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Ubiquitylation is a three-step process, which results in the attachment of the small protein ubiquitin (Ub) to lysine residues on a substrate protein. SUMO proteins are ubiquitin (Ub)-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. During my PhD I isolated and characterized SUMO1 and SUMO2 binding motifs. Using Yeast Two Hybrid system, bioinformatics and NMR spectroscopy we defined a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a β-strand that could bind in parallel or anti-parallel orientation to the β2-strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions. Mutation of the SUMO interacting motif of TTRAP (TRAFS and TNF receptor associated protein) influences both its localization and dynamic behaviour in living cells. Ubiquitin (Ub)-binding domains (UBDs) are key elements in conveying Ub-based cellular signals. UBD-containing proteins interact with ubiquitylated targets and control numerous biological processes including receptor trafficking, DNA repair, virus budding and gene transcription. They themselves undergo UBD-dependent monoubiquitylation, which promotes intramolecular binding of the UBD to the attached Ub and consequently leads to their functional inhibition. During the second part of my PhD I could show that, in contrast to the established ubiquitylation pathway, the presence of UBDs allows the monoubiquitylation of host protein independently of classical E3 ligases. UBDs of different types including UBA, UIM, UBM, NFZ and UBZ, can directly cooperate with E2 Ub-conjugating enzymes to promote monoubiquitylation of their host proteins. Using FRET technology I verified that the E2 enzyme and the substrate directly interact in cells. Moreover, UBD-containing proteins Stam2 and Sts2 promote self-ubiquitylation and not ubiquitylation of other targets or form polyUb chains from free Ub. Our study revealed a yet unappreciated role of E2 enzymes in ubiquitylation reactions of UBD containing proteins.
The chemiosmotic theory suggested by Peter Mitchell (Mitchell, 1961, Nature 191:144-148; see Mitchell, 1979, Science 206:1148-1159 for review) postulated that the energy released upon the oxidation of electron donor substrates is transiently stored as electrochemical proton potential, delta-p across energy-transducing membranes, which acts then as the driving force for the ATP synthesis. Membrane protein complexes can both generate and utilise a transmembrane electrochemical proton potential, either by transmembrane proton transfer or by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane. The dihaem-containing membrane protein complex quinol:fumarate reductase (QFR) from the anaerobic epsilon-proteobacterium Wolinella succinogenes apparently combines both of these mechanisms (Haas et al, 2005, Biochemistry 44:13949-13961; Lancaster et al, 2005, PNAS 102:18860–18865; Mileni et al, 2005, Biochemistry 44:16718-16728; Madej et al, 2006, EMBO J 25:4963-4970). QFR is the terminal enzyme of anaerobic fumarate respiration that allows bacteria to use fumarate as the terminal electron acceptor (Kröger, 1978, Biochim Biophys Acta 505:129-45; Lancaster, 2004, In: Respiration in Archaea and Bacteria Volume 1:57-85). QFR couples the two-electron reduction of fumarate to succinate to the two-electron oxidation of quinol to quinone. QFR contains two haem b groups bound by the transmembrane subunit C, which are termed the ‘proximal haem’, bP, and the ‘distal haem’, bD, according to the relative proximity to the hydrophilic subunits A and B (Lancaster et al, 1999, Nature 402:377-85). The two-electron transfer via the two haem groups has been proposed (Lancaster, 2002, Biochimica et Biophysica Acta 1565:215-231) and demonstrated (Madej et al, 2006, EMBO J 25:4963-4970) to be coupled to a compensatory, parallel transfer of two protons via a transmembrane proton transfer pathway. The two most prominent constituents of the proposed pathway were suggested to be the haem bD ring C propionate and the side chain of amino-acid residue Glu C180, after which the proton transfer pathway was named the ‘E-pathway’ (Lancaster, 2002, Biochimica et Biophysica Acta 565:215-231). The essential role of Glu C180 was supported by site-directed mutagenesis and structural and functional characterization of the enzyme E180Q, where the Glu C180 was replaced with a Gln residue (Lancaster et al, 2005, PNAS 102:18860–18865). Moreover, multiconformer continuum electrostatics (MCCE) calculations (Haas and Lancaster 2004, Biophys J 87:4298-4315) and Fouriertransformed infrared (FTIR) spectroscopy experiments (Haas et al, 2005, Biochemistry 44:13949-13961) indicated the Glu C180 side chain to undergo a combination of a conformational change and protonation upon haem reduction. The contribution of haem bD propionate is less clear, however, a combination of 13C labelling of the haem propionates with redox-induced FTIR experiments (Mileni et al, 2005, Biochemistry 44:16718-16728) and MCCE calculations (Haas and Lancaster, 2004, Biophys J 87:4298-4315) support a change in protonation, possibly accompanied by a change in environment upon haem reduction. These experiments and their results strongly support the existence of the ‘E-pathway’ which is transiently open during the reduction