Jiayu Liao

Quantitative Förster resonance energy transfer analysis for kinetic determinations of SUMO-specific protease.

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and it is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO [small ubiquitin-like modifier] conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. Here, we report a novel development of a quantitative FRET-based protease assay for SENP1 kinetic parameter determination. The assay is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor (CyPet-SUMO1) emission, acceptor (YPet) emission, and FRET signal during the digestion process. Subsequently, we developed novel theoretical and experimental procedures to determine the kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP1 toward pre-SUMO1. Importantly, the general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases.

Chemoselective fabrication of high density peptide microarray by hetero-bifunctional tetra(ethylene glycol) linker for click chemistry conjugation †

A hetero-bifunctional tetra(ethylene glycol) molecule with silane and azide termini was synthesized, and this molecule was used to prepare azide-derivatized glass surface in one step. The resulting glass surface was available for fabricating peptide microarray by the conjugation with alkyne-containing peptide using click chemistry, which proceeded to the completion at low temperature and in aqueous solution. A high density of peptide on the surface was achieved due to concise overall procedure and highly efficient conjugation reaction. Immobilized peptides were highly bio-functional on the surface, as demonstrated by the ability to detect protease activity. Due to the biologically orthogonal manner of conjugation, peptide conjugated by site-specific immobilization was more accessible by protease than that conjugated by random amide conjugation. This site-specific and high efficient immobilization technique could be expanded to large scale development of biocompatible peptide and protein arrays for use in various applications.

Internal Calibration Förster Resonance Energy Transfer Assay: A Real-Time Approach for Determining Protease Kinetics

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research. This powerful tool can elucidate protein interactions in either a dynamic or steady state. We recently developed a series of FRET-based technologies to determine protein interaction dissociation constant and for use in high-throughput screening assays of SUMOylation. SUMO (small ubiquitin-like modifier) is conjugated to substrates through an enzymatic cascade. This important posttranslational protein modification is critical for multiple biological processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate SUMO from its substrate. Here, we describe a novel quantitative FRET-based protease assay for determining the kinetics of SENP1. Our strategy is based on the quantitative analysis and differentiation of fluorescent emission signals at the FRET acceptor emission wavelengths. Those fluorescent emission signals consist of three components: the FRET signal and the fluorescent emissions of donor (CyPet) and acceptor (YPet). Unlike our previous method in which donor and acceptor direct emissions were excluded by standard curves, the three fluorescent emissions were determined quantitatively during the SENP digestion process from onesample. New mathematical algorithms were developed to determine digested substrate concentrations directly from the FRET signal and donor/acceptor direct emissions. The kinetic parameters, kcat, KM, and catalytic efficiency (kcat/KM) of SENP1 catalytic domain for pre-SUMO1/2/3 were derived. Importantly, the general principles of this new quantitative methodology of FRET-based protease kinetic determinations can be applied to other proteases in a robust and systems biology approach.

Quantitative analysis of FRET assay in biology—new developments in protein interaction affinity and protease kinetics determinations in the SUMOylation cascade

Förster resonance energy transfer (FRET) techniques have been widely used in biological studies in vitro and in vivo and are powerful tools for elucidating protein interactions in many regulatory cascades. FRET occurs between oscillating dipoles of two fluorophores with overlapping emission and excitation wavelengths and is dependent on the spectroscopic and geometric properties of the donor-acceptor pair. Various efforts have been made to develop quantitative FRET methods to accurately determine the interaction affinity and kinetics parameters. SUMOylation is an important post-translational protein modification with key roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENP) act as endopeptidases to process the pre-SUMO or an isopeptidase to deconjugate SUMO from its substrate. Here we also summarize recent developments of theoretical and experimental procedures for determining the protein interaction dissociation constant, Kd, and protease kinetics parameters, kcat and Km, in the SUMOylation pathway. The general principles of these quantitative FRET-based measurements can be applied to other protein interactions and proteases.

β-Deuterium Isotope Effects on Firefly Luciferase Bioluminescence

Abstract A 5,5‐d2‐luciferin was prepared to measure isotope effects on reactions of two intermediates in firefly bioluminescence: emission by oxyluciferin and elimination of a putative luciferyl adenylate hydroperoxide to dehydroluciferin. A negligible isotope effect on bioluminescence provides further support for the belief that the emitting species is the keto‐phenolate of oxyluciferin and rules out its excited‐state tautomerization, one potential contribution to a bioluminescence quantum yield less than unity. A small isotope effect on dehydroluciferin formation supports a single‐electron‐transfer mechanism for reaction of the luciferyl adenylate enolate with oxygen to form the hydroperoxide or dehydroluciferin. Partitioning between the dioxetanone intermediate (en route to oxyluciferin) and dehydroluciferin is determined, not by the fate of the hydroperoxide, but by that of the radical formed from luciferyl adenylate, and the kinetic isotope effect (KIE) reflects H‐atom abstraction by superoxide.

