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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:

thyrotropin-releasing hormone (TRH)
gonadotropin-releasing hormone (GnRH)
growth hormone-releasing hormone (GHRH)
corticotropin releasing hormone (CRH)
somatostatin
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

Somatrotopes - GH - acidophil
Lactotropes - PRL - acidophil
Thyrotropes - TSH - basophil
Corticotropes - ACTH - basophil
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

separation - membranes form a semi permeable diffusion barrier
exchange - membranes allow transport of metabolites and macromolecules in and out of cell compartments
integration - membranes mediate cell to cell communication , cell adhesion and signalling
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 :

length of hydrocarbon tails
degree of unsaturation
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

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

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

donor membrane docking
lipid extraction
donor membane undocking
diffusion
acceptor membrane docking
lipid deposition
acceptor membrane undocking
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

catalytic proteins
binding proteins
signalling proteins
transport proteins
structural p roteins
motor proteins
storage proteins
hormonal proteins
chaperone proteins

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

Hsp40 delivers unfolded or misfoleded proteins to ATP-bound Hsp70
ATP hydrolysis results in Hsp70 conformational change : the lid closes, leading to tight binding of the substrate and Hsp40 dissociates
NEF (Nucleotide Exchange Factor) binds to Hsp70
ADP dissociates and is replaced by ATP
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

GroEL binds to ATP and captures an unfolded or misfolded substrate protein
GroES associates with the open end of the cylinder creating a closed space
the substrate protein undergos folding in the protected chamber
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

Phosphorylation
acylation
alkylation
glycoslyation
oxidation
Methylation
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

Regulation of thyroid gland function
TSH and its structure
effects of TSH on thyroid gland function
effects of TSH on thyroid follicle
synthesis of T3 and T4
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:

adenylate cyclase activation
cAMP activation of protein kinase A
iodide uptake and transport
thyroglobulin synthesis
thyroglobulin iodination
differentiation of apical surface pseudopodia
thyroglobulin resorption
thyroglobulin digestion
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

control of basal metabolic rate
growth-regulating roles of thyroid hormones
role of thyroid hormone in foetal development
cardiovascular effect
musculoskeletal effects

secretion and transport

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

Vertical interactive image

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