L-06 Neurochemistry: Peptides

Front 1

Neuropeptide signaling history

Back 1

Evolutionarily old - hydra uses peptidergic signaling instead of classical and yeast communicate with peptides (alpha-mating factor)

Front 2

Neuropeptide families

Back 2

Families are formed either from different genes (via gene duplication and divergence) or from related sequences within same gene product due to splicing.

Front 3

Opioid peptide gene family members

Back 3

1. POMC

2. Prodynorphin

3. Proenkephalin

4. Proorphanin/FQ

Front 4

Evolution of RF-Amide subfamilies

Back 4

Useful motif (arginine-phenylalanine) was useful. Gene duplication lead to different precursors with C terminus RF with other sequences that bind to receptors. Involved in various functions including signaling, pain, reproduction.

Front 5

Neuropeptide size

Back 5

Less than 10 kD

Front 6

Neuropeptide and convential transmitter relationship

Back 6

Both are found at majority of CNS synapses.

Can be in same vesicle or different ones.

Peptides can modulate conventional signaling

Front 7

LHRH modulation of ACh activity

Back 7

ACh has rapid action and does not diffuse far because rapidly broken down and removed.

LHRH diffuses further than ACh and acts on receptors at more distanct postsynaptic terminals - influences activity at many terminals.

Front 8

Strength of neuronal activity and type of vesicle released

Back 8

Single pulse releases small vesicles with classical transmitter

High frequency pulse releases large dense core vesicles with peptide neurotransmitters (and some classical neurotransmitters).

Front 9

Endogenous opioid peptides

Back 9

Discovered using assay of ileum in water bath. Shortens when electrically stimulated.

Apply fractionated spleen extract - inhibits electrically stimulated contraction.

Found Met-enkephalin and Leu-enkephalin, which have same sequence except terminal C terminus amino acid.

Front 10

Radioreceptor assay

Back 10

Also used to discover endogenous opioid peptides. Stereo-specific assay

Radiolabel drug and bind to brain membranes. Add fractionated brain extracts and follow fractions able to displace radioactive stereoisomer from membrane (eliminate non-specific binding).

Front 11

Correlation between guinea pig ileum contraction assay and radioreceptor affinity assay

Back 11

Perfect correlation

Both are measuring opioid receptor

Front 12

Opioid peptides

Back 12

Beta-endorphin

Met-enkephalin

Leu-enkephalin

Dynorphin (extended leu-enkephalin)

alpha-neo-endorphin

Share common Tyr-Gly-Gly sequence (business end) and other end which directs peptide to specific receptors.

Front 13

Structure of "business end" of opioid peptides

Back 13

Resembles morphine in space filling model

Explains why humans have receptor for plant compounds

Front 14

Endorphin

Back 14

Entire class of endogenous morphines including enkephalins

Front 15

Enkephalin precursors

Back 15

Preproenkephalin B arose from duplication of preproenkephalin A.

Preproenkephalin A has 4 met-enkephalins and two extended enkephalins (Arg-Gly-Leu, Arg-Phe)

Preproenkephalin has two Leu-enkephalins.

All are bounded by basic pairs.

Front 16

Precursor of beta-endorphin

Back 16

C-terminus of POMC

Betaendorphin starts with Tyr-gly-gly but no paired basic after Tyr-gly-gly-phe-met so not processed to met-enkephalin

But active in opioid receptors because tyrosine is liberated (free tyrosine = opioid activity)

Front 17

Brain distributions of beta endorphin and enkephalin

Back 17

Majority of endorphins are in hypothalamus and pituitary

Majority of enkephalins are in inner neurons of striatum

But, opioid systems are present in many nuclei and in dopaminergic system.

Front 18

Neuropeptide trafficking

Back 18

Begins in cell body. Secreted protein goes through ER, through golgi stacks. Packaged into vesicles at trans-Golgi and then shuttled to terminal.

Takes long time to synthesize and reach docked stage

Front 19

Regulation of neuropeptide expression

Back 19

Transcriptional

Activity at synapse sends signal to cell body.

Transcription factors such as CREB are phosphorylated to add additional precursors or enzymes.

Front 20

Regulated secretory pathway of neuropeptides

Back 20

Few post-translational modifications. Commonly amidated in trans-golgi.

Cleaved at trans-golgi and granules by prohormone convertases 1/3 and 2. These enzymes are not present in organs that do not do neuropeptide signaling (e.g. liver).

