npb 107 Flashcards

Question

Understanding: Gram pos vs Gram neg

Answer 1

Quorum sensing = bacteria sending message-- can occur through dint ,mechanism House analogy------> 1. Gram-negative bacteria = houses with open windows: -They send small messages -The messages can slip in and out easily -These messages are AHLs. So: *** Gram-negative bacteria just let the message float in and out. Gram-negative → small signal → goes in/out by itself 2. Gram-positive bacteria = houses with thick walls & locked doors -Small slips can’t get through -They send big messages -The message must be handed to a doorbell (receptor) -These messages are AIPs (peptides). So: ***Gram-positive bacteria need a doorbell to get the message. Gram-positive → big signal → needs a receptor *** BUT BOTH BACTERIA---do signal, release, and sense it ---> JUST DIFFRNTLY

Answer 2

Gram-positive bacteria: - Thick cell wall - Stain purple in Gram stain - Use AIPs (autoinducing peptides) as signals -AIPs are larger Cannot diffuse freely Must be: Exported out Detected by surface receptors ➡️ Receptor-based signaling

Answer 3

between cells

Answer 4

within cells

Answer 5

Intercellular signaling: - Between cells - Examples on slide: Hormones Neurotransmitters, Cytokines, Cell–cell contact signals

Answer 6

Within a cell Examples: Second messengers (Ca²⁺, cAMP) Protein–protein interactions Conformational changes

Answer 7

Allostery means: - Something binds to one site on a protein -That causes a shape change - Which affects a different site (often far away) Result: protein activity changes So: Binding → shape change → function change

Answer 8

PROTEIN: - A local binding event can cause--> A distant functional change -This is why proteins can: Receive a signal Process it Produce an output They behave like input–output devices.

Answer 9

mRNA is short-lived Protein is more stable and plentiful

Answer 10

no Some proteins are stable Some are degraded quickly Protein amount ≠ protein activity

Answer 11

There is an assumption --> More MRNA --> more protein If biology were simple: Every mRNA makes protein at the same rate Every protein lasts the same amount of time Then: double the mRNA → double the protein every dot would lie on one straight line * wrong only correlation BC WHAT PLOT SHOWS: Two genes can have the same amount of mRNA but very different amounts of protein

Answer 12

Protein level depends on two extra things that mRNA does not capture: 1. How fast the mRNA is translated Thing 1: Speed of making protein -- Some mRNAs: are read fast → lots of protein --Others: are read slow → little protein Even if mRNA amount is the same 2. How fast the protein is destroyed Thing 2: Speed of protein destruction Some proteins: last a long time Others: get destroyed quickly So: Same mRNA ≠ same protein ****Different genes ≠ same rates.

Answer 13

A kinase protein: is already present in the cell inactive Signal arrives → add phosphate → kinase becomes active → signal transmitted ****No new mRNA needed.

Answer 14

initial local conformation change at one site is ---> coupled to local conflation change at the second sit meaning initial binding chnages shape of the second binding site

Answer 15

What the protein picture is doing (the right side) The protein is being treated like a tiny “device” with: -- an input site (often a regulatory binding site) --an output site (often an active site or binding site) What happens: -A signal binds the input site (or a modification happens there) -The protein’s shape shifts -That shift spreads through the structure -The output site changes what it does (turns on/off, binds/doesn’t bind, opens/closes) So: the protein itself is the “wire. *porotien transi tj info interlly through shae changes

Answer 16

What a signal actually does (this is the key leap) --> does not create a new ON state does not force the protein to change Instead, it: makes the ON shape more likely or the OFF shape more likely Because: the ON shape already exists the protein doesn’t need to be rebuilt A signal just: stabilizes one shape over the other That happens in milliseconds to seconds. This is why signaling is fast **

Answer 17

allosteric drugs. These drugs: bind away from the active site don’t block function directly Instead, they: push the equilibrium toward ON or OFF So drugs work by biasing probability, not flipping switches.

Answer 18

Allosteric equilibrium - means a protein naturally fluctuates between inactive and active shapes, - and signals regulate activity by shifting how often the protein occupies each shape.

Answer 19

Changing protein amount requires: transcription translation time Changing protein activity requires: a shape change a phosphate a binding event Much faster. So cells: keep proteins around switch them ON or OFF as needed

Answer 20

1. Non covalent moditififcations ----> non covalent binding of small molecules - case conformation --. activity in print 2. Intermolecular interactions: -one protein binds to another protein - can activate/ unit/ stabilize / bloc part gf it - nothing ia chemically chnages *** touch and separate 3. Covalent modification ( phosphorylation) - covalentt = chemical group attached to protein EX: PHOSPHYRYALIGTON - reversible ( bc of phosphatase) \ - changes charge/ shape/ interactions 4. Other - volaTeeg ( voltage gated ion channels) -mecganincla force - proteolysis ( capsases) -->cuts protein

Answer 21

Proteins can be controlled through the Q: Does the protein exist? Is it allowed to function? Is it in the right place? 1. Control strategy 1: Make it or destroy it - Protein synthesis - Protein degradation If the protein doesn’t exist: it can’t be active This is: slow long-lasting Used for: developmental decisions long-term changes 2. Control strategy 2: Change its activity directly This is the fast signaling layer. Includes: ligand binding covalent modification (phosphorylation) binding of regulatory proteins intramolecular “unmasking” The protein exists already — you’re just changing what it does. 3. Control strategy 3: Move it to where it can work Some proteins: only work in the nucleus or at the membrane or in a specific compartment So: protein can be present but inactive because it’s in the wrong place **Translocation turns activity ON or OFF without changing shape much.

Answer 22

( when protein in wrong place and can't function in its current location) If the protein is in the cytoplasm( but wants nucleus): -it is OFF Even though: it exists - it’s fully functional ------> What “translocation” means A signal causes the protein to: move into the nucleus (or membrane, etc.) Now: same protein same shape now active because it’s in the right place

Answer 23

Non covalent: - nothing chemically attached - binds --> but can leave - protein shape changes 9 during bing )--> activity changes - molecule unbind --> proti9n returs * fast * reversible * temporary

Answer 24

Covalent regulation: - chem group- attached to protein - protein changes behavior - still reversible but * more stable * longer lasting ( than non covalnet) Like putting sticker on switch ---> switch behaves ddiifnre until sticker removed .

Answer 25

( ligand/ drug) - activator

Answer 26

( ligand, drug) - incactivates it

Answer 27

Closed: - specific -reliable - ensures signal ecived bay apporto9oat target * direct contact * no didffudoion

Answer 28

Open - signal broadcast widely - many cells exposed - critical fro cells --> in distant target organs

Answer 29

Each cell contributes half channel --> connexion -lined up - form continuous tunnel - allows synchronization between cells for electoral and mecganila ouput ( ex; cardiomycete) ** cell to cell contact form gap junction --> making. it a closed methodd of intercellular communiaaiot

Answer 30

Only small things, such as: ions metabolites (glucose, amino acids) ATP small signaling molecules < 1,000 DA ❌ Not proteins ❌ Not RNA ❌ Not DNA

Answer 31

- cytoplasm stay separate - no molcuels pass ( no size rule) How contact-dependent signaling works (literally) Cell A has a ligand stuck in its membrane Cell B has a receptor in its membrane The two cells must touch Ligand binds receptor → signal happens * still a form of close communication

Answer 32

immune cellse gametes mobile cells forming tissues --> during development

Answer 33

Synaptic signaling The presynaptic cell releases neurotransmitter The molecule diffuses across a tiny gap (synaptic cleft) It binds receptors on the postsynaptic cell Signal is rapidly terminated (reuptake or degradation) The key is the tiny distance. Why synaptic signaling is NOT “open” Even though a molecule diffuses: - the gap is extremely small -receptors are concentrated exactly opposite release sites - neurotransmitter is removed quickly So: only the intended target cell responds This keeps it highly specific. **Synaptic signaling often uses: high local concentration low-affinity receptors Why? signal must act fast then disappear quickly

Answer 34

A cell releases a signal that affects nearby cells in the same tissue What physically happens - Cell releases a signaling molecule -Molecule diffuses through extracellular space -Multiple nearby cells are exposed -Only cells with the right receptor respond Key change: no targeting to one specific cell range is limited by diffusion and degradation * more open communicant bc now it just depend on who apse the receptor * low affinity / High conc

Answer 35

Local mediator = a signal meant to act nearby, not far away.