of the haem groups and blocked in the oxidized state of the enzyme (Lancaster, 2002b, Biochim Biophys Acta 1565:215-231). All available crystal structures of the QFR, however, are those of the oxidized enzyme. Therefore, it is advantageous to perform simulations of various redox states of the enzyme to determine for instance, how the side-chain of Glu C180 and haem bD ring C propionate behave upon changes of the redox states of the haem groups and why is the ‘E-pathway’ blocked in the oxidized state of the enzyme. Although the distal haem ring C propionate and Glu C180 were identified as the most prominent components of the proton transfer pathway, it was not clear, on the basis of the structure, how proton transfer could occur between them. In addition, two constituents are not enough to span the membrane region and the additional participants in the proton transfer pathway must be identified. Since an atomistic investigation of proton transfer in this system is not yet possible experimentally, I used available theoretical methods such as classical molecular dynamics (MD) simulation (Alder and Wainwright, 1959, J Phys Chem 31:459-466; McCammon et al, 1977, Nature 267:585-590) and Q-HOP molecular dynamics (Q-HOP MD) simulation (Lill and Helms, 2001, J Chem Phys 115:7993-8005) to investigate the postulated mechanism of electron coupled proton transfer in QFR. MD simulations allowed us to move away from static difference pictures obtained from FTIR experiments and MCCE calculations. The advantage of the MD simulations over the experiments and the simulations performed so far is that the time-dependent properties could now be analyzed. The behaviour of various residues and their side-chains and any environmental changes may be directly observed during MD simulations. Although classical MD simulations cannot be used to study proton transfer reactions, they can provide information on formation of configurations that would allow either direct proton transfer between donor and acceptor residues or indirect proton transfer mediated by water molecules. To avoid the static protonation of residues which is inherent in classical MD simulations, Q-HOP MD simulations were performed which explicitly describe proton transfer reactions by allowing the change of the protonation state of residues ‘on the fly’. The structures obtained after classical molecular dynamics simulations ....
The aim of the study was to investigate the role of the CX3C chemokine FKN in the role of platelet adhesion. The presence of the FKN receptor CX3CR1 in platelets is demonstrated and G-protein dependent activation of platelets with soluble FKN results in the increased adhesion of platelets to collagen and fibrinogen under flow 228 and adhesion of leucocytes to firmly attached platelets 231. Whether membrane-bound FKN is capable to promote the direct adhesion of platelets in flowing blood analogue to leucocytes was completely unknown. The adhesion mechanisms of FKN in mediating the adhesion of leucocytes under flow are well characterised and represent a novel unique mechanism of leucocyte capture and firm adhesion: FKN is responsible for immediate arrest of flowing CX3CR1 expressing leucocytes without the participation of additional adhesion receptors and ligands. This is in contrast to the classical leucocyte adhesion pathways, which are multistep processes involving leucocyte arrest, rolling and subsequent cell activation prior to firm arrest. In leucocytes, the FKN – CX3CR1 axis is sufficient to allow rapid arrest of leucocytes at low shear flow conditions 67, 101, 115, 122, 261. The set of data from this study demonstrates that immobilised FKN was capable to mediate the adhesion of platelets under low shear conditions, whereas there was no interaction in the absence of shear flow. In the presence of vWf in the adhesion matrix, FKN mediated the potent increased adhesion of platelets. This was in parts due to the activation of flowing platelets via CX3CR1 and the augmented translocation of platelets on FKN via the vWf receptor GPIbα. With respect to platelet activation, the function of endothelial FKN was comparable to leucocytes: in both cell types, the FKN dependent activation is mediated by its cognate receptor CX3CR1. This is in contrast to the adhesive capacity: in leucocytes, FKN dependent adhesion is mediated by CX3CR1, whereas in platelets, the adhesive capacity was mostly mediated by the vWf receptor GPIbα with only minor contribution from CX3CR1. In platelets, activation and adhesion by FKN were mediated by two distinct receptors, whereas in leucocytes, CX3CR1 is solely responsible for FKN dependent activation and adhesion. The presented results point out to a role of platelets in early stage of atherosclerosis. The in vivo expression of both, FKN and vWf is regulated by TNF-α, which is released in early stages of inflammation. The presence of vWf and FKN in the endothelial lining of blood vessels during these conditions is sufficient to initiate the capturing and translocation of platelets on the tunica interna. The rolling of platelets on the endothelium can induce endothelial damage and inflammation of the vessel, which might advance to the generation of clinically significant atherosclerotic plaques and fibrous atheroma.
