1. Molecular Basis of Peptide Hormone Production
Understanding Regulation of Hormone Levels How to Make a Peptide: Basic Steps Cell Structures Involved in Peptide Production Gene Structure and Transcription Processing of RNA Transcripts Translation of mRNA into Peptide Post-translational Processing of Peptides Secretion of Peptide Hormones

2. Peptide/protein hormones
Range from 3 amino acids to hundreds of amino acids in size.
Often produced as larger molecular weight precursors that are proteolytically cleaved to the active form of the hormone.
Peptide/protein hormones are water soluble.
Comprise the largest number of hormones– perhaps in thousands

3. Peptide/protein hormones
Are encoded by a specific gene which is transcribed into mRNA and translated into a protein precursor called a preprohormone
Preprohormones are often post-translationally modified in the ER to contain carbohydrates (glycosylation)
Preprohormones contain signal peptides (hydrophobic amino acids) which targets them to the golgi where signal sequence is removed to form prohormone
Prohormone is processed into active hormone and packaged into secretory vessicles

4. Peptide/protein hormones
Secretory vesicles move to plasma membrane where they await a signal. Then they are exocytosed and secreted into blood stream
In some cases the prohormone is secreted and converted in the extracellular fluid into the active hormone: an example is angiotensin is secreted by liver and converted into active form by enzymes secreted by kidney and lung

5. Relation of Hormone Production to Regulation of Hormone Levels
Endocrine feedback is dependent upon the level of hormone available to act on the target tissue, and the number of receptors for that hormone in the target tissue.
The amount of available hormone is determined by several factors:
- rate of hormone synthesis
- rate of hormone release (from endocrine gland)
- presence of binding proteins in blood
- speed of degradation/removal (circulating half-life)
Today will study how peptide hormones are synthesized

6. What are the Basic Steps in Making a Peptide Destined for Secretion from the Cell?
gene for peptide (DNA)
transcription
primary RNA transcript
post-transcriptional
modification
messenger RNA
translation
prepeptide/prepropeptide
post-translational
modification
mature (active) peptide
secretion

7. Peptide/protein hormone synthesis

8. Protein and Polypeptide Hormones: Synthesis and Release

9. Protein and Polypeptide Hormone Receptors
Binds to surface receptor
Transduction
System activation
Open ion channel
Enzyme activation
Second messenger systems
Protein synthesis

10. Peptide hormones
Amino acids/ modified amino acids/ peptide/glycoprotein or protein
The receptors are on the plasma membrane
When hormone binds to receptor
Activates an enzyme to produce cyclic AMP (cAMP)
This activates a specific enzyme in the cell, which activates another………and so on
Known as an enzyme cascade

11. Peptide hormones:
Each enzyme can be used over and over again in every step of the cascade.
So more and more reactions take place.
The binding of a single hormone molecule can result in a 1000X response.
Fact acting, as enzymes are already present in cells.

12. Amplification via 2nd messenger

13. Why so many steps?? At each step, you can get:
- regulation: you can control whether you proceed to the next step or not
- variation: you can change not only whether or not a step occurs, but the way in which it occurs. This can result in production of peptides with different activities, from a single gene.
Example: By regulating how luteinizing hormone is glycosylated (post-translational modification step), you can create LH molecules with different biological activities.

14. Gene Transcription: The Structure of Nucleic Acids and Genes
The genetic information for protein structure is contained within nucleic acids
Two types: DNA and RNA
The basic building block is the nucleotide
phosphate group + sugar + organic base
In RNA the sugar is ribose, in DNA its deoxyribose
PO4 + ribose + organic base = RNA
The organic bases are adenine, guanine, cytosine, thymine (DNA only), and uracil (RNA only)
DNA is double-stranded, RNA is single-stranded

15. The Structure of Genes
A eukaryotic gene encodes for one (or more) peptides and is typically composed of the following:
intron
exon
5’-flanking region
CAT
CRE ERE TATA
BOX
regulatory
region
Transcriptional region

16. Regulation of Transcription by Regulatory Regions
In the 5’-flanking region reside DNA sequences which regulate the transcription of gene into RNA
Examples:
- TATAA box: bases upstream from initiation start site. Binds RNA polymerase II. Basic stuff required for transcription.
- CCAAT (CAT) box: binds CTF proteins
- Tissue-/cell-specific elements: limit expression to certain cell types
- response elements (enhancers): allow high degree of regulation of expression rate in a given tissue (ie, steroid response elements, cAMP-response element [CRE])

17. Transcriptional Regulation by Cyclic AMP
Some hormones bind to their receptor and increase cellular levels of cyclic AMP.
Cyclic AMP activates protein kinase A, which phosphorylates cyclic AMP response element-binding protein (CREB)
CREB binds to a response element on the 5’flanking region of target genes, turning on their transcription.

