- A series of point mutations were made to NPC2 to neutralize charged residues on its surface. Kinetic studies of these mutants showed that certain residues are necessary for NPC2's cholesterol transport function.
- Turbidity assays suggested that NPC2 may bind to more than one membrane simultaneously. This led to the hypothesis that NPC2 transports cholesterol by forming inter-membrane bridges between the inner endo/lysosomal membranes and other membranes, facilitating cholesterol egress from the compartment.
- Methods like tryptophan fluorescence quenching and resonance energy transfer were used to quantify sterol transfer rates of NPC2 and its mutants between membranes. Preliminary results support the hypothesis that NPC2 can bind two membranes at once to
Presentation made by Dr. Paul Taylor on October 30, 2015 at the Alzforum-hosted live webinar titled "Fluid Business: Could “Liquid” Protein Herald Neurodegeneration?"
More information and the recording of the session available at http://www.alzforum.org/webinars/fluid-business-could-liquid-protein-herald-neurodegeneration
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Ther...星云 王
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function
>, only for private study use, please do not use it for profit or public.
Presentation made by Dr. Paul Taylor on October 30, 2015 at the Alzforum-hosted live webinar titled "Fluid Business: Could “Liquid” Protein Herald Neurodegeneration?"
More information and the recording of the session available at http://www.alzforum.org/webinars/fluid-business-could-liquid-protein-herald-neurodegeneration
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Ther...星云 王
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function
>, only for private study use, please do not use it for profit or public.
and lifelong absence of FXR as occurs inthe FXR-null model, .docxjustine1simpson78276
and lifelong absence of FXR as occurs in
the FXR-null model, can result in
abnormal metabolic effects that are quite
different from those caused by acute,
transient antagonism of this receptor.
Because the FXR-null mouse was
produced using Cre–loxP technology,
conditional disruption of this allele after
normal development has occurred can
now be used to help resolve this issue. An
alternative explanation is that the site(s)
of pharmacological action of
guggulsterone do not include all of the
tissues in which FXR is functional, such as
the liver and gut (i.e. although FXR
synthesis is uniformly absent from all
tissues of the FXR-null mouse model,
guggulsterone might antagonize FXR only
within a subset of these sites). In the
absence of in vivo data regarding the
modulation of FXR target gene expression
by guggulsterone, this is difficult to judge.
Thus, it remains a possibility that the
effects of orally-administered
guggulsterone occur primarily at the level
of the gut (i.e. versus gut and liver), for
instance, by affecting cholesterol
absorption and bile-acid reuptake
processes regulated by FXR, rather than
the hepatic biosynthesis and transport of
bile acids. Again, the conditional nature of
the strategy used to create the FXR-null
mouse model allows for tissue-specific
deletion of the FXR gene and might help
resolve this issue.
As reinforced by the recent work of
Urizar et al. [3], as well as by the present
therapeutic use of bile-acid binding
resins for hypercholesterolemia, there
exists an intimate linkage between bile
acid and cholesterol metabolism. Recent
demonstrations that FXR is also involved
in the regulation of genes (e.g. encoding
apolipoprotein A-I, apolipoprotein C-II
and phospholipids transfer protein) [4–6]
more closely linked with lipid rather
than bile-acid homeostasis, presents
additional avenues by which FXR
ligands could be beneficial for the
treatment of disorders of lipid
metabolism. As suggested by the work of
Urizar et al. [3] and others (e.g. [7]),
careful and comprehensive study of the
effects of natural products, such as
guggulsterone, on the function of nuclear
hormone receptors, is likely to yield
additional agents with desirable
therapeutic effects.
References
1 Sinal, C.J. et al. (2000) Targeted disruption of the
nuclear receptor FXR/BAR impairs bile acid and
lipid homeostasis. Cell 102, 731–744
2 Singh, R.B. et al. (1994) Hypolipidemic and
antioxidant effects of Commiphora mukul as an
adjunct to dietary therapy in patients with
hypercholesterolemia. Cardiovasc. Drugs Ther. 8,
659–664
3 Urizar, N.L. et al. (2002) A natural product that
lowers cholesterol as an antagonist ligand for
FXR. Science 296, 1703–1706
4 Claudel, T. et al. (2002) Bile acid-activated nuclear
receptor FXR suppresses apolipoprotein A-I
transcription via a negative FXR response
element. J. Clin. Invest. 109, 961–971
5 Kast, H.R. et al. (2001) Farnesoid X-activated
receptor induces apolipoprotein C-II
transcription: a molecular mech.
