MM ALL

LECTURE 13

Intro 

Cell division: cells pass through a series of defined stages, how cells reproduce 

• Doesn’t stop with formation of mature organism… continues throughout life 

Mitosis: cells are genetically identical to their parent, basis for producing new cells

Meiosis: only in sex cells, half genetic content of parent, bases for producing new sex reproducing organisms

Cell cycle

2 cells from one, 2 major phases (M and Interphase)

• M-phase: when duplicated chromosomes separate into 2 nuclei 

• Cytokinesis: when entire cell divides into 2 daughter cells 

• Interphase: majority of cell cycle, lasts longer than M phase 

Interphase: G1, S and G2

G1: end of mitosis and beginning of DNA replication 

• Cells grow and carry out normal metabolism… organelles duplicate 

S: DNA replication and chromosome duplication

• Length can be determined directly with percentage of cells nuclei are radioactively labeled (3H Thymidine)  

G2: cell grows, prepares for mitosis

Cells in vivo 

3 cell types distinguished on how they grow and divide

1. Nerve, Muscle, RBC

• Highly specialized and lack ability to divide

• Once differentiated, remain in that state until they die 

2. Liver cells and Lymphocytes

• Normally don't divide but can be induced to begin DNA synthesis and divide when given an appropriate stimulus 

3. Hematopoietic stem cells (in bone marrow)

• Cells possess a relatively high level of mitotic activity (divide very fast and 24/7)

• Stem cells have asymmetric division which the daughter cells have different fates (only one differentiated cell and one stem cell)

*cell cycles can range in length from 30 min (frog embryo) to several months (liver)

Control of the cell cycle 

• Cells contain factors that stimulate entry into mitosis

• Cytoplasm contains factors that regulate the state of the nucleus

In replicating cell: contain factors that stimulate initiation of DNA synthesis

In mitosis: contain factors that trigger chromosomal condensation 

• G1-S and G2-M both under positive control 

Cdks

• Engines that drive the cell through stages

• Activities are regulated by brakes and accelerators 

Cdk Inhibitors

• Sic1 controls cell cycle progression in budding yeast 

Controlled proteolysis of cyclins 

• Loose cyclin = no division

• Occurs via ubiquitin-proteasome pathway 

• Two classes function as ubiquitin ligases

• SCF ligase enables the entry into S phase by promoting degradation of the S phase cyclin dependent kinase inhibitor Sic1, subcellular localization of cell regulators 

Protein Kinases 

Role: entry into M phase is triggered by activation of one called Maturation Promoting Factor (MPF)

• Consists of 2 subunits, a kinase and a cyclin 

• Increase concentration of cyclin activates kinase 

Role: Cdks occur in yeast cells

• Product of the CDC2 gene in fission yeast and CDC28 in budding yeast is a cyclin dependent kinase responsible for passage through both control points 

• Cdks must be activated by specific cyclins for cells to pass through a point 

• Control is at 2 points… START, and at the G2-M transition

Cyclin binding

• Cyclin binds to catalytic subunit of Cdk

• Cdk phosphorylate other proteins 

Cdk Activation

• Cdc-activating Kinase (CAK) phosphorylates both threonine and tyrosine on the Cdk subunit

• Double phosphate one is inactive

• A phosphate(cdc25)  removes one phosphate 

• The singly phosphorylated cyclin is active, driving the cell to mitosis

• CAK and cdc25 are activated by other kinases and phosphatases

Localization 

• Movement of cyclins between the cytoplasm and the nucleus 

Cyclin B1: serine phosphorylation of NES sequence allows it to enter nucleus 

• If accumulation of cyclin is blocked, cells fail to initiate mitosis

Checkpoints, Kinase Inhibitors and Cellular responses

Ataxia-telangiectasia (AT): inherited recessive disorder with diverse symptoms including increased risk of cancer 

• Patients with AT are extremely sensitive to ionizing radiation 

MADE IT TO HERE FOR FLASHCARDS

Checkpoints: surveillance mechanisms that halt the progress of cell cycle if 

• Any of the chromosomal DNA is damaged

• Certain critical processes ex. DNA replication during S phase or chromosome alignment during M phase were not properly done 

P27

• A cdk inhibitor that arrests cell cycle progression 

• No p27 makes rat bigger, and increases size if the thymus gland 

Crucial functions of cdks and cyclins 

• Mice unable to synthesize cdk1, cyclin b1 or cyclin a2 die as early embryos, suggesting the proteins encoded by these genes are essential for normal cell cycle 

• Mice lacking cdk4 develop without insulin producing cells in their pancreas

• Mice lacking cdk2 appear to develop normally but exhibit specific defects during meiosis 

M phase: Mitosis and Cytokinesis

• Mitosis maintains the chromosome number, the chromatids of each chromosome are split apart and separate into 2 daughter nuclei in a single division 

• Can occur in haploid or diploid cells 

Phases include: Prophase, Metaphase, Anaphase, Telophase

Meiosis

• Chromosome number is halved and 4 daughter haploid cells are formed

• 2 divisions 

  • Homologous chromosomes pair and then segregate ensuring that daughter cells receive full haploid set of chromosomes 

  • Two chromatids are separated and DNA is replicated prior to meiosis during prophase stage 

Male gametes 

• Occurs prior to differentiation of spermatozoa 

• Sperm comes from spermatozoa 

• Spermatogonia that undergo meiosis become primary spermatocytes 

• Then 2 divisions of mitosis produce 4 undifferentiated spermatids 

• Each spermatid undergoes differentiation to become sperm cell 

Female gametes 

• Oogonia become primary oocytes then a greatly extended meiotic prophase 

• Vertebrate eggs are fertilized at a stage before the completion of meiosis it’s completed after fertilization while the sperm resides in egg cytoplasm 

MITOSIS VS MEIOSIS TABLE 

Mammalian fertilization role of PCSK4 (PC4) enzyme

PCSK4 (only found in reproductive)

• 4th member of the family enzymes

• Located in testicular germ cells, sperm plasma membrane, ovary and placenta 

• Function is fertility and reproduction 

• Knock out mice are subfertile in both genders… more in males 

Capacitated sperm: activated sperm capable of interacting and fusion with egg (into jelly ike substance) 

Role in acrosome reaction during fertilization 

• Binding of sperm to egg surface signals release of acrosome 

• Released acrosome contains hydrolyzing enzyme that digests the jelly coat to make way for sperm 

• Sperm and egg membranes fuse together and the nucleus enters 

1. Contact

2. Acrosomal reaction

3. Growth of acrosomal process

4. Fusion of plasma membranes 

5. Entry of sperm nucleus 

6. Cortical reaction 

The placenta

• Required for fetal survival and growth

• Villous tree = maternal fetal exchange region of placenta

• Contains fetal blood vessels, bathed in maternal blood 

• Point of convergence 

• Primary cell is trophoblast cell 

Villous compartment: 

• Underlying cytotrophoblast terminally differentiate (ct) – syncytiotrophoblast (syn)

Extra-villous compartment: 

• Cytotrophoblast invade uterus and remodel uterine spiral arteries (SA) 

Issues in placenta 

• Perturbations in placenta development have impacts on health of both fetus and mother 

  • Preeclampsia 

  • Intrauterine growth restriction (IUGR)

• These conditions often co exist 

Preeclampsia 

• Hypertensive disorder of pregnancy 

• Hypertension, renal dysfunction 

• Affects approx 5% of all pregnancies, leading cause of fetal and maternal morbidity and mortality 

• Impaired trophoblast differentiation likely caused by reduced expression of placenta GCM1 (missing transcription factors which bind to DNA)

• NO CURE

GCM1

• Expression reduced by up to 50% in placenta 

• A critical regulator of trophoblast differentiation 

• Mouse model showed normal fetal growth but increased feto-placental vascularity, abnormal pro angiogenic expression, dysregulated trophoblast differentiation within labyrinth… when its not there it leads to preeclampsia 

IUGR disorder (Intrauterine growth restriction)... placenta isn't growing properly

• Leading cause of perinatal mortality 

• Caused by aberrant development of placenta and fetus growth 

• IGF-2 is important regulator of feto-placenta growth

• IGF2 is active form derived from Pro-IGF2 following cleavage by PCSK4 enzyme 

• Lack of PCSK4 enzyme is main cause of IUGR

Cause:

• Lack of PCSK4 activity which can be due to 

  • Mutation in PCSK4 gene

  • Increased level of inhibitor

  • Decreased level or activator or promoter  

LECTURE 15

Cell signaling 

• The process with cells communicate with each other to carry out one+ functions or tasks

• Cells respond adequately and in an appropriate manner to specific external stimuli to survive

• Involved in the regulation of cell growth and division (main process involved in tumors) 

• When cells lose the ability to control cell division leading to malignant tumor 

Signal transduction 

• Cell to cell communication is mediated by signal molecules 

• can enter the cells or bind to cell surface receptors 

• Response within the intracellular molecules involving a series of steps known as signal transduction pathway 

Types of intercellular signalling 

• Autocrine (same cell)

  • Cell has receptors on its surface that respond to EM molecules produced by same cell 

• Paracrine (adjacent cell)

  • Only travel short distance through extracellular space 

• Endocrine (cell far away via bloodstream)

  • Called hormones 

Signal molecules (extracellular molecules/EM)

• Carry messages, majority are small

• Small compounds (steroids, neurotransmitters)

• Small soluble protein hormones (glucagon, insulin)

• Huge glycoproteins, bound to surface of other cells

• Cells can only respond to message if they express receptors (membrane bound proteins) 

• The molecule that binds to receptor is called ligand 

Receptors 

• Cells have different receptors that allow them to respond to different EM molecules

• Cells that share a receptor may respond differently to the same EM molecules 

• Liver and Smooth muscle cells share b2-Adrenergic receptor… activation of receptor by adrenaline in liver cells leads to glycogen breakdown but it also causes relaxation in a smooth muscle cell 

  • Due to interactions with different intracellular proteins 

Signal Transduction 

• 2 pathways 

• Via a second messenger 

• Via recruitment of a protein 

• Some surface receptors generate an intracellular second messenger through an enzyme called effector 

