Day 3 :
- Track 11: Structural Biology in Cancer Research
Gordon Life Science Institute, Boston, USA
Guo-Ping Zhou is currently a Distinguished Professor of Gordon Life Science Institute, USA. He is also an Adjunct Professor of several academics in the United States and China. Dr. Zhou received his Ph.D in Biophysics from University of California at Davis, and completed his postdoctoral training at Stanford University and Harvard University, respectively. Dr. Zhou determined the 3D NMR structures of some important proteins, protein-DNA complexes, and super lipids. He has successfully introduced the elegant wenxiang diagrams to elucidate the biological mechanisms of protein-protein/ligands interactions observed by NMR. Meanwhile, he has also published many papers in bioinformatics, and edited some special issues on structural biology for several influential scientific journals.
The α2,8-sialyltransferase (ST8Sia) family consists of 6 sia-lytranseferases, which are related to forms of polysialic acid chains (PSA) on neural cell adhesion molecule (NCAM) and NCAM polysialylation, and have important effects on formation of sialic acid storage diseases, neural system diseases and invasive cancers. It has been known that synthesis of PSA chains is catalysed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST). In addition, a polybasic motif of 32 amino acids in both ST8Sia II and ST8Sia IV has been designated as “polysialytransferase domain” (PSTD), which is essential for NCAM polysialylation. In this study, we have determined the 3D structure of the PSTD peptide containing 22 amino acids (22AA) in ST8Sia IV using NMR spectroscopy. This NMR-based model displays that the PSTD domain consists of an α-helical segment, two unstructured domains in both N- and C-terminus, and two three-residue-loops near the C-terminus of the peptide. Our overlaid 2D 1H-15N-HSQC spectra of the 22AA-PSTD peptide show that the amide proton chemical shifts of some amino acids such as I260, I261, H262, R265, L269 and K272 have been changed after polySA was mixed with the PSTD peptide. In addition, the peak intensity of A263, V264, R265, Y267, L269 and K272 were also decreased after adding polySA. However, there is no any change in both chemical shift and the amide proton peak intensity for all other residues located on outside of the helix. Above NMR results indicate a weak interaction exists between the helix of the PSTD and the PolySA, which may play a vital roles in modulating biosynthesis of polySA chain and NCAM polysialylation.
Academia Sinica, Taiwan
Tzu-Ching has completed his PhD from University of Nebraska Medical Center in 1999 and postdoctoral studies from Cold Spring Harbor Laboratory in 2003. Since then, he has been working at Academia Sinica, the premier government-funded institution in Taiwan. He is now a Research Fellow with professorship jointly appointed by National Taiwan University. He has published more than 40 papers in reputed journals and has been serving as an advisory board member of competitive journals.
The Ras signaling cascade acts as a key driver in human colon cancer progression. Among the modules in this pathway, p38gamma (MAPK12) and its specific protein tyrosine phosphatase PTPN3 (PTPH1) are critical regulators responsible for Ras oncogenic activity. However, the molecular basis for their interaction is completely unknown. Here we report the unique architecture of the PTPN3-p38gamma complex by employing an advanced hybrid method integrating X-ray crystallography, small-angle X-ray scattering (SAXS) and chemical cross-linking/mass spectrometry (CX-MS). Our crystal structure of PTPN3 in complex with the p38gamma phosphopeptide presented a unique feature of the E-loop that defines the substrate specificity of PTPN3 towards fully activated p38gamma. The low-resolution structure demonstrated the formation of an active-state or a resting-state complex of PTPN3-p38gamma. We showed a regulatory function of PTPN3’s PDZ domain, which stabilizes the active-state complex through interaction with the PDZ-binding motif of p38gamma. Using SAXS and CX-MS approaches, we found that binding of the PDZ domain to the PDZ-binding motif lifts the atypical auto-inhibitory constraint of PTPN3, enabling efficient tyrosine dephosphorylation of p38gamma to occur. Our findings emphasize the potential of structural approach for PTPN3-p38gamma complex that may deliver new therapeutic strategies against Ras-mediated oncogenesis in colon cancer.