Control of Local Protein Synthesisand Initial Events in Myel.docxrichardnorman90310
Control of Local Protein Synthesis
and Initial Events in Myelination by
Action Potentials
Hiroaki Wake, Philip R. Lee, R. Douglas Fields*
Formation of myelin, the electrical insulation on axons produced by oligodendrocytes, is
controlled by complex cell-cell signaling that regulates oligodendrocyte development and myelin
formation on appropriate axons. If electrical activity could stimulate myelin induction, then
neurodevelopment and the speed of information transmission through circuits could be modified
by neural activity. We find that release of glutamate from synaptic vesicles along axons of
mouse dorsal root ganglion neurons in culture promotes myelin induction by stimulating formation
of cholesterol-rich signaling domains between oligodendrocytes and axons, and increasing local
synthesis of the major protein in the myelin sheath, myelin basic protein, through Fyn
kinase-dependent signaling. This axon-oligodendrocyte signaling would promote myelination of
electrically active axons to regulate neural development and function according to
environmental experience.
Myelin, the multilayered membrane of
insulation wrapped around axons by
oligodendrocytes, is essential for ner-
vous system function and increases conduction
velocity by at least 50 times (1, 2). Unique to
vertebrates, formation of the myelin sheath must
be highly regulated temporally during develop-
ment and targeted specifically to appropriate
axons. Many axon-derived signals regulate my-
elination, but there is great interest in the pos-
sibility that electrical activity could provide an
instructive signal, because activity-dependent
regulation of myelinogenesis could control my-
elination during development according to en-
vironmental experience, contribute to learning,
and guide regeneration after injury according to
functional efficacy (3). Electrical activity has
been shown to affect proliferation and differen-
tiation of myelinating glia (4–7), but if electrical
activity could regulate subcellular events neces-
sary for myelin induction, then myelin could
form preferentially on electrically active axons.
Here we test this hypothesis, beginning with the
question of how electrical activity in axons might
signal to oligodendrocytes to control myelination.
Both neurotransmitters adenosine 5′-triphosphate
(ATP) and glutamate (glu) have been implicated in
signaling to oligodendrocyte progenitor cells
(OPCs). Glutamatergic synapses can form tran-
siently between axons and someOPCs (8, 9). It has
been proposed that such synaptic communication
Nervous System Development and Plasticity Section, The Eunice
Kennedy Shriver National Institute of Child Health and Human
Development, Bethesda, MD 20892, USA.
*To whom correspondence should be addressed. E-mail:
[email protected]
Fig. 1. Release of synaptic vesicles from axons
promotes myelination. (A) Synaptic vesicle release
from DRG neurons was blocked by adding BnTX or
TnTX to neuron cultures, and OPCs were added
after washing.
and lifelong absence of FXR as occurs inthe FXR-null model, .docxjustine1simpson78276
and lifelong absence of FXR as occurs in
the FXR-null model, can result in
abnormal metabolic effects that are quite
different from those caused by acute,
transient antagonism of this receptor.
Because the FXR-null mouse was
produced using Cre–loxP technology,
conditional disruption of this allele after
normal development has occurred can
now be used to help resolve this issue. An
alternative explanation is that the site(s)
of pharmacological action of
guggulsterone do not include all of the
tissues in which FXR is functional, such as
the liver and gut (i.e. although FXR
synthesis is uniformly absent from all
tissues of the FXR-null mouse model,
guggulsterone might antagonize FXR only
within a subset of these sites). In the
absence of in vivo data regarding the
modulation of FXR target gene expression
by guggulsterone, this is difficult to judge.
Thus, it remains a possibility that the
effects of orally-administered
guggulsterone occur primarily at the level
of the gut (i.e. versus gut and liver), for
instance, by affecting cholesterol
absorption and bile-acid reuptake
processes regulated by FXR, rather than
the hepatic biosynthesis and transport of
bile acids. Again, the conditional nature of
the strategy used to create the FXR-null
mouse model allows for tissue-specific
deletion of the FXR gene and might help
resolve this issue.
