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Redefining the Role of Metallothionein within the Injured
Brain
EXTRACELLULAR METALLOTHIONEINS PLAY AN IMPORTANT ROLE
IN THE ASTROCYTE-NEURON RESPONSE TO INJURY.
Roger S. Chung1, Milena Penkowa2, Justin Dittmann1, Carolyn E. King3, Carole
Bartlett3, Johanne W. Asmussen2, Juan Hidalgo4, Javier Carrasco4, Yee Kee J.
Leung1, Adam K. Walker1, Samantha J. Fung1, Sarah A. Dunlop3, Melinda
Fitzgerald3, Lyn D. Beazley3, Meng I. Chuah1, James C. Vickers1, and Adrian K.
West1
1)NeuroRepair Group, Menzies Research Institute, University of Tasmania, Private
Bag 58, Hobart, Tasmania 7001, Australia,
2)Section of Neuroprotection, Faculty of Health Sciences, University of
Copenhagen, Copenhagen DK2200, Denmark,
3)School of Animal Biology, University of Western Australia, Nedlands, Western
Australia 6907, Australia, and
4)Institute of Neurosciences and Department of Cellular Biology, Physiology and
Immunology, Animal Physiology Unit, Faculty of Sciences, Autonomous University
of Barcelona, Bellaterra, Barcelona 08193, Spain
Journal of Biological Chemistry, Vol. 283, Issue 22, 15349-15358, May 30,
2008.
Abstract: A
number of intracellular proteins that are protective after brain injury are
classically thought to exert their effect within the expressing cell. The
astrocytic metallothioneins (MT) are one example and are thought to act via
intracellular free radical scavenging and heavy metal regulation, and in
particular zinc. Indeed, we have previously established that astrocytic MTs are
required for successful brain healing. Here we provide evidence for a
fundamentally different mode of action relying upon intercellular transfer from
astrocytes to neurons, which in turn leads to uptake-dependent axonal
regeneration. First, we show that MT can be detected within the extracellular
fluid of the injured brain, and that cultured astrocytes are capable of actively
secreting MT in a regulatable manner. Second, we identify a receptor, megalin,
that mediates MT transport into neurons. Third, we directly demonstrate for the
first time the transfer of MT from astrocytes to neurons over a specific time
course in vitro. Finally, we show that MT is rapidly internalized via the cell
bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal
regeneration through the inhibitory environment of the completely severed mature
optic nerve. Our work suggests that the protective functions of MT in the
central nervous system should be widened from a purely astrocytic focus to
include extracellular and intra-neuronal roles. This unsuspected action of MT
represents a novel paradigm of astrocyte-neuronal interaction after injury and
may have implications for the development of MT-based therapeutic agents.
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Metallothionein-IIA promotes neurite growth via the megalin receptor
Melinda Fitzgerald (1) , Pia Nairn (1),
Carole A. Bartlett (1), Roger S. Chung (3), Adrian K. West (3) and Lyn D.
Beazley (1, 2)
(1) Experimental and Regenerative Neurosciences, School of Animal Biology,
University of Western Australia, Hackett Drive, Crawley, 6009, WA, Australia
(2) Western Australian Institute of Medical Research, University of Western
Australia, Hackett Drive, Crawley, 6009, WA, Australia
(3) Neurorepair Group, Menzies Research Institute, University of Tasmania,
Private Bag 58, Hobart, TAS, 7001, Australia
Journal: Experimental Brain Research, Volume 183, Number 2 / November, 2007, p. 171-180
Open: Entire document (Subscription to the journal required).
Received: 20 February 2007 Accepted: 13 June 2007 Published online: 19 July 2007
Abstract: Metallothionein (MT)-I/II has been shown to be neuroprotective
and neuroregenerative in a model of rat cortical brain injury. Here we examine
expression patterns of MT-I/II and its putative receptor megalin in rat retina.
