• The endocrine regulation of testosterone and estrogen production occurs through the hypothalamic-pituitary-gonadal ( HPG ) axis

1. The hypothalamus releases gonadotropin-releasing hormone ( GnRH ) in a pulsatile manner.

2. GnRH stimulates the anterior pituitary to release luteinizing hormone ( LH ) and follicle-stimulating hormone ( FSH )

3. LH stimulates Leydig cells in testes ( in males ) and theca cells in ovaries ( in females ) to produce testosterone.

4. FSH works synergistically with LH to stimulate Sertoli cells in males ( spermatogenesis ) and granulosa cells in females ( estrogen synthesis )

Feedback Mechanisms :

• Testosterone and estrogen exert negative feedback on the hypothalamus and pituitary to reduce GnRH , LH , and FSH secretion

• In females , estradiol has a dual role :

  • low levels provide negative feedback

  • but high levels during the late follicular phase cause positive feedback , triggering the LH surge and ovulation

Male System ( Spermatogenesis ) :

• LH in Leydig Cells :

  LH ➡️ cAMP ➡️ PKA ➡️ Cholesterol ➡️ Pregnenolone ➡️ Testosterone
  

  • Explanation : LH activates cAMP and PKA , driving gene transcription of enzymes that convert cholesterol to testosterone in Leydig cells.

• FSH in Sertoli Cells :

  xxxxxxxxxx
  FSH ➡️ cAMP ➡️ PKA ➡️ Gene Transcription ➡️ Androgen-Binding Protein ( ABP )
  

  • Explanation : FSH activates gene transcription for ABP , aiding sperm maturation and transport.

Female System ( Oogenesis and Follicle Development ) :

• LH in Theca Cells :

  xxxxxxxxxx
  LH ➡️ cAMP ➡️ PKA ➡️ Cholesterol ➡️ Pregnenolone ➡️ Androgens
  

  • Explanation : LH activates gene transcription for enzymes that convert cholesterol to androgens in theca cells.

• FSH in Granulosa Cells :

  xxxxxxxxxx
  FSH ➡️ cAMP ➡️ PKA ➡️ Gene Transcription ➡️ Aromatase ➡️ Androgens ➡️ Estrogens
  

  • Explanation : FSH in granulosa cells transcribes aromatase , converting androgens ( from theca cells ) to estrogens , completing estrogen production.

• Hormones :

  • FSH ( Follicle-Stimulating Hormone ) : Stimulates follicle development in the ovaries.

  • LH ( Luteinizing Hormone ) : Triggers ovulation through the LH surge.

  • Estrogen : Rises during the follicular phase, supporting uterine lining growth; drops after ovulation.

  • Progesterone : Rises post-ovulation during the luteal phase to support the endometrium for potential implantation.

• Sequence of Hormone Events :

  • Follicular Phase ( Days 1-14 ) : FSH and estrogen levels gradually increase , supporting follicle development.

  • Ovulation ( Day 14 ) : Estrogen hits a threshold, causing a positive feedback loop that triggers the LH surge. This surge leads to ovulation.

  • Luteal Phase ( Days 14-28 ) : Progesterone rises as the corpus luteum forms , maintaining the uterine lining. If no fertilization occurs, progesterone drops , leading to menstruation.

• Cellular Composition :

  • Zygote : The single-cell stage resulting from the fusion of a sperm and an egg, marking the beginning of the embryo.

  • Blastomeres : As the zygote undergoes mitotic divisions ( cleavage ) , it forms smaller, undifferentiated cells called blastomeres.

  • Embryonic Stem Cells : These are pluripotent cells that arise during the early stages ( e.g., inner cell mass of the blastocyst ) and have the potential to differentiate into various cell types.

