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. Office/lab: 224 Lumbers E-mail: [email protected]

The Cell Cycle and Cancer 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 Integral in molecular biology Often involved in diseases

What is Cell Signaling? Extracellular Signaling often involves a receptor membrane or intracellular receptors Extracellular Plasma membrane Intracellular

What is Cell Signaling? 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

What is Cell Signaling? Transmembrane domain: can form a pore or ion channel eg. acetylcholine receptors Intracellular domain: relays signal to cytoplasm via interactions with effector proteins and 2nd messengers associated enzyme activities, kinase activities

What is Cell Signaling? 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

What is Cell Signaling? 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

How Does a Protein Become Phosphorylated? phosphorylation uses cellular ATP

negligible rates in the absence of enzymes

phosphorylation and dephosphorylation are not the reverse of one another

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/T MAPK: 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 Aberrant

Development/healing disease/cancer

External growth factors Mis-regulation of cellular pathways Not a “REAL” signal; inappropriate growth

The Mammalian Cell Cycle Rb Dephosphorylation

M p21 p27 p57

Cyclin B/A

+

G2

Cdk1

S Cyclin A + Cdk2

INK4: p15 GO p16 p18 p19 D Cyclins + G1 Cdk4/6 KIP: p21 p27 p57 Rb Phosphorylation Cyclin E + Cdk2

p21 p27 p57

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

not true

Preparation of cells for chromosome replication 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 D/cdk4-6

Cyclin E/cdk2

Cyclin A or B/cdk1

Each phase has a checkpoint

Cyclin A/cdk2

ensure proper progression

The Cyclins Cell cycle proteins that are expressed cyclically Cyclin A

Cyclin B Cyclin D

G1

Cyclin E

S

G2

M

Activities of the Cyclin/cdk Complexes Cyclin E/cdk2

Cyclin D/cdk4-6

G1

S

G2

Cyclin A/cdk2

M

Cyclin B/ cdk1

The Cyclins and Cdks Cyclin-dependent kinases (cdks) are the motor that makes the cell cycle go Ser/thr kinases 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)

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) Binds only to cdk4 or 6 Sole inhibitor of cyclin D/cdk4 or 6

Cdk4/6 INK4 Cyclin D

Cdk4/6 Cyclin D Cdk4/6 INK4

cdk2 cyclin E

ARRESTED

p27

p27 Cyclin D

cdk2 cyclin E CYCLING

Cdk4/6

P

CAK

tyr15 P

P

inactive

Cdk4/6 INK4 Cyclin D

thr172

Cdk4/6

p27 P cdc 25A

Wee 1

P

INK4

Cdk4/6

P

Cdk4/6

active

p27 Cyclin D

The Mammalian Cell Cycle Rb Dephosphorylation

M p21 p27 p57

Cyclin B/A

+

G2

Cdk1

S Cyclin A + Cdk2

INK4: p15 GO p16 p18 p19 D Cyclins + G1 Cdk4/6 KIP: p21 p27 p57 Rb Phosphorylation Cyclin E + Cdk2

p21 p27 p57

Cyclin E Similar to cyclin D in function Transcribed by E2F transcription factors Integral to G1/S transition Binds to cdk2 to form active complex Broader spectrum of proteins phosphorylated than cyclin D

Cyclin E Cyclin D/cdk4-6 phosphorylates Rb only Cyclin E/cdk2 phsophorylates: 1. Rb

activates its own transcription

2. Histone H1 3. p27

removes its inhibitor enhances its own activity

Necessary for G1-S transition

thr 160 P

P tyr 15

cdk2

cyclin E P cdc 25A

Wee 1

thr 160 P

cdk2

thr 160 P

active

cyclin E

cdk2

cyclin E

Cdk4/6

active

p27 thr 160 P

cdk2

P

cdk2

cyclin E

Cyclin D

cyclin E p27

inactive/growth arrest

p27

P

degradation

The Mammalian Cell Cycle Rb Dephosphorylation

M p21 p27 p57

Cyclin B/A

+

G2

Cdk1

S Cyclin A + Cdk2

INK4: p15 GO p16 p18 p19 D Cyclins + G1 Cdk4/6 KIP: p21 p27 p57 Rb Phosphorylation Cyclin E + Cdk2

p21 p27 p57

p27 KIP1 Cell cycle inhibitor Inhibits:

