Molecular Basis of Peptide Hormone Production

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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

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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

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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
secretion
mature (active) peptide
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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.

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Anatomy of the CellComponents involved in
Peptide Synthesis

• The nucleus contains the genetic information
  (genes) in DNA.
• The nucleus has a nuclear membrane, which
  separates contents of nucleus from cytoplasm.
• The nuclear membrane has pores, which allow the
  passage of RNA and proteins.
• The nucleolus is the site of ribosome synthesis.

Nucleolus
Nucleus
Cytoplasm
Plasma Membrane
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Organelles Associated with Peptide Synthesis and
Secretion

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Ribosomes

• Sites of protein synthesis.
• Composed of two subunits, made of rRNA and
  proteins.
• Assembled in the nucleolus, moves to cytoplasm.
• Found free, or attached to endoplasmic reticulum
  (ER).

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Rough Endoplasmic Reticulum

• Series of interconnecting membranous tubules.
• Interior spaces of the ER are called the
  cisternae.
• The rough ER has attached ribosomes site of
  protein synthesis.

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Golgi Apparatus

• Composed of stacked, flattened membranous sacs
  with cisternae.
• Continuous with RER.
• Role in modification and packaging of proteins
  and lipids for transport.
• Numerous in cells which secrete lots of proteins.

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Secretory Vesicles

• Membrane-bound sacs from the Golgi, carrying
  proteins and lipids for release from the cell.
• Fuse with the plasma membrane and release
  contents by exocytosis.
• Release of secretory vesicle contents may depend
  upon a signal to the cell.

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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

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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
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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)

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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
  5flanking region of target genes, turning on
  their transcription.

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Transcriptional Regulation by Cyclic AMP
CREB
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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

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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)

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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)
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Regulation of mRNA Stability

• In general, mRNA stability is regulated by
  factors binding to the 3- untranslated region
  (3-UT) of mRNAs.
• The 3UT 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 inhibins effects reflect decreased
  stability (half-life) of FSHb subunit mRNA.

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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.

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Translation

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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
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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.

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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)

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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!
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Prehormone vs. Preprohormone vs. Prohormone

• Prehormone signal sequence mature peptide
• Preprohormone signal sequence prohormone
• Prohormone precursor form of peptide (inactive,
  usually)

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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.

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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.

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Other Post-translational Modifications

• In addition, peptide hormones may be
  phosphorylated, acetylated, and sulfated,
  influencing their tertiary/quaternary structure
  and thus their biological activity.

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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.

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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.