1. Zygote : Day 0 ( fertilization )

2. Blastomeres : Day 1-3 ( cleavage )

3. Morula : Day 3-4 ( solid ball of cells )

4. Blastocyst : Day 5-6 ( hollow structure , first differentiation )

5. Bilaminar Disc : Week 2 ( epiblast and hypoblast form )

6. Trilaminar Disc : Week 3 ( gastrulation creates germ layers )

7. Organogenesis : Weeks 4-8 ( development of organs and systems )

8. Fetal Stage : Week 9 onward ( growth and maturation )

By Week 9 , it is no longer referred to as an embryo but as a fetus

• Acts as the conduit connecting the embryo/fetus to the placenta.

• Directly connects to the fetal liver and subsequently to the fetal heart via the umbilical vein.

• Carries oxygenated blood from the placenta to the fetus.

• Carries deoxygenated blood and waste from the fetus to the placenta via the umbilical arteries.

• Umbilical Cord Stem Cells :

  • Multipotent

  • you bank them at birth and then don't have to worry about donor rejection problems

  • expensive over time

Route of Maternal Blood Oxygen to Baby

1. Maternal Lungs → Pulmonary veins → Left atrium → Left ventricle → Aorta → Uterine arteries → Placenta ( intervillous space )

2. Placenta → Oxygen diffuses into fetal capillaries → Umbilical vein :

   • Liver Branch : A portion of oxygenated blood enters the fetal liver via smaller branches of the umbilical vein

   • Bypass Liver ( Ductus Venosus ) : The rest bypasses the liver through the ductus venosus , merging with the inferior vena cava ( IVC )

3. IVC → Right atrium → Foramen ovale → Left atrium → Left ventricle → Aorta → Fetal tissues

Route of CO₂ from Baby Back to Mother

1. Fetal Tissues ( CO₂ produced ) → Fetal veins → Right atrium → Right ventricle → Pulmonary artery :

   • Bypass Lungs ( Ductus Arteriosus ) : Most blood bypasses the lungs via the ductus arteriosus , joining the descending aorta

2. Descending Aorta → Umbilical arteries → Placenta

3. Placenta ( CO₂ diffuses into maternal blood ) → Uterine veins → Inferior vena cava → Right atrium → Right ventricle → Pulmonary arteries → Maternal lungs ( CO₂ exhaled )

• $G_i$$G_i$ = inhibitory

  1. Signal Molecule binds and Activates GPCR ( $G_i$$G_i$ )

  2. binds and inhibits adenylyl cyclase

  3. reduces PKA activation

  4. reduces cell response ( gene transcription )

• $G_s$$G_s$ = stimulatory

  1. Signal Molecule binds and Activates GPCR ( $G_s$$G_s$ )

  2. binds and stimulates adenylyl cyclase

  3. increases PKA activation

  4. increases cell response ( gene transcription )

• $G_q$$G_q$ = activates PLC via DAG and IP3

  1. Signal Molecule binds and Activates GPCR ( $G_q$$G_q$ )

  2. $PLC\beta$$PLC \beta$ becomes activated

  3. $\mathrm{PIP}{\phantom{A}}_2\underset{\mathrm{PLC}\beta }{\overset{}{\to }}DAG+I{P}_3$$\ce{PIP_2 ->[][PLC \beta]} DAG + IP_3$

     • this depletes levels of PIP2 in the membrane

     • but voltage gated M-channels ( potassium channels ) need PIP2 in order to open

       • so M-channels close

       • this is a SIGNALING event !

       • the removal of PIP2 closed potassium channels , depolarizes the membrane ,

         • makes it very easy to fire action potential now

         • only a little bit of sodium is need to reach the action potential threshold

  4. DAG then activates PKC

  5. IP3 causes calcium to be released from endoplasmic reticulum

     • the calcium can also reinforce / enhance signaling of PKC

  • 3 Different Ways $G_q$$G_q$ can signal :

    1. Depletion of $PI{P}_2$$PIP_2$ in the membrane

    2. Liberation of $I{P}_3$$IP_3$ and hence intracellular calcium release

    3. Libration of DAG and hence activation of PKC

• Describe one alternative pathway that G-protein activation may signal via

  • Conventional Way = producing signaling molecules

  • Alternative Way = reduction ! of $PI{P}_2$$PIP_2$

Cell Junctions

• Tight Junctions :