1. Cyclin E/cdk2

G1-S transition S phase

2. Cyclin A/cdk2

G2-M

3. Cyclin A-B/cdk1 Highly regulated by phosphorylation Prevents cell cycle progression Assembles cyclin D/cdk4-6

cell cycle progression

p27 KIP1 Levels high in G1 Regulated at the protein level (not transcription like cyclins) Increased synthesis Degraded by proteasome

p27 protein levels G1

S

G2

M

The Mammalian Cell Cycle Rb Dephosphorylation

M p21 p27 p57

Cyclin B/A

+

G2

Cdk1

S Cyclin A + Cdk2

INK4: p15 GO p16 p18 p19 D Cyclins + G1 Cdk4/6 KIP: p21 p27 p57 Rb Phosphorylation Cyclin E + Cdk2

p21 p27 p57

The Retinoblastoma (Rb) Protein Tumor suppressor gene 3 isoforms Inhibit the E2F transcription factors Function is regulated by phosphorylation on multiple sites Phosphorylated by active cyclin E and cyclin D complexes

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 Family of transcription factors 8 different isoforms Important for cell cycle progression Important structural domains: 1. DNA binding 2. Pocket protein binding domain 3. Dimerization domain

The E2F Transcription Factors Bind to “pocket” region on Rb

inhibits activity

Hyperphosphorylation of Rb dissociates complex E2F can become active Promoter binding site:

Binds to other transcription factors (DP-1) (T/C)TT(C/G)(G/C)CG(G/C)

Gene targets involved in: 1. Cell cycle (cyclin E) 2. DNA replication (DNA polymerase α ) 3. DNA damage repair (BRCA1) 4. DNA synthesis (thymidine kinase) 5. Apoptosis

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

The Mammalian Cell Cycle Rb Dephosphorylation

M p21 p27 p57

Cyclin B/A

+

G2

Cdk1

S Cyclin A + Cdk2

INK4: p15 GO p16 p18 p19 D Cyclins + G1 Cdk4/6 KIP: p21 p27 p57 Rb Phosphorylation Cyclin E + Cdk2

p21 p27 p57

p53 Discovered ≈ 25 years ago Family of proteins (p63 and p73) Tumor suppressor 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

p53 Protein level regulated post-translationally p53 levels/activity low in unstressed cells 2 Main Functions: Cell Cycle Control Induces cell cycle arrest during DNA damage (UV) 1. Transcribes p21

Inhibits cyclin E/cdk2 G1 arrest

p53 2. Transcribes Gadd45

Inhibits cdk1 (G2/M)

3. Inhibits c-myc transcription (G1)

Apoptosis (cell death) During DNA damage p53 up-regulates pro-apoptotic genes Including:

Bax PUMA Noxa

Counteract Bcl-2

p53 p53 function regulated by: 1. Regulation of p53 protein levels 2. Cellular localization of the protein 3. Modulation of activity Mdm2 main inhibitor of p53 function 1. Degrades p53 2. Shuttles p53 out of nucleus 3. Binds and inhibits p53

Prevents interaction with other transcription factors

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

The Ubiquitin Proteasome Pathway E1-Ubiquitin activating enzyme

E3-Ubiquitin ligase

E2-Ubiquitin conjugating enzyme

The Ubiquitin Proteasome Pathway Two main classes of E3 enzymes (ubiquitin ligases) HECT-domain Single protein HECT domain is ≈ 350 amino acids (40 kDa) Forms thiol-ester bond with Ub before transfer of Ub to target protein Smurf2, Nedd4 and E6AP

The Ubiquitin Proteasome Pathway RING-finger ligases 1. Single protein RING domain binds target proteins Transfers Ub to target protein MDM2, Cbl 2. Multi-protein complex SKP1/Cullin1/F-box protein Also includes ROC1

RING-finger

F-Box protein gives complex specificity Transfers Ub to target protein SKP2, FBW7

binds to target

The Ubiquitin Proteasome Pathway

HECT-type E3 Ligases E2

Ub

HECT

Ub

RING-type E3 Ligases

A E2

Target Ub

Ub

Target Ub

Ring Finger

B

Ub

E2 Cullin 1 SKP1 ROC1

F-Box

Target Ub

The Cell Cycle and Cancer Cancer is a disease of the cell cycle Starts out as a normal cell Can be caused by 1. Genetic mutation or deletion Environmental impact; mutagens/carcinogens Pollution, UV radiation 2. Misregulation of normal cell cycle processes Hit the gas or take foot off the brakes