Sodium proton antiporters are ubiquitous membrane proteins found in the cytoplasmic and organelle membranes of cells of many different origins, including plants, animals and microorganisms. They are involved in cell energetics, and play primary roles in the homeostasis of intracellular pH, cellular Na+ content and cell volume. Adaptation to high salinity and/or extreme pH in plants and bacteria or in human heart muscles requires the action of such Na+/H+ antiporters. NhaA is the essential Na+/H+ antiporter for pH and Na+ homeostasis (at alkaline pH) in Escherichia coli and many other enterobacteria. NhaA is an electrogenic Na+/H+ antiporter that exchanges 2H+ for 1Na+ (or Li+). NhaA shares with many other prokaryotic and eukaryotic antiporters a very strong dependence on pH. In order to achieve three-dimensional structure of NhaA, the previously described NhaA protein preparation was modified: (i) the wild type bacterial strain (TA16) used for homologous over-expression of NhaA was replaced with a delta nhaA strain (RK20). As a result, the purity and homogeneity of the sample was significantly improved; (ii) the previously two-step purification procedure was shortened to a single step affinity chromatography purification; (iii) a wide-range screening of crystallisation conditions, more than 20,000, was performed; (iv) a Seleno-L-methionine (SeMet) NhaA derivative was produced in order to solve the phases during structure determination. In parallel, attempts of production and crystallisation of co-complexes composed of NhaA and antibody fragments have been made. Four different monoclonal antibodies were available against NhaA. Selected antibody fragments were produced and the stability of the complex analysed. Here, the crystal structure of the pH down-regulated secondary transporter NhaA of Escherichia coli is presented at 3.45 Å resolution. A negatively charged ion funnel opens to the cytoplasm and ends in the middle of the membrane at the putative ion-binding site. There, a unique assembly of two pairs of short helices connected by crossed, extended chains creates a balanced electrostatic environment. A possible mechanism is proposed: the binding of charged substrates causes electric imbalance inducing movements, which allow for a rapid alternating access mechanism. This ion exchange machinery is regulated by a conformational change elicited by a pH signal perceived at the cytoplasmic funnel entry. The structure represents a novel fold that provides two major insights: it reveals the structural basis for the mechanism of Na+/H+ exchange and its unique regulation by pH in NhaA and in many other similar antiporters. Furthermore, it is also important for the understanding of the architecture of membrane proteins in general. However, although many aspects of the ion-translocation mechanism and pH regulation are clarified by the NhaA structure, higher resolution structures with Li+ or Na+ bound are required for understanding the ligand binding and the translocation mechanism at the atomic level. The alkaline pH-induced conformation is essential to further understand the pH-control and proton access to the binding site.
Membranes are essential for life, because a cell must separate itself from the environment to keep its molecules from dissipating away and also must keep out foreign molecules that disturb them or their cell components. However, the cell must communicate with the environment and adapt to the external conditions, needs to pump in nutrients and release toxic products of its metabolism. Membrane proteins present in the membranes of the cell and cell organelles, help the cell to gather information about the environment and perform various biological processes. Membrane proteins perform a wide range of biological functions including respiration, signal transduction and transport. Despite their high importance in biological function, only few structures have been determined because of the difficulties in producing high amounts of membrane proteins and obtaining good quality crystals. This Ph. D. thesis involves the study of different kinds of cytochrome oxidases and a membrane anchored cytochrome oxidase electron donor. Though structures of many cytochrome oxidases are known to date, there exist many different types of oxidases in different organisms, which help the organism to survive under unfavorable environmental conditions. The structural differences between these terminal oxidases which make the organism to survive in extreme environments are unclear. To investigate these, structures of different types of oxidases are necessary. Therefore, we are interested in revealing the structural details of different types of oxidases. The different types of oxidase I worked with were the caa3 HiPIP:oxygen oxidoreductase from Rhodothermus marinus, the aa3-type quinol oxidase from Acidianus ambivalens and bd-type quinol oxidase from three different organisms (Escherichia coli, Bacillus thermodenitrificans and Aquifex aeolicus). Besides the protein from E. coli all other proteins are from thermophilic organisms from which the proteins obtained are generally believed to be highly stable. The presence of a high content of charged amino acids that enhances the occurrence of salt bridges contributes to the stability of thermophilic proteins. ....
