B.Sc. CUHK; Ph.D. UBC,Canada
Chong Yuet Ming Chemistry Building
The University of Hong Kong
|Tel: (852) 2859 7915
Determine the structures and functions of biologically important molecules using NMR and other physical methods
NMR spectroscopy and other physical methods (CD, mass spectrometry, etc.) will be used to investigate the structures, dynamics, functions and interactions of biologically or clinically important biomolecules and their binding ligands. Such studies are not only essential for elucidating structure-function relationship and better understanding of the underlying mechanisms of action for these biomolecules, but also necessary for engineering better and more potent drugs. Existing programs included:
| Improve the
capability and efficiency for determining high-resolution structures by
This work involves the development of new NMR pulse sequences and computer software for the rapid and automatic analysis of biomolecule structures from NMR based on current structural modeling methods. Significant progress in this area includes the development of computer programs for automated analysis of NMR data for biomolecules, and the development of improved NMR pulse sequences for structural analysis. The results from this work will enable the development of high throughput technologies, i.e. to build a technical platform suitable for determining many new structures of biomolecules for the structural genomics and proteomics project. Existing programs included:
studies of the SWIRM domain in Lysine-Specific
Although the recent discovery of the first lysine demethylase LSD1 by Shi et al. (2004) represents a major breakthrough in chromatin biology, it also raises many questions. For example, how can the regulatory complexity of histone demethylation be accomplished with only ten LSD1-related proteins (Kubicek and Jenuwein, 2004)? For comparison, the corresponding regulation of histone methylation requires over 70 proteins that contain the specific motif (SET domain) responsible for methylation. Three recent papers demonstrate that the specificity and activity of LSD1 is indeed modulated by physical association with specific cofactors, including androgen receptor (AR), CoREST, and BHC80 (Lee et al., 2005; Metzger et al., 2005; Shi et al., 2005). This also reinforces the general view that histone methylation is a dynamic process and a reversible modification with far-reaching implications for human disease.
Although most research has focused on the role of histone modifications, such as acetylation and methylation in gene regulation and other epigenetic phenomena, there is increasing recognition that histone modifications are an important component of human disease, notably cancer (Ayton et al., 2001). A recent study reported the potential application of global changes in specific histone acetylation and methylation marks as predictors of clinical outcome for certain low-grade prostate cancers (Seligson et al., 2005). The importance of lysine methylation in human disease is also underscored by the wide range of different methyltransferases that correlate with carcinogenesis when mutated (Schneider et al., 2002; Hamamoto et al., 2004).
LSD1 contains a carboxyl-terminal amine oxidase domain and a centrally located SWIRM domain, which is a putative protein-protein interaction motif composed of 102 residues. In order to characterize the structure and function of SWIRM domain in LSD1, we propose to address the following objectives in this project:
|2.||Structural and Functional
Studies of the Kringle 1 domain of hepatocyte growth factor
(HGFK1) by NMR Spectroscopy
Hepatocyte growth factor (HGF) (1-7), also known as scatter factor (SF), is a vertebrate-specific polypeptide growth factor that plays an important role in complex biological processes such as embryogenesis, tissue regeneration, cancerogenesis and angiogenesis. HGF promotes cell proliferation, survival, migration and morphogenesis of endothelial cells (EC) through binding to tyrosine kinase MET receptor. Interest in HGF/SF and its receptor MET (8) stems from their unique biological roles in embryogenesis, tissue regeneration, and cancer (9¡V14). These activities have led to a strong interest in the structure of the molecules as this knowledge may underpin the development of MET-based therapeutics.
HGF consists of six domains: an N-terminal domain (N), four copies of the kringle domain (K1¡VK4), and a C-terminal domain (sp) structurally related to the catalytic domain of serine proteinases. The factor is synthesized as a precursor protein of single-chain HGF (sc-HGF) and is proteolytically processed to a two-chain form (tc-HGF) by cleavage of the linker at a trypsin-like site connecting the K4 and sp domains by hepatocyte growth factor activator (HGFA). Sc-HGF binds MET (15, 16) but is unable to induce biological responses. MET consists of an N-terminal sema domain, which is responsible for ligand binding, and four copies of Ig-like domains. The sema and Ig-like domains are joined by cystine-rich domain (cr). MET receptor is initially synthesized as a partially glycosylated single-chain intracellular precursor. The precursor is subsequently cleaved by furin, yielding an extracellular α-chain and a β-chain which spans the membrane.
