Redox regulation of mammalian heat shock factor 1 is essential for

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Redox regulation of mammalian heat shock factor 1
is essential for Hsp gene
activation and protection from stress Sang-Gun
Ahn and Dennis J. Thiele1 Department of
Biological Chemistry, The University of Michigan
Medical School, Ann Arbor, Michigan 48109-0606,
USA Presented by Santhosh Kumar
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Introduction to Heat Shock Proteins

• Hsp are a family of proteins found in all cells
• Hsp are induced when a cell undergoes various
  types of environmental
• stressors like heat, cold, oxygen
  deprivation
• Under normal conditions they act like
  chaperones they make sure that
• cells proteins are in the right shape in
  the right place at the right time
• Hsp help new or distorted proteins fold into
  shape which is essential for
• their function
• Hsp shuttle proteins from one compartment to
  another inside the cell,
• transport old proteins to garbage disposals
  inside the cell
• One of the most crucial function attributed to
  Hsp is that it presents pieces
• of proteins on the cell surface to help the
  immune system recognize
• diseased cells

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Stress Response As I Understand
Stress
Changes in Redox potential
Activation (homotrimerization) of Hsp
HSF
Acts as a transcription factor for Hsp gene
Anti-apoptotic other cell saving process
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Introduction to the Paper

• Heat shock response originally identified in
  salivary gland of Drosophila
• under stress
• Characteristic feature noted in most cells is
  the dramatic increase in Hsp
• response to stress
• Hsp genes is turned on by cis-acting promoter
  elements Heat Shock
• Elements (HSE) a transcription factor,
  Heat Shock Factor (HSF)
• Although 3 genes code for HSF, the one involved
  in Hsp gene
• transcription is HSF1 isoform
• Observed that activation of Hsp occurs in
  absence of new protein
• synthesis hence stress signal is not a
  multi-step cascade
• In this study, the effect of heat oxidative
  stress invitro on HSF1
• hence Hsp genes is shown

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Hypothesis

• Hsp is normally present in cells
• Hsp is activated during stress
• Trimerization of Hsp is an essential step for
  activation
• Hsp translocates to nucleus after activation
• No intermediary proteins involved in this stress
  response
• Cis acting elements activate Hsp gene and cause
  transcription of
• Hsp gene
• Crucial role of Cysteine residues in the
  activation of HSF

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Structure of Heat Shock Factor . N-terminal DNA
binding domain of the helix-turn-winged helix
type . One or more coiled-coil trimerization
domains . Nuclear localization domains .
C-terminal trans-activation domain
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Results
SDS-PAGE Assay
A
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B
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C
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D
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Fig 2a
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Fig 2B
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Fig 2C
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Fig 2D
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Fig 3
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Fig 4
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A
Fig 5
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Fig 5B
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Fig 7
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Fig 8
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Materials Methods

• Cells used were Mouse Embryonic fibroblasts
• MEF cells were transfected by a process called
  electroporation
• Cells were incubated for 48hrs before use
• For Heat Shock- cultured plates were wrapped
  with parafilm
• immersed in water bath for the required time
  temperature
• For H2O2 stress-H2O2 was added directly into the
  culture dishes
• For HSE-HSF1 reaction-32P labeled HSE were used
• Apoptosis was measured using a cell death
  detection ELISA kit for
• apoptotic antigens

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Discussion

• Homotrimerization of mammalian HSF1 is directly
  activated in vitro
• by both heat stress H2O2, in a
  redox-regulated, reversible fashion
• Cysteine residues play a crucial role in the
  activation of HSF
• Cysteine residues are required for
  multimerization, nuclear localization
• Hsp gene activation, and protection from
  apoptosis
• Nuclear localization-due to exposing of an
  otherwise quiescent nuclear
• localization signal (due to trimerization)

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Future Studies

• Study mechanism of direct heat activation of HSF
  1
• Studies with other stressors like changes in pH,
  mechanical stress etc.,
• on activation of stress response

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Clinical Implications

• Cancer Vaccination a concept similar to normal
  vaccination. Very
• successful in animals but in the preliminary
  stages with respect to
• humans
• Mechanism Hsp acts by presenting abnormal
  intracellular proteins
• to the outside hence triggering a immune
  response-destroying the
• cancer cells

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