Phospholipids and a protein-conducting channel regulate cotranslational protein targeting

Signal recognition particle (SRP) and signal recognition particle receptor (SR) are evolutionarily conserved GTPases that deliver secretory and membrane proteins to the protein-conducting channel Sec61 complex in the lipid bilayer of the endoplasmic reticulum in eukaryotes or the SecYEG complex in the inner membrane of bacteria. Unlike the canonical Ras-type GTPases, SRP and SR are activated via nucleotide-dependent heterodimerization. Upon formation of the SR•SRP targeting complex, SRP and SR undergo a series of discrete conformational changes that culminate in their reciprocal activation and hydrolysis of GTP. How the SR•SRP GTPase cycle is regulated and coupled to the delivery of the cargo protein to the protein-conducting channel at the target membrane is not well-understood. Here we examine the role of the lipid bilayer and SecYEG in regulation of the SRP-mediated protein targeting pathway and show that they serve as important biological cues that spatially control the targeting reaction.

In the first chapter, we show that anionic phospholipids of the inner membrane activate the bacterial SR, FtsY, and favor the late conformational states of the targeting complex conducive to efficient unloading of the cargo. The results of our studies suggest that the lipid bilayer acts as a spatial cue that weakens the interaction of the cargo protein with SRP and primes the complex for unloading its cargo onto SecYEG.

In the second chapter, we focus on the effect of SecYEG on the conformational states and activity of the targeting complex. While phospholipids prime the complex for unloading its cargo, they are insufficient to trigger hydrolysis of GTP and the release of the cargo from the complex. SecYEG modulates the conformation of the targeting complex and triggers the GTP hydrolysis from the complex, thus driving the targeting reaction to completion. The results of this study suggest that SecYEG is not a passive recipient of the cargo protein; rather, it actively releases the cargo from the targeting complex. Together, anionic phospholipids and SecYEG serve distinct yet complementary roles. They spatially control the targeting reaction in a sequential manner, ensuring efficient delivery and unloading of the cargo protein.

In the third chapter, we reconstitute the transfer reaction in vitro and visualize it in real time. We show that the ribosome-nascent chain complex is transferred to SecYEG via a stepwise mechanism with gradual dissolution and formation of the contacts with SRP and SecYEG, respectively, explaining how the cargo is kept tethered to the membrane during the transfer and how its loss to the cytosol is avoided.

In the fourth chapter, we examine interaction of SecYEG with secretory and membrane proteins and attempt to address the role of a novel insertase YidC in this interaction. We show that detergent-solubilized SecYEG is capable of discriminating between the nascent chains of various lengths and engages a signal sequence in a well-defined conformation in the absence of accessory factors. Further, YidC alters the conformation of the signal peptide bound to SecYEG. The results described in this chapter show that YidC affects the SecYEG-nascent chain interaction at early stages of translocation/insertion and suggest a YidC-facilitated mechanism for lateral exit of transmembrane domains from SecYEG into the lipid bilayer.

Development of protein-catalyzed capture (PCC) agents with application to the specific targeting of the E17K Point mutation of Akt1

This thesis describes the expansion and improvement of the iterative in situ click chemistry OBOC peptide library screening technology. Previous work provided a proof-of-concept demonstration that this technique was advantageous for the production of protein-catalyzed capture (PCC) agents that could be used as drop-in replacements for antibodies in a variety of applications. Chapter 2 describes the technology development that was undertaken to optimize this screening process and make it readily available for a wide variety of targets. This optimization is what has allowed for the explosive growth of the PCC agent project over the past few years.

These technology improvements were applied to the discovery of PCC agents specific for single amino acid point mutations in proteins, which have many applications in cancer detection and treatment. Chapter 3 describes the use of a general all-chemical epitope-targeting strategy that can focus PCC agent development directly to a site of interest on a protein surface. This technique utilizes a chemically-synthesized chunk of the protein, called an epitope, substituted with a click handle in combination with the OBOC in situ click chemistry libraries in order to focus ligand development at a site of interest. Specifically, Chapter 3 discusses the use of this technique in developing a PCC agent specific for the E17K mutation of Akt1. Chapter 4 details the expansion of this ligand into a mutation-specific inhibitor, with applications in therapeutics.

