Transcript HRP-12: A Novel HSP,its Distribution and Function

HRP-12: A Novel HSP;Its
Distribution and Function.
Syed Muzamil H Zaidi. PGY-3
Mentor: Dr S. Jayanth Samuel M.D., Ph.D
VAMC Buffalo,NY
Heat Shock Proteins
• A group of molecular Chaperones.
• Chaperones are molecules mostly
proteins with diverse functions
especially protein folding.
• While the polypeptide chain is being
synthesized, the intermediary forms are
stabilized by these proteins before they
can assume their final conformation.
How are they induced?
• Heat shock proteins
are induced by
various stresses
including heat,
Toxins,Radiation,
Infection etc.
What do they do?
• HSP are involved in:
–
–
–
–
–
–
Protein Synthesis.
Protein translocation.
Apoptosis.
Protein Degradation.
Signaling.
Antigen Presentation
How and where do they work?
• HSP 70 - Cytosol,
mitochondria,ER
• HSP 90 - Cytosol
ER
• HSP 40 - Cytosol.
• HSP 60 - Cytosol
and mitocondiria
• HSP 47 - ER
Clinical Implications:
• In Cystic Fibrosis,
mutation delays the
dissociation of Cl
channel protein from
its chaperones so
resulting in
abnormal folding
and loss of function.
Inflammatory Bowel Disease
• Inducible Hsps have a significant role in
gut inflammation in IBD.
• Cytoprotective agents, protecting IECs
from inflammation induced changes in
function.
• Immunomodulatory agents stimulating
lymphocytes in target specific manner.
Hu S, Ciancio MJ, Gastroenterology 2007;133:1893-1904
Tumor Immunity
• Tumor cells release
various Hsps.
• These are
immunogenic.
• HSP peptide complexes
are distinct from tumor
to tumor.
• Possibility of new
treatments
Heat Responsive Protein - 12
• Homology with Hsp
90 and Hsp 70.
• Both Hrp-12 mRNA
and Protein increase
with Stress.
• Molecular weight is
12 KD.
Discovery
• While the LDIF (Liver derived
immunoinhibitory factor) was purified, an
unexpected 12 kd protein was co purified.
• Although LDIF is 28 kd protein, hrp was
shown to purify with this column and later be
reducible to 12 kd size, indicating a possible
dimeric form.
Tzung SP et al,Hepatology 1991; 14:888-894
Methodology
• Purification: Livers from C57BI/6 mice were
prepared by homogenization and delipidation
and set to column chromatography.
• The column was further purified by Reversed
Phase Liquid chromatography.
• The sample was concentrated and
suspended in SDS-PAGE sample buffer and
ran on a 15% Polyacrylamide gel.
Sameul SJ et al,1997 Hepatology 25,1213-1222
Electrophoresis
• Under reducing
conditions (ME), most
of Hrp12 is in 12 kd
conformation
• Under non reducing
conditions, a minor 28
kd band was visualized.
• Lane 1,Liver Cytosol; 2,
Hrp Red;Hrp Non Red
Cloning & Sequencing
• The 12 kd bands were excised and subjected
individually to N terminal sequencing.
• The determined amino acid sequence was
used to design a pool of 128 oligonucleotides,
each 20 bp in length.
• These represented all possible nucleotide
sequences for first 7 amino acids.
• These were now applied to commercially
available murine cDNA library.
Sameul SJ et al,1997 Hepatology 25,1213-1222
Evaluation of cDNA
• 1240 bp long.
• Open Reading frame was 521bp long.
• It codes for 173 amino acids with mol mass
18.5 kd.
• It was variably phosphorylated and no bands
were present in 12 kd range if reaction did not
contain PKC and phosphatidyl serine.
Similarity
• Search of amino acids and nucleotide
sequences in Gene bank data identified
two rat proteins: PSP and LKPS
• 38-136 residues are identical.
• With Hsp70/DnaK, Hrp has a significant
homology too.
