Real-time Measurement of Glial Progenitor Chemotactic Migration
Year of Dissertation:
Gliomas are the most commonly diagnosed form of central nervous system tumors, occurring primarily in adults. Like many malignant cancers, gliomas pathologically exhibit very aggressive spreading and lead to an average diagnosed survival expectancy of twelve months. This prognosis is due in large part to the uncontrolled division and migration of malignant tumor cells within healthy brain, which makes complete surgical resection impossible. Gliomas are known to contain numerous genotypic and phenotypic alterations that affect cell proliferation and survival. Previous research has indicated that both gliomas and their precursor cells exhibit distinct migration patterns in brain tissue, which may be induced by specific cytokines and their concentration gradients. Here, we investigated the migration of four brain tumor cell (BTCs) lines (U-87 MG; U-251 MG; Daoy; and XFMPDGF) and three RCAS-infected glial progenitor cell (GPCs) populations (GPCLacZ, GPCPDGF, and GPCkRas) toward various growth factors, including but not limited to: EGF, HGF/SF, PDGF-BB, and TGF-α. Mouse neural cultures of nestin expressing glial progenitor cells that have been engineered to display the Avian Leukosis Virus (ALV) target receptor, tv-a, were infected with the RCAS viral vectors RCAS-LacZ, RCAS-PDGF-B, and RCAS-kRas, and are referred to as Ntv-a glial progenitor cells (Ntv-a-GPCs). Upon successful infection, the Ntv-a-GPCs were capable of constitutively producing β-galactosidase, PDGF-B, and kRas, respectively. Additionally, when these viral vectors were injected into the brains of transgenic Ntv-a mouse pups, only the RCAS-PDGF-B viral titer induced gliomas (Dai et al, 2001; Holland et al, 2000). For the purpose of this work it is relevant to note that the Ntv-a cell lines used, with the exception of the XFMPDGF cells, were never injected into the mouse brain. Conversely, the XFMPDGF cell population represents an excised tumor from the brain of a ≤ 12-week-old Ntv-a transgenic mouse having the Ink4a-Arf -/- genetic background. After cranial injection with a RCAS-PDGF-B viral titer, tumors of the oligodendroglioma classification developed within 1-12 weeks in 100% of the injected mice (Tchougounova et al, 2007). We use these oligodendrogliomas as our mouse tumor model. Additionally, Daoy medulloblastomas were used to model the migration of the most common childhood human brain tumor, while the U-87 MG and U-251 MG were used to model glioblastomas, the most common adult brain tumor. The design and characterization of our stand-alone bridged µLane system demonstrated sustained steady-state concentration gradients over 2-3 days while enabling the diffusivity measurement of 0.82 ± 0.01 X 10-6 cm2 / s for Dextran molecules having a molecular weight similar to growth factors examined here. A modified version of this device was then used to examine the migratory response of human MB-derived Daoy, glioblastomas, and neonatal mouse glial progenitors, which has led to the characterization of differential invasion patterns that each cell line utilizes when exposed to various growth factor concentration gradients. This data is fundamental for understanding how BTCs and GPCs use cytokines to communicate with each other and alter cellular functions, specifically migration. Additionally, we demonstrate EGF-induced Akt activation in the migration of MB-derived Daoy cells, and the induced recruitment GPCs towards picomolar concentrations of both HGF and TGF-α. We suggest based on our results that glial progenitors consisting of varying genetic background can migrate in very distinct patterns and may contribute to the selectivity of glioma recruitment/or progenitor sorting.
