Cell Signaling

Niketa A. Patel, Ph.D.

Department of Molecular Medicine

Office: USF Connect, 303-4
E-mail: [email protected]
 Summary of previous lecture

• Steps involved in cell signaling: Reception, Transduction, Response

– Reception of signal can be contact dependent or independent
– Transduction of signal is via receptors. Receptors can be intracellular or cell-surface

• Targets of signaling pathways- example, transcription factors

• Steroid hormones have intracellular receptors target for therapy

– Estrogen receptor and SERM

• Growth factors, peptide hormones have cell surface receptors Enzyme-

linked receptors leading to protein phosphorylation cascade and Ras GTPase
pathway
Major Classes of Transmembrane
Receptors
1. Ligand Gated Ion
Channels

2. Enzyme-linked
receptors

3. Cytokine receptors

4. GPCR
 3. Cytokine Receptors

Some receptors do not have intrinsic kinase

activity. These cytokine receptors are associated
with a protein kinase family, Janus family (JAKs)
which when autophosphorylated recruit STATs
(signal transducers and activators of transcription).
3. Cytokine Receptors
 JAK STAT Pathway
Cytokines: eg IL6, IFNa,b,g; NFkB

Growth, Proliferation, cell

fate determination, stem
cell renewal
 4. G Protein Coupled Receptors (GPCRs)

© 2002 Nature Publishing Group Li, J. et al. The Molecule

Pages database. Nature 420, 716-717 (2002).
GPCR
 G Protein Coupled Receptors (GPCRs)

Classification Scheme of GPCRs. Class A (Rhodopsin-like),

Class B (Secretin-like),Class C (Glutamate Receptor-like),
Others (Adhesion (33), Frizzled (11), Taste type-2 (25),
unclassified (23)).[6]

https://gpcrdb.org
Different G proteins mediate distinct
responses
GPCR regulation of Adenylyl Cyclase
 GPCR

Associated enzyme activates second messengers

Phosphorylation of other
targets

Biochim. Biophys. Acta (1998). 1422, 207-234.

Common second messengers
cAMP regulated Protein Kinase A
Complexities of GPCR
GPCR in Diseases
 Drug Discovery
Targeting Signaling Pathways
 Drug Discovery- Targeting Signaling
Pathways
Example 1. Block the receptor-e.g. Herceptin (trastuzamab)
 Drug Discovery- Targeting Signaling
Pathways
Example 2. Target cancer cell with antibody – toxin conjugate
 Drugs targeting Growth Factor Pathway

Figure 16.8 The Biology of Cancer (© Garland Science 2007)

Drug Targets: G Protein Coupled Receptors (GPCRs)

~60% of all pharmaceuticals target GPCRs

Signal transduction pathway cross-talk
 Signaling Cascade

• A hormone binding to its receptor activates

the signaling cascade amplification of signal
– Hormone can bind to multiple receptors before
being degraded
– Each receptor can activate several enzymes
– These enzymes produce second messengers
– Result in transcription of several genes, and
other metabolic effects
Modes of down regulation of signaling
 Summary

• General Principles of Cell Signaling: Reception,

Transduction, Response
• Intercellular signaling between cell originating signal and
target cell
• Intracellular signaling involves transduction of signal
within cells to elicit response
• Components of intracellular signaling: Types of receptors-
Difference between Enzyme linked receptor (activation of
kinase activity e.g. Ras) and cytokine receptors
(autophosphorylation of JAK and recruitment of STAT) and
GPCR (activate enzymes e.g. adenylyl cyclase, PLC)
• Second messengers: cAMP, IP3, DAG, Calcium
• Cross-talk and Integration
• Medical and Pharmaceutical Implications
 Lipoproteins

Niketa A. Patel, Ph.D.

Department of Molecular Medicine

Office: USF Connect, 303-4
E-mail: [email protected]
 Objectives

• Understand the distribution (Transport and Cellular Uptake) of lipids

• Know the major constituents of Lipoproteins and their function

• Know the major enzymes of lipoprotein metabolism: Lipoprotein Lipase (LPL),

Lecithin:Cholesterol Acyl Transferase (LCAT), and Cholesterol ester transfer
Protein (CETP)

• Understand the inter-conversion of lipoprotein particles

• Know the difference between LDL and HDL, and why HDL is considered the
“good” form of cholesterol

• Understand the basis for the LDL hypothesis of atherosclerosis.