Answer 36

Cells release hormones into the bloodstream to act on distant target cells. What physically happens 1. Endocrine cell releases hormone into blood 2. Hormone is carried throughout the body 3.Many cells are exposed 4. Only cells with the receptor respond So delivery is non-specific, but response is specific * most open * low conc of signaling * high affinity between ligand and receptor

Answer 37

- no charge - non polar (mostly c-c or c-h bond)

Answer 38

- charged -polar

Answer 39

1. Mixed: Amino acids and derivatives 2. Hydrophilic: Peptides/Proteins ( hella amino acids so even fi side chain are hydrophobic it soonest matter) 3. Hydrophobic: Steroids (derived from cholesterol, hydrophobic molecule)

Answer 40

Mediate intercellular signaling Many different chemical forms Most are water-soluble (cannot cross plasma membrane), require an interaction with a transmembrane receptor to transmit signal to cell interior

Answer 41

--> Receptors are very accurate, need to be able to sense changes in concentration of a specific signal present at very low levels receptors must be: high affinity highly specific structurally precise

Answer 42

Two major functions of cell surface receptors: 1. bind correct signal/ligand present in --> extracellular fluid through an extracellular domain 2. transmit information that ligand is bound to the interior of cell by a---> change in activity of an intracellular receptor domain

Answer 43

Receptors --> operate via Allosteric conformational changes - ligand binding --> change and then transducer eeffectio in cell

Answer 44

Signal binding site ( outside of cell) - initial local conformational change - response to direct outpour Linker / coupling domain ( ALLOSTERC coupling) - regulatory domain - catalytic domain Function effector ( intracelleruate0 - output signal

Answer 45

WT MUscaranic Receptor - initial local conformational change - local conformational change -> gen output Mutant receptor ***point mutation différence 1 amino acid ( asparatic acid --> asparagine) - initial local change - no G protein signaling aka conformance change in output

Answer 46

1. Transcription factors: steroid hormones and thyroid hormones 2. Ion channels: neurotransmitter receptors 3. G protein-coupled: huge number (>90% of all receptors) 4. Enzymes: mostly growth factor receptor tyrosine kinase 5. Other: a wide array that couple to other signaling systems 2-5 Should be ≥ 1 transmembrane segment in here somewhere

Answer 47

A module = - a specific region of a protein - That region has one main job -The same type of region appears in--> many different proteins * autonomous ( meaning if removed can it still do its job in nosher location ). EX: kinase enzyme domain , ligand binding domain

Answer 48

“Autonomous: self-contained, existing or capable of existing independently” In protein terms, this means: - A domain can fold on its own -it can retain its function --> even if moved to a different protein - It does not need the whole protein to work

Answer 49

SAME signal → different outcomes Acetylcholine (ACh) is the ligand. 1. Receptor A: Nicotinic receptor Modules: ACh-binding module Ion-channel module Outcome: Ion flow Fast electrical response 2. Receptor B: Muscarinic receptor Modules: ACh-binding module G-protein–coupling module Outcome: Second messengers Slower, amplified signaling Key insight Same ligand-binding module Different intracellular module → Completely different function

Answer 50

Steroid hormones - derived from the membrane component cholesterol - mostly hydrocarbon rings -few polar or charged groups -unusual among extracellular signals as they can cross membranes. ( bc there component is basically like a lipid membrane ) - diffuse directly through membrane * no transporter / pore /surface receptors ***** intracellular receptors

Answer 51

yes Steroid hormones are: hydrophobic lipid-like So in blood (water): they do not dissolve they would clump or precipitate they could not circulate efficiently Carrier proteins: bind the hormone shield it from water keep it soluble in blood

Answer 52

Act primarily through effects on gene expression” Steroid hormones: do not mainly change activity of existing proteins instead, they change which proteins are made Mechanism: hormone enters cell binds receptor receptor binds DNA transcription changes So the output of the signal is: 👉 altered gene expression

Answer 53

Steroid hormone receptors are transcription factors” Most receptors: - -detect signals -pass the message to other proteins Steroid hormone receptors: \ - are the final effector themselves When activated, they: -bind specific DNA sequences -directly regulate transcription So they are: -receptors and -transcription factors

Answer 54

Have separate, allosterically-coupled, steroid-binding domains and DNA-binding domains” 🔹 Steroid-binding domain binds the hormone determines ligand specificity 🔹 DNA-binding domain binds specific DNA sequences determines which genes are regulated 🔹 Linker / coupling domain physically connects the two transmits conformational change

Answer 55

no same hormone + diffrnt receptor = different time - some steroid hormones also cause rapid responses -these are mediated by membrane receptors many of these receptors are GPCRs Examples given on the slide: rapid stress responses to glucocorticoids fast thyroid hormone effects in heart acute uterine/vaginal responses to estrogen So steroid hormones can act via: intracellular receptors → slow, genomic membrane GPCRs → fast, non-genomic

Answer 56

Provide basis for fast synaptic signaling in nervous system and skeletal muscle What happens step-by-step (mechanism) - Ligand binds outside the cell -Binding causes a local conformational change -Shape change propagates through linker -Ion channel opens or closes -Ions move across membrane -Membrane potential changes → response

Answer 57

Why this class is so fast (important contrast) Compare to other receptor types: GPCRs → G proteins → enzymes → second messengers → slower Steroid receptors → transcription → very slow VS Ion-channel receptors: -skip intermediates -directly change ion flow That’s why they are used for: synapses muscle contraction rapid reflexes

Answer 58

GPCRs do not directly carry out the response — they activate G proteins, which then control downstream effectors. Sequence: -Ligand binds GPCR -GPCR changes conformation -GPCR activates a heterotrimeric G protein G protein regulates: -enzymes (e.g., adenylyl cyclase) i-on channels ***Cellular response occurs This is why GPCR signaling is: indirect flexible amplifiable

Answer 59

Left cartoon: - receptor has its OWN catalytic domain (common for RTKs) -- Start: two receptors are separate → inactive catalytic domains ---Ligand often brings them together (dimerization) ----Dimerization changes shape → activates the intracellular enzyme domains Key idea: the ligand’s job is often to force proximity + proper alignment, which turns the enzyme on. B) Right cartoon: receptor activates an ASSOCIATED enzyme ---Receptor itself may not be the enzyme ---Ligand binding makes the receptor recruit/activate an intracellular enzyme partner Key idea: the receptor is like an “adapter” that switches on a nearby enzyme. ***** impt in cancer bc enzyme-coupled receptors control “big decisions” like: growth survival division

Answer 60

oo many receptors → even normal signal levels produce an abnormally strong response

Answer 61

the receptor/enzyme is mutated so it’s overly active (sometimes even without ligand).

Answer 62

yes - diffrnt cells can have ---> diffrnt intracellular modular domains ---> ie diffrnt output

Answer 63

Biology needs interactions that are: Specific (right partner, not random) Reversible (can turn on, turn off, and be regulated) Fast (form and break quickly) Tunable (weak/strong depending on what the cell needs) Covalent bonds are usually too permanent for signaling and regulation.

