Transcript Lecture 5(2-2-16) - Edward A. Dennis

BIOM 209/CHEM 210/PHARM 209
Sphingolipid and Sterol Metabolism,
Signaling and Lipidomics
Professor Edward A. Dennis
Department of Chemistry and Biochemistry
Department of Pharmacology, School of Medicine
University of California, San Diego
Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or individual
slides (without alteration) for academic, non-profit and non-commercial purposes. Attribution: Edward A.
Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org
E.A. DENNIS 2016 ©
Sphingolipid definitions
Sphingosine: a family of compounds, with the most common found in mammals being
this 18-carbon amino alcohol with a trans double bond; the starting point for ceramides.
Ceramide: a sphingosine molecule connected to a fatty acid by an amide bond.
Ceramides are the starting point for sphingomyelin, cerebrosides and gangliosides.
Sphingomyelin: a ceramide that has a phosphorylcholine head group in place
of its hydroxyl. Present in most mammalian cells, and rich in myelin sheaths
around nerves.
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Palmitoyl CoA + Serine = Sphingosine
Serine
Palmitoyl-CoA
1. Serine donates 2 carbons
and an amino group
2. Reduction of the carbonyl to
a hydroxyl
3. Acyl group added to convert
to a dihydroceramide
4. Then, oxidation to add a
double bond
Look familiar?
Resembles both b-oxidation
and D4 desaturation
N-acyl-Sphingosine (Ceramide)
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Sphingosine-1-phosphate
+H N
3
Sphingosine
kinase
Sphingosine
phosphatase
• Sphingosine can be phosphorylated by
sphingosine kinases, ubiquitous
enzymes in the cytosol, ER and nucleus
to make sphingosine-1-phosphate
(S1P).
• Sphingosine-1-phosphate, a
lysophospholipid, acts as a potent
messenger molecule that operates both
intra- and inter-cellularly.
• Within the cell, it promotes mitosis and
inhibits apoptosis. It also regulates
calcium mobilization and cell growth in
response to a variety of extracellular
stimuli.
+H N
3
•
•
Outside the cell, S1P exerts many of its
effects through interaction with five specific
G protein-coupled receptors on cell
surfaces. Different cells have different
receptor profiles.
S1P is vital to the function of several
immune cells. It is a major regulator of T
cell development, B and T cell
recirculation, tissue homing patterns, and
chemotactic responses to chemokines.
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Sphingosine + Fatty Acid = Ceramide
Sphingosine
Fatty acyl-CoA
R = (CH2)n-CH3
Simple acyl transfer,
but to an amide
bond instead of the
typical ester
Ceramide
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Ceramide + Choline = Sphingomyelin
Phosphocholine head
group gives sphingomyelin
a hydrophilic end
Choline carries a positive
charge the whole molecule
becomes more amphoteric
The phosphocholine
headgroup is transferred
to ceramide from PC
Choline
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Comparison of Sphingomyelin and PC
At least one fatty acid of PC is usually unsaturated or polyunsaturated,
whereas, SM is usually saturated or mono-unsaturated; therefore,
SM rich membranes are less “fluid” than typical PC-rich membranes.
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Comparison of S-1-P and LPA
Sphingosine-1-phosphate
(neutral zwitterion; net charge 0)
+H N
3
Lysophosphatidic acid
(Example: 1-myristoyl-sn-glycerophosphate)
(net negative charge)
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More Definitions
Galactose (polar head)
Ceramide (non-polar tail)
Glycosidic
bond
Cerebrosides: a ceramide
that has a sugar added to the
head group. Most commonly,
the sugar is glucose (Glu) or
galactose (Gal).
Sialic acid
Gangliosides: a ceramide
that has multiple sugars
including at least 1 sialic
acid residue added to the
head group. Increased
variety and complexity.
