small ubiquitin-like modifier (SUMO), plays a crucial role in many cellular activities, such as regulation of cell cycle, transcription, apoptosis and DNA damage response. SUMOylation pathway is an important mechanism in modifying protein function and regulates protein activities in eukaryotes. Deregulation of SUMOylation pathway is found in many diseases, for instance, neurodegenerative disease, human cancers, heart disease and diabetes. SUMOylation pathway is a three-step process involves a multi-enzyme catalyzed cascade which consists of the following enzymatic steps: activation (E1), conjugation (E2) and ligation (E3). Only some E3 ligation proteins are understood. One of the E3 ligase types have extensively studied is PIAS. It is a protein inhibitor of activated STAT (PIAS1). Intracellular expression of PIAS1 with the bacterial cells does not work well due to the toxicity PIAS1 can create within the cells.

We established two secretion systems to test SUMO- related recombinant proteins expression: Bacteria secretion system and yeast secretion system. We used type II secretion system which includes Sec, Tat and SRP- pathways to secreted recombinant protein production in E.coli K-12 strain. Recombinant proteins also been tested on Pichia pastoris yeast cell line. Since yeast is a eukaryote, it is a suitable host for produce eukaryotic heterologous protein like SUMO-related proteins.

Förster resonance energy transfer (FRET) is a technology that analyzes protein-protein interactions using fluorescent proteins tags, such as cyanoprotein (CyPet) and yellow fluorescent protein (YPet). In our lab, we developed a mature quantitative FRET technology to measure protein-protein interactions, especially the interactions of some proteins which be involved in cancer induction and regulation process. Cancer is a disease that when cells and tissues on any part of the body without control. These cells can continue to grow without limitation, invade nearby tissues, and interfere with organ functions. There is one important mechanism involved in cancer induction process which is SUMOylation pathway. SUMOylation pathway has been extensively studied for a long time but the accurate components of SUMOylation are still not very clear. There are lots of induction factors of cancer, one of them has been found recently is influenza virus. Influenza, an infectious disease caused by an influenza virus which can kill many people every year and spread in various methods .It has three different types which are Type A, Type B, and Type C. H1N1 virus is a subtype influenza A virus. It including 8 RNA segments coding for 12 proteins. According to recent reports, some of H1N1 virus proteins can be SUMOylated so that it makes H1N1 has a high investigation value. There are some other proteins can be potential SUMO targets that contribute to cancer induction such as PD-1 and PD-L1.Programmed cell death protein1 (PD-1 or CD279), is known as a very important cell surface receptor of T- cell and plays an important role in the immune reaction mechanisms, such as it can suppress T-cell immune activities. Programmed death-ligand1 (PD-L1 or CD274 or B7-H1) plays an important role in immune system too. In this study, I applied our FRET technology to measure SUMOylation reactions of some H1N1 proteins and do quantitative Kd determination of PD-1/PD-L1. Through with construct design, gene clone, transformation and protein purification to get proteins with high purity. Then I measured Kd and SUMOylation reactions with these proteins. As results, I got the Kd of YPet-NP and CyPet-Ubc9, PD-1 extra-cellular domain and PD-L1 extra-cellular domain. And I found that NP virus protein which is one of H1N1 proteins can be SUMOylated. These results can contribute to protein-protein interaction studies of H1N1 virus and potential drug target detection of PD-1 and PD-L1 pathway.

SUMOylation is an important post-translational protein modification mechanism which plays important roles in a variety of biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. SENP, SUMO specific protease, can either mature pre-SUMO or deconjugate SUMO from its target proteins. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. FRET is an energy transfer phenomenon which occurs between two spectrum-overlapping fluorophores in close proximity. The efficiency of FRET is highly dependent on the distance between the donor and acceptor fluorophores, which makes FRET very powerful in the detection of molecular interactions and biomolecular conformational changes.

