Biochemie, Chemie und Pharmazie
In vivo selection of retroviral display libraries for tumor homing
Lydia Jasmin Dürner
- The display of foreign polypeptides and proteins on the surface of viruses or cells provides an important tool for the engineering of biomolecules and the analysis of their interactions with binding partners. The most extensively used display platform is the coat protein of the filamentous bacteriophage (Smith, 1985). Phage display libraries have often been selected for polypeptides, e.g. single chain (sc) antibodies that bind to a protein of interest, but in vivo selection could only be demonstrated for peptides so far. An alternative display platform is the retrovirus murine leukemia virus (MLV). Here, polypeptides are displayed at the N-terminus of the viral envelope glycoprotein. Proof of principle for this platform was demonstrated for protease substrate libraries, which can be selected through coupling proteolytic activation with viral infectivity (Buchholz et al., 1998). Selection of the library CX4A on living cells resulted in viruses with more than three orders of magnitude improved spreading efficiency through tumor cells (Hartl et al., 2005). Also scAb libraries have recently been displayed and selected using retroviruses (Urban et al., 2005). The library scFvlibxMo displays the repertoire of phage display preselected sc antibodies for laminin-1 binding. The retrovirus based selection process resulted in laminin-specific sc antibodies with improved expression levels in mammalian cells.
This thesis describes the in vivo (i.e. in mouse tumor models) selection of the C-X4-A and scFvlibxMo for tumor homing upon systemic delivery.
For selection of the protease substrate library C-X4-A a subcutaneous tumor was induced in SCID mice followed by three systemic injections of the library. The selection process was monitored over a period of 34 days. After the incubation period mice were sacrificed and virus load in organs and tumor determined. PCR analysis after 34 days showed that virus from the library had preferentially infected the tumor. Sequence analysis showed the selection of protease substrates with the most prominent one with a frequency of over 65%. The four most prominent protease substrate variants where reconstituted into the original viral backbone for further investigation (C-SK-A, C-HI-A, C-HM-A and C-HS-A). Interestingly, these viruses exhibited a reduced spreading capacity in vitro on HT1080 cells as compared to the C-AK-A virus, which had previously been selected on HT1080
cells. When assayed for tumor homing, however, viruses C-HI-A and C-HS-A had clearly improved in comparison to C-AK-A. Tumor tissue had been infected at rates of over 55% while virus load of extratumoral organs was very low (infection rates <0.7 for C-HS-A and <0.02 for C-HI-A). Tumor targeting capacity had thus been improved over 10-fold by the in vivo selection of the C-X4-A library.
The experimental set up for the in vivo selection of the scFvlibxMo library was performed according to that of the C-X4-A library. Fingerprint analysis of the selected viruses that infected tumor tissue resulted in the identification of seven antibody variants showing unique CDR3 sequences. Two prominent clones (M49T-A and M49T-B) were cloned back into the MoMLV genome for further analysis of the reconstituted viruses. While variant B bound laminin-1 efficiently, variant A was unable to do so, although it was selected at highest frequency (76%). Both reconstituted viruses were equally well infectious and spread through HT1080rec1 cells at a similar efficiency as MoMLV. In an in vivo competition experiment the selected viruses clearly out-competed a laminin-1 binding reference virus L36xMo for tumor homing. To understand the molecular driving forces behind the in vivo selection process the epitope of the selected scFv M49T-A was identified using a phage peptide library approach. In silico analysis led to the identification of a small group of possible antigens, including tenascin, fibronectin and collagen.
The data described in this thesis demonstrate that the retrovirus display platform is capable of allowing the in vivo selection of protease substrates and scFvs. Notably, the replication competence of the system introduced an additional level of complexity to the library. The performed in vivo selections significantly enhanced tumor tropism. Selective infection of tumor cells combined with transfer of anti-tumoral genes is an attractive strategy for cancer therapy being in focus of current research. The viruses selected in this thesis build prime candidates for targeted retrovirus based tumor therapy.
Structural characterization and interaction studies of ubiquitin-like proteins
- Ubiquitin is a highly conserved protein involved in several cellular processes like protein degradation, endocytosis, signal transduction and DNA repair. The discovery of ubiquitin-like proteins (UBL) and ubiquitin-like domains (ULD) increases the number of regulation pathways where the property of the ubiquitin-fold is profitable.