A linker strategy for trans-FRET assay to determine activation intermediate of NEDDylation cascade.

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a valuable tool for elucidating molecular interactions in vitro and in vivo. Quantitative FRET analysis is a powerful method for determining biochemical parameters and molecular distances at nanometer levels. Recently, we reported theoretical developments and experimental procedures for determining the dissociation constant, Kd and enzymatic kinetics parameters, Kcat and KM, of protein interactions with the engineered FRET pair, CyPet and YPet. The strong FRET signal from this pair made these developments possible. However, the direct link of fluorescent proteins with proteins of interests may interfere with the folding of some fusion proteins. Here, we report a new protein engineering strategy for improving FRET signals by adding a linker between the fluorescent protein and the targeted protein. This improvement allowed us to follow the covalent conjugation of NEDD8 to its E2 ligase in the presence of E1 and ATP, which was difficult to determine without linker. Three linkers, LAEAAAKEAA, TSGSPGLQEFGT, and LAAALAAA, which are alpha helix or random coil, all significantly improved the FRET signals. Our results show a general methodology for improving trans‐FRET signals to effectively determine biochemical reaction intermediates. Biotechnol. Bioeng. 2014;111: 1288–1295.

Determination of SUMO1 and ATP affinity for the SUMO E1by quantitative FRET technology.

SUMOylation plays important roles in many key physiological and pathological processes. The SUMOylation cascade involves a heterodimer of activating enzyme, E1 (Aos1/Uba2); a conjugating enzyme, E2 (Ubc9); and many ligase enzymes, E3. Focusing on the activation step of the SUMOylation process, we examined the interaction of E1 with its substrates. Previous studies reported the Km of E1 enzymes in ubiquitin and other ubiquitin‐like pathways, but the Km of the SUMO paralogs (SUMO2 and SUMO3) is unknown. Here, by using quantitative FRET to measure the SUMO E1 enzyme kinetics of SUMO1, 2, and 3 and ATP under steady state conditions, we found that the enzyme kinetics from the quantitative FRET method are comparable to those from conventional radioactive assays. Additionally, the kinetic constants, Km, of SUMO2 (3.418 ± 0.9131 μM) and SUMO3 (2.764 ± 0.75 μM) [FW1] are approximately four to five times higher than that of SUMO1 Km (0.7458 ± 0.1105 μM). These results demonstrate the advantages of FRET technology for determining Km, including the ability to monitor reaction progress in real‐time with high‐throughput and high‐sensitivity in an environmentally friendly manner. The processes discussed here extend the utility of quantitative FRET in characterizing protein–protein interactions and enzyme kinetics. Biotechnol. Bioeng. 2015;112: 652–658.

Dissecting Distinct Roles of NEDDylation E1 Ligase Heterodimer APPBP1 and UBA3 Reveals Potential Evolution Process for Activation of Ubiquitin-related Pathways

Despite the similar enzyme cascade in the Ubiquitin and Ubiquitin-like peptide(Ubl) conjugation, the involvement of single or heterodimer E1 activating enzyme has been a mystery. Here, by using a quantitative Förster Resonance Energy Transfer (FRET) technology, aided with Analysis of Electrostatic Similarities Of Proteins (AESOP) computational framework, we elucidate in detail the functional properties of each subunit of the E1 heterodimer activating-enzyme for NEDD8, UBA3 and APPBP1. In contrast to SUMO activation, which requires both subunits of its E1 heterodimer AOS1-Uba2 for its activation, NEDD8 activation requires only one of two E1 subunits, UBA3. The other subunit, APPBP1, only contributes by accelerating the activation reaction rate. This discovery implies that APPBP1 functions mainly as a scaffold protein to enhance molecular interactions and facilitate catalytic reaction. These findings for the first time reveal critical new mechanisms and a potential evolutionary pathway for Ubl activations. Furthermore, this quantitative FRET approach can be used for other general biochemical pathway analysis in a dynamic mode.