Targeting DNA repeat sequences with Py-Im polyamides

Hairpin pyrrole-imdazole polyamides are cell-permeable, sequence-programmable oligomers that bind in the minor groove of DNA. This thesis describes studies of Py-Im polyamides targeted to biologically important DNA repeat sequences for the purpose of modulating disease states. Design of a hairpin polyamide that binds the CG dyad, a site of DNA methylation that can become dysregulated in cancer, is described. We report the synthesis of a DNA methylation antagonist, its sequence specificity and affinity informed by Bind-n-Seq and iteratively designed, which improves inhibitory activity in a cell-free assay by 1000-fold to low nanomolar IC50. Additionally, a hairpin polyamide targeted to the telomeric sequence is found to trigger a slow necrotic-type cell death with the release of inflammatory molecules in a model of B cell lymphoma. The effects of the polyamide are unique in this class of oligomers; its effects are characterized and a functional assay of phagocytosis by macrophages is described. Additionally, hairpin polyamides targeted to pathologically expanded CTG•CAG triplet repeat DNA sequences, the molecular cause of myotonic dystrophy type 1, are synthesized and assessed for toxicity. Lastly, ChIP-seq of Hypoxia-Inducible Factor is performed under hypoxia-induced conditions. The study results show that ChIP-seq can be employed to understand the genome-wide perturbation of Hypoxia-Inducible Factor occupancy by a Py-Im polyamide.

Investigating the Role of the GET3-GET4/GET5 Interaction During Tail-Anchor Protein Targeting

The proper targeting of membrane proteins is essential to the viability of all cells. Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C-terminus, are post-translationally targeted to the endoplasmic reticulum (ER) membrane by the GET pathway (Guided Entry of TA proteins). In the yeast pathway, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 (Get4/5) complex, which tethers the co-chaperone Sgt2 to the central targeting factor, the Get3 ATPase. Although binding of Get4/5 to Get3 is critical for efficient TA targeting, the mechanisms by which Get4 regulates Get3 are unknown. To understand the molecular basis of Get4 function, we used a combination of structural biology, biochemistry, and cell biology. Get4/5 binds across the Get3 dimer interface, in an orientation only compatible with a closed Get3, providing insight into the role of nucleotide in complex formation. Additionally, this structure reveals two functionally distinct binding interfaces for anchoring and ATPase regulation, and loss of the regulatory interface leads to strong defects in vitro and in vivo. Additional crystal structures of the Get3-Get4/5 complex give rise to an alternate conformation, which represents an initial binding interaction mediated by electrostatics that facilitates the rate of subsequent inhibited complex formation. This interface is supported by an in-depth kinetic analysis of the Get3-Get4/5 interaction confirming the two-step complex formation. These results allow us to generate a refined model for Get4/5 function in TA targeting.

Delivery of Targeted Nanoparticles Across the Blood-Brain Barrier Using a Detachable Targeting Ligand

Chronic diseases of the central nervous system are poorly treated due to the inability of most therapeutics to cross the blood-brain barrier. The blood-brain barrier is an anatomical and physiological barrier that severely restricts solute influx, including most drugs, from the blood to the brain. One promising method to overcome this obstacle is to use endogenous solute influx systems at the blood-brain barrier to transport drugs. Therapeutics designed to enter the brain through transcytosis by binding the transferrin receptor, however, are restricted within endothelial cells. The focus of this work was to develop a method to increase uptake of transferrin-containing nanoparticles into the brain by overcoming these restrictive processes.

To accomplish this goal, nanoparticles were prepared with surface transferrin molecules bound through various liable chemical bonds. These nanoparticles were designed to shed the targeting molecule during transcytosis to allow increased accumulation of nanoparticles within the brain.

Transferrin was added to the surface of nanoparticles through either redox or pH sensitive chemistry. First, nanoparticles with transferrin bound through disulfide bonds were prepared. These nanoparticles showed decreased avidity for the transferrin receptor after exposure to reducing agents and increased ability to enter the brain in vivo compared to those lacking the disulfide link.

Next, transferrin was attached through a chemical bond that cleaves at mildly acidic pH. Nanoparticles containing a cleavable link between transferrin and gold nanoparticle cores were found to both cross an in vitro model of the blood-brain barrier and accumulate within the brain in significantly higher numbers than similar nanoparticles lacking the cleavable bond. Also, this increased accumulation was not seen when using this same strategy with an antibody to transferrin receptor, indicating that behavior of nanoparticles at the blood-brain barrier varies depending on what type of targeting ligand is used.