• Second messengers are small things that activate or inactivate specific proteins 

• Other surface receptors recruit proteins to their intracellular proteins to their intracellular domains at the plasma membrane

Basic elements: Cell signaling systems 

• Series of proteins which alters conformation of next one 

• Usually altered by phosphorylation 

• Kinases ADD phosphate groups, phosphates REMOVE them 

• Target proteins receive a message to alter cell activity 

• Overall process is called signal transduction (info carried by EM molecules translated into changes that occur inside a cell) 

Example

1. PK2 is activated by PK1

2. PK2 then phosphorylates PK3 which phosphorylates a transcription factor

3. Increasing its affinity for a site in DNA leading to alteration in gene expression 

Signal transduction 

• Protein phosphorylation adds phosphate group on SER, THR, and TYR 

  • Can activate or inactivate an enzyme

  • Increase or decrease protein-protein interactions

  • Change the subcellular location of the protein

  • Can trigger protein degradation 

• Patterns differ between cell types (ex. Two different types of breast cancer cells)

  • Triple negative breast cancer is very aggressive… it’s missing estrogen receptor, progesterone receptor, and growth factor receptor, HER2)

  • Tyrosine phosphorylation is significantly enhanced 

EM and their receptors 

EM:

• Small molecules such as amino acids and their derivatives

• Gases such as NO and CO

• Steroids 

• Eicosanoids which are lipids derived from fatty acids 

• Various peptides and proteins 

Receptor types:

• GPCRs, largest family of receptors, affect protein synthesis, protein transport, and variety of bioactivities 

• RTKS, after cytoplasmic protein activities 

• Ligand-gated channels are third type of cell surface receptors that bind to extracellular ligand or messengers… regulate flow of ions

• Steroid hormone receptors (regulate hormonal activity of steroids)

• B and T receptors (act with response to foreign antigens)

GCPRs

• Largest superfamily of receptors encoded by animal genomes 

• Have 7 a-helical transmembrane domains and interact with G proteins 

• Transmit signals from variety of stimuli outside cell into inside of cell

• When bound to GTP its ON when bound to GDP its OFF

Natural ligands: hormones, neurotransmitters, opium derivatives, chemo-attractants

Model of GPCR Rhodopsin in Vision Cycle 

• Rhodopsin contains opsin and retinal(covalently bound cofactor)

• Opsin has 7 transmembrane helices connected to each other by protein loops

• Binds with retinal (a photoreactive chromophore) which lies horizontally to the membrane 

• About half the opsin is within the lipid bilayer 

• Retinol is produced in the retina from vitamin A which comes from beta carotene

• Isomerization of 11-cis-retinal into all-trans-retinal by light created a change in opsin which activates the G protein transducin, and triggers a cyclic guanosine monophosphate second messenger cascade 

*the photoisomerization of 11-cis retinal into all trans retinal in photoreceptor (rhodopsin) is the first step in vision 

Structural features of GPCR

• 7 membrane bound helices 

• N-terminal in the extracellular part 

• C-terminal in cytoplasmic (intra-cellular) part

• 3 loops outside

• Another 3 loops inside

• Each loop connects 2 membrane segments 

• Loops are of varying lengths 

• Outer loops together to form the ligand binding pocket 

• Different GPCRs have different pocket structures 

• Inner loops provide binding sites for intracellular signaling proteins 

GPCR type receptors 

• GPCR superfamily is composed of 750 genes in humans (code for GPCR) 

• Are targeted by about half the drugs prescribed for human diseases 

• Their dysfunction or absence is cause for disease 

GPCR and their second messengers 

Signal transduction:

• Ligand binding on the extracellular domain changes the intracellular domain

• Affinity for G proteins increases and the receptor binds a G protein intracellularly

• GDP is exchanged for STP on the G protein, activating it 

• One ligand-bound receptor can activate many G proteins 

*adenylyl cyclase is effector in this event 

Termination of the response:

Desensitization - by blocking active receptors from turning on additional G proteins 

• GRK activated a GPCR via phosphorylation 

• Proteins called arrestins compete with G proteins to bind GPCRs

• Termination of the response is accelerated by regulators of RGSs 

Arrestin

• Phosphorylation of GPCRs sets the stage for the binding of arrestins 

• Upon binding, the GPCRs become desensitized even though ligands are still bound extracellularly 

• If receptors are recycled and returned to the cell surface, the cells remain sensitive to the ligand and are said to be resensitized 

AP2: recruits clathrin proteins to form catherine coated vesicles

ERK: extracellular signal regulated kinases leading to tumorigenesis 

Bacterial toxins 

• GPCRs provide excellent targets for bacterial pathogens 

• Cholera toxin exerts its effect by modifying the Ga subunits which are stimulated by beta adrenergic receptors 

• Caused inhibition of GTPase activity in cells if intestinal epithelium 

• As a result, churns out cAMP which causes epithelial cells to secrete large volumes of fluid into intestinal lumen which locks in activation state causing diarrhea

• Similar effects were notes with pertussis toxins 

• Alpha adrenergic receptors stimulate gai to inhibit adenylate cyclase. Gai is target of pertussis toxin, locks in inhibitory state causing excessive coughing 

Human perspective: Disorders associated with GPCRs

Retinitis pigmentosa - progressive degeneration of the retina, can be caused by mutations in rhodopsin ability to activate a G protein 

• Gain of function mutations (highly activated receptor) may create an always activated G protein

  • Some benign thyroid tumors are caused by mutation in receptor 

• Loss of function mutation can regulate the binding activity of g protein leading to disease or disorder condition 

• Certain polymorphisms in g protein related genes may cause increased susceptibility to asthma or HBP as well as decreased susceptibility to HIV

Polymorphism: existence of many forms of DNA sequences at a locus within the population as well as a discontinuous genetic variation 

G/GPCR related diseases 

1. Albright's hereditary osteodystrophy

• Lack of response to PTH, leading to low serum CS, high serum phos[hate and PTH… low mental retardation  

2. Mccune albright syndrome 

• Genetic disorder of bones, skin pigmentation, hormonal problems and premature puberty 

3. Precocious puberty 

• Onset of early puberty (7-8 for girls, 9 for boys)

4. Hypocalciuric/Hypercalciuric 

• High Ca in serum and low Ca in urine… risk factor for preeclampsia during pregnancy 

5. Parahyperthyroidism 

• High PTH level which regulated Ca and phosphate levels, harmful to bones 

• Symptoms: weakness and fatigue, depression, bone pain, muscle soreness, decreased appetite, feelings of nausea, cognitive impairment, kidney stones, osteoporosis 

6. Familial Glucocorticoid deficiency 

• Elevated ACTh, low serum cortisol level

• Symptoms: lethargy, muscle weakness, decreased consciousness level 

Orphan GPCR - Ex from Melatonin

• Melatonin in involved in circadian rhythm regulation, reproduction, and sleep

• Three members of melatonin receptor have been cloned MT1, MT2, and GPR50

• Melatonin targets MT1 and MT2 receptors (binds with them)

• GPR50 doesn’t bind to melatonin or any other logan so can be classified as orphan GPCR 

LECTURE 16

• G-proteins become activated when GDP→GTP

• Switched on by cAMP

• GPK blocks/prevents 

GPCRs… Senses

• Phosopsin is a photosensitive protein for black and white vision that is also GPCR

• Several color receptors in cones of retina are GPCRs 

• Distal tips of neurons contain odorant receptors GPCRs that bind various chemicals 

• There are more than 400 types of these receptors in the human nose

• Each taste receptor cell in tongue transmits a sense of only 5 basic taste qualities 

  • Salty

  • Sour

  • Sweet

  • Bitter

  • Savory

• Perception that food is bitter, sweet, or savory depends on compound interacting with GPCR at surface of receptor cell 

Second messenger molecules 

PIs: phospho inositols

DAG: diacylglycerides 

cAMP: cyclic adenosine monophosphate 

cAMP

• Presence was discovered in 1971 in aplysia 

• It's a second messenger which is released into the cytoplasm after binding with an extracellular ligand with a GPCR

• Messengers amplify the response due to a single extracellular ligand

•  5-HT also called serotonin 

cAMP continued…

• Synthesized from ATP by adenylate cyclase on inner side of the plasma membrane 

• Adenylate cyclase is activated by a range of signalling molecules through activation of Adenylate Cyclase Stimulatory G protein coupled receptors and inhibited by agonists of Adenylate cyclase inhibitory G protein coupled receptors called cAMP-Dependent Pathways 

• Liver adenylate cyclase responds more strongly to glucagon and muscle adenylate cyclase responds and muscle adenylate cyclase responds more strongly to adrenaline (epinephrine) 

• cAMP decomposition into AMP is catalyzed by phosphodiesterase 

• It transfers the effect of hormones like glucagon and adrenaline cannot pass through cell membrane

• Involves the activation of protein kinases and regulates effects of adrenaline and glucagon 

• Also binds to and regulated ion and protein channels 

Prefrontal cortex disorders

• cAMP affects the function of higher order thinking in the prefrontal cortex through its regulation of ion channels called HCN

• When cAMP stimulates the HCN, the channels open, closing the brain cell to communication interfering with the function of the prefrontal cortex 

Human carcinoma: cAMP induces apoptosis to multiple myeloma (cancer of blood cells)

Glucagon(29 aa peptide): produced by PCSK2 mediated cleavage followed by CpE cleavage to remove terminal extra basic aa residues

• Produced by pancreatic alpha cell, it increases sugar level in blood… opposite effect of insulin 

• Pancreas releases glucagon when blood sugar levels fall too low, it causes the liver to convert stored glycogen into glucose which is released into the bloodstream

• High blood glucose levels stimulates the release of insulin (pancreatic beta cells)

• Insulin allows glucose to be taken up and used by insulin dependent tissues 

• Glucagon and insulin are part of a feedback system that keeps blood glucose levels at stable level 

Proglucagon: gives you glucagon in pancreas 

Regulation of serum glucose level 

Glucose: source of energy 

• Oxidized to CO2 and H2O, providing cells with STP used for all energy driven cellular reactions

