Molecular Basis of Selected Diseases: Cancer, HIV, Alzheimer's

KINE 4518

The Molecular Basis Of Selected Diseases

F09

Mon/Wed 11:30 AM-12:50 PM

Dr. Michael Connor Ph.D.

The Cell Cycle and Cancer

Office/lab: 224 Lumbers

E-mail: [email protected]

The Immune System and A.I.D.S.

Alzheimer’s Disease

Grade Breakdown

Mid-term #1: Monday October 5, 2009 (35%)

Mid-term #2: Monday November 16, 2009 (35%)

Final Exam: T.B.A. (30%)

Fill in the blank, short answer and essay questions.

For downloading slides: WebCT.yorku.ca

What is Cell Signaling?

1. A means whereby cells can adapt to changing conditions

2. A means to regulate cellular behaviour and activity

3. A mechanism to turn responses off and on

Vital to proper maintenance of cell function

Often involved in diseases

Integral in molecular biology


often involves a receptor

membrane or intracellular receptors

Extracellular Signaling

Extracellular

Plasma membrane

IIntracellular


Extracellular domain:

where ligand (signal) binds to

binds specific ligands

neurotransmitters, hormones, growth factors

Transmembrane domain:

links the outside with the inside

often involves a change of shape


Intracellular domain:

eg. acetylcholine receptors

can form a pore or ion channel

Transmembrane domain:

relays signal to cytoplasm via interactions with effector proteins and 2nd messengers

associated enzyme activities, kinase activities


Intracellular signaling:

from the cytoplasmic domain inside the cell

signal has been “converted” from an extracellular one to an intracellular one

2nd messengers (i.e. cAMP, Ca2+)

leads to cellular response

Figure 05.05

Figure 5-6 Vander et al, Human Physiology, 10th edition

A Cellular Signaling Pathway


Cytoplasmic receptors:

some ligands/signals can pass through the plasma membrane (i.e. estrogen)

contain similar domains as transmembrane receptors

ligand binding domain (extracellular domain)

kinase domain (intracellular domain)

The Estrogen Receptor Signaling Pathway

phosphorylation and dephosphorylation are not the reverse of one another

negligible rates in the absence of enzymes

How Does a Protein Become Phosphorylated?

phosphorylation uses cellular ATP

How Does a Protein Become Phosphorylated?

Protein Kinases: a family of proteins that reversibly adds a covalent phosphate group to amino acids on target proteins

occurs on serine (S), threonine (T) or tyrosine (Y)

either activates or inactivates target protein

mediated by receptors (membrane or cellular)

varied specificity

PKB/AKT: RXRXXS/TMAPK: PXS/TP

How Does a Protein Become Dephosphorylated?

Phosphatases:

action directly opposite to kinases

hydrolyses phosphate group from protein

this removes the phosphate group from the protein

far fewer phosphatases than kinases

Phosphorylation Affects Protein Function

adds negative charges to a modified protein

causes conformational changes

exposes enzyme catalytic/active site

sends original signal “downstream” to other effector proteins

The PKB/AKT Pathway

The MAP Kinase Pathway

The JAK/STAT Pathway

The Estrogen Receptor Signaling Pathway

What Initiates Cell Division/Proliferation?

Growth signals: Natural/normal Development/healing

Aberrant disease/cancer

External growth factors

Mis-regulation of cellular pathways

Not a “REAL” signal; inappropriate growth

RbPhosphorylation

D Cyclins+

Cdk4/6

Cyclin E+ Cdk2Cyclin A + Cdk2

Cyclin B/A

+Cdk1

p15p16p18p19

INK4:

p21KIP:

p21p27p57

p21p27p57

p27p57

The Mammalian Cell Cycle

GOM

S

G1G2

Rb Dephosphorylation

Phases of the Cell Cycle

M phase: Cell separation/division

S phase: DNA synthesis/replication

G1 phase: Gap 1 phase

Originally thought as a rest phase

Preparation of cells for chromosome replication

not true

Cell grows in size

Cell surveys environment to see if conditions are right

Phases of the Cell Cycle

G2 phase: Gap 2 phase

Also originally thought as a rest phase

Preparation of cells for mitosis

Cell grows in size

Check integrity of DNA

Important Factors in G1, S, G2 and M Phases

Cyclin A/cdk2Cyclin A or B/cdk1

Cyclin D/cdk4-6

Cyclin E/cdk2

Each phase has a checkpoint ensure proper progression

The Cyclins

Cell cycle proteins that are expressed cyclically

Cyclin D

G1 S G2 M

Cyclin A

Cyclin B

Cyclin E

Activities of the Cyclin/cdk Complexes

G1 S G2 M

Cyclin D/cdk4-6Cyclin E/cdk2 Cyclin A/cdk2

Cyclin B/cdk1

The Cyclins and Cdks

Cyclin-dependent kinases (cdks) are the motor that makes the cell cycle go

Unlike cyclins they are not cyclically expressed

Need to be bound with their associated cyclin to become functional

Often activated by phosphorylation

Are inhibited by cdk inhibitors (CKIs)

Ser/thr kinases

Cyclin D

G1 cyclin

Three isoforms: cyclin D1, D2, D3

Each is somewhat dispensable

Binds to either cdk4 or cdk6

2 main functions: 1) Phosphorylate Rb (retinoblastoma)

