A Small Molecule Inhibitor of Redox-Regulated Protein Translocation into Mitochondria

SummaryThe mitochondrial disulfide relay system of Mia40 and Erv1/ALR facilitates import of the small translocase of the inner membrane (Tim) proteins and cysteine-rich proteins. A chemical screen identified small molecules that inhibit Erv1 oxidase activity, thereby facilitating dissection of the disulfide relay system in yeast and vertebrate mitochondria. One molecule, mitochondrial protein import blockers from the Carla Koehler laboratory (MitoBloCK-6), attenuated the import of Erv1 substrates into yeast mitochondria and inhibited oxidation of Tim13 and Cmc1 in in vitro reconstitution assays. In addition, MitoBloCK-6 revealed an unexpected role for Erv1 in the carrier import pathway, namely transferring substrates from the translocase of the outer membrane complex onto the small Tim complexes. Cardiac development was impaired in MitoBloCK-6-exposed zebrafish embryos. Finally, MitoBloCK-6 induced apoptosis via cytochrome c release in human embryonic stem cells (hESCs) but not in differentiated cells, suggesting an important role for ALR in hESC homeostasis

Public health in Thailand: Emerging focus on non‐communicable diseases

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86815/1/hpm1078.pd

Top-Down Mass Spectrometry Characterization of Protein-Ligand Complexes Important to Neurodegenerative Diseases

Mass spectrometry (MS) has made significant contributions to protein and proteomics analysis during the past decades from its advantages of speed, sensitivity, specificity, and low sample consumption. While the proteomics field grows rapidly to identify thousands of proteins in a single analysis, “native” mass spectrometry, exploiting the unique features of electrospray ionization (ESI) for delivering large macromolecules to the mass spectrometer, has provided many potential exciting capabilities and applications to structural biology and biochemistry. It can analyze proteins in their native states, i.e., structures present in their native configurations from physiological pH solutions, with minimal sample preparation.In this thesis, I describe the application of native ESI combined with top-down MS using electron capture dissociation (ECD) and ion mobility (IM) to characterize the molecular features of protein-ligand complexes. Binding and structural information can be comprehensively obtained from this experimental platform. Native ESI-MS alone provides molecular mass, stoichiometry, and binding affinity, all from a single analysis. We demonstrate that top-down MS, the fragmentation of intact proteins and protein complexes using MS, offers a powerful capability to elucidate the location of ligand binding on a protein’s structure and for probing the surface topology of proteins. Ion mobility mass spectrometry, a recently developed technique that yields information on the structural conformation of molecules, was used to reveal structural changes of proteins upon ligand binding.My thesis focuses on several proteins, including α-synuclein (AS), which is a small protein related to Parkinson’s disease. AS is natively unfolded at physiological pH, which makes it difficult to study by standard methods such as X-ray crystallography or NMR. Using our mass spectrometry techniques, transition metal binding (copper, cobalt, and manganese) to AS that is associated with accelerating fibril formation was monitored. The binding of a small molecule amyloid inhibitor called molecular tweezer (MT or CLR01) on two model proteins important in neurodegenerative diseases, AS and superoxide dismutase (SOD1), was studied. Tandem mass spectrometry (MS/MS) techniques such as collisionally activated dissociation (CAD) along with ECD were used to characterize the sites of binding of small molecule ligands to proteins. Ion mobility mass spectrometry was implemented to reveal the conformational changes of AS upon metal binding. It was demonstrated that copper can induce the AS protein to collapse into a more compact state, which may provide a hint of the mechanisms behind amyloid fibrillation.Additionally, two new methods to extend the application of top-down MS for protein structure characterization were developed. First, the same molecular tweezer molecule, which has a specificity to bind lysine residues, was used to probe surface residues of proteins. The lysines found to bind to the molecular tweezers identified by top-down MS correlates well with solvent accessibility values, suggesting that the MT compound can be applied as a molecular probe to pinpoint surface active lysine residues. Lastly, supplemental activation methods by ultraviolet and infrared laser irradiation prior to ECD was applied to assist disulfide bond cleavage of complex multiple intermolecular and intramolecular disulfide bond-containing proteins. Backbone bond cleavage from top-down MS was significantly increased when the disulfide bonds were cleaved, allowing more sequence information to be obtained. The new methods described in this thesis extend the applicability of mass spectrometry to provide a more complete picture of a protein’s structure

Significance of filamin A in mTORC2 function in glioblastoma

BACKGROUND: Glioblastoma multiforme (GBM) is one of the most highly metastatic cancers. GBM has been associated with a high level of the mechanistic target of rapamycin complex 2 (mTORC2) activity. We aimed to observe roles of mTORC2 in GBM cells especially on actin cytoskeleton reorganization, cell migration and invasion, and further determine new important players involved in the regulation of these cellular processes. METHODS: To further investigate the significance of mTORC2 in GBM, we treated GBM cells with PP242, an ATP-competitive inhibitor of mTOR, and used RICTOR siRNA to knock down mTORC2 activity. Effects on actin cytoskeleton, focal adhesion, migration, and invasion of GBM cells were examined. To gain insight into molecular basis of the mTORC2 effects on cellular cytoskeletal arrangement and motility/invasion, we affinity purified mTORC2 from GBM cells and identified proteins of interest by mass spectrometry. Characterization of the protein of interest was performed. RESULTS: In addition to the inhibition of mTORC2 activity, we demonstrated significant alteration of actin distribution as revealed by the use of phalloidin staining. Furthermore, vinculin staining was altered which suggests changes in focal adhesion. Inhibition of cell migration and invasion was observed with PP242. Two major proteins that are associated with this mTORC2 multiprotein complex were found. Mass spectrometry identified one of them as Filamin A (FLNA). Association of FLNA with RICTOR but not mTOR was demonstrated. Moreover, in vitro, purified mTORC2 can phosphorylate FLNA likewise its known substrate, AKT. In GBM cells, colocalization of FLNA with RICTOR was observed, and the overall amounts of FLNA protein as well as phosphorylated FLNA are high. Upon treatments of RICTOR siRNA or PP242, phosphorylated FLNA levels at the regulatory residue (Ser2152) decreased. This treatment also disrupted colocalization of Actin filaments and FLNA. CONCLUSIONS: Our results support FLNA as a new downstream effector of mTORC2 controlling GBM cell motility. This new mTORC2-FLNA signaling pathway plays important roles in motility and invasion of glioblastoma cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12943-015-0396-z) contains supplementary material, which is available to authorized users

Revealing Ligand Binding Sites and Quantifying Subunit Variants of Noncovalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment

"Native" mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains

Native Top-Down Mass Spectrometry and Ion Mobility MS for Characterizing the Cobalt and Manganese Metal Binding of α-Synuclein Protein

Structural characterization of intrinsically disordered proteins (IDPs) has been a major challenge in the field of protein science due to limited capabilities to obtain full-length high-resolution structures. Native ESI-MS with top-down MS was utilized to obtain structural features of protein-ligand binding for the Parkinson's disease-related protein, α-synuclein (αSyn), which is natively unstructured. Binding of heavy metals has been implicated in the accelerated formation of αSyn aggregation. Using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, native top-down MS with various fragmentation methods, including electron capture dissociation (ECD), collisional activated dissociation (CAD), and multistage tandem MS (MS3), deduced the binding sites of cobalt and manganese to the C-terminal region of the protein. Ion mobility MS (IM-MS) revealed a collapse toward compacted states of αSyn upon metal binding. The combination of native top-down MS and IM-MS provides structural information of protein-ligand interactions for intrinsically disordered proteins. Graphical Abstract ᅟ