COMPUTATIONAL STUDIES ON INTERACTION BETWEEN SOME RECEPTORS AND LIGANDS OF TRANSFORMING GROWTH FACTOR-BETA SUPERFAMILY: DESIGN OF INHIBITOR(S) TO PROMOTE OSTEOGENESIS
Year of Dissertation:
Transforming growth factor beta (TGF-β) superfamily members execute distinct and intricate roles in numerous biological events such as cell growth, differentiation, embryogenesis, immune responses, and morphogenesis etc. Diverse cellular responses are instigated by binding of these superfamily members to specific transmembrane serine/threonine kinase receptors on the cell surface and thereby activating specific pathways. As a result receptor-regulated Smad proteins are phosphorylated, followed by their complex formation with Co-Smad that together translocates into nucleus and leads to the initiation of transcription of target genes. Bone morphogenic proteins (BMPs), activins, Inhibins are the most important members of TGF-β family. When they exert their biological activity, they are sternly regulated by extracellular antagonists such as noggin, follistatin, CV2, and so forth that are expressed in close temporal and spatial proximity. Blocking these antagonists' interaction with their target receptor proteins helps to promote their respective biological responses when needed. This work is an attempt to use the current understanding of some of the receptors and ligands of TGF- superfamily members, namely BMPs, activins and inhibins and their antagonists such as noggin, follistatin, and crossveinless 2 (CV2) interactions through the analysis of their available complex structures to design small molecular inhibitors with an ultimate goal of promoting their respective biological responses. Noggin is a major natural extracellular antagonist to BMPs which binds to BMPs and blocks binding of them to BMP-specific receptors and thus negatively regulates BMP-induced osteoblastic differentiation. BMPs signal through heteromeric protein complexes composed of type I and type II serine/threonine kinase receptors. Preventing the BMP-2/noggin interaction will preserve free BMP-2 and enhance the efficacy of BMP-2 to induce bone formation. One part of this work is an attempt to use the current understanding of BMP-2, and its interaction with its receptors and antagonists, through the analysis of known structures in protein data bank (PDB), to design inhibitors of BMP-2/noggin interaction with the goal of lowering the dose of BMP-2 required in clinical applications to promote osteogenesis. Another TGF- superfamily member, the activin, exerts pivotal roles in male and female reproduction, and has powerful actions in growth and differentiation in various tissues. The ability of activins to assemble their receptor complex, however, is regulated by a number of extracellular binding proteins. Follistatin is one of the main inhibitors among them. Follistatin acts primarily by binding to activin and preventing its interaction with its receptor there by bio-neutralizes activin-mediated responses. Here our main hypothesis is to understand the activin interaction with its receptors and antagonist follistatin, through the available structures in PDB, to design small molecular binder that blocks activin/follistatin interaction with the goal of promoting BMP responsiveness of the cells. Crossveinless 2 (CV2), a member of Chordin family, is an extracellular modulator of BMPs which has a unique feature of having both the anti- and pro-BMP activity depending on the cellular context. The anti-BMP activity is directed by excessive dosage of CV2 that impedes the BMP-dependent differentiation of osteoblast and chondrocyte in cell culture and in certain developmental stages. Crystal Structure of BMP-2/CV2-VWC1 domain is available. Here, our main goal is to design a potential inhibitor of BMP-2/CV2 interaction to promote osteoblast differentiation leading to promotion of bone healing. We analyzed the available complex structures to identify contact region of CV2 with BMP-2. By using the binding information of BMP with its receptors from BMP-2/noggin studies, we performed virtual screening and identified several compounds that are likely to bind to CV2 and block BMP-2/CV2 interaction that may enhance the ectopic bone formation. Finally, by using the BMP-2 sequence fragments that are in contact with noggin, from the BMP/noggin complex structure, we designed several, di-, tri- and tetra-peptide combination structures and docked them onto respective binding sites on noggin with future goal to develop modified peptide mimetic compounds that may help blocking BMP/noggin binding to promote osteogenesis.
