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Experiments + research

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Parathyroid gland

Hypothalamus

Hypothalamus is the main link between your endocrine system and nervous system

the hypothalamus reacts to many inputs such as:

  • light
  • olfactory
  • steroids
  • autonomic inputs
  • stress
  • blood-borne stimuli

the hypothalamus has 6 main releasing hormones:

  1. thyrotropin-releasing hormone (TRH)
  2. gonadotropin-releasing hormone (GnRH)
  3. growth hormone-releasing hormone (GHRH)
  4. corticotropin releasing hormone (CRH)
  5. somatostatin
  6. dopamine

these hormones stimulate release of anterior pituitray hormones

the hypothalamus is involved in a range of feedback loops

  • HPA axis
  • HPT axis
  • control of CRH and ACTH
  • control of GH secretion
  • contol of PRL secretion
  • feedback effects of GnRH + FSHLH secretion (male+ female)
  • stimulation of uterine smooth muscle contraction at birth
  • hypothalamic circuits in hunger
  • gut-brain axis

  • recieves information from the hypothalamus via the hypothalamic-portal system in the form of releasing hormones
  • small parvocellular neurons pass to median eminence
  • contains 5 cell types that secrete six hormones

  1. Somatrotopes - GH - acidophil
  2. Lactotropes - PRL - acidophil
  3. Thyrotropes - TSH - basophil
  4. Corticotropes - ACTH - basophil
  5. Gonadotropes - LH and FSH - basophil

Anterior pituitary hormones

  • Neurons come from the supraoptic nuclei (SON) and paraventricular nuclei (PVN)
  • magnocellular neurons in anterior hypothalamus extend all the way into the posterior pituitray gland
  • supraoptic-hypothalamic tract - stores and secretes twonhormones ( oxytocin and antidiuretic hormone)
  • also contains glial-like supportive cells called pituicytes which aid in the storage and secretion of the hormones

Anterior pituitary glands hormones

  • GnRH causes release of => gonadotrophins
  • GHRH causes release of => growth hormone
  • somatostatin causes => inhibition of growth hormone
  • TRH causes release of => TSH and prolactin
  • Dopamin causes => inhibition of prolactin
  • CRH causes release of => adrencorticotropic hormone

  • Follicle stimulating hormone (FSH)
  • Luteinising hormone (LH)
  • Adrenocorticotropic hormone (ACTH)
  • Thyroid stimulating hormone (TSH)
  • Prolactin (PRL)
  • Growth hormone (GH)

FAT (tropic) PiG ( non-tropic)

Glycoprotein corticotropin-lipotropin somatomammotropin(2 subunits , a = identical B= biological specificity)

Hypothalamic-pituitary-adrenal axis (HPA)

  • Main stress response system
  • primary function is to release glucocorticoids that activate short-term physiological responses to stress
  • cortisol has several roles including : increasing glucose levels, supressing the immune system and aiding in metabolilsm
  • negative feedback loop to maintain balance

In vitro test

Hypothalamic- pituitary-thyroid axis (HPT)

  • stresses (mental,physical , starvation etc. ) decrease TRH release
  • T3 is the main hormone for negative feedback
  • TSH stimulates nearly every aspect of thyroid function
  • TSH secretion is decreased mainly by repressing TSH-B subunit gene expression
  • at low levels of iodide intake the rate of thyroid hormone synthesis is directly related to iodide availability
  • if the levels rise the intraglandular conc of iodide reaches a level that suppresses NADPH oxidase activity and the mechanism of hormone biosynthesis
  • wolf-chaikoff effect

Control of CRH and ACTH action

  • negative feedback is coming from cortisol
  • ADH increases ACTH release
  • ACTH binds to the melanocortin-2 receptor on cells in the adrenal cortex
  • ACTH acutely increases cortisol and adrenal androgen production
  • it also increases expression of steroidogenic enzyme genes and in the long term promotes growth and survival of two zones of the adrenal cortex

  • diurnal control mediated via suprachiasmatic nucleus (SCN) of anterior hypothalamus
  • endogenous clock genes in many peripheral genes which are entrained by ligh/dark cycles
  • important for medicine so you know when receptors are more present to be able to bind to drug

Control of GH secretion

  • somatostatin reduces amount of GH produced and released
  • it binds to the somatostatin receptor which lowers cAMP
  • negative feedback comes from IGF-1
  • GH stimulates IGF-1 production at the liver and IGF-1 then inhibits GH synthesis and secretion at the pituitary and hypothalamus
  • liver and kidney are major sights of degradation for GH
  • GH secretion is pulsatile
  • falling blood glucose levels are a very effective stimuli to increase GH secretion and can be used as a provocative test of a persons ability to secrete GH
  • GH and IGF-1 have many actions

issues associated

Control of prolactin (PRL) secretion

  • don't need prolactin in day to day life
  • TRH has an effetc but is very weak
  • production and secretion is predoinantly under inhibitory control through dopamine
  • PRL circulates unbound to serum proteins adn thus has a short half-life
  • in humans the predominant physiological role of PRL is the regulation of essentially every aspect of postnatal breast development and function

Feedback effects on GnRH + FSHLH secretion

  • FSH and LH are segregated to a large degree into different secretory granules and are not co-secreted in equimolar amounts
  • this allows for the modulation of the ration of FSH:LH
  • FSH and LH receptors are both GPCRs which are primarily coupled to Gs-cAMP-PKA signalling pathways

Feedback effects on GnRH + FSHLH secretion : Male

  • gonadotropins promote testosterone production
  • FSH also increases teh secretion of a TGF-B related protein hormone - inhibin
  • GnRH is released in a pulsatile manner
  • continuous infusion of GnRH downregulates the GnRh receptor resulting in a decrease in FSH and LH secretion
  • at a quicker frequency GnRH preferentially increases LH secretion
  • at a slower frequency GnRH preferentially increases FSH secretion

Feedback effects on GnRH + FSHLH secretion: Females

  • can be effected by both positive and negative feedback
  • in general it promotes estrogen and porgesterone secretion
  • FSH also increases the secretion of inhbin
  • GnRh is released in a pulsatile manner
  • progesterone and testosterone negatively feedback on function at the level of hypothalaus an dpituitary
  • at low doses estrogen also exerts a negative feedback on FSH and LH secretion
  • positive feedback is observed at the hypothalamus and pituitary when high estrogen levels are maintained for 3 days , causing a surge of LH and to a lesser extent FSH secretion

Stimulation of uterine smooth muscle contraction at birth

  • oxytocin is released during labour from the hypothalamus when the fetus stimulates the cervix
  • synthetic oxytocin may be used to further stimulate uterine contractions
  • have to do it carefully to avoid ruptuing the uterus
  • or an oxytocin antagonist may be used to inhibit premature labour

Hypothalamic circuits

  • Arc neurons project to anorectic neurons in the PVH
  • POMC neurons release aMSH which activates MC4R neurons in the PVH
  • AgRP is an inverse agonist of MC4R and hyperpolarises these neurons
  • NPY binds to Y-receptors to inhibit PVH neurons
  • GABA is an inhibitory neurotransmitter
  • PVH neurons project to : brain stem , SNS , higher brain regions , thyroid axis