Comparison with PSP/LKPS &
HSP
•*…..Sites for phosphorylation by Protein
kinase C
• IIRKVIS……mitochondrial targeting
signal
• Hsp90-like sequence between L(84)
and Y(138
Response to Heat
• Hepa1-6 cells (murine hepatoma) in
monolayers were exposed to heat and RNA
was harvested at various stages.
• There was an unexpected oscillatory pattern
of mRNA expression as the cells were
exposed to different temperatures.
• The most consistent response was at 44.5O
Steady State levels of Hrp12 and Hsp90
mRNA normalized with Actin mRNA
Normalized Hrp12/Hsp90 at 44.5
C
Heat Respose Of HRP and HSP
3
2.5
2
HR P 12
1.5
HSP 90
1
0.5
0
0 H
0.5 H
1 H
2 H
R 1 H R 2 H R 4H
Lane Number
Protein folding abnormalities
• Implicated in
Alzheimer’s.
• Huntington’s
Chorea.
• Parkinson’s
Disease.
• Type 2 Diabetes.
• Prion Disease
Tissue Distribution of Hrp12
• Major signal in Liver and Kidney on Northern
Blots.
• This detected the relatively lower presence of
Hrp12 mRNA in Brain and lung.
• A zooblot revealed that Hrp gene is highly
conserved in all mammalian species.
Tissue specificity of HRP12
Northern blot of mouse tissues
Western blot of mouse tissues
Hrp12
Actin
liv
tes kid ske liv
lun spl brn hrt
kid
int
hrt
lun
spl
ske
brn
Cellular Distribution
• Methodology:
– Construction of bacterial expression vectors
pGST-Hrp12 and pGST-C, its expression and
preparation of antisera.
– Preparation of mouse tissue extracts.
– Western blots of these extracts.
– Subcellular fractionation of Liver.
– Immunohistochemistry
– Immunofluorescence
Liver
• IHC and IF showed
intense staining in
periportal region.
• A coarsely granular
staining in cytoplasm
was the most consistent
pattern.
• Laser Confocal
Microscopy of mouse
Liver
Kidneys
• Intense staining on
the luminal border
due to Alkaline
Phosphatase.
• Not removed by
levamisole.
• Most staining in PCT
and DCT to a lesser
extent.
What does it do?
• The yeast-based 2 hybrid system is a way to identify
protein-protein interactions.
• Chimeric proteins are created by fusing the binding
domain of an Transcription Activator to a bait protein
(hrp12) and co-expressing this protein with a prey
protein fused to the activation domain of the same
Transcription Activator in yeast cells.
• Interaction of the bait and prey proteins re-constitutes
the Transcription Activator and a reporter gene is
expressed
Results of sequencing (3)
Secreted (8 clones)
1.
2.
3.
4.
5.
Inter-alpha trypsin inhibitor, heavy chain 4 (2)
Contrapsin (1)
Serine peptidase inhibitor, clade A, member Ia (1)
Pregnancy Zone Protein (1)
Transthyretin (3)
Mitochondrial/Peroxi- 1.
somal (8)
2.
3.
4.
SDH beta (2)
Acyl CoA dehydrogenase, member 11 (4)
Voltage Dependent Anion Channel 1 (1)
Cytochrome P450 (1)
Cytosolic (1)
Phosphoglucomutase
1.
From Bench To Bedside!
• TTR is a Homotetrameric protein secreted by the liver.
• It binds retinol-binding protein and weakly to Thyroxine.
• Much interest centers on how it multimerizes in the ER
and how it is secreted.
• 2 disorders of TTR tetramer stability are known: senile
systemic amyloidosis (SSA) and familial amyloidotic
polyneuropathy (FAP).
• TTR can form amyloid in CSF (synthesized by the only
other organ that makes it, i.e. choroid plexus, and has
been implicated in depression and Alzheimer’s.
Acknowledgements
• Dr Michael Sitrin M.D.
• Valerie Anderson.
Thank you all for your attention.