Enzyme enhancement therapy based on pharmacological chaperones (PCs) represents a promising new therapeutic strategy for the treatment of rare genetic disorders associated with protein misfolding. PCs are small extrinsic molecules that activate, stabilize and promote folding as a way to rescue mutant enzymes from endoplasmic reticulum-associated protein degradation. To date, high throughput drug screening has relied on fluorescence-based inhibition and/or thermal stability assays for putative PC selection from large chemical libraries with confirmatory testing on patient-derived cell-based assays or animal models. However, conventional primary screening methods do not directly measure for chaperone activity that may contribute to high attrition rates in drug discovery. The major aim of this thesis is to develop and validate a high quality screening strategy for the discovery of novel PCs that restore the activity of denatured/mutant enzymes associated with Gaucher disease (GD) and phenylketonuria (PKU). Chapter II introduces a simple yet selective capillary electrophoresis (CE)-based inhibition assay for improved characterization of previously-approved FDA drugs that function as putative PCs for β-glucocerebrosidase (GCase), a lysosomal enzyme associated with GD. A novel in-vitro assay based on restoration of enzyme activity via denaturation (READ) was developed in Chapter III for unambiguous characterization of the chaperone activity of previously identified PCs for GCase when using CE with UV detection. Chapter IV subsequently adapted READ to a fluorescence-based high throughput screening platform to discover novel stilbene derivatives as PCs from a chemical library comprising structural unique compounds after in silico assessment of drug-like activity. Chapter V then used this two-tiered screening strategy to discover plant-derived natural products that enhance the activity of phenylalanine hydroxylase (PAH), the enzyme associated with PKU. In summary, an integrated two-tiered strategy for high quality screening of PCs has been developed in this thesis, which is anticipated to enhance drug discovery while reducing false discoveries for treatment of various human diseases associated with deleterious protein misfolding. / Thesis / Candidate in Philosophy

No description available.

Tau is a protein which regulates microtubule stability and is heavily involved in axonal transport. This stability is dynamically controlled in part by over 40 phosphorylation sites across the tau protein which allows for binding and release from the microtubules. However, if abnormal hyperphosphorylation occurs, tau dissociates from the microtubules. Once released, the microtubules become unstable and the aberrant tau mislocalizes from the axon to the somatodendric compartment, where it aggregates. These aggregates are made of many pathological forms of tau including oligomeric species, paired helical filaments, and neurofibrillary tangles, all of which have associated toxicities. Tau pathology is a hallmark of Alzheimer's disease, one of over 15 diseases known as tauopathies which present with tau pathology, all of which lack effective treatments. Heat shock protein 90 kDa (Hsp90) is a major adenosine triphosphate (ATP)-dependent regulator of non-native proteins, like misfolded tau. Although Hsp90 is able to effectively refold and degrade many aberrant proteins, it has been associated with preserving aberrant tau. In fact, inhibiting the Hsp90 ATPase activity leads to the degradation of tau, which has been demonstrated in a number of models with the use of various Hsp90 inhibitors. However, there are many side-effects associated with the use of these inhibitors including toxicity and heat shock factor 1 (HSF1) activation. Although improvements on Hsp90 inhibitors are still in progress, this study explores targeting Hsp90 through a slightly different mechanism, by targeting Hsp90 co-chaperones. Hsp90 is involved in almost every pathway in each cell throughout the body. Co-chaperone proteins assist Hsp90 in these various processes, but are each only involved in a subset of the total Hsp90 interactome. Therefore, targeting Hsp90 co-chaperones could lead to improved efficacy, potency, and safety of drugs designed toward Hsp90 for the treatment of tauopathies. We previously showed one of these co-chaperones, FK506 binding protein 51 kDa (FKBP51), a tetratricopeptide repeat (TPR) domain containing immunophilin, coordinates with Hsp90 to regulate tau metabolism. More specifically, we found that increases and decreases in FKBP51 levels correlated with increases and decreases in tau levels, respectively. FKBP51 knockout mice have been extensively studied and have shown no negative phenotypes in these characterizations. In this study, we found that this mouse model has decreased endogenous tau levels. Furthermore, this study demonstrates that FKPB51 colocalizes with pathological tau in the AD brain, and synergizes with Hsp90 to preserve tau from proteasomal degradation. Additionally, FKBP51 overexpression in mouse model of tau pathology leads to the preservation of tau. We went on to characterize this accumulated tau as being neurotoxic and oligomeric in nature, while being low in silver positive, β-sheet structure. In the human brain, we found that FKBP51 is strikingly increased with aging and even further in the AD brain. In support of these findings, we also found age-associated decreased methylation in the FKBP5 gene, which encodes FKBP51. Moreover, we found that increasing levels of FKBP51 caused other co-chaperone to have reduced Hsp90 binding and led to tau preservation. This supports a model where age-related increases in FKBP51 lead to the preservation of misfolded tau species and ultimately disease. In order to model the high FKBP51 expression found in the aging brain, we generated the first FKBP5 overexpressing mouse model, which is tet-regulatable. This mouse, rTgFKBP5, was made by targeted, single insertion of the human FKBP5 gene into the HIP11 locus of the mouse genome crossed with CamKIIα tTa mice. We have now confirmed high FKBP51 levels in the forebrain and hippocampus of this mouse, which will serve as a testing platform for FKBP51 regulating drugs. Overall, this work exemplifies FKBP51 as an important regulator of tau metabolism through Hsp90. With the absence of a negative phenotype in mice ablated of FKBP51 and the development of this novel, FKBP51 overexpressing mouse model, strategies designed to decrease FKPB51 levels or to disrupt the FKBP51/Hsp90 complex could be relevant for the treatment of tauopathies, like AD.