  • contain strands of cloudin and occludin

  • example = blood-brain-barrier

• Adherens :

  • strong mechanical

  • peristalsis

  • uses classical cadherins to link cytoskeleton ➡️ actin

• Desmosomes :

  • stronger than adherens

  • tissue rigidity

  • uses non-classical cadherins to link cytoskeleton ➡️ intermediate filaments

  • cardiac muscle , skin

• Hemidesmosomes :

  • uses integrins to link cytoskeleton ➡️ basal lamina intermediate filaments

• Gap Junctions :

  • facillitate communication

  • synchronized heart beat

  • allow electricity , ATP , misc things to pass

  • 6 trans membrane domains

  • connected by connexins

• Nuclear Localization Sequences ( NLS ) :

  • General Trend :

    • Imports = basic : lysine ( K ) , arginine ( R ) , histidine ( H )

    • Exports = aliphatic : isoleucine ( I ) , leucine ( L )

      • LxxxLxxLxL

  • Specific :

    • Nuclear Import = continuous basic

    • Mitochondria Import = spaced basic

    • ER Import = continuous hydrophobic

• Nuclear Import Process :

  1. cargo-NLS is complexed with an importin

  2. the complex travels through the Nuclear Pore Complex ( NPC ) from the cytosol , into the nucleus

  3. Ran-GTP binds to the complex , allowing the cargo to be released from the importin

  4. Ran-GTP bound to the important exits the nucleus back into the cytosol

  5. Ran-GTP is hydrolyzed into Ran-GDP and the importin is released

• Nuclear Localization Signals use the adapters like importins to facilitate movement into the nucleus

• Mitochondria also use signal localization sequences

  • instead of importins , they use TOM / TIM pore complexes

  • the localization sequence gets cleaved once its inside

  • depends on heat shock proteins as chaperones

• Peroxisomes also use short signal sequences to get proteins from ER ➡️ peroxisome

• Signal sequences are also used to direct partially started ribosomal complexes to the ER wall itself

  • here it can stabilize , and continue translation , dumping the contents into the ER lumen

  • N-terminal domain enters into the ER lumen first

• Glycosylation is very common for rough ER synthesized proteins

  • the tags mark the state of protein folding

• Improperly folded proteins get ubiquitinated , and then exported into the cytosol , to the be broken down in peroxisomes

• Accumulation of Improperly folded proteins ,

  • triggers gene transcription of chaperones to assist in folding

• Vesicle Coatings :

  1. Clathrin = normal endocytosis

  2. COPI = golgi ➡️ ER

     • retrograde , backward direction

  3. COPII = ER ➡️ golgi

     • anterograde , forward direction , for export

• Dynamin = GTPase that pinches off vesicles

  • uncoats clathrin

• Rab = used to guide endosomes cargo to its target location in the cell

• SNARES mediate membrane fusion

  • they need to be separated after

  • used in exocytosis

• Endocytosis :

  • clathrin = normal

    • Process : Cargo binds to receptors, adaptors recruit clathrin, and vesicle formation begins. Dynamin constricts the vesicle neck, enabling release.

    • Uncoating : ATP-driven disassembly of the clathrin coat for vesicle integration with endosomes.

  • pinocytosis = water

  • LDL fats = also use clathrin

• Exocytosis :

  • Continuous : Supplies membrane and secretory proteins without regulation

  • Stimulus-Dependent : Triggered by external signals, often involving calcium influx binding to synaptotagmin, facilitating vesicle fusion with the plasma membrane.