The Cell Cycle and Cancer Cell will often interpret continuous growth signals 1) Lose the ability to regulate normal growth 2) Lose the ability to detect/repair damaged DNA 3) Lose the ability to get rid of damaged cells (apoptosis) End result: Unwanted proliferation of cells Tumour development Numerous potential underlying causes

Cyclin D Transcriptionally up-regulated in response to mitogenic stimuli Initiated by MAP kinase pathway (AP-1; jun-fos) 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 Assembled by Kip proteins (p21 or p27) Displaces INK4 proteins

Cyclin D Complex translocates to nucleus and phosphorylates Rb Very specific to phosphorylating Rb (no other targets) 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

Binds with cdk 4 or cdk 6

cyclin D protein

via p27 displaces INK4

Functions of Complex 1. Phosphorylates Rb

2. Removes p27 from cyclin E/cdk2

Summary of cdk4/6 Inactivated by phosphorylation (WEE1)

removed by cdc25

B Inactivated by INK4

displaced by p27

C Activated by phosphorylation (CAK)

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

A

Cyclin D Deactivated by phosphorylation by GSK-3β on thr 286

Induces cyclin D nuclear export

Targets protein for degradation by the proteasome (ubiquitin)

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

Cyclin D E3 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 Deactivated by phosphorylation by GSK-3β on thr 286 thr 286 not mutated in any cancers 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 Tumours can have activated Ras Ras activates the MAP kinase pathway 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

PD 0183812

Binds competitively to ATP-binding region Inhibits growth of tumour cells that are cdk4-6 dependent Nothing specific for cyclin D yet

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 infrequent (2-20%) Endometrial, ovarian, colorectal, breast and gastric

Cyclin E 2. Disrupted Degradation A) Loss of Fbw7 2-step process A. Loss of one allele (gene copy) B. Point mutations of remaining allele B) Cyclin E phosphorylated at T380 by GSK3-β Mutation of this site would prevent degradation Not found yet

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 1. p27 Loss How is p27 degraded? 2-mechanisms A. Cyclin E/cdk2 (late G1) Cdk2 phosphorylates p27 on thr187 Targets p27 for degradation by SCF ubiquitin ligase complex Promotes interaction with F-box protein (Skp2)

p27 A. Cyclin E/cdk2 (cont.) Degrades most of cellular p27 (80%) Skp2 elevated in cancer Oral, lung, colorectal, lymphoma, breast Inconsistencies

p27 Skp2

p27

Protein Level

???

G0

G2/M Time

p27 A. Cyclin E/cdk2 (cont.) Degrades most of cellular p27 (80%) Skp2 elevated in cancer Oral, lung, colorectal, lymphoma, breast Inconsistencies Cyclin E/cdk2 activated before Skp2 is present Why does this occur??

p27 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

may be AKT

p27 B. Nuclear export (cont.) Degraded by newly identified E3 complex Kip1 ubiquitination Promoting Complex (KPC) 2 proteins: 1. Ring-finger protein 2. Ubiquitination protein ≈ 20% of all p27 degradation Helps allow for activation of cyclin E/cdk2 initially MAP kinase and AKT pathways activated in many cancers

p27 Other Mechanisms of p27 Degradation 2 other p27 phosphorylation sites important Discovered in 2007 1) tyr88 BCR-ABL kinase Disrupts inhibitory “pocket” of p27 p27 binds cyclin E/cdk2

no kinase inhibition

p27 2) thr198 AMP-kinase (AMPK) “energy-sensing” kinase Stabilizes p27: prevents Ub-dependent degradation 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 No reduction in p27 levels p27 located in cytoplasm p27 phosphorylated on thr 157 by AKT Prevents entry of p27 into the nucleus p27 can’t inhibit cyclin E/cdk2 AKT implicated in numerous cancers

p27 Therapy NONE To date no therapeutic strategy has been directed at p27 Possibly because of the many different pathways involved 1 study in animals Link p27 to HIV-TAT protein p27 “delivered” to all cells in the body

p27 Therapy

p27 Therapy (contd.) Stops cell cycle progression Problems: Protein based, continuous dosage Expensive Side effects not different from chemotherapy Plausibility?????