Autophagy is the catabolic pathway used in cells to deliver cytosolic components and dysfunctional organelles to the lysosome for degradation. MAP1LC3 proteins are ubiquitin-like proteins involved in one hand for the expansion of the autophagosome, which sequesters cytosolic substrates. In the other hand, these proteins (LC3- and GABARAP- subfamilies) bind to autophagic receptors linked to polyubiquitinated proteins aggregates. For this project, the 3D structure of the GABARAPL-1/NBR1-LIR complex was determined and confirmed that GABARAPL-1 belongs to the MAP1LC3 proteins family, structurally characterized by an ubiquitin-fold, consisting of a central beta-sheet formed by four beta-strands and two alpha-helices on one side of the beta-sheet, preceded N terminally by two alpha-helices, resulting in the formation of two hydrophobic pockets, hp1 and hp2. The autophagic receptor NBR1 interacts with GABARAPL-1 through the hp1 and hp2 with its LIR motif taking an extended beta conformation upon binding, forming an intermolecular beta-sheet with the second beta-strand of GABARAPL 1. This LC3- interacting region (LIR) consists of an Theta XX Gamma sequence preceded by acidic amino acids, with Theta and Gamma represented by any aromatic and hydrophobic residues, respectively. Interaction studies of the LIR domains of p62, Nix and NBR1 with different members of the MAP1LC3 proteins family indicate that the presence of a tryptophan in the LIR motif increases the binding affinity. Substitution to other aromatic amino acids or increasing the number of negatively charged residues at the N-terminus of the LIR motif, however, has little effect on the binding affinity due to enthalpy-entropy compensation, suggesting that effector proteins can interact with a wide variety of different sequences with similar and moderate binding affinities.
Additionally to be present in proteins dealing with protein folding and degradation, ubiquitin-like domain were found protein involved in the regulation of signal transduction like TBK1, a serine/threonine kinase responsible for induction of immune response. In this second project, based on the NMR chemical shifts of the TBK1 domain contained between amino acids 302 and 383, secondary structure prediction programs (TALOS and CSI) confirmed the presence of an Ubiquitin-like domain in TBK1 by identifying one alpha-helix and four beta-strands sequentially aligned like following beta-beta-alpha-beta-beta. This alignment corresponds perfectly with the secondary structure elements of Ubiquitin and proved that TBK1_ULD belongs to the UBL protein superfamily. The similarity to ubiquitin was even bigger by the presence in addition of a small beta-strand and a short helix, which are observed as the beta 5-strand and a 310-helix in Ubiquitin, respectively. The first attempts on the 3D structure determination confirmed the Ub-fold but due to the lack of assignment in TBK1_ULD, only a structure based on ubiquitin as a model was determined. Interaction studies of TBK1_ULD with the IAD-SRR domain of IRF3 showed that both side of the molecule seems involved and that the TBK1/IRF3 interaction is more complex than a one to one binding process. Unfortunately, the instability of TBK1_ULD associated to the difficulty in the purification of IAD-SRR did not allow to further study this interaction more precisely.
Finally, to overcome the difficulty encountered in NMR experiments because of low expression and/or poor solubility, an expression vector using the intrinsic property of ubiquitin was designed. Fused to proteins or peptides targets, this construct produced proteins and peptides in a larger amount than with traditional expression vectors and also with a less cost than chemical synthesis for pure labeled peptides for NMR structural studies. The presence of a hexa histidine tag was useful for the isolation and the purification of the constructs. The existence of a TEV cleavage site was created to keep the possibility of releasing the ubiquitin moiety from the expressed protein or peptide. Moreover, the ubiquitin-tag could also still be attached to the protein/peptide of interest when biophysical methods like NMR, ITC or CD spectroscopy are applied, providing the same results than for the protein/peptide moiety alone.
Structural and functional characterization of the triplet acyl carrier protein in the curacin cluster and its interaction partners
- According to the World Health Organization (WHO) bacterial resistance to antibiotic drug therapy is emerging as a major public health problem around the world. Infectious diseases seriously threaten the health and economy of all countries. Hence, the preservation of the effectiveness of antibiotics is a world wide priority. The key to preserving the power of antibiotics lies in maintaining their diversity. Many microorganisms are capable of producing these bioactive products, the so called antibiotics. Specifically in microorganisms, polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS) produce these natural bioactive compounds. Besides being used as antibiotics these non-ribosomal peptides and polyketides display an even broader spectrum of biological activities, e.g. as antivirals, immunosuppressants or in antitumor therapy. The wide functional spectrum of the peptides and ketides is due to their structural diversity. Mostly they are cyclic or branched cyclic compounds, containing non-proteinogenic amino acids, small heterocyclic rings and other unusual modifications such as epimerization, methylation, N‐formylation or heterocyclization. It is has been shown that these modifications are important for biological activity, but little is known about their biosynthetic origin.