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Ceramide + Sugar = Cerebroside
Ceramide
UDP-Glucose
Sugar is activated
by UDP
UDP
Addition of sugar
occurs at the C1
OH group of
ceramide
Cerebroside
(Example:glucosyl-ceramide)
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Ceramide + (Many Sugars) = Gangliosides
GM1
GM2
GM3
Sugars are activated
by UDP
(sialic acid by CMP)
Each sugar is added
individually
Gangliosides can
have varied,
complex structures
They often function
as antigens and
surface markers
Stearic acid (C18) N-acyl chain
Trivia: Do you know your blood type? Is it A+? B-? O? The letters refer to the
specific multi-sugar structures are attached to gangliosides and proteins on the
surface of your red blood cells.
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Degradation of Sphingolipids
• The amide bond of sphingolipids does not break down easily
– which is why they make good membrane components
• Enzymatic degradation is used for turnover
– LOTS of degradation enzymes exist
• it’s a long, complicated bunch of pathways
• Genetic defects in these enzymes cause a long list of
diseases
–
–
–
–
all involve unhealthy accumulation of some sphingolipid
most are rare, but more common in specific ethnicities
key diseases: Gaucher’s, Tay-Sachs’, Fabry’s and Niemann-Pick
Resources: (Online Mendelian Inheritance in Man)
• OMIM Web site: www.ncbi.nih.gov/OMIM/searchomim.html
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Degradation of Sphingolipids
GM1
Sulfatide
Globoside
GM1 b-galactosidase
Hexosaminidase A/B
GM1 Gangliosidosis
Gal
GM2
GM3
Metachromatic leukodystrophy
GalNAc
Trihexosylceramide
GalNAc
Fabry’s disease
Lactosylceramide
Ganglioside neuraminidase
Galactocerebrosidase
Krabbe’s disease
Gal
Glucocerebroside
b-galactosidase
NANA
Sphingomyelin
SO42-
Galactocerebroside
a-galactosidase A
Hexosaminidase A
Tay-Sachs disease
Sandhoff’s disease
Arylsulfatase A
Gal
Gal
Ceramide
Glucocerebrosidase
Gaucher’s disease
Glc
Sphingomyelinase
Phosphocholine
Fatty acid
+
Niemann-Pick disease
Sphingosine
Ceramidase
Farber’s disease
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Tay-Sachs’ Disease
• Incidence: Like Gaucher’s but rarer
– ~1:30 Ashkenazi Jews are carriers
– ~1:500 carriers in general population
• Symptoms:Neurodegenerative
– mental retardation and seizures
– listlessness, fixed gaze, hypotonia
– cherry-red spot on retina (see picture)
• Mechanism: Genetic
– Lack of GM2 hexosaminidase A
Cherry-red spot on a patient’s retina, a
common finding in patients with TaySachs’ disease.
• Auto recessive, OMIM #272800
– Ganglioside GM2
• Builds up in CNS
• Treatments: No good therapy yet
Trivia: Injections of recombinant hexosaminidase A do not
help Tay-Sachs’ patients because it cannot cross the
blood-brain barrier.
– Supportive and symptomatic
– Patients die by age 5
– Gene therapy target (future)
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Gaucher’s Disease
• Incidence: Uncommon in most groups
– ~1:13 Ashkenazi Jews are carriers
• Symptoms:
– enlarged liver and spleen (see picture)
– easy bruising and bone fractures
– hyperpigmentation of skin
– sometimes: anemia
• Mechanism: Genetic
– Lack of working b-glucosidase
• Auto recessive, OMIM #230800
– Glucosyl acylsphingosine
• Builds up in liver, spleen & bone
Magic marker outlines of the enlarged
liver and spleen in a school-aged boy with
Gaucher’s disease. Note also the
hyperpigmented skin.