2) Sequester Kip proteins

INK4 proteins

INK4 (Inhibitor of cdk4)

Cyclin D

Cdk4/6 INK4

Binds only to cdk4 or 6

Sole inhibitor of cyclin D/cdk4 or 6

Cdk4/6

p27

Cyclin D

INK4

cdk2

cyclin E

ARRESTED

p27

cdk2

cyclin E

Cdk4/6

Cyclin D

CYCLING

Cdk4/6

CAK

Cdk4/6

P

cdc 25AP

Wee 1

P

Cdk4/6

P

tyr15Cyclin D

Cdk4/6

P

INK4inactive

p27

Cyclin D

Cdk4/6

P

active p27

INK4

thr172

RbPhosphorylation

D Cyclins+

Cdk4/6


Cyclin B/A

+Cdk1

p15p16p18p19

INK4:

p21KIP:

p21p27p57

p21p27p57

p27p57


GOM

S

G1G2


Cyclin E

Similar to cyclin D in function

Integral to G1/S transition

Binds to cdk2 to form active complex

Transcribed by E2F transcription factors

Broader spectrum of proteins phosphorylated than cyclin D

Cyclin E

Cyclin E/cdk2 phsophorylates:

2. Histone H1

3. p27

1. Rb activates its own transcription

removes its inhibitor

enhances its own activity

Cyclin D/cdk4-6 phosphorylates Rb only

Necessary for G1-S transition

cdk2

cyclin E

P

p27

cdk2

cyclin E

P Pthr 160 tyr 15

cdk2

cyclin E

Pthr 160

Wee 1 cdc 25AP

p27P

cdk2

cyclin E

Pthr 160

degradation

inactive/growth arrest

active

cdk2

cyclin E

Pthr 160

active

Cyclin D

Cdk4/6p27

RbPhosphorylation

D Cyclins+

Cdk4/6


Cyclin B/A

+Cdk1

p15p16p18p19

INK4:

p21KIP:

p21p27p57

p21p27p57

p27p57


GOM

S

G1G2


p27 KIP1

Cell cycle inhibitor

Inhibits: 1. Cyclin E/cdk2

2. Cyclin A/cdk2

3. Cyclin A-B/cdk1

G1-S transition

S phase

G2-M

Prevents cell cycle progression

Assembles cyclin D/cdk4-6 cell cycle progression

Highly regulated by phosphorylation

p27 KIP1

Levels high in G1

Regulated at the protein level (not transcription like cyclins)

p27 protein levels

G1 S G2 M

Degraded by proteasome

Increased synthesis

RbPhosphorylation

D Cyclins+

Cdk4/6


Cyclin B/A

+Cdk1

p15p16p18p19

INK4:

p21KIP:

p21p27p57

p21p27p57

p27p57


GOM

S

G1G2


Inhibit the E2F transcription factors

Tumor suppressor gene

3 isoforms

Function is regulated by phosphorylation on multiple sites

Phosphorylated by active cyclin E and cyclin D complexes

The Retinoblastoma (Rb) Protein

The Retinoblastoma (Rb) Protein

Hypo-and hyper-phosphorylated forms

Hyperphosphorylated Rb:

1. Dissociates from E2F transcription factors

2. Is targeted for degradation by proteasome

Results in S-phase entry

Sherr & Roberts, Genes Dev, 1999

The E2F Transcription Factors

Important for cell cycle progression

Important structural domains:

1. DNA binding

2. Pocket protein binding domain

3. Dimerization domain

Family of transcription factors

8 different isoforms

Hyperphosphorylation of Rb dissociates complex

2. DNA replication (DNA polymerase )

3. DNA damage repair (BRCA1)

4. DNA synthesis (thymidine kinase)

5. Apoptosis

The E2F Transcription FactorsBind to “pocket” region on Rb inhibits activity

E2F can become active

Gene targets involved in:

1. Cell cycle (cyclin E)

Binds to other transcription factors (DP-1)

Promoter binding site: (T/C)TT(C/G)(G/C)CG(G/C)

The E2F Transcription Factors

3 main functions:

1. DNA replication in S-phase

2. Ensure DNA integrity

3. Prepare for cell destruction if DNA isn’t intact

Phosphorylated by cyclin A/cdk2 Increases affinity for Rb

Prepares E2F to bind new hypophosphorylated Rb

Inhibits its activity

RbPhosphorylation

D Cyclins+

Cdk4/6


Cyclin B/A

+Cdk1

p15p16p18p19

INK4:

p21KIP:

p21p27p57

p21p27p57

p27p57


GOM

S

G1G2


p53

Discovered ≈ 25 years ago

Involved in cell cycle and apoptosis (cell death)

p53 is responsible for decision of cell to “live or die”

Transcription factor

Gene targets induce cell cycle arrest or apoptosis

Family of proteins (p63 and p73)

Tumor suppressor

p53

Protein level regulated post-translationally

2 Main Functions:

Cell Cycle Control

Induces cell cycle arrest during DNA damage (UV)