The structural basis of the conversion of single-chain to two-chain HGF is unknown. The whole picture of HGF-induced MET signaling is unclear until the overall architecture of HGF and MET are obtained by cryo-EM and SAXS recently (17). Interestingly, the low resolution structures of single-chain HGF precursor (sc-HGF) and two-chain HGF (tc-HGF) reviewed by cryo-EM differ markedly, where sc-HGF appears as ring-shaped, closed structure, while tc-HGF appears as elongated, open conformation. HGF is in equilibrium between the closed and elongated conformation. In the presence of MET receptor, the equilibrium shifts toward the open form (i.e. tc-HGF). The receptor binding sites of HGF are located in K1 and sp domains. For sc-HGF, the receptor binding sites are too close to each other, thus sc-HGF is unable to bind MET due to steric hindrance. In contrast, the elongated form of tc-HGF allows it to wrap around the α-chain of MET928 (full-length MET) in form of a monomeric 1:1 complex. The N and K1 domains bind the ¡¥a¡¦ face of MET928. K2 and K3 domains cross the side face, leaving K4 on top of sp domain which binds on the ¡¥b¡¦ face of MET928.
The soluble recombinant protein Kringle 1 domain of human hepatocyte growth factor (HGFK1), which includes 88 residues from 127-214 of HGF, has been reported to inhibit the proliferation of bovine aortic endothelial (BAE) cell stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner (18). It has been shown that the recombinant HGFK1 protein is a much more effective anti-angiogeneis molecule than angiostatin in vitro in cell culture system (18).
We have recently evaluated the utility of a recombinant Adeno-associated virus carrying the HGFK1 gene in treating hepatocellular carcinoma (HCC), a highly metastatic cancer. Hepatocellular carcinoma (HCC) is a hypervascular tumor associated with a poor prognosis and a lack of effective treatments. Consequently, identifying novel therapeutic strategies are urgently needed. We constructed a recombinant adeno-associated virus carrying the HGFK1 (the kringle 1 domain of human hepatocyte growth factor) gene (rAAV-HGFK1) and administered it to a syngenic orthotopic rat model of HCC by intra-tumoral and intra-portal injections.
Results showed that rAAV-HGFK1 inhibited tumor growth, decreased microvessel density in the tumor, and completely prevented intra-hepatic, lung, and peritoneal metastasis. rAAV-HGFK1 was able to mediate long-term expression of HGFK1 and markedly prolong the median survival time of HCC-bearing rats. Moreover, high doses of rAAV-HGFK1 were not toxic to the animal. In vitro experiments further demonstrated that rAAV-HGFK1 affected mice microvessel endothelial cells (EC) by inducing apoptosis and inhibiting EC proliferation and tube formation. Furthermore, rAAV-HGFK1 exerted its effect by a mechanism entirely different from that previously reported for endostatin. It induced EC apoptosis via the up-regulation of β-amyloid binding protein (BBP), as knocking down BBP gene expression prevented rAAV-HGFK1 induced EC apoptosis. In conclusion, this study demonstrates that rAAV-HGFK1 anti-angiogenic gene therapy is a novel and promising approach for the treatment of HCC, and that the anti-angiogenic effect of HGFK1 is dependent on the BBP signal apoptotic transduction pathway.
We further showed that HGFK1 is essential and sufficient to induce growth signals transduced by HGF, EGF, and VEGF. Since HGFK1 is a fragment of HGF, which potentially has an oncogenic effect, the therapeutic efficacy, possible hepatic cytotoxicity, and the molecular mechanism of action requires careful evaluation. In particular, we aim to:.
|3.|| Structural and functional
studies of human X-linked Inhibitor of Apoptosis Protein-Associated
factor 1 (XAF1) by NMR Spectroscopy
Apoptosis, referring to the biological process of programmed cell death, plays an essential role in developmental and cellular homeostasis of mammalian cells (1). Dysfunction of the process leads to a number of human pathologies, including cancer, autoimmune diseases, and neurodegenerative disorders (2-6). The molecular pathway of apoptosis is evolutionarily conserved and the initiation, execution, and regulation of apotosis pathway are governed by hundreds of genes. The process culminates in the activation of caspases cascade to degrade the cellular machinery (e.g. Chromosome) (7,8). Research in the past two decades has led to the identification of several caspases-inhibiting proteins. Deregulation of these proteins may confer apoptosis resistance and results in low sensitivity of cancer cell to therapeutic agents (1).