Dissecting the role of human PPR motif proteins in mitochondrial gene expression
Year of Dissertation:
Pentatricopeptide repeat (PPR) motif proteins constitute a growing superfamily of proteins that are broadly defined by the presence of one or more copies of a conserved 35 amino acid sequence, the PPR motif. They are particularly abundant in plants, and those whose function has been characterized have been implicated in several aspects of RNA metabolism in mitochondria and chloroplasts. In humans, PPR motif proteins are fewer in number. They include LRPPRC (Leucine-Rich PPR-motif -Containing protein), an RNA-binding protein that is a component of nuclear ribonucleoprotein (RNP) complexes that contain spliced mRNAs. Most of the LRPPRC, however, localizes predominantly to mitochondria, where it binds polyadenylated RNAs. Mutations in the lrpprc gene cause cytochrome c oxidase deficiency in Leigh Syndrome (LSFC), which is accompanied by a decrease in COXI and COXIII mitochondrial mRNAs. Our hypothesis is that LRPPRC is an essential trans-acting factor in mitochondrial mRNA metabolism. In order to address the function of LRPPRC in mitochondria, we isolated LRPPRC-associated mitochondrial RNP complexes (mtRNPs). Analysis of isolated mtRNPs shows that the mitochondrially-encoded mRNAs associate with LRPPRC. A reduction in LRPPRC levels using RNAi causes a parallel reduction in steady-state levels of mitochondrially-encoded mRNAs, but not of nuclear-encoded mRNAs. Thus, LRPPRC is an important factor for mitochondrial gene expression and is necessary for the accumulation of the mitochondrial mRNAs to which it binds. Using LRPPRC as a paradigm, we sought and analyzed other members of the PPR motif family in humans. Four other human PPR-motif proteins, PTCD1, PTCD2, PTCD3 and PTCD4, also localize in mitochondria. Moreover, some of these proteins also bind RNA and exist in the same complexes as LRPPRC. This indicates that the human PTCD proteins, as is the case with LRPPRC, are also involved in mitochondrial RNA metabolism, pointing to PPR motif proteins in humans as a novel family of trans-acting factors in mitochondrial gene expression. These findings open the way for an expanded and more detailed understanding of human mitochondrial gene expression, and for an exploration of the potential involvement of human PPR motif proteins in mitochondrial diseases, as has already been determined for LRPPRC.
Boronic Acids as Penicillinase Inhibitors
Year of Dissertation:
Abstract Boronic Acids as Penicillinase Inhibitors by Juan F. Barquero Advisor: Dr. Manfred Philipp. £]-lactamases are enzymes produced by bacteria resistant to antibiotics. A common feature on beta lactam antibiotics is the beta-lactam ring. £]-lactamases hydrolyze the £]-lactam ring leaving the antibiotic inoperative. The advent of bacteria that are resistant to £]-lactams has impelled researchers to find inhibitors for £]-lactamases that mimic the lactam ring but do not get hydrolyzed. One group of these new antibiotics is the aryl boronic acids. The main reason the boronic acids have been chosen as potential drugs is their lack of toxicity and their easy excretion in the urine. One of the most important structural features of these compounds is their chemical and geometric fitness in the active site of £]-lactamases. Boronic acids mimic the tetrahedral intermediate formed in the half-acylation reaction that occurs during the hydrolysis of the fÒ-lactam ring. The major goal of the research presented here was to discover new aryl boronic acids inhibitors of penicillinases from the class A £]-lactamases. To accomplish this goal, commercially available boronic acids that are manufactured for the Suzuki reaction were used. These compounds included fluorinated, chlorinated, brominated, carboxylated, nitrophenylated, pinacol-esterified and thiophene-carboxylated aryl boronic acid derivatives. Kinetic evaluations of each class of compounds were performed under pseudo first-order enzymatic reaction conditions and the inhibitory constants (Ki) were reported using nitrocefin as substrate for two enzymes: the in-house expressed £]-lactamase BlaC and the £]-lactamase from Bacillus cereus 569/H9 (Calbiochem) identified as TEM-116. The structure-activity relationship (SAR) showed that the most potent inhibitors of BlaC £]-lactamase were 2-carboxythiophene-5-boronic acid; 3,4,5-trifluorophenylboronic acid; 3-nitrophenytlboronic acid and 2,3,4,5-tetrafluorophenylboronic acid, Ki values of 1.2, 175.7, 213.9 and 228.6 micromolar respectively. In addition, SAR revealed that the most potent inhibitors for Bacillus cereus £]-lactamase I were 2-carboxythiophen-5-boronic acid, 3-carboxyphenylboronic acid, 2-carboxythiophene-4-boronic acid, and 3-carboxy-4-fluorophenylboronic acid having Ki values of 1.1, 19.4, 46.5, and 47.1 micromolar respectively. To gain further insight into the molecular interactions between each class of inhibitors and their targeted enzymes docking experiments were performed using Autodock Vina program combined with Sculpt from MDL and followed by the molecular visualization of the protein-ligand complexes using Swiss-PdbViewer and DiscoveryStudio from Accelerys. The results conclusively show that some selective classes of aryl boronic acids are potent competitive inhibitors of BlaC and Bacillus cereus £]-lactamase I and that they should be further considered for advanced drug discovery and improvement of treatment against antibiotic resistant bacteria. Furthermore, the discovery that 4,4¡¦-DDT is an inhibitor of Mycobacterium tuberculosis £]-lactamase, combined with in silico studies, suggests that further elaboration of this molecule may be one route to new inhibitors.