Institute of Biophysics at the National Research Council (IBF-CNR), Italy
Title: Type I BIR domain inhibitors in cancer therapy: designing drugs to modulate the NF-κB pathway
Federica Cossu has always been interested in the field of cancer research, being fascinated by structural studies of crucial macromolecules and protein complexes involved in the cellular processes of cell death/survival. She gathered experience in cloning, expression, purification and crystallization of recombinant proteins, mainly belonging to the field of cancer. During the last years, she focused on the structure-based design of small molecules to be developed as drug candidates directed to pre-clinical studies. The success of this activity is proven by one patent, one award for her PhD thesis and several publications in the field. She progressively improved her knowledge on biophysical techniques for the study of proteins and on the in silico analysis of protein structures. She collected experiences in European laboratories, including several short visits/experiments at the ESRF synchrotron in Grenoble and at SOLEIL in Paris. She has been working in lab equipe for ten years, covering different positions within the research group, from master degree student to post-doc. She has been the supervisor of students, also giving lessons on the structural approaches applied to cancer therapy.
Inhibitors of apoptosis proteins (IAPs) constitute a family of conserved proteins whose over-expression enhances cell survival and resistance to anticancer agents. IAPs are E3 ligases, ubiquitylating substrates for the regulation of NF-kB; furthermore, they sequester caspases to prevent apoptosis. IAPs interactions occur through type I and type II BIR (Baculovirus IAP repeat) domains. Smac-mimetics (SM) mimicking the active N-terminal peptide of Smac-DIABLO, the natural antagonist of IAPs, have been shown to sensitize cancer cells to apoptosis. SM interact with type II BIR domains of IAPs, thus relieving caspases from X-linked IAP (XIAP) inhibitory activity and leading to cellular IAPs (cIAPs) auto-ubiquitylation and proteasomal degradation within minutes from exposure. Although SM are currently promising candidates for cancer therapy, some cancer cell lines present SM-resistance due to renewed cIAP2 activity and re-activation of NF-κB. IAPs-mediated regulation of NF-kB signaling is based on the formation of different protein-protein complexes, regulating ubiquitin-dependent signal transduction cascades. The type I BIR domain from different IAPs has been recognized as a pivotal platform for the assembly of such complexes.
We analyzed the surface of type I BIR domains (X- and cIAP-BIR1) to identify the hot-spots for the relevant protein-protein interactions. Virtual docking using libraries of compounds returned hits (NF023 and analogues) able to impair BIR1-based complexes with predicted low micromolar affinities that were experimentally confirmed. To this purpose, in vitro assays include fluorescence-based and biophysical techniques (Thermofluor, Microscale Thermophoresis, SEC, DLS, SLS). Crystallography on the protein-ligand complexes is the core of the structure-driven approach used for the iterative optimization of specific and selective drug candidates. Treatment of cancer cell cultures with the selected compounds will verify their effects on the modulation of IAPs-dependent signaling cascades. This represents a novel strategy to promote apoptosis in cancer and will unravel new insights on the regulation of NF-kB pathway.
ETH Zurich, Switzerland
After her PhD at the University of Padua (Italy) Paola Picotti joined the group of Ruedi Aebersold at ETH Zuerich (Switzerland), where she developed novel targeted proteomic techniques. In 2011 she was appointed assistant professor at ETH Zurich. Her group develops structural and chemoproteomics methods and uses them to study the consequences of intracellular protein aggregation. Paola Picotti’s research was awarded an ERC Starting grant, a Professorship grant from the Swiss National Science Foundation, the Latsis Prize, the Robert J. Cotter Award, the SGMS Award and the EMBO Young Investigator Award. Main contributions of the Picotti group are the development of a structural method to analyze protein conformational changes on a system-wide level, the discovery of novel allosteric interactions, the analysis of the determinants of proteome thermostability and the identification of a novel neuronal clearance mechanism for a protein involved in Parkinson’s disease.
Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. Mass spectrometry (MS)-based proteomic techniques are routinely used to measure changes in protein abundance, post-translational modification and protein interactors, but much less is known about protein structural changes, owing to the lack of suitable approaches to study global changes in protein folds in cells.
In my talk I will present a novel structural proteomics technology developed by our group that enables the analysis of protein structural changes on a proteome-wide scale and directly in complex biological extracts. The approach relies on the coupling of limited proteolysis (LiP) tools and an advanced MS workflow. LiP-MS can detect subtle alterations in secondary structure content, larger scale movements such as domain motions, and more pronounced transitions such as the switch between folded and unfolded states or multimerization events. The method can also be used to pinpoint protein regions undergoing a structural transition with peptide-level resolution. I will describe selected applications of the approach, including 1. The identification of proteins that undergo structural rearrangements in cells due to a nutrient shift; 2. The analysis of in vivo protein aggregation; 3. The cell-wide analysis of protein thermal unfolding; and 4. The identification of protein-small molecule interactions (e.g drug-target deconvolution).
I will discuss the power and limitations of the method and possible new directions in structural biology enabled by this emerging approach to protein structure analysis.