Answer 64

electrostatic interaction Vand der walls interaction hydrogen bonds

Answer 65

Binding of Ligands ) to Receptors --->Typically Involves Multiple Types of Non-Covalent Bond ----Having multiple bonds that are each weak, will yield a Stable Comple

Answer 66

Kd is the ligand concentration at which 50% of receptors are occupied' KD = [A] x [B] / [AB] (moles/liter) aka KD = [L]x[R] / [LR] Concentrations of [L] = ligand [R] = free receptor [LR] = bound receptor ***

Answer 67

If [L] ≪ Kd → fraction bound ≈ 0 ( not bound) If [L] = Kd → fraction bound = 0.5 If [L] ≫ Kd → fraction bound ≈ 1 (compt bind)

Answer 68

1 / (1 + KD/[L] )

Answer 69

Many pathogens stay avirulent until dense enough Why? -Avoid immune detection -Maximize damage Phase transition includes: -biofilms -toxins -enzymes

Answer 70

Purpose: prevent a classic confusion. KD = - thermodynamic binding affinity. ( where system ends up, ya equilibrium or forward or reverse ) - At equilibrium, KD is the ligand concentration where 50% of sites are bound. Km = -kinetic. -Substrate concentration where enzyme runs at ½ Vmax, -depends on binding and turnover. (Why Km is NOT pure affinity Km depends on: how well substrate binds (like KD) AND how fast it is converted to product) Connection: For receptors we care about “how much receptor is bound/occupied” → KD is the right tool.

Answer 71

(Figure shows three curves with same plateau (same Bmax = 100,000) but different KD (10⁻¹², 10⁻¹⁰, 10⁻⁸).) -----Bmax = total receptors available (max bound at saturation). aka Plateau height. ---KD = how much ligand you need to bind half of them. So: Lower KD (10⁻¹²) = higher affinity = curve shifts left (needs less ligand to reach same binding). Higher KD (10⁻⁸) = lower affinity = curve shifts right. Purpose: isolate “affinity changes” from “receptor number changes.” Connection to physiology: If your body lowers receptor affinity (or ligand affinity), you need higher signal ---> concentration to get the same response. Exam skill: “Left shift = higher affinity (lower KD). Right shift = lower affinity (higher KD).”

Answer 72

KD is intrinsic, but can be regulated ( by molecular structure) KD depends on: - amino acids in the receptor - binding pocket - chemical groups on the--ligand -shape, charge, hydrogen bonds, van der Waals force 👉 If nothing about the ligand or receptor changes, KD is fixed. KD changes cann occur by; A) Physiological regulation - Same receptor gene, -different conformation or modification. Examples: phosphorylation interaction with another protein receptor switching between active/inactive conformations These can subtly change: how tightly ligand binds how often ligand dissociates B) Pharmacological regulation (drugs) Drugs can: bind tighter than the natural ligand---stabilize a high-affinity conformation---- compete differently at the binding site This changes the effective KD you observe. Example: a high-affinity agonist → lower KD → left shift a weak partial agonist → higher KD → right shift C) Pathological regulation (mutations) This is the cleanest case. A mutation in the receptor: alters the binding pocket changes non-covalent interactions changes how stable LR is That directly changes KD.

Answer 73

Acetylcholine (ACh): -high affinity (low KD) for muscarinic receptors -lower affinity (higher KD) for nicotinic receptors So if you inject ACh into the body:(at normal physiological concentrations) -it binds muscarinic receptors much more effectively Result: effects look muscarinic unless you block them with ATROPINE ------ Same ligand. Different receptors. Different KD values. That’s drug selectivity explained by KD.

Answer 74

“Bmax differs dramatically by cell type” - reg---physiologically/pathologically - receptor number is a primary determinant of response. Not just presence—also how many * cell surface repscetor expression ----> highly regulated ( transcription/ translation / post tranalsationally )

Answer 75

Binding is random collision + stability depends on dissociation - association often driven by random encounters -stability depends mainly on dissociation rate ( ak can they withstand thermal jolting) Connection: In real tissues, ligand concentrations fluctuate. Receptors with low KD stay bound more at low ligand levels.

Answer 76

When you are dehydrated, DEC your blood water volume (plasma) INC. ion. conc Why Ion Concentration Increases? Reduced Volume: Dehydration reduces the liquid part of your blood (plasma volume), making the blood thicker and more concentrated. Increased Electrolytes: When you lose free water through sweat or urine, the absolute amount of salt (sodium) in your blood remains the same, but it is dissolved in less water, leading to a higher concentration, or hypernatremia. Internal Shift: water is drawn out of cells to balance the concentration, causing cellular shrinkage. Why saline (not pure water)? Saline ≈ 0.9% NaCl, close to plasma osmolarity. --If you gave pure water IV: blood osmolarity would drop water would rush into cells cells (especially neurons) would swell → brain edema → death So saline: restores volume preserves ion gradients keeps membrane potentials stable

Answer 77

What happens in DKA: Insulin deficiency → cells can’t take up glucose Cells switch to fat metabolism → ketoacids -Blood becomes acidic -Potassium shifts out of cells into blood Blood K⁺ may look normal or high But total body K⁺ is actually low When treatment starts (insulin + fluids): Insulin drives K⁺ back into cells Blood K⁺ can crash Low extracellular K⁺ → cardiac arrhythmias → death So clinicians give KCl to: maintain extracellular K⁺ preserve cardiac membrane potentials When treatment starts (insulin + fluids): -Insulin drives K⁺ back into cells - Blood K⁺ can crash Low extracellular K⁺ → cardiac arrhythmias → death So clinicians give KCl to: maintain extracellular K⁺ preserve cardiac membrane potentials

Answer 78

delta G = - RT ln ( outsis cone /inside con) + ZFdelta V Purpose: show the total driving force = chemical term + electrical term G= fr energy of transport ( driving force behind transport of charged molecules) R= gas constant T = temp kelvin Z= electrical charge of solute ( valence of ion) F = faraday constant V = membrane portent inside / outside relative

Answer 79

Ion channel: -transmembrane proteins -form gated pores -usually multi-subunit selectivity filter -most ion channels ---> the ion permeation pore resides in the center of the complex - 230 ion channel genes in human genome ( third largest family) -some chanels only pass one kind of ions -- other pass mroe.

Answer 80

excitatory channels: - tend to pass ions with positive reversal potentials (Na⁺, Ca²⁺) ------>if opening a channel moves Vm toward a more positive value, it is excitatory inhibitory channels: - pass ions with negative reversal potentials (K⁺, Cl⁻ 👉 If opening a channel moves Vm toward a more negative value, it is inhibitory

Answer 81

CA2+ and NA+

Answer 82

The receptor can be in a few distinct states: State 1 — Resting (closed, no ACh bound) --Channel is closed. --Receptor is ready to respond. State 2 — Bound but still closed (ACh binds first) ACh binds → “induced fit” (local shape change at binding site). -This is not yet the full open state — it’s the setup. State 3 — Open (activated) -Allosteric coupling spreads the shape change to the pore. -Activation gate opens → ions flow. --This is what causes the fast EPSP (excitation) at many synapses. State 4 — Desensitized (closed even though ligand may be present) -With continued ligand exposure, the receptor shifts into a conformation where the pore is closed again. Functionally: “I’m overstimulated; I’m going offline.” State 5 — Recovery back to resting -Ligand must unbind and/or the receptor must relax back. -Now it can respond again. That return to the start is why it’s a cycle. ***** impt bc shows Receptors can shut themselves down (desensitize), which matters in drugs, addiction, synaptic physiology.