The X-linked Inhibitor of Apoptosis (XIAP), belonging to the family of intrinsic inhibitor apoptosis protein (IAP), is a newly discovered key caspases-inhibiting protein (9). The XIAP was demonstrated to be an endogenous repressor, which blocks the activity of the caspases in the terminal part of the cascade in vitro (9). In turns, the caspases-inhibiting activity of the XIAP is negatively controlled by two XIAP-binding proteins, namely Smac/DIABLO (10-15) and XAF1 (XIAP-associated factor 1)(16).
In contrast to Smac/DIABLO, the structural and biochemical basis of XAF1 remain to be determined. It was reported that XAF1, which antagonizes recombinant XIAP in vitro, normally reside in the nucleus of the cell. Overexpression of XAF1 was shown to trigger the redistribution of cytoplamic XIAP to the nucleus, leading to XIAP-suppression, caspases activation and apoptosis (9). On the other hands, the loss of endogenous XAF1, by utilizing the adenovirus infection of antisense XAF1 mRNA, was reported to enhance cellular resistance to apoptosis (16). It is believed that XAF1 was a putative tumor suppressor gene (17).
XAF1 is ubiquitously expressed in normal tissue, but present at low level or undetectable levels in nucleus and cytoplasm of different cancer cell lines (18). It was recently showed that the aberrant reduction of XAF1 transcription, but not Smac/DIABLO, in gastric adenocarcinomas might be attributed to the hypermethylation of seven CpGs in the promoter region of XAF1 gene locus (17). A low concentration of cellular XAF1 transcription was also observed in human colorectal cancer (19). In addition to the tumor suppressing behavior, XAF1 was reported to enhance neuronal susceptibility toward degeneration in reperfusion injury after ischemia (20). Nevertheless, the pathophysiological mechanism of XAF1 is still not clear.
XAF1 is a 37kDa nuclear protein, comprising of 317 amino acids and having two mRNA spicing variants (16). The domain architecture and structure of XAF1 is unknown. Sequencing analysis of the nuclear protein revealed the existence of TRAF zinc finger within H23-E99. Overexpression of the Zinc-finger portion blocked INF (Interferon)-dependent sensitization of A375 melanoma cell to the pro-apoptotic effect of TRAIL, which is a tumor necrosis factor related apoptosis inducing ligand(21). The domains responsible for XIAP binding and compartmental translocation are yet to be identified.
The objectives of this research are:
Studies of Thermophilic Acylphosphatase (ACP)
Thermophilic enzymes adapted to perform catalysis at elevated temperatures are often sluggish enzymes at lower temperatures, when comparing to mesophilic homologues. Reduced flexibility is often regarded as the culprit behind the reduced catalytic efficiency of thermophilic enzymes. We propose to use an acylphosphatase from Pyrococcus horikoshii (PhAcP) as a model system to study the stability, activity, and flexibility relationships of enzymes. Acylphosphatase (AcP, ~90-100 residues) is one of the smallest enzymes known. We have recently solved the crystal structure of PhAcP, and measured its stability and kinetics parameters. We have found that while PhAcP is extremely stable, with a melting temperature of ~112¢XC, and a free energy of unfolding of ~54 kJ/mol. Kinetics studies showed that PhAcP is a less efficient enzyme than other mesophilic AcP, for its kcat value of ~95 s-1 is much lower than the value of ~1500 s-1 reported for mesophilic AcP. As there is no structural difference between the active site of PhAcP and mesophilic AcP, we hypothesize that the reduced activity of PhAcP is due to reduced flexibility of the active site. We will determine the structure of PhAcP complexed with substrate-analogs by crystallography or NMR spectroscopy. The protein dynamics of PhAcP and a mesophilic AcP will be characterized by NMR relaxation experiments. We will compare protein dynamics, stability and activity to find out if there is any correlation among them. We will create mutants designed to perturb flexibility or stability of PhAcP. If reduced flexibility and catalytic efficiency are necessary consequences of enhanced stability, we should be able to observe a correlation among stability, activity and flexibility.