• Treatments:
– Recombinant acid b-glucosidase
– Symptomatic support
– Gene therapy target (future)
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Niemann-Pick Disease Type A
• Incidence: Type A is the most severe of
the 5 subtypes of Niemann-Pick Disease
– ~1:90 Ashkenazi Jews are carriers
• Symptoms: Neurodegenerative
– Large abdomen within 3-6 mos. and jaundice
– Progressive loss of early motor skills,
progressive spasticity, developmental delay
– Cherry red spot in the eye
– (Generally) a very rapid decline leading to
death by two to three years of age.
• Mechanism: Genetic
– Lack of Sphingomyelinase
• Auto recessive, OMIM #257200
– Sphingomyelin
Patient with Niemann Pick Disease
• builds up in CNS, liver and lungs
• Treatments:
– Supportive and symptomatic
– Patients die by age 3
– No effective therapy to date
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Summary of Today’s Sphingolipids
Molecule(s)
Synthesis Scheme
Significance
Sphingosine
Palmitoyl CoA + Serine
Brings in the amine group
Important signaling molecule
Ceramides
Sphingosine + Fatty Acid
Amide bond, hydrophobicity
Important signaling molecule
Sphingomyelins
Ceramide + PhosphoCholine
Amphoteric & charged, diseases
Membrane component
Cerebrosides
Ceramide + (Mono)saccharides
Amphoteric & neutral, diseases
Rich in brain
Gangliosides
Ceramide + Polysaccharides
+ Sialic acid
Complexity, diseases
Rich in brain
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Lipid Biochemistry - The Big Picture
Today’s
Topic
Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of
Biochemistry: Life at the Molecular Level, 2nd ed. Reprinted with
permission of John Wiley & Sons, Inc.
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Why Do We Care about Cholesterol?
Heart Disease
– #1 killer in US
– largely preventable
– strongly linked to
cholesterol
– overall deaths linked
as well (see graph)
Figure: Levine, NEJM, 332, 512-21 (1995).
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Cholesterol Structure and Numbering
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Cholesterol Stereochemistry
Stereochemical diagram of sterol nucleus
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Cholesterol Synthesis in a Nutshell
3 acetyl CoA’s
HMG-CoA
HMG-CoA
reductase
Mevalonate
Isopentyl
Pyrophosphate
Squalene
Cholesterol
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The Business End of Cholesterol Synthesis
thiolase
HMG-CoA
synthase
Regulated
Step!
HMG-CoA
reductase
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The Later Steps in Cholesterol Synthesis
Isoprene group
• Steps are irreversible
after isopentenyl
pyrophosphate
• Starting with
squalene, everything
is hydrophobic
– carrier proteins!
Isopentyl
Pyrophosphate
Squalene
Squalene
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Cholesteryl Esters - ACAT Style
Cholesterol
Acyl-CoA-cholesterol
acyl transferase
(ACAT)
Fatty Acyl-CoA
CoA-SH
• ACAT = “acyl CoA
cholesterol acyl
transferase”
• It acts mainly inside cells
• Donor acyl comes from a
free fatty acyl CoA
Cholesteryl ester
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Cholesteryl Esters - LCAT Style
Cholesterol
Phosphatidylcholine
(lecithin)
Lecitincholesterol
acyltransferase
(LCAT)
Cholesterol Ester
Lyso-phosphatidylcholine (lysolecithin)
• LCAT = “lecithin-cholesterol acyl transferase”
• Two-thirds of plasma cholesterol is esterfied by LCAT
• Acyl chain is donated from sn-2 position of phosphatidylcholine
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Bile Acids and Bile Salts
Cholesterol
7a-hydroxylase
Choline
Cholic Acid
• Made in the Liver (where the
cholesterol is being made)
• Made directly from
cholesterol
• Secreted into the bile ducts
and GI tract
• Reabsorbed and recycled
Cholic Acid – a bile acid
Glycine
Glycocholic Acid – a bile salt
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Steroid Hormone Synthesis
• A wide range of
hormones are
made from
cholesterol
–
–
–
–
Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of Biochemistry, 2 nd ed. Worth Publishers, Inc.
estrogen
testosterone
cortisol
aldosterone
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Regulation of Cholesterol Synthesis
• HMG-CoA reductase is
the regulated enzyme
• Inhibitors
– Glucagon
– XOL (negative feedback)
• Promotor: Insulin
• Intracellular synthesized
cholesterol downregulates
LDL receptors so cell
takes up less extracellular
LDL cholesterol
Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of Biochemistry, 2 nd ed. Worth Publishers, Inc.