Answer 83

Nucleur receptors matter bc: -they regulate gene expression -Gene expression controls long-term physiology -Many diseases involve misregulated gene expression - Therefore, nuclear receptors are high-value drug targets

Answer 84

sigignling mocleyle that work through members of the nuclear receptor of gene superfamily: A. Steroid receptor ligands (top group): -17β-estradiol (ER) -Testosterone (AR) -Progesterone (PR) -Cortisol (GR) -Aldosterone (MR) What do they visibly share? Four fused carbon rings (steroid backbone) Very few charged groups Mostly C–C and C–H bonds A few –OH or =O groups, but no long charged chains B. RXR-heterodimer receptor ligands (bottom group) -T3 (thyroid hormone) -All-trans retinoic acid (RAR) -9-cis retinoic acid (RXR) -Ecdysone (EcR) -1,25-dihydroxy vitamin D₃ (VDR) These look different from steroids, but notice: Long hydrocarbon backbones Rings + chains Very few ionizable groups Still mostly nonpolar

Answer 85

Common chemical propter: - are lipophilic (hydrophobic) molecules with -high membrane permeability. -These molecules do not -----------> need transporters -------->They do not bind extracellular receptors ---------> diffuse through lipid bilayers what this means mechanistically: (Because lipophilic) - They cross the plasma membrane -They encounter receptors inside the cell -Those receptors can access DNA ( nucleus reactors). -The signal regulates transcription

Answer 86

Nuclear receptors: - don’t just respond to “hormones” — - many of them act as sensors of endogenous lipid molecules what does it mean for them to be seonsors: -The ligand reflects the metabolic state of the cell or organism -When lipid levels change → receptor activity changes -That change alters gene transcription ***(So the receptor is not just responding to an external signal — it is monitoring internal chemistry.)

Answer 87

normal metabolic intermediates: fatty acids cholesterol derivatives bile acids oxysterols retinoids

Answer 88

Impt bc: Nuclear receptors link metabolism → gene expression Cells can adjust transcription based on: nutrient availability lipid abundance energy state So nuclear receptors help maintain: metabolic homeostasis lipid balance long-term adaptation This is why NRs show up in: metabolism obesity diabetes cardiovascular disease 6️⃣ Exam depth calibration (CRITICAL) You ARE expected to: ✅ Understand that many nuclear receptors bind endogenous lipids ✅ Explain what it means for a receptor to act as a metabolic sensor ✅ Connect ligand binding to transcriptional regulation of metabolic genes You are NOT expected to: 7️⃣ How this slide is commonly tested Example question Why are some nuclear receptors described as lipid sensors? Correct reasoning: Because they bind endogenous lipid molecules whose concentrations reflect metabolic state, allowing regulation of gene expression in response to lipid availability.

Answer 89

Nuclear receptors detect circulating hormone levels and ----> convert them into transcriptional responses in target tissues

Answer 90

Because these hormones are lipophilic: - they circulate systemically -they cross cell membranes -they do not need membrane receptors -they act inside target cel

Answer 91

Answer 92

Ecdysone - molting hormone - insect --> steroidd hormone -acts through nuclear receptors - use as a model for other steroid hormone ( bc visible puffing)

Answer 93

Puffs” --- are regions of chromatin that have become transcriptionally active. So: No puff = DNA tightly packed, low transcription Big puff = DNA opened up, high transcription ***Puff size = how actively genes are being transcribe

Answer 94

Panel A: + Ecdysone What you see: Early puffs appear quickly Later, new puffs appear Even later, additional puffs appear What this panel PROVES: -Hormone causes visible chromatin changes -Those changes happen in a time-ordered sequence - Gene activation is not simultaneous This already tells you: -->The hormone is not flipping one switch I. ---->t is triggering a program Panel B: + hormone + transcription inhibitor ( block RNA poll 11) What you see: NO PUFFS AT ALL Chromosomes stay compact What this panel is ASKING Is hormone-induced puffing dependent on transcription? If transcription is blocked → nothing happens Therefore: Puffing = transcription This eliminates entire classes of mechanisms: ❌ Ion chnanels/ second mesnegers/ phosphate Panel C: + hormone + translation inhibitor What you see: Early puffs STILL appear Late puffs do NOT appear What this panel is ASKING Does hormone-induced transcription require new protein synthesis? Step 1: Early puffs still appear ---> This means: The hormone does not need new proteins The receptor itself must be: already present capable of activating transcription directly Step 2: Late puffs disappear This means: Late genes require new proteins Those proteins must come from: early genes 👉 This proves a transcriptional cascade.

Answer 95

Ashburger model: Step 1: Hormone enters the cell It diffuses across the membrane No surface receptor No second messenger Step 2: Hormone binds the nuclear receptor -conformational change in the receptor Activation of its transcriptional function ***This explains why: No new protein synthesis is required for early genes 5️⃣ Step 3: Activated receptor binds DNA The hormone–receptor complex: -Binds specific regulatory DNA sequences - These are near early genes What happens: Chromatin opens Transcription machinery is recruited Early puffs appear --->This corresponds directly to: Panel A (early puffs) Panel C (early puffs still present with translation blocked Step 4: Early genes are transcribed Early genes typically encode: transcription factors regulatory proteins Important: These are not the final physiological effect. They are intermediate regulators. (logic behind the phrase: early vs late genes) Step 5: Early gene products activate late genes early gene products: -- are proteins --they take time to be synthesized Late genes: turn on later require translation disappear when translation is blocked This corresponds to: Panel C (late puffs gone with cycloheximide) Step 6: Late genes produce the phenotype Late genes encode: enzymes structural proteins transporters These cause: developmental changes metabolic changes long-lasting physiological effects This explains: why nuclear receptor signaling is slow why it is long-lasting

Answer 96

physiology diagram (what talks to what): - Estrogen produced by ovary -Estrogen travels in blood - Estrogen acts on liver cells -Liver transcribes vitellogenin gene -Vitellogenin protein enters blood -Taken up by developing eggs * so transcription regulation matter bc helps reproductive capacity Why this matters: estrogen is endocrine target tissue ≠ source tissue effect is gene-specific This is already telling you: Estrogen is not “excitatory” like a neurotransmitter —> it reprograms liver cells.

Answer 97

What the axes are X-axis: time after estrogen exposure (hours → days) Y-axis: vitellogenin level in blood What you see: -Nothing happens immediately -After hours, vitellogenin rises -Levels increase over days What this proves Estrogen action is slow The effect is sustained This is NOT second-messenger signaling ***This directly reinforces: nuclear receptor → transcription → delayed protein accumulation

Answer 98

TABLE — cycloheximide experiment (MOST IMPORTANT LOGIC) The table shows: Treatment |Relative vitellogenin transcription None baseline Estrogen (12 hr) ↑↑ Cycloheximide + Estrogen |. ↑↑ (still high) What cycloheximide does: Blocks translation Prevents new protein synthesis ****The key observation Vitellogenin transcription still increases even when translation is blocked. Transcription happens in the liver ********** Transcription happens in the liver Translation happens in the liver Only the finished protein enters the blood This is where your mental picture was getting flipped. EARLY response gene

Answer 99

(Nuclear receptors regulate transcription by binding specific DNA sequences in regulatory regions of target genes. aka ERE) Estrogen response elements: -a short DNA sequence -located near or upstream of estrogen-regulated genes -recognized by the estrogen receptor (ER) Think of it as: A docking site for the estrogen–ER complex If a gene does not have an ERE: estrogen cannot directly regulate it ******This is why vitellogenin is estrogen-responsive and most genes are not

