Molecular Basis of Peptide Hormone Production

Understanding Regulation of Hormone LevelsHow to Make a Peptide: Basic Steps

Cell Structures Involved in Peptide ProductionGene Structure and Transcription

Processing of RNA TranscriptsTranslation of mRNA into Peptide

Post-translational Processing of PeptidesSecretion of Peptide 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

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)

secretionmature (active) peptide

prepeptide/prepropeptide

messenger RNA

post-translationalmodification

translation

post-transcriptionalmodification

primary RNA transcript

transcription

Peptide/protein hormone synthesis

Protein and Polypeptide Hormones: Synthesis and Release

• Binds to surface receptor

• Transduction• System activation

– Open ion channel– Enzyme activation

• Second messenger systems

• Protein synthesis

Protein and Polypeptide Hormone Receptors

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

CRE ERE TATA BOX

CAT

5’-flanking region

regulatoryregion

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

cyclic AMP

protein kinase A

CREBmRNA

proteinP

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:

exon 1 2 3

Alternative Splicing

1 3

1 2 3

exon 1 2 3

Normal Splicing

RNA

(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 FSH 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

ASP

-...AUGGAGGAC...

MET GLU

ASP

-...AUGGAGGAC...

MET GLU-

-...AUGGAGGAC...

MET

GLU

mRNA on ribosome

Protein Sorting: Role of Post-translational Processing

• How does a cell know where a translated peptide is supposed to go?

50,000 proteinsproduced

plasma membrane

mitochondria, other organelles

nucleus

export from cell

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

Inhibin alpha

processing

Example: POMC

• The Proopiomelanocortin (POMC) peptide can be processed to give several different peptides, depending on regulation:

MSH MSH clip LPH Endorphin}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.