• Body maintains level within narrow range 

• Excess glucose is stored as glycogen 

Regulation

• Controlled by hormones glucagon, insulin and epinephrine (adrenaline)

• Both glucose and epinephrine bind to different GPCR receptors on the same cell

• Both stimulate glycogen breakdown and release of glucose into blood stream

• Higher glucagon and epinephrine mean higher blood glucose 

• Hormones upon binding with receptors, activate G protein which then stimulates cAMP

• Responses are amplified by signal cascades 

• Insulin stimulated glucose uptake and storage as glycogen 

Glucose storage or mobilization 

• Activities of glycogen phosphorylase (GP) and glycogen synthase (GS) are controlled by hormones that act through signal transduction pathway

• GP is activated in response to glucagon and epinephrine and increases glucose level 

• GS is activate by insulin and destroys sugar 

Glucose metabolism

• cAMP is synthesized by adenylyl cyclase 

• Evokes a reaction cascade that leads to glucose mobilization 

• Once formed cAMP molecules diffuse into cytoplasm where they bind a cAMP dependent protein kinase (PKA)

Other aspects

• Some PKA molecules phosphorylate nuclear proteins 

• Phosphorylated transcription factors regulate gene expression

• Phosphates halt the reaction cascade 

• cAMP is produced as long as external stimulus is present 

Liver: epinephrine and glucagon, glycogen breakdown, glucose synthesis, inhibition of glycogen synthesis

Cardiac muscle: epinephrine, increased contractility 

PI (phospho-inositol)... part of the cell membrane

• Derived from inositol phospholipid structure 

• Helps with cell growth, apoptosis, cell migration, endocytosis, and cell differentiation 

• Cleaves by 4 types of phospholipases 

• PLC splits phosphorylated head group from diacylglycerol

• All 4 enzymes can be activated in response to a signal molecule and the products they produce can act as second messengers 

Cell membrane types

1. Phospholipids (Major): polar head+phospho+glycerine+fatty acid 

2. Glycolipids

3. Cholesterol 

Inositol: brain, bone marrow, eyes, and intestine membranes 

Oleate: double bond, an be cis or trans

Palmitate: straight 

PI-derived second messengers

• Some phospholipids of cell membranes are converted into second messenger by activated phospholipases 

*phosphorylations of PI by PI kinases followed by cleavage of phospho-glycerol bond by PLC enzyme, leading to PI-P3 and DAG

Phosphatidylinositol phosphorylation

• PI are derivatives of Phosphatidylinositol

IP3 and DAG

• Result of ligand-induced breakdown of phospho-inositides in the lipid bilayer 

Net effect: release of Ca2+ into cytosol 

• Linked to stroke, alzheimer and huntington disease 

• Stroke is linked to how much IP3 produced, higher levels of calcium into cytosol 

DAG: activated protein kinase C which phosphorylates Ser and Thr residues on target proteins that play role in cell differentiation, cell growth, etc

IP3: 

• One receptor is a calcium channel located at surface of smooth ER

• Binding of IP3 opens the channel and allows Ca2+ ions to diffuse out 

PI-PL-Cb: phospho iositol specific phospholipase Cb enzyme (becomes activated)

Lipid kinases: add phospho group to lipids

Liquid phosphates: PTEN removes phospho group 

Activation of protein kinase C by DAG biological consequences

• PKC is a member of protein kinase enzymes (15 types and 3 sub families)

• Protein kinases control the function of other proteins through protein phosphorylations via Ser and Thr residues

• Many play important roles in the control of cell growth and differentiation

• PKC are activated by DAG as well as by Ca+2 and phospholipid like phosphatidyl-Serine

Synopsis (remember all)

• Cell signaling is a thing where info is relayed across the plasma membrane into the cell and often to the nucleus

• Many extracellular stimulus (first messengers) initiate responses by interacting with a GPCR on outer cell surface and stimulating the release of a second messenger within the cell

• Phospholipase C is important effector on inner surface of plasma membrane that can be activated by heterotrimeric G proteins 

• PI C splits POP2 into 2 different second messengers; IP3 and DAG

• Utilization of glucose is controlled by a signaling pathway that begins with an activated GPCR

• Many extracellular stimuli initiate a cellular response by binding to the extracellular domain of a RTK, which activated the tyrosine kinase domain located at the inner surface of the plasma membrane 

LECTURE 17

Intracellular receptors (IR)

2 types 

• Both bind to extracellular signaling molecules that migrate or penetrate through cell membrane or are transported across the membrane by other molecules 

• Signalling molecules for IR are lipid soluble 

1. Cytoplasmic receptors

• Present in the cytoplasm

• Also called nuclear receptor type 1 

2. Nuclear receptors 

• Present inside the nucleus 

• Also called nuclear receptor type 2 

• Are used by steroid hormones 

• Steroid hormones are lipid soluble and diffuse through the cell membrane and bind to steroid receptors 

• This complex then binds to parts of DNA in nucleus called hormone responsive elements leading to alteration in gene expression 

1. Steroid hormone diffuses through the cell membrane (also called ligands)

2. Hormone binds to intracellular receptor either in cytoplasm or nucleus, forming a hormone-receptor complex 

3. The complex interacts with DNA in the nucleus, altering gene expression and cell function 

Nuclear Receptors (NR)

• Special class of soluble cytosolic proteins that function as receptors and bind to 

  • Steroid hormones

  • Thyroid hormones

  • Certain molecules such as vitamins

• All known as NR-ligands are found in animals but not in plants, algae, or fungi 

• The NR number in human 48 which 24 have been described with their ligands

• Other are orphan receptors meaning no known ligands 

• C-elegans have 270 NRs 

Cont.

• NR bind to DNA and regulate the expression of adjacent genes… classified as transcription factors

• NR mediated gene expression happens when it binds to a specific ligand, resulting in a conformational change and activation of the receptor

• This leads to increase or decrease regulation of gene expression 

• NRs possess the ability to directly interact with and control the expression of genomic DNA

• Also known as nuclear receptors or ligand activated transcription factors 

NR ligands

• Molecules that bind to NR

• They are mostly steroid, thyroid, hormones and vitamins 

• Are lipophilic/highly hydrophobic and readily diffuse across cell membranes via hydrophobic interaction 

Steroid hormones as NR-Ligands

5 major types…

Sex hormones: 

• Estrogen (female), major: Estrone, estradiol, estriol 

• Androgen (male), major: testosterone, androstenedione

  • Much longer 10-terminal than estrogen… very close together 

• Progesterones (female… little bit in male)

Non sex: 

• Glucocorticoids 

• Mineralocorticoids 

Estrone: 12x weaker than estradiol, one OH

Estradiol: strongest estrogen, 2 OH

Estriol: 80x weaker than estradiol, 3 OH

Testosterone: not aromatic, ketone, methyl group 

Cortisol: 2 carbons added 

Corticosterone: Aldehyde group 

Non-steroid hormones as receptors

RAR, TR(bad for you), VDR 

Types of nuclear receptors

• Classified according to either mechanism or sequence homology 

• Monomore type: mostly steroid hormone receptors 

• Homo or hetero-dimer type: mostly non steroid receptors, only active when become dimer 

Homodimer: 2 identical NR homo-dimerizes

Heterodimer: 2 different types of NR hetero-dimerizes 

Ex. PPAR heterodimerize with retinoid x receptor… interferes with action of diabetes

AD: auto-activation domain

NLS: nuclear localisation signal 

Both work together with DNA binding to alter … flexible hinge domain 

DBD: DNA binding domain

LBD: ligand binding domain

AF-1: Ligand independent transactivation domain 

AF-2: Ligand dependent transactivation domain
RAR (retinoic acid receptor):
N-terminal domain (A/B), contains a transcriptional activation function, the DNA binding domain, a hinge region, the ligand-binding domain, and the c terminal domain

• Regions c and e are most conserved with the RAR family

A-B regulatory domain (variable): regulates transcriptional activity cia interaction of its AF-1 region with AF-2 region of E-domain to produce a robust gene-up regulation 

C DNA-binding domain: highly conserved domain containing two zinc fingers that binds to specific sequences of DNA called HRE

D Hinge domain: flexible domain that connect the DBD with the LBD… influences intracellular trafficking and subcellular distribution 

E Ligand binding domain: Conserved in sequence and structure, 3 antiparallel alpha helices flanked by 2 alpha helices on one side and three on the other… the ligand binding cavity is within the interior of LBD… contains activation function 2 whose action is dependent on presence of bound ligand 

F C-terminal domain: highly variable in sequence between various nuclear receptors 

Heat Shock Proteins (HSP) as co-receptors to steroid receptors 

• Are chaperones that assist proper folding of other proteins

• Present in low levels but increase in response to stress like sudden temp increase

• Stabilize other proteins against heat stress

• Play roles in protein maturation, activation, translocation and degradation

• Other HSP proteins act as co-chaperones 

*steroid receptor (glucocorticoid receptor or GR) activity depends on their binding with Hsp90 which maintains in a state capable of binding with hormone 

• Binding with HSP90, Immunophilin FKBP-51/52 and dynein proteins allows GR to translocate into the nucleus 

• Once inside nucleus, GR dimerizes and binds with DNA thereby upregulating gene expression 

Translocation of GR into the nucleus 

• Huge complex

• Must be formed before it crosses membrane then all released and forms a dimer to bind to DNA

• Immunophilins are peptidyl-pro isomerase enzymes which are involved in immunosuppressive events

• They are targeted by immunosuppressive drugs to prevent Cis-Trans isomerisation of X-Pro bond 

Releasing hormones: Steroid or Peptide Hormones 

Releasing hormones: produced by hypothalamus which are capable of accelerating the secretion of a given hormone by anterior pituitary gland 

• Luteinizing hormone (LH)

• Follicle stimulating hormone (FSH)

• Adrenocorticotropic hormone (ACTH) 

*steroid hormones have receptors that are nuclear or cytoplasmic

Biological roles of NRs and disease implications 

• Regulate specific gene expression, controlling cellular development, metabolism and homeostasis

• Homeostatic state (Maintain pH, salt conc., temp, oxygen level… many illnesses are caused by disturbances in this as well as aging)