G1 Gene Mutations/defects and Cancer

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

p53 1. Mutations of 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

p53 2. Inactivation Mdm2 overexpression is most-often the culprit Degrades, mislocalizes or prevents activity of p53 HPV infection

E6AP; E3-ligase

All prevent p53 function and compromise DNA integrity and apoptosis

p53 Therapy Wild-type p53 tumours Have to target the underlying cause Mdm2-directed therapies: 1. Non-peptide inhibitor “syc-7” 2. Nutlins 3. RITA

displace p53 from Mdm2 displace p53 from Mdm2

4. Mdm2 antisense

prevents transcription of Mdm2

in vitro

p53 Therapy (contd.) p53 mutant tumours Introduce wild-type p53 back into tumours Adenoviral vectors Encouraging results (Phase I and phase II clinical trials) New “smart” virus Viral proteins not expressed in cells with normal p53 Specifically target tumour cells

p53 Therapy (contd.) Small molecules that can re-establish normal p53 function Changes 3-d structure, binds targets complicated Prima-1

so many possible mutations first generation toxic at higher concentrations promising start

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

Kinesiology & Health Science

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

Kinesiology & Health Science

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

Kinesiology & Health Science

Tumour Types

http://www.wisc.edu/wolberg/breast.ht ml

Kinesiology & Health Science

Mammary fat Nipple Montgomery’s tubercles

Ampulla Lactiferous ducts

Areola

Alveoli

Suspensory ligaments

Lobules

Subcutaneous fat Nipple and subareolar musculature http://www.breastcancer.org/breast_anatomy_picture.html

Kinesiology & Health Science

Lobe Interlobular connective tissue Adapted from: http://www.breastdiagnostic.com/anatomy2.html

Breast Cancer

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

Kinesiology & Health Science

Adipocytes as Paracrine Cells

Mammary duct

LEPTIN AND ADIPONECTIN Kinesiology & Health Science

What is Leptin? A peptide hormone coded for by the ob gene Recently discovered – 1994; >13,690 ref. (14,263) Influences quantity of food consumed relative to energy expended –Leptin : appetite and energy expenditure Most abundant in adipose tissue

Kinesiology & Health Science

Leptin Signaling

P

Jak2

Tyr985

P Extracellula r

Tyr1138 STAT3

Intracellula r

Kinesiology & Health Science

P

Jak2

P

Tyr985

P Tyr1138

P STAT3

Nucleu s

What is Adiponectin? • Improves insulin resistance • Inversely related to adiposity • Low serum adiponectin may lead to a more aggressive phenotype

Kinesiology & Health Science

Obesity and Cancer - >30% of North American population is obese - obesity-cancer link known for over 40 years - molecular mechanism is unclear

Kinesiology & Health Science

Adiposity and Adipokines Increased adiponectin

Decreased adiponectin

Insulin sensitivity

Insulin insensitivity

Cell Cycle effects??

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

Kinesiology & Health Science

Hypotheses

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

2. Paracrine effects of adipocytes change with adiposity

Kinesiology & Health Science

Adipokines Alter Cell Cycle Status A

Leptin (nM) 0

25

B

Adiponectin (nM)

50 100 150 200

0

p27

p27

GAPDH

GAPDH

Kinesiology & Health Science

3

6

12

24

36

Adipokines Alter Cell Cycle Status Leptin (50 nM)

A

Hours

0

4

8

p27 GAPDH

16

24

30

B

Adiponectin (9 nM) Hours p27 GAPDH

Kinesiology & Health Science

0

4

8

16

24 30

Adipokines Antagonize Each Other A

Adip (nM)

0

6

12

24

48

Lep (nM)

