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.