Home

CHEMISTRY-BIOLOGY INTERFACE TRAINING PROGRAM  

Updates on Trainee Activities

1. The Arrowhead Retreat         Photos from the Retreat

Chem/Bio Interface Trainees and Molecular Biology Institute Members Retreat to Arrowhead

On October 17-19, 2003, the Trainees, PI Ken Houk, and Trainee Advisor Jon Fukuto traveled the two hours to UCLA's Arrowhead Conference Facility, in the mountains just above the spot where fires ravaged the forests a week later. The conference included a talk by Prof. Houk on "Why Enzymes Are Proficient," as well as poster presentations by the trainees.

Why Enzymes Are Proficient - Professor K. N. Houk and Xiyun Zhang

Noncovalent host-guest binding affinities are usually in the range of 103-106 M-1. Protein- ligand binding affinities are similar in magnitude, but can be tuned to > 109 M-1 in potent reversible enzyme inhibitors. The binding of transition states by enzymes, as measured by WolfendenÕs proficiency, kcat/KM/kuncat, is generally much larger, commonly exceeding 108 M-1 and achieving 1027 M-1 in some cases. Many explanations have been offered, such as electrostatic stabilization, ground state destabilization and desolvation, entropy traps, orbital steering, low barrier hydrogen bonds, covalent bonding with cofactors, tunneling, the "Circe effect", induced fit, dynamic coupling of protein fluctuations to motions in the transition state, reduction of reorganization energy by binding in near attack conformations (NACs), noncovalent cooperativity, and the spatiotemporal hypothesis.

We propose that proficient enzymes, those with kcat/KM/kuncat > 1011 M-1, achieve 15-38 kcal/mol of transition state binding not by a concatenation of noncovalent effects, but by covalent bond formation between enzyme and transition state, involving a change in mechanism from that in aqueous solution. These interactions involve covalently bound transition states and intermediates, general acid/base catalysis, metal-substrate binding, and covalent binding to cofactors. A survey of established enzyme mechanisms for proficient enzymes is consistent with this conclusion. Estimations of uncatalyzed rates in water have extended proficiencies to over 1000 examples for which proficiencies are now known to within several orders of magnitude.

Ionizing Radiation and Restriction Enzymes Induce Microhomology-Mediated Illegitimate Recombination in Trans in Saccharomyces Cerevisiae - Cecilia Chan, Palaniyandi Manivasakam, and Robert H. Schiestl

DNA double strand breaks are repaired by two pathways, homologous recombination in the presense of sequence homology and nonhomologous or illegitimate recombination in the absence of extended sequence homology. One mechanism of illegitimate recombination occurs between several basepairs of homology, also called microhomology-mediated recombination which is conserved from bacteria to mammalian cells. Here, we report that both ionizing radiation and restriction enzymes induce microhomology-mediated integration in trans at sites that are probably not damaged. Both, irradiation and restriction enzymes increased the frequency of illegitimate integrations. Irradiated yeast cells displayed 82% of microhomology-mediated illegitimate integration, compared to 23% of microhomology usage in spontaneous integration. Restriction enzymes mediated integration events not only at genomic restriction sites, but also at microhomologies at random non-restriction sites. These results suggest that DSBs caused by ionizing radiation and restriction enzymes may induce a genome wide microhomology-mediated illegitimate recombination pathway and facilitate integration at non-targeted sites.

Progress Towards the Total Synthesis of Guanacastepene A - Hiufung Chu, Xiaohui Du, and Ohyun Kwon

Guanacastepene A was recently isolated by Jon Clardy and his colleagues from an unidentified fungus CR115 growing on the branch of Daphnopsis americana in Guanacaste, Costa Rica. This natural product exhibits antibiotic activity against drug resistant strains of Staphylococcus aureus and Enterococcus faecalis, which has been a tremendous threat to human health. Therefore, guanacastepene A is a potential candidate as a new antibacterial agent. However, guanacastepene A also displays hemolytic activity against human red blood cells and thus, has limited its therapeutic application. Analogs of guanacastepene A that possess antibacterial activity without hemolytic effect will be a great value. Our goal is to complete the total synthesis of guanacastepene A and to establish a general synthetic scheme that allows us to elaborate the skeleton framework of guanacastene A to its analogs.