RNA interference (RNAi) is triggered by recognition of double-stranded RNA (dsRNA), and elicits the silencing of gene(s) complementary to the dsRNA sequence. RNAi is thought to have emerged as a way of safeguarding the genome against mobile genetic elements and viral infection, thus maintaining genomic integrity. dsRNA is first processed into small interfering RNAs (siRNA) by the enzyme Dicer. siRNAs are ~21 to 25 -nt long, and contain a signature 5’ phosphate group and a two nucleotide long 3’ overhang (Bernstein et al., 2001). The siRNA is then loaded into the RNA-induced si-lencing complex (RISC), of which Argonaute is the primary catalytic component (Liu et al., 2004). Energetic asymmetry of the siRNA ends allows for its directional loading into RISC (Khvorova et al., 2003; Schwarz et al., 2003). Argonaute cleaves the passen-ger strand of the siRNA, leaving the guide strand of the siRNA bound to RISC (Gregory et al., 2005; Matranga et al., 2005; Rand et al., 2005). This single-stranded guide strand siRNA bound to Argonaute is able to recognize target mRNA in a sequence-specific manner, and cleaves the mRNA. Argonaute 2 in complex with single-stranded siRNA is sufficient for mRNA recognition and cleavage, thus forming a minimal RISC (Rivas et al., 2005). miRNAs, endogenously expressed small RNA genes which typically contain mismatches and non-Watson-Crick base pairing, are processed by this general pathway, although typically modulate gene expression by translational repression as opposed to cleavage of their target mRNA. The number of Argonaute genes is highly variable between species, ranging from one in S. pombe to twenty-seven in C. elegans. Earlier crystal structures of Argonaute apoen-zymes show the architecture of Argonaute to be a multidomain protein composed of N terminal, PAZ, MID, and PIWI domains (Song et al., 2004; Yuan et al., 2005). These multi-domain proteins are present in both prokaryotic and eukaryotic organisms. The role of Argonaute proteins in prokaryotes is still unknown, but based similarity to eu-karyotic Argonautes, they may also be involved in nucleic acid-directed regulatory pathways. These proteins have served as excellent models for learning about the struc-ture and function of this family of proteins. RNAi has found a widespread application for the simple yet effective knockdown of genes of interest. The catalytic cycle of RISC requires the binding of a number of different nucleotide structures to Argonaute, and we expect Argonaute to undergo a number of conforma-tional changes during the cycle of mRNA recognition by RISC (Filipowicz, 2005; Tom-ari and Zamore, 2005). Nevertheless, it remains unclear how the multi-domain ar-rangement of Argonaute recognizes and distinguishes between single-stranded and dou-ble-stranded oligonucleotides, which correspond to the Dicer-processed siRNA product, guide strand siRNA, and the guide strand / mRNA duplex. The Argonaute protein from Aquifex aeolicus was cloned, expressed, crystallized and solved by molecular replacement. Relative to earlier Argonaute structures, a 24° reorientation of the PAZ domain in this structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. A 5.5-ns molecular dynamics simulation of Argonaute showed a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute was monitored by total internal reflection fluorescence spectroscopy (TIRFS). The experi-ments showed biphasic kinetics indicative of large conformational changes, and re-vealed a hotspot of binding energy corresponding to the first 9 nucleotides, the so-called “seed region” most crucial for sequence-specific target recognition. As RNAi may have evolved as a way of safeguarding the genome viral infection, it is not surprising that viruses have evolved different strategies to suppress the host RNAi response in the form of viral suppressor protein. (Hock and Meister, 2008; Lecellier and Voinnet, 2004; Rashid et al., 2007; Song et al., 2004; Vastenhouw and Plasterk, 2004). These viral suppressors are widespread, having been identified in a number of different viral families. Not surprisingly, they generally share little sequence homology with one another, although they appear to exist as oligomers built upon a ~ 100-200 amino acid protomer. Tomato aspermy virus, a member of the Cucumoviruses, encodes for protein 2B (TAV 2B, 95 a.a., ~11.3 kDa) that acts as an RNAi suppressor. Intriguingly, a similar genomic arrangement is seen in RNAi suppressors in the Nodaviruses, a family of viruses that can infect both plants and animals, such as Flock house virus b2 (FHV b2). The 2B and b2 proteins are both derived from a frameshifted ORF within the RNA polymerase gene (Chao et al., 2005). In spite of this genomic similarity, the 2B and b2 proteins share little sequence identity, and it is not well understood how the Cucumovirus 2B proteins suppress RNAi. To address how TAV 2B suppresses RNAi, the oligonucleotide-binding properties of TAV 2B were studied. TAV 2B shows a preference for double-stranded RNA oligonucleotides corresponding to siRNAs and miRNAs, and also binds to single-stranded RNA oligonucleotides. A stretch of positively charged residues between amino acids 20-30 are critical for RNA binding. Binding to RNA oligomerizes and induces a conformational change in TAV 2B into a primarily helical structure. These studies sug-gest that suppression of RNAi by TAV 2B may occur by targeting different stages of the RNAi pathway. TAV 2B falls under the category of more general RNAi suppres-sors, with potentially multiple targets for suppression.
