Molecular Basis of Peptide Hormone Production

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

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: 25-30 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. cyclic AMP protein kinase A mRNA P protein pCREB Transcriptional Regulation by Cyclic AMP CREB

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. exon 1 2 3 -AAAAAAA... methy-G- 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)

21. Normal Splicing RNA exon 1 2 3 Alternative Splicing exon 1 2 3 1 2 3 1 3 Alternative Splicing • By varying which exons are included or excluded during splicing, you get can more than one gene product from a single gene: (occurs in nucleus)

22. 3’ UT 5’ UT AAAAAAAA... coding region binding protein 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. • 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. ASP GLU MET MET GLU -...AUGGAGGAC... -...AUGGAGGAC... mRNA on ribosome MET GLU- ASP -...AUGGAGGAC... Translation

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 15-30 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 15-30 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. Inhibin alpha processing Inhibin alpha 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)

28. gMSH aMSH clip bLPH bEndorphin } ACTH Example: POMC • The Proopiomelanocortin (POMC) peptide can be processed to give several different peptides, depending on regulation: 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.