- STEP levels are elevated in Fragile X mice and inhibit long-term potentiation.
- Researchers tested the STEP inhibitor TC-2153 in wild type and Fragile X mice.
- TC-2153 treatment led to increased phosphorylation of STEP substrates like NR2B, Pyk2 and ERK1/2 in both wild type and Fragile X mice by inhibiting STEP.
- This suggests that TC-2153 may be an effective treatment for cognitive deficits in Fragile X Syndrome by blocking the elevated STEP activity.
A new effector pathway links ATM kinase with the DNA damage responseCostas Demonacos
The related kinases ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) phosphorylate a limited number of downstream protein targets in response to DNA damage. Here we report a new pathway in which ATM kinase signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue, stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53 acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can be assigned to ATM through the DNA damage-dependent phosphorylation of Strap.
A new effector pathway links ATM kinase with the DNA damage responseCostas Demonacos
The related kinases ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) phosphorylate a limited number of downstream protein targets in response to DNA damage. Here we report a new pathway in which ATM kinase signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue, stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53 acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can be assigned to ATM through the DNA damage-dependent phosphorylation of Strap.
KDM5 epigenetic modifiers as a focus for drug discoveryChristopher Wynder
A summary presentation of my scientific work.
My laboratory focused on an enzyme KDM5b (aka PLU-1, JARID1b) that was widely expressed during development and played a key role in progression of breast cancer through HER-2.
My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
Our results were confirmed through the use of ES cells, adult stem cells and mouse models.
Much of this work remains unpublished, please contact me for more information and/or access to any reagents that I still have as part of this work.
crwynder@gmail.com
Regulation of KDM5 by multiple cofactors regulates cancer and stem cellsChristopher Wynder
Presentation of data regarding proteins that regulate the activity of KDM5b.
The studies use multiple disciplines including in vitro enzymology, ES cell studies of differentiation, Mass spectrometry to detect protein protein interactions.
These studies resulted in a comprehensive view of KDM5b function. It required development of at least three novel assays that are focused on moving epigenetic research from yeast and HeLa cell types to primary, clinically relevant cell types.
The techniques have been successfully used in Embryonic stem cells (human and mouse), Neural stem cells (mouse and patient derived as well as iPSCs.
Oskar Fernández-Capetillo - Centro Nacional de Investigaciones Oncológicas (C...Fundación Ramón Areces
El martes 26 de septiembre del 2017 organizamos en la Fundación Ramón Areces un Simposio Internacional sobre nuevas perspectivas en la investigación sobre el cáncer. En colaboración con el Centro Nacional de Investigaciones Oncológicas (CNIO) y Weizmann Institute for Science.
Dietary Administration of Diquat for 13 Weeks Does Not Result in a Loss of Do...EPL, Inc.
Dietary Administration of Diquat for 13 Weeks Does Not Result in a Loss of Dopaminergic Neurons in the Substantia Nigra Pars Compacta (SNpc) of C57BL/6J Mice
KDM5 epigenetic modifiers as a focus for drug discoveryChristopher Wynder
A summary presentation of my scientific work.
My laboratory focused on an enzyme KDM5b (aka PLU-1, JARID1b) that was widely expressed during development and played a key role in progression of breast cancer through HER-2.
My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
Our results were confirmed through the use of ES cells, adult stem cells and mouse models.
Much of this work remains unpublished, please contact me for more information and/or access to any reagents that I still have as part of this work.
crwynder@gmail.com
Regulation of KDM5 by multiple cofactors regulates cancer and stem cellsChristopher Wynder
Presentation of data regarding proteins that regulate the activity of KDM5b.
The studies use multiple disciplines including in vitro enzymology, ES cell studies of differentiation, Mass spectrometry to detect protein protein interactions.
These studies resulted in a comprehensive view of KDM5b function. It required development of at least three novel assays that are focused on moving epigenetic research from yeast and HeLa cell types to primary, clinically relevant cell types.
The techniques have been successfully used in Embryonic stem cells (human and mouse), Neural stem cells (mouse and patient derived as well as iPSCs.
Oskar Fernández-Capetillo - Centro Nacional de Investigaciones Oncológicas (C...Fundación Ramón Areces
El martes 26 de septiembre del 2017 organizamos en la Fundación Ramón Areces un Simposio Internacional sobre nuevas perspectivas en la investigación sobre el cáncer. En colaboración con el Centro Nacional de Investigaciones Oncológicas (CNIO) y Weizmann Institute for Science.
Dietary Administration of Diquat for 13 Weeks Does Not Result in a Loss of Do...EPL, Inc.
Dietary Administration of Diquat for 13 Weeks Does Not Result in a Loss of Dopaminergic Neurons in the Substantia Nigra Pars Compacta (SNpc) of C57BL/6J Mice
Cells respond to nutrient deprivation a variety of ways. In addition to global down regulation of cap-dependent protein
synthesis mediated by the GCN2 and mTO RC1 signaling pathways, a catabolic process autophagy is upregulated to
provide internal building blocks and energy needed to sustain viability. It has recently been shown that during nutrient
deprivation tRNAs accumulate in the nucleus, but the functional role of this accumulation remains unknown. This study
investigates whether subcellular localization of tRNAs plays a role in signaling nutritional stress and autophagy. We report
that human fibroblasts that accumulate tRNA in the nucleus due to downregulation of their transportin, Xpo-t, show
reduced mTO RC1 activity and upregulated autophagy. This suggests that sub-cellular localization of tRNAs may regulate
an unicellular response to starvation independently of the cellular nutritional status.