1. Transcribes p21 Inhibits cyclin E/cdk2

G1 arrest

p53 levels/activity low in unstressed cells

p53

3. Inhibits c-myc transcription (G1)

Apoptosis (cell death)

During DNA damage p53 up-regulates pro-apoptotic genes

Including:

2. Transcribes Gadd45

Bax

PUMA

Inhibits cdk1 (G2/M)

Noxa

Counteract Bcl-2

p53

1. Regulation of p53 protein levels

p53 function regulated by:

2. Cellular localization of the protein

3. Modulation of activity

Mdm2 main inhibitor of p53 function

3. Binds and inhibits p53 Prevents interaction with other transcription factors

2. Shuttles p53 out of nucleus

1. Degrades p53

The Ubiquitin Proteasome Pathway

Mechanism for targeting proteins for degradation

Addition of ubiquitin to target proteins on lys

Ubiquitin 76 amino acids (8 kDa)

Multiple ubiquitin molecules added in a chain (polyubiquitination)

Can also just add 1 ubiquitin (monoubiquitination)

Modifies protein function

E1-Ubiquitin activating enzymeE2-Ubiquitin conjugating enzyme

E3-Ubiquitin ligase


Two main classes of E3 enzymes (ubiquitin ligases)

HECT-domain

HECT domain is ≈ 350 amino acids (40 kDa)

Single protein

Forms thiol-ester bond with Ub before transfer of Ub to targetprotein

Smurf2, Nedd4 and E6AP


The Ubiquitin Proteasome PathwayRING-finger ligases

2. Multi-protein complex

SKP1/Cullin1/F-box protein

F-Box protein gives complex specificity binds to target

Transfers Ub to target protein

SKP2, FBW7

1. Single protein

RING domain binds target proteinsTransfers Ub to target protein

MDM2, Cbl

Also includes ROC1 RING-finger

HECT-type E3 Ligases


HECT Ub

E2 Ub

Target Ub

RING-type E3 Ligases

Target Ub

F-Box

SKP1

Cullin 1

ROC1

E2 UbB

A

E2 Ub

Ring FingerTarget Ub


Cancer is a disease of the cell cycle

Can be caused by

1. Genetic mutation or deletion

2. Misregulation of normal cell cycle processes

Environmental impact; mutagens/carcinogens

Pollution, UV radiation

Hit the gas or take foot off the brakes

Starts out as a normal cell


Cell will often interpret continuous growth signals

1) Lose the ability to regulate normal growth

3) Lose the ability to get rid of damaged cells (apoptosis)

2) Lose the ability to detect/repair damaged DNA

End result: Unwanted proliferation of cells

Tumour development

Numerous potential underlying causes

Cyclin D

Transcriptionally up-regulated in response to mitogenic stimuli

Translates to increase in protein

Must combine with cdk4 or cdk6 for effect

Cdk must be phosphorylated by Cyclin Activating Kinase (CAK)on thr 172

Initiated by MAP kinase pathway (AP-1; jun-fos)

Assembled by Kip proteins (p21 or p27)

Displaces INK4 proteins

Very specific to phosphorylating Rb (no other targets)

Complex translocates to nucleus and phosphorylates Rb

Cyclin D

Phosphorylation of Rb dissociates it from E2F proteins

E2F transcription factors become active

Transcribe genes that advance the cell cycle

Summary of Cyclin D

in mRNA induced by MAPK pathway cyclin D protein

Binds with cdk 4 or cdk 6 via p27

1. Phosphorylates Rb

2. Removes p27 from cyclin E/cdk2

displaces INK4

Functions of Complex

Summary of cdk4/6

Inactivated by phosphorylation (WEE1)

Inactivated by INK4

Need A, B and C to have active cdk4/6

removed by cdc25A

Activated by phosphorylation (CAK)C

displaced by p27B

Induces cyclin D nuclear export

Deactivated by phosphorylation by GSK-3 on thr 286

Cyclin D

Targets protein for degradation by the proteasome (ubiquitin)

Decreases protein stability (half-life 25 min down to 10 min)

Cyclin DE3 ligase for Cyclin D degradation recently identified

Fbx4

Cytoplasmic degradation of cyclin D

Contains N-Terminal dimerization domain

Dimerization regulated by phosphorylation on Ser12

Mutations of Fbx4 decrease ubiquitination of Cyclin D1

Cyclin D

Mostly increased expression of cyclin D1 (not D2 or D3)

1. Chromosomal Translocations

Can cause “new” improper regulation/expression

Lymphoma, parathyroid adenoma, myeloma

2. Gene Amplification

Gene transcribed too often

Lung, head & neck, pancreatic, bladder, pituitary and breast

Cyclin D

3. Mutation

thr 286 not mutated in any cancers

Deactivated by phosphorylation by GSK-3 on thr 286

gly 870 ala

3-d structure change may interfere with thr 286 phosphorylation

Mutations of Fbx4 discovered:

ser, 8, ser 12, pro 13, lys 23, pro 76

Cyclin D

4. Misregulation

Ras activates the MAP kinase pathway

Tumours can have activated Ras

MAP kinase pathway regulates transcription of cyclin D

cyclin D transcription and protein level

Perpetual cell cycle entry

Cyclin D

Therapy

Specific inhibitor of cdk4 and cdk6

Binds competitively to ATP-binding region

Inhibits growth of tumour cells that are cdk4-6 dependent

Nothing specific for cyclin D yet

PD 0183812

Cyclin E

Many cancers overexpress cyclin E mRNA or protein

Breast, lung, cervix, endometrial, GI, lymphoma, leukemia

Anything that affects Rb may increase cyclin E by E2F

1. Gene amplification

Endometrial, ovarian, colorectal, breast and gastric

infrequent (2-20%)

