Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) plays important roles in EBV-mediated B cell transformation, development of EBV-associated malignancies, and exacerbation of certain autoimmune conditions. LMP1 functionally mimics tumor necrosis factor receptor (TNFR) superfamily member CD40, but LMP1 signals are amplified and sustained compared to those induced by CD40. CD40 and LMP1 rely on TNFR-associated factors (TRAFs) to mediate signaling, but use TRAFs differently. TRAF6 is important for CD40 signaling, and was implicated in LMP1 signaling in non-B cells. Here, we addressed the hypothesis that TRAF6 is a critical regulator of a subset of LMP1 signals in B cells. We found that TRAF6 was required for LMP1-mediated kinase activation and costimulatory molecule upregulation, and associated with the LMP1 TRAF1/2/3/5 binding site (TBS). Additionally, TRAF6 and the TBS contributed to LMP1-induced autoreactivity and antibody (Ab) production in vivo. Finally, in contrast to CD40, LMP1 required the TRAF6 receptor-binding domain to mediate TRAF6-dependent pathways. Thus, TRAF6 is critical for LMP1 signaling and requires LMP1 interaction to propagate signals. Importantly, TRAF6 associates with LMP1 in a manner distinct from CD40, raising the possibility of disrupting LMP1 functions while leaving normal CD40 signaling intact. We next investigated roles of the kinase TAK1 in TRAF6-dependent LMP1 functions. TAK1 was required for CD40- and LMP1-mediated JNK activation in B cells, leading to IL-6 and Ab production. Understanding mechanisms of CD40 and LMP1 signaling provides important insights into normal regulatory control of CD40 functions and how LMP1-mediated pathogenesis escapes or subverts these regulatory mechanisms. LMP1 itself may be a difficult therapeutic target, because it lacks an extracellular domain and is continually processed from the cell surface. Thus, it is important to elucidate similarities and differences between CD40 and LMP1 signals to identify therapeutic targets to block LMP1-mediated pathogenesis. Comparing and contrasting CD40 and LMP1 also increases our understanding of the critical mechanisms used to regulate normal CD40 signaling.

Pseudomonas aeruginosais an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosaundergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitrodue to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosaopen reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped tolptD(PA0595) encoding lipopolysaccharide transport protein. E. coliLptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+phenotype was restored upon complementation with P. aeruginosa lptDalone, suggesting that sap27likely harbor a chromosomal mutation inlptD. Sequencing analysis of sap27showed the presence of a mutation not in lptDbut in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass analgO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6.P. aeruginosagrown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptDoperon plays a role in the P. aeruginosavirulence by regulating alginate and pyocyanin production.

Rare lipoprotein A (RlpA) has been studied previously only in Escherichia coli, where it localizes to the septal ring and scattered foci along the lateral wall, but mutants have no phenotypic change. In this thesis, we show rlpA mutants of Pseudomonas aeruginosa form chains of short, fat cells when grown in media of low osmotic strength. These morphological defects indicate RlpA is needed for efficient separation of daughter cells and maintenance of rod shape. Analysis of peptidoglycan sacculi from a ΔrlpA mutant revealed increased tetra and hexasaccharides that lack stem peptides (hereafter called "naked glycans"). Incubation of these sacculi with purified RlpA resulted in release of naked glycans containing 1,6-anhydro N-acetylmuramic acid ends. RlpA did not degrade sacculi from wild-type cells unless the sacculi were subjected to a limited digestion with an amidase to remove some of the stem peptides. Collectively, these findings indicate RlpA is a lytic transglycosylase with a strong preference for naked glycan strands. We propose that RlpA activity is regulated in vivo by substrate availability, and that amidases and RlpA work in tandem to degrade peptidoglycan in the division septum and lateral wall. Our discovery that RlpA from P. aeruginosa is a lytic transglycosylase motivated us to reinvestigate RlpA from E. coli. We confirmed predictions that RlpA of E. coli is an outer membrane protein and determined its abundance to be about 600 molecules per cell. However, multiple efforts to demonstrate that E. coli RlpA is a lytic transglycosylase were unsuccessful and the function of this protein in E. coli remains obscure.

