Richard Anderson

UW Madison Press Release

JCB Online Article

The Science Coalition Article

J Cell Biol. 2007 Jan 29;176(3):343-53.

Type Ig phosphatidylinositol phosphate kinase modulates adherens junction and E-cadherin trafficking via a direct interaction with m1B adaptin.

Assembly of E-cadherin-based adherens junctions (AJ) is obligatory for establishment of polarized epithelia and plays a key role in repressing the invasiveness of many carcinomas. Here we show that type Igamma phosphatidylinositol phosphate kinase (PIPKIgamma) directly binds to E-cadherin and modulates E-cadherin trafficking. PIPKIgamma also interacts with the mu subunits of clathrin adaptor protein (AP) complexes and acts as a signalling scaffold that links AP complexes to E-cadherin. Depletion of PIPKIgamma or disruption of PIPKIgamma binding to either E-cadherin or AP complexes results in defects in E-cadherin transport and blocks AJ assembly. An E-cadherin germline mutation that loses PIPKIgamma binding and shows disrupted basolateral membrane targeting no longer forms AJs and leads to hereditary gastric cancers. These combined results reveal a novel mechanism where PIPKIgamma serves as both a scaffold, which links E-cadherin to AP complexes and the trafficking machinery, and a regulator of trafficking events via the spatial generation of phosphatidylinositol-4,5-bisphosphate.

Ling K, Bairstow SF, Carbonara C, Turbin DA, Huntsman DG, Anderson RA.

Emery Bresnick

Mol Cell Biol. 2007 Jan 12

Glucocorticoid and Growth Factor Synergism Requirement for Notch4 Chromatin Domain Activation.

The Notch signaling pathway modulates cell fate in diverse contexts, including vascular development. Notch4 is selectively expressed in vascular endothelium and regulates vascular remodeling. The signal-dependent transcription factor Activator Protein 1 (AP-1) activates Notch4 transcription in endothelial cells, but other factors/signals that regulate Notch4 are largely unknown. We demonstrate that, unlike the established transrepression mechanism in which the glucocorticoid receptor (GR) antagonizes AP-1, AP-1 and GR synergistically activated Notch4 transcription in endothelial cells. Fibroblast Growth Factor-2 (FGF-2) and cortisol induced AP-1 and GR occupancy, respectively, at a Notch4 promoter composite response element, consisting of an imperfect half GRE and an AP-1 motif, which mediated signal-dependent activation. Analysis of Notch4 promoter complex assembly provided evidence that GR and AP-1 independently occupy the composite response element, but AP-1 stabilizes GR occupancy. In multipotent 10T1/2 cells, FGF-2 and cortisol induced a histone modification pattern at the Notch4 locus mimicking that present in endothelial cells and reprogrammed Notch4 from a repressed to an active state. These results establish the molecular basis for a novel AP-1/GR - Notch4 axis in vascular endothelium. PMID: 17220278

Wu J, Bresnick EH. University of Wisconsin School of Medicine, Department of Pharmacology, 1300 University Avenue, Madison, WI 53706.

Emery Bresnick

UW-Madison Press Release

Journal of Cell Biology, 2006

An antiangiogenic neurokinin-B/thromboxane A2 regulatory axis

Establishment of angiogenic circuits that orchestrate blood vessel development and remodeling requires an exquisite balance between the activities of pro- and antiangiogenic factors. However, the logic that permits complex signal integration by vascular endothelium is poorly understood. We demonstrate that a "neuropeptide," neurokinin-B (NK-B), reversibly inhibits endothelial cell vascular network assembly and opposes angiogenesis in the chicken chorioallantoic membrane. Disruption of endogenous NK-B signaling promoted angiogenesis. Mechanistic analyses defined a multicomponent pathway in which NK-B signaling converges upon cellular processes essential for angiogenesis. NK-B–mediated ablation of Ca2+ oscillations and elevation of 3'–5' cyclic adenosine monophosphate (cAMP) reduced cellular proliferation, migration, and vascular endothelial growth factor receptor expression and induced the antiangiogenic protein calreticulin. Whereas NK-B initiated certain responses, other activities required additional stimuli that increase cAMP. Although NK-B is a neurotransmitter/ neuromodulator and NK-B overexpression characterizes the pregnancy-associated disorder preeclampsia, NK-B had not been linked to vascular remodeling. These results establish a conserved mechanism in which NK-B instigates multiple activities that collectively oppose vascular remodeling.