Gut-brain axis

  • during food intake , peripheral mechanoreceptors and chemoreceptors activate the vagus nerve which projects to the brain stem
  • GI hormones act in an endocrine manner in the CNS to modulate food intake and metabolic proccesses they also stimualte ascending vagal afferent nerve fibres
  • adipostiy has a major influence on gut-brain intercations
  • leptin has a dimmer-switch-effect causing a redution in long-term food intake
  • gut-brain intercations also influence adiposity

Cell membrane

provides 4 functions

  1. separation - membranes form a semi permeable diffusion barrier
  2. exchange - membranes allow transport of metabolites and macromolecules in and out of cell compartments
  3. integration - membranes mediate cell to cell communication , cell adhesion and signalling
  4. metabolism - membranes are an integral part of metabolic pathwas , also contain machinery for membrane synthesis , remodelling and degradation

lipid rafts

membrane proteins

Proteins

Lipids

Membrane lipids

Sphingolipid

Cholesterol

  • Animal - cholesterol
  • yeast - ergosterol
  • plants - stigmasterol

  • contributes 20-40% of total lipid
  • neutral charge
  • hydroxyl group
  • cycliic structure and increases bulkiness
  • it affects fluidity - associated with sphingomyelin and gangliosides in lipid rafts

synthesis of isopentenyl pyrophosphate (an activated isoprene unit needed to produce cholesterol) takes place in cytosol condenstaion of six molecules of isopentenyl pyrophosphate to form squalene cyclisation of squalene tetracyclic product converted into cholesterol

Biosynthesis of cholesterol

Sterols are syntehsised in the lumenal leaflet of the ER membrane

Carbohydrate

Membrane lipids

Phospholipids

4 main types

  • phosphatidyl-ethanolamine
  • phosphatidyl-choline - most common in phospholipid bilayer

  • phosphatidyl-serine
  • phosphatidylinositol

Neutral

Negative

size of the head group changes the shape of the phospholipid bilayerhead groups determine what category they fit inhead group can be - choline,ethanolamine,polyalcohols and serine

acyl chains

Synthesis of phospholipids

  • Fatty acids bind with CoA to form fatty acyl-CoA
  • glycerol-3-phosphate is generated through phosphorylation of glycerol by glycerol kinase
  • fatty acyl-CoA molecules are attached to glycerol-3-phosphate resulting in formation of phosphatidic acid
  • the addition of the head group forming a strong phosphodiester linkage ,determines the end phospholipid

Acyl chains

  • shorter the chain - the more 'fluid' the lipid
  • chains can be saturated (no double bonds)-free rotation
  • chains can be unsaturated (double bonds) - kink at each bond
  • more unsaturated - more fluid

in a biological membrane

contains phospholpids where 1 chain saturated and 1 unsaturateddouble bonds invariably cis not trans (kinked)chain length is 18 carbons long on average

can change the acyl chain composition - number of C and double bondsto accomodate for organisms that grow at different temperatures

Fatty Acids

can differ from one another in :

  1. length of hydrocarbon tails
  2. degree of unsaturation
  3. position of the double bonds in the chain

most fatty acids have 12 to 22 carbons !!

Sphingolipids

If R is :

H phosphocoline or -ethanolamine gluocse or galactosecomplex oligosaccharide

ceramidesphingomyelincerebrosideganglioside

known to function as intercellular signalling molecules and can affect cell growth , differentiation and programmed cell death

Ceramides

common lipid in bilayer ( head group being phosphocoline)found in lipid raftsassociate with cholesterol and regulate cell surface location of membrane proteins

have a ceramide unit linked by glycosidic bond at carbon 1 of long chain base to glucose or galactose

have sialic acid components they have larger more complex head groups and numbered according to how many sialic acids they have

Sphingolipids that form rafts tend to have more saturated and longer side chains which makes them less fluid with a higher melting temperature

Sphingomyelin

Cerebroside

Ganglioside

Synthesis begins in the cytoslic leaflet of the ER and finishes in the lumenal leaflet of the golgi

Glycolipids

Present on the outer cell surface membranecontain a carbohydrate attached to a lipid moleculeplay a key role in cell recognition and cell signallingthey are ampipathicdifferent glycolipids have different carbohydratesdiversity of glycolipids helps the specificity of cellular interactionscan have a sphingosine ( sphingolipids) or glycerol backbone

Read more

Triglycerides (TAGs)

  • Primary storage form of fat in the body
  • consist of 3 fatty acids esterified to a glycerol molecule
  • they are hydrophobic
  • suitable for storage in non-aqueous environments
  • dietary fats and oils are composed of triglycerides
  • when consumed triglycerides are broken down and the fatty acids absorbed

Phospholipid bilayer

What drives the formation of the bilayer?

  • membranes tend to form spontaneously
  • due to -ve free energy
  • comes down to hydrophobic effect
  • change in gibbs free energy during self assembly at ambient temperatures is -ve and spontaneous
  • it is -ve because water gains mores stronger molecular interactions

Hydrophobic effect

polar solutes :non-polar solutes:

can organise in water without affecting water structure

would force adjacent water molecules to organise into a more ordered lattice , decreased entropy ,higher free energy

Membrane fluidity

  • Influenced by temperature and composition
  • more fluid when heat applied
  • mobility increases tiwards bilayer centre
  • Tm depends upon
- legnth of chain - degree of unsaturation - headgroup size

Also effected by cholesterol , more cholesterol = less fluidrigid steroid interacts with outer region of the hydrophobic core and makes the bilateral more rigid

in a planar phospholipid dbilayers , hydrophobic tails are exposed to water along the edges so the bilayers spontaneously close in on themselves to form sealed compartmetns

Lipid distribution and movement

Read more

Vesicle budding and membrane curvature

  • need to generate positive curvature for vesicle
  • extreme negative curvature at site of fission

Generating curvature

  • need more lipid on cytoplasmic leaflet of a vesicle
  • aminophospholipid translocator - flippase
  • uses ATP to flip lipid (PE/PS) from outer to inner leaflet of plasma membrane
  • ABC transporters (ATP-binding cassette ) - floppase
  • uses ATP to move lipid from inner to outer leaflet of plasma membrane

Lipids can help promote membrane curvature as they have different 'shapes' - relative size of head to tail

  • Cone shaped (PE) lipids inside
  • Inverted-cone shaped (LPC or SM) lipids outside

at bud site : enzymes can act on lipids to alter their shape and hence promote membrane curvature

Lipid distribution and movement

  • Lateral diffusion
  • transverse diffusion
  • rotation + flexion
  • non-vesicular transport
  • bi-direcitonal scrambling

membranes have asymmetric lipid distributions:- important for membrane function - asymmetric synthesis , specific lipid transport/translocation- minimal spontaneous flip-flop

PhosphatidylcholineSphingomyelinPhosphatidylserinePhoshpatidylinositolsGlycolipidsCholesterol

Plasma membrane : largely asymmetric , mainly cholesterol , contains sphingolipids

Endosomes/Lysosomes : similar to plasma membrane

Mitochondria : similar to ER ( no SM or glycolipids)

ER : mainly phosphatidylcholine, largely symmetric , low cholesterol and little SM

Movement

Lateral diffusion

  • movement of lipids within the plan of the biological membrane
  • key to membrane fluidity
  • allows exchange of lipids between adjacent regions of the membrane
  • very rapid process

FRAP

Transverse diffusion (flip-flop)

  • Very slow
  • once every several hours as it has to go through a hydrophobic region
  • diffuses from one monolayer to the other monolayer