• NRs are important mediators of many diseases

• Play key roles in Embryonic development and liver disease 

• Mutations in androgen receptor can cause infertility and prostate cancer

• Mutations in PPAR can lead to colon cancer, DM, and mutations in estrogen receptor leading to breast cancer  

Endocrinology (IMPORTANT KNOW ALL)

• Defined as the branch of biology and medicine which deal with the endocrine rather than autocrine or paracrine systems

• Particularly associated with the study of hormones, their specific secretions, and the associated disorders and diseases

• Diseases associated with is are 

  • Cell proliferation

  • Growth

  • Differentiation and coordination of metabolism

  • Respiration

  • Excretion

  • Movement

  • Reproduction

  • Sensory perception

• These events depend on chemicals and substances synthesized and secreted by specialized cells via endocrine systems and signal molecules called hormones 

• Specific glands secrete specific hormones 

Protein hormones 

• Bind cell surface receptors 

• Triggering an intracellular signalling cascade 

Ex. GPCR and its hormone ligands 

Endocrine glands (secrete signalling molecules)

• Regulate body's growth and development

• Control function of various tissues

• Support pregnancy and other reproductive functions 

• Regulate metabolism 

Major organs include:

• Hypothalamus

• Pituitary gland

• Thyroid gland

• Parathyroid gland

• Islets of pancreas

• Adrenal glands

• Testes

• Ovaries

• Mammary gland

• Adipose tissue

*also placenta during pregnancy 

Hypothalamic-pituitary-adrenal- axis 

CRH: corticotropin releasing hormone released by hypothalamus

ACTH: AdrenoCorticoTropic hormone released by pituitary 

Cortisol: a steroid released by adrenal cortex 

HPA play a major part in neuroendocrine system that controls

• Stress response

• Digestion

• Immune system

• Mood

• Emotions 

• Sexuality

• Energy storage and expenditure 

Protein/Peptide hormones 

• Derived from inactive precursor molecules 

• Act as ligands/signaling molecules 

Too much or too little hormones = disease or dysfunction  

LECTURE 18

Cell Death 

2 types

1. Necrosis 

2. Apoptosis 

3. Autophagy(destruction of cell by itself)

Necrosis

• Cell death caused by damage due to a physical trauma, external force, or biochemical insult like 

  • Poison

  • Body injury

  • Infection

  • Blockade of blood supply (heart attack or stroke)

• When cells die from this, it causes inflammation leading to further distress or injury within the body 

• It is generally considered as regulated but much less orderly than apoptosis

• Swelling of cell and its internal organs

• Membrane breakdown

• Leakage of cell contents into the medium

• Induction of inflammation leading to cell death

Apoptosis

• Cell death that won't cause damage to other good cells around it 

• Programmed cell death, when needed, sequence of events take place in orderly fashion, safe

• May be compared with controlled implosion of a building using carefully placed explosives as compared to simply blowing up the structure without concern for what happens to flying debris 

Autophagy (swallowing itself)

• Cellular degradation 

• Autophagocytosis (self eat)

• Destruction of unnecessary or dysfunctional cellular components 

• Allows the degradation and recycling of cellular components 

• Targeted cytoplasmic constituents are isolated from the rest of the cell within a double-membrane vesicle known as auto-phagosome

• The auto-phagosome then fuses with a lysosome and its cargo is degraded and recycled 

Apoptosis characteristic structural changes

• Loss of adhesion to neighboring cells 

• Formation of blebs: irregular bulge in plasma membrane of a cell caused by breakdown of cytoskeleton at call surface 

• Rapid engulfment of corpse (solid particles) by phagocytosis leading to destruction of wbc that protect the body by ingesting harmful foreign particles  

Karyorrhexis: dissection of the chromatin into small fragments

Pyknosis: Overall shrinkage in volume of the cell and its nucleus 

1. Normal cell

2. Pyknosis

3. Formation of blebs

4. Karyorrhexis

5. Phagocytosis

6. Cell destruction/death 

Necrosis: passive cell death

Exhaustion of oxygen or nutriment → exhaustion of ATP in cell → impairment of cell membrane → enzyme release causes inflammation

Apoptosis

Triggering of the death program → intracellular signaling (caspases) → fragmentation of the cell into vesicles → phagocytosis by neighbouring cells 

• Cell shrinkage, loss of adhesion to other cells, dissection of chromatin, engulfment by phagocytosis 

Apoptosis and its role 

• 1010-1011 cells in human body die everyday by it 

• Important because damage to genetic blueprints can result in unregulated cell division and cancer development 

• First discovered in 1972 in scotland 

• Notes in nematode worm… loss of 131 cells by apoptosis out of 1090 cells during embryonic development 

Apoptotic changes are activated by proteolytic enzymes, CASPASES, which target:

• Protein kinases, some cause detachment of cells

• Lamins, which line the nuclear envelope

• Proteins of the cytoskeleton

• Caspase activated DNase (CAD)

Apoptosis during embryonic development

• Forms structures, organs, and tissues 

Ex. carves out the structure of mammalian digits… spaces between fingers 

• Also active in adults where cells die

• Reduced or elevated apoptosis is linked to:

  • Cancer

  • Parkinsons, alchiemers, huntington's disease

  • Diabetes type 1 

Apoptosis Clearance 

• Antiapoptotic proteins promote survival

• Cell fate depends on balance between the two 

• The death occurs without spilling cellular contents to prevent inflammation

• A protein SCRAMBLASE facilities the rapid movement of lipids between the 2 layers of a cell membrane, promoting lipid equilibration across the bilayer 

• Apoptotic cells are cleared by phagocytosis 

Capsae enzymes in apoptosis

• In 1986, CED-3 was discovered… its a promote of apoptosis or cell killer

• A mutation in it caused no loss of cells by apoptosis (C358, D221, D371 and D374) so it becomes inactive (apoptosis wont take place, can cause cancer) 

• Discovery of CED-3 gene in nematodes led to discovery of caspases which are proteolytic enzymes 

Caspases (equivalent of CED of nematodes) 

• Distinctive class of cysteine proteases with a key cysteine residue in their catalytic pockets that are activated at an early stage of apoptosis and responsible for triggering the changes noted during cell death 

• They are made initially as a zymogen which gets activated following prodomain cleavage by itself 

• They accomplish this by cleaving and activating a select group of essential proteins (substrates) that are implicated in cell destruction and killing 

Caspase enzymes

• At least 12 are known in human 

2 types 

• Initiator (Apical) caspases

• Incluse caspase 2, 8, 9, 10 which cleave inactive pro-forms activating them

• Effector (Executioner) caspases

  • Include caspase 3, 6, 7, and cleave other protein substrates within the cell to trigger the apoptotic process 

  • These caspase cleave carboxy terminal to aspartic acids

  • Ex. caspase-3 likes the sequence Asp-Glu-Val-Asp X-X- or D-E-V-D X-X

Caspase substrates, final targets

Protein kinases: include FAK, PKB, PKC, PAK2, and Raf1. 

• Inactivation of FAK disrupts cell adhesion, leading to detachment of the apoptotic cell from its neighbors 

Nuclear lamins: make up the inner lining of nuclear envelope

• Cleavage of lamin proteins lead to disassembly of the nuclear lamina, and shrinkage of the nucleus 

Cytoskeleton Proteins: such as intermediate filaments, actin, tubulin, and gelsolin

• Cleavage and inactivation lead to changes in cell shape 

Inhibitor of ICAD: 

• Cleavage by Caspase-3 activates CAD and endonuclease

• Once activated, CAD translocated from cytoplasm to nucleus where it attacks DNA severing it into fragments

Poly ADP Ribose Polymerase (PARP): 

• Cleavage results in loss of catalytic activity and may prevent deletion of ATP 

Pathway of apoptosis

• Triggers by internal stimuli such as abnormalities in DNA, and external stimuli, such as certain cytokines, released by immune system

Extrinsic pathway

1. Receptor mediated event

2. Requires stimulant molecule 

3. Stimuli in this case is a cellular messenger protein called TNF (Tumor necrosis factor)

4. TNF is produced by cells of immune system which have been exposed to 

• Ionizing radiation

• Elevated temp 

• Viral infection, or exposure to toxic chemicals used in chemo 

• TNF binds to TNFR1, that turns on the apoptotic process by binding with cytoplasmic adapter proteins (TRADD and FADD) 

• Procaspase-8 (2 molecules) binds with FADD (2 molecules) 

• One procaspase-8 activates the other which then carry out the death event 

• TNF receptor is present in the plasma membrane as a pre-assembled trimer 

• Cytoplasmic domain of each TNF receptor subunit contains a segment of 70 aa called death domain that mediates protein-protein interactions 

• Binding of TNF to the trimeric receptor produces a change in conformation of receptors death domain, which leads to recruitment of number of proteins 

Intrinsic pathway of apoptosis 

1. Mitochondria mediated events

2. Triggered by an internal stimulant

3. Stimuli include

• Irreparable genetic damage

• Lack of oxygen (hypoxia)

• Extremely high concentrations of cytosolic+2

• Viral infection

• Severe oxidative stress (production of large numbers of destructive free radicals)

• Activation is regulated by Bcl-2 (b-cell lymphoma) family proteins such as Bac which contains one or more BH (BCL2 Homology) somains

• Upon binding of Bax to mitochondria, the latter releases cytochrome C into cytosol where it forms multimeric complex with a cytosolic protein (Apaf1) and procaspase-9

• The latter becomes highly activated, cleaves and activates executioner caspases (ex. CASP-3) which then carry out apoptotic response 

LECTURE 19

Basic properties of cancer cells

Normal: 

• Bound together

Cancer

• Irregular shape

• May have 2 nucleus

• Enlarged nucleus

• Every cell is different 

• Loss of cytoplasm 

Intro

Cancer: an abnormal growth of cells which tend to proliferate in an uncontrolled manner and in some cases to metastasize

Neoplasm: an abnormal mass of tissue that grows and divides more than they should. They may be benign (not cancer) or malignant (cancer)

