the programmed formation of DNA double-strand breaks (DSBs). These DSBs are repaired by homologous recombination to promote crossover formation that ensures proper chromosomal segregation in meiosis. hRad51 and hDmc1 are two human recombinases present during meiosis that are homologous to the RecA recombinase from Escherichia coli. The hRad51 and hDmc1 recombinases bind the nucleolytically processed ends of the DSB forming a presynaptic filament. Formation of the presynaptic filament is necessary for the search for homology and the progression of recombination. In this chapter, we provide a method to purify hDmc1 and prepare samples for visualizing hDmc1 nucleoprotein presynaptic filaments via transmission electron microscopy.

The budding yeast Mei5-Sae3 complex interacts with Rad51 and preferentially binds a DNA fork structure.

Meiotic homologous recombination in Saccharomyces cerevisiae involves formation of nucleoprotein filaments of Rad51 and Dmc1 that mediate DNA strand exchange between homologous chromosomes. The Mei5-Sae3 protein complex functions as a recombination mediator to promote nucleation of the Dmc1 recombinase onto replication protein A-coated single-stranded DNA. Here, we have expressed and purified the Mei5 protein, Sae3 protein and the Mei5-Sae3 complex for biochemical studies. We show the Mei5-Sae3 complex preferentially binds a fork-like DNA substrate to 3' overhanging DNA, single-stranded DNA or double-stranded DNA. We demonstrate that Mei5 confers DNA binding activity to the Mei5-Sae3 complex. We determined Mei5-Sae3 interacts with the Rad51 recombinase through the N-terminal domain of Mei5. Unlike Rad52, Mei5-Sae3 lacks recombination mediator activity for Rad51. Importantly, we find that the Mei5-Sae3 complex does not harbor single-strand DNA annealing activity. These properties of the Mei5-Sae3 complex distinguishes it from the Rad52 protein, which serves as the mediator of Rad51 and is involved in the single-strand DNA annealing pathway of homologous recombination.

Selective imaging and killing of cancer cells with protein-activated near-infrared fluorescing nanoparticles.

We present a general approach for the selective imaging and killing of cancer cells using protein-activated near-infrared emitting and cytotoxic oxygen generating nanoparticles. Poly(propargyl acrylate) (PA) particles were surface modified through the copper-catalyzed azide/alkyne cycloaddition of azide-terminated indocyanine green (azICG), a near-infrared emitter, and poly(ethylene glycol) (azPEG) chains of various molecular weights. The placement of azICG onto the surface of the particles allowed for the chromophores to complex with bovine serum albumin when dispersed in PBS that resulted in an enhancement of the dye emission. In addition, the inclusion of azPEG with the chromophores onto the particle surface resulted in a synergistic ninefold enhancement of the fluorescence intensity, with azPEGs of increasing molecular weight amplifying the response. Human liver carcinoma cells (HepG2) overexpress albumin proteins and could be employed to activate the fluorescence of the nanoparticles. Preliminary PDT studies with HepG2 cells combined with the modified particles indicated that a minor exposure of 780 nm radiation resulted in a statistically significant reduction in cell growth.

Spidroin N-terminal domain promotes a pH-dependent association of silk proteins during self-assembly.

Spider silks are spun from concentrated solutions of spidroin proteins. The appropriate timing of spidroin assembly into organized fibers must be highly regulated to avoid premature fiber formation. Chemical and physical signals presented to the silk proteins as they pass from the ampulle and through the tapered duct include changes in ionic environment and pH as well as the introduction of shear forces. Here, we show that the N-terminal domain of spidroins from the major ampullate gland (MaSp-NTDs) for both Nephila and Latrodectus spiders associate noncovalently as homodimers. The MaSp-NTDs are highly pH-responsive and undergo a structural transition in the physiological pH range of the spider duct. Tryptophan fluorescence of the MaSp-NTDs reveals a change in conformation when pH is decreased, and the pH at which the transition occurs is determined by the amount and type of salt present. Size exclusion chromatography and pulldown assays both indicate that the lower pH conformation is associated with a significantly increased MaSp-NTD homodimer stability. By transducing the duct pH signal into specific protein-protein interactions, this conserved spidroin domain likely contributes significantly to the silk-spinning process. Based on these results, we propose a model of spider silk assembly dynamics as mediated through the MaSp-NTD.

Functional interactions of meiotic recombination factors Rdh54 and Dmc1.

Genetic studies in budding and fission yeasts have provided evidence that Rdh54, a Swi2/Snf2-like factor, synergizes with the Dmc1 recombinase to mediate inter-homologue recombination during meiosis. Rdh54 associates with Dmc1 in the yeast two-hybrid assay, but whether the Rdh54-Dmc1 interaction is direct and the manner in which these two recombination factors may functionally co-operate to accomplish their biological task have not yet been defined. Here, using purified Schizosaccharomyces pombe proteins, we demonstrate complex formation between Rdh54 and Dmc1 and enhancement of the recombinase activity of Dmc1 by Rdh54. Consistent with published cytological and chromatin immunoprecipitation data that implicate Rdh54 in preventing the non-specific association of Dmc1 with chromatin, we show here that Rdh54 mediates the efficient removal of Dmc1 from dsDNA. These functional attributes of Rdh54 are reliant on its ATPase function. The results presented herein provide valuable information concerning the Rdh54-Dmc1 protein pair that is germane for understanding their role in meiotic recombination. The biochemical systems established in this study should be useful for the continuing dissection of the action mechanism of Rdh54 and Dmc1.

A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments.

The eukaryotic RecA homologs Rad51 and Dmc1 are essential for strand exchange between homologous chromosomes during meiosis. All members of the RecA family of recombinases polymerize on DNA to form helical nucleoprotein filaments, which is the active form of the protein. Here we compare the filament structures of the Rad51 and Dmc1 proteins from both human and budding yeast. Previous studies of Dmc1 filaments suggested that they might be structurally distinct from filaments of other members of the RecA family, including Rad51. The data presented here indicate that Rad51 and Dmc1 filaments are essentially identical with respect to several structural parameters, including persistence length, helical pitch, filament diameter, DNA base pairs per helical turn and helical handedness. These data, together with previous studies demonstrating similar in vitro recombinase activity for Dmc1 and Rad51, support the view that differences in the meiotic function of Rad51 and Dmc1 are more likely to result from the influence of distinct sets of accessory proteins than from intrinsic differences in filament structure.