Flippases

Floppases

Flippases

  • couple ATP hydrolysis to movement of lipids
  • help to establish and maintain the asymmetric distribution of phospholipids
  • selectively remove PS and PE from the noncystolic monolayer and flip them to the cystolic side
  • transfer leaves PC and SM concentrated in the noncystolic monolayer
  • the resulting curvature of the membrane may help drive subsequent vesicle budding

  • P family - P1-P5
  • P4 ATPase - needs interaction with CDC50 (cell division control)
  • binding of these leads to phosphorylation of P4ATPase
  • hydrolysis of ATP- energy needed for movement
  • bidning of lipid - dephosphorylation

P4-ATPaseA- actuator P-phosphorylationN - nucleotide binding10 membrane spanning helices

CDC502 membrane spanning domainslarge extracellular loopcontains 4 possible N-linked glycosylation

Floppases

ABC transporterslarge family of membarne transporters with ATP Binding Cassettetransport range of small molecules including lipids transport lipids from cytolic to extracellular leaflet (exception is ABCAA- found in photoreceptor cells)

  • binding of lipid and ABC transporter
  • ATP then binds at cytoplasmic side of the potein
  • following binding of each , ATP hydrolysis provides energy for flopping
  • releas of phosphate from original ATP molecule
  • ADP is released and a new molecule of ATP binds to secondary ATP-bidning site
  • Hydrolysis and release of ADP and a phosphate molecule resets the protein so process can start again

ABCB1 (sub-family B member 1)

Scramblases

  • PC and PE rapidly equilibrate between the two leaflets
  • far too fast to be explained by flip-flop
  • ATP-energy independent
  • bidirectional

In ER :

TMEM16

  • The scrambling domain between transmembrane regions IV and V
  • Ca2+ binding site comprised of Aspartate and Glutamate in transmembrane regions VI-VIII are shown by red asteriks

Flipping and scrambling mechanism

Exposing PS on cell surface

  • PS exposure in cells with high Ca2+
  • intracellular Ca2+ increase and activates TMEM16F to scramble phospholipids
  • It inactivates P4-ATPases and reduces their flipping activity
  • When Ca2+ returns to normal TMEM16F stops scrambling phospholipids
  • while P4-ATPase resumes flipping PS
  • constant flipping of PS prevents PS-exposing cells to be engulfed by macrophages

Non-vesicular transport

  • Not connected to ER via vesicular transport system
  • Can't synthesise lipids
  • lipids must be imported from ER

  • show a suprising amount of non-vesicular lipid transport

Mitochondria and peroxisomes:

Golgi , endosomes , plasma membrane

Lipid transfer proteins

  1. donor membrane docking
  2. lipid extraction
  3. donor membane undocking
  4. diffusion
  5. acceptor membrane docking
  6. lipid deposition
  7. acceptor membrane undocking
  8. further diffusion

Example of a lipid transfer protein - Tubular Lipid Binding Domains (TULIP) proteins

long hydrophobic tunnel open seam present down the middle

Flexion of lipids

Rotation of lipids

the bending or flexing of lipid molecules

  • unsaturated fatty acids introduce kink and increase flexibility
  • shorter fatty acid chains are more flexible than longer ones
  • temperature influences the degree of flexion with higher temperatures promoting greater flexibility

  • temperature : higher temperature increases kinetic energy , facilitating rotational motion
  • the amphipathic nature of lipids allows them to rotate within the hydrophobic core of the bilayer

spinning or rotational movement of lipid molecules around their own axis within the lipid bilayer

Lipid rafts

specialised dynamic microdomains within the cell membrane that are enriched in certain lipids and proteins

  • enriched in sphingolipids, cholesterol and specific proteins
  • has a more ordered and tightly packed environment compared to the surrounding membrane
  • act as platforms for the assembly of signalling molecules
  • implicated in membrane trafficking processes
  • they are dynamic and can change in composition and size in response to various cellular signals and environmental conditions
  • formation due to physico-chemical forces - increase H-bonding
  • has an increased bilayer thickness

Fluorescence photobleaching after recovery - FRAP

  • can be used to measure the rate of lateral diffusion of a membrane protein
  • specific type of protein can be labeled with a fluorescent antibody or tagged with a fluourescent protein such as GFP
  • a small area of the membrane containing these fluorescent protein molecules is then bleached using a laser beam
  • as the bleached molecules diffuse away and unfleached diffuse into the area the intensity of the fluorescence is recovered
  • the diffusion coeffcieint is then calculated from a graph of the rate of fluorescence recovery
  • the greater the diffusion coefcient of the membrane protein the faster the recovery

Membrane proteins

Transmembrane proteins

  • Span across hydrophobic core of bilayer
  • can be small or quite large
  • they can connect two aqeuous compartments-outside and inside cell
  • function as transporters , cell surface receptors , cell adhesion molecules and enzymes

Example : Rhodopsin

  • GPCR coupled receptor
  • usually found in the voids of the retina and quite light sensitive
  • Each TMD has 20 hydrophobic residues that form alpha helices
  • flanked by charged residues - these are useful and help to provide H bonding between helices to stabilise protein

Surface-bound membrane proteins

  • Dont span across the membrane
  • almost entirely extramembranous on one side of bilayer only

Interact with the membrane in 2 ways :

1. Monotopic

2.Lipid-anchored

  • membrane-embedded domain that anchors proteins with nearest monolayer

  • these are found entirely outsisde of hydrophobic core
  • but have a lipid anchor that holds protein close to membrane surface on one side of bilayer

Example : COX 2ampipathic helix catalyses reaction of arachidonic acid to prostaglandin H2 which causes inflammation

Peripheral membrane proteins

  • surface bound peripheral proteins don't have any attachment to a hydrophobic anchor
  • they are called extrinsic membrane proteins
  • associate with membrane by interacting with head groups of lipids
  • or can associate with extramembranous domains of integral membrane proteins through H bonds and charged groups

How do surface bound proteins differ from peripheral membrane proteins?

SURFACE bound Peripheral membrane

  • location
  • association
  • attachment
  • examples

generally located on the surface of the membrane , either on the extracellular side , the cytoplasmic side or both

often bound to the membrane through non-covalent interactions with integral membrane proteins or throguh electrostatic interactions with membrane lipids

Receptors , some enzymes and cell adhesion molecules ( integrins , GPCRs, cadherins)

may not fully transverse the membrane , may be attached to one side of the membrane or associated wiwth the membrane through peripheral interatcions

can be anchored to the membrane thorugh various interactions such as electrostatic interactions or association with other membrane proteins

are not embedded within the lipid bilayer , they are loosely associated with the membrane surface

can be attached to the membrane temporarily and can be removed without disrupting the membranes integrity

many signalling proteins (Src kinase , Ras GTPase , PKC)

Anchoring to the outside of the membrane

Anchoring to the inside of the membrane

  • Glycosyl phosphatidyl inositol (GPI) modification
  • quite flexible
  • essential for protein to remain attached to cell
  • specific phospholipase can release protein in response to signals

  • N-terminal myristoylation
  • palmitoylation of cysteine residues
  • farnesylation
  • geranylgeranylation

Example : PrPcPrion protein associated with BSE(mad cow disease)misfolding and aggregatino of the prion protein

N-terminal myristoylation

Palmitoylation of cysteine residues

  • added to the N-terminus as the protein is synthesised on the ER
  • Enzyme : N-myristoyltransferase
  • stable and reversible giving it a dynamic process