EPR Iron Homeostasis Studies in Organisms Lacking Cu/Zn Superoxide Dismutase - Matthew H. Clement, Chandra Srinivasan, Sailaja Mantha, Ting-Ting Huang, Joan S. Valentine, and Edith B. Gralla

Previous studies in yeast (S. cerevisiae) lacking CuZn superoxide dismutase (sod1D) have shown elevated Fe(III) levels under aerobic growth conditions. This altered iron homeostasis can be observed by a whole cell electron paramagnetic resonance (EPR) method designed to quantitate EPR detectable iron (high spin, rhombic Fe(III), at g = 4.3) levels. The solvent exposed 4Fe-4S clusters of aconitase are one of the suspected sources of the elevated Fe(III) levels exhibited in sod1D cells. This ÔfreeÕ or Ôjunk ironÕ liberated from 4Fe-4S clusters is then capable of participating in Fenton-type reactions which can lead to the formation of reactive oxygen species (ROS) causing oxidative stress. In collaboration with Sailaja Mantha and Ting-Ting Huang at Stanford, we have adapted the EPR method to study the Fe(III) levels in organ tissue homogenates of mice lacking SOD1. Preliminary studies by Mantha and Huang have investigated changes in iron homeostasis as well as aconitase activity in the knockout mice. Our collaborative study will aid in the elucidation of the source of elevated Fe(III) levels as well as its relationship to ROS production and oxidative stress in organisms lacking SOD1.

Single Molecule Detection of Protein-Protein Interactions - Natalie Gassman, Achillefs N. Kapanidis, Nam Ki Lee, Ted A. Laurence, Xiangxu Kong, and Shimon Weiss

Characterization of protein-protein interactions has become of great interest in the post-genomic era, and a variety of methodologies have been developed to assign activity to sets of proteins. Although these methodologies have greatly expanded working knowledge of protein functions and their interacting partners, there is still a large gap between proteins in the genome and their known interactions. By developing optical methods based on fluorescence burst and fluctuation spectroscopies to screen protein-protein interactions; we hope to detect oligomerization in identical macromolecules, as well as interactions between two or more different macromolecules. Detection of oligomerization can occur with a single photon channel where photon bursts emitted by free molecules can be differentiated from complexes, which have slower diffusion times and increased brightness. For protein-protein interaction studies, a greater sensitivity of detection is required and can be achieved by labeling the two different macromolecules with two distinct and separable fluorophores. Single molecule detection of the two fluorophores with distinct emission results in a more sensitive binding assay due to the ability to separate the emissions into two detection channels and resolve subpopulations of complexes, which display simultaneous photon bursts on both channels. These methods are applied to the elucidation of transcription in Shewanella oneidensis MR-1. Our lab is pursuing the protein-protein interactions involved in transcription of electron transport proteins by the prokaryotic enhancer binding protein, nitrogen regulatory protein (NtrC).

Biological Activity of Nitroxyl (HNO) - Matthew Jackson and Jon Fukuto

Hydrogen Peroxide (H2O2) and alkyl peroxides are now recognized to be intramolecular signalling agents, produced in response to ligand induced receptor activation. H2O2 modifies thiols present on signaling proteins to effect changes in enzymatic activity and gene expression. The protein sulfenates and disulfides (PSOH and PSSR/PSSP respectively) are recycled back to protein thiols (PSH) by a family of thioredoxin enzymes. The bolus H2O2 produced is then disposed of by Glutathione peroxidase (GPx) and Catalase (CAT). Although both CAT and GPX degrade H2O2; they do so via mutally distinct mechanisms. CAT contains a heme iron with a +3 resting state, while GPx contains a selenocysteine that is involved in catalysis.

Our hypothesis is that nitroxyl (N+1, HNO) will inhibit the cellular H2O2 disposal systems CAT and GPx, due to the selective reactivity of HNO with ferric and seleno species respectively. HNO should reduce the FeIII heme iron of CAT to an inactive FeII-NO complex. HNO should form a covalent N-Hydroxy selenenamide adduct with GPx active site selenol (selenocysteine), this product rearranging to a seleninamide or producing a GPx-Se-S-G adduct. Therefore the presence of HNO in a cellular milieu will lead to enhanced sensitivity to the presence of H2O2 (more pronounced peak effect, eg signaling events or toxicity) as well as temporal extension of peroxide mediated effects.