Answer 100

Transient transfection = temporarily putting DNA into cells. You are not changing the genome You are not studying development You are doing a short-term experiment Why short-term is good: You only care about regulation Not long-term effects Not cell division

Answer 101

The setup The cell: It’s a normal cell It already has estrogen receptors But nothing is activated yet The DNA you add: You add extra DNA (temporarily) That DNA has: a control region (maybe an ERE) a reporter gene (a fake gene that makes light/color) ****This reporter gene is just a readout. If it turns on → transcription happen ------------FLOW 1️⃣ DNA goes into the cell: You put the reporter DNA into the cell. It sits in the nucleus like any other gene. Nothing happens yet. 2️⃣ You decide whether to add estrogen: ----You now choose: No estrogen → ER stays inactive + estrogen → estrogen enters the cell and binds ER This is the switch. 3️⃣ What happens INSIDE the nucleus (the key moment) If estrogen is present: ER becomes active ER looks for a DNA sequence it recognizes Now two possibilities: ✅ If the DNA contains an ERE ----> ER binds that ERE ----> transcription of the reporter gene starts ❌ If the DNA does NOT contain an ERE---> ER cannot bind----> transcription does not start This is the decision point of the whole experiment. 4️⃣ Time passes: If transcription started --reporter mRNA is made ------reporter protein is made If transcription didn’t start: -----nothing is made ---You don’t interfere here — you just wait. 5️⃣ You look for the reporter signal Now you check: --is there light? color? ----fluorescence? If yes → that DNA responded to estrogen If no → it didn’t That’s the readout. * test necessityI f I keep this DNA piece and throw everything else away, does hormone regulation still occur?”

Answer 102

intact response element → reporter ON with hormone mutated response element → reporter OFF with hormone If mutation kills response: ➡️ the sequence is necessary

Answer 103

Is this response element alone enough to make ANY gene hormone-responsive?” ---->Make DNA sequence with only the suspected response element + a different gene’s promoter + reporter gene What is being tested Now you take: --ONLY the response element ---Put it next to a completely different promoter ( ex minimal promoter) ---Attach reporter gene Why this is done: This tests sufficiency. If reporter turns on: ➡️ the response element is sufficient by itself

Answer 104

A minimal promoter: -barely drives transcription -produces almost no reporter signal alone Why it’s used It ensures: -any increase in reporter activity is due to the response element not due to strong basal transcription -This isolates regulatory DNA function **isolates regulatory DNA function..

Answer 105

Construct A: Promoter + intact response element + reporter What this construct contains: Promoter Intact ERE Reporter gene What happens – hormone → low reporter +hormone → high reporter What this tells you This proves: estrogen + ER can activate transcription activation depends on the response element This is your positive result.

Answer 106

Construct 1: Promoter + reporter only (NO response element) What this construct contains: Minimal promoter Reporter gene No ERE What happens: – hormone → low reporter +hormone → still low reporter What this tells you: This is the negative control. It proves: hormone alone does NOT activate transcription ER does NOT activate promoters nonspecifically ***So estrogen does nothing unless a response element is present.

Answer 107

What this construct contains: Promoter Mutated ERE Reporter gene What happens: – hormone → low reporter +hormone → still low reporter What this tells you This proves: ---the specific ERE sequence matters -------ER cannot bind mutated DNA -----response element is necessary This rules out: ****general DNA effects random hormone activati

Answer 108

Construct 4: Response element + minimal promoter + reporter What this construct contains: ERE alone Minimal promoter Reporter gene What happens: – hormone → low reporter +hormone → high reporter What this tells you This proves: the ERE alone is enough it can confer hormone responsiveness to another gene response element is sufficient

Answer 109

1) The top-left squiggle: receptor mRNA That wavy line is receptor mRNA transcribed from the receptor gene The little “AAAAA” is the poly-A tail—a hallmark of mature eukaryotic mRNA. Why it matters: you can’t easily “sequence a receptor gene” if you don’t know its genomic structure (introns etc.). Instead, you start from the mRNA that already has introns removed.

Answer 110

cDNA is: ----DNA copy of the mRNA ----contains ONLY coding sequence ----no introns-->stable ---easy to clone and manipulate WHY this step matters You cannot: ----sequence RNA easily ---express RNA directly in cells You can: -----sequence DNA ----insert DNA into cells -----mutate DNA ***So cDNA is the working form of the receptor gene for experiments

Answer 111

CDNA ( can do three things) cDNA is: a DNA copy of mRNA contains only exons (no introns) stable easy to manipulate Once you have cDNA, you can: ✅ Sequence it → learn the amino acid sequence ✅ Insert it into cells → force cells to make the receptor ✅ Mutate it → delete domains, change amino acids ✅ Express parts of it → test which regions bind DNA or ligand **So cDNA is the experimental version of the gene 👉 We take the cDNA for the estrogen receptor (ER) and put it into other cells in the lab so those cells can now make the ER protein. 👉 This lets us study what ER does, even in cells that normally wouldn’t respond to estrogen.

Answer 112

Big picture (how everything fits): What the DBD is A single folded protein domain Stabilized by two zinc fingers Always exists as one structure Contains: P-box D-box 1️⃣ The DNA-binding domain (DBD): (highly conserved region of all nuclear receptors) Its job: recognize and bind hormone response elements (HREs) in DNA For estrogen receptor → binds ERE For glucocorticoid receptor → binds GRE, 2️⃣ Zinc fingers (core structural idea) The DBD contains two zinc fingers: Each zinc finger is formed when: 4 cysteine residues chelate (hold) one zinc ion On the slide: Brown spheres = zinc ions The loops formed = zinc fingers 🔑 Zinc is structural, not catalytic — it holds the protein in the correct shape so it can sit in DNA’s major groove. 3️⃣ P-box amino acids → DNA specificity: ( which DNA to read) P-box = “positioning / recognition” These amino acids: Directly contact specific base pairs in the response element Determine which DNA sequence the receptor binds ➡️ This is why ER binds EREs and not GREs ➡️ Change the P-box → you change DNA specificity 4️⃣ D-box amino acids → dimerization D-box = dimerization These amino acids: Help two receptors bind together (homo- or heterodimers) Example: ER–ER homodimer RXR–RAR heterodimer ➡️ Many nuclear receptors must dimerize to bind DNA properly. Think: D-box = “Who do I pair up with?

Answer 113

Step-by-step: what happens when it hits DNA 🧱 Step 1 — Structural readiness Zinc ions are already bound 4 Cysteines already chelate zinc The domain is pre-folded ➡️ Nothing activates yet. This is just struct Step 2 — DNA encounter ---The receptor dimer approaches DNA. Now both boxes act at the same time: 🔵 P-box action ---Inserts into the major groove of DNA -----“Reads” specific base pairs Answers: Is this the correct response element? If NO → receptor falls off If YES → binding stabilizes 🟢 D-box action (simultaneous) ---Locks receptor A to receptor B ---Aligns the two zinc fingers correctly Ensures the dimer matches the spacing of the response element ➡️ This is mechanical alignment, not signaling Step 3 — Stable docking The receptor is now: Correct sequence ✔ Correct spacing ✔ Correct orientation ✔ 🛑 Still no transcription yet.