  • SNARE Complex :

    • Components: Synaptobrevin ( V-SNARE ) , Syntaxin , and SNAP-25 ( T-SNAREs )

• Parkinson's Disease :

  • can't release or reuptake dopamine

  • Parkin 1 = accumulates and prevents synaptic vesicle release

  • 𝛼-synuclein = blocks SNARE complexes , aka vesicle fusion

• Golgi = sorting device :

  1. lysosomes

  2. secretory vesicles ( signal mediated )

  3. secretory vesicles ( constitutive secretion )

• Pathways to a Lysosome :

  • enocytose , phagocytose , micropinocytose , or autophagy of mitochondrion

• Mannose-6-Phosphate Receptors = on golgi , used in early endosome formation

• Neurons :

  • golgi ➡️ synaptic vesicle ( endosome ) ➡️ fuses , offloads contents into synaptic cleft ➡️ contents "re-uptook" / endocytosed back into pre-synaptic terminal

Hydroxylation

1. Definition : Adds hydroxyl ( -OH ) groups , primarily to proline or lysine residues.

2. What Occurs :

   • Example: Hypoxia-Inducible Factor ( HIF-α )

     • Under normal oxygen levels ( normoxia ) :

       • Prolyl hydroxylase enzymes hydroxylate HIF-α.

       • Hydroxylation enables binding to von Hippel-Lindau ( VHL ) protein

       • HIF-α is ubiquitinated and degraded by the proteasome.

     • Under low oxygen ( hypoxia ) :

       • Hydroxylation does not occur.

       • HIF-α stabilizes , binds with HIF-β , and activates gene transcription for hypoxia response.

3. Effect on Protein Function :

   • Regulates protein stability.

   • Hydroxylation under normoxia ensures degradation of HIF-α , preventing hypoxia response.

   • Lack of hydroxylation under hypoxia allows HIF-α to activate survival genes.

• Normoxia = HIF gets hydroxylated ➡️ becomes ubiquinated ➡️ proteosomal degradation

• Hypoxia = HIF doesn't get hydroxylated ➡️ becomes nuclear transcription factor ➡️ hypoxia response elements ( HREs ) are transcribed

Ubiquitination

1. Definition : Attaches ubiquitin proteins to lysine residues on target proteins.

2. What Occurs :

   • Marks proteins for degradation by the 26S proteasome.

   • Example : HIF-α ubiquitination under normoxia.

     • Ubiquitination is triggered by hydroxylation and VHL protein binding.

3. Effect on Protein Function :

   • Regulates protein turnover by targeting them for degradation.

   • Maintains cellular homeostasis by removing damaged or unnecessary proteins.

Lipidation

1. Definition : Adds lipid groups ( e.g., palmitate, myristate ) to cysteine residues.

2. What Occurs :

   • Anchors proteins to membranes, altering localization and function.

   • Examples :

     • AMPA Receptor ( GluA1-4 ) :

       • Lipidation at TM2 ➡️ Golgi retention.

       • Lipidation at TM4 ➡️ Membrane insertion; enables PKC phosphorylation.

     • Potassium Channel ( Kcnma1 ) :

       • Lipidation at S0-S1 loop ➡️ ER and Golgi exit.

       • Lipidation at STREX site ➡️ Regulates kinase activity ( e.g., PKA inhibition )

3. Effect on Protein Function :

   • Alters membrane localization , which impacts protein interactions and signaling.

   • Specific lipidation sites determine whether proteins are retained in organelles or inserted into membranes , influencing downstream activity.

Phosphorylation

1. Definition : Adds phosphate groups to serine, threonine, or tyrosine residues.

2. What Occurs :

   • Carried out by kinases and reversed by phosphatases.

   • Example: AMP-Activated Protein Kinase ( AMPK ) :

     • Activated under stress ( e.g., low ATP/high AMP )

     • Phosphorylates glucose transporters to increase glucose uptake

   • Energy regulation :

     • AMP rises during low ATP ➡️ Activates AMPK.

3. Effect on Protein Function :

   • Alters protein activity , stability , and interactions.

   • Phosphorylation of glucose transporters enhances glucose uptake , helping maintain energy balance under stress.

• Low ATP / High AMP ➡️ Activates AMPK ➡️ phosphorylates serine and threonine residues on glucose transporters ➡️ increases glucose import

Glycosylation

1. Definition : Addition of sugar groups ( glycans ) to proteins , primarily on extracellular domains.

2. What Occurs :

   • Facilitates cell-cell adhesion and protein-protein interactions.

