Cellular Secretion: Essential for establishment and maintenance of pregnancy.

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Cellular SecretionEssential for establishment
and maintenance of pregnancy.

• Chapter 9 in Cooper Hausman

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What cells secrete?

• Mucus/Anitmicrobials
• Protection for mucosal surfaces
• Mucin
• ß-defensins
• Ions
• Regulate membrane excitability
• Calcium
• Neurotransmitters
• Transmit neural signals
• Acetylcholine
• Enzymes
• Involved in digestive processes
• Trypsin
• Hormones
• Regulate cellular functions
• Insulin
• Estrogen/Testosterone
• Antibodies
• Immune cells

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How cells secrete

• Classical secretory pathway
• ER-Golgi-TGN-Plasma membrane
• Mediated by signal peptide on protein
• Membrane-bound vesicles transported on
  cytoskeletal highways
• microtubules
• Constitutive and regulated
• microfilaments

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How cells secrete

• Non-classical Unconventional secretory pathways
• Not well-described
• Proteins translated on free ribosomes in
  cytoplasm
• Lysosomes
• Plasma membrane pores
• Multivesicular bodies
• Membrane blebbing.

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Pathways of cellular secretion.
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Figure 9.5
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Figure 9.7
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Figure 9.8
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Figure 9-10
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Figure 9-28
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Development of PorcineConceptuses from Day 10 to
12
5 mm Spherical
10 mm Spherical
Inner Cell Mass
14 mm Tubular
150 mm Filamentous
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Senger, 1999
letters to nature                                
                                                  
                                                  
      Nature 330, 377 - 379 (1987)
doi10.1038/330377a0 Interferon-like sequence of
ovine trophoblast protein secreted by embryonic
trophectoderm Kazuhiko Imakawa, Russell
V. Anthony, Mohammad Kazemi, Keith R. Marotti, H.
Gregory Polites  R. Michael Roberts
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Interferon tau-induced proteins

• 25 oligoadenylate synthetase
• ß2-microglobulin, MHC Class I
• Interferon regulatory factor-1, IRF-2, IRF-9
• Granulocyte chemotactic protein-2
• ISG15 (ISG17/UCRP)
• IP-10
• Mx protein (Charleston and Stewart, 1993, Ott et
  al., 1998)
• Secretions called Histotrophe or uterine milk

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Mx Proteins

• Named for ability to inhibit Orthomyxo viruses
• Member of Dynamin Superfamily
• Inhibitors of viral replication

From Praefcke McMahon 2004
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Mx Protein Structure

• Dynamin-Like monomeric GTPases
• Contain GTP-binding domain leucine zipper

From Haller Kochs 2002
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The Function of Dynamin
(Alberts et al., 2002)
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Mx Protein Folding

• Mx is present in both monomeric and multimeric
  forms.

From Kochs et al 1998
From Praefcke McMahon 2004
From Haller Kochs 2002
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Mx Protein Function

• GTPase activity

www.rpi.edu/.../MBWeb/mb1/part2/signals.htm
From Praefcke McMahon 2004
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Mx Protein Function

• Antiviral activity

From Janzen et al, 2000
From Samuel 2001
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-Influenza -Thogoto -Vesicular Stomatitis -Measles
-Bunya -Semliki -Hepatitis B
Antiviral Activity
Mx Protein
Normal Cellular Function?
-Cattle -Sheep -Pigs -Horses -Rats -Primates
Reproduction
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Could Ovine Mx1 be Involved in Secretory
Processes?

• oMx1 is highly expressed in response to conceptus
  produced IFN-t
• oMx1 is expressed in
• luminal and glandular
• epithelium

Luminal Epithelium
Lumen
Uterine Gland
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So what are the function(s) of Mx proteins in
pregnancy?

• Can we ascribe a new function to these antiviral
  proteins?
• Intracellular transport/vesicle trafficking
• Secretion
• GTPase

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One approach to determine Mx functionKnock it
out and see what happens.

• RNAi Short interfering RNA (siRNA)
• Transient gene-specific knockdown without
  activation of antiviral pathways.

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How does RNAi work?
-Evolutionarily conserved system against viral
infections-
- Invertebrates -
Long dsRNA (gt30bp)
Endogenous mRNA
Specific-gene silencing
(Elbashir et al, Nature, 2001)
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- Vertebrates -
Long dsRNA (gt30bp)
Mx (Protein GTPase)
Non-specific effects
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Secretion Summary

• Proteins destined for secretion possess a signal
  sequence that associates with a SRP and targets
  them to the ER.
• Following insertion into the ER through a
  translocon, proteins are folded and modified
  (glycosylation).
• Secreted proteins move in vesicles to the Golgi
  stacks.
• Vesicles derived from the TGN move on microtubule
  tracks to the plasma membrane.
• Secretory vesicles fuse with the PM to release
  contents into the extracellular space.

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Mx Summary

• The ruminant uterus expresses Mx proteins in
  response to IFN tau from the embryo.
• Mx1 is a mechanochemical enzyme.
• Mx1 is a high molecular weight GTPase that can
  form oligomers and bind cellular proteins.
• Mx1 is a molecular motor protein that mediates
  secretion by endometrial cells.
• Mx1 associates with microtubules and may be
  involved with cell division.
• Mx1 is critical to establishment and maintenance
  of pregnancy.

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Mx1
Mx1
Mx1
Mx2