1. TC-2153 Inhibits STEP in Fmr1KO mice
Amy Lee1,2, Clare Ham1, Robert Samples1, Mitchell Powell1, Manavi Chatterje1, Paul Lombroso1
1Child Study Center Yale University, New Haven, CT 2The University of the South, Sewanee, TN
STEP (STriatal-Enriched protein tyrosine Phosphatase) is a
neuron-specific phosphatase that is overactive in several
neuropsychiatric disorders, including Fragile X Syndrome and
Alzheimer’s Disease. STEP opposes long term potentiation (LTP)
and promotes synaptic weakening by its phosphatase activity.
Dephosphorylation of STEP substrates ERK1/2, Pyk2 and
GluN2B cause some cognitive deficits observed in these
diseases. Protein translation disregulation in Fmr1KO mice results
in increased levels of STEP; increased STEP levels are correlated
with Fragile X behaviors. Inhibiting STEP may be effective in
treating Fragile X Syndrome. In search of a STEP inhibitor,
researchers have discovered TC-2153; a known small-molecule
STEP inhibitor that has been shown to increase tyrosine
phosphorylation of STEP substrates and improve cognitive
deficits. We first asked how TC-2153 affects STEP and its
substrates at various subcellular fractions in Wild Type animals.
From this we chose to focus on the P2 crude membrane that
appeared most responsive to TC treatment and where STEP61
substrates reside. We then administered the drug to Fmr1KO
mice. To address these questions we performed subcellular
fractionation and Western Blot analysis. Our data shows
increased Tyr phosphorylation of STEP stubstrate in WT and
Fmr1 KO mice after TC treatment. Leading us to conclude TC-
2153 could be effective treatment for Fragile X syndrome.
Crude Membrane Fraction Most Responsive to TC-2153
Treatment in WT mice
Fragile X Genotype
Conclusion
Subcellular Fractionation: Cellular compartments were sequentially
extracted (Figure 1). Hippocampus tissue samples of Fmr1KO mice
were homogenized using Tepfon in glass. Samples were centrifuged
10 min at 28000 rpm to obtain S1 and P1. S1 was centrifuged 15
minutes at 10400 rpm to obtain S2 and P2 fractions. P2 was
suspended and centrifuged 20 minutes at 18000 rpm to obtain LS1 and
LP1. S2 and LS1 were centrifuged for 2 hours at 70000rpm to obtain
S3 and P3 and LS2 and LP2 respectively.
Western Blotting: Samples were prepared and resolved by SDS-
PAGE, transferred to a PVDF membrane and incubated in
corresponding antibodies (anti-STEP23E5, pGluN2B:Tyr1472,
pPyk2:Tyr402, and pERK1/2:Tyr204/187), overnight at 4°C .
Immunoreactivity was visualized with Pierce Biotechnology
Chemiluminescent substrate kit and G: Box. Quantifications were
determined using ImageJ program.
Genotyping: DNA samples were extracting using Qiagen DNA Mini
Prep kit. Real-Time PCR was performed on the samples. Samples
were run on a 2% Agarose Gel and imaged using GeneSnap program
with G:Box.
TC Treatment: WT and Fmr1 KO mice were treated with vehicle
or10mg/kg TC-2153 IP three hours prior to sacrifice and immediate
harvest of tissue (Figure 2).
Figure 1: Levels of Subcellular Fractionation.
H, whole brain homogenate; P1, nuclei,
unbroken cells, blood vessels; P2, crude
membrane; S3, cytosol; P3, small organelles;
LP1, lysed synaptosomal membranes and
mitochondria; LP2, synaptic vesicles and small
organelles; LS2, synaptic vesicle supernatant.
Western Blot analysis of vehicle and TC-2153 treated WT mice normalized to B-actin.
Samples of hippocampal subcellular fractionation showing no significant decrease of STEP
in crude membrane (P2) fractions of WT mice upon TC-2153 treatment. TC-2153 activity in
the P2 fraction corresponds with the membrane region enriched with substrates of STEP61
specifically pNR2B..
TC-2153 Increases the Tyr phosphorylation of STEP
substrates in vivo
P2 fractionation and Western blot of cortex brain region of c57B1/6 WT mice three
hours post vehicle or 10mg/kg TC-2153 treatment. Tyr phosphorylation of STEP
substrates were determined as a function of immunoreactivity normalized to total
protein signal and GAPDH. Increased Tyr phosphorylation of STEP substrates upon
TC-2153 treatment was indicative of effective STEP inhibition in the P2.
Representative gel of PCR reactions
screened for the presence or
absence of Fmr1 alleles. This
technique is used to confirm the
genotype of mice subject to
experiment.
Western blot analysis normalized to B-actin.
Samples of STEP levels in WT and Fmr1KO
animals each treated with vehicle and TC-2153.
As shown in the figure STEP levels are
significantly elevated in the Fmr1KO mice, but
shows no change in STEP levels after TC
treatment due to inactivation