Safety concerns associated with the use of viral vectors in gene therapy applications have attracted considerable attention towards the development of nonviral vectors as alternatives for DNA delivery. While nonviral vectors are commonly not associated with safety problems, they are still very inefficient compared to viral vectors, and require significant improvements to approach the efficiency of their viral counterparts. Meanwhile ligands or single-chain antibody fragments that bind to cell surface receptors for increased and/or specific cellular uptake, endosome escape activities, and nuclear localization sequences (NLSs) to enhance transport of plasmid DNA into the nucleus, have become available that can be incorporated into nonviral vectors to improve their efficacy. However, as gene delivery is a multistep process, the challenge is to incorporate multiple of these functional elements into a single nonviral vector system, while retaining their specific activities. A promising method to attach such entities to plasmid DNA is the use of multifunctional fusion proteins that bind to DNA through a DNA-binding domain. In principle, two types of DNA-binding domains/proteins can be used to anchor additional functional domains or peptides to a plasmid, namely sequence-specific DNA-binding domains, described in the first part of this thesis, or those that bind DNA independent of its sequence, exemplified in the second part of this work by a derivative of the human HMGB2 protein. The first fusion protein constructed and analyzed contained the E. coli LexA repressor as a sequence-specific DNA-binding domain. In addition, this DNA-carrier protein, termed TEL, included a bacterial translocation domain as an integrated endosome escape activity, and human TGF-a for specific targeting to the EGF-receptor (EGFR). TEL was expressed in E. coli and purified under both native and denaturing conditions. Purified, denatured TEL was refolded and subsequently shown to bind specifically to EGFR-expressing cells. However, inclusion of TEL in complexes of plasmid DNA and poly-L-lysine (pL) did not lead to increased gene delivery into EGFR-expressing COS-1 cells. Most likely this was due to the absence of DNA-binding activity of the LexA moiety in TEL. In contrast, native TEL was able to interact specifically with DNA. Nevertheless, since this interaction was rather weak, and refolding of denatured TEL had not resulted in functional activity of all of its protein domains, it seemed unlikely that fusion proteins containing LexA would exhibit gene transfer capabilities superior to those of similar DNA-carrier proteins previously constructed in our group. Further work therefore focused on the use of the E2C-Sp1C protein as an alternative sequencespecific DNA-binding domain. This artificial zinc-finger protein was fused to the single-chain antibody fragment scFv(FRP5), directed against the human ErbB2 growth factor receptor. The resulting 5-E2C fusion protein was expressed in E. coli and purified under native and denaturing conditions. Refolded and native 5-E2C were found to bind specifically to ErbB2-expressing cells, indicating that scFv(FRP5) in 5-E2C was functional in both preparations. In contrast, whereas refolded 5-E2C bound DNA only weakly, significant DNA binding was observed for native 5-E2C. In addition, it could not only be shown that the interaction of native 5-E2C with DNA containing its recognition sequence was specific, but also that this protein was able to bind DNA and recombinant ErbB2 simultaneously, demonstrating the functionality of both domains in native 5-E2C. Despite these encouraging results, the inclusion of native 5-E2C in pL- or polyethyleneimine (PEI)-DNA complexes did not lead to an (5-E2C-specific) enhancement of gene transfer efficiency, irrespective of the presence of the endosome-disruptive reagent chloroquine during transfection. In the second part of this thesis an alternative approach for the development of DNA-carrier proteins for nonviral gene delivery is described, based on human HMGB2, a DNA-binding protein without sequence specificity. HMGB2 contains an acidic C-terminus that has been found to decrease the affinity of the protein for DNA. Therefore, this C-terminal tail was deleted, resulting in an HMGB2-variant consisting of amino acids 1-186. HMGB2186, purified under native conditions from E. coli lysates, was able to interact with DNA and bound to the surface of different cell lines. Importantly, after binding to plasmid DNA HMGB2186 mediated gene delivery into COS-7 cells with higher efficiency than pL. In addition, HMGB2186-mediated gene transfer was strongly enhanced in the presence of chloroquine, indicating that the endocytic pathway was involved in cellular