|| Structure and
Studies of Antimicrobial peptides: Loloatins
Loloatin B (LoB) is a cyclic decapeptide originally isolated from the laboratory cultures of a tropical marine bacterium collected from the Great Barrier Reef at the southern coast of Papua New Guinea. Loloatin family contains four analogs Loloatin A, B, C, and D (1). Loloatin B, the most abundant element of the four analogs, inhibits the growth of methicillin resistant Staphylococcus aureus, vancomycin resistant Enterococcus sp, and penicillin resistant Streptococcus pneumoniae and some other gram positive bacteria with MICs of 1-2μg /ml (2), which are at least comparable to the antimicrobial potency of tyrocidine C, the most potent antibiotic in tyrocidine family. Loloatins can be structurally classified as a family of antimicrobial cyclic decapeptides composed of gramicidin S (3), tyrocidines (4), and the recently isolated streptocidins (5). This class of natural peptide product is broad-spectrum antibiotics with high potency. The small size and structural simplicity of these peptides make them attractive targets for drug developments (6). In order to investigate the functional importance of the D amino acids, we have synthesized the all-L LoB by diastereomeric substitutions of the two D residues with two L residues. Unlike natural LoB, which exhibits a broad spectrum of antimicrobial activities, all-L LoB was found to possess no antimicrobial activity. Since both diastereomers have the same chemical composition, sequence, and intrinsic hydrophobicity, their difference in antibiotic potency is likely attributed to conformational and structural variations. In the present study, NMR spectroscopy, one of the best methods for determining the solution structures of small peptides, is used to depict the 3D solution conformations of natural LoB and all-L LoB peptides to elucidate the structural changes induced by introduction of diastereomeric substitutions. The characterization, at the molecular level, the detail conformations as well as the charges and amphipathicity dispersions of these peptides is essential for the development of a structure-function profile for this potential new class of antimicrobial therapeutic agents.
Perturbation Method for Mapping Protein-Protein Interaction Sites
Currently, most nuclear magnetic resonance (NMR) studies on biomolecules rely on measurements of NMR signals originated directly from the biomolecules or their interacting ligands. In living systems, all biological macromolecules are essentially surrounded by a hydration shell of water molecules. Water-related phenomena are occurring in restricted geometries in cells, and at active sites of proteins and membranes or at their surfaces. Therefore, water is not only playing a major role in the stability and function of biological macromolecules but also can be utilized as a useful probe for studying these biological systems. Recently, an elegant NMR method, SEA-TROSY (Solvent Exposed Amides with TROSY), based on magnetization transfer from the bulk water to selective interacting solvent-exposed amide protons, is developed. We have involved in the development of Clean SEA-HSQC to extend the application of SEA-type experiments to more commonly used and less costly non-deuterated NMR samples. This proposal represents our continuous efforts in this emerging new field of development of NMR methods based on SEA-TROSY and Clean SEA-HSQC experiments and their applications on proteins in the following area:
Kringle 1 Domain of
Hepatocyte Growth Factor Has Antiangiogenic
and Antitumor Cell Effects on Hepatocellular Carcinoma”, Zan Shen, Zhen
Fan Yang, Yi Gao, Cheng Li, Hai Xiao Chen, Ching Chiu Liu, Ronnie T.P.
Poon, Sheung Tat Fan, John M. Luk, Kong Hung Sze, Tsai Ping Li, Ren Bao
Gan, Ming Liang He, Hsiang Fu Kung, and Marie C.M. Lin, Cancer
Research, (2008) 68: 404-414.
protein reveals the role of the central inactivation Structure-function
study of maize loop and shows the active site pocket is too small to
accommodate a back-up negative charged glutamate residue”. Mak, A.N.S.,
Wong, Y.T., An, Y.J., Cha, S.S., Sze, K.H., Au, S.W.N., Wong, K.B. and
Shaw, P.C. (2007) Prot. Sci. 16(s1): 203-204.
Stereochemistry of 2-Succinyl-5-Enolpyruvyl-6-Hydroxy-3-Cyclohexene-1-
Carboxylic Acid (SEPHCHC), a Key Intermediate in Menaquinone Biosynthesis" Guo, Zhihong; Jiang, Ming; Chen, Minjiao; Cao, Yang; Yang, Yinhua; Sze, Kong Hung; Chen, Xiaolei, Organic Letters (2007) 9 (23): 4765-4767 NOV 8 2007.
|4.||“1H, 13C and
and side chain resonance assignments of a 28 kDa Active Mutant of Maize
Ribosome-Inactivating protein (MOD)”, Yinhua Yang, Amanda Nga-Sze Mak,
Pang-Chui Shaw, Kong Hung Sze, Biomol. NMR Assignments 1 (2007) 187-189.
Cyclase-Activating Polypeptide (PACAP) as a Growth Hormone (GH)
-Releasing Factor in Grass Carp: II. Solution Structure of a
Brain-Specific PACAP by Nuclear Magnetic Resonance Spectroscopy and
Functional Studies on GH Release and Gene Expression”, Kong Hung Sze,
Hong Zhou, Yinhua Yang, Mulan He, Yonghua Jiang and Anderson O. L.