Cyclin E2. Disrupted Degradation

2-step process

A. Loss of one allele (gene copy)

B. Point mutations of remaining allele

Mutation of this site would prevent degradation

Not found yet

Loss of Fbw7A)

Cyclin E phosphorylated at T380 by GSK3-B)

Cyclin E

Clinical marker

Predictive of patient outcome:

Breast, lung, laryngeal, adrenocortical

May be indirect effect

Overexpression may cause genetic instability

Elevated cyclin E activity impedes S-phase progression

Impaired replication, DNA breakage and premature entry into M

Effects more dramatic when coupled with p53 loss

Cyclin E

Therapy

cdk2 inhibitors (not cyclin E)

Problem:

If cyclin E overexpression leads to genetic instability……..

inhibiting cdk2 won’t treat what is wrong, unless…….

cyclin E overexpression detected before genetic problems start

p27

Loss of p27 expression in 60-70% of ALL cancers

Predictor of patient outcome

No p27 genetic mutations detected yet

No alterations in gene transcription detected yet

Post-transcriptional alterations

p27

How is p27 degraded?

2-mechanisms

1. p27 Loss

A. Cyclin E/cdk2 (late G1)

Cdk2 phosphorylates p27 on thr187

Promotes interaction with F-box protein (Skp2)

Targets p27 for degradation by SCF ubiquitin ligase complex

p27

Degrades most of cellular p27 (80%)

Skp2 elevated in cancer

Oral, lung, colorectal, lymphoma, breast

A. Cyclin E/cdk2 (cont.)

Inconsistencies

p27

Time

Pro

tein

Lev

el

???

p27Skp2

G0 G2/M

p27

Degrades most of cellular p27 (80%)

Skp2 elevated in cancer

Oral, lung, colorectal, lymphoma, breast

A. Cyclin E/cdk2 (cont.)

Inconsistencies

Cyclin E/cdk2 activated before Skp2 is present

Why does this occur??

B. Nuclear export (early G1)

No activation of cyclin E at this point

Phosphorylation on ser10 by hKIS

p27 exported to cytoplasm

Mediated by MAP kinase pathway

p27

may be AKT

p27

B. Nuclear export (cont.)

≈ 20% of all p27 degradation

Helps allow for activation of cyclin E/cdk2 initially

MAP kinase and AKT pathways activated in many cancers

Degraded by newly identified E3 complex

Kip1 ubiquitination Promoting Complex (KPC)

2 proteins: 1. Ring-finger protein

2. Ubiquitination protein

p27

Other Mechanisms of p27 Degradation

Discovered in 2007

2 other p27 phosphorylation sites important

1) tyr88

BCR-ABL kinase

Disrupts inhibitory “pocket” of p27

p27 binds cyclin E/cdk2 no kinase inhibition

p272) thr198

AMP-kinase (AMPK)

Stabilizes p27: prevents Ub-dependent degradation

“energy-sensing” kinase

Leads to autophagy

Cells “chew-up” organelles to generate energy

Can lead to non-apoptotic cell death if prolonged

Tumours CAN have “too little” and/or “too much” p27!!

p27

2. Mislocalization

p27 phosphorylated on thr 157 by AKT

Prevents entry of p27 into the nucleus

No reduction in p27 levels

p27 can’t inhibit cyclin E/cdk2

AKT implicated in numerous cancers

p27 located in cytoplasm

p27

Therapy

To date no therapeutic strategy has been directed at p27

Possibly because of the many different pathways involved

NONE

1 study in animals

Link p27 to HIV-TAT protein

p27 “delivered” to all cells in the body

p27

Therapy

p27

Therapy (contd.)

Problems:

Protein based, continuous dosage

Stops cell cycle progression

Expensive

Side effects not different from chemotherapy

Plausibility?????

G1 Gene Mutations/defects and Cancer

Malumbres & Barbacid. Nature Rev. Cancer, 1, 222-231, 2001

p53

p53 is most frequently mutated gene in cancer (>50%)

More than 18,000 mutations identified in 150 cancer types

85% of these involve a single amino acid mutation

>90% are located in DNA binding domain (190 amino acids)

p53 can’t bind DNA

1. Mutations of p53

p53

Mdm2 overexpression is most-often the culprit

Degrades, mislocalizes or prevents activity of p53

HPV infection

All prevent p53 function and compromise DNA integrity and apoptosis

2. Inactivation

E6AP; E3-ligase

p53

Wild-type p53 tumours

Have to target the underlying cause

Mdm2-directed therapies:

Therapy

1. Non-peptide inhibitor “syc-7”

2. Nutlins displace p53 from Mdm2

4. Mdm2 antisense prevents transcription of Mdm2

3. RITA displace p53 from Mdm2

in vitro

p53

Adenoviral vectors

Encouraging results (Phase I and phase II clinical trials)

New “smart” virus

Specifically target tumour cells

Therapy (contd.)