uptake. To improve internalization and intracellular routing of HMGB2186 as a DNA-carrier, a derivative containing the TAT47-57 cell-penetrating peptide (CPP), reported to facilitate cell entry independent of endocytosis, was constructed. Since this peptide also contains an NLS, in addition an HGMB2186-variant containing the SV40-NLS was constructed to investigate the effect of a peptide that has only nuclear localizing properties. Interestingly, the resulting TAT-HMGB2186 and SV40-HMGB2186 fusion proteins displayed DNA-binding activities similar to HMGB2186, but mediated gene delivery into different cell lines clearly more efficiently than the parental molecule. Furthermore, the efficacy of both fusion proteins was enhanced markedly in the presence of chloroquine, an indication that endocytosis was involved in the transfection process mediated by these proteins. This suggests that the increased transfection efficiency observed for TAT-HMGB2186 was more likely due to the NLS function present in the TAT47-57 peptide, rather than to its ‘cell penetrating properties’. Finally, the incorporation of functional peptides derived from human proteins into HMGB2186 was investigated. An uncharged CPP originating from Kaposi-FGF, reported to facilitate efficient cellular uptake of fused protein domains in an endocytosis-independent manner, was fused to HMGB2186 together with the SV40-NLS. Interestingly, the resulting KSV40-HMGB2186 fusion protein bound DNA similarly as previously tested DNA-carrier proteins, but did not mediate enhanced transfection compared to HMGB2186. In addition, the importin-b-binding (IBB) domain derived from human importin-a2 was investigated as a component of a DNA-carrier protein. Since the IBB domain can function as an NLS, it was fused to HMGB2186 resulting in the DNA-carrier protein IBBHMGB2186. Although IBB-HMGB2186 bound DNA in a similar manner as the other HMGB2186-derivatives, gene delivery mediated by IBB-HMGB2186 was only as effective as HMGB2186 mediated transfection, suggesting no significant role of the IBB domain. However, addition of chloroquine resulted in a remarkable enhancement of IBB-HMGB2186-mediated gene transfer, which was now more efficient than with any other HMGB2186-variant tested, and not much lower than gene transfer mediated by PEI, one of the most efficient transfection reagents available to date. To enhance nonviral gene delivery even further, the HMGB2186-based DNA-carrier proteins described in this thesis might now serve as building blocks for novel fusion proteins that include additional complementing activities. In this respect it seems particularly promising that, under conditions of effective end some escape, IBB-HMGB2186, which consists entirely of protein domains of human origin, was the most efficient of all proteins tested in this work.
The respiratory chain is composed of protein complexes residing in the inner mitochondrial membrane of eukaryotes or in the cytoplasmic membrane of prokaryotes. This cellular energy converter transforms a redox potential stored in low potential substrates into an electrochemical potential across the respective membrane. Typical respiratory chains contain the complexes I, II, III and IV named according to their sequence in the respiratory chain reaction. Electrons of low potential substrates enter at complex I or II and are passed via complex III to complex IV where they are transferred to oxygen. The transport of electrons between the complexes is mediated by small electron shuttles like quinol or cytochrome c. Two different models describe their exchange either by (1) random collision of freely diffusible electron shuttles and membrane protein complexes or (2) arrangement of the complexes in supercomplexes enabling direct channeling of electron shuttles. In the Gram positive bacterium Corynebacterium glutamicum, the complex III to complex IV electron shuttle cytochrome c is not diffusible but a covalently bound part of the diheme cytochrome subunit QcrC of complex III. Therefore, the complexes III and IV have to form a supercomplex for electron transduction. The aim of this thesis was to purify and characterise this obligatory supercomplex III/IV of C. glutamicum. To gain sufficient biomass of C. glutamicum as starting material for purification, a phosphate buffered minimal medium was developed that enabled yield of total 120 g wet cell mass (38 g dry mass) in 12 L (6×2 L) shaking cultures. The determined conversion factor of glucose into biomass was 0.46 g/g indicating an intact respiratory chain. The yield was increased by bioreactor cultivation to ~690 g wet cell mass (~220 g dry mass) in ~10 L culture volume. A previously described homologous expression system was applied that