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

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

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

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

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

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

Peptide/protein hormone synthesis

Protein and Polypeptide Hormones: Synthesis and Release

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

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

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.

Amplification via 2nd messenger

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.

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

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

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])

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.

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

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

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

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

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.

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.

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

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

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.

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

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!

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

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.

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.

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

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.

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.