“Textbook of Biochemistry with Clinical Correlations” (Thomas M. Devlin) Chapters 3 and 18

 Lipids

• Derived from fatty acids

• E.g. fats, oils, waxes, steroids and sterols, hormones
• Function
– Energy source
– Component of cell membranes
– Hormones; cell:cell communication
– Insulation for nerves
– Protection and insulation for organs
– Vitamins A, D, E, K are lipid soluble
– Buoyancy
 Transport of Lipids
Why do lipids present a special challenge?
Store energy Fuel source

Triacylglycerols
(Fats,
Triglycerides)

Fatty Acids

How does the body transport lipids: cholesterol and triglycerides?

Transport of Lipids: Lipoproteins

Composition of a lipoprotein particle

Anatomy of a Lipoprotein Particle

Phospholipid
 Lipoproteins were discovered and
named by their Density

Figure 3.46 Correspondence of plasma lipoprotein density classes with electrophoretic mobility in
a plasma electrophoresis.
Reprinted with permission from Soutar, A.K., and Myant, N.B. In Offord, R.E. (Ed) Chemistry of Macromolecules, IIB. Baltimore, MD:
University Park, Press, 1979.
Textbook of Biochemistry with Clinical Correlations, 7e edited by Thomas M. Devlin © 2011 John Wiley & Sons, Inc.
 Generalized structure of plasma lipoproteins

Chylomicron VLDL
Cholesterol: 3% Cholesterol : 10%
Phospholipid: 5% Phospholipid: 10%
Triacylglycerol: 90% Triacylglycerol: 70%

HDL
Cholesterol : 20%
Phospholipid: 25%
Triacylglycerol: 5% LDL
Cholesterol : 26%
Phospholipid: 15%
Triacylglycerol: 10%

* Relative to total weight

Lipoproteins Fractions- Density x Diameter
 Apoproteins are on the surface, providing
structure and function (binding, enzyme activation)
 Composition of Major Lipoproteins

ApoA-1: unique to HDL -- Structural and enzyme activator

ApoB: unique to non-HDL particles -- Structural and ligand for receptor binding
 Mechanism of Apolipoprotein B mRNA Editing
G7m -CAA-
14.1 kb Apolipoprotein B mRNA

INTESTINE
Cytidine Deaminase
NH4+ Editing Complex
(ACTIVE)
Stop
G7m -UAA-
Translation
LIVER
INTESTINAL ApoB48 protein

Gln
G7m -CAA-
Translation

LIVER ApoB100 protein

 AS A RESULT OF RNA
EDITING:

ApoB 48 is expressed in the

intestinal mucosal cells and
involved in receptor mediated
Chylomicron Pathway chylomicron remnant uptake by the
liver.

Apo B100 is expressed in the liver

and involved in receptor mediated
LDL uptake by the liver.

VLDL-LDL Pathway
 Chylomicrons

TG: Triglyceride CHOL: Cholesterol

Lipoprotein Lipase (LPL) Reaction

LPL
VLDL and LDL

C
LCAT
CE

LCAT

LCAT: Lecithin:cholesterol acyl transferase

 LCAT Reaction

/lecithin /lysolecithin

LCAT

LCAT
Acyl-coenzyme A:cholesterol acyltransferases
(ACAT)
HDL Particles- Reverse Cholesterol
Transport

CETP: Cholesterol ester transfer protein

CETP: Cholesterol ester transfer protein

CE
HDL
CE
C
E

C
T C

E
P E

T
T

P
P
CE CETP CE
TG
LDL VLDL
CE CETP CE
 HDL Particles- Reverse Cholesterol
Transport

• HDL removes cholesterol from tissue and

transports it to liver
• Liver disposes cholesterol as bile acids
• HDL is called “good” cholesterol
 Cholesterol is taken up via LDL
receptors

Joseph L. Goldstein, and Michael S. Brown Arterioscler

Thromb Vasc Biol. 2009;29:431-438
Copyright © American Heart Association, Inc. All rights reserved.
 Organs and pathways involved in plasma
lipoprotein metabolism

Sources
Target Organs
Triglyceride
and
cholesterol
 Organs and pathways involved in plasma
lipoprotein metabolism

Sources
Target Organs
Triglyceride
and
cholesterol

Lipoprotein
Transport
Particle
 Organs and pathways involved in plasma
lipoprotein metabolism

Sources

Triglyceride
and
cholesterol
Lipoprotein
Transport Protein component: Apolipoproteins
Particle Functions
1. Solubility, structural integrity
2. receptor binding- targeting
3. Enzyme activation
4. Exchange
Organs and pathways involved in lipoprotein metabolism
 Chylomicrons

