Producing green energy has become the main goal in our society in the search of reducing or eliminating the carbon emission from fossil fuels. In this project, proteins were used as a tool to... Show moreProducing green energy has become the main goal in our society in the search of reducing or eliminating the carbon emission from fossil fuels. In this project, proteins were used as a tool to develop an artificial system capable of promoting the most demanding reaction in photosynthesis, water oxidation, as a new concept of producing energy from a green source. For this purpose, a screening study of the interaction between selected proteins and a library photocatalytic ruthenium and cobalt complexes was carried out. The results from such study allowed the production of two types of artificial metalloenzymes (ArM) which were proven to be able to perform water oxidation reaction via blue light irradiation. As well, an unusual interaction between proteins which promote the photocatalytic O2 evolution from water. These findings lead to develop ruthenium and cobalt metalloenzymes as promising candidates for artificial photosynthesis in bio-inspired systems Show less
Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and... Show moreEarly recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the mu(s) heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD. Show less
By utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and... Show moreBy utilizing paramagnetic NMR techniques, the structure and dynamics of the P450cam system were investigated. The analysis of PCS and RDC illuminated the stereo-specific final complex of Pdx and P450cam, while the results of PRE demonstrated the presence of a transient encounter complex. Furthermore, the significant insights of the interaction in the interface were uncovered by X-ray crystallography. Currently, the nature of Pdx effector activity is under debate. Since paramagnetic NMR experiments are applicable to solution studies at ambient temperature, PCS, RDC and PRE methods can further resolve the molecular mechanism of P450cam in the future. Show less
The majority of the work presented in this thesis involves the design and synthesis of paramagnetic NMR probes, including lanthanoids caged probes and spin labels. An overview of the development of... Show moreThe majority of the work presented in this thesis involves the design and synthesis of paramagnetic NMR probes, including lanthanoids caged probes and spin labels. An overview of the development of different types of lanthanoids caged probes is given. Among all of the reported lanthanoid probes, the caged lanthanoid NMR probe version 5 (CLaNP-5) shows the largest paramagnetic effects due to the fact that it is tagged to proteins via two-point attachment and its free complex presents a single conformation. Although CLaNP-5 is successfully applied to study proteins and protein complexes, the net charge of Ln CLaNP-5 complex and the weak disulfide linker are the drawbacks. A new paramagnetic probe, CLaNP-7, was synthesized, for which the net charge of lanthanoids complexes was reduced to +1 by introducing p-nitrophenol. The __-tensor of CLaNP-7 is pH-dependent when a histidine residue is located close to the attachment site. It is proposed that the pH dependence is due to the fact that the histidine forms a hydrogen bond with a water that acts as the ninth ligand of the lanthanoid. In order to enhance the stability of the tag linkers, two approaches, thioether and bioorthogonal reactions, were investigated. The results showed that a new thio-reactive CLaNP-9 was successfully attached to protein and the reaction product was stable in the reductive conditions. Moreover, inhibitor-based paramagnetic probes were also synthesized and the co-crystal structures of protein with inhibitor-based probes were also presented. Show less
Text-mining is a challenging field of research initially meant for reading large text collections with a computer. Text-mining is useful in summarizing text, searching for the informative documents... Show moreText-mining is a challenging field of research initially meant for reading large text collections with a computer. Text-mining is useful in summarizing text, searching for the informative documents, and most important to do knowledge discovery. Knowledge discovery is the main subject of this thesis. The hypothesis that knowledge discovery is possible started with the work done by Swanson. He made, as a first finding, links between Raynaud__s disease and fish oil using intermediate medical terms to relate them to each other. This principle was formalized in the AB- C concept. A and C are not directly related to each other but via an intermediate concept B that needs to be discovered. Tex data can be extended by adding other non textual data such as microarray experiments. Then we are in the field of data-mining. The final goal is to do all kinds of discoveries with computer (in silico) using data sources in order to assist biology research to save time and discover more. Show less
The interactions between proteins are of central importance for virtually every process in a living cell. It has long been a mystery how two proteins associate to form a complex in a complicated... Show moreThe interactions between proteins are of central importance for virtually every process in a living cell. It has long been a mystery how two proteins associate to form a complex in a complicated cellular context. Recently, it was found that an intermediate state called encounter state, of a protein complex, exists briefly before a final protein complex is formed. In the encounter state, one protein is rolling over on the surface of its partner, searching for the optimal orientation. In my PhD thesis, a transient electron transfer complex formed between a heme protein and an iron-sulfur protein was found to be trapped in this intermediate state, existing as a pure encounter complex. Thus, characterization of this dynamic complex by nuclear magnetic resonance spectroscopy advances our understanding of the general mechanism of protein-protein interaction Show less
The main objective of this research was to increase our knowledge of the pathological mechanisms underlying LGMD 2B, to better characterize each member of the dysferlin complex and, more... Show moreThe main objective of this research was to increase our knowledge of the pathological mechanisms underlying LGMD 2B, to better characterize each member of the dysferlin complex and, more importantly, to try to identify potential modifier genes that might modulate the clinical course in patients. In chapter 2, by diverse selection methodologies on different truncated forms of recombinant dysferlin, VHH antibody fragments with specificity for two different dysferlin domains were selected from a non-immune phage display library. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation. Using these antibody fragments, we found that CAPN3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.In order to gain functional insight into the molecular mechanisms of dysferlin, we have searched for yet unknown proteins that interact with dysferlin in skeletal muscle. By co-immunoprecipitation coupled to mass spectrometry, we demonstrated that dysferlin interacts with AHNAK. Furthermore, the binding domains in dysferlin and AHNAK were mapped and the AHNAK expression in normal skeletal muscle dysferlinopathy was assessed by immonofluorescence analysis (Chapter 3).Chapter 4 describes the proteolysis of AHNAK by CAPN3. Direct interaction of AHNAK and CAPN3 was studied by GST-pull down assay and specific AHNAK domains that were cleaved by CAPN3 were identified in a cell model. The biological relevance of this cleavage was also studied at cellular level.Finally, in chapter 5 the selection strategies of antibody phage display and the biomedical application of VHH will be discussed. This chapter will mainly focus on the role of dysferlin complex in LGMD 2B and future perspectives will be discussed in this chapter. Show less