18. Transcriptional Regulation by Cyclic AMP
protein kinase A
mRNA
protein
CREB P
pCREB

19. What is Transcribed into RNA?
Both exons and introns are transcribed into RNA.
Exons contain:
- 5’ untranslated region
- protein coding sequence
- 3’ untranslated region
Why bother with introns?
- allows alternative splicing of RNA into different mRNA forms (stay tuned…).
- introns may regulate process of transcription

20. Post-transcriptional Processing
Three major steps:
- splicing of primary RNA transcript: removal of intronic sequences
- Addition of methyl-guanine (cap) to 5’-UT
- Addition of poly-A tail to 3’-UT(at AAUAA or AUUAAA)
exon
methy-G-
-AAAAAAA...

21. Alternative Splicing
By varying which exons are included or excluded during splicing, you get can more than one gene product from a single gene:
exon
Normal Splicing
RNA
exon
Alternative Splicing
(occurs in nucleus)

22. Regulation of mRNA Stability
In general, mRNA stability is regulated by factors binding to the 3’- untranslated region (3’-UT) of mRNAs.
The 3’UT often has stem-loop structures which serve as binding sites for proteins regulating stability.
5’ UT
coding region
3’ UT
AAAAAAAA...
binding protein
This regulation occurs in the cytoplasm.
Example: Inhibin acts on pituitary to decrease FSH synthesis and release.
Part of inhibin’s effects reflect decreased stability (half-life) of FSHb subunit mRNA.

23. Translation
Translation from mRNA into protein occurs in ribosomes (RER, in the case of peptide hormones)
Codons of RNA match anticodons of tRNA, which bring in specific amino acids to ribosome complex
Example: AUG = methionine (first amino acid; translation start site)
Other “special” codons: UAA, UAG, UGA = termination codons (translation ends)
At end of translation, you get a prehormone, or preprohormone.

24. Translation -...AUGGAGGAC... -...AUGGAGGAC... -...AUGGAGGAC... MET GLU
mRNA on ribosome
ASP
-...AUGGAGGAC...
MET
GLU
ASP
-...AUGGAGGAC...
MET
GLU-

25. Protein Sorting: Role of Post-translational Processing
How does a cell know where a translated peptide is supposed to go?
plasma membrane
mitochondria,
other organelles
nucleus
export from cell
50,000 proteins
produced

26. Signal Sequences
At the amino terminus of the prepeptide, there is a signal sequence of about amino acids, which tells the cell to send the peptide into the cisterna of the endoplasmic reticulum.
Inside the ER, the signal sequence is cleaved off.
Thus, the first amino acids translated do not encode the functional peptide, but are a signal for export from the cell.
After removal of the signal sequence, you have a hormone or prohormone.

27. Processing of Prohormones
Some hormones are produced in an “immature” form, and require further cutting to get the active peptide hormone.
Prohormones are cut into final form by peptidases in the Golgi apparatus.
Cutting usually occurs at basic amino acids (lysine, arginine)
Inhibin alpha
processing
Inhibin alpha

28. Example: POMC
The Proopiomelanocortin (POMC) peptide can be processed to give several different peptides, depending on regulation:
gMSH
aMSH clip bLPH bEndorphin }
ACTH
Get: melanocyte-stimulating hormone, lipoprotein hormone, beta endorphin, or ACTH, depending on how you cut it!

29. Prehormone vs. Preprohormone vs. Prohormone
Prehormone: signal sequence + mature peptide
Preprohormone: signal sequence + prohormone
Prohormone: precursor form of peptide (inactive, usually)

30. Post-translational Modification of Peptide Hormones
Glycosylation: addition of carbohydrates to amino acids on the peptide, utilizing specific enzymes (transferases)
Function: Carbohydrate side chains play roles in subunit assembly, secretion, plasma half life, receptor binding, and signal transduction.
Each carbohydrate side chain is composed of several simple sugars, with a special arrangement.
Two types: N-linked and O-linked, which differ in the amino acids that they are attached to.

31. N-linked and O-linked Glycosylation
N-linked sugars are bound to an asparagine residue, if the coding sequence Asn-X-Thr or Asn-X-Ser is present (X = any amino acid).
O-linked sugars are bound to serine/threonine residues.
Glycosylation begins in the RER, and is completed in the Golgi.

32. Other Post-translational Modifications
In addition, peptide hormones may be phosphorylated, acetylated, and sulfated, influencing their tertiary/quaternary structure and thus their biological activity.

33. Subunit Assembly
If a peptide hormone is composed of two subunits, they must be joined in the Golgi apparatus.
Disulfide bridges may form between subunits or between parts of a protein to reinforce natural conformation.

34. Secretion from Cells
Following production of the mature peptide hormone in the Golgi, the peptide is then packaged into secretory vesicles.
Secretory vesicles can stay within the cell until signaled to migrate to the plasma membrane.
Fusing of secretory vesicle with the plasma membrane releases hormone to outside of the cell.