  • added post-translationally
  • reversible
  • consensus not well defined
  • particullarly common among membrane-associated proteins
  • Enzyme : palmitoyltransferase

Example: Src kinase non-receptor tyrosine kinase undergos this process to be able to become anchored to the cell membrane where it can interact with other signalling molecules and participate in varius cellular signalling pathways

Example: Rasreaction occurs in the ERpalmitoylation enhances the hydrophobic nature of Ras helping it become more associated with the membrane and aiding in Ras signalling

Prenylation of C-terminus

Farnesylation

Gernaylgeranylation

  • post-translational , thioether linkage of farnesyl group
  • Enzyme-linked ( farensyl transferase)
  • stable
  • consensus motif : CaaXcooh
  • after modification aaX residues removed and new cooh methylated

Example:GTPase RhoAcontrols local actin assemblyhelps with anchoring it to the membrane

Example:GTPase Rasactivates ERK signalling pathwayallows it to anchor to the membrane

  • Post-translational , thioether linkage of geranylgeranyl group
  • enyzyme-linked (geranylgeranyl transferase I and II)
  • stbale
  • consenus motifs:
  • for GGT I : CaaXcooh
  • for GGT II : CCcooh , CXCcooh , CCXXcooh

Measurement of the pituitary response to hypothalamic extracts in an in vitro pituitarysystem

To test the theory:

Chemical messengers (i.e.“releasing hormones”),synthesised within the hypothalamus, stimulate the release of hormones from the pituitary gland into the general circulation

  • ACTH release from isolated anterior pituitary cells
  • hypotahalmic cells produce and release a soluble factor that can stimulate the release of ACTH from pituitary cells
  • hypothalamus releases CRH which causes cortioctropes to release ACTH which goes to the adrenal cortex
  • this then releases glucocorticoid that causes a negative feedback loop

conclusion : hypothalamic cells produce and release a soluble factor that can stimulate the release of ACTH from pituitary cells

Steroid hormones

  • Lipids
  • cannot be stored in cell
  • rate of release = rate of synthesis
  • H2O-insoluble : transported in protein-bound form
  • act via intracellular receptors
  • alter rate of (specific) gene expression:
  • delayed,prolonged action
  • all derived from cholesterol
  • must be carried protein bound as insoluble in blood serum

9 types of proteins

  1. catalytic proteins
  2. binding proteins
  3. signalling proteins
  4. transport proteins
  5. structural p roteins
  6. motor proteins
  7. storage proteins
  8. hormonal proteins
  9. chaperone proteins

8

9

Proteins

protein trafficking

modifications

degradation/turnover

Peptide hormones

  • proteins (polypeptides or modified amino acids)
  • stored in secretory granules
  • rate of release >> rate of synthesis
  • H2O -soluble : transported 'free' in circulation
  • act at cell surface receptors
  • exert rappid , acute and also long-term effects

Synthesis

  • synthesised on ribosomes as pre-hormone : initial signal sequence of 15-25 hydrophobic amino acids
  • SS binds to signal recognition particle SRP
  • SS-SRP complex binds to receptor in RER membrane
  • nascent peptide transported into RER lumen
  • SS cleaved off => hormone or prohormone => further processing

Secretion

  • secretory granules translocation to plasma membrane - involved cytoskeleton : microfilaments and microtubules
  • docking and fusion of SG with PM - involves docking fusion proteins : SNAPs,SNAREs,VAMPs (SNARE hypothesis)
  • SV contents released to extracellular space
  • secretion is regulated e.g. by cyclic AMP

Action

  • binds with high affinity and specificity to cell surface receptors on target cell
  • receptor : plasma membrane proteins with 3 functional domains
  • extracellular 9hormone binding )
  • 1-7 membrane -spanning domains - hydropathy plot
  • intracellular (associated with 'effector' function )
  • hormone binding => conformational change in receptor , relayed to effecetor => generation of intracellular signals => response that is excitatory or inhibitory

Chaperone proteins

  • a large group of proteins whose role is to :
  • stabilize unfolded proteins
  • unfold them for translocation across membrane or for degradation
  • assist in their correct folding and assembly

Chaperonins

Heat shock proteins

Co-chaperones

Assist in the folding of newly-synthesized proteins or the refolding of denatured proteinsATP-dependent

Group I - found in bacteria and mitochondria

  • TRAP1 -mitochondria
  • GroEL and GroES - bacteria

Group II - found in eukarya and archaea (single-cell organisms)

  • HSPD1/CCT
  • Hsp60 and Hsp10

Produced by cells in response to stressclassified into families based on their molecular weight

  • sHsps
  • Hsp60
  • Hsp70

Assist chaperones in protein folding

  • Hsp10
  • Hsp40
  • PFDN

Small Heat shock proteins (sHSPs)

  • Role in suppressing protein aggregation in vivo and in vitro
  • bind and stabilise denatured proteins under conditions of cellular stress, ageing and degenerative disease
  • do not appear to have unfolding or refolding activity
  • high capacity for protein binding - up to 2 per subunit
  • upregulated under cellular stress
  • smaller in moleular weight ( 12 to 43 kDa in size) and form oligomeric complexes
  • their presence in different cellular locations reflects their adaptability to respond to stress in diverse cellular environments

Hsp70 family

  • different Hsp70 proteins are found in prokaryotic and eukaryotic cytosol , ER , mitochondria and chloroplasts
  • bind to hydrophobic parts of partyly synthesised proteins
  • protect cells from thermal or oxidative stress by preventing protein aggregation
  • binding and refolding is driven by ATP hydrolysis , stimulated by Hsp40 protein

Reaction cycle

  1. Hsp40 delivers unfolded or misfoleded proteins to ATP-bound Hsp70
  2. ATP hydrolysis results in Hsp70 conformational change : the lid closes, leading to tight binding of the substrate and Hsp40 dissociates
  3. NEF (Nucleotide Exchange Factor) binds to Hsp70
  4. ADP dissociates and is replaced by ATP
  5. Lid opens , NEF and substrate are released

Dysregulation of Hsp70

Can cause neurodegenerative diseases:Alzheimers - aggregation of misfolded proteins such as beta-amyloid and tau , lack of Hsp70 means these aren't degraded And in cancer:Hsp70 is abundantly present in cancer , provides malignant cells an advantage by suppressing multiple apoptotic pathways

GroEL/GroES and Hsp60/Hsp10

  • GroEL or Hsp60 - 14 x 60kD subunits ( chaperonin)
  • GroES or Hsp10 - 7 x 10kD subunits (co-chaperonin)
  • found in eubacteria , mitochondria and chloroplast
  • very abundant and non-specific
  • can intercact with most non-native proteins
  • provides a box for proteins to safely fold inside
  • protein structure is determined solely by its amino acid seqeuence not by a chaperonin

Chaperonin cycle

  1. GroEL binds to ATP and captures an unfolded or misfolded substrate protein
  2. GroES associates with the open end of the cylinder creating a closed space
  3. the substrate protein undergos folding in the protected chamber
  4. ATP is hydrolyzed causing the release of GroES and the substrate protein

Similarities

  • same chaperonin function
  • same complex formation
  • both ATP-dependent mechanisms
  • both undergo conformational changes that are essential for the chaperonin cycle

Differences

Location GroEL/GroES is present in the cytoplasn of bacteria Hsp60/Hsp10 is present in the mitochondria of eukaryotic cells