The role of Mdm2 in estrogen-mediated breast cancer cell proliferation
Year of Dissertation:
Estrogen signaling is important in breast cancer development and progression. Mdm2, a negative regulator of the p53 tumor suppressor, is often over-expressed in estrogen receptor positive breast cancers. To study the role of Mdm2 in the estrogen-mediated breast cancer cell proliferation, we examined the effect of estrogen on the p53-Mdm2 pathway in estrogen receptor positive and p53 wild-type MCF-7 breast cancer cells. Estrogen-mediated increase in cell proliferation correlated with increased Mdm2, but no concomitant decrease in the p53 protein level. Blocking Mdm2 expression with inducible shRNA inhibited estrogen-mediated cell proliferation and colony formation in soft agar. Mdm2 knockdown in the presence of estrogen increased p21 and the percent of cells in the G1 phase. Interestingly, knockdown of p53 had no effect on the estrogen-mediated cell proliferation. Estrogen also up-regulated the Mdm2 protein levels in cells exposed to the DNA damaging agent, etoposide, and the Mdm2 inhibitor, Nutlin-3. In turn, estrogen inhibited etoposide- and Nutlin-3-induced transcription of puma, a pro-apoptotic p53 target gene, without changing the p53 protein levels or p53 recruitment to the chromatin. The decrease in puma gene transcription correlated with a decrease in Puma protein and an increase in Bcl-2 protein, an anti-apoptotic estrogen receptor target. Overall, our findings suggest that estrogen signals to an Mdm2-mediated pathway to provoke cell proliferation and that this pathway is associated with inhibition of the G1 checkpoint.
Expression, Purification, Circular Dichroism, and NMR Analysis of Triple Transmembrane Domain Containing Fragments of a GPCR
Year of Dissertation:
G protein-coupled receptors (GPCRs) are the largest class of signaling molecules in eukaryotes and are important pharmacological targets. Structural characterization of GPCRs is of paramount importance to the discovery of more efficient drugs; however, these studies are hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Since their flexibility makes crystallization difficult, stabilizing mutations or substitutions are required to facilitate crystal-packing contacts. The size of the receptor/membrane mimetic complex required for solution-state nuclear magnetic resonance (NMR) analysis is too large to enable efficient isotropic tumbling. For these reasons, high-resolution structural information is available for only thirteen of the ~1000 GPCRs identified to date. Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. This approach is relevant because functional GPCRs can be reconstituted from fragments. Our lab has been using the yeast α-factor receptor as a paradigm for methods development for GPCR structural characterization. We have studied isolated fragments of this GPCR containing 1 and 2 transmembrane domains (TMs). For my dissertation project, I set out to characterize 3TM containing fragments of Ste2p. The goals of my project were 1) to determine whether 3TM containing fragments form more definied tertiary structures than a 2TM containing counterpart and 2) to determine whether chemical shifts from smaller fragments could be used to assign larger fragments. Two 3TM-containing fragments of Ste2p were recombinantly expressed, purified by reverse phase high performance liquid chromatography (RP-HPLC), and subjected to extensive biophysical analysis by circular dichroism (CD) and NMR spectroscopy. A 131-residue fragment containing the first 3TMs of Ste2p, TM1-TM3 (G31-R161) and a 151-residue fragment containing the first, second, and seventh TMs, TM127 (G31-T114,T274-L340) were cloned and expressed as TrpΔLE fusion proteins in Escherichia coli. The expressed proteins were subjected to CNBr cleavage to remove the fusion tag and TM1-TM3 and TM127 were purified by reverse-phase HPLC. The cleavage products were isolated in yields of up to 20 mg per liter of culture in a variety of isotopic-labeled forms. The secondary structure of TM1-TM3 and TM127 was determined to be helical in a number of membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE):water and detergent micelles by CD spectroscopy. Preliminary HSQC analysis in 50% TFE:water and detergent micelles revealed that these