Viral proteins not expressed in cells with normal p53

Introduce wild-type p53 back into tumours

p53 mutant tumours

p53

Small molecules that can re-establish normal p53 function

Changes 3-d structure, binds targets

Therapy (contd.)

Prima-1

complicated so many possible mutations

first generation

toxic at higher concentrations

promising start

Kinesiology & Health Science

Cancer Statistics

General Cancer Stats

• ~149,000 new cases of cancer and ~69,500 deaths will occur each year

• ~72,800 Canadian women will be diagnosed and ~32,800 will die

• ~76,200 Canadian men will be diagnosed and ~36,700 will die

2006 Canadian Cancer Society


Cancer Statistics

Breast Cancer Stats

• Most common cancer among Canadian women

• <1% of men will be diagnosed

• 1 in 9 women will develop breast cancer. 1 in 27 will die of it

2006 Canadian Cancer Society


Malignant Tumours

• Evading apoptosis• Insensitive to anti-growth factors• ↑ Rate of cell division• Altered ability to differentiate• No ability for contact inhibition• Invade neighbouring tissues• Metastisize• Promote angiogenesis


Tumour Types

http://www.wisc.edu/wolberg/breast.html


http://www.breastcancer.org/breast_anatomy_picture.html

Mammary fat

Lobules

Lobe

Interlobular connective tissue

Subcutaneous fat

Nipple and subareolar musculature

Nipple

Areola

Montgomery’s tubercles Lactiferous

ducts

Ampulla

Suspensory ligaments

Alveoli

Adapted from: http://www.breastdiagnostic.com/anatomy2.html


Breast Cancer

http://www.breastcancer.org/type_breast_cancer_picture.html


Adipocytes as Paracrine Cells

LEPTIN AND ADIPONECTIN

Mammaryduct

A peptide hormone coded for by the ob gene

Recently discovered – 1994; >13,690 ref.

Influences quantity of food consumed relative to energy expended

Most abundant in adipose tissue


What is Leptin?

–Leptin : appetite and energy expenditure

(14,263)


Leptin Signaling

Jak2

Tyr985

Tyr1138

P

P

P

Tyr1138

Tyr985

Jak2P

P

P STAT3PSTAT3 P

Nucleus

Intracellular

Extracellular


What is Adiponectin?

• Improves insulin resistance

• Inversely related to adiposity

• Low serum adiponectin may lead to a more aggressive phenotype


Obesity and Cancer

- >30% of North American population is obese

- obesity-cancer link known for over 40 years

- molecular mechanism is unclear


Cell Cycle effects??

Adiposity and Adipokines

adapted from: www.eurodiabesity.org/Leptin.htm

Increasedadiponectin

Decreasedadiponectin

Insulin insensitivity


Insulin sensitivity


Hypotheses

1. The leptin:adiponectin ratio affects mammary epithelial cell cycle status

Paracrine effects of adipocytes change with adiposity2.


p27

GAPDH

Leptin (nM)

0 25 15050 100 200

A

Adipokines Alter Cell Cycle Status

B

GAPDH

p27

0 3 6 12 24 36

Adiponectin (nM)


Adiponectin (9 nM)

Hours

GAPDH

p27

B300 4 8 16 24

A Hours

Leptin (50 nM)

0 4 248 16 30

GAPDH

p27

Adipokines Alter Cell Cycle Status


B

p27

GAPDH

Lep (nM)Adip (nM)

0 25 50 100 150 2000 9 9 9 9 9

A

Lep (nM)

Adip (nM)

0 50 50 50 50

0 6 12 24 48

p27

GAPDH

Adipokines Antagonize Each Other


p27

GAPDH

Lep (nM)Adip (nM)

0 200 100 50 250 0 6 12 24 36

150

Adipokine Ratio Regulates the Cell Cycle


Adipocyte Isolation


Adipocyte Isolation


Subcutaneous

GAPDH

p27

0 1 2 3 4 3+Adipocyte Number (x106)

A BVisceral

GAPDH

p27

0 3 6 9 12 9+Adipocyte Number (x105)

Adipocytes Affect Proliferation

GAPDH

Adipocyte Number (x105)

0 0.4 0.8 1.2 1.6 2.0+

cyclin E

0 0.4 0.8 1.2 1.6 2.0+0

25

50

75

100

*

**

Number of Adipocytes (x105)

p27

Pro

tein

Lev

el

(per

cent

of

cont

rol)

GAPDH

p27p27

0 0.4 0.8 1.2 1.6 2.0+

cyclin E

0 0.4 0.8 1.2 1.6 2.0+0

25

50

75

100

*

**

p27

Pro

tein

Lev

el

(per

cent

of

cont

rol)


Adipocytes Affect ProliferationA

BAdipocyte Number (x105)

0 0.5 1.0 1.5 2.0 2.5+

GAPDH

cyclin E

0 0.5 1.0 1.5 2.0 2.5+0

25

50

75

100 *

**

Number of Adipocytes (x105)

p27

Pro

tein

Lev

el(p

erce

nt o

f co

ntro

l)