At neonatal stages, MT-I/II was present in retinal ganglion cells (RGCs) but not
glial or amacrine cells; megalin was present throughout the retina. Whilst
MT-I/II was absent from adult RGC in normal animals and after optic nerve
transection, the constitutive megalin expression in RGCs was lost following
optic nerve transection. In vitro MT-IIA treatment stimulated neuritic growth:
more RGCs grew neurites longer than 25 μm (P < 0.05) in dissociated retinal
cultures and neurite extension increased in retinal explants (P < 0.05). MT-IIA
treatment of mixed retinal cultures increased megalin expression in RGCs, and
pre-treating cells with anti-megalin antibodies prevented MT-IIA-stimulated
neurite extension. Our results indicate that MT-IIA stimulates neurite outgrowth
in RGCs and may do so via the megalin receptor; we propose that neurite
extension is triggered via signal transduction pathways activated by the NPxY
motifs of megalin’s cytoplasmic tail.
Keywords Metallothionein - Retinal ganglion cells - Neuroregeneration
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Metal binding of metallothionein-3 versus
metallothionein-2: lower affinity and higher plasticity
Peep Palumaa (1), , Indrek Tammiste (2), Keiu Kruusel (1), Liina Kangur (1),
Hans Jörnvall (3) and Rannar Sillard (3)
(1) Department of Gene Technology, Tallinn Technical University Akadeemia tee
23, 12618 Tallinn, Estonia
(2) National Institute of Chemical Physics and Biophysics, Akadeemia tee 23,
12618 Tallinn, Estonia
(3) Department of Medical Biochemistry and Biophysics, Karolinska Institutet,
S-171 77 Stockholm, Sweden
Journal: Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics. Volume 1747, Issue 2, 14 March 2005, Pages 205-211
Received 19 August 2004; revised 16 November 2004; accepted 16 November 2004.
Available online 8 December 2004.
Abstract
Mammalian metallothioneins (MTs) are involved in cellular metabolism of zinc
and copper and in cytoprotection against toxic metals and reactive oxygen
species. MT-3 plays a specific role in the brain and is down-regulated in
Alzheimer's disease. To evaluate differences in metal binding, we conducted
direct metal competition experiments with MT-3 and MT-2 using electrospray
ionization mass spectroscopy (ESI-MS). Results demonstrate that MT-3 binds Zn2+
and Cd2+ ions more weakly than MT-2 but exposes higher metal-binding capacity
and plasticity. Titration with Cd2+ ions demonstrates that metal-binding
affinities of individual clusters of MT-2 and MT-3 are decreasing in the
following order: four-metal cluster of MT-2>three-metal cluster of
MT-2≈four-metal cluster of MT-3>three-metal cluster of MT-3>extra metal-binding
sites of MT-3. To evaluate the reasons for weaker metal-binding affinity of MT-3
and the enhanced resistance of MT-3 towards proteolysis under zinc-depleted
cellular conditions, we studied the secondary structures of apo-MT-3 and
apo-MT-2 by CD spectroscopy. Results showed that apo-MT-3 and apo-MT-2 have
almost equal helical content (approximately 10%) in aqueous buffer, but that
MT-3 had slightly higher tendency to form α-helical secondary structure in TFE–water
mixtures. Secondary structure predictions also indicated some differences
between MT-3 and MT-2, by predicting random coil for common MTs, but 22%
α-helical structure for MT-3. Combined, all results highlight further
differences between MT-3 and common MTs, which may be related with their
functional specificities.
Keywords: MT-3; Alzheimer's disease; ESI-MS; Secondary structure
prediction; CD spectroscopy
Abbreviations: MT, metallothionein; DTT, dithiothreitol; TFE,
2,2,2,-trifluoroethanol; CD, circular dichroism; ESI MS, electrospray ionization
mass spectroscopy
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Metal binding to brain-specific metallothionein-3 studied by electrospray ionization mass spectrometry.
Palumaa P (1), Eriste E (2), Kruusel K (1), Kangur L, (1) Jörnvall H (2), Sillard R (2).