<p>The major aim of the studies that this thesis is based on has been to better define the topological determinants of the formation of so-called helical hairpins during membrane protein assembly in the ER membrane.</p><p>The helical hairpin is a basic folding unit in membrane proteins. It is composed of two closely spaced transmembrane helices with a short connecting loop and it is believed to be inserted into the membrane as one compact unit. It is becoming increasingly clear that the helical hairpin is a very common structural element in membrane proteins and a detailed understanding of its properties is of central importance.</p><p>We demonstrate that the efficiency of formation of helical hairpins depends both on the overall length of the hydrophobic segment, on the amino acids flanking the transmembrane segment, and on the identity of the central, potentially turn-forming residues. We also show that interhelical hydrogen bonds between pairs of Asn or Asp residues can induce helical hairpin formation.</p><p>A detailed topology mapping is also reported for the <i>Escherichia coli </i>inner membrane chloride channel YadQ, a protein for which the X-ray structure is known. Our results provide a critical test of the reporter fusion approach and offer new insights into the YadQ folding pathway.</p><p>In summary, the results present in this thesis have increased our understanding of the determinants of membrane protein topology and structure. Furthermore, the information obtained can be used to improve current models for predictions of membrane protein topology.</p>

Environmental signals are sensed by membrane-spanning receptors that communicate with the cell interior. Bacterial chemoreceptors modulate the activity of the CheA kinase in response to binding of small ligands or upon interaction with substrate-bound periplasmic-binding proteins. The mechanism of signal transduction across the membrane is a displacement of the second transmembrane domain (TM2) a few angstroms toward the cytoplasm. This movement repositions a dynamic transmembrane helix relative to the plane of the cell membrane. The research presented in this dissertation investigated the contribution of TM2-membrane interactions to signaling by the aspartate chemoreceptor (Tar) of Escherichia coli. Aromatic residues that reside at the cytoplasmic polar-hydrophobic membrane interface (Trp-209 and Tyr-210) were found to play a significant role in regulating signaling by Tar. These interactions were subsequently manipulated to modulate the signaling properties of Tar. The baseline signaling state was shown to be incrementally altered by repositioning the Trp-209/Tyr-210 pair. To our knowledge, this is the first example of harnessing membrane-protein interactions to modulate the signal output of a transmembrane receptor in a controlled and predictable manner. Potential long-term applications include the use of analogous mutations to elucidate two-component signaling pathways, to engineer the signaling parameters of biosensors that incorporate chemoreceptors, and to predict the movement of dynamic transmembrane helices in silico.

The major aim of the studies that this thesis is based on has been to better define the topological determinants of the formation of so-called helical hairpins during membrane protein assembly in the ER membrane. The helical hairpin is a basic folding unit in membrane proteins. It is composed of two closely spaced transmembrane helices with a short connecting loop and it is believed to be inserted into the membrane as one compact unit. It is becoming increasingly clear that the helical hairpin is a very common structural element in membrane proteins and a detailed understanding of its properties is of central importance. We demonstrate that the efficiency of formation of helical hairpins depends both on the overall length of the hydrophobic segment, on the amino acids flanking the transmembrane segment, and on the identity of the central, potentially turn-forming residues. We also show that interhelical hydrogen bonds between pairs of Asn or Asp residues can induce helical hairpin formation. A detailed topology mapping is also reported for the Escherichia coli inner membrane chloride channel YadQ, a protein for which the X-ray structure is known. Our results provide a critical test of the reporter fusion approach and offer new insights into the YadQ folding pathway. In summary, the results present in this thesis have increased our understanding of the determinants of membrane protein topology and structure. Furthermore, the information obtained can be used to improve current models for predictions of membrane protein topology.

Membrane proteins comprise around 20-30% of a typical proteome and play crucial roles in a wide variety of biochemical pathways. Apart from their general biological significance, membrane proteins are of particular interest to the pharmaceutical industry, being targets for more than half of all available drugs. This thesis focuses on prediction methods for membrane proteins that ultimately rely on their amino acid sequence only. By identifying soluble protein domains in membrane protein sequences, we were able to constrain and improve prediction of membrane protein topology, i.e. what parts of the sequence span the membrane and what parts are located on the cytoplasmic and extra-cytoplasmic sides. Using predicted topology as input to a profile-profile based alignment protocol, we managed to increase sensitivity to detect distant membrane protein homologs. Finally, experimental measurements of the level of membrane integration of systematically designed transmembrane helices in vitro were used to derive a scale of position-specific contributions to helix insertion efficiency for all 20 naturally occurring amino acids. Notably, position within the helix was found to be an important factor for the contribution to helix insertion efficiency for polar and charged amino acids, reflecting the highly anisotropic environment of the membrane. Using the scale to predict natural transmembrane helices in protein sequences revealed that, whereas helices in single-spanning proteins are typically hydrophobic enough to insert by themselves, a large part of the helices in multi-spanning proteins seem to require stabilizing helix-helix interactions for proper membrane integration. Implementing the scale to predict full transmembrane topologies yielded results comparable to the best statistics-based topology prediction methods.