0 0.5 1.0 1.5 2.0 2.5+

GAPDH

p27p27

cyclin E

0 0.5 1.0 1.5 2.0 2.5+0

25

50

75

100 *

**

p27

Pro

tein

Lev

el(p

erce

nt o

f co

ntro

l)

Subcutaneous Visceral


Adipocytes Affect Proliferation

Ctl Obese Adipocytes Obese Adipocytes +

A B C

G1:S:

G2:SUB-G1:

G1:S:

G2:SUB-G1:

G1:S:

G2:SUB-G1:

72.81.525.70

55.816.026.31.9

60.15.630.63.6

G1:S:

G2:SUB-G1:

47.95.620.426.1


Adiponectin:Leptin With Obesity

Phenotype Depot Adiponectin (ng/ml)

Leptin (ng/ml)

Adiponectin:Leptin

Lean Subcutaneous 293.3 ± 3.7 1.11±0.18 281:1

Visceral 278.8 ± 4.15 1.56 ± 0.06 180:1

Obese Subcutaneous 174.0 ± 3.4 2.21 ± 0.24 81:1

Visceral 176.8 ± 2.0 3.56 ± 0.15 50:1



Adiposity and Adipokines

adapted from: www.eurodiabesity.org/Leptin.htm

Increasedadiponectin

Decreasedadiponectin

Insulin insensitivity


Insulin sensitivity


GAPDH

AG490 (10 µM)

p27

25+0 + + + +

0Leptin (nM) 50 100 1500

A Novel Leptin Signaling Pathway?


A Novel Leptin Signaling Pathway?

0 50 100 1500

25

50

75

100 + AG490- AG490

Leptin (nm)

p27

Pro

tein

Lev

el(p

erce

nt

of

un

trea

ted

) (10 M)

B

-- -

0 1 1 2

+

4 8

+ + + +

+ + + +

Lep (100 nM)

AG490 (10 µM)

Time (hrs)

Stat3

GAPDH

p27

phospho-Stat3

A


Obesity-Cancer Molecular LinkLeptin

Y1138

Y985 P

P

JAK2

STAT3

Metabolic Effects

Cell Cycle Effects&

???



Y1138

Y985 P

P

JAK2

STAT3

Metabolic Effects

Cell Cycle Effects&

Adiponectin

?????

AMPK/T198 p27??

???



Y1138

Y985 P

P

JAK2

STAT3

Metabolic Effects

Cell Cycle Effects&

Adiponectin

?????

AMPK/T198 p27??

???



Y1138

Y985 P

P

JAK2

STAT3

Metabolic Effects

Cell Cycle Effects&

Adiponectin

?????

AMPK/T198 p27??

???


Summary

1. Leptin and adiponectin stoichiometrically antagonize each other

2. Paracrine leptin cell cycle effects involve a novel pathway

3. Modification of the leptin:adiponectin ratio may provide new therapeutic avenues

80%

The Immune System and AIDS

Immune system: Body’s defense against “invaders”

Non-specific and Specific mechanisms

Non-specific: don’t have to “recognize” foreign substances

Specific: foreign substance identified by lymphocytes

Vander, Sherman Luciano Chapter 18

T cells (thymus):

Cells Of The Immune System

1. Leukocytes

B Cells (bone marrow)

2. Lymphocytes

Natural Killer (NK) cells

helper

cytotoxic

3. Macrophages

4. Mast cells

Specific Immune Defenses

Lymphocytes are the essential cells in specific immunity

Lymphocytes recognize specific foreign matter (antigens)

Basis behind immunization

3 stages

1. The encounter and recognition of antigen by lymphocytes

Lymphocytes contain receptors that recognize antigens

1 antigen per lymphocyte (≈ 100 million unique receptors)


2. Activation of lymphocytes

Binding of antigen to receptor causes cell division

Multiple cell divisions lead to increased numbers of antigen-specific lymphocytes (clonal expansion)

Can all recognize original antigen

Some cells carry out attack and some will serve as “memory” cells


3. Attack on Invader by Activated Lymphocytes

B cells (activated) differentiate into plasma cells

Plasma cells secrete antibodies that bind to antigen

Antibodies attract other cells to carry out killing

Cytotoxic T cells can directly attack invader

After invader is gone activate lymphocytes apoptose

Functions of B cells and T cells

Basically 4 types of cells that initiate/mediate the response

1. B cells:

2. Helper T cells:

3. Cytotoxic T cells:

Recognize foreign antigens and secrete antibodies

Help activate B cells and cytotoxic T cells

Carry out attack response

Secrete toxic chemicals

CD4-positive

CD8-positive4. Macrophages

Macrophage

NK cell

Lymphocyte

Invader Recognition

Invader antigen “presented” to helper T Cell

Macrophages or B cells

Helper T cells secrete cytokines

Co-ordination of Response

Invader engulfed by a) macrophages

b) B cells

Invader digested and antigens presented on cell (Macrophage or B cell) surface

Antigen recognized by helper T cell activation

differentiation to plasma cellspresentation to helper T cellsmemory B cells

Activated helper T cells help to increase number of plasma cells and cytotoxic T cells