Proliferation: multiply rapidly to produce new cells, tissues uncontrollably producing malignant tumors that have the ability to invade surrounding healthy tissues 

Metastasis: when spawning cancer cells break away form parent mass, enter the lymphatic or vascular circulation, and spread to distant sites in body 

Stages of cancer determined by TNM score: T=tumor size, N=spread to lymph nodes, M=metastasis or spread

Popularity 

Basis: cancer is a genetic disease, but not always an inherited disease 

• Cancer is the number one killer in canada 

• Prostate and breast are most survivable cancer

• Pancreatic is most lethal with lung being second most lethal 

Distribution/gender

Both gender: liver, lung, bone, skin, colon, brain, rectal, pancreatic, kidney, bladder, blood

Male: prostate, testicular

Female: cervical, ovarian, breast, vaginal

Histological types of cancer

Epithelial = carcinoma

Glandular = adenocarcinoma

Connective = sarcoma

WBC = leukemia

Lymphocyte = lymphoma 

Nerve = glioma 

Plasma = myeloma 

Basic properties of a cancer cell

• Malignant cells are not responsive to influence that cause normal cells to stop growth and division

• The capacity for growth and division is similar between cancer cells and normal cells

• When there are no growth factors in the medium or when cells contact surrounding cells… normal cells stop growing, malignant cells continue to grow → immortality 

• Cancer cells grow in clumps (foci), normal cells grow in monolayer

• Cancer cells will grow without a growth factor, normal cells grow and then stop 

Phenotype

• Normal cells can be changed into cancerous by chemicals or viruses

• Karyotype can have differences in numbers, missing, morphology, shape, size, etc

• Different types of cancer cells share similarities

  • Aberrant chromosome numbers (aneuploidy)

  • Fail to elicit apoptosis

  • High metabolic requirements 

6 main characteristics of cancer cells

1. Self-sufficiency in growth signals (immortal)

2. Evading apoptosis (don't die)

3. Sustained angiogenesis (make new blood vessels for their survival)

4. Limitless replicative potential (repeat lots)

5. Tissue invasion and metastasis

6. Insensitivity to anti-growth signals (don't care about them)

Causes of cancer

• Most are still unknown

• Mutagenic agents, such as carcinogenic chemicals, radiation or virus can cause cancer by altering genome → asbestos

• DNA tumor viruses and RNA tumor viruses carry genes that interfere with cell growth regulation

• Diet can also influence risk of developing cancer 

• Asbestos and mesothelioma (lungs) very aggressive and die very quickly 

Biological carcinogens

• Viruses, parasites, bacteria

• DNA virus

• Papovirus – HIV

• Herpes virus

• Adenovirus

• Hepatitis B virus 

Genetics of cancer 

• Cancer results from an uncontrolled proliferation of a single cell

• Tumorigenesis occurs by a cumulative progression of genetic alterations 

• Cells become less responsive to growth regulation and better able to invade normal tissues 

• First step is the formation of a benign tumor, which is composed of cells that proliferate uncontrolled but cannot metastasize to other sites 

• Products of the genes involved in carcinogenesis are usually responsible for cell cycle regulation, cell adhesion, and DNA repair 

*sequence in which genes mutate influences the development of cancer 

1. Mutation inactivates tumor suppressor gene

2. Cells proliferate

3. Mutation inactivates DNA repair gene

4. Mutation of proto-oncogene creates an oncogene

5. Mutation inactivated several more tumor suppressor genes 

6. CANCER

Genetics of cancer: Stem cells 

Proposed cells of origin of malignant tumors: cells can either arise from tissue stem cells or progenitor cells 

Stem cells (pluripotent/totipotent): differentiate into progenitor cells, which are more committed to cell line than stems, but are still undifferentiated

Pluripotent: can become any cell type except placenta and reproductive

Totipotent: form any cell type plus the extra embryonic or placental cells

Progenitor cells are multipotent (more than one cell type but limited) or unipotent (one cell type)

• stem cells can self-replicate and produce progenitors that differentiate into more mature cell types 

• A cancer stem cell is thought to self replicate and produce progenitors that generate all cell types that make up a tumor 

• CSCs adapt and resist chemotherapy-radiation therapy 

Genetics of cancer

• Genetic changes that occur are often accompanied by histological changes or changes in appearance of cells

• Initial changes often produce precancerous cells that have gained some cancer cell properties but lack the capability to invade normal tissues or metastasis to distant sites 

• Cervical cancer typically progresses over a period of more than 10 years… very slow progressing and is characterized by cells that appear very abnormal, and big nuclei 

Malignant vs benign tumors 

Benign: not cancer, tumor cells grow only locally and cannot spread

Malignant: cancer, cells invade neighbouring tissues, enter blood vessels, and metastasize to different sites 

Proto-oncogene: genes that encode normal proteins involved in regulation of cell signaling and proliferation (normal genes)

Oncogene: mature proto-oncogenes, activated by gain of function mutations resulting in uncontrolled cellular proliferation and transformation 

Oncogenes

Proto-oncogenes can be converted into oncogenes by several mechanisms:

• Gene can be mutated to alter properties of gene product so it doesn't function normally

• Gene can be duplicated resulting in gene amplification and excess protein production

• Chromosome rearrangement brings a DNA sequence into close proximity of the gene to alter expression or nature of gene product 

Oncogenes

• Encode proteins that promote loss of growth control and the conversion of a cell to a malignant state 

• Were first discovered in genomes of tumor viruses

• Is an alters cellular gene

• Oncogenes act dominantly

• For a cell to become malignant, both allele of a tumor-suppressor gene must be lost, and a proto-oncogene must be converted into oncogene 

Oncogenes that encode growth factors or their receptors 

• Simian sarcoma virus contains the oncogene (sis) which is derived from a cellular gene which encodes for growth factor PDGF

• Oncogene (erbB) directs the formation of an altered EGF receptor that stimulated the cell regardless of the presence of growth factor 

• Some malignant cells contain lots of surface receptors which make them sensitive to low concentrations of growth factors 

Oncogenes that encode cytoplasmic protein kinases

• Raf, a serine-threonine protein kinase in the MAP kinase cascade, can be converted into an oncogene by mutations that turn it always on

• Oncogene product Src is a protein tyrosine kinase which phosphorylates proteins involved in signal transduction, control of cytoskeleton, and cell adhesion 

Oncogenes that encode nuclear transcription factors

• Myc protein stimulated cells to re enter cell cycle form G0 stage

• Overexpression of myc may cause cells to proliferate uncontrollably 

Oncogenes that encode for proteins that modify chromatin 

• Several oncogenes encode proteins that affect DNA methylation or histone modifications 

• DNA methyltransferases, histone acetylases and deacetylases, histone methyltransferases and demethylases and proteins in the chromatin remodeling complexes 

Oncogenes that encode metabolic enzymes 

• Tumor cells are more reliant on glycolysis compared to normal cells 

• TCA mutations cause conversion of isocitrate to an abnormal metabolite that impacts histone demethylation and DNA methylation, leading to aberrant gene expression 

Oncogenes that encode products that affect apoptosis 

• Overexpression of Bcl-2 gene leads to suppression of apoptosis allowing abnormal cells to proliferate into tumors 

Extracellular mitogen: a form of protein which encourages cell to undergo cell division triggering mitosis 

Tumor suppressor genes 

• Tumor suppressor genes encode proteins that restrain cell growth

• A normal cell fused to a cancer cell can suppress malignant characteristics in the latter

• Specific regions of chromosomes are deleted in cells of certain cancers 

• Most proteins encoded by them act as negative regulators of cell proliferation

• Products of them can help mainatin genetic stability 

APC - colorectal

BRCA1 - breast 

MSH2, MLH1 - colorectal 

E-Cadherin - breast, colon, etc

INK4a - melanoma, pancreatic

RB - retinal (blindness)

Retinoblastoma 

• RB was the first tumor suppressor gene to be discovered

• It is inherited in certain families, and occurs sporadically in the population at large 

• Cells of children with inherited RB have a deletion in one copy of the RB gene 

• Development requires both copies of RB to be altered or eliminated 

Role of pRB(protein encoded by RB gene) in regulating cell cycle

• Regulated the G1 - S transition 

• E2F family are targeted by pRB

• The arrest of cell cycle in G1, required for normal cell differentiation is directed by pRB

• Animals with one mutated copy of RB gene have elevated risk of cancer 

P53

• Guardian of genome

• Blocks by binding to DNA

• Suppressed formation of tumors and maintains genetic stability 

• Most important tumor suppressor gene

• Proper functioning is very sensitive to even slight changes in the aa sequence 

• Particularly sensitive to mutations in DNA binding domain 

• P53 acts as a transcription factor, activating the expression of a gene that inhibits the G1-S transition

• When a cell has damage to its DNA, the concentration of p53 rises so that the damage can be repaired before initiating DNA replication

• P53 protein triggers apoptosis in cells whose DNA is damaged beyond repair 

%mutations in gene lead to increased pancreatic, ovarian and colon cancer 

In cells with chemo agents

• Normal cells undergo growth arrest and apoptosis, cells lacking p53 continue to proliferate 

Tumor suppressor genes

• Colon cancer is often caused by an inherited deletion in a tumor suppressor gene called APC

• Inherited breast cancer is caused by mutations in BRCA tumor suppressor genes, which may act as transcription factors and in DNA repair 

Cancer treatment options 

• As long as growth of tumor remains localized, disease can be treated and cures by surgical removal 

• Malignant tumors tend to metastasize and spawn cells that break away from parent mass, enter lymphatic or vascular circulation and spread to different sites in body where they establish secondary tumors METASTASIS that are no longer amenable to surgical removal 

Current treatments 

1. Surgery (removal of cancer if localized)

2. Chemotherapy (using chemical agents to destroy cancer cells… may also kill good ones)

3. Radiation (to mutate cancer cells so that they lose their property.. may also for good cells ) 

*these treatments often lack specificity needed to kill cancer cells without damaging good cells, as evident with the serious side effects that come with them 

New treatment strategies 

Conventional cancer therapies may be replaced by targeted therapies based on the molecular basis of malignancy