Finally, polymeric nanoparticles loaded with dopamine and utilizing a superior acid-cleavable targeting chemistry were investigated as a potential treatment for Parkinson’s disease. These nanoparticles were capable of increasing dopamine quantities in the brains of healthy mice, highlighting the therapeutic potential of this design. Overall, this work describes a novel method to increase targeted nanoparticle accumulation in the brain.

Identification of treatments targeting PMLexpressing breast cancers

The main aim of this project is to find a suitable ATO concentration to combine with already approved chemotherapeutic agents to find that synergistic effect in triple negative breast cancer MDA-MB 231 cell line, as a new strategy to treat the disease.

D77, one benzoic acid derivative, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular LEDGF/p75

Integration of viral-DNA into host chromosome mediated by the viral protein HIV-1 integrase (IN) is an essential step in the HIV-1 life cycle. In this process, Lens epithelium-derived growth factor (LEDGF/p75) is discovered to function as a cellular co-fa

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Development of a positive-negative selection procedure for gene targeting in fish cells

The gene targeting technique is a powerful tool for analyzing functions of cloned genes and for generating transgenic animals with site-directed integration of foreign genes. In order to develop this technique in fish, positive-negative selection (PNS) and homologous recombination vectors were constructed, and their expression was examined in fish cells. A vector (pNK) for PNS consists of the neomycin resistance gene (neo) as a positive selectable marker gene and the herpes simplex virus (HSV) thymidine kinase (tk) gene as a negative selectable marker gene. Positive selection with geneticin (G418) of epithelioma papulosum of carp (EPC) cells transfected with linearized pNK vector yielded 350 colonies, while double selection of transfected EPC cells with G418 and gancyclovir (Gc) resulted in nearly complete cell death, demonstrating that the PNS procedure is effective in fish cells. Homologous recombination vectors consist of the Xiphophorus melanoma receptor kinase (X mrk(Y)) gene as homologous sequence in addition to the neo and tk genes. Conditions for homologous recombination vector transfection and drug selection were established. After verification of the feasibility of expression of homologous recombination vectors in EPC cells, the first gene targeting experiments were attempted in the Xiphophorus melanoma cell line, PSM. Positive-negative selection of the targeting vector-transfectants led to a low enrichment in this particular cell line. The reasons for the low enrichment in PSM cells were discussed. (C) 2002 Elsevier Science B.V. All rights reserved.

A switch-on mechanism to activate maize ribosome-inactivating protein for targeting HIV-infected cells

Maize ribosome-inactivating protein (RIP) is a plant toxin that inactivates eukaryotic ribosomes by depurinating a specific adenine residue at the a-sarcin/ricin loop of 28S rRNA. Maize RIP is first produced as a proenzyme with a 25-amino acid internal inactivation region on the protein surface. During germination, proteolytic removal of this internal inactivation region generates the active heterodimeric maize RIP with full N-glycosidase activity. This naturally occurring switch-on mechanism provides an opportunity for targeting the cytotoxin to pathogen-infected cells. Here, we report the addition of HIV-1 protease recognition sequences to the internal inactivation region and the activation of the maize RIP variants by HIV-1 protease in vitro and in HIV-infected cells. Among the variants generated, two were cleaved efficiently by HIV-1 protease. The HIV-1 protease-activated variants showed enhanced N-glycosidase activity in vivo as compared to their un-activated counterparts. They also possessed potent inhibitory effect on p24 antigen production in human T cells infected by two HIV-1 strains. This switch-on strategy for activating the enzymatic activity of maize RIP in target cells provides a platform for combating pathogens with a specific protease.

Functional gold nanoparticle-peptide complexes as cell-targeting agents

In this paper, we report a novel approach using peptide CALNN and its derivative CALNNGGRRRRRRRR (CALNNR(8)) to functionalize gold nanoparticles for intracellular component targeting. The translocation is effected by the nanoparticle diameter and CALNNR8 surface coverage. The intracellular distributions of the complexes are change from the cellular nucleus to the endoplasmic reticulum by increasing the density of CALNNR8 at a constant nanoparticle diameter. Additionally, increasing the nanoparticle diameter at a constant density of CALNNR8 leads to less cellular internalization.