A novel highly sensitive FRET-based protease assay was designed and developed to study the activities and specificities of SENP in SUMOylation signaling pathway and to identify bioactive chemical compounds which can specifically inhibit SENPs' activities. The endopeptidase and isopeptidase activities of SENP1/2/5/6/7c toward SUMO1/2/3 were studied and verified by western-blot assay. A novel quantitative FRET analysis was performed to study the protease kinetics to compare the specificities to substrate. The direct emissions of donor and acceptor were considered in FRET signal analysis. The mathematical algorithm dependent on pre-established standard curves or later optimized internal calibration method related the hydrolyzed substrate concentration to detected fluorescent signals during the dynamic processes, and thus derived the kinetics constants. Then, the developed protease assay and protease kinetics analysis methods were applied to study the pre-SUMO4's maturation and the product inhibition in pre-SUMO's maturation, which indicated the potential application in biomechanism study and chemical compounds' inhibition characterization. Finally, the FRET-based assay was developed to HTS assay to screen small chemical SENP inhibitors. The screening conditions were optimized to achieve satisfactory Z' factor value and high signal-to-noise ratio. 55,000 compounds were screened by the developed HTS assay. The general principles of the quantitative FRET analysis in protease kinetics, including the characterization of inhibition, can be applied to other substrate-protease with inhibitors. The FRET-based protease assay as well as the HTS assay provided a powerful tool for large-scale and high-throughput applications.

SUMOylation is one of the most important post-translational modifications, which plays pivotal roles in many physiological processes. SUMOylation is a multi-step enzymatic cascade, which involves multiple protein-protein interactions and regulates protein activity in many aspects. Misregulation of the SUMO pathway has been associated with many types of diseases, including viral infection, tumorigenesis, and neurodegenerative diseases. Thus, SUMOylation has great potential to be a target for the development of novel antiviral and anticancer agents. Förster Resonance Energy Transfer (FRET) is an energy transfer process that occurs between two interacting fluorophores with overlapping spectra, and is widely used to study protein-protein interactions. Previously, our group has developed quantitative FRET assay for determining protein interaction affinity and enzymatic kinetics. Here, my research goal is to further develop quantitative FRET (qFRET) technology into a robust and reliable method for determining biochemical parameters, and to investigate the interplay between SUMOylation and influenza replication. Specifically, I have proven our high-sensitive FRET technology can be used to determine protein interaction affinity even without protein purification, and utilizing this strategy I have for the first time measured the Kd between SUMO E3-PIAS1 with SUMO E2 or SUMO substrate-NS1 which elucidates the mechanism of how SUMO E3 regulates SUMOylation of different substrates. Then, I have developed a systematic method for determining mechanism of enzyme inhibition through our qFRET technology, and characterized the inhibition type and inhibition constant of our newly found SUMOylation inhibitor- STE. We have also identified Lys131 of influenza NS1 protein as the SUMOylated lysine residue which is important for virus replication through an in vitro FRET-based SUMOylation assay. And lastly, I have shown our SUMOylation inhibitor-STE exhibits great inhibition on influenza growth and can serve as a potential new anti-influenza drug. In summary, these findings prove our qFRET technology provides a powerful tool for determining biochemical parameters and dissects the role of SUMOylation in influenza viral life cycle.

Disease progression is often facilitated by protein-protein interaction. Reports of commandeering host protein mechanisms by cancer and viral proteins has armed scientist with novel drug targets. The challenge arises when attempting to quantitatively evaluate the variety of covalent and non-covalent interactions that occur during disease progression. This thesis covers the development of a quantitative Förster resonance energy transfer (qFRET) assay for the evaluation of covalent SUMOylation of viral proteins and non-covalent interaction of programmed cell death 1 (PD1) with programmed cell death ligand 1 (PDL1). The interaction of PD1 and PDL1 results in the negative regulation of T cell immune response and is reported to be used by cancer cells to circumvent the immune checkpoint mechanism. Immune checkpoints inhibitors, that target PD1-PDL1 interaction, have been the focus for cancer therapies in the past decade, but recent development of novel treatments has plateaued. Reported here is a FRET based reporter for the in vitro characterization of the PD1 and PDL1 interaction. The results of the work produced a qFRET based KD of full-length custom codon optimized PD1 and PDL1 of 0.82 µM with standard error of 0.13 µM and determined a qFRET based Ki of PermbrolizumabTM to be 1.14 nM with a 95 % confidence interval of 0.94 to 2.14 nM. The remainder of the work focuses on the development of an in vitro SUMOylation of viral proteins with qFRET as a reporter, in combination with mass spectrometry to identify SUMO1 modified lysine sites. The qFRET assay for the in vitro SUMOylation of IAV-Matrix Protein 1 in tandem with MS, resolved 5 SUMO1 modification. The evaluation of the novel K21 modification proved fatal to the viral pathogenesis, and M1 K242R mutant reported diminished IAV infectivity. The qFRET based in vitro SUMOylation of severe acute respiratory syndrome coronavirus 2 Nucleocapsid protein (SARS-CoV- 2 N) coupled with MS analysis identified 4 novel SUMO modified lysine residues. The evaluation of identified lysine in vitro and in cell mutant studies found lysine 61 and 65 to modulate SUMOylation activity and modulate cellular translocation.