   • Example - Sialic acid :

     • Negative charges repel but allow connections with positively charged glycans.

     • Example in podocytes: Glycosylation maintains podocyte-endothelial cell linkages.

3. Effect on Protein Function :

   • Enhances structural integrity and adhesion in cellular networks.

   • Modulates interactions with other proteins or cells , crucial for processes like immune responses and tissue stability.

Disulfide Bond Formation

1. Definition : Covalent linkage between two cysteine residues , forming disulfide bonds ( S-S )

2. What Occurs :

   • Stabilizes protein conformation by maintaining structural integrity.

   • Influenced by cellular redox states , which are regulated by reactive oxygen species ( ROS ) and antioxidants like glutathione.

   • Complex I and III of the mitochondrial electron transport chain can leak ROS , altering the redox environment and influencing disulfide bond formation.

     • This can lead to oxidative stress and misfolding if the redox balance is disrupted.

3. Effect on Protein Function :

   • Ensures correct protein folding and structural stability.

   • Misregulation can lead to misfolded proteins, resulting in dysfunction and disease.

   • Role of Glutathione : Glutathione maintains the proper redox environment for forming and breaking disulfide bonds.

     • It ensures that disulfide bonds are formed correctly by reducing incorrect linkages and preventing oxidative damage to cysteine residues.

SUMOylation

1. Definition : Addition of SUMO ( Small Ubiquitin-like Modifier ) proteins to target proteins.

2. What Occurs :

   • Modulates nuclear transport , transcriptional regulation , and DNA repair.

   • SUMO is attached to lysine residues , similar to ubiquitin.

3. Effect on Protein Function :

   • Alters protein localization and interactions.

   • Enhances or suppresses transcriptional activity , depending on the target.

Methylation

1. Definition: Adds methyl groups , primarily to lysine or arginine residues on histones.

2. What Occurs :

   • Regulates chromatin structure and gene expression.

   • Example: Histone methylation :

     • Specific methylation patterns can activate or repress gene transcription.

3. Effect on Protein Function :

   • Modulates gene expression by altering chromatin accessibility.

   • Plays a role in epigenetic regulation , influencing long-term cellular behavior.

Pyruvate Kinase Deficiency in Red Blood Cells

• RBCs don't have mitochondria

• They rely on anaerobic glycolysis for energy

  • no TCA or ETC

1. Pyruvate Kinase Deficiency ➡️

2. Slow Glycolysis ➡️

3. Loss of ATP ➡️

4. Can't Power/Run Sodium-Potasium-ATPase ( pump ) ➡️

5. Sodium ions accumulate in cytosol ➡️

6. Osmotic pressure increases ➡️

   • cell looses shape here , becomes "spiculated"

7. Cell lyses ➡️

8. Anemia

9. Build up of 2,3-BPG

Major Protein Kinase Families

• Serine / Threonine

  • example = AMPK

• Tyrosine

  • example = insulin receptor

• Serine / Threonine and Tyrosine ( dual-specificity )

  • can phosphorylate both serine/threonine and tyrosine residues

  • example = MEK1 / MEK2 ( Mitogen-Activated Protein Kinase Kinase )

• Histidine

  • example = CheA ( involved in bacterial chemotaxis )

Know 3 Receptors that Act via Tyrosine Kinase Signaling

• RTKs are generally membrane-anchored to facilitate ligand binding and signal transduction

1. Insulin

   • stimulates carbohydrate utilization and protein synthesis

   • rate = seconds to minutes

     • direct ligand to receptor binding

     • immediate release of pre-stored granules

     • but also has longer transcriptional effects

2. Platelet Derived Growth Factor

   • stimulates survival , growth , proliferation , and migration of various cell types

   • rate = minutes to hours

     • needs time for secondary signaling cascades to occur

3. Nerve Growth Factor

   • stimulates survival and growth of some neurons

   • rate = hours to days

     • transcriptional changes take a while