Attack Response

Cytotoxic T cells and natural killer (NK) cells

Cytotoxic T cells recognize antigen presenting cells

- virus infected cells and cancer cells

NK cells attack same cells, no antigen recognition

- unknown mechanism

- enhanced by antibodies and cytokines from helper T cells

Figure 18.13

HIV and AIDS

AIDS – acquired immune deficiency syndrome

Loss of immune response

Makes individual susceptible to benign infections

Patients do not die from AIDS, they die from other infections

First cases seen/diagnosed in early 80s

HIV and AIDS

Major Routes of HIV Transmission

2. Sexual intercourse

3. From mother to fetus

4. Breast milk during nursing

1. Blood transfer (including needle sharing)

AIDS Statistics

Living with AIDS: 40.3 million

New Cases (2005): 4.9 million

Deaths (2005): 3.1 million

Sub-saharan Africa: 25.8 million (7.2)

North America: 1.2 million (0.7%)

Sub-saharan Africa: 3.2 million

North America: 43,000 million

Sub-saharan Africa: 2.4 million

North America: 18,000

HIV

HIV – human immunodeficiency virus

gp120

gp41

Viral RNA

Reverse transcriptase

Viral core

HIV

Key Features of HIV Genome

1. Genes are encoded by RNA NOT DNA

2. Genes that cause reduction of CD4-positive (helper T) cells

Nef: negative factor

Env: encodes gp120 and gp41; binds to CD4

Vpu: Viral protein unknown; downregulates CD4

Important for virus release from infected cells

uses hosts translational machinery to produce proteins

one of the HIV genes makes DNA from RNA

HIV

3. Genes that cause nuclear import

Important for integration of the viral genes into host DNA

4. Genes that cause T cell activation

Tat: trans-activator of viral transcription

Nef

gp120

Promotes HIV replication; high levels of transcription factors

HIV

5. Genes that inhibit antibody formation

Prevents recognition of foreign substances

6. Nef

The Nef gene is critical for disease induction

HIV

HIV gains entry into helper T cells by gp120 binding to CD4

Need more than this binding

Chemokine receptor on helper T cell also necessary

People with mutations in this chemokine receptor are resistant to HIV infection

THERAPY!!! chemokine receptor blockers/antagonists

CC-chemokine receptor 5 (CCR5) or CXCR4

HIV

After HIV entry into cell:

Nuclear import and integration into host DNA

Viral mRNA converted into DNA by reverse transcriptase

DNA selectively integrated into transcriptionally active regions

Transcription of viral DNA host and viral proteins

Integrase enzyme (viral)

HIVViral Transcription

1. TATA box; recruits host RNA polymerase II

Key control elements

very important in active cells

3. Modular elements; binding sites for host proteins

4. Tat-response element (TAR)

2. Enhancer elements; use host proteins to enhance transcription

Tat-mediated effects

HIVViral Transactivators

1. Tat: TransActivator of Transcription (≈ 100 amino acids)

Tat and Rev

Binds to TAR

Increases the efficiency of elongation

Needs host proteins to do its job

Essential for viral replication

HIVOther Tat Functions

Domain within Tat (RKKRRQRR) allows entry into cells

Translocated across plasma membrane

May “prime” T-cells for infection and replication

Induces apoptosis in T cells and neurons

Neuropathogenesis and immune evasion

Exploited for therapyR = argK = lysineQ = glutamine

HIV2. Rev: REgulator of Viral expression (≈ 116 amino acids)

RRE in env (viral) gene

Acts through Rev-responsive element (RRE)

Also needs cellular co-factors

Effects of Rev

Nuclear export of “late” mRNAs

Encodes viral envelope (incl. gp120 and gp41)

Encode for viral structural proteins

HIV

Other Viral Proteins

Help in packaging new viruses

Core proteins (3):

Generating DNA from RNA (reverse transcriptase; RT)

Cleavage/activation of viral proteins (viral protease; PR)

DNA integration into host (integrase; IN)

Enzymes (4):

HIV


Many functions essential for disease induction

Nef (Negative Factor):

2. Enhancement of viral infectivity

3. Killing of cytotoxic T cells (indirectly)

1. Downregulation of CD4

HIV


4. Modulation of host-cell signaling

Nef (Negative Factor):

Impairs normal T cell function

THERAPY??

Disease can still develop in absence of Nef

Interferes with normal cellular signaling pathways

HIV Life Cycle

HIV

End Effects of HIV on Body Function

Viral proteins downregulate CD4 expression and causes apoptosis in infected cells

1. Helper T-cell suppression

Insufficient immune response

Susceptibility to multiple normally benign infections

Loss of helper T cells and their function

HIV

End Effects of HIV on Body Function

HIV also infects microglial cells in brain (CD4 positive)

2. Dementia

Decreased cognitive and motor function

Induces apoptosis/cell loss

HIV

Treatment Strategies

Unsuccessful so far

Vaccines

Reverse transcriptase highly prone to errors

Highly mutated

No check on integrity of DNA sequence

End result: many mutations get through

Original vaccine memory T cells don’t recognize mutations

HIV

Treatment Strategies

1. Protease inhibitors

Antiviral Cocktails

3. Non-nucleoside inhibitors of RT

2. Nucleoside inhibitors of RT (AZT, Zidovudine)

4. Immune system “boosters”

5. Anti-infective drugs

Effectiveness??