Thesis (Ph. D.)--University of Chicago, Dept. of Pathology, Dec. 2003. / Includes bibliographical references. Also available on the Internet.

Iron is important for human health and serves as a co-factor in a variety of proteins and enzymes. Human serum transferrin (hTF) is an Fe(III) transporter in blood plasma which delivers metal to cells via a receptor-mediated endocytosis. In the first part, crystal structures of FeNFeC-hTF and BiNFeC-hTF have been characterized. The N-lobes of the two structures adopt “partially opened” conformations between holo-hTF’s “closed” and apo-hTF’s “fully-opened” states. The N-lobe of BiNFeC-hTF opens wider than FeNFeC-hTF. Their metal-bound C-lobes are totally closed. Rigid-body movement and different inter-lobal hydrogen bonds for the “partially opened” conformations are observed. The binding affinities of four putative binding residues are in the order: Tyr188>Tyr95>Asp63~His249. In the N-lobe of BiNFeC-hTF, Tyr188, bicarbonate and a nitrilotriacetate (NTA) ion bind to Bi(III), whilst Tyr95 and Asp63 interact with NTA ligand. One (BiNFeC-hTF) or two (FeNFeC-hTF) glycan molecules are identified on the surface area of C-lobe. In the second part, biocoordination chemistry of selected metal ions was investigated using hTF as a template. The Al(III), Fe(III), Ga(III), Dy(III) and Yb(III)-bound hTF exhibit closed conformations in the C-lobe and “fully-opened” conformations in the N-lobe. In these structures, malonate serves as an anion in the C-lobe and provides two tunable ligation sites that lead to a less distorted octahedral coordination geometry. As a result, the large lanthanide ions (Dy(III) and Yb(III)) turn from their favored high coordination numbers (8~12) and fit into the protein’s hexadental pocket. Unexpectedly, in the presence of malonate ion and the excess amount of Dy(III) ion, the Ga(III) can be partially replaced by Dy(III), although Ga(III) has a much higher affinity than Dy(III) towards the protein. The chaperone system in Helicobacter pylorithat helps protein refold is assembled with HspB and HspA. In the third part, preliminary crystallographic work is reported for HspB and HspA. The chaperone HspB has been crystallized under various conditions and currently the diffraction resolution is 6.8Å. The co-chaperone HspA, which binds Bi(III) tightly, although its crystals diffract to 1.6Å, still needs improvement for data collection due to radiation damage.The crystal structure of HspB revealed that HspB presents as a single-ring heptamer, though it is a mixture of dimer, tetramer and a higher oligomer in solution. The interactions between HspB monomers in crystal structure are significantly weaker than that of GroEL (counterpart in Escherichia coli) monomers which may makes the HspB heptamer easier to dissociate in solution. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--Biology, Georgia Institute of Technology, 2005. / Dr Yury Chernoff, Committee Member ; Dr Jung Choi, Committee Member ; Dr Nick Hud, Committee Member ; Dr Roger Wartell, Committee Member ; Dr Harish Radhakrishna, Committee Member. Vita. Includes bibliographical references.