In this thesis several important proteins are investigated from a structural perspective. Some of the proteins are disease related while other have important but not completely characterised functions. The techniques used are general as demonstrated by applications on metabolic proteins (CYP21, CYP11B1, IAPP, ADH3), regulatory proteins (p53, GDNF) and a transporter protein (ANTR1). When the protein CYP21 (steroid 21-hydroxylase) is deficient it causes CAH (congenital adrenal hyperplasia). For this protein, there are about 60 known mutations with characterised clinical phenotypes. Using manual structural analysis we managed to explain the severity of all but one of the mutations. By observing the properties of these mutations we could perform good predictions on, at the time, not classified mutations. For the cancer suppressor protein p53, there are over thousand mutations with known activity. To be able to analyse such a large number of mutations we developed an automated method for evaluation of the mutation effect called PREDMUT. In this method we include twelve different prediction parameters including two energy parameters calculated using an energy minimization procedure. The method manages to differentiate severe mutations from non-severe mutations with 77% accuracy on all possible single base substitutions and with 88% on mutations found in breast cancer patients. The automated prediction was further applied to CYP11B1 (steroid 11-beta-hydroxylase), which in a similar way as CYP21 causes CAH when deficient. A generalized method applicable to any kind of globular protein was developed. The method was subsequently evaluated on nine additional proteins for which mutants were known with annotated disease phenotypes. This prediction achieved 84% accuracy on CYP11B1 and 81% accuracy in total on the evaluation proteins while leaving 8% as unclassified. By increasing the number of unclassified mutations the accuracy of the remaining mutations could be increased on the evaluation proteins and substantially increase the classification quality as measured by the Matthews correlation coefficient. Servers with predictions for all possible single based substitutions are provided for p53, CYP21 and CYP11B1. The amyloid formation of IAPP (islet amyloid polypeptide) is strongly connected to diabetes and has been studied using both molecular dynamics and Monte Carlo energy minimization. The effects of mutations on the amount and speed of amyloid formation were investigated using three approaches. Applying a consensus of the three methods on a number of interesting mutations, 94% of the mutations could be correctly classified as amyloid forming or not, evaluated with in vitro measurements. In the brain there are many proteins whose functions and interactions are largely unknown. GDNF (glial cell line-derived neurotrophic factor) and NCAM (neural cell adhesion molecule) are two such neuron connected proteins that are known to interact. The form of interaction was studied using protein--protein docking where a docking interface was found mediated by four oppositely charged residues in respective protein. This interface was subsequently confirmed by mutagenesis experiments. The NCAM dimer interface upon binding to the GDNF dimer was also mapped as well as an additional interacting protein, GFRα1, which was successfully added to the protein complex without any clashes. A large and well studied protein family is the alcohol dehydrogenase family, ADH. A class of this family is ADH3 (alcohol dehydrogenase class III) that has several known substrates and inhibitors. By using virtual screening we tried to characterize new ligands. As some ligands were already known we could incorporate this knowledge when the compound docking simulations were scored and thereby find two new substrates and two new inhibitors which were subsequently successfully tested in vitro. ANTR1 (anion transporter 1) is a membrane bound transporter important in the photosynthesis in plants. To be able to study the amino acid residues involved in inorganic phosphate transportation a homology model of the protein was created. Important residues were then mapped onto the structure using conservation analysis and we were in this way able to propose roles of amino acid residues involved in the transportation of inorganic phosphate. Key residues were subsequently mutated in vitro and a transportation process could be postulated. To conclude, we have used several molecular modelling techniques to find functional clues, interaction sites and new ligands. Furthermore, we have investigated the effect of muations on the function and structure of a multitude of disease related proteins.

Membrane proteins are involved in a number of disease pathologies and thus comprise a large number of drug targets. Determination of the high-resolution three-dimensional structure is essential for rational drug design, but several hurdles need to be overcome, primarily the over-expression and purification of said membrane proteins. Human leukotriene C4 synthase (hLTC4S), an 18 kDa integral membrane protein localized in the outer nuclear membrane of eosinophils and basophils, catalyzes the conjugation of LTA4 and reduced glutathione to produce LTC4. LTC4 and its metabolites LTD4 and LTE4 are the cysteinyl leukotrienes implicated in bronchoconstriction and inflammation pathways. The focus of my project involves optimizing the over-expression and purification of hLTC4S, which was heterologously expressed in Schizosaccharomyces pombe, purified by immobilized affinity chromatography, and finally "polished" with a buffer exchange step to remove excess co-purified lipids. The optimized protocol yielded ~1 mg of ~90% homogenous, pure protein per liter of cell culture. The finalized purified protein can then be used for further investigation of two-dimensional crystals by electron crystallography with the overall goal of structure determination.

Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography. / <p>De två (2) första författarna delar förstaförfattarskapet.</p>