Molecular Basis of Peptide Hormone Production

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Transcript Molecular Basis of Peptide Hormone Production

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
cyclic AMP
protein kinase A
mRNA
CREB
P
protein
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
methy-G-
2
3
-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:
Normal Splicing
RNA
exon 1
2
3
Alternative Splicing
1
2
3
exon 1
2
1
(occurs in nucleus)
3
3
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.
3’ UT
5’ UT
coding region
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
ASP
GLU
MET
MET GLU
-...AUGGAGGAC...
-...AUGGAGGAC...
mRNA on ribosome
MET GLU- ASP
-...AUGGAGGAC...
Protein Sorting: Role of
Post-translational Processing
• How does a cell know where a translated peptide is
supposed to go?
plasma membrane
50,000 proteins
produced
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
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 AsnX-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.