Misfolded protein - not in its natural form

Protein trafficking

Eukaryotes

more complex problem than for prokaryotesproteins need to be sent to the right environment

  • proteins carry codes in their sequences that are ready by targetting machinery at every stage
  • proteins targeted to cytosol, mitochondria , peroxisomes or chloroplasts are synthesized on free ribosomes
  • proteins destined for secretion , the ER , golgi , lysosomes or any membrane are syntehsized on membrane bound ribosomes in the RER and then targeted to the correct location

Signal sequences

proteins are directed to the ER by a signal sequence , a stretch of 9 or more hydrophobic residues the signal sequence is recognised by the signal recognition particle (SRP)SRP binds to SRP receptor in the ER and protein is transported in where the sigal sequence is then removed

Diseases

  • mutatn cargo proteins fold inefficiently and fail to exit the ER causing a loss of function phenotype
  • misfolded porteins are detectd and degraded by the ubiquitin proteasome system

Example : Cystic Fibrosismajority of CF patients have a missing Phe (F508-CFTR) folding defect inhibits transport to the golgi . Instead the misfolded mutant protein is targeted to the ER assisted degradation pathway

Protein trafficking

Prokaryotes

  • either secreted to cytoplasm , cytoplasmic membrane , cell wall or extracelllar environment
  • cell wall is made of peptidoglycan
  • gram-negative bacteria ( two membranes , thin cell wall)
  • gram-positive ( one membrane , thick cell wall)

Sec-dependent pathway

  • most common and is found in gram-postive and -negative bacteria , archaea and eucarya
  • translocates proteins across the membrane or integrates them into it
  • signal peptide at the begging of a protein when it is synthesized shows protein is to be exported

  • SecB proteins (chaperones) binds to preprotein to keep it from folding too soon
  • SecA protein binds to the SecY/SecE complex of proteins and translocates the preprotein through the plasma membrane
  • driven by ATP hydrolysis
  • on the other side of membrane the signal peptide is removed by the enzyme signal peptidase
  • the protein then folds into its proper shape and carriers out its function

In Eukaryotes

Protein modifications

Covalent addition to side chains

Cleavage of protein backbone at a specific site

5 most common ways to modify a side chain

  1. Phosphorylation
  2. acylation
  3. alkylation
  4. glycoslyation
  5. oxidation
  6. Methylation
  7. Hydroxylation

Phosphorylation in PTM

  • usually to an OH ( ser,thr,tyr)
  • occasionally to His , Asp ,Lys
  • very common
  • about 1/3 of proteins can be phosphorylated by kinase enzymes to change their function
  • many drugs act on kinases
  • known to be important to functional activity of proteins involved in intracellular signalling

  • phosphate group can add one or two negative charges
  • forms strong non-covalent bonds especially with +ve groups
  • Protein structure often changes after phosphorylation
  • can simply block access to a binding site

Structural changes caused by phosphorylation in cysteine string

  • conversion of a neutral Ser10 OH to PO3^2- by PKA or PKB kinase induces conformational change , forming a new salt bridge to Lys58
  • neurotransmitter release affected

Acetylation

Acetylation of lysines in histones or the p53 transcription factor control gene transcription - epigenetics

15 Lys residuesin the N-terminal tails of H2A,H2B,H3, and H4 that are sites for possible enzymatic acetylation

N-terminal Acetylation and why it occurs

  • most eukaryotic proteins are N-acetylated
  • takes place during protein synthesis and is irreversible
  • its poorly understood why it happens but it is thought that it is to protect against degradation
  • for example : blocking the N-terminus by acetylation prevents ubiquitin being added there instead
  • however the opposite has been found in yeast
  • N-acetylation also prevents post-translational translocation throguh the ER membrane so could be used for targetting protein to membranes

Methylation

  • usually on Lys or Arg
  • forcing Lys to be positive all the time instead of maybe being neutral , makes it bind more tightly to DNA
  • often on histone proteins (bound to DNA in chromatin) to control gene expression
  • Acetylation can have the opposite effect
  • S-adenosyl methionine co-factor (SAM) commonly used

Parathyroid gland

  • 5% of people > 4 glands
  • PT gland derived from pharyngeal pouch endoderm driven by Gcm-2
  • Gcm-2 is also expressed in pharyngeal pouches => internal gill buds in zebrafish and dogfish
  • Gcm-2 required for gill bud formation in zebrafish
  • thus PT glands are in the neck because they are derived from gill

Parathyroid hormone (PTH)

  • produced in chief cells of parathyroid gland
  • 84 aa hormone
  • PTH secretion is inversely proportional to serum Ca2+
  • low plasma Ca2+ = increase in PTH secretion
  • feedback regulation of PTH secretiion via Ca2+-sensing receptor (CaR)

PTH elevates plasma Ca2+ levels by :increasing bone resorptionincreasing renal Ca2+ reabsorption increasing production of 1,25(OH)2D3 (vitD)

Ca2+ - sensing receptor (CaR)

  • CaR is a GPCR responsive to Ca2+ (and Mg2+)
  • CaR is also present in kidney where it limits calcium reabsorption
  • CaR monitors blood Ca2+ levels continuously and serves as the ultimate control point for Ca2+ homeostasis

Increasing bone resoprtion

  • PTH binds to receptors on the surface of osteoblasts and osteocytes
  • promotes the differentiation and activation of osteoclast precursors into mature osteoclasts
  • PTH mobilizes calcium from the bone tissue when blood calcium levels are low through resorption

Osteoporosis

Increasing renal calcium reabsorption

  • PTH binds to its receptors on the renal tubular cells in the distal convoluted tubules
  • stimulates the renal tubular cells to increase the reabsorption of calcium from the urine
  • PTH promotes the reabsopriton of calcium while inhibiting the reabsorption of phosphate
  • this effect helps maintain an appropriate balance between calcium and phosphate levels in the blood
  • stimulates the kidneys to convert inactive vitamin D into its active form which enhances the absorption of calcium in the intestines

Renal faillure and Calcium stones

Increased production of 1,25(OH)2D3 (vit D)

  • Vit D increases net intestinal Ca2+ uptake
  • it increase serum Ca2+ levels by increasing bone resorption and renal Ca2+ reabsorption (as for PTH)

RICEKTS

Intestinal Ca2+ absorption

TRPV makes a calcium sized pore high calcium levels is a death signal so you have calcium binding protein (CaID9K) that shuffels calcium along the cell so free ionized caclium levels never go up too much

  • 90 % of dietary calcium absorption occurs in the small intestine via 2 ways
  • passive - paracellular diffusion down its electrochemical gradient
  • active transcellular transport under the control of vit D ( most calcium reabsorbed this way
  • TRPV6 predominant

Calbindins

Calbindin-D28K - mainly present in kidneys Calbindin-D9K - expressed primarily in gut

calbindin binds to calcium so theres no free calcium in the cellcalbindin expression is primarily independent on vitamin D

Calcitonin

  • secreted from parafollicular thyroidal C cells
  • half life is 5 minutes

  • CT secretion decreased plasma Ca2+ following a Ca2+ load
  • decrease osteoclast activity => decrease bone resorption => allows rapid bond deposition

  • CT secretion also rises post-prandially ( after a meal) as blood calcium rises (gastrin may be involved in this secretion)
  • it is inhibited by LOW calcium levels in the blood
  • contirbution of CT to mammalian calcium homeostasis is very modest and alot more important in fish