As reinforced by the recent work of
Urizar et al. [3], as well as by the present
therapeutic use of bile-acid binding
resins for hypercholesterolemia, there
exists an intimate linkage between bile
acid and cholesterol metabolism. Recent
demonstrations that FXR is also involved
in the regulation of genes (e.g. encoding
apolipoprotein A-I, apolipoprotein C-II
and phospholipids transfer protein) [4–6]
more closely linked with lipid rather
than bile-acid homeostasis, presents
additional avenues by which FXR
ligands could be beneficial for the
treatment of disorders of lipid
metabolism. As suggested by the work of
Urizar et al. [3] and others (e.g. [7]),
careful and comprehensive study of the
effects of natural products, such as
guggulsterone, on the function of nuclear
hormone receptors, is likely to yield
additional agents with desirable
therapeutic effects.
References
1 Sinal, C.J. et al. (2000) Targeted disruption of the
nuclear receptor FXR/BAR impairs bile acid and
lipid homeostasis. Cell 102, 731–744
2 Singh, R.B. et al. (1994) Hypolipidemic and
antioxidant effects of Commiphora mukul as an
adjunct to dietary therapy in patients with
hypercholesterolemia. Cardiovasc. Drugs Ther. 8,
659–664
3 Urizar, N.L. et al. (2002) A natural product that
lowers cholesterol as an antagonist ligand for
FXR. Science 296, 1703–1706
4 Claudel, T. et al. (2002) Bile acid-activated nuclear
receptor FXR suppresses apolipoprotein A-I
transcription via a negative FXR response
element. J. Clin. Invest. 109, 961–971
5 Kast, H.R. et al. (2001) Farnesoid X-activated
receptor induces apolipoprotein C-II
transcription: a molecular mech.
Control of Local Protein Synthesisand Initial Events in Myel.docxrichardnorman90310
Control of Local Protein Synthesis
and Initial Events in Myelination by
Action Potentials
Hiroaki Wake, Philip R. Lee, R. Douglas Fields*
Formation of myelin, the electrical insulation on axons produced by oligodendrocytes, is
controlled by complex cell-cell signaling that regulates oligodendrocyte development and myelin
formation on appropriate axons. If electrical activity could stimulate myelin induction, then
neurodevelopment and the speed of information transmission through circuits could be modified
by neural activity. We find that release of glutamate from synaptic vesicles along axons of
mouse dorsal root ganglion neurons in culture promotes myelin induction by stimulating formation
of cholesterol-rich signaling domains between oligodendrocytes and axons, and increasing local
synthesis of the major protein in the myelin sheath, myelin basic protein, through Fyn
kinase-dependent signaling. This axon-oligodendrocyte signaling would promote myelination of
electrically active axons to regulate neural development and function according to
environmental experience.
Myelin, the multilayered membrane of
insulation wrapped around axons by
oligodendrocytes, is essential for ner-
vous system function and increases conduction
velocity by at least 50 times (1, 2). Unique to
vertebrates, formation of the myelin sheath must
be highly regulated temporally during develop-
ment and targeted specifically to appropriate
axons. Many axon-derived signals regulate my-
elination, but there is great interest in the pos-
sibility that electrical activity could provide an
instructive signal, because activity-dependent
regulation of myelinogenesis could control my-
elination during development according to en-
vironmental experience, contribute to learning,
and guide regeneration after injury according to
functional efficacy (3). Electrical activity has
been shown to affect proliferation and differen-
tiation of myelinating glia (4–7), but if electrical
activity could regulate subcellular events neces-
sary for myelin induction, then myelin could
form preferentially on electrically active axons.
Here we test this hypothesis, beginning with the
question of how electrical activity in axons might
signal to oligodendrocytes to control myelination.
Both neurotransmitters adenosine 5′-triphosphate
(ATP) and glutamate (glu) have been implicated in
signaling to oligodendrocyte progenitor cells
(OPCs). Glutamatergic synapses can form tran-
siently between axons and someOPCs (8, 9). It has
been proposed that such synaptic communication
Nervous System Development and Plasticity Section, The Eunice
Kennedy Shriver National Institute of Child Health and Human
Development, Bethesda, MD 20892, USA.