Saumen Pal, Jing Wu, Justin K. Murray, Samuel H. Gellman, Michele A. Wozniak, Patricia J. Keely, Meghan E. Boyer, Timothy M. Gomez, Sean M. Hasso, John F. Fallon, and Emery H. Bresnick

Ed Chapman

UW Madison press release

Nature News and Views

Nature News and Views 2

HHMI press release

Nature (in press). (2006)

Structural basis of receptor recognition by botulinum neurotoxin B. 

Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking neurotransmitter release. A 'double receptor' model has been proposed in which BoNTs recognize nerve terminals via interactions with both gangliosides and protein receptors that mediate their entry. Of seven BoNTs (subtypes A - G), the putative receptors for BoNT/A, BoNT/B, and BoNT/G have been indentified, but the molecular details that govern recognition remain undefined. Here we report the crystal structure of full-length BoNT/B in complex with the synaptotagmin II (Syt-II) recognition domain at 2.6 A resolution. The structure of the complex reveals that Syt-II forms a short helix that binds to a hydrophobic groove within the binding domain of BoNT/B. In addition, mutagenesis of amino acid residues within this interface on Syt-II affects binding of BoNT/B. Structural and sequence analysis reveals that this hydrophobic groove is conserved in the BoNT/G and BoNT/B subtypes, but varies in other clostridial neurotoxins. Futhermore, molecular docking studies using the ganglioside GT1b indicate that its binding site is more extensive than previously proposed and might form contacts with both BoNT/B and synaptotagmin. The results provide structural insights into how BoNTs recognize protein receptors and reveal a promising target for blocking toxin-receptor recognition.

*Chai, Q., *Arndt, J.W., *Dong, M., Tepp, W.H., Johnson, E.A., #Chapman, E.R. and #Stevens, R.C.. (2006). Structural basis of receptor recognition by botulinum neurotoxin B.  Nature 444: 1096-1100.

* equal contribution
# corresponding authors

Science 312: 592-596. (2006)

A Neuronal Receptor for Botulinum Toxin ("Perspective") - Science

How the widely used botulinum neurotoxin A (BoNT/A) recognizes and enters neurons is poorly understood. We found that BoNT/A enters neurons by binding to the synaptic vesicle protein SV2 (isoforms A, B, and C). Fragments of SV2 that harbor the toxin interaction domain inhibited BoNT/A from binding to neurons. BoNT/A binding to SV2A and SV2B knockout hippocampal neurons was abolished and was restored by expressing SV2A, SV2B, or SV2C. Reduction of SV2 expression in PC12 and Neuro-2a cells also inhibited entry of BoNT/A, which could be restored by expressing SV2 isoforms. Finally, mice that lacked an SV2 isoform (SV2B) displayed reduced sensitivity to BoNT/A. Thus, SV2 acts as the protein receptor for BoNT/A.

Dong, M., Yeh, Y., Tepp, W.H., Dean, C., Johnson, E.A., Janz, R. and Chapman, E.R..

Ed Chapman

UW Madison News Article

Wisconsin State Journal Article

Faculty of 1000: 1 - 2 - 3 - 4 - 5 - 6 - 7

Proc. Natl. Acad. Sci. USA 101: 14701-14706. (2004).

Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells.

Dong, M., Goodnough, M.C., Tepp, W.H., Johnson, E.A. and Chapman, E.R..