Front 21

Neuropeptide precursor maturation

Back 21

Pairs of basic residues between final neuropeptides are cleaved.

Carboxypeptidases get rid of overhanging bases at C terminus.

In half of neuropeptides, glycine of pair of bases is turned by amidating enzyme into an amide group that provides stability and may engage in signaling.

Front 22

Can a neuropeptide precursor generate peptides acting at different places?

Back 22

Yes

POMC generates ACTH (adrenal medulla) and beta-endorphin (opioid receptors).

Front 23

Tissue-specific processing of POMC

Back 23

In anterior pituirary, PC1/3 are only present to make ACTH

In intermediate pituitary, PC2 is present which is not as specific as PC1/3. Cleaves ACTH into alpha MSH.

Beta-endorphin can be acetylated to remove exposed tyrosine, inactivating.

Front 24

Neuropeptide GPCRs

Back 24

Big peptides bind to extracellular N terminus
Smaller peptides bind to extracellular loops between domains

Front 25

Opioid receptor analogousness

Back 25

Mu, kappa, delta

Mostly same in transmembrane areas

Different in G protein coupled C terminus and ligand binding N terminus

Front 26

Correlation between enkephalin and opiate receptors

Back 26

Opiate receptors are present where enkephalin is present.

Lack of correspondence between mRNA and opiate receptors because mRNA is located in cell body, not nerve terminal.

Front 27

Ligands for opioid rceptors

Back 27

Mu - binds mostly beta-endorphins

Delta - binds mostly met-enkephalins

Kappa - binds mostly dynorphins

Each can also bind other opioids.

All are GPCRs, linked to inhibition of adenylate cyclase.

Each has subtypes as well.

Front 28

Opioid precursor processing and receptor affinity

Back 28

If process into larger peptides (PC1/3 generated), work on mu receptor. If smaller peptides (PC2 generated), work on delta opioid receptor.

Front 29

Opioid receptor function

Back 29

Mu reeceptors - analgesia, respiratory effects

Delta receptors - analgesia in spinal cord

Kappa receptors - analgesia in spincal cord and psychoactive effects

Front 30

Ways to generate diversity in neuropeptide action

Back 30

1) Genetic - from various families

2) Splicing variants - transcriptional

3) Processing variants - differential processing

4) Receptor variants and heterodimers

5) Receptor modulators

Front 31

Tracts that mediate pain transmission

Back 31

Spinoreticular tract - conveys information from medulla to mid brain to cortex.

Spinomesenphalic tract - similar pathway but terminates in periaqueductal grey.

Front 32

Ascending pain pathway

Back 32

Afferents with cell bodies in DRG release excitatory neurotransmitters - substance P, CGRP, and glutamate

Enkephalin on interneurons inhibit release of neurotransmitter.

Front 33

Cone snail toxins

Back 33

Blocks flow of Ca2+ required for fusion of vesicles and transmission of pain information.

Blocks pain transmission to higher center through tracts.

Front 34

Presynaptic inhibition of signaling by opioid peptides

Back 34

Interneuron releases enkephalins which binds to opioid receptors to modulate release of excitatory neurotransmitter i.e. substance P

Front 35

Imaging changes in patients with central neuropathic pain

Back 35

Observe reduced binding of radioactive substance in thalamic and cortical areas - opiate signaling is reduced.

Front 36

Levels of substance P and cGRP in arthritis

Back 36

Treat animal with experimental arthritis.

Stain dorsal horn for excitatory neurotransmitter.

Glutamate, Substance P, and cGRP is increased compared to control.

Front 37

Migraine therapeutic

Back 37

Inject antibodies against cGRP to nerves in subarachnoid space to relieve migraines

Peptides haven't been successful because those that relieve pain are addictive.

Front 38

Enkephalinases

Back 38

Neuropeptidases in synaptic cleft are cleaved by exopeptidases (neprilysin, aminopeptidase N)

Front 39

DENK inhibitors

Back 39

Dual enkiphalase inhibitors

Block action of neprilysin and aminopeptidase, increase concentration of enkephalins so it inhibits substance P.

Treat intractable pain.

Front 40

Difference between classical and peptide transmitters

Back 40

Much lower concentrations released of peptide transmitters

Peptides synthesized in cell body and released from large dense-core vsicles.

Peptides act slower

Never reuptaked and reused.

Always GPCR receptor.