Diseases of Calcium homeostasis

  • Primary hyperparathyroidism
overproduction of PTH elevated levels of PTH result in increased calcium release from bones , increased calcium absorption from the intestines and reduced calcium excretion by the kidneys leading to high blood calcium levels
  • Secondary Hyperparathyroidism ( renal failure)
occurs as a response to chronic kidney diseasekidneys are unable to properyl regulate calcium and phosphorus levelsleads to low blood calcium levelsin response more PTH is released to increase calcium levels , leading to bone resorption
  • osteoporosis
weakened bones and decreased bone density excessive brone breakdown
  • rickets (vitD deficiency )
insufficeint calcium absorption in the intestines results in low blood calcium levelsPTH increases , leading to increased bone resoprtioncharacterized by soft and deformed bones
  • calcium stones
excessive calcium in the urine can contribute to formation of kidney stones
  • receptor mutations PTH receptor , CaR
genetic mutations leading to impaired sensing of calcium levels or altered responsivenss to PTH

Thyroid gland

  • consists of multiple thyroid follicules which consist of a cavity in the centre surrounded by a single cell of epithelial cells held together on a basal lamina
  • thyroid follicle is one cell thick
  • parafollicular c cells - responsible for secreting calcitonin

  1. Regulation of thyroid gland function
  2. TSH and its structure
  3. effects of TSH on thyroid gland function
  4. effects of TSH on thyroid follicle
  5. synthesis of T3 and T4
  6. abnormalities in thyroid hormone balance

Thyroid hormones

TSH and it's structure

\

  • Thyroid Stimulating Hormone
  • Secreted by the anterior pituitary
  • composed of a and b subunits
  • alpha subunit is identical for all four glycoprotein hormones within a single species

  • the beta subunit imparts a uniquie biological specificty to each hormone
  • beta subunit has glycoproteins attached to them
  • CHO side cahins determine the stability and bioligical acitivity

Subtitle

Effects on thyroid gland

Opposite occurs for high TSH:

  1. adenylate cyclase activation
  2. cAMP activation of protein kinase A
  3. iodide uptake and transport
  4. thyroglobulin synthesis
  5. thyroglobulin iodination
  6. differentiation of apical surface pseudopodia
  7. thyroglobulin resorption
  8. thyroglobulin digestion
  9. thyroid hormone release to circulation

Effects of TSH on thyroid follicle

Synthesis of T3 and T4

  • thyroid peroxidas (TPO) enzyme complex spans the apical membrane
  • responsible for iodination of thyroglobulin (Tg)

  • usually occurs in a 90% T4 to 10% T3 ratio
  • the thyroglobulin backbone needs to be removed to make the enzyme active
  • Tg with the T3 or T4 attached is endocytosed and undergos proteolysis which frees T3 and T4

  • It will break down any tyrosines that haven't been attached to an iodine and they will also be recycled

T3 = tri-iodothyronineT4 = thyroxine

Thyroid disorders

hypothyoidism

development of a goitre in severe iodine deficiency

hyperthyroidism

why does a goitre develop in severe iodine deficiency ?

  • iodide is actively concentrated in the thyroid gland , salivary glands and many more
  • most iodide in the thyroid gland is in the form of iodothyronines
  • of the 1% released from the gland daily , about 75% is secreted as a thyroid hormone and the remainder as free iodide
  • thyroid becomes overactive desperately trying to extract iodide from the blood
  • no break on the anterior pituitary gland to stop the production of TSH
  • this will cause the thyroid gland to increase in size due to its attempt to produce mroe thyroid hormones and lead to a goitre

Hypothyroidism

(thyroid hormone DEFICIENCY)

Potential causes

  • inadequate dietary iodide
  • unresponsive thyroid
  • inhibition of TPO by goitrogens ( food rich in thiocyanate)
  • impaired conversion of T4 to T3 in target tissues
  • Iatrogenic ( surgery or radiotherapy)
  • autoimmune destruction of the thyroid gland

  • overweight
  • hairloss
  • sleepiness
  • cold
  • dry

Characteristics

Treatment

  • restore iodide supply
  • thyroxine tablets (T4)
  • triiodothyronine tablets (T3)

Hyperthyroidism

(thyroid hormone EXCESS)

Potential causes

  • hyperstimulated thyroid gland (TSH-secreting tumour in pituitary -RARE)
  • autonomously-functioning thyrroids (TSH receptor function even in absence of TSH)
  • autoimmune sitmulation of throid by TSH-receptor antibodies (Graves disease - most common)

Characteristics

  • weightloss
  • sometimes a goitre
  • warm
  • tremor
  • osteoporosis

Treatment

  • surgical removal of thyroid galnd - radioactive iodine treatment
  • thioamide drugs - competitively inhibit thyroperoxidase enzymes

Problems with drugs

  • slow result
  • results in hypothyroidism
  • may result in a goitre due to decreased feedback on TSH

Thyroid hormone binding proteins

Thyroxine-binding globulin (TBG)

  • binds 70-75% of plasma T4
  • increased by T4 and by oestrigens/androgens
  • involved in thyroid hormone transport
  • lowered by corticosteroids , illness , stress
  • synthesised in the liver
  • stability and half-life are extended after T4 binding
  • prevents loss of T4 and T3 in urine through binding an therefore helps conserve iodide
  • has the highest affinity but lowest capactiy
  • also binds T3

Transthyretin (TTR)

  • binds 20% of plasma T4
  • important for delivery to CNS
  • has low affinity and high capacity
  • ONLY binds T4

Albumin

  • binds 5-10% of plasma T4
  • lowest affinity but highest capcity
  • also binds T3

Secretion and transport of thyroid hormones

  • T4 and some T3 produced from thyroid gland
  • TBG leads to bound T4
  • transport to peripheral tissue
  • bound T4 and T3 cannot enter cells
  • free T4
  • T3 and T4 enter the cell via specific transporters
  • T3 is biologically active and T4 is inactive
  • conversion of T4 to T3 by intracellular iodothyrine deiodinases (DIO)
  • metabolic effect

Thyroxine (T4)

  • hydrophobic
  • insoluble i nserum
  • can only be transported in serum with binding proteins that are synthesised in the liver
  • liver disease can lead to loss of effective T4 transport to peripheral tissue

iodothyronine deiodinases (DIO)

DIO1

DIO2

DIO3

  • preodminates in liver , kidney and muscle
  • also found in thyroid
  • produces most of circulating T3

  • predominates in areas of the CNS. pituitary thyrotropes
  • controls intracellular T3 conc
  • important for feedback regulation
  • found in skeletal muscle in somes species

  • produces inactive rT3
  • prevents thyroid hormone access to specific tissue

Thyroid hormone action

  • Thyroid hormone receptor (TR)
  • THRA and THRB produce proteins TRa and TRb which are found in the nucleus
  • here they form heterodimers with retinoid X receptor (RXR)
  • function as transcription factors
  • has a higher affinity for T3 than T4 and can affect gene transcription

5 biological actions

  1. control of basal metabolic rate
  2. growth-regulating roles of thyroid hormones
  3. role of thyroid hormone in foetal development
  4. cardiovascular effect
  5. musculoskeletal effects

secretion and transport

T4

control of basal metabolic rate

growth-regulating roles

  • increase in Na+ , K+ ATPase
  • increase in mitochondria respiratory enzymes
  • you get an increase in oxygen consumptino which leads to an increase in metabolic rate
  • hyperthyroidism leads to an INCREASE in metabolic rate

