Circadian Rhythms: A CRY for Help

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Circadian Rhythms A CRY for Help

• By Rikard Fred

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Content

• Part I Introduction
• What is The Circadian Clock
• How Does The Circadian Clock Work
• Why is it Important

• Part II Experimental Plan
• Approach
• Finding the Target protein
• Expression and Purification
• Manipulation of CRY

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What is The Circadian Clock?

• Circadian originates from circa and dies which
  means about a day
• It is the biological clock that keeps us on a 24
  hour day

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The Circadian Clock

• Helps organisms predict daily and seasonal
  changes in their environment
• Give rise to changes in behavior and physiology
• Is located to the suprachiasmatic nucleus in the
  brain
• Is controlled by the environmental Light and Dark
  cycle

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How Does the Circadian Clock Work?

• It has three major components
• A photoactive pigment i.e. Cryptochrome (CRY)
• The translation/transcription clock that
  oscillates with a 24 hour periodicity
• The genes controlled by the clock that generate
  the physiological response

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• The genes involved are per 1, 2, 3 and cry 1 and
  2
• Which are controlled by the transcriptional
  activators CLOCK and BMAL
• Results from cry knockouts indicates that the CRY
  protein is important for the maintenance of the
  circadian clock

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The Clock Feedback Loop
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Cryptochromes (CRYs)

• Are blue light receptors
• Are flavoproteins and have a Flavin (FAD) group
  as functional element
• The Flavin group can transfer electrons
• The FAD photogroup binds non-covalently to the
  CRY protein

The FAD Group
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Why is this important?

• We have today largely abandoned the natural day
  and night activity pattern
• A lot of people suffers from difficult working
  hours
• Being out of phase can affect both mental
  alertness as well as eating habits and therefore
  have a great impact on overall health

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Approach

• Light is the primary stimulus for the clock
• The effect is cumulative but slow
• Therefore a chemical manipulation of the
  photoreceptor might help resetting the clock
  faster then with light therapy
• My approach is to silence the CRY for a short
  period of time to reset the clock

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Finding the Target protein

• Both cry1 and cry2 essential
• But in the SCN only cry1 mRNA levels oscillates
• cry1 is also the only one with an E-box
• This makes CRY1 my primary target
• But if possible the experiments should be carried
  out with both CRY1 and CRY2

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Expression and Purification

• Escherichia coli will be used as expression host
  (the SY2 cell line)
• With the pGEX-4T-2 vector from Amersham biotech
• The protein will be expressed as a glutathione
  S-transferase fusion protein and
• Purified using a gluthatione-Sepharose column
• If necessary the protein could be concentrated
  using available concentration columns

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Manipulation of CRY

• Two different approaches
• Find a protein that binds to (and hopefully
  inhibits) CRY
• Find/Construct a non-functional FAD molecule that
  will still bind to CRY

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Finding an Interacting Protein

• Perform co-precipitation using Western blot to
  find protein-protein interactions.
• Use a cDNA library to randomly express possible
  target proteins
• Transfer proteins to a nitro-cellulose membrane
  and incubate with labeled CRY
• Sequence possible interacting proteins to
  identify them

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Construct a FAD molecule

• This will require some collaboration.
• The FAD group is fairly simple
• Synthesizing an analogue lacking a few electron
  bonds might be enough

The FAD Group
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End of Presentation and Time for Questions