Resume

• 1997

  Ph.D.

  Molecular and Cell Biology

• 1992

  B.A.

  Biology

  Chemistry (minor)

  Robert D. Clarke Honors College

  Phi Beta Kappa

• Molecular Cloning

  Animal Models

  SDS-PAGE

  Immunoprecipitation

  Protein Chemistry

  Stem Cells

  PCR

  Protein Purification

  Cell Biology

  Western Blotting

  Immunohistochemistry

  Cell Culture

  Biochemistry

  Protein Expression

  Molecular Biology

  Transfection

  Fluorescence Microscopy

  Confocal Microscopy

  qPCR

  Cancer Research

  A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells.

  A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells.

  The nuclear receptor NR4A1 induces a form of cell death dependent on autophagy in mammalian cells.

  A calpain-like protease inhibits autophagic cell death.

  A variety of studies have implicated the lipid PtdIns(4

  5)P2 in endocytic internalization

  but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4

  5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p

  a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4

  5)P2 binding regulates actin-dependent endocytosis

  we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4

  5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4

  5)P2 binding showed significant defects in cell growth

  actin organization

  and endocytic internalization. These defects could be rescued by increasing PtdIns(4

  5)P2 levels in vivo. Strikingly

  mutant Sla2p defective in PtdIns(4

  5)P2 binding localized with the endocytic machinery at the cell cortex

  establishing that the ANTH-PtdIns(4

  5)P2 interaction is not necessary for this association. In contrast

  multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4

  5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4

  5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.

  Interaction of Sla2p's ANTH domain with PtdIns(4

  5)P2 is important for actin dependent endocytic in- ternalization.

  We have developed an assay to monitor the assembly of the COPII coat onto liposomes in real time. We show that with Sar1pGTP bound to liposomes

  a single round of assembly and disassembly of the COPII coat lasts a few seconds. The two large COPII complexes Sec23/24p and Sec13/31p bind almost instantaneously (in less than 1 s) to Sar1pGTP-doped liposomes. This binding is followed by a fast (less than 10 s) disassembly due to a 10-fold acceleration of the GTPase-activating protein activity of Sec23/24p by the Sec13/31p complex. Experiments with the phosphate analogue BeFx suggest that Sec23/24p provides residues directly involved in GTP hydrolysis on Sar1p.

  Dynamics of the COPII coat with GTP and stable analogues

  SEC16 encodes a 240-kD hydrophilic protein that is required for transport vesicle budding from the ER in Saccharomyces cerevisiae. Sec16p is tightly and peripherally bound to ER membranes

  hence it is not one of the cytosolic proteins required to reconstitute transport vesicle budding in a cell-free reaction. However

  Sec16p is removed from the membrane by salt washes

  and using such membranes we have reconstituted a vesicle budding reaction dependent on the addition of COPII proteins and pure Sec16p. Although COPII vesicle budding is promoted by GTP or a nonhydrolyzable analogue

  guanylimide diphosphate (GMP-PNP)

  Sec16p stimulation is dependent on GTP in the reaction. Details of coat protein assembly and Sec16p-stimulated vesicle budding were explored with synthetic liposomes composed of a mixture of lipids

  including acidic phospholipids (major-minor mix)

  or a simple binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Sec16p binds to major-minor mix liposomes and facilitates the recruitment of COPII proteins and vesicle budding in a reaction that is stimulated by Sar1p and GMP-PNP. Thin-section electron microscopy confirms a stimulation of budding profiles produced by incubation of liposomes with COPII and Sec16p. Whereas acidic phospholipids in the major-minor mix are required to recruit pure Sec16p to liposomes

  PC/PE liposomes bind Sar1p-GTP

  which stimulates the association of Sec16p and Sec23/24p. We propose that Sec16p nucleates a Sar1-GTP-dependent initiation of COPII assembly and serves to stabilize the coat to premature disassembly after Sar1p hydrolyzes GTP.

  Sec16p potentiates the action of COPII proteins to bud transport vesicles

  Cells exposed to sustained endoplasmic reticulum (ER) stress undergo programmed cell death and display features typical of apoptosis

  such as cysteine aspartyl protease (caspase) activation

  cytochrome c release

  and DNA fragmentation. Here

  we show that the execution of cell death induced by ER stress is mediated via the proteasome. Inhibition of the proteasome by lactacystin prevented ER stress-induced degradation of Bcl-2

  release of cytochrome c

  processing of effector caspase-3

  and exposure of phosphatidylserine. Owing to the ability of lactacystin to inhibit cytochrome c release

  we propose that the pro- apoptotic activity of the proteasome lies upstream of mitochondrial activation. Thus

  the proteasome serves as a principal mediator of ER stress-induced cell death in this system.