PKS and NRPS are multidomain protein assembly lines which function by sequentially elongating a growing polyketide or peptide chain by incorporating acyl units or amino acids, respectively. The growing product is attached via a thioester linkage to the 4’-phosphopantetheine (4’-Ppant) arm of a holo acyl carrier protein (ACP) in PKSs or holo peptidyl carrier protein (PCP) in NRPSs and is passed from one module to another along the chain of reaction centers. The modular arrangement makes PKS and NRPS systems an interesting target for protein engineering. More than 200 novel polyketide compounds have already been created by module swapping, gene deletion or other specific manipulations. Unfortunately, however, engineered PKS often fail to produce significant amounts of the desired products. Structural studies may faciliate yield improvement from engineered systems by providing a more complete understanding of the interface between the different domains. While some information about domain-domain interactions, involving the most common enzymatic modules, ketosynthase and acyltransferase, is starting to emerge, little is known about the interaction of ACP domains with other modifying enzymes such as methyltransferases, epimerases or halogenases.
To further improve the understanding of domain-domain interactions this work focuses on the curacin A assembly line. Curacin A, which exhibits anti-mitotic activity, is from the marine cyanobacterium Lyngbya majuscula. This outstanding natural product contains a cyclopropane ring, a thiazoline ring, an internal cis double bond and a terminal alkene. The biosynthesis of curacin A is performed by a 2.2 Mega Dalton (MDa) hybrid PKS-NRPS cluster. A 10-enzyme assembly catalyzes the formation of the cyclopropane moiety as the first building block of the final product. Interestingly, for these enzymes the substrate is presented by an unusual cluster of three consecutive ACPs (ACPI,II,III). Little is known about the function of multiple ACPs which are supposed to increase the overall flux for enhanced production of secondary metabolites.
The first task in this work was to elucidate the structural effect of the triplet ACP repetition by nuclear magnetic resonance (NMR). The initial data show that the excised ACPI, ACPII or ACPIII proteins resulted in [15N, 1H]-TROSY spectra with strong chemical shift perturbations (CSPs), suggesting an effect on the structure. The triplet ACP domains display a high sequence identity (93- 100%) making structural investigation using usual NMR techniques due to high peak overlap impossible. To enable the investigation of the triplet ACP in its native composition we developed a powerful method, the three fragment ligation. Segmental labeling allows incorporating isotopes into one single domain in its multidomain context. As a result we could prepare the triplet ACP with only one domain isotopically labeled and therefore assign the full length protein. In this way our method paved the way to study the structural effects of the triplet ACP repetition. We could show unexpectedly, that, despite the fact that the triplet repeat of CurA ACPI,II,III has a synergistic effect in the biosynthesis of CurA, the domains are structurally independent.
In the second part of this work, we studied the structure of the isolated ACPI domain. Our results show that the CurA ACPI undergoes no major conformational changes upon activation via phosphopantetheinylation and therefore contradicts the conformational switching model which has been proposed for PCPs. Further we report the NMR solution structures of holo-ACPI and 3-hydroxyl-3-methylglutaryl (HMG)-ACPI. Data obtained from filtered nuclear overhauser effect (NOE) experiments indicate that the substrate HMG is not sequestered but presented on the ACP surface.
In the third part of this work we focussed on the protein-protein interactions of the isolated ACPI with its cognate interaction partners. We were especially interested in the interaction with the halogenase (Cur Hal), the first enzyme within the curacin A sub-cluster, acting on the initial hydroxyl-methyl-glutaryl (HMG) attached to ACPI. Primarily we studied the interaction using NMR titration and fluorescence anisotropy measurements. Surprisingly no complex between ACPI and Cur Hal could be detected. The combination of an activity assay using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and mutational analysis revealed several amino acids of ACPI that strongly decrease the activity of CurA Hal. Mapping these mutations according to their effect on the Cur Hal activity onto the structure of HMG-ACPI displays that these amino acids surround the substrate and form a consecutive surface. These results suggest that this surface is important for Cur Hal recognition and selectivity. Our research presented herein is an excellent example for protein-protein interactions in PKS systems underlying a specific recognition process.