• Synthesis in: the small intestines in the fed

state
• Secreted into: the lymph vessels, then -->
moves into the blood
• Contains apo B48
• Rich in: TGs
• Function: Deliver TG’s to body cells to be used
as fuel
 VLDL
• Synthesis in the liver from excess dietary
carbohydrate and protein along with the
Chylomicron remnant
• Secreted into: the bloodstream
• Rich in: TGs
• Function: Deliver TGs to body cells
• Contains apo B100
 LDL
• Synthesis in the Liver as VLDL
• Results from: VLDL once it has lost a lot of its TG’s
and CCE
• Secreted into: the bloodstream
• Rich in: Cholesterol
• Function: Deliver cholesterol to all body cells
 HDL

• Synthesis in : the Liver and Small Intestine

• Secreted into: the bloodstream
• Function: Pick up cholesterol from body
cells and take it back to the liver = “reverse
cholesterol transport”
• Potential to help reverse heart disease
 Overview: Key Enzymes
Site of Metabolic
Enzyme Substrate Reaction
Action Role

LCAT
Cholesterol and Cholesterol Plasma lipoproteins Reverse
Lecithin-cholesterol
PC esterification HDL Cholesterol
acyltransferase
transport

LPL
TG, VLDL, VLDL and chylomicron
Lipoprotein hydrolysis Capillary surface
chylomicrons degradation
Lipase

Hepatic
TG, HDL hydrolysis Liver HDL catabolism
Lipase

CETP
Plasma lipoproteins Lipoproteins
Cholesterol ester Cholesterol, TG transfer
VLDL/LDL to HDL remodeling
Transfer protein
ACAT
Cholesterol Cholesterol Liver Cholesterol uptake
AcylCoA-cholesterol
esterification LDL
acyltransferase
Overview
 Lipoproteins and
Cardiovascular diseases
 Diseases associated with
Lipoprotein dysfunction

• Hypolipidemia/ • Hyperlipidemia/
hypolipoproteinemia hyperlipoproteinemia

• Increased risk of
atherosclerosis and
cardiovascular diseases

*increased levels of plasma cholesterol and LDL

 Familial Hypercholesterolemia

• FH patients have high

LDL and plasma
cholesterol levels
• Lack LDL receptors
– Decreased degradation
of LDL
•Mutations in LDL
receptor gene

Reading: Management of FH: J Manag Care Spec Pharm, 2013 Mar;19(2):139-149.

 Atherosclerosis

Found in animal food only

NOT in fruits, vegetables,

grains, nuts (plants contain
other sterols)

Liver synthesizes
cholesterol
Atherosclerosis:
LDL hypothesis
Oxygen radicals in endothelial lining of the arteries oxidize LDL and retain them
Activated endothelial cells have increased inflammation
Macrophages take up LDL and are saturated with cholesterol thus forming foam cells
 Atherosclerosis:
LDL hypothesis

• Elevated levels of LDL cholesterol and

apoB100 directly contribute to atherosclerosis
• Oxygen radicals in endothelial lining of the
arteries oxidize LDL and retain them
• Activated endothelial cells have increased
inflammation
• Macrophages take up LDL and are saturated
with cholesterol thus forming foam cells
LDL hypothesis- Cholesterol deposits/inflammation

Nature Reviews Endocrinology 10, 659-672; 2014

 Healthy Lipid Profile
Fasting Blood Level

Total Cholesterol < 200 mg/dl

LDL-Cholesterol < 100 mg/dl

HDL-Cholesterol ≥ 60 mg/dl

Triglycerides < 150 mg/dl

 The Metabolic Syndrome

Abdominal Obesity
Men > 40 inch waist
Women > 35 inch waist

Triglycerides ≥ 150 mg/dL

HDL cholesterol
Men < 40 mg/dL
Women < 50 mg/dL
Blood Pressure ≥ 130/ 85 mm Hg
Fasting Blood Glucose >110-125 mg/dL
 Summary
• Distribution (Transport and Cellular Uptake) of lipids (Fatty Acids and Cholesterol) is
via Lipoproteins

• Major constituents of Lipoproteins: TG, cholesterol, cholesteryl ester,

Apolipoproteins

• Major enzymes of lipoprotein metabolism: Lipoprotein Lipase (LPL),

Lecithin:Cholesterol Acyl Transferase (LCAT), and Cholesterol ester transfer
Protein (CETP)

• Interconversion of lipoprotein particles

• VLDL,LDL, HDL are ALL needed to maintain healthy metabolism

However, excess LDL is considered “bad” form of cholesterol as it
contributes to plaque formation
HDL is considered “good” form of cholesterol as it helps remove cholesterol
from tissues and arteries

• Basis for the LDL hypothesis of atherosclerosis: foam cell formation and deposition