Answer 114

two approaches are shown: EMSA (in vitro) ChIP (in vivo – next slide)

Answer 115

(Nuclear receptors bind response elements (like EREs) --->The DNA-binding domain does this---> Now we ask: Can we see this binding directly in a test tube? That’s EMSA.) EMSA = Electrophoretic: What you start with A short piece of DNA containing: -----A known response element (e.g., vitellogenin ERE) ----The DNA is radioactively or fluorescently labeled Nuclear receptor protein (e.g., estrogen receptor) What you do Mix: DNA alone DNA + receptor protein Run the mixture on a gel What happens on the gel Free DNA: Small Moves fast DNA + receptor bound: Bigger complex Moves slower ➡️ This difference in movement is the “shift” ******Competitor DNA (PART) ( test specificity)

Answer 116

What is “competitor DNA” really? Competitor DNA is: --->A different DNA sequence ---->Same length, same chemistry --->But NOT a hormone response element (AP-1 and SP-1 are common transcription factor sites) Key idea: It looks like DNA, but it’s the wrong address. ----- What they test (this is the logic) Condition 1: DNA + receptor You see a shifted band → receptor binds the response element So far so good. Condition 2: DNA + receptor + competitor DNA Now we ask: Will the receptor leave the response element and bind the competitor instead? ----- Two possible outcomes (THIS is the whole point) ❌ Outcome A (bad / nonspecific) -----Receptor binds competitor DNA ---Shifted band disappears That would mean: “This receptor doesn’t care what sequence it binds.” ❌ Bad science ❌ No specificity ✅ Outcome B (what we WANT) Receptor ignores competitor DNA Shifted band stays This means: “The receptor ONLY binds the correct response element.” ✅ Specific ✅ Real biological binding

Answer 117

“Does the receptor bind this DNA sequence inside a real cell, on real chromatin? EMSA = test tube ChIP = inside living cells ****Why EMSA wasn’t enough (1 sentence) EMSA shows can the receptor bind DNA, but cells have chromatin, histones, cofactors, and competition — ChIP proves binding is biologically real. -----Flow 🧩 STEP 1 — Binding happens naturally Cell is treated with: hormone antagonist/ or drug with unknown activity Nuclear receptor binds its response elements---> in chromatin Nothing experimental yet — just biology happening. 🔗 STEP 2 — Freeze interactions (CRITICAL) Add a crosslinker (usually formaldehyde) This: Locks receptor to DNA Locks histones to DNA 🧠 Think: “Take a snapshot of protein–DNA interactions.” ✂️ STEP 3 — Chop DNA(sonicatipn) Chromatin is fragmented You now have: tiny DNA pieces each still attached to whatever protein was bound 🎯 STEP 4 — Immunoprecipitation (the “IP” part) ----Add an antibody specific to the receptor e.g., anti–estrogen receptor ***Antibody pulls down: receptor plus whatever DNA it was attached to This is the key logic step. 🧪 STEP 5 — Identify the DNA Now ask: What DNA sequences came down with the receptor? Methods: PCR for known response elements Or genome-wide sequencing (ChIP-seq) ***If a sequence is enriched → receptor was bound there in the cell\

Answer 118

Key idea up front: DNA binding puts the receptor in the right place. Ligand binding decides what the receptor DOES. LBD: -- made of 12 α-helices ---helices fold into a pocket that binds hormone ---One helix is special and constantly tested on exams: ⭐ Helix 12 (H12) Flow of the ligand-binding domain (step-by-step): 🔹 Step 1 — No ligand (inactive) ---Helix 12 is in the wrong position - --Coactivators cannot bind Transcription = low The receptor may already be on DNA — but it’s doing nothing useful. 🔹 Step 2 — Agonist binds (e.g., estradiol) ---Ligand fits into the pocket ---Helix 12 snaps into place This creates a coactivator binding groove Now: Coactivators bind Histones get acetylated RNA pol II is recruited ➡️ High transcription 🔹 Step 3 — Antagonist binds (e.g., tamoxifen): Ligand still binds the pocket --- BUT it forces helix 12 into the wrong position --Coactivator binding site is blocked So: DNA binding still happens But activation does NOT ➡️ Receptor is bound but silent (or inhibitory)

Answer 119

Coactivators are proteins that: ---Open chromatin ----Recruit transcription machinery If they can’t bind: Nothing downstream happens Gene stays off

Answer 120

TWO DNA constructs (this is crucial) 🧬 Construct 1: Reporter gene Contains: A response element (e.g. ERE) A minimal promoter A reporter gene (luciferase, GFP, β-gal) This answers: “If transcription happens, can I see it?” 🧬 Construct 2: Receptor cDNA Contains: A strong promoter The receptor gene (ER, GR, etc.) This answers: “What happens when THIS receptor is present?” ---------- The experimental flow (step-by-step) 🔹 Step 1 — Transfect cells Put in: Reporter gene ± Receptor cDNA 🔹 Step 2 — Treat with ligand Conditions: No hormone Hormone (agonist) Antagonist (sometimes) 🔹 Step 3 — Measure reporter output: Light (luciferase) Fluorescence (GFP) Color (β-gal) More signal = more transcription. What this assay actually tells you If reporter turns ON only when: receptor is present AND ligand is present ➡️ The receptor is a ligand-dependent transcription factor If receptor is present but NO ligand: ➡️ Low signal (bound ≠ active) If ligand is present but NO receptor: ➡️ No signal (ligand alone does nothing)

Answer 121

Why use cells that don’t normally respond? This is subtle but important. They use cells that: Do not express the receptor naturally So: No background signal Any response you see = because of the receptor you added

Answer 122

BEFORE hormone (OFF state) What exists already: --Receptor is present (often in nucleus or cytoplasm) --Heat shock proteins (hsp) are bound to the receptor --Chromatin is packed (tight nucleosomes) What the receptor is doing: --May be near DNA or even weakly bound ---NOT activating transcription Why transcription is low: Coactivators cannot bind Histones are not acetylated RNA pol II can’t access DNA -------- AFTER hormone (ON state) Step-by-step flow (this is the money): 1️⃣ Hormone enters cell (lipophilic → crosses membrane) 2️⃣ Hormone binds ligand-binding domain ----Helix 12 moves ----Heat shock proteins dissociate 3️⃣ Receptor is now active ----Proper conformation ---Can recruit coactivators 4️⃣ Coactivator complex binds -----These enzymes acetylate histones 5️⃣ Chromatin loosens ----DNA becomes accessible 6️⃣ RNA polymerase II is recruited ➡️ High transcription

Answer 123

1️⃣ Ancient receptors Some receptors are found in: Humans/ Fish/Frogs/nsects This tells us: These receptors existed before vertebrates They regulate fundamental biology: (development, metabolism) Example: RXR-like receptors → very ancient 2️⃣ Vertebrate-specific receptors: Some receptors appear only in: Vertebrates (fish → humans) This tells us: They evolved later They regulate things like: Stress physiology Reproduction Mineral balance Example: Glucocorticoid receptor (GR) Thyroid hormone receptor (TR 3️⃣ Insect-specific receptors Some receptors appear only in: Insects Example: Ecdysone receptor (EcR) Key insight: Different ligands, same basic receptor architecture. **the receptors all share: DNA-binding domain (very conserved) Ligand-binding domain (diversified)

Answer 124

Endocrine-disrupting chemicals: are substances in the environment that interfere with normal hormone signaling.' That can mean: mimic block exaggerate shut down *They bind nuclear receptors even though they are not natural hormones.