0

50

50

50

50

B Adip (nM) Lep (nM)

p27

p27

GAPDH

GAPDH

Kinesiology & Health Science

0 0

9 25

9 50

9 100

9 150

9 200

Adipokine Ratio Regulates the Cell Cycle Adip (nM) Lep (nM) p27 GAPDH

Kinesiology & Health Science

0 0

0 200

6 150

12 100

24 50

36 25

Adipocyte Isolation

Kinesiology & Health Science

Adipocyte Isolation

Kinesiology & Health Science

Adipocytes Affect Proliferation A

Subcutaneous

B

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

Adipocyte Number (x106) 0 1 2 3 4 3+ p27

p27

GAPDH

GAPDH

Kinesiology & Health Science

Adipocytes Affect Proliferation A

B

Subcutaneous

Visceral

Adipocyte Number (x105) 0

0.4

0.8

1.2

1.6

Adipocyte Number (x105) 2.0+

0

p27 cyclin

1.0 1.5

2.0 2.5+

p27

E

cyclin E

GAPDH

GAPDH

100

75

*

50

**

25 0

0

0.4

0.8

1.2

1.6

Number of Adipocytes (x105)

Kinesiology & Health Science

2.0+

p27 Protein Level (percent of control)

100

p27 Protein Level (percent of control)

0.5

*

75 **

50 25 0

0

0.5

1.0

1.5

2.0

Number of Adipocytes (x105)

2.5+

Adipocytes Affect Proliferation A

B

Ctl G1: S: G2: SUB-G1:

72.8 1.5 25.7 0

Kinesiology & Health Science

C

Obese Adipocytes G1: S: G2: SUB-G1:

55.8 16.0 26.3 1.9

G1: 47.9 S: 5.6 G2: 20.4 SUB-G1: 26.1

Obese Adipocytes + G1: S: G2: SUB-G1:

60.1 5.6 30.6 3.6

Adiponectin:Leptin With Obesity Phenotype Lean

Obese

Depot

Adiponectin (ng/ml)

Leptin (ng/ml)

Adiponectin:Leptin

Subcutaneous

293.3 ± 3.7

1.11±0.18

281:1

Visceral

278.8 ± 4.15

1.56 ± 0.06

180:1

Subcutaneous

174.0 ± 3.4

2.21 ± 0.24

81:1

Visceral

176.8 ± 2.0

3.56 ± 0.15

50:1

Kinesiology & Health Science

Adiposity and Adipokines Increased adiponectin

Decreased adiponectin

Insulin sensitivity

Insulin insensitivity

Cell Cycle effects??

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

Kinesiology & Health Science

A Novel Leptin Signaling Pathway? Leptin (nM) AG490 (10 µM) p27 GAPDH

Kinesiology & Health Science

0 0

0 +

25 +

50 +

100 150 + +

A Novel Leptin Signaling Pathway? A

AG490 (10 µM) Lep (100 nM) phospho-Stat3

-

+

0

1

+ +

+ +

+ +

+ +

Stat3 p27 GAPDH 1 2 Time (hrs)

4

8

p 2 7 P r o te in L e v e l (p e rc e n t o f u n tre a te d )

B + AG490 (10 µM) - AG490

100 75 50 25 0

0

50

100

Leptin (nm)

Kinesiology & Health Science

150

Obesity-Cancer Molecular Link Leptin

Y985

Y1138

JAK2

P

P

STAT3

??? Metabolic Effects & Cell Cycle Effects

Kinesiology & Health Science

Obesity-Cancer Molecular Link Leptin

Y985

Y1138

Adiponectin

JAK2

P

P

STAT3

?????

??? Metabolic Effects & Cell Cycle Effects

Kinesiology & Health Science

AMPK/T198 p27??

Obesity-Cancer Molecular Link Leptin

Y985

Y1138

Adiponectin

JAK2

P

P

STAT3

?????

??? Metabolic Effects & Cell Cycle Effects

Kinesiology & Health Science

AMPK/T198 p27??

Obesity-Cancer Molecular Link Leptin

Y985

Y1138

Adiponectin

JAK2

P

P

STAT3

?????

??? Metabolic Effects & Cell Cycle Effects

Kinesiology & Health Science

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

Kinesiology & Health Science

80%

The Immune System and AIDS Vander, Sherman Luciano Chapter 18 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

Cells Of The Immune System 1. Leukocytes 2. Lymphocytes B Cells (bone marrow) Natural Killer (NK) cells T cells (thymus): 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)

Specific Immune Defenses 2. Activation of lymphocytes Binding of antigen to receptor causes cell division Multiple cell divisions lead to increased numbers of antigenspecific lymphocytes (clonal expansion) Can all recognize original antigen Some cells carry out attack and some will serve as “memory” cells