Tail anchored (TA) proteins constitute an evolutionarily-conserved group of integral membrane proteins that are characterised by the presence of a single C-terminal transmembrane segment (TMS), which acts as both a membrane anchor and a targeting signal. In eukaryotes, TA-proteins localise to most intracellular membranes with the endoplasmic reticulum (ER) being the entry site for TA-proteins destined for the compartments of the secretory pathway and the plasma membrane. Notably, distinct routes for TA-protein delivery to the ER have been identified, and the pathway preference seems to be determined by a relative hydrophobicity of the TMS.In the present study I demonstrate that two major routes for TA-protein delivery to the ER membrane, the TRC40-dependent and “unassisted”/chaperone-mediated pathways, both rely on the action of cytosolic factors which are extremely flexible and can accommodate substrates with TMSs that have been extensively modified (Chapters 2.1 – 2.3). Moreover, the ability of PEGylated forms of the TRC40 client Sec61b to become membrane-integrated correlates very well with the calculated changes in free energy that are associated with its partitioning into a lipid bilayer, supporting a thermodynamics-driven mode of membrane insertion for TA-proteins (Chapter 2.1). The use of fluorescently-labelled recombinant cytochrome b5 (Cytb5), a model TA-protein exploiting the “unassisted”/chaperone-mediated pathway, strongly suggests the involvement of cytosolic components during its biogenesis, whilst the accessibility of novel cysteine residues to the reagent mPEG-5000 indicates a role for peripheral membrane proteins during Cytb5 membrane integration (Chapter 2.2). Importantly, pull down assays using recombinant TA-proteins as bait, followed by mass spectrometric analysis, allowed me to identify a number of cytosolic interacting partners of TA-proteins (Chapters 2.3 and 2.4). The function of one such a factor, Bat3, was further investigated, and it was found to act prior to TRC40 and facilitate the loading of TA-protein substrates onto this targeting factor (Chapter 2.3). Based on these results and available published data, a hypothetical protein-protein interaction network is presented, and I speculate about the role of individual components during TA-protein biogenesis (Discussion).

Doctor of Philosophy / Division of Biology / Jeroen Roelofs / The ubiquitin-proteasome pathway is the major pathway of selective protein degradation in the cell. Disruption of this pathway affects cellular protein homeostasis and contributes to diseases like cancer, and neurodegeneration. The end point of this pathway is the proteasome, a complex protease formed by 66 polypeptides. Structurally, it can be subdivided into the Core Particle (CP) and the Regulatory Particle (RP). The CP harbors the proteolytic sites, whereas, the RP contains six orthologous AAA-ATPases, the Rpt proteins. These Rpt’s are essential for proteasome function and are at the interface between RP and CP. The work in this thesis focuses on the Rpt subunit Rpt5 from yeast. The C-terminal tail of Rpt5 has been shown to contribute to the binding with the CP. However, our study showed it is also essential for the interaction with Nas2, one of nine proteasome-specific chaperones. Thus, Nas2 might function as a regulator of the Rpt5-CP interaction. Further analyses suggested that Nas2 has an additional function in assembly, and that mutating the tail of Rpt5 results in increased binding of the proteasome-associated protein Ecm29 to the proteasome. We showed that Ecm29 binds Rpt5 directly, thereby inducing a closed conformation of the CP substrate entry channel, and inhibiting proteasomal ATPase activity. Consistent with these activities, several proteasome mutant strains showed Ecm29-dependent accumulation of unstable substrates. Thus, Ecm29 is an inhibitor of the proteasome in vivo and in vitro. Interestingly, besides the Rpt5 mutants, several other proteasome mutants show increased levels of Ecm29, suggesting Ecm29 has a role in quality control. Consistent with this, we observed that Ecm29 associates preferably with specific mutants and nucleotide-depleted proteasomes. Based on our data we propose a model, where early in assembly Nas2 binds to the Rpt5 tail inhibiting the Rpt5-CP interaction directly. Later in assembly Ecm29 performs a quality control function, where it recognizes and remains bound to defective proteasomes. By inhibiting these proteasomes Ecm29 prevents the aberrant degradation of proteins.

In mammalian cells newly synthesised proteins are translocated across the ER membrane and their subsequent folding is facilitated by an array of folding factors present in the lumen. These include the lectins calreticulin and calnexin, which form complexes with ERp57 to generate glycoprotein specific molecular chaperones. ERp57 is a member of the protein disulphide isomerase (PDI) family and its binding to ER lectins can be reconstituted in vitro. I have exploited this approach to define the regions of ERp57 that are necessary and sufficient for its specific interaction with calreticulin and calnexin. Truncated forms of ERp57, chimeric proteins containing various domains of ERp57 and PDI (which does not interact with calreticulin) and ERp57 b' domain point mutants have been constructed. By analysing the interactions of ERp57 derivatives with calreticulin using both cross-linking and binding assays I have been able to provide detailed insights into the molecular basis for the specific assembly of these components within the ER lumen. My results indicate that the b and b' domains of ERp57 are necessary, but not sufficient for binding to both calreticulin and calnexin. The more stringent binding assay revealed that the a' domain of ERp57 significantly enhanced binding to biotin-tagged calreticulin. The ERp57 C-terminal extension also increased binding to biotin-tagged calreticulin, perhaps by playing a role in the overall stability of the ERp57. In addition, the ERp57 b' domain point mutants show that certain amino acids in this domain, in particular residues F280, V283 and F299, may be crucial for binding to calreticulin, consistent with the principal lectin-binding site being located in the b' domain. However, the binding region clearly extends into other domains, in particular the b and a' domains.