Small Ubiquitin-like Modifier (SUMO) modification is a post-translational modification affecting many cellular processes, including nuclear transport, transcriptional regulation, cell cycle progression, and protein stability. Upregulation of SUMO, as well as other components of the SUMOylation pathway, has been observed in human cancers such as lung adenocarcinomas, anaplastic large-cell lymphoma, hepatocellular carcinoma, and ovarian tumors. Since SUMO modification changes the activity of a large number of proteins, SUMOylation pathway is one of the cancer-supportive cellular machineries responsible for many aspects of abnormal cell functions in cancer including increased survival of cancerous cells. We hypothesized that targeting SUMOylation may decrease cancer cells' viability. Recently, our lab used FRET-based high-throughput screening to identify STE as an inhibitor of the SUMOylation pathway.

To elucidate the mechanism of action of STE, we performed SUMO E1 and E2 thioester formation assays and the enzyme kinetic assays. STE inhibits E1~SUMO thioester conjugate formation in a dose dependent manner and this inhibition is specific to the SUMOylation pathway. We developed a quantitative FRET technology to measure the E1 enzyme kinetics and employed this method to measure the inhibition constant of the STE. We found the results of enzyme kinetics assays using the quantitative FRET methods are comparable to those of conventional radioactive assays. The Km of SUMO2 (3.42 ± 0.91 µM) and SUMO3 (2.76 ± 0.75 µM) are about four to five times higher than the Km of SUMO1 (0.75 ± 0.11 µM). In the presence of STE, the Km for the E1 enzyme is unaltered whereas the Vmax is decreased as STE concentration increases which indicates that STE is a non-competitive inhibitor of the E1 enzyme. Based on these experiments, the inhibition constant of the STE was calculated to be 1.90µM. Our results also indicated that STE induces cell death in the both Non-Small Cell Lung Cancer (NSCLC) cell lines NCI-H358 and NCI-H460 and inhibit cell cycle progression in HEK293.

We identified and characterized STE, a small molecule inhibitor of the E1 enzyme of the SUMOylation pathway that is active in the cells. Due to the diverse roles of SUMO, the identification of selective inhibitors of the SUMOylation pathway may lead to pharmacological tools for cancer treatment.

Cell death is a major process in a biological cell that occurs during development, homeostasis and immune regulation in multicellular organisms. Dysregulation of cell death pathway has been implicated in many diseases. Principle cell death pathways include

apoptosis, autophagy, necrosis, mitotic catastrophe etc. Knowledge of cell death pathways and the reason the cell chooses to die one way or the other, are key factors to understand the disease, the way it affects the cellular system and subsequent drug discovery.

This study is focused on developing genetically encoded Förster Resonance Energy Transfer (FRET) based biosensors to identify apoptosis and autophagy pathways in vitro.

FRET is an energy transfer phenomenon which occurs between two spectrum-overlapping fluorophores that are in close proximity. The design of the sensor is based on enzyme substrate dynamics and consists of a reporter gene fused between fluorescent proteins. Additionally, FRET based protease assay has been used to determine the kinetics of Atg4A, an enzyme involved in autophagy. The kinetic parameters km, kcat, kcat /km were derived using real time detection method. To take this forward, the sensor will be transfected in H460 lung cancer cell line to identify the type of death the cell chooses on treatment with sumoylation inhibitors that were previously developed in the lab. MTS assay was conducted to establish the supremacy of sumoylation inhibitors over other commercially available drugs.