• Target attack only cancer cells, leaving normal cells fine

• Can be targeted to a particular protein whose inactivation leaves cancer cells unable to grow or survive

• Targeted to the cancer cells of a particular patient based on their unique pattern of somatic mutations 

New strategies include:

• Antibodies against tumor cells (immunotherapy)

• Inhibition of cancer promoting proteins

• Preventing the growth of blood vessels that nourish tumor 

• Cancer killing virus 

1. Immunotherapy

• Passive immunotherapy uses the patient's own antibodies to respond to tumor cells 

• Herceptin is an antibody against growth factors that stimulates proliferation of breast cancer cells

• Rituxan is an antibody that binds to cell surface proteins of non-hodgkin's lymphomas 

• Vectibix is an antibody directed against the EGF receptor or colon cancer 

2. Targeting cancer proteins 

• Inhibiting the activity of cancer promoting proteins

• It might stop the uncontrolled growth and invasive properties of malignant cells

• Studies with these drugs have shown moderate success

• A possible reason for the success is that agents are not targeting appropriate cells within tumor

• Tumors with BRAF have an altered site inhibited by the drug Zelbocaf 

• These inhibitors target cancer promoting growth factor,s their receptors, protein kinases and their promoters, proteins that inhibit apoptosis, proteins involved in tissue invasion, metastasis, p53 interaction with other proteins 

Ex. Gleevec 

3. Blocking angiogenesis 

• Inhibiting formation of new blood vessels 

• Compounds that inhibit it are promising treatments 

• Interruption of VEGF

• An angiogenesis inhibitor denies the tumor access to nutrients and oxygen needed to grow 

Oncolytic virus therapy for cancer

*breaking oncogenes, harmless and wont attack other good cells 

• Type of virus that preferentially infects or breaks down cancer cells

• If modified they can destroy cancer cells

• Most current are engineered for tumor selectivity through there are a few naturally occurring ones such as seneca valley virus 

Applications (on trial):

Colorectal cancer and hepatocellular carcinoma 

Calcium in prostate cancer

• Intake of calcium and/or dairy products is associated with increased risk of prostate cancer 

• One mechanism is that the highest intake, down regulated 1,25 dihydroxyvitamin D3 which may increase cell proliferation in the prostate (VITAMIN D THEORY) 

• The second mechanism is through IGF-1 

  • Peptide is known as a mitogen for cells of prostatic gland

  • Studies established a positive correlation between plasma levels of hormone and prostate cancer risk 

  • IGF-1 uses calcium ion as second messenger 

LECTURE 20

Diabetes 

• 285 million worldwide, might hit 438 million by 2030

Diabetes Mellitus (DM) 

• Glucose in urine 

• Increased volume of urine, frequent urination (polyuria)

• Increase thirst (polydipsia)

• Increased blood glucose levels

*as urine becomes higher in glucose, water from bloodstream diffuses into urine leading to frequent urination 

Diabetes Insipidus

• Different disease

• Produces large volume of urine 

• Deficiency of antidiuretic hormone (vasopressin), which controls amount of water in urine

• No sugar in urine 

Diabetes Mellitus 

• High level of sugar in blood concentration 

• Maintenance of sugar levels in blood is crucial and any imbalance may cause serious health issues leading to:

  • Increased thirst

  • Increased hunger

  • Kidney failure

  • Fatigue

  • Loss of eyesight

  • Complications involving kidney, muscle, bone

  • Infections (because bacteria love sugar)  

*Highest prevalence in NL,NA and ON lowest in NU, AB and QC

Types of diabetes mellitus 

Type 1: body produces very little or no insulin, as all cells that produce insulin have been destroyed by autoimmune reaction 

• Insulin replacement by daily injection is only treatment besides diet

• It's also called Juvenile diabetes as its common in children OR Insulin dependent diabetes mellitus 

Type 2: body produces insulin but insulin doesn’t work 

• Referred to as insulin resistance 

• Also called non insulin dependent diabetes OR adult-onset diabetes 

Type 3: occurs when pregnant women develop a high blood glucose level

• May precede development of type 2 diabetes

• Also called gestational diabetes 

• Body can't produce enough insulin to handle effects of growing baby and changing hormone levels 

Glucose homeostasis

• Balanced by: 

• Insulin (pancreatic beta hormone)

• Glucagon (pancreatic alpha hormone)

• Hyperglycemia is the typical feature of DM

Hormones of pancreas

4 types:

• Beta cells secrete insulin

• Alpha cells secrete glucagon

• Delta cells secrete somatostatin which inhibit GH and TSH

• Gamma cells secrete a polypeptide of unknown function 

Beta cells of pancreas

• Produce insulin… required for glucose homeostasis

• Contain Glut2 Transporter (transmembrane carrier protein) which transport glucose from blood into beta cell 

• Glucose triggers intracellular events which result in insulin secretion (high level of glucose → beta cells releasing insulin) 

• Trigger for insulin secretion from beta cells: rising level of circulating glucose or blood sugar 

Insulin release 

GLUT 2 (glucose transporter 2)

• Mediate entry of glucose into beta cells of pancreas 

• Glucose is phosphorylated by glucokinase and metabolized to produced ATP

ATP triggers closure of K+ channels

• K+ can no longer leave cell

• Membrane potential increases 

Increase in membrane potential activated Ca2+ channels 

• Ca2+ moved into cell 

• Increase in intracellular Ca2+ triggers exocytosis of insulin granules 

• Calcium-mediated exocytosis

• Insulin release, activates insulin containing vesicles 

Complete cascade 

1. Glucose uptake, GLUT2

2. ATP-sensitive potassium channel 

3. Voltage-gated calcium channel

4. Insulin release 

Insulin action

• Insulin promotes the uptake of glucose into skeletal muscle and fat tissues that contain Type 4 Glucose Transporters (GLUT4) 

• Insulin action is transduction by insulin receptor and a signal transduction cascade 

Outcomes: 

• Activation of GLUT4 transporters

• Increased synthesis of GLUT4

• Results in increased uptake of glucose from blood, reducing blood glucose levels 

Insulin action with its receptor 

• Insulin binds to insulin receptor (consists of 2 alpha and 2 beta chains forming a dimer of alpha-beta) 

Stimulates gene expression 

• Increased synthesis of GLUT4

• Stimulation of glycogen-glucose pathways 

Features of diabetes 

Type 1: 

• May become an autoimmune disease with cell mediated autoimmune attack on beta cells 

• Beta cells produce little or no insulin 

• Progressive insulin deficiency 

Type 2: 

• Represents 90% of all DM cases

• Often leads to obesity 

• Cardiovascular disease

• Become insulin resistant 

Effects of diabetes upon health 

Retinopathy: damage to capillaries in retina 

Neuropathy: nerve damage

Nephropathy: kidney damage 

Vascular damage: muscle weakness and infection 

Processing of proglucagon 

GLP-1: Regulates insulin biosynthesis 

GLP-2: potent growth-promoting and cytoprotective effects in the GI tract

Insulin resistant: disrupted signaling (insulin receptor)

IRS-1: Insulin receptor substrate 1 

PI3K: phosphatidylinositol 3 kinase

pS/T: phosphorylated ser/thr residues

pY: phosphorylated tyr 

*activation of PI3K leads to increased glucose uptake and glycogen synthesis 

Insulin resistance

• Inflammatory mediators trigger increased expression of IRE1 kinase (a Ser/Thr Kinase), which activated JKN1, which phosphorylates Ser of the IRS1 complex 

• Phospho-ser is inhibitory to the IRS1 complex, thereby disrupting normal insulin signaling 

Drugs to treat type 2 diabetes 

Metformin: very common and effective, enhance insulin action in peripheral tissues 

GLP-1 analogues DPP-IV inhibitors: enhance insulin secretion

Metformin, thiazolidinediones: Drugs that suppress glucose production 

Alpha glucosidase inhibitors: delay absorption of carbohydrate from the GI tract

Obesity 

• Condition of overweight 

• Accumulation of fat and fat storage cells called adipose tissues

• Central adiposity is most harmful and is a risk factor for cardiovascular disease, diabetes, and insulin resistance 

• BMI is commonly used as an indicator of obesity 

Normal: < 25 kg/m2 

Overweight: > 25kg/m2

Obese: >30 kg/m2 

• It’s not an ideal parameter, other more predictive methods are 

  • Skinfold thickness 

  • Hydrostatic weighing (underwater weighing)

  • Dual energy x-ray absorptiometry (DEXA)

  • Bone mineral density 

  • CT (computerized tomography) or MRI imaging

  • Waist-hip ratio measurements 

Adipocyte lineage (adipose tissues) 

• High estrogen or low testosterone promote obesity 

Estradiol: stimulated adipocyte differentiation 

Testosterone/DHT: stimulate differentiation of stem cells to muscle cells 

E2: estradiol 

T: testosterone 

DHT: dihydroxy testosterone 

SHBG: sex hormone, binding, globulin 

Single X chromosome - Turner Syndrome 

Metabolic syndrome: 

• Central obesity (apple shape) 

• High blood pressure

• High triglycerides 

• Low HDL-cholesterol

• Insulin resistance 

J-shaped curve showing high disease risk and fat accumulation 

T2DS resistant to anti-lipolytic effect of insulin 

• Type 2 diabetes syndrome 

Significance of the big 2: 

• Insulin resistance

• Obesity (increased risk of T2DB and CVD) 

Obesity health risks

• Cancer

• Gallbladder disease

• Renal failure 

• Stroke

• Heart failure 

• Atherosclerosis

• NIDDM

• Hypertension 

Adipose tissue

• Consist of adipocytes (cells)

• Loose connective tissue composed of adipocytes 

• Play role in controlling triglyceride and free fatty acid levels

• Comprise 15-25% of body weight in lean individuals and much higher proportions in obese individuals 

Function:

• Store energy in the form of fat

• cushion/insulation

• Endocrine

• Regulate triglyceride and free fatty acid levels 

• Determine insulin resistance 

Adipocytokines

• Synthesize a number of chemical messengers 

Examples

Adipocytokines: leptin, ghrelin

• Estrogens by the aromatization of androgens using aromatase enzyme 

Leptin and its receptors 

• 16 kDa protein (leptin)