Specific Immune Defenses 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: Recognize foreign antigens and secrete antibodies 2. Helper T cells:

Help activate B cells and cytotoxic T cells CD4-positive

3. Cytotoxic T cells:

Carry out attack response Secrete toxic chemicals CD8-positive

4. Macrophages

Lymphocyte

Macrophage

NK cell

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

differentiation to plasma cells presentation to helper T cells memory B cells

Invader digested and antigens presented on cell (Macrophage or B cell) surface Antigen recognized by helper T cell

activation

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 First cases seen/diagnosed in early 80s Loss of immune response Makes individual susceptible to benign infections Patients do not die from AIDS, they die from other infections

HIV and AIDS Major Routes of HIV Transmission 1. Blood transfer (including needle sharing) 2. Sexual intercourse 3. From mother to fetus 4. Breast milk during nursing

AIDS Statistics Living with AIDS: 40.3 million Sub-saharan Africa: 25.8 million (7.2) North America: 1.2 million (0.7%) New Cases (2005): 4.9 million Sub-saharan Africa: 3.2 million North America: 43,000 million Deaths (2005): 3.1 million Sub-saharan Africa: 2.4 million North America: 18,000

HIV HIV – human immunodeficiency virus gp120 gp41 Reverse transcriptase

Viral RNA

Viral core

HIV Key Features of HIV Genome 1. Genes are encoded by RNA NOT DNA uses hosts translational machinery to produce proteins one of the HIV genes makes DNA from RNA 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

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 CC-chemokine receptor 5 (CCR5) or CXCR4 People with mutations in this chemokine receptor are resistant to HIV infection

THERAPY!!!

chemokine receptor blockers/antagonists

HIV After HIV entry into cell: Viral mRNA converted into DNA by reverse transcriptase Nuclear import and integration into host DNA Integrase enzyme (viral) DNA selectively integrated into transcriptionally active regions Transcription of viral DNA

host and viral proteins

HIV Viral Transcription Key control elements 1. TATA box; recruits host RNA polymerase II 2. Enhancer elements; use host proteins to enhance transcription very important in active cells 3. Modular elements; binding sites for host proteins 4. Tat-response element (TAR) Tat-mediated effects

HIV Viral Transactivators Tat and Rev 1. Tat: TransActivator of Transcription (≈ 100 amino acids) Essential for viral replication Binds to TAR Increases the efficiency of elongation Needs host proteins to do its job

HIV Other Tat Functions Translocated across plasma membrane Domain within Tat (RKKRRQRR) allows entry into cells R = arg K = lysine Exploited for therapy Q = glutamine May “prime” T-cells for infection and replication Induces apoptosis in T cells and neurons Neuropathogenesis and immune evasion

HIV 2. Rev: REgulator of Viral expression (≈ 116 amino acids) Acts through Rev-responsive element (RRE) RRE in env (viral) gene Encodes viral envelope (incl. gp120 and gp41) Also needs cellular co-factors Effects of Rev Nuclear export of “late” mRNAs Encode for viral structural proteins

HIV Other Viral Proteins Core proteins (3): Help in packaging new viruses Enzymes (4): Generating DNA from RNA (reverse transcriptase; RT) Cleavage/activation of viral proteins (viral protease; PR) DNA integration into host (integrase; IN)

HIV Other Viral Proteins Nef (Negative Factor): Many functions essential for disease induction 1. Downregulation of CD4 2. Enhancement of viral infectivity 3. Killing of cytotoxic T cells (indirectly)

HIV Other Viral Proteins Nef (Negative Factor): 4. Modulation of host-cell signaling Interferes with normal cellular signaling pathways Impairs normal T cell function THERAPY?? Disease can still develop in absence of Nef

HIV Life Cycle

HIV End Effects of HIV on Body Function 1. Helper T-cell suppression Viral proteins downregulate CD4 expression and causes apoptosis in infected cells Loss of helper T cells and their function Insufficient immune response Susceptibility to multiple normally benign infections

HIV End Effects of HIV on Body Function 2. Dementia HIV also infects microglial cells in brain (CD4 positive) Induces apoptosis/cell loss Decreased cognitive and motor function

HIV Treatment Strategies Vaccines Unsuccessful so far Highly mutated Reverse transcriptase highly prone to errors 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 Antiviral Cocktails 1. Protease inhibitors 2. Nucleoside inhibitors of RT (AZT, Zidovudine) 3. Non-nucleoside inhibitors of RT 4. Immune system “boosters” 5. Anti-infective drugs Effectiveness??