In conclusion, the biosensor developed in this study is highly sensitive in detecting apoptosis and autophagy and can be used to derive quantitative data using FRET technology. It can be used both in vitro and in mammalian cells and can differentiate between apoptosis and autophagy. The results of this study can help to expand biomedical knowledge by illuminating the mechanisms of different cell death pathways. This will pave way for simple and non-invasive ways to modulate cell death pathways for therapeutic intervention in the future.

Influenza, which is also commonly known as "the flu", is an infectious disease caused by an influenza virus. The symptoms of influenza could be mild to severe, including fever, headache, sore throat, feeling tired, headache, coughing, and muscle pains. The influenza virus infects and kills over 100,000 people in the world every year. Due to the fast pace in which influenza virus mutates, the virus itself would develop resistances to the current FDA approved drugs. The influenza non-structural protein (NS1) is related to the SUMOylation pathway, a post-translational modification involved in various cellular processes.

The first part of this thesis is mainly focus on finding out the SUMOylation site of the NS1 influenza protein by using site-directed mutagenesis and Förster Resonance Energy Transfer (FRET) based technology. Since the SUMO protein is conjugated to the lysine residue of the NS1 protein, by mutate each lysine to Alanine and using FRET-based SUMOylation assay to test every mutants of NS1, the Lysine residue K131 was founded to be the SUMOylation site of NS1. This result was further proved by Plaque assay, which showed the mutation of Lysine 131 would affect the viral replication in MDCK cells. Identification of essential SUMOylation site(s) of NS1 can provide supporting evidence to develop new medicine for treating Influenza virus.

The second part of this thesis is focus on designing a new strategy for fluorescence protein expression. Nowadays, using E.coli bacteria as the host for protein production is a very common strategy. However, there are several challenges that needs to be overcome by using E.coli protein production, such as the degradation of the target protein, the toxic of protein towards the host, the misfolding and instability of the protein, and the complex purification procedures. By expression the proteins in inclusion bodies (IBs) would help alleviating these problems. This thesis conducted a signal sequence TMAO-reductase (ssTorA) to drive the target protein Ypet-Uba2, into the inclusion bodies and overcame the degradation of the protein. Also, a buffer screening method was designed to purify the protein from inclusion bodies. As a result, trehalose was proved to be a high efficient additive for the Ypet-Uba2 protein purification.

SUMOylation is an important post-translational protein modification mechanism which plays important roles in a variety of biological processes. Conjugation and deconjugation of SUMO is a cascade event requiring multiple protein-protein interactions between SUMO, SUMO E1-E3 enzymes and SUMO substrates. The nature of the SUMOylation network indicates a great potential for small chemical inhibitors to be used in the investigation and manipulation of this important pathway. FRET is an energy transfer phenomenon which occurs between two spectrum-overlapping fluorophores in close proximity. The efficiency of FRET is highly dependent on the distance between the donor and acceptor fluorophores, which makes FRET very powerful in the detection of molecular interactions. The goal of my research is to develop FRET-based techniques to study protein-protein interactions in the SUMOylation network and to identify bioactive chemical compounds which can inhibit the activity of this pathway. Using engineered fluorescent proteins to modify protein components involved in the SUMOylation pathway, I set up a FRET-based assay and developed mathematical algorithms to determine the binding affinity of non-covalent protein-protein interactions. I then developed this FRET-based assay into high-throughput screening platforms to look for small chemical inhibitors which can disrupt the interaction of SUMO1 and its E2 enzyme, Ubc9. I further used FRET-based strategy to set up a whole-pathway screening assay to identify SUMOylation/deSUMOylation inhibitors. Over 200,000 compounds were screened and the positive hits were characterized for their activities in vitro and in cells. Finally, I was able to use FRET to detect the formation of intermediate protein complex during SUMOylation reactions, and used the assay to map the targets of the SUMOylation inhibitors identified from the high-throughput screening. These FRET-based applications provide powerful tools for the study of the SUMOylation pathway and can be used in the investigation of protein-protein interactions in other biological pathways.