Answer 125

What happened: 1980s pesticide spill (DDT, dicofol) Lake Apopka alligators: Massive population decline Mostly female Males feminized Nearby lake = normal alligators This rules out: ❌ genetics ❌ temperature ❌ chance Why this screamed “endocrine disruption”: Alligators use hormone-dependent sexual development. The chemicals: Looked “estrogen-like” Were present during development Caused permanent developmental effects ➡️ Classic nuclear receptor prob

Answer 126

Part 1: the biological setup (why males were affected) Alligator sex determination basics: Sex is temperature-dependent BUT hormones still matter: Male-favoring temps → low estrogen Female-favoring temps → high estrogen So estrogen signaling is decisive during developmen What they did (step-by-step) 1️⃣ Took cells in culture 2️⃣ Transfected them with: Estrogen receptor cDNA (human or alligator) Estrogen-responsive reporter gene 3️⃣ Treated cells with: 17β-estradiol (natural estrogen) DDT Dicofol What they measured Reporter gene activity More signal = more ER activation The result (why this matters) Estradiol → strong activation ✅ DDT → activation ✅ Dicofol → activation ✅ ➡️ Same receptor. Same output. Different ligands

Answer 127

Answer: 👉 The same transient transfection What they put into cells: Estrogen receptor cDNA Estrogen-responsive reporter gene (luciferase, etc.) What they treat cells with Extracts from: Toys Plastics Household products Often mixtures, not pure chemical What they measure Reporter gene activity Interpretation: ↑ reporter activity → estrogenic (agonist-like) ↓ reporter activity → anti-estrogenic (antagonist-like) No change → likely no ER interaction

Answer 128

(STEROIDS) Estrogens and androgens regulate normal growth and differentiation — cancer happens when these same pathways are overactivated or misregulated. Androgens vs estrogens: Androgens → act through androgen receptor (AR) Estrogens → act through estrogen receptor (ER) But important twist: Androgens can be converted to estrogens via aromatase ➡️ Same precursor, different signaling outcomes. 3️⃣ Why cancer enters the picture Steroid receptors: Drive cell proliferation Promote gene expression programs If signaling is: Too strong Too long Not shut off ➡️ Cells divide when they shouldn’t. That’s the cancer link.

Answer 129

Estrogen-linked conditions (ER signaling): 1.When estrogen signaling is TOO HIGH or unregulated --Breast cancer --Uterine cancer --Fibroids Logic: ER drives transcription of growth genes → excess signaling → excessive cell division 2. When estrogen signaling is TOO LOW: Osteoporosis Infertility Logic: Estrogen is required for bone maintenance and reproductive function 3. Dysregulated signaling PCOS Infertility Not just “more or less hormone,” but mis-timed or mis-localized signaling.

Answer 130

1. Too much androgen signaling: Prostate cancer Hair loss / hirsutism AR drives proliferation in prostate tissue → overactivation = cancer risk. 2.Too little androgen signaling: Muscle atrophy with aging Infertility Androgens normally maintain muscle mass and reproductive function.

Answer 131

📈 Incidence increases with age: Breast cancer rates are low in young women Increase steadily with age Rise sharply around and after menopause This tells you: Cancer risk accumulates over time and is linked to long-term hormone exposure. Estrogen: Promotes cell proliferation in breast tissue Increases chances of: DNA replication errors Mutations So over decades: ➡️ More estrogen signaling = higher cumulative risk Why menopause shows up on this slide: Even though estrogen levels drop after menopause: Lifetime exposure has already happened ER-positive tumors can: Still respond to low estrogen Or produce estrogen locally This sets up: 👉 why ER status matters for treatment

Answer 132

1️⃣ Correlation First show: Disease is associated with hormone levels or receptor presence Example: ER-positive breast cancers grow faster when estrogen is present 2️⃣ Receptor expression in diseased tissue Show that: The receptor is present in affected cells Ideally: High receptor expression = worse disease 3️⃣ Mechanistic action Demonstrate: Hormone binding → receptor activation → gene transcription This uses: Reporter assays ChIP Target gene expression This links ligand → receptor → genes 4️⃣ Gain-of-function Ask: “If I ADD hormone or receptor, does disease behavior increase?” 5️⃣ Loss-of-function Ask: “If I REMOVE hormone signaling, does disease improve?” 6️⃣ Therapeutic response The strongest proof: Target the hormone or receptor Disease outcome improves

Answer 133

Doctors noticed breast cancer growth seemed tied to ovaries and hormones — but they didn’t know how.” The key historical observations 1️⃣ Early clinical observations: Removing ovaries sometimes slowed breast cancer Giving estrogen sometimes made tumors grow faster But at the time: No receptors known No molecular explanation So people were arguing: Is estrogen really doing this, or is it indirect? 2️⃣ Enter Elwood Jensen (important idea, not memorization) Jensen proposed something radical for the time: “Estrogen must act through a specific protein receptor, not just dissolve into cells.” 3️⃣ Why this mattered If estrogen works through a receptor, then: Only tumors with that receptor should respond You could: Detect the receptor Target it with drugs This idea changed cancer treatment foreve

Answer 134

Luminal breast cancer (ER+): ---Expresses estrogen receptor (ER): --Often also progesterone receptor (PR) ---Still relatively differentiated 🔵 HER2-positive breast cancer: ER may be present or absent Overexpresses HER2 (a growth factor receptor) ⚫ Triple-negative breast cancer ER− PR− HER2− What the graph shows: Often occurs earlier More aggressive Higher metastatic potential Why this matters mechanistically: No nuclear receptor signaling to target No growth factor receptor to block Treatment implication: Hormone therapy ❌ HER2 therapy ❌ Chemotherapy is main option

Answer 135

Tamoxifen is an --estrogen receptor antagonistt --targets the receptor, not estrogen production.

Answer 136

Tamoxifen - used as antagonist therapy for er - only work son ER+ Cells - In er: -- PR ( progesterone receptor) = gene regulated by er - if Pr present this indicates ER active ---* there from viable for as therapeutic target Correct: TAmoxifen ✔ Competes with estrogen ✔ Blocks activation via helix 12

Answer 137

Agonist-bound ER (LEFT side idea): What estradiol does --Fits neatly into the ligand-binding pocket ---Allows helix 12 to fold over the pocket like a lid Result: Creates the coactivator binding groove Coactivators bind Histones acetylated Transcription ON Antagonist-bound ER (RIGHT side idea): What tamoxifen does Binds the same pocket Has a bulky side chain That bulky group: Pushes helix 12 out of position Helix 12 now blocks the coactivator groove Result Coactivators cannot bind DNA may still be bound Transcription OFF

Answer 138

SERM — Selective Estrogen Receptor Modulator: Example: tamoxifen/ raloxifene/ droloxifene Same receptor Same ligand-binding pocket Different effects depending on tissue

Answer 139

SERD — Selective Estrogen Receptor Degrader: ex: fluvestrant Binds ER Causes ER to be destroyed Less receptor = less signaling

Answer 140

CERAN — Complete Estrogen Receptor Antagonist Blocks ER activity everywhere No agonist activity in any tissue

Answer 141

1️⃣ SERM binds ER Same ligand-binding domain Same helix 12 displacement everywhere 2️⃣ Tissue context decides outcome In each tissue: Different coactivators Different corepressors Different chromatin environments So: In breast tissue Coactivators can’t bind Corepressors dominate ➡️ Antagonist effect (good for cancer) In bone tissue Different cofactors available Partial recruitment possible ➡️ Agonist-like effect (prevents osteoporosis)

Answer 142

Tamoxifen -16-49 reduction droloxifene -10 -60x greater Er affinity raloxifene- no acidify in resting breast cancer / nut effective at preventing beret cancer

Answer 143

Raloxifene - prevents endometrial cancer

Answer 144

tamoxifen - induces endometrial cell growth - assoc with endometrial cancer droloxifene - minimal effects

Answer 145

Boen tamoxifen -partical agonism droloxifene - full er agnosticism raloxifene - treatment of postmenopausal osteproissi

Answer 146

What if the estrogen receptor no longer needs estrogen to turn ON? No estrogen required Receptor activates transcription by itself Cancer grows even without hormone If that happens: Blocking estrogen ❌ Blocking ligand binding ❌ Some drugs stop working ❌ This is endocrine therapy resistance.