Abstract 21: Quantitative FRET technology for SUMOylation cascade and high-throughput screening assay for SUMOylation inhibitor in cancer drug discovery

The ubiquitin–proteasome system and ubiquitin-like protein pathways, such as SUMOylation, are critical in protein homeostasis and activities in vivo and are emerging as a new strategy to treat many acute and chronic human diseases, such as cancers. Although various kinase inhibitors have been developed as target-based therapy, solid tumors are still challenges in clinical therapy because various resistant are developed after kinase inhibitor treatments, and therapeutic agents with novel mechanisms are urgently needed. SUMO has been shown to modify various critical proteins, such as p53, MDM2, Estrogen receptor and androgen receptors. More recently, a genome-wide siRNA screening shown that inhibition of SUMO E1 ligases can lead to synergistically lethality of c-Myc overexpressed breast cancer cells. However, so far, specific inhibitor of SUMOylation is still not available for the community. Fig. 1. SUMOylation in human diseases and quantitative systems biology approach for basic and translational research of SUMOylation. We developed a novel quantitative Forster resonance energy transfer (FRET) technology platform for both basic kinetics parameter determinations and high-through screening(HTS) assays for SUMOylation cascade. The novel theoretical and experimental procedures for protein interactions affinity(Kd) determinations in the SUMOylation cascade, including the interaction between SUMO1 and its E2 ligase, Ubc9, E1 heterodimers(Aos1 and Uba2), E1 and E2 interactions(Uba2 and Ubc9), and E2 and substrate interactions(Ubc9 and RanGap1c) and protease kinetics, Kcat/KM of SENP1 endopeptidase activity have been developed in a systems biology manner. The data are in good agreement with traditional methods. Multiple FRET-based HTS assays have also been developed and HTS campaigns have led to very promising hit that can preferentially kill Non-small cell lung cancer cells. The novel SUMOylation inhibitor can be used for cancer treatments by synergistically lethality strategy. Citation Format: Jiayu Liao, Hilda Wiryawan, Yang Li, Yang Song, Yan Liu, Jiacong You, Ling Jiang, Harbani Kaur Malik, Amanda N. Saavedra, Sophie Qu. Quantitative FRET technology for SUMOylation cascade and high-throughput screening assay for SUMOylation inhibitor in cancer drug discovery. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 21. doi:10.1158/1538-7445.CANSUSC14-21

Abstract A39: Quantitative FRET technology for SUMOylation cascade and high-throughput screening assay for SUMOylation inhibitor in cancer drug discovery

The ubiquitin—proteasome system and ubiquitin-like protein pathways, such as SUMOylation, are critical in protein homeostasis and activities in vivo and are emerging as a new strategy to treat many acute and chronic human diseases, such as cancers. Although various kinase inhibitors have been developed as target-based therapy, solid tumors are still challenges in clinical therapy because various resistant are developed after kinase inhibitor treatments, and therapeutic agents with novel mechanisms are urgently needed. SUMO has been shown to modify various critical proteins, such as p53, MDM2, Estrogen receptor and androgen receptors. More recently, a genome-wide siRNA screening shown that inhibition of SUMO E1 ligases can lead to synergistically lethality of c-Myc overexpressed breast cancer cells. However, so far, specific inhibitor of SUMOylation is still not available for the scientific or pharmaceutical communities. We developed a novel quantitative Forster resonance energy transfer (FRET) technology platform for both basic kinetics parameter determinations and high-through screening (HTS) assays for SUMOylation cascade. The novel theoretical and experimental procedures for protein interactions affinity (Kd) determinations in the SUMOylation cascade, including the interaction between SUMO1 and its E2 ligase, Ubc9, E1 heterodimers(Aos1 and Uba2), E1 and E2 interactions(Uba2 and Ubc9), and E2 and substrate interactions(Ubc9 and RanGap1c) and protease kinetics, Kcat/KM of SENP1 endopeptidase activity have been developed in a systems biology manner. The data are in good agreement with traditional methods. Multiple FRET-based HTS assays have also been developed and HTS campaigns have led to very promising hit that can preferentially kill Non-small cell lung cancer cells. The novel SUMOylation inhibitor can be used for cancer treatments by synergistically lethality strategy. Note: This abstract was not presented at the conference. Citation Format: Jiayu Liao, Hilda Wiryawan, Yang Song, Yan Liu, Ling Jiang, Harbani Kaur Malik, Amanda N. Saaredra. Quantitative FRET technology for SUMOylation cascade and high-throughput screening assay for SUMOylation inhibitor in cancer drug discovery. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr A39.