• Fat burning hormone (good hormone)

• Product of the ob gene 

• Leptin receptors (OB-R): hypothalamus, pituitary, reproductive system 

• Many isoforms of leptin receptors are known Ra, Rb, Rc, Rd, Re 

• Most common is Rb, except Re all contain a tyr-kinase 

Ob/Ob mouse (leptin deficient mouse) and Fa/Fa obese zucker rat 

• Mouse without leptin were fat 

Leptin: Satiety, energy balance 

• Binding of leptin signals to the brain that the body has had enough to eat… a sensation of satiety 

Decreased leptin: increased food intake, decreased energy expenditure 

Increased leptin: decreased food intake, increased energy expenditure 

Leptin action 

• Inhibits the activity of neurons that contain neuropeptide NPY and AgRP

• Both of them stimulate feeding

• Increases the activity of neurons expressing alpha-melanocyte-stimulating hormone (a-MSH)

• Those neurons mediate satiety… feeling of satisfaction 

Estrogen: Adipocytes 

• Aromatase critical enzyme converts androgens to estrogen 

• Estrogens reduce food intake, appetite and less body weight 

• It promotes expression of POMC and decreases the neuropeptide NpY

Ghrelin/obestatin and obesity

Ghrelin: acylated ghrelin (AG), unacylated ghrelin (UAG)

Obestatin (Ob)

• Derived from a common precursor protein 

AG

• Mainly prude in the stomach, exerts its central and peripheral effects through GH secretagogue receptor type 1a 

UAG

• Devoid of GHS-Ra1-binding affinity, it is an active peptide sharing with AG many effects through an unknown receptor

• Promotes more food intake, stimulates appetite (orexigenic hormone)

Ob

• Was discovered as the g-protein coupled receptor 39 ligand, but its physiological actions remain unclear

Ag, UAG, and Ob 

• Express din the pancreatic islets from fetal to adult life, and pancreas is major source of ghrelin in perinatal period

GHs-R1a and GPR-39

• Expression has been shown in beta cells and islets as well as binding sites for AG, UAG, and Ob

Ob 

• Promotes less food intake (anorexic or anorectic hormone)

Ghrelin and obestatin from precursor, Prepro-ghrelin 

• 28-aa ghrelin derives from N-terminal cleavage of prepro-ghrelin by PCSK1 enzyme

• 23-aa obestatin originates from the C-terminal cleavage by PCSK1 or PCSK2

• Ghrelin exists in 2 main forms.. Acylated and unacylated 

• Ghrelin acylation on serine 3, promoted by GOAT is essential for binding to GHS-R1a and for its endocrine functions  

• Ghrelin peptide relative to the preprohormone that also is the source for obestatin (upper), and growth hormone secretagogue receptor 

Lect 21

1.     Ligand (various types): Protein interaction

Ligand: molecule that binds/interacts or forms complex with proten leading to change in conformation and activity of logan to initiate or alter cell responses 

• Peptide, lipid, carb, nucleic acid, small organic molecules

2.     The example of “STRING” network in Trp pathway of bacterial gene

• The colour saturation of the edges represents that confidence score of a functional association 

• Thicker the line means stronger the interaction

• Strongest between a,b,c,d,s

3.     Protein:Protein interaction, how to detect?

• Interactions that regulate the cell processes and are significant points of intervention for development of therapeutics and drugs for disease intervention 

1. Fluorescence method

2. Co-immunoprecipitation method

3. Labeled protein method

4. Native gel electrophoresis method

5. Immobilization method 

6. Surface plasmon resonance method 

4.     Papilloma virus infection and the role of E1 and E2 proteins

HPV

• Most commonly sti (skin to skin)

• Many types 

• Also called condylomata 

E1 and E2

• Required for replication of HPV genome

• 650 and 370 aa in length 

• Replication is initiated by recruitment of E1, by E2 to the viral origin 

• Recruitment involves an interaction between TAD of E2 and helicase domain of E1 

• E2 recruits additional E1 and the ATP stimulates E1 and is needed to power the helicase activity

• E1 interacts with host cell factors to promote bidirectional replication of viral genome

5.     PCSK9 and LDL-R interaction

• They bind together which leads to LDL-R degradation in lysosome, leading to accumulation of cholesterol in blood 

6.     Types of protein:protein interaction

7.     Protein lipid interaction in AD

• Lipid rafts promote interaction of the APP with BACE-1

• This leads to increased generation of the amyloid beta peptide

• It promotes alzheimer's disease

LECTURE 22

Drug design and therapeutics

• Strategies 

• Various steps and stages

• Challenges

• Various issues

• Approval

• Summary 

Epigenetics/epigenome

• Definition

• Causes 

• Role in diseases

Drug development process/stages

1. Disease 

2. Understand the mechanism

3. Identify target

4. Design molecules for intervention (in vitro study)

5. Select the most potent molecule

6. Design strategy for cellular delivery for intracellular target

7. Demonstrate efficacy in cell culture (ex. Vivo work)

8. Testing in animal (in vivo study)

9. Toxicity test

10. Clinical human trial (4 phases)

11. Mode of admin: oral, intravenous injection, inhalation or external application 

12. Safety study/side effects (short and long term)

13. FDA (food and drug admin) approval 

Clinical trials for drug development (12 years from drug invention to market)

Phase 1: 20-100 subjects (healthy volunteers or people with disease)

• Study length: several months

• Purpose: safety and dose

• 70% of drugs going to next phase 

Phase 2: several hundred subjects with the disease

• Study length: several months to 2 years

• Purpose: efficacy and side effects

• 33% of drugs going to next phase

Phase 3: 300-3000 volunteers with disease/condition

• Length: 1-4 years 

• Purpose: efficacy and monitoring adverse reactions

• 25-30% of drugs going to next phase

Phase 4: several thousand volunteers with disease/condition

• Purpose: safety and efficacy 

Rule of Five (Lipinskis or Pfizers)

• In 1997, Lipinski from Pfizer found a simple rule which he called the rule of 5, which determines the drug like property of a compound

• Describes molecular properties best suited for a drugs pharmacokinetics in the human bosy that contains parameters such as Absorption, distribution, metabolism, and excretion (ADME)

• Cannot predict is a compound is pharmacologically active

• Rule states that an orally active drug compound never violated more than one of the 4 rules:

1. More than 5 H-bond donors

2. The molecular weight is over 500 

3. The log p is over 5 (lipophilicity value)

4. The sum of amine and hydroxyl groups (N+O) is over 10 

Small molecule chemical entities as drugs

N: unmodified natural product

ND: modified natural product

S: synthesis compound with no natural product conception (largest part)

S/NM: synthetic compound showing competitive inhibition of the natural product material substrate 

S*: a synthetic compound with a natural product pharmacophore 

S*/NM: A synthetic compound with a natural product pharmacophore showing competitive inhibition of the natural product substrate 

Natural products: Present and future 

Ways natural products can contribute to the search for new drugs

1. By acting as new drugs that can be used in a unmodified state (anticancer)

2. By providing chemical building blocks used to synthesize more complex molecules (contraceptive pill, anticancer as well)

3. By indicating new modes of pharmacological action that allow complete synthesis of novel analogs (antibiotic)

They will continue to be considered as one of the major sources of new drugs because:

1. They offer incomparable structural diversity

2. Many of them are relatively small (<2000 Da)

3. They have drug like properties 

Value of natural products

• Provide drugs that are hard to produce commercially by synthetic means 

• Supply basic compounds that can be changed to become less toxic or more effective

• Some contain compounds that demonstrate no activity themselves but can be modified to produce potent drugs not easily obtained by other methods 

Leaves of Taxus Baccata (Yew Plant).. Taxol 

TAXOL

• Most important member of clinically useful natural anticancer is paclitaxel (taxol)

• Approved in USA in 1992 for refractory breast cancer 

• Currently being tested against variety of different cancers… also best selling anticancer drug in history 

Development

1989: Bristol-Myers Squibb (BMS)

1991: Activity observes against breast cancer

1992: NDA approval for ovarian cancer

1994: FDA approve for breast cancer 

• Now used for lung and other cancers

• Produced by cell culture methods 

Poor yield problem

• Major problems in natural product isolation

Ideas to help included:

1. Finding a better source for supply, such as different species or a cultivar, or different plant part or cultivation conditions

2. Semisynthesis of taxol from a more abundant precursor

3. Total synthesis of taxol

4. Tissue culture production of taxol or close relative 

• Most successful was semisynthesis, total synthesis haven't been proven to be economically better 

Major challenges in drug development process

Cell permeability

• How to make a bioactive drug compound cross cell membrane in order to target intracellular molecule

Common Cell permeable or penetrating peptides (CPP)

• Tryptophan… rich, neutral, basic domain 

• Most potent so far (PEP-1), trp rich, neutral, basic 

Potential mechanism of cell entry of CPP

• Recruits negatively charged phospholipid circles and induced the formation of an inverted micelle

• The hydrophilic cavity of that accommodates the peptide and its cargo cna be released into the cytoplasmic compartment 

Bioavailability and stability

• How to make the drug stable and survive for a long time before degradation in harsh physiological condition 

Novel strategy for design of cell permeable drugs using CPP

• Furin inhibitors exhibit anticancer and antiviral activity in models 

• One can attach CPP to furin inhibitors to make them cell permeable 

• Using fluorescent and CPP labeled peptide one can monitor cell entry 

SASS10 is derived from SAAS protein and has been shown to be an inhibitor of PCSK1 enzyme 

Cell impermeable bioactive compounds (drugs)

Applications:

• Needed when the drug is targeting a molecule on cell surface or in the extracellular space 

• Will not affect intracellular target 

• Selective target 

• Cell impermeable furin inhibitors (won't go inside), may find therapeutic applications for intervention of viral infections 

Epigenetics 

Heredity: passing of traits to offspring 

What is epigenetics 

Epigenetics: study of heritable changes in gene expression or phenotype caused by mechanisms other than changes in underlying DNA sequence 

Epigenome: overall epigenetic state of a cell 

• Functionally relevant modifications to the genome that don't involve a change in nucleotide sequence 

• Epigenetic changes are preserved when cells divide(mitosis)… can be maintained throughout a lifetime 

• They can also be preserved in the next generation of germ cells (meiosis) → can be transferred to the next generation 

Causes for epigenetic changes 

• Diet

• Exercise

• Stress

• Toxins

• biochemistry/physiology ex hormones 

• Inheritance 

What is epigenetics?