Alzheimer’s Disease What is Alzheimer’s? Progressive neurodegenerative disease Dementia: Cognitive impairment

not unique

Many non-Alzheimer’s forms of dementia Commonly manifests as memory loss Short-term memory first Then long term memory Loss of inhibition

Alzheimer’s Disease

Alzheimer’s Disease 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

Alzheimer’s Disease

Alzheimer’s Disease 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

Alzheimer’s Disease β -Amyloid precursor protein (APP) Integral transmembrane protein Natural neuroprotective peptide Protects against glutamate toxicity Cleaved into peptide fragments by 3 secretases (α , β and γ )

Alzheimer’s Disease

Alzheimer’s Disease α −secretase or β -secretase cleaves APP α product is cleaved into harmless by-product β product is cleaved into 40 or 42 amino acid peptide by γ secretase β -amyloid Secreted into extracellular space Forms expanding protein mass β -amyloid plaques

Alzheimer’s Disease

Alzheimer’s Disease γ -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: Not well known 4. Pen-2:

Alzheimer’s Disease β −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

Alzheimer’s Disease Plaques are normally only detected post mortem New technologies helping to get around this

Compound binds to plaques

Allows for detection by PET scan

Alzheimer’s Disease Apoptosis in neurons induced by β -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 β -amyloid Activates p38MAPK and jun n-terminal kinase (JNK) Possibly through inactivation of Bcl-2

Alzheimer’s Disease 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

Alzheimer’s Disease 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

Alzheimer’s Disease Phosphorylation balance of a protein determined by actions of kinases vs. phosphatases Tau phosphorylated by many kinases GSK-3β cdk5

p38MAPK JNK

Protein kinase A MAP kinase Calcium and calmodulin-dependent kinase II

Alzheimer’s Disease Associations between kinases and Alzheimer’s Nothing definitive yet New kinase identified: Brain-derived Tau kinase (BDTK) Found only in humans May explain resistance to animals to disease development Therapy…..??

Alzheimer’s Disease Phosphatases Activities of PP1 and PP-2A are compromised in Alzheimer’s brain Account for >90% of phosphatase activity Impairing phosphatase activity leads to phosphorylation of Tau similar to Alzheimer’s May be an increase in naturally occurring phosphatase inhibitors Okadaic acid, Inhibitor-1, Darp-32 Time will tell!!

Alzheimer’s Disease Mechanism of Neurofibrilatory Degeneration Normal tau + tubulin phosphatase

microtubules

kinase

hyperphosphorylated tau microtubule dissasembly

polymerization of hyperphosphorylated tau

compromised axoplasmic flow retrograde degeneration neuronal death DEMENTIA

Neurofibrilatory tangles

Alzheimer’s Disease 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

Alzheimer’s Disease Therapies for Alzheimer’s EXPERIEMNTAL 1. γ -secretase inhibitors Bristol-Myers in 2001 Stopped due to side effects Eli-Lily

just beginning clinical testing

Maybe β -secretase is a better target

Alzheimer’s Disease Therapies for Alzheimer’s 2. Alzhemed Neurochem Pharmaceuticals Prevent β -amyloid from “sticking” together Moving to phase 3 trials

Alzheimer’s Disease Therapies for Alzheimer’s 3. Vaccine (Elan Pharmaceutical) Use immune system to find and destroy β -amyloid Inject mice with β -amyloid Prevents formation of plaques Wiped away existing plaques

in mice

Alzheimer’s Disease

normal

“vaccinated”

Alzheimer’s Disease Therapies for Alzheimer’s 3. Vaccine (Elan Pharmaceutical) Use immune system to find and destroy β -amyloid Inject mice with β -amyloid Prevents formation of plaques

in mice

Wiped away existing plaques Move to humans

brain swelling

Alzheimer’s Disease Prevention 1. Anti-oxidants Mitochondrial defects in Alzheimer’s patients Oxidative damage Nutritional supplements/alterations 2. Keep mind active 3. Diet Low fat diet to keep cholesterol levels low

Alzheimer’s Disease Prevention

4. Exercise/fitness