What is Alzheimer’s?

Progressive neurodegenerative disease

Dementia: Cognitive impairment

Commonly manifests as memory loss

Short-term memory first

Then long term memory

Loss of inhibition

Many non-Alzheimer’s forms of dementia

not unique



First reported in 1906 by Lois Alzheimer

Genetic and sporadic causes

Genetic only accounts for a small proportion of patients

Sporadic make up ≈ 99% of all cases

Complex interaction between genetic, environmental and lifestyle risk factors



Molecular/biochemical Nature of Alzheimer’s

Surprisingly little is known about the molecular etiology

2 main hypotheses:

1. Amyloid plaques

Build-up of protein deposits within the brain/synapses


-Amyloid precursor protein (APP)

Integral transmembrane protein

Natural neuroprotective peptide

Protects against glutamate toxicity

Cleaved into peptide fragments by 3 secretases (, and )



Forms expanding protein mass

-amyloid plaques

secretase or -secretase cleaves APP

product is cleaved into harmless by-product

product is cleaved into 40 or 42 amino acid peptide by -secretase

-amyloid

Secreted into extracellular space



-secretase is a multiprotein complex

Made up of 4 proteins

1. Presenilin (50 kDa):Forms catalytic core

2. nicastrin:

Binds to presilin and APP

3. Aph-1:

4. Pen-2:Not well known


amyloid plaques are toxic to cells

Cause apoptosis in neurons

Responsible for loss in neuronal function and number

Involves cholesterol (similar to atherosclerotic plaques)

Apolipoprotein E4 (ApoE4) seems to correlate with disease

May cause increased susceptibility plaque formation


Plaques are normally only detected post mortem

New technologies helping to get around this

Compound binds to plaques

Allows for detection by PET scan


Apoptosis in neurons induced by -amyloid plaques occurs by:

1. Activation of caspase 3

2. Recruitment of p53

3. Release of cytochrome c from mitochondria

Appears to be a direct effect of -amyloid

Activates p38MAPK and jun n-terminal kinase (JNK)

Possibly through inactivation of Bcl-2


2. Tau filaments

Microtubule associated proteins (MAPs) are important for cellular structue

3 main proteins:

a) Tau (757 amino acids)

b) MAP 1

c) MAP 2

redundancy in function


Essential for axoplasmic flow: neurotransmitters

neurotrophic factors

Tau promotes assembly AND stability of microtubules

Regulated by phosphorylation (Iqbal et al. BBA 1739, p198, 2005)

Hyperphosphorylation of Tau inhibits microtubule binding and assembly

Hyperphosphorylation results in neurofibrilatory tangles


Tau phosphorylated by many kinases

GSK-3

cdk5

MAP kinase

Protein kinase A

Calcium and calmodulin-dependent kinase II

Phosphorylation balance of a protein determined by actions of kinases vs. phosphatases

p38MAPK

JNK


New kinase identified: Brain-derived Tau kinase (BDTK)

May explain resistance to animals to disease development

Found only in humans

Therapy…..??

Associations between kinases and Alzheimer’s

Nothing definitive yet


Impairing phosphatase activity leads to phosphorylation of Tau similar to Alzheimer’s

May be an increase in naturally occurring phosphatase inhibitors

Time will tell!!

Phosphatases

Activities of PP1 and PP-2A are compromised in Alzheimer’s brain

Okadaic acid, Inhibitor-1, Darp-32

Account for >90% of phosphatase activity

Alzheimer’s DiseaseMechanism of Neurofibrilatory Degeneration

Normal tau + tubulin microtubules

phosphatase kinase

hyperphosphorylated tau

microtubule dissasembly

compromised axoplasmic flow

retrograde degeneration

DEMENTIA

polymerization of hyperphosphorylated tau

Neurofibrilatory tangles

neuronal death


Therapies for Alzheimer’s

CURRENT

1. Acetylcholinesterase inhibitors

Alzheimer’s brains have low levels of acetylcholine

Decreased neural transmission

Helpful in early stages

Does not prevent disease progression



EXPERIEMNTAL

1. -secretase inhibitors

Bristol-Myers in 2001

Stopped due to side effects

Eli-Lily

Maybe -secretase is a better target

just beginning clinical testing



2. Alzhemed

Neurochem Pharmaceuticals

Prevent -amyloid from “sticking” together

Moving to phase 3 trials



Prevents formation of plaques

Inject mice with -amyloid

Use immune system to find and destroy -amyloid

3. Vaccine (Elan Pharmaceutical)

Wiped away existing plaques

in mice


normal

“vaccinated”



Prevents formation of plaques

Inject mice with -amyloid

Use immune system to find and destroy -amyloid

3. Vaccine (Elan Pharmaceutical)

Wiped away existing plaques

Move to humans

in mice

brain swelling


Prevention

Oxidative damage

1. Anti-oxidants

Mitochondrial defects in Alzheimer’s patients

3. Diet

Nutritional supplements/alterations

2. Keep mind active

Low fat diet to keep cholesterol levels low


Prevention

4. Exercise/fitness

Molecular Basis of Selected Diseases: Cancer, HIV, Alzheimer's

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