Wong, Endocrinology 148(10):5042-5059, October 2007.
of maize ribosome-inactivating protein: implications for the internal
inactivation region and the sole glutamate in the active site”, Mak,
Amanda; Wong, Yuen-Ting; An, Young-Jun; Cha, Sun-Shin; Sze, Kong-Hung;
Au, Shannon; Wong, Kam-Bo; Shaw, Pang-Chui, Nucleic Acids Research, 35
trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk
protein P2 by chemical shift perturbation and mutagenesis analyses”,
Denise S.B. Chan, Lai-On Chu, Ka-Ming Lee, Priscilla H.M. Too, Kit-Wan
Ma, Kong-Hung Sze, Guang Zhu, Pang-Chui Shaw, and Kam-Bo Wong, Nucleic
Acids Research, 35 (2007) 1660-1672.
studies of the
small subunit of EcoHK31I DNA methyltransferase”, Wai-To Fung,
Kong-Hung Sze, Kai-Fai Lee and Pang-Chui Shaw, Biological
Chemistry.387 (2006) 507-513.
Dimerization Behavior of Quadruple Hydrogen
Bond-Containing Oligoether Dendrons”, Chun-Ho Wong, Hak-Fun Chow,
Sin-Kam Hui, Kong-Hung Sze, Organic Letters 8 (2006) 1811-1814.
and dynamics of
metallothionein-3 (MT-3)”, by Wang H., Zheng Q., Cai B., Li H., Sze
K.H., Huang Z.X., Wu H.M. and Sun H, FEBS Letters 580, (2006), 795-800
structure note - Solution structure of
a bacterial BolA-like protein XC975 from a plant pathogen Xanthomonas
campestris pv. campestris", by KH Chin , FY Lin , YC Hu , KH Sze , PC
Lyu , SH Chou , J. Biomol NMR 31 (2005), 167-172.
Loloatin C by NMR Spectroscopy and Molecular Simulation", by H. Chen,
R.K. Haynes, J. Scherkenbeck, K.H. Sze and G. Zhu, Eur. J.
Org. Chem. 2004 (2004), 31-37.
structure of the
domain of the ciliary neurotrophic factor (CNTF) receptor and ligand
free associations among components of the CNTF receptor complex", by
Man D, He W, Sze KH, Gong K, Smith DK, Zhu G, Ip NY, J. Biol. Chem. 278
3D and 4D
15N/15N Separated NOESY Experiments of 15N Labeled Proteins", by Y.L.
Xia, K.H. Sze and G. Zhu, J. Biomol. NMR, 18 (2000) 261-268.
Measurement of the
pKa of Aspartic
Acid 26 in Lactobacillus casei Dihydrofolate Reductacse: Implications
for the Catalytic Mechanism", by M.G. Cassarotto, J. Basran, R. Badii,
K.H. Sze and G.C.K. Roberts, Biochemistry, 38 (1999) 8038-8044.
Sensitive 3D J-Resolved
Experiment for Spectral Assignment and Measurement of Long-Range
Heteronuclear Coupling Constants", by K.H. Sze, X.Z. Yan, X.M. Kong, C.
Che and G. Zhu, Tetrahedron Letters, 40 (1999) 5587-5591.
Relaxation Optimized NMR Spectroscopy", by G. Zhu, X.M. Kong, X.Z. Yan
& K.H. Sze, Angew. Chem. Int. Ed. Engl., 37 (1998) 2859-2861.
(Selected as Hot Paper)
conformation of coenzyme A bound to
chloramphenicol acetyltransferase determined by transferred NOE
experiments", by I.L. Barsukov, L.Y. Lian, J. Ellis, K.H. Sze,
W.V. Shaw and G.C.K. Roberts, J. Mol. Biol 262 (1996) 543-558.
Volumes in Multi-Demensional Spectra by Non-linear Least-Squares
Curve-Fitting", by K.H. Sze, I.L. Barsukov and G.C.K. Roberts, J. Magn.
Reson., Series A, 113 (1995) 185-195.
Processes and the Determination of Ligand Conformation and
Protein-Ligand Contacts", by L.Y. Lian, I.L. Barsukov, M.J.
Sutcliffe, K.H. Sze and G.C.K. Roberts, "Methods in Enzymology",
V. 239, Ed. James Oppenheimer, Academic Press, 1994, p657-701.