Doctora en Bioquímica / Autorizada por el autor, pero con restricción para ser publicada a texto completo hasta diciembre de 2015, en el Portal de Tesis Electrónicas / El catabolismo de proteínas es un proceso celular fundamental que ha captado la atención de distintos investigadores en los últimos años. Existen dos mecanismos por los cuales la célula degrada proteínas defectuosas: uno extralisosomal, mediado esencialmente por el proteosoma, y otro denominado lisosomal, en el cual este organelo tiene un papel protagónico en la degradación de proteínas, especialmente en las de vida media prolongada. La célula utiliza tres vías para degradar las proteínas a través del lisosoma: macroautofagia, microautofagia y autofagia mediada por chaperonas (AMC). Esta última se activa bajo condiciones de estrés fisiológico tales como la privación de nutrientes. Proteínas citosólicas con una secuencia aminoacídica particular son reconocidas por un complejo de proteínas chaperonas y destinadas al lisosoma para ser degradadas vía AMC, esta última se distingue de la macroautofagia principalmente en que no requiere tráfico vesicular. Las proteínas sustrato a ser degradadas se unen a la proteína receptora LAMP-2A, presente en la membrana lisosomal por lo que tanto sus niveles como los de Hsc70 (chaperona requerida para este proceso proteolítico) en el lisosoma se relacionan directamente con la velocidad de degradación de AMC. El recambio de proteínas intracelulares es de particular importancia en células terminalmente diferenciadas como son los cardiomiocitos y las neuronas, pues cualquier desequilibrio induce la acumulación de proteínas anormales. Diferente es lo que ocurre en células con alta capacidad proliferativa, en las cuales este efecto se mitiga por dilución a través de múltiples divisiones celulares. La oxidación de proteínas es una consecuencia del metabolismo aeróbico, así como la producción de especies reactivas de oxígeno (EROs). Paralelamente, también se ha observado aumento de EROs en estados de estrés fisiológico, modificando las proteínas y favoreciendo su agregación al interior de la célula. Finalmente, este último proceso se asocia a diferentes estados patológicos por lo cual su remoción o la prevención de su formación son fundamentales para la sobrevida celular. Siendo la AMC un mecanismo involucrado en la degradación de proteínas especialmente bajo condiciones de estrés, se requiere establecer si ella se activa en cardiomiocitos privados de nutrientes, conocer cómo se regula y cuál es su interdependencia con la formación de EROs. Con esta finalidad, esta tesis tiene como hipótesis: “La privación de nutrientes estimula la autofagia mediada por chaperonas en el cardiomiocito como un mecanismo protector frente a daño oxidativo”. / The catabolism of proteins is a fundamental cellular process that has captured the attention of several researchers in the recent years. There are two mechanisms by which the cell degrades defective proteins: one extralysosomal, mediated primarily by the proteasome, and another called lysosomal, in which this organelle has a key role in protein degradation, especially in the long half-life proteins. In the last case, the cell may use three mechanisms to degrade proteins: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). The latter is activated under physiological stress conditions such as nutrient deprivation. Cytosolic proteins with a specific amino acid sequence are recognized by a chaperone protein complex and destined into the lysosome for degradation via CMA, the latter is distinguished mainly from macroautophagy by requiring no vesicular traffic. The substrate protein to be degraded bind to the receptor protein LAMP-2A present in the lysosomal membrane, therefore substrate protein and Hsc70 levels (chaperone required for this proteolytic process) in the lysosome are directly related to the rate of degradation AMC. The turnover of intracellular proteins is of particular importance in terminally differentiated cells such as cardiomyocytes and neurons, since any imbalance induces the accumulation of abnormal proteins. In those cells with high proliferative capacity, this effect is mitigated by dilution through multiple cell divisions. The protein oxidation is a consequence of aerobic metabolism and the production of reactive oxygen species (ROS). In parallel, it has been shown that the increase in ROS during physiological stress, modifying proteins and promoting their aggregation into the cells. Finally, this latter process is associated with various disease states for which removal or prevention of their formation are essential for cell survival. Being the AMC a mechanism involved in protein degradation, especially under stress, it is important to establish whether AMC is activated in nutrient-deprived cardiomyocytes, how is regulated and its interdependence with ROS generation. To this end, we propose the following hypothesis: "The deprivation of nutrients stimulates chaperone-mediated autophagy in cardiomyocytes as a protective mechanism against oxidative damage." / CONICYT FONDAP Anillo ACT 1111