produces the complex IV subunit CtaD with a fused Strep-tag II to facilitate purification. Affinity purifications using the Strep-tag II affinity to Strep-Tactin resin yielded a mixture of complexes and supercomplexes. Two supercomplex III/IV versions named supercomplex A and B and free complex IV were identified in this mixture by size exclusion chromatography, redox difference spectroscopy and two dimensional polyacrylamide gel electrophoresis including blue native polyacrylamide electrophoresis. The here presented downscaled blue native polyacrylamide electrophoresis method with analysis times of ~1 h enabled efficient screening of factors influencing the stability of supercomplex III/IV. The screening resulted that the integrity of supercomplex III/IV is preserved by using neutral detergents at minimal detergent to protein ratios for solubilisation and low detergent concentrations for purification and storage slightly above the required critical micellar concentration. Furthermore, pH <=7.5 is required for stability of supercomplex III/IV. Large biomass yields enabled upscaling of supercomplex III/IV affinity purification. Application of the identified stability conditions resulted in affinity purified samples free of supercomplex B. The major component supercomplex A was efficiently separated from residual free complex IV by preparative size exclusion chromatography. Concentration of purified supercomplex A by ultracentrifugation resulted in integrity of the supercomplex for several days at 4 °C. Purified supercomplex A contains ten different previously described subunits. The heme content of supercomplex A relative to the protein mass is heme A: 6.0 μmol/g, heme B: 6.5 μmol/g, and heme C: 5.8 μmol/g determined by redox difference spectroscopy and biochemical protein quantification. This indicates an equimolar ratio of complex III and complex IV in supercomplex A. Supercomplex A has quinol oxidase activity that is inhibited by stigmatellin or sodium azide. The turnover number of transferred electrons per complex III monomer is 148 s−1 at 25° C. The homogeneity and stability of the prepared supercomplex A enabled the growth of threedimensional crystals of up to 0.1 mm in length. Their composition of supercomplex A was verified by redox difference spectroscopy of intact crystals and blue native polyacrylamide electrophoresis of dissolved crystals. The crystals diffracted X-rays corresponding to a resolution of ~10 Å. Electron microscopy of negative stained samples revealed the uniform shape of purified supercomplex A particles with dimensions of 22 × 9 nm in the view plane. Combined heme quantification, size determination, determined activity, symmetry considerations, and particle shape indicate that supercomplex A has a central dimer of complex III and two monomers of complex IV on opposite sides. This conformation is functionally reasonable because it provides each complex III monomer with one complex IV monomer as electron acceptor. Therefore, the stoichiometry of supercomplex A is most likely III2IV2. The sensitivity of supercomplex A to detergents indicated a role of phospholipids in its stability. Therefore, a method for phospholipid identification and quantification was developed that is suitable for detergent solubilised crude and purified membrane protein samples. The analysis combines separation of phospholipid classes according to their head group by normal phase high performance liquid chromatography with evaporative light scattering detection. Calibration with external standard allows quantification of phospholipid amount in the range of 0.25-12 μg. The method is verified by analysing the phospholipid content of the well characterised complex III of Saccharomyces cerevisiae. The reduction of its phospholipid content during its purification steps is monitored. The complex III sample purified to crystallisation quality contains the phospholipid content that was also observed in previously reported structures determined by X-ray crystallography. Purified stable supercomplex A from C. glutamicum revealed a large content of bound phospholipids. The main differences between intact supercomplex A and a mixture of potentially disintegrated smaller complexes is that intact supercomplex A has a doubled phosphatidic acid content and an increased phosphatidyl glycerol content. The importance of the small anionic phosphatidic acid for mediation of contacts between complexes in a supercomplex is discussed. The total phospholipid content of stable supercomplex A is sufficient for a complete belt surrounding the supercomplex in the membrane plane. This indicates that also all essential internal phospholipid binding positions are occupied and potentially stabilise supercomplex A.