Botulinum neurotoxins (BoTNs) act as zinc-dependent endopeptidases that cleave proteins required for neurotransmitter release. To detect toxin activity, fragments of the toxin substrate proteins, synaptobrevin (Syb) or synaptosome-associated protein of 25 kDa (SNAP-25), were used to link cyan fluorescent protein (CFP) to yellow fluorescent protein (YFP). Cleavage of these fusion proteins by BoNTs abolished fluorescence resonance energy transfer between the CFP and YFP, providing a sensitive means to detect toxin activity in real-time in vitro. Furthermore, using full-length SNAP-25 and Syb as the linkers, we report two fluorescent biosensors that can detect toxin activity within living cells. Cleavage of the SNAP-25 fusion protein abolished fluorescence resonance energy transfer between CFP and YFP, and cleavage of SYb resulted in spatial redistribution of YFP fluorescence in cells. This approach provides a means to carry out cell-based screening of toxin inhibitors and to study toxin activity in situ. By using these biosensors, we found that the subcellular localizations of SNAP-25 and Syb are critical for efficient cleavage by Banta/A and B, respectively.

Ed Chapman

Meyer Jackson

Science 304:289-292. (2004)

Transmembrane segments of syntaxin line the fusion pore of Ca2+-triggered exocytosis.

Han, X., Wang, C.T., Bai, J., Chapman, E.R. and Jackson, M.B.

The fusion pore of regulated exocytosis is a channel that connects and spans the vesicle and plasma membranes. The molecular composition of this important intermediate structure of exocytosis is unknown. Here, we found that mutations of some residues within the transmembrane segment of syntaxin (Syx), a plasma membrane protein essential for exocytosis, altered neurotransmitter flux through fusion pores and altered pore conductance. The residues that influenced fusion-pore flux lay along one face of an a-helical model. Thus, the fusion pore is formed at least in part by a circular arrangement of 5 to 8 Syx transmembrane segments in the plasma membrane.

Shigeki Miyamoto

Cell, Vol. 115, 1-20, November 26, 2003, Copyright 2003 by Cell Press

Sequential Modification of NEMO/IKKgamma by SUMO-1 and Ubiquitin Mediates NF-kappaB Activation by Genotoxic Stress.

Huang TT, Wuerzberger-Davis SM, Wu ZH, Miyamoto S.

The transcription factor NF-kappaB is critical for setting the cellular sensitivities to apoptotic stimuli, including DNA damaging anticancer agents. Central to NF-kappaB signaling pathways is NEMO/IKKgamma, the regulatory subunit of the cytoplasmic IkappaB kinase (IKK) complex. While NF-kappaB activation by genotoxic stress provides an attractive paradigm for nuclear-to-cytoplasmic signaling pathways, the mechanism by which nuclear DNA damage modulates NEMO to activate cytoplasmic IKK remains unknown. Here, we show that genotoxic stress causes nuclear localization of IKK-unbound NEMO via site-specific SUMO-1 attachment. Surprisingly, this sumoylation step is ATM-independent, but nuclear localization allows subsequent ATM-dependent ubiquitylation of NEMO to ultimately activate IKK in the cytoplasm. Thus, genotoxic stress induces two independent signaling pathways, SUMO-1 modification and ATM activation, which work in concert to sequentially cause nuclear targeting and ubiquitylation of free NEMO to permit the NF-kappaB survival pathway. These SUMO and ubiquitin modification pathways may serve as anticancer drug targets.

Edwin Chapman

Meyer Jackson

Thomas Martin

Emery Bresnick

Germany - "Innovations Report"

United States - New release web site

Spanish Medical Society of Anti-aging and Longevity

Italian news server

California Blood Bank Society

University of Wisconsin Medical School Web site

ASBMB Today

from the University of Wisconsin Medical School Web site:

Researchers Find Mechanism That May Determine Early Blood

Cell Fate Hematopoietic stem cells, the mother of all blood cells, face a fundamental dilemma in their lives. Each must either remain a hematopoietic stem cell (HSC) by renewing itself or it must transform into one of eight specialized types of blood cells, such as a red blood cell, a white blood cell or a platelet.

Until recently, scientists didn’t know how the essential cells, which exist in limited amounts in the body, decide which direction to go. Now, researchers in the University of Wisconsin Medical School Department of Pharmacology have found a mechanism that might determine what each HSC will be. The mechanism involves an unexpected interaction between two related proteins.

Timothy Kamp