  • most bodily functions affected
  • often synergise with other hormones
  • deficienceies lead to abnormal , growth , devlopment , reproduction and metabolism
  • exert effects on all organs and tissues throughout life
  • arrest of bone elongation - delayed bone maturation , reduction in growth hormone secretion

foetal development

cardiovascular effect

musculoskeletal effects

  • thyroid hormones play a key role in developing neural and skeletal systems
  • loss of T4 supply to foetus leads to irreversible intellectual disability and dwarfism
  • if this happens during foetal growth - miscarriage,still births
  • neoneatal and adolescent time - goiter , delayed physical development

  • T3 increases
  • cardiac contraction and output,
  • HR
  • oxygen supply to tissues
  • CO2 removal

  • T3 has a potent stimulatory effect on bone turnover increasing bone formation and resorption
  • it inscreases linear bone growth after birth
  • increases the rate of muscle relaxation
  • normal skeletal muscle function requires T3

Assay of hormones

  • using a given test system to compare the response produced by the hormone in the sample
  • with the response produced by a known conc of the same hormone
  • can plot a graph which can be used to compare other peoples hormone levels to the normal range

IMMUNOASSAY : measure the amount of a given molecular structure or mass

  • immunoassay - competitive
  • radioimmunoassay
  • fluoroimmunoassay
  • enzyme-linked immunoassay
  • immunometric assay - mainly non-competitive
  • ELISA

BIOASSAYS - measure the biological activity

  • ligand binding assay
  • receptor assay

Immunoassay

Immunometric assay

Bioassays

Immunoassay

  • hormone + specific antibody <=> hormone:antibody complex
  • reaction is reversible and will reach an equillibrium state
  • a 'competitve binding assay' as there's competition between labelled (tagged) and unlabelled forms of a hormone for a limiting amound of antibody
  • highly-sensitive , specific , precise and convenient

  • at equilibrium , antibody-bound hormone must be seperated from 'free' hormone using charcoal
  • charcoal is porous but the hormones that are bound can't enter
  • solution is centrifuged so that charcoal goes to the bottom of the tube
  • decant the liquid phase into a new tube - this will contain the antibody-bound hormone
  • determine the amount of radioactivity present
  • if there is high hormone level in a blood sample - lower level of labelled hormone will be bound to the antibody

  • for the results plot a graph of signal against increasing dose of UNLABELLED hormone
  • need it to see the conc of hormone in the serum sample can be collected
  • you need to known the conc of the unlabelled hormone , this is know as assay standards

Immunometric assay

  • non-competitive assay
  • use two different antibodies which bind to different regions (epitope) of a sinngle hormone molecule
  • monoclonal antibdoy is bound to the surface of a test tube
  • antibody will bind to a particular epitope of our horone of interest
  • the second antibody then binds to a different epitope
  • this antibody has been chemically modified to contain a label
  • very specific for the assay

The Enzyme-Linked Immunosorbent Assay (ELISA)

  • capture antibody binds to the target antigen
  • a detection antibody binds to the target antigen causing a colour change of the substrate added at the end of the experiment
  • colour change is measured using spectrophotometry
  • this is a non-competitive assay so the amount of colour change is directly proportional to the amount of hormone present in the sample

ELISA - a competitive version

  • the antibody is fixed to the surface
  • we add a known conc of a lebelled hormone and our sample into the mixture
  • here there is competition to bind to the antibody

Bioassay

measure the magnitude or intensity of a biological effect typicall bioassay effects : cell growth , release of a metabolite , uptake of a radioisotope

In vivo bioassays

  • involve administration of a test or standard hormone to an animal , with quantification of the response
DISADVANTAGES
  • laborious , technically demanding , expensibe , poorly reproducible
In vitro bioassays
  • addition of a test or standard hormon to cell cultures or tissue fragments with quantification of the response
ADVANTAGES
  • cost -effective , robust , reliable , sensitive , precise

Ligand-binding assays

  • hybrid assays
  • isolate the hormone receptors from the whole tissue by fractionation or solubilisation and use this instead of using antibodys
  • procedure is the same as that for a competitive immunoassay

ADVANTAGES

  • uses 'natural' binding site for hormone
  • high affinity , specifity and sensitivity for hormone
  • good precision
DISADVANTAGES
  • labour intensive
  • requires extensive tissue processing
  • usually dependent on availability of animal material

Adrenal glands

Steroid biogenesis

  • cholesterol is the main precursor
  • rate limiting step is the conversion of cholesterol to pregnenolone
  • cholesterol is brough to the tissue through the LDL receptors on the tissue
  • natural steroid synthesis depends on delivery of cholesterol to the tissue

Synthetic steroids

Experiments and research

  • Assay of hormones

Corticosteroids

Genomic mechanism of action of steroids

Non-genomic mechanism of action of steroids

  • cortisol is released into the blood stream bound to globulins
  • in cell receptor is complexed with heat shock protein
  • binding of cortisol to receptor displaces HSP
  • steroid-receptor complex migrates to nucleus and binds to steroid-response element (SRE) on target gene
  • dissociates rapidly and alters gene transcription/translation

  • relates to rapid response to stress
  • binds to receptors in the plasma membrane - GPCR and activates kinases
  • results in decreased excitability or secretion of ions

Glucocorticoids

  • controls metabolic effects
  • has anti-inflammatory effects
  • have immunosuppressive effects

Release

  • stress,excitement , changes in temp - stimulate release
  • hypothalamus releases corticotropin releasing hormone (CRH)
  • acts on the anterior pituitary to release (ACTH)
  • this acts on the adrenal cortex which then releases cortiosl
  • cortisol has a negative feedback system

Anti-inflammatory effects

Metabolic effects

  • permissive action - indicates to catecholamines to exert lipolytic effects , indicates to glucagon to exert calorigenic effects
  • increase gluconeogenesis particularly in the liver
  • increases storage of glycogen in liver and muscle
  • reduction in protein stores
  • increase liver and plasma amino acids and proteins

  • reduces release of proteolytic enzymes - stabilising lysosomal membranes
  • reduces oedema - decreasing permeability of capillaries
  • reduces prostaglandins - decreasing migration of WBC
  • reduction in T cells - suppresing the immune system
  • reduces production of IL-1 - reduces fever

endogenous glucocorticoid = cortisol

Cortisol

Excessive cortisol release = cushings syndrome

Inadequate cortisol release = Addisons disease

Cushings syndrome

  • tumour of the pituitary gland , adrenal glands
  • excessive cortisol release

  • glutocorticoid receptors (GR) antagonists counteract the effects of cortisol at the tissue level by blocking the GR
  • mifepristone is the only GR antagonsit for clinical use
  • dose adjustment is difficult and mifepristone is also a progesterone receptor antagonist
  • relacorilant is a new GR antagonist that selectively binds to GR without anti-progesterone receptor effects and is in trials now

Treatment

Symptoms

  • purple stretch marks on the abdomen area
  • weight gain in the trunk and face
  • thin , frail skin that bruises easily

Addisons disease

Treatment

  • hydrocorticsone , predninsone or methylpredninsolone are used to replace cortisol
  • hormones are given on a 24 hour schedule to mimic the circadian fluctuation of cortisol levels
  • fludrocortison acetate to replace aldosterone
  • limited side effects as plasma levels mimic natural situation