A generic drug product (World Health Organization (WHO) terminology: multisource product) is usually marketed and manufactured after the expiry date of the innovator’s patent. Generic drugs are less expensive than the innovator products because generic manufacturers do not have to amortize the investment costs of research, development, marketing, and promotion. Multisource products must contain the same active pharmaceutical ingredients (APIs) as the original formulation and have to be shown to be interchangeable with the original formulation. Multisource products have to be shown bioequivalent to the innovator counterpart with respect to pharmacokinetic and pharmacodynamic properties. Multisource products are therefore identical in dose, strength, route of administration, safety, efficacy, and intended use. Bioequivalence can be demonstrated by in vitro dissolution, pharmacokinetic, pharmacodynamic or clinical studies. Since 2000, the U.S. Food and Drug Administration (FDA) allows the approval of certain multisource products solely on the basis of in vitro studies, i.e. by waiving in vivo studies in humans (“Biowaiver”), based on the Biopharmaceutics Classification Scheme (BCS). The BCS characterizes APIs by their solubility and permeability in the gastrointestinal tract (GIT). The different BCS Classes I-IV (Class I: high solubility, high permeability; Class II: low solubility, high permeability; Class III: high solubility, low permeability and Class IV: low solubility, low permeability) result from all possible combinations of high and low solubility with high and low permeability. Since the adoption of the BCS by the FDA in 1995, the BCS criteria have been under continuous development. In 2006, the WHO has released the most recent bioequivalence guidance including relaxed criteria for bioequivalence studies based on modified BCS criteria. According to this guidance, APIs belonging to the BCS classes I – and under defined conditions - II and III – are eligible for a biowaiver-based approval. The principal objective of this work was to characterize the first-line anti tuberculosis APIs, isoniazid, pyrazinamide, ethambutol dihydrochloride and rifampicin, according to their physicochemical, biopharmaceutical, pharmacokinetic and pharmacological properties and to classify them according to the BCS. Ethambutol dihydrochloride and isoniazid were classified as borderline BCS class I/III APIs. Pyrazinamide was classified as a BCS class III and rifampicin as a BCS class II API. Based on the BCS classification and the additional criteria defined in the WHO bioequivalence guidance, the possibility of biowaiver-based approval for immediate release (immediate release) solid oral dosage forms containing the first-line antituberculosis drugs was evaluated. A biowaiver-based approval with defined constraints was recommended for immediate release solid oral dosage forms containing isoniazid (interaction with reducing sugars), pyrazinamide and ethambutol dihydrochloride (relative narrow therapeutic index). Rifampicin was classified as a BCS class II API, and it was concluded that rifampicin containing solid oral immediate release drug products as well as Scale-Up and Post-Approval Changes (SUPAC) changes should not be approved by a biowaiver on the following basis: (i) its solubility and dissolution are highly variable due to polymorphism and instability, (ii) concomitant intake of food and antacids reduces its absorption and bioavailability, (iii) no in vitro predictive dissolution test has been found which correlates to in vivo absorption and (iv) several publications reporting cases of non-bioequivalent and bioinequivalent rifampicin products have been located in the literature. Thus, it is recommended that bioequivalence of rifampicin containing solid oral immediate release drug products should be established by in vivo pharmacokinetic studies in humans. This risk-benefit benefit assessment of a biowaiver-based approval was presented as a poster at the American Association of Pharmaceutical Scientists (AAPS) 2005 and subsequently published as “Biowaiver Monographs” in the Journal of Pharmaceutical Sciences. Based on the assessment of the dissolution properties of the antituberculosis drugs for a biowaiver approval, quality control dissolution methodologies for the International Pharmacopoeia (Pharm. Int.) were developed, presented at the WHO expert meeting and adopted in the Pharm. Int. (http://www.who.int/medicines/publications/pharmprep/OMS_TRS_948.pdf). Additionally, preliminary biowaiver recommendations were also developed for four firstline antimalarial drugs listed on the WHO Essential Medicines List (EML): Quinine, as both the hydrochloride and sulphate, and proguanil hydrochloride were classified as borderline BCS class I/III APIs. Since quinine is a narrow therapeutic index drug and many cases of non-bioequivalence have been reported in the literature, a biowaiverbased approval was not recommended. For solid oral immediate release dosage forms containing proguanil a biowaiver-based approval was recommended under the condition that they dissolve very rapidly. Primaquine phosphate was classified as a BCS class I API. Therefore, a biowaiver-based approval was recommended for immediate release solid oral dosage forms containing primaquine phosphate. Mefloquine hydrochloride was classified as a basic, BCS class IV/II API, making it ineligible for the biowaiver. Additionally, reports of non-bioequivalence and a narrow therapeutic index were found in the scientific literature. Consequently, bioequivalence of solid oral immediate release dosage forms containing mefloquine hydrochloride should be established by in vivo pharmacokinetic studies. The results for quinine hydrochloride and sulphate, proguanil hydrochloride, primaquine diphosphate and mefloquine hydrochloride were presented as a poster at the Pharmaceutical Sciences World Congress (PSWC) 2007 and published as a WHO Collaborating Center Report in June 2006. The aim of this project was to collect, evaluate, generate and publish relevant information for a biowaiver-based approval of essential medicines in order to provide a summary to local regulatory authorities. This information complements the selected list of essential medicines by providing information about the biopharmaceutical properties and pharmaceutical quality of solid oral immediate release dosage forms containing these APIs. The aim of the biowaiver project, inspired by the WHO and brought in life by the International Pharmaceutical Federation (FIP), is to enable access to essential medicines in standardized quality at an affordable price. In this work, a significant contribution to this aim in the form of four biowaiver monographs for the antituberculosis drugs and several reports on the antimalarials has been achieved.