fragments were suitable for structural analysis by NMR spectroscopy. Complete backbone and side chain assignments and a detailed localization of the secondary structural elements of TM1-TM3 in 50% TFE:water have been achieved. Under these conditions, an NMR structure was determined to have low convergence, and no tertiary contacts were observed. Attempts are currently being made to ascertain tertiary contacts by paramagnetic relaxation enhancement (PRE). NMR structural characterization of both TM1-TM3 and TM127 in detergent micelles is being conducted at the University of Zurich. To determine the transferability of chemical shifts for small to large fragments of Ste2p, two smaller constructs were analyzed. A 1TM containing fragment of Ste2p, TM1 [Ste2p(G31-T78)] was cloned into a direct expression vector and expressed with an N-terminal histidine tag in E.coli. The fragment was purified by RP-HPLC. This construct and a previously expressed TM7CT40 construct [Ste2p(S267-S339)](1) were subject to extensive heteronuclear NMR analysis in lysopalmitoylphosphatidylglycerol:dodecylphosphocholine (LPPG:DPC) micelles for backbone and side chain assignment. Backbone and side chain assignments for the TM1 fragment have been completed. Comparison of amide chemical shifts obtained for this fragment to those obtained for the TM1-TM2, TM1-TM3, and TM127 fragments of Ste2p suggests that chemical shifts are transferable for all regions except the flexible GXXXG region. A calculated NMR structure for TM1 reveals a large kink at this region. Comparison of the amide TM7 chemical shifts to those of the TM127 fragment reveals that all obtained chemical shifts are similar for this region. Detailed computational analysis is currently being performed at the Frankfurt Institute of Advanced Studies of Goethe University in order to assess complete chemical shift transferability for these fragments.
Computational Studies of the Functional States Associated with Epidermal Growth Factor Receptor Activation
Year of Dissertation:
Epidermal growth factor receptors (EGFR) belong to the ErbB family of receptor tyrosine kinases. ErbB signaling is involved in a wide range of biological processes including cell motility, migration and adhesion as well as gene transcription, differentiation, proliferation and apoptosis. ErbB receptors are known to dimerize upon ligand binding. This event is thought to promote intracellular transactivation of the receptors and consequently trigger a number of signaling cas- cades. Mutation and over expression of ErbB receptors have been associated with the onset of many human malignancies, making ErbB receptors central targets in cancer therapy research. Most of these mutations are localized in the intracellular tyrosine kinase domain but recently mutations have also been identified in the extracellular region of the receptors. EGFRs are present on the cell surface in a tethered conformation (believed to correspond to an auto inhibited state). In this tethered conformation the so called "dimerization arm" (a beta hairpin protruding from the extracellular D2 domain) is masked by intra molecular contacts. Upon ligand binding a dramatic conformational change reorients the extracellular domains exposing the dimerization arm, promoting the dimerization and eventually triggering the signaling cascades. The main objective of this thesis is to investigate computationally the conformational events that lead to the extension of the extracellular region of EGFR and to analyze the energetics of the process. Most biological processes in the cell occur at time-scales and involve macromolecular assembly sizes that are often beyond the current limits of classical all-atom computational simulation approaches. One possible solution to overcome these time- and size-scale limitations is to move from all-atom to coarse-grained representations of molecules. We have undertaken the development of structural representations that can enable an accurate description of the conformational dynamics and known structural transitions of protein macromolecules. The long-term objective is to be able to elucidate in a realistic environment (including both lipids and whole transmembrane protein receptors) the molecular mechanisms underlying EGFR-mediated transmembrane signaling events.