The Biological Significance of RSNOs and Testing Decomposition Mechanisms Computationally - Patrick R. McCarren and K. N. Houk

Nitric oxide is an important signaling agent in physiological systems with important roles in the cardiac, respiratory, immune, and neurological systems as well as controlling cell destruction through caspase. S-nitrosothiols (RSNO's) are believed to transport and store nitric oxide by stabilizing it to reactions that usually lead to NO's short lifetime (~10 seconds). The RSNO concentration in rabbits and humans has been found to be in the nM to µM range with R being protein or peptide in nature. In vitro experiments show that simple alkane RSNO's decompose rapidly (seconds to minutes) to NO and disulfide as do oligopeptides or proteins but with a longer half-life. The mechanism frequently reported for this mechanism is a simple homolytic mechanism forming thiyl radical and NO. However, quantum mechanical calculations at the CBS-QB3 level, which have an approximate error of 2 kcal/mol, predict bond dissociation energies of 30 kcal/mol with virtually no dependence on the alkyl R group. This would be an unlikely process at room temperature used in the in vitro experiments. Bimolecular reactions could be another explanation of this key NO generation mechanism where the S of one RSNO attacks the N of the other RSNO. They lead to slightly lower activation energies than homolysis and produced the expected R dependence using CBS-QB3 calculations. Transnitrosation is another mechanism of importance to the biological action of RSNO's. It could lead to the specific direction of NO to important areas of action. Transnitrosation is the effective transfer of NO+ from one RS- to another. CBS-QB3 calculations in H2O solvent found an interesting symmetrical intermediate and an activation energy within the range of known experimental energies.

Probing the Protein Partners in Huntington's Disease - Dyna I. Shirasaki, Allan J. Tobin, and Joseph A. Loo

Although the function of huntingtin (Htt) currently remains elusive, Òguilt by associationÓ with a number of proteins has implicated a possible role in such cellular events as vesicular trafficking, transcriptional regulation, and the endosome/lysosome pathway. While previously discovered interactions have provided valuable insight, it is questionable as to how well they portray actual occurrences within the human brain. In order to obtain a more accurate picture, we are searching for Htt-protein interactions in PC12 cells have been stably transfected with a construct containing exon-1 of the huntingtin gene (containing either 25 or 103 glutamines) fused to a C-terminal EGFP tag. Immunoprecipitated Htt was separated on an SDS-PAGE gel and several bands were excised and the proteins digested with trypsin. Peptide mass fingerprints were obtained by MALDI-TOF mass spectrometry, and peptide sequencing was accomplished by nanoLC-MS/MS, nanoelectrospray-MS/MS and MALDI-MS/MS with a QqTOF-MS and an ion trap mass spectrometer. Preliminary sequence information has identified some of the interacting proteins to be GAPDH, tyrosine hydroxylase and HSP27. Western blots are currently being performed to confirm our results. Proteomics and mass spectrometry offer an alternative means for detecting huntingtin-associated proteins that enables us to search for interactions occurring throughout the cytoplasm as well as in the nucleus. It may also allow for increased detection sensitivity to proteins that may be in low abundance. Revealing the nature of huntingtinÕs protein partners may provide valuable clues as to the function of huntingtin, and possibly lead to a testable hypothesis for the disease mechanism.

Novel HNO Donors and HNO Traps - Jay Wang and Jon Fukuto

HNO is a novel biological molecule with novel and potentially important pharmacological properties. Since HNO is a highly reactive molecule that readily dimerizes, directly studying HNO is very difficult. One part of the project is to design small molecules that can trap HNO released in cells, in order to determine if a cell is generating HNO. The second part of the project involves the synthesis of small molecules that release HNO under various conditions (HNO donors). By making these donors, we can further study the biological effects of HNO. This will further our understanding of HNO and its interaction in biological systems.