(1) Department of Gene Technology, Tallinn Technical University, Ehitajate tee
5, EE-19086 Tallinn, Estonia.
(2) Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Journal: Cell Mol Biol (Noisy-le-grand). 2003
Jul;49(5):763-768.
Abstract
Metallothionein-3 (MT-3) is a brain-specific isoform of metallothioneins,
which is down-regulated in Alzheimer's disease (AD), inhibits the growth of
neurons in vitro, and differs from common MTs also in gene regulation. To
elucidate the differences in structure and function between MT-3 and common MTs,
Zn2+ and Cd2+ binding to MT-3 and MT-1 were studied using electrospray
ionization time of flight mass spectrometry (ESI TOF MS) at pH values between
7.5 and 2.7. The metal binding properties of MT-3 differ considerably from those
of MT-1. After reconstitution with a metal excess, metallated MT-3 exists as a
mixture of Zn7MT-3 (or Cd7MT-3, respectively) and several metalloforms with
stoichiometries below and above seven. In contrast, MT-1 exists as a single
Zn7MT-1 (or Cd7MT-1). Lowering of pH leads to a stepwise release of metals from
metallated MT-3, first from extra sites, then from the 3-metal cluster and
finally from the 4-metal cluster. At acidic pH values the 4-metal cluster of
MT-3 is slightly more stable than that of MT-1. The results demonstrate higher
structural plasticity, dynamics and metal binding capacity of MT-3 than of MT-1,
which makes MT-3 suitable as a zinc buffer-transfer molecule in zinc-enriched
neurons functioning at conditions of fluctuating zinc concentrations.
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Brain-Specific Metallothionein-3 Has Higher Metal-Binding
Capacity than Ubiquitous Metallothioneins and Binds Metals Noncooperatively
Peep Palumaa, Elo Eriste, Olga Njunkova, Lesja Pokras, Hans Jörnvall, and
Rannar Sillard
Centre for Gene Technology, Tallinn Technical University, Ehitajate tee 5,
EE-19086 Tallinn, Estonia,
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden,
National Institute of Chemical Physics and
Biophysics, Akadeemia tee 23, EE-12618 Tallinn, Estonia
Journal: : Biochemistry. 2002 May
14;41(19):6158-6163.
Received February 11, 2002, Revised Manuscript
Received March 26, 2002
Abstract:
Zinc metabolism in the cells is largely regulated by ubiquitous small
proteins, metallothioneins (MT). Metallothionein-3 is specifically expressed in
the brain and is down regulated in Alzheimer's disease. We demonstrate by mass
spectrometry that MT-3, in contrast to common MTs, binds Zn2+ and Cd2+ in a
noncooperative manner and can also bind higher stoichiometries of metals than
seven. MT-3 reconstituted with seven metals exists in a dynamic equilibrium of
different metalloforms, where the prevalent metalloform is Me7MT-3, but
metalloforms with 6, 8, and even 9 metals are also present. The results from pH
and stability studies demonstrate that the heterogeneity of metalloforms
originates from the N-terminal -cluster, whereas the C-terminal -cluster of MT-3
binds four metal ions such as that of common MTs. Experiments with EDTA
demonstrate that the -cluster of ZnMT-3 has a higher metal transfer potential
than the -cluster of Zn7MT-2. Moreover, ZnMT-3 loses metals during
ultrafiltration. MT-3, reconstituted with an excess of Zn2+ or Cd2+, exists as a
dynamic mixture of metalloforms with higher than 7 metal stoichiometries (8-11).
Such forms of ZnMT-3 are unstable and decompose partly already during a rapid
gel filtration, whereas CdMT-3 forms are more stable. Extra metal ions may bind
to the -cluster region as well as to the carboxylates of MT-3. The specific
metal-binding properties of MT-3 could be functionally implemented for buffering
of fluctuating concentrations of zinc in zincergic neurons and for transfer of
zinc to synaptic vesicles.
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