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Removal of Bile Acids Upregulates 7α-Hydroxylase
Cholesterol
7a-hydroxylase
Choline
• Net result: decrease in
intracellular cholesterol in
liver
• Causes upregulation of
LDL receptors so increases
removal of circulating LDL
• Results in overall lowering
of plasma cholesterol
Cholic Acid
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Statin-Class Drugs
(Zocor)
• Lovastatin was first
• They inhibit HMG-CoA
reductase
• Decreases intracellular
cholesterol and upregulates
LDL receptors
• Mimic HMG-CoA; competitive
inhibitors
• Very effective at lowering
cholesterol
– 25-40% drop is common
• Widely prescribed
– Currently, 30% of people
over 65 years use a statin.
This is up from 12% in
1997.
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Sterol Regulated Promoters
SRE (Sterol regulatory element)
SREBP (SRE binding protein)
Figure: Brown, Nature, 343, 425-30 (1990 ).
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Proteolytic Release of SREBPs and Role of SCAP
SCAP: SREBP cleavage activating protein
Figure: Horton, JCI 109:1125-31 (2002)
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Regulation of Cellular Sterol Content
• HMG CoA reductase is controlled in several ways:
– The sterol regulated element binding protein (SREBP)
controls the rate of synthesis of HMG CoA reductase RNA.
This transcription factor binds to the Sterol regulatory
element -1 (SRE-1), a short DNA sequence on the 5’ side of
the gene. When inactive, it is in the ER associated with
SCAP (SREBP cleavage activating protein), a cholesterol
sensor. When the cholesterol level falls, SCAP escorts
SREBP into small membrane vesicles in the Golgi, and it is
released via two cleavages. Then, it migrates to the nucleus
and binds SRE to enhance transcription. As the cholesterol
level rises, cleavage is blocked and SREBP in the nucleus is
degraded, halting transcription.
– Translation of HMG CoA reductase mRNA is inhibited by
nonsterol metabolites derived from mevalonate and dietary
cholesterol.
– Degradation of HMG CoA reductase is strictly controlled.
– Phosphorylation decreases the activity of the reductase.
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SREBP Regulated Promoters:
Different Architecture, Distinct “Co-regulators”
LDL Receptor
5’ -
- 3’
HMG CoA Synthase
5’ -
- 3’
HMG CoA Reductase
5’ -
- 3’
Farnesyl Diphosphate Synthase
5’ -
- 3’
Squalene Synthase
5’ -
- 3’
Acetyl CoA Carboxylase
5’ -
- 3’
Fatty Acid Synthase
5’ -
- 3’
SREBP-2
5’ -
- 3’
SREBPs =
Sp1 =
NF-Y/CBF =
CREB/ATF =
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Role of SREBPs in Global Regulation of Lipid Metabolism
SREBP1a works equally at all promoters
Figure: Horton, JCI 109:1125-31 (2002)
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Summary: Regulation of Cellular Sterol Content
• Sterol control of transcription affects more than
30 genes involved in the biosynthesis of
cholesterol, triacylglycerols, phospholipids and
fatty acids.
• The regulation of these events is primarily due to
sterol-regulated transcription of key rate limiting
enzymes and by the regulated degradation of
HMG CoA reductase.
• Activation of transcriptional control occurs via the
cleavage of the membrane-bound transcription
factor sterol regulated element binding protein
(SREBP).
• Sterol regulatory element -1 (SRE-1) is in a
gene that is required for transcriptional control.
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