• Critical to morphogenesis 

• can cause gene silencing, or gene activation 

Morphogenesis: the biological process that causes a cell, tissue, or organism to develop its shape 

Proof?

Twin studies:

Monozygotic twins: identical genetic makeup at birth and death

• Across a lifespan they can differ in environmental exposures

• Share a genome but not an epigenome, not destined to same fate 

• Differences in health and disease 

  • Cancer

  • Diabetes

  • Cardiovascular disease

  • Asthma 

• Differences become more apparent over time → longer exposure time 

• Research on 80 sets of identical twins

• DNA is marked in different ways by methyl

• Much more pronounced in older twins 

Agouti mouse model 

• Genetically identical mice

• Agouti gene 

  • Activated: yellow fur coat, obesity

  • Silenced: brown fur coast, normal weight 

• Expression of the agouti gene in offspring through enrichment of maternal diet with methyl rich supplements (i.e folic acid)

Mechanisms: DNA methylation 

• NO methylation of CpG: gene “on”

  • Tissue specific genes, housekeeping genes 

• Methylation of CpG - gene “off”

  • Non tissue-specific genes, silent DNA

• Methylation of CpG’s done through DNA methyltransferases 

  • Knock it out: lethal in animals 

  • Over-express: cancers in humans 

• Methyl groups acquired through diet 

  • Folate, methionine, selenium 

• Incorporation of methyl groups can be influenced by environmental toxins 

*on C but not G, but need G beside it 

Cont..

• It’s a crucial part of normal organismal development and cellular differentiation in higher organisms 

• It involves the intro of methyl group to position 5 of

  • Cytosine © pyrimidine od the number 6 nitrogen of the Adenine (A) purine ring 

• C and A are 2 of the 4 bases of DNA that can undergo methylation  

• This modification can be inherited through cell division 

• DNA methylation stably changes the gene expression pattern in cells such that cells can remember where they were or decrease gene expression 

Cont… 

• Occurs mostly at Cytosine 5-position 

• Between 60%-90% of all CpGs are methylated in mammals 

• Methylated-C can spontaneously deaminate to form Thymine over evolutionary time (several generations)

• So CpG may transform to TpG sequence 

DNA is complexed with histones to form chromatin 

• DNA methylation is carried out by two generational classes of enzymatic activities 

1. Maintenance methylation 

2. De novo methylation 

• Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle and the enzyme responsible DNMT1

• It’s through that DNMT3a and DNMT3b are the de novo methyltransferases that set up DNA methylation patterns early in development 

*DNA demethylases removes methyl group 

Adenine Methylation 

• N6-Methyladenine (m6A) has been found in DNAs of various eukaryotes (Algae, Fungi, Protozoa, and higher plants) 

• Like bacteria DNA, DNAs of these organisms are subject to enzymatic modification not only at cytosine but also at adenine bases

• The first higher-eukaryotic adenine DNA N6-methyltransferase was isolated from vacuolar vesicles 

• The enzyme helps to methylate the first adenine in the sequence TGATCA 

Histone modificationx

• Addition of methyl groups to lysine or arginine residues on histone tails

• Typically a marker for gene silencing 

• Sometimes a marker of gene activation 

• Methylation of histones done through histone methyltransferases (HMTs/KMTs) 

Acetylation of histones

• Acetylation of lysine residues on histone tails

  • Removes positive charge of histone tail, loosening the histone-DNA complex

• Marker of gene activation 

• Regulated by histone acetyltransferases (HAT/KAT) and histone deacetyltransferase (HDAC)

  • Activity of these enzymes can be regulated by several environmental factors 

• Acetyl Co-A is donor for Acetyl groups

• Can change rapidly within a cell cycle 

Long non-coding RNA (IncRNA)

• Sequence-specific molecules that can guide protein complexed to specific sites in the chromatin and orchestrate transcriptional repression 

• Important role in X-chromosome inactivation, imprinting 

• Transcriptionally active epigenetic mark, methyl group removed from H3K4

• Transcriptionally repressive epigenetic mark, methyl group added to H3K27 

Clinical consequences of epigenetic errors 

• Following fertilization, the paternal genome undergoes rapid DNA demethylation and histone modifications 

• The maternal genome is demethylated gradually and eventually a new wave of embryonic methylation is initiated that established the blueprint for the tissues of the developing embryo 

• Perturbations in patterns of DNA methylation and histone modifications can lead to congenital disorders and multisystem pediatric syndromes, as well as predispose people to acquired disease states such as sporadic cancers and neurodegenerative disorders 

Epigenetic therapies 

• Are few but several are being developed or approved for specific cancer types 

• Nucleoside analogues such as azacitidine are incorporated into replicating DNA, inhibit methylation, and reactivate previously silenced genes 

• Azacitidine has been effective in phase 1 clinical trials in treating myelodysplastic syndrome and leukemia characterized by gene hypermethylation 

• The antisense oligonucleotide MG98 that downregulates DNMT1 is showing promising results in phase 1 clinical trials 

• Small molecules such as valproic acid downregulate HDACs are being used to induce growth arrest and tumor cell death 

• Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis, there have been attempts to re-express these by inhibiting the DNMTs

• 5-Aza-2-deoxycytidine (decitabine) is a nucleoside analog that inhibits DNMTs leading to its degradation 

• For decitabine to be active, it must be incorporated into the genome, which can cause mutations in the daughter cells if the cell doesn’t die 

• Decitabine is toxic to the bone marrow, which limits the size of its therapeutic window 

• Pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their mRNAs and preventing their translation 

Huntington's Disease (HD)

• A fatal neurodegenerative disorder, transcriptional dysregulation is a key pathogenic feature 

• Histone modifications are altered in multiple cellular and animal models of HD suggesting a potential mechanism for the observed changes in transcriptional levels 

• Link between decreased histone acetylation, particularly acetylated histone H3 (H3-K9K14-ac) and down regulated gene expression 

DNA methylation 

• Methylation of CpG sites within the promoters of genes can lead to their silencing, a feature found in a number of human cancers… ex. Silencing of tumor suppressor genes 

• The hypermethylation of CpG sites has been associated with the over-expression of oncogenes within cancer cells 

It is crucial for normal development and is linked to a number of key processes. Its abnormality leads to:

• Genomic imprinting

• X-chromosome inactivation

• Suppression of repetitive elements

• Carcinogenesis 

Histone acetyl ation 

• Acetylation of the lysine residues at the N terminus of histone proteins removes positive charges, thereby reducing the affinity between histones and DNA 

• This makes RNA polymerase and transcription factors easier to access the promoter region 

• In most cases houston acetylation enhances transcription while histone deacetylation represses transcription 

• It’s catalyzed by histone acetyltransferases (HATs) and histone deacetylation is catalyzed by histone deacetylases (HDs or HDACs) 

• Different forms of HATs and HDs have been identified 

• Acetylation occurs on the N-terminal tail Lys as well as nucleosome core surface 

• Source of acetyl group in histone acetylation is acetyl-coenzyme A, and in histone deacetylation the acetyl group is transferred Coenzyme A 

Lect 23

1.     What reagents can precipitate protein from its solution?

• Ammonium sulphate 

• PEG

• TCA

• Acetone

• Ethanol… less efficient 

• Heavy metal ions (Pb+2, Ag+, Hg+)... less efficient 

2.     Light microscopy and the use of Feulgen staining

Feulgen staining: specific for DNA, showing the chromosomes of a cell 

3.     What is FRET and its application

FRET: fluorescence resonance energy transfer 

• With a GFP variant, uses fluorochromes to measure changes in distance between cellular components 

4.     Various techniques of light (DIC, etc) and laser as well as electron (TEM and SEM) microscopy

5.     Radiolabeling of proteins (example with I¹³¹ ) and its application.

• One protein is labelled with radioactivity (Ex. I131, H3, C14)

• Second protein not labelled 

• Upon interaction, radioactivity can be seen in complex 

6.     Use of sucrose gradient buffer

• Used in a continuous sucrose-density gradient, different organelles sediment according to density, where they form bands 

7.     What do you need for western blot?

• Antibodies can be used in conjunction with various types of fractionation procedures to identify an antigen among a mixture of proteins 

8.     What are needed to carry out Mass spectrometry (MALDI and SELDI techniques) analysis of a sample

• MALDI: clean, purified sample, chemical that helps sample absorb laser energy and ionise, mass spectrometer, software 

• SELDI: clean, purified sample, chip or surface to capture specific molecules, mass spectrometer, software 

9.     Protein staining dyes

• Fluorochrome: stains cause cell components to glow 

• Coomassie blue, silver ion or cypro ruby (most sensitive)

10.  What is RP-HPLC and how is it carried out?

• Used for separating, identifying, and quantifying components in a mixture 

• Need two buffer systems 

  • TFA in water (aqueous)

  • TFA in acetonitrile (solvent B, organic)

  • A clear and completely clear sample solution 

  • Injection of sample system 

  • Good gradient system 

  • Uses high pressure to push the mobile phase through tightly packed columns, resulting in high resolution and fast separations

11.  Specific activity of an enzyme, how do you calculate? 

• Nanomol AMC released/min from a fluorogenic substrate 

• Specific: nanomole AMC released/min per mg of protein 

12.  Compare Native gel (it can detect noncovalent protein:protein interaction) vs SDS PAGE gel electrophoresis (noncovalent interactions cannot be detected as it is disrupted).

Native gel: 

• Can detect noncovalent protein:protein interaction 

• Gel electrophoresis is performed in absence of SDS 

SDS: 

• Noncovalent interactions cannot be detected as it is disrupted

• SDS disrupts binding 

13.  Isoelectric point of protein. What do you mean by that?

• A pH when the number of positive and negative charges is equal