Magnetic Mesoporous Silica Spheres for Drug Targeting and Controlled Release

Magnetically functionalized mesoporous silica spheres with different size (average diameter, A.D.) from 150 nm to 2 mu m and pore size distribution were synthesized by generating magnetic FexOy nanoparticles onto the mesoporous silica hosts using the sol-gel method. The X-ray diffraction (XRD), field emission scanning electron microscope (FESEM), N-2 adsorption/desorption results show that these composites conserved regular sphere morphology and ordered mesoporous structure after the formation of FexOy nanoparticles. XRD and X-ray photoelectron spectroscopy (XPS) analysis confirmed that the FexOy generated in these mesoporous silica hosts is mainly composed of gamma-Fe2O3. Magnetic measurements reveal that these composites with different gamma-Fe2O3 loading amounts possess super-paramagnetic properties at 300 K, and the saturation magnetization increases with increasing Fe ratio loaded.

Synthesis and characterization of magnetic FexOy@SBA-15 composites with different morphologies for controlled drug release and targeting

Magnetic functionalization of the ordered mesoporous SBA-15 (SiO2) aggregate blocks and rice grain-like particles were realized by using a sol-gel method, resulting in the formation of FexOy@SBA-15 composite materials. The X-ray diffraction (XRD), N-2 adsorption/desorption, and transmission electron microscopy (TEM) results show that these composites conserved ordered mesoporous structure after the formation of FexOy nanoparticles in the pores and on the outer surface of SBA-15. It was confirmed by the XRD and X-ray photoelectron spectroscopy (XPS) analysis that the FexOy generated in these mesoporous silica hosts is mainly composed of gamma-Fe2O3. Magnetic measurements reveal that these composites possess superparamagnetic properties at 300 K. The saturation magnetization of these composites increased with the increasing loading amount of gamma-Fe2O3. These composites, which possess high surface area and high pore volume, show magnetic response sufficient for drug targeting in the presence of an external magnetic field.

Prostate cancer cell-specific VEGF siRNA delivery system using cell targeting peptide conjugated polyplexes

A polymeric gene carrier was developed to deliver vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) for prostate cancer cells in a target-specific manner. Prostate cancer-binding peptide (PCP) was conjugated with polyethylenimine (PEI) via a poly(ethylene glycol) (PEG) linker (PEI-PEG-PCP). The PEI-PEG-PCP conjugate could effectively condense siRNA to form stable polyelectrolyte complexes (polyplexes) with an average diameter of approximately 150 nm in an aqueous solution. VEGF siRNA/PEI-PEG-PCP polyplexes exhibited significantly higher VEGF inhibition efficiency than PCP-unmodified polycationic carriers (PEI-PEG or PEI) in human prostate carcinoma cells (PC-3 cells). The enhanced gene silencing activity of VEGF siRNA/PEI-PEG-PCP was maintained even under serum conditions, owing to the steric stabilization of the polyplexes with hydrophilic PEG grafts. Confocal microscopic studies revealed that the siRNA/PEI-PEG-PCP polyplexes were delivered into PC-3 cells in a PCP ligand-specific manner.

Reversible addition-fragmentation chain transfer mediated radical polymerization of asymmetrical divinyl monomers targeting hyperbranched vinyl polymers

Reversible addition-fragmentation chain transfer (RAFT) mediated radical polymerizations of allyl methacrylate and undecenyl methacrylate, compounds containing two types of vinyl groups with different reactivities, were investigated to provide hyperbranched polymers. The RAFT agent benzyl dithiobenzoate was demonstrated to be an appropriate chain-transfer agent to inhibit crosslinking and obtain polymers with moderate-to-high conversions. The polymerization of allyl methacrylate led to a polymer without branches but with five- or six-membered rings. However, poly(undecenyl methacrylate) showed an indication of branching rather than intramolecular cycles. The hyperbranched structure of poly(undecenyl methacrylate) was confirmed by a combination of H-1, C-13, H-1-H-1 correlation spectroscopy, and distortionless enhancement by polarization transfer 135 NMR spectra. The branching topology of the polymers was controlled by the variation of the reaction temperature, chain-transfer-agent concentration, and monomer conversion. The significantly lower inherent viscosities of the resulting polymers, compared with those of linear analogues, demonstrated their compact structure,