Polyadenylation/Deadenylation/Tumor Suppressor Factors Regulate 3' End Processing Under Different Cellular Conditions
Year of Dissertation:
Under DNA damaging conditions the steady-state levels of cellular mRNAs change as a result of regulation of either or both their biosynthesis and turnover. mRNA 3' end cleavage, involved in the regulation of mRNA stability, is strongly but transiently inhibited upon UV treatment. This inhibition is mediated by both the formation of the BRCA1/BARD1/CstF complex and the proteasomal-mediated degradation of RNA polymerase II (RNAP II). As CstF-50 interacts with the tumor suppressor BARD1 to inhibit 3' processing and with RNAP II to activate 3' cleavage, it has been proposed that this cleavage factor plays a coordinating role in the DNA damage response. BARD1 is modified by ATM kinase-dependent phosphorylation at the consensus site T734 upon UV treatment. Here we show that the T734A mutation abrogates the UVinduced BARD1/CstF complex formation; the UV-induced degradation of RNAP II and the UV-induced inhibition of 3' cleavage. Chromatin immunoprecipitation reactions revealed that BARD1, CstF and RNAP II, involved in the UV-induced inhibition of 3' cleavage, associate at sites of DNA damage. Together these results indicate that BARD1 with the 3' processing factor CstF play a role in the DNA damage response. To further understand the role of CstF-50 in the DNA damage response; we analyzed other CstF-50 interactors and found that DNA damage not only induces the formation of the BARD1/CstF-50/poly(A) specific ribonuclease (PARN) complex but also the expression levels of PARN. Based on the nature of the factors, it is possible to hypothesize that the PARN/CstF-50/BARD1 interaction regulates mRNA turnover in different cellular responses. Consistent with this, the CstF-50/PARN/BARD1 complex plays a role in inhibition of 3' cleavage and activation of deadenylation upon DNA damage. CstF-50/BARD1 can revert the cap binding protein 80-mediated inhibition of PARN activity. Importantly, it is shown that PARN affects both polyadenylation and stability of different mRNA precursors, such as housekeeping genes and some clinically significant genes, under different cellular conditions. These studies indicate that the PARN/CstF/BARD1 complex plays a role in the regulation of gene expression upon DNA damage, representing an alternative mechanism to prevent the processing of premature terminated messengers and to control the expression of oncogenes and DNA repair factors.