  • Bulleted list
  • Bulleted list

  • adrenal insufficiency due to a disorder of the adrenal glands themselves
  • inadequate secretion of ACTH by pituitary gland
  • means inadequat levels of cortisol

Symptoms

Mineralcorticoids

  • spironolactone acts as a competitive inhibitor of MC receptors
  • diuretic and antihypertensive effects
  • increased salt retention causes increased blood pressure

aldosterone effects in renal tubular epithelial cells

  • incerases absorption of Na+
  • increases secretion of K+
also controls Na+ and K+ transport in
  • salivary glands
  • sweat glands
  • intestines

Control of release

MC receptors

  • increased release in response to
  • increased K+ in kidney tubules
  • increased activity of renin-angiotensin syste
  • ACTH ( necessary but doesn't affect rate of release)
  • increased Na+ in kidney tubules ( much smaller effect)

  • genomic and non-genomic effects - similar to GCs
  • genomic effects mainly through
  • increased Na+ channel proteins and Na-K-ATPase in renal tubular cells
  • Na+ channel proteins insertion into luminal side
  • pump that exchanges Na+ and K+ in basolateral membrane
  • more limited expression of MC receptors

Synthetic steroids

  • selectivity depends on structure
  • hydrocortisone (short acting) has equal GC/MC activity
  • prednisolone , prednisone (intermediate acting)- mixed GC/MC activity
  • dexamethasone , betamethasone ( long acting)- pure GC activity
  • fludrocortione ( short acting) - mainly MC activity

  • can alter certain groups to change the balance of activity
  • also to influence potency

Growth hormone (GH)

  • part of a cluster of 5 closely related genes
GH-N - expressed in pituitary gland
  • gives rise to a peptide hormone
  • is synthesised as a precursor protein
  • N terminal signal peptid is cleaved when secreted
  • secreted in pulses ( more pronounced in males than females )
GH-V - expressed in placenta (important for pregnancy)

GH receptor

  • homodimer that exists in the plasma membrane
  • GH binding induces conformation change
  • activation of JAK2 phosphorylates the GHR and this allows transcription factor stat5 to bind
  • stat5 then gets phosphorylated and becomes activated
  • GHBP is the extracellular portion of the GHR

GH and IGF-1 actions

Growth plate struture

Reserve zone

  • small clusters of porgenitor cells which are sat in a matrix of collagen and proteoglycans
  • provides cells for the proliferative zone
Proliferative zone
  • massive expansion of chondroblasts
  • organise themselves into columns

Maturation zone

  • cells differentiate into chondrocytes where they secrete a matrix
Hypertrophic zone
  • grow in size and eventually go through apoptosis
  • as long as there are cells in the reserve zone , growth can happen and it happens at the growth plates on both ends

In metabolism

  • GH acts on the GHR in adipose tissues and stimulates lipolysis
  • IGF-1 stimulates lipogenesis
  • GH and IGF-1 stimulates muscles to take up amino actis and then to convert them to proteins and stimulate protein synthesis
  • in liver GH increases glucose output and stimulates gluconeogenesis increasing levels of glucose in the circulation
  • IGF-1 stimulates uptake of glucose into muscle and tissues to reduce glucose levels in circulation

Intracellular signalling

  • both protein hormones so theyre hydrophillic so can't cross the plasma membrane
  • work through enzyme-coupled receptors

IGF axis

IGF-BP

  • IGFBP-3 main IGFBP in circulation - storage of IGF
  • ALS used to prolong IGF halfllife
  • IGFBP has much higher affinity for IGF then IGF has for receptor
  • inhibits IGF action as it stops IGF from binding to receptor
  • proteolysis is the main way of releasing IGF from the BP

Hormone excess

  • there can be issues with the hypothalamic prodution of GHRH
  • issue with GH gene
  • can lead to short stature or adiposity
  • there could be issues with IGF-1 or IGF receptor

  • usually due to a tumour in the anterior pituitary causing too much GH
  • if tht occurs before the growth plate closes it leads to gigantism
  • if it occurs after the growth plast closes it leads to acromegaly
  • treatment - surgery or GH receptor antagonists

Hormone deficiency

Glycosylation

common monosaccharide added is GlcNAc many proteins are anchored to the lipid membrane by GPI groups GPI groups consist of an array of mannose,galactose , galactosamine, ethanolamine and phosphatidyl inositol groups

O-glycosylation

  • adding gylcans (oligosaccharides) to protein oxygens
  • usually Ser,Thr or Tyr
  • can cause branching and create complex polysaccharides

N-glycosylation

  • more common than O-glycoslyation
  • takes place in ER
  • sugars are attached to Asn in Ser/Thr - X - Asn sequence motif
  • complex saccharides are added to proteins destined for secretion in the ER and golgi
  • interact with chaperone proteins to help folding
  • if they don't fold properly they will be degraded
  • mature glycoproteins on cell surface can be found with huge diverstiy in mature N-glycan chains

Hydroxylation

  • hydroxylated amino acid residues
  • all use Fe(III) monoxygenase enzymes to add O
  • 5-OH-Lys usually then glycoslated

very commonn in collagen

  • main protein in bone,skin,ligaments etc.
  • it is a triple helix structure made out of hydroxypoline
  • requires Vitamin C which oxidises Pro to Hyp - this stabilises the triple helix structure
  • hydroxylation of proline residues stabilises collagen by favouring additional H bonding
  • in its absence microfibrils are weaker

also seen in Asparagine

  • 3-hydroxyasparagine
  • Asn is hydroxylated in transcription factor HIF
  • initiated when O2 pressure is low
  • induces transcription of hundreds of genes
  • popular with cheating distance athletes as it increases level of red blood cells to carry more oxygen

Alkylation

  • addition of alkyl groups
  • transfer of methyl or ethyl groups to reactive sites on bases or to phosphates in the DNA backbone
  • most commonly occurs at cysteine residues
  • can lead to wrong base pairing
  • if it itsn't corrected it can cause DNA replication to be incorrrect

- Alkylation is an essential step in many protein analysis techniques, - it helps to preserve the native structure of proteins during experiments.- it aids in accurate and reproducible mass spectrometric analysis by preventing artifacts related to cysteine reactivity.

  • primary amino acids susceptible to oxidation are cysteine, methionine, tyrosine, and tryptophan.
  • cysteine residues can undergo oxidation as part of their formation of disulphide bonds
  • reactive oxygen species , generated by ionising radiation and by chemical agents
  • OxoG can wrongly base pair with adenine - most common mutation in cancer

Oxidataion

Disulphide bonds

  • oxidation reaction between cysteine residues
  • cytoplasm and nucleus are reducing environments - SS bonds are rare
  • conditions become more oxidising passing through secretory pathway
  • extracllular proteins usually have SS bonds
  • oxidation of dithiols to disulphides by oxygen
  • to get reduction to happen you need an enzyme (disulfide oxidoreductases) and NADPH
  • covalent bond restricts confromational mobility in a protein and normally occurs between residues widely separated in the primary sequence

Protein disulfide isomerase (PDI)

  • a chaperone enzyme
  • disulphides first form randomly in the ER so usually the wrong Cys side chains pair up
  • PDI catalyses the random exchange of SS bonds
  • PDI doesn't know which SS bonds to make - it just randomly swaps them
  • crorect SS bonds are the most stable so you end up with the native folded protein
  • acts as a dithiol oxidase