  Endoplasmic reticulum stress-induced cell death mediated by the proteasome.

  Simon Melov

  Diana Davila-Kruger

  Human Embryonic Stem Cells Express Elevated Levels of Multiple Pro-Apoptotic BCL-2 Family Members

  Valosin-containing protein gene mutations: cellular phenotypes relevant to neurodegeneration.

  Endogenously EGFP-labeled mouse embryonic stem cells.

  By improving the expression and purification of Escherichia coli methionine aminopeptidase (eMetAP) and using slightly different crystallization conditions

  the resolution of the parent structure was extended from 2.4 to 1.9 A resolution. This has permitted visualization of the coordination geometry and solvent structure of the active-site dinuclear metal center. One solvent molecule (likely a mu-hydroxide) bridges the trigonal bipyramidal (Co1) and octahedral (Co2) cobalt ions. A second solvent (possibly a hydroxide ion) is bound terminally to Co2. A monovalent cation binding site was also identified about 13 A away from the metal center at an interface between the two subdomains of the protein. The first structure of a substrate-like inhibitor

  (3R)-amino-(2S)-hydroxyheptanoyl-L-Ala-L-Leu-L-Val-L-Phe-OMe

  bound to a methionine aminopeptidase

  has also been determined. This inhibitor coordinates the metal center through four interactions as follows: (i) ligation of the N-terminal (3R)-nitrogen to Co2

  (ii

  iii) bridging coordination of the (2S)-hydroxyl group

  and (iv) terminal ligation to Co1 by the keto oxygen of the pseudo-peptide linkage. Inhibitor binding occurs with the displacement of two solvent ligands and the expansion of the coordination sphere of Co1. In addition to the tetradentate

  bis-chelate metal coordination

  the substrate analogue forms hydrogen bonds with His79 and His178

  two conserved residues within the active site of all MetAPs. To evaluate their importance in catalysis His79 and His178 were replaced with alanine. Both substitutions

  but especially that of His79

  reduce activity... ...These results also suggest that inhibitors analogous to that reported here may be useful in preventing angiogenesis in cancer and in the treatment of microbial and fungal infections.

  Escherichia coli Methionine Aminopeptidase: Implications of Crystallographic Analyses of the Native

  Mutant

  and Inhibited Enzymes for the Mechanism of Catalysis

  Sophisticated mechanisms that preserve genome integrity are critical to ensure the maintenance of regenerative capacity while preventing transformation of somatic stem cells (SCs). Intestinal Stem Cells (ISCs) in Drosophila serve as productive models for the understanding of regenerative activity of somatic SCs

  yet little is known about mechanisms regulating genome maintenance in these cells. Here we show that ISCs induce the piRNA binding protein Piwi in response to JAK/STAT signaling during acute proliferative episodes. Piwi function is critical to ensure heterochromatin maintenance

  suppress retrotransposon activation

  and prevent DNA damage in homeostasis and under regenerative pressure. Accordingly

  loss of Piwi results in rapid loss of actively dividing ISCs by apoptosis. We further show that aging ISCs exhibit a progressive increase in retrotransposon expression and activity and that increasing Piwi expression is sufficient to allay age-related TE expression

  DNA damage

  apoptosis and mis-differentiation phenotypes in the ISC lineage

  improving epithelial homeostasis. Our data identify a novel role for Piwi in the regulation of somatic SC function and highlight the importance of retrotransposon control in somatic SC maintenance.

  Piwi is required to limit exhaustion of aging somatic stem cells.

  Madden

  Buck Institute for Research on Aging

  Touro University California

  BioMarin Pharmaceutical Inc.

  Buck Institute for Research on Aging

  Assistant Professor

  Touro University California

  Project Manager

  United States

  BioMarin Pharmaceutical Inc.

  Buck Institute for Research on Aging

  Novato

  Adjunct Assistant Professor

  Buck Institute for Research on Aging

  Touro University California

  Vallejo

  CA 94592

  Associate Professor