A Role of Yeast Adhesin Amyloids in Force-Dependent Adhesion and Biofilm Formation
Year of Dissertation:
Candida albicans adhesins have amyloid-forming sequences (Ramsook et al. 2010, Otoo et al. 2008). Similarly, Tango and Waltz predicted that amyloid-forming sequences are also present in Saccharomyces cerevisiae flocculins, Flo1p and Flo11p. The cell surface of Flo1p- and Flo11p-expressing cells have ordered domains that are thioflavin T fluorescent and Congo red birefringent, two hallmarks of amyloids. Flo1p and Flo11p amyloids were important for activities of the flocculins including cell-to-cell adhesion, cell-to-substrate adhesion, and agar invasion. In addition, amyloid-perturbing dyes thioflavin S and Congo red inhibited aggregation, biofilm formation and agar invasion. Force-induced formation and propagation of adhesion nanodomains in Als5p-expressing cells were demonstrated with single-molecule atomic force microscopy (Alsteens et al. 2010). Because amyloid formation can be triggered by force, we investigated whether laminar flow and mechanical stress could activate amyloid formation and therefore increase adhesion. Shearing S. cerevisiae cells expressing Als5p or C. albicans at 0.8 dyne/cm2 increased quantity and strength of cell-to-surface and cell-to-cell binding, compared to 0.02 dyne/cm2. Mechanical stress from vortex-mixing at 2500 rpm also increased the aggregation of S. cerevisiae cells expressing Als5p or C. albicans. Similarly, cells expressing Flo1p and Flo11p also showed shear-and mechanical stress-dependent binding, and biofilm formation. I report here for the first time that catch bonding behavior in yeast cells was mediated by amyloid formation. Amyloids mediate both sensing and response in the presence of force. Adhesin-expressing cells binding to surfaces under shear stress were less likely to be washed off from the substrate than cells that were not stressed. This is characteristic of catch bonding. Catch bonding behavior was accompanied by the formation of amyloid nanodomains through the clustering at homotypic amyloid sequences. Thus, these nanodomains increased binding avidity of the adhesin-expressing cells to other cells (flocculation and aggregation assays) and to substrate surfaces. Furthermore we have devised ways of quantifying forces needed to activate aggregation, cell adhesion, and amyloids on the surface of yeast cells. Two different types of force, mechanical stress from vortex-mixing and shear stress from laminar flow increased adhesion and biofilm formation. Additionally, we quantified amyloid formation in live whole cell yeast suspensions in response to force. Fluorescent confocal microscopy and flow cytometry were used to quantify surface amyloids. Force-activated cells had punctate nanodomains with increased thioflavin T staining. Collectively, the assays can now be used to quantify amyloids in other fungal adhesins. These results demonstrate that 1. there are functional amyloids present in fungal adhesins Flo1p and Flo11p from S. cerevisiae, 2. amyloid formation mediates adhesion, agar invasion and biofilm, 3. amyloid nanodomains mediate force-sensitive catch-bonding, and 4. force-sensitive amyloid formation on the yeast cell wall surface can be quantified.
STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF RHOMBOID INTRAMEMBRANE PROTEASES
Year of Dissertation:
Intramembrane proteases are involved in multiple biological processes including cell growth and development, and apoptosis. There is no conservancy between hydrosoluble and membrane proteases. However, the catalytic residues and surrounding amino acids are absolutely conserved, suggesting that they both protein families share catalytic mechanisms but with two remarkable differences. (1) The ability of intramembrane proteases to cleave their substrates in the hydrophobic interior of the lipid bilayer, and (2) to do so in regions where the substrate displays a-helical conformation. D. melanogaster rhomboid-1 cleaves within the transmembrane domain region of epidermic growth factor receptor (EGFR) ligands Gurken, Keren and Spitz, resulting in their extracellular export. We designed substrate chimeras in which the transmembrane and cytoplasmic regions of Gurken, Keren and Spitz were preserved, while their EGFR ligand ectodomain was replaced by maltose binding protein. In vitro activity assays in detergent using purified components showed that rhomboid-1, H. sapiens RHBDL2, P. aeruginosa PA3086 and E. coli GlpG display comparable activity against these substrate chimeras. Mass spectrometry analysis of the N-terminal reaction product identified a single cleavage site after Ala138 for the Spitz chimeras, after Ala122 for the Keren chimeras, and after Ala245 for the Gurken chimeras that was identical for all rhomboids tested, suggesting a conservation of proteolytic profiles among prokaryotic and eukaryotic rhomboids. The identified cleavage site was located towards the N-terminal end of the transmembrane domain of each substrate. Positions that were sensitive to alanine scanning were further studied by introducing additional mutations to show that aside of ala in position P1, amino acids with low-helical propensities are necessary in the positions P2 and P1'. Finally, a bulky hydrophobic residue with a high helical propensity is important in P2' position to control the location of cleavage. We also carried out structural work and solved the N-terminal domain of Rhomboid and showed it displays high-affinity for membranes. Our work is put in a more general context by comparison with other intramembrane proteases and future work to unravel the mechanism of substrate binding and unwinding is also discussed.