Answer 147

Mutations cluster in three regions of ER, all affecting: ⭐ Helix 12 positioning These mutations: Force helix 12 into the agonist-like position Mimic estrogen binding Create a permanent coactivator-binding surface 🧠 Translation: The receptor pretends estrogen is bound even when it isn’t. * somem effect of mutation can ve revers -------- 🔵 ZONE 1 (labeled “spring” on the image) WHERE it acts: At the N-terminal side of the LBD At the hinge that allows helix 12 to move WHAT it acts on: The mobility of helix 12 WHAT the mutation does: Removes flexibility The “spring” can’t recoil RESULT (directly on helix 12): ➡️ Helix 12 cannot move back to OFF ➡️ It stays locked ON 🔵 ZONE 2 — CHARGE REPULSION Where it is: Helix 5 (H5) Adjacent to the C-terminal region of helix 12 What it normally does: Participates in intramolecular stabilization of helix 12 Only stabilizes helix 12 when ligand is bound What mutation does: Strengthens H5–H12 interaction Mimics ligand-induced stabilization 🔵 ZONE 3 — INTRAMOLECULAR INSTABILITY: H9–H10 THIS is the one I skipped earlier — and you were 100% correct to call it out What the slide explicitly shows Helix 9 and helix 10 (H9–H10) are labeled This zone is described as intramolecular instability These helices are part of the structural core of the LBD What this zone normally does Maintains structural tension in the OFF state Prevents spontaneous stabilization of helix 12 Mutation effect Alters H9–H10 interactions Destabilizes the OFF conformation Structural changes propagate through the LBD ➡️ Helix 12 becomes stabilized in the agonist (ON) position ➡️ Ligand-independent activation

Answer 148

RXR heterodimers do not wait for ligand to bind DNA. They are already there\ FLOW --What is happening mechanistically 1. In the absence of ligand: --RXR heterodimer binds DNA --Corepressors (NCoR / SMRT) bind the receptor ---Corepressors recruit HDACs --Histones are deacetylated --Chromatin is closed ---RNA pol II cannot engage **actively repressing transcription. What ligand actually does Ligand binding causes: --Helix 12 repositioning ---Loss of corepressor binding ---Gain of coactivator binding --Recruitment of HATs ---Open chromatin ---Transcription ON extraceelula ligand = thyroid hormone / vitamin d / reitnoic acid RXR ligand intracellular ligand = fary acid, chollesota dervidatice , toci chemical s

Answer 149

Advantages Tight control Prevents leaky expression Developmentally precise Disadvantages Mutations cause dominant repression Hard to override pharmacologically

Answer 150

Thyroid hormone recprtos: Mutant TR: * TH resistance, hyper and hypo Nurr1: drug target for Parkinson disease other neurological disease

Answer 151

Key points: TRα and TRβ genes Different tissue expression AF-1 domain differs → functional specificity

Answer 152

T3 is essential for - brain, -visual system, -auditory system and -intestinal development - and bone growth, --as well as normal heart rate ---and force of contraction, --body temperature, and --overall metabolic rate

Answer 153

Mechanism: Helix 12 mutation Poor T3 binding Can’t recruit coactivators Still binds corepressors → repression Mutation : TRA 1 ( RHTA) = low affinity ( high KD, cant recut coacticl tors0 - bidn corepressors well Clinical challenges: -Neurological problems cannot be corrected after a week or two after birth -Can’t really treat with a very high dose of --> T3 because you would hyperstimulate -The RTHa mutant allele interferes with--> a normal TRa in heterozygous individuals (and TRb where expression overlaps) due to constitutive repression activity

Answer 154

** progresi los of dopamine symptoms Motor symptoms: Tremor (hands, arms, legs, jaw, head) → classic resting tremor Muscle stiffness (rigidity) → due to loss of fine motor control Slowness of movement (bradykinesia) → patients struggle to initiate movement Impaired balance & coordination → falls Non-motor symptoms Depression and emotional changes Difficulty swallowing, chewing, speaking Urinary problems, constipation

Answer 155

rogressive loss of dopamine-producing neurons in the substantia nigra (S Substantia nigra: Region in the midbrain Sends dopamine to the striatum Part of the basal ganglia motor circuit Function “Involved in suppressing unnecessary movement, refining motor outpu

Answer 156

L-Dopa = dopamine precursor that crosses the blood–brain barrier --a dopamine precursor that -crosses blood - brain barrier Why not dopamine itself? Dopamine is hydrophilic Cannot cross the blood-brain barrier Why L-Dopa works Transported into the brain Converted into dopamine by surviving neurons 📌 Crucial limitation (implied, not stated outright): L-Dopa only works if dopamine neurons are still alive **** Tyrosine → L-Dopa → Dopamine Enzymes involved: Tyrosine hydroxylase (TH) Aromatic amino acid decarboxylase (AADC)

Answer 157

A nuclear receptor Classified as an orphan receptor Essential for: Development of dopaminergic neurons Maintenance and survival of those neurons Why “exploiting the RXR side”? Nurr1’s ligand-binding pocket is crowded Hard to activate directly But Nurr1 forms a heterodimer with RXR -----> RXR can be drugged related receptors: Nur77 and NOR-1 All part of the NR4A subfamily Parkinson → Enter Nurr1–RXR nuclear receptors

Answer 158

Response Elements Shown Above: NBRE = (Nurr1 monomer binding site) NurRE= (Nurr1 homodimer site) DR5 =(RXR heterodimer site) Why this matters: Nurr1 can act: Alone As a homodimer As an RXR heterodimer 📌 This is how RXR enters the PD story. *****Nurr1 is functionally ligand-independent

Answer 159

Human Nurr1 polymorphisms are linked to early onset PD ---Multiple exons ----Polymorphisms and mutations associated with PD Mice with reduced Nurr1 expression show increased risk for PD-like symptoms” ---Loss-of-function experiments ---Establish necessity, not just correlation his slide is proving that: Dopaminergic neurons require Nurr1 Nurr1 loss → neuron vulnerability → PD Nurr1 cannot be easily drugged directly But Nurr1 forms heterodimers with RXR RXR is druggable

Answer 160

Bexarotene is a high affinity and specific RXR ligand: High affinity Binds RXR tightly Low Kd → effective at low concentrations Specific: Selective for RXRs ( in nigra) , not random NRs Already FDA-approved (originally for cancer) so binding RXR--> activity NUrr1 ( more dopamine 0 lees Parkinson * dopamine returns resotres

Answer 161

hese cells express: Tyrosine hydroxylase (TH) Nurr1 RXRα Why this toxin is used MPP⁺ = 1-methyl-4-phenylpyridinium This is a classic Parkinson’s disease model toxin. What MPP⁺ does: ---Enters dopaminergic neurons selectively I----nhibits mitochondrial complex I ----Causes energy failure ----Leads to neuron death Why this matters: ----MPP⁺ reproduces PD-like dopaminergic neuron loss It is not a generic toxin Green – TH (Tyrosine Hydroxylase): Marker of dopaminergic neurons Loss of green = neuron loss Preservation of green = neuron survival Blue – DNA stain: Labels nuclei Confirms cells are present Helps distinguish death vs differentiation _______ Control: Strong green TH signal Healthy neuron morphology MPP⁺ alone: Dramatic loss of green signal Dopaminergic neurons are gone Confirms toxin works MPP⁺ + BRF110: TH signal largely restored Neuron structure preserved Indicates protection, not replacement

Answer 162

gluacoma mutlole screlpros Parkinson disease stroke psychiatric disorders alzehiemrs disease

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