*To whom correspondence should be addressed. E-mail:
[email protected]
Fig. 1. Release of synaptic vesicles from axons
promotes myelination. (A) Synaptic vesicle release
from DRG neurons was blocked by adding BnTX or
TnTX to neuron cultures, and OPCs were added
after washing.
Bridging length and time scales in biomolecular systemsuvacolloquium
Speaker: prof. dr. Peter Bolhuis
Date: 27-11-2015
Abstract: Biomolecular systems, whether in living cells or in complex biomaterials, often undergo rare but important conformational changes; for instance, proteins can fold and (partially) unfold, bind and dissociate again, lipid membranes can phase separate, or transform shape and topology, DNA can hybridize, supercoil, and so on. Molecular dynamics simulation can in principle provide useful predictions of such processes on an atomistic level that are complementary to experiment. However, in practice, molecular dynamics is far from fulfilling this promise due to the large size of a cellular system (billions of atoms) and the long times involved (at least on the order of seconds). The multiscale modeling framework aims to circumvent this problem by envisioning hierarchical levels of description, each appropriate for certain length and time scales of a biophysical system. The challenge for the computer simulator is to develop the right description for each length or time sc!
ales, to link different scales together and to develop efficient sampling algorithms. One approach is to start with the atomistic level molecular dynamics description and use this to define a coarser grained description. Another approach is to realize that many processes such as (un)folding and other conformational changes in proteins in fact are rare events caused by high free energy barriers between stable states. To overcome such barriers, many techniques have been developed, e.g. replica exchange, metadynamics, and transition path sampling. In this presentation I will focus on the need for such simulation methods, and exemplify these methods on interesting applications such as proton transfer in a protein environment, conformational changes in (signalling) proteins, chaperone function, protein and fibril self-assembly, cooperativity in G-coupled receptors. In each case, we can gain novel insight from these simulations.
1. Structure-function analysis of NPC2 suggests a mechanism of cholesterol transport in the late
endo/lysosomal compartment
Richa Mehta, Leslie McCauliff, Judith Storch
Department of Nutritional Sciences and Rutgers Center for Lipid Research, Rutgers, the State University of New Jersey, 96 Lipman Drive, New Brunswick, NJ 08901
Abstract
Niemann-Pick C disease is a rare, inherited disease
in which cholesterol builds up to toxic levels in the
late endo/lysosomes (LE/LYs). Deficiencies in either
of two lysosomal proteins, NPC1 or NPC2 have
been implicated in the disease. NPC2 is a soluble
intralysosomal protein that binds cholesterol in vitro.
In previous studies shown by our lab NPC2 exhibits
cholesterol transport properties, likely explaining its
role in normal LE/LY cholesterol egress. Cholesterol
transfer was shown to occur via direct interaction of
NPC2 with donor or acceptor membranes, and
transfer rates were markedly enhanced by the
inclusion of the unique lysosomal phospholipid, bis-
monoacylglycerol phosphate (BMP/LBPA). To
elucidate the structural basis of the NPC2
cholesterol transfer function, a series of point
mutations that neutralize charged residues on the
surface of NPC2 are constructed. These mutants
bind cholesterol normally. An examination of the
cholesterol transfer properties of eleven mutants
using fluorescence quenching and resonance
energy transfer assays suggest s that there may be
more than one domain on the NPC2 surface that is
involved in cholesterol trafficking. To test this
hypothesis we will use a turbidity assay which
monitors the aggregation or fusion of large
unilamellar vesicles in the presence of NPC2
protein. Preliminary results from membrane
aggregation studies suggest that NPC2 may in fact
bind greater than one membrane simultaneously,
effectively forming a “bridge” for rapid transport of
cholesterol from internal LE/LY membranes,
eventually leading to efflux of cholesterol from the
LE/LY.
Introduction
Niemen Pick C (NPC) is an inherited lipid storage disorder in
which the intracellular trafficking of cholesterol is disturbed,
resulting in the accumulation of the unesterified cholesterol
and glycolipids in the late endosome/lysosomes. This results
in physiological consequences, almost always including
neurodegeneration, which is thought to secondary to the
specific absence of normal post- lysosomal cholesterol
metabolism and the effects of general lysomal dysfunction.
This dysfunction arises from the build in up of cholesterol and
other lipids in LE/LY. Defects in either NPC1 or NPC2 gene
product causes the accumulation of cholesterol, yet the exact
function of NPC1 and NPC2 in lysosomal cholesterol
trafficking remains unknown. In previous studies, we
examined the transfer of cholesterol from NPC2 protein to
membranes in vitro, using a fluorescence quenching assay.
This experiment showed that NPC2 could be rapidly
transporting cholesterol. To further investigate this
hypothesis we are currently studying the structure-function
relation of NPC2 in the transport of cholesterol in the late
endo/lysosomals.
Acknowledgment
We thank Dr. Matt Scott for providing plasmid for the K32A, D72A and K75A mutants. Also, I would like to
thank every one who helped me in the research and would like to thank my parents for their
support
Funding source
Summary
• A series of NPC2 point mutants were prepared by site directed mutagenesis. Kinetic studies
on these mutants show that certain charged residues on the surface of NPC2 appear
necessary for the sterol transport property of NPC2.
• Turbidity assay suggests that NPC2 may bind to more than one membrane simultaneously.
• It is hypothesized that cholesterol transfer by NPC2 may involve the formation of inter-
membrane bridges that effectively move cholesterol out of the inner endo/lysosomal
membranes for eventual egress from the compartment.
Various Sites on the NPC2 surface Mutated in the study
Wavelength, nm
290 300 310 320 330 340 350 360 370
TrpEmission
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
2 uM NPC2
2 uM NPC2 + 1 uM Chol
2 uM NPC2 + 2 uM Chol
Time (sec)
0 10 20 30 40 50
0.0071
0.0072
0.0073
0.0074
0.0075
0.0076
0.0077
RelativeFluorescence
Wavelength, nm
320 360 400 440 480 520 560
Fluorescenceintensity
0
5000
10000
15000
20000
25000
30000
Asp85
Lys97
Glu99
Lys115
Lys6
Lys97
Lys16
Glu97
Glu70
Asn39
Lys75
Asp72
Arg32
Time, sec
0 200 400 600 800 1000 1200
Tryptophanemission
0.990
0.995
1.000
1.005
1.010 wt NPC2
K97A
K75A
K32A
D72A
D85A
no WT D72AK75AD85AK115AN39AK32AK16AE70A K6A E99AK97A
DHEintramembranetransferrate,s
-1
0.000
0.002
0.004
0.006
0.008
0.010
0.012
[Protein], uM
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
A350
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
Time, sec
0 2 4 6 8 10
A350
0.015
0.016
0.017
0.018
0.019
0.020
These are tryptophan
fluorescence of NPC2 with
different amounts of cholesterol
Cholesterol transfer from NPC2 protein
(to membranes is monitored by
dequenching of tryptophan fluorescence.
DHE is a florescent cholestrol analyse.
A fluorescence resonance energy
transfer (FRET) assay is utilized to
monitor DHE transfer. DHE transfer
from donor to acceptor membranes is
monitored by energy transfer quenching
of DHE by acceptor membrane Dansyl-
phosphatidylethanolamine.
Structure-Function Analysis of NPC2
Effect of specific mutations on DHE
intermembrane transfer rates
Turbidity assay: To determine whether NPC2 can cause
membrane-membrane aggregation
BSA
NPC2NPC2 with LUV
containing LBPA
NPC2 with EPC
LUV
BSA with
EPC and LUV
Point mutants of NPC2 were obtained by
site directed mutagenesis and sterol
transport properties were examined. For this
study, wt and all mutants contained a myc-
6xhis tag
Cholesterol transfer from NPC2
protein (wt and mutants) is very
slow for some of the mutants.
NPC2 causes aggregation of
membranes, as indicated by the
increase of A350.
The results shows NPC2, but not
BSA, caused aggregation/fusion
of LUV, and LBPA in LUV makes
this process much faster.
Methods for quantifying sterol transfer rates
Ara Parseghian Medical Research Foundation
These results suggest that there may be more than one membrane binding domain, implying
that NPC2 could be binding to two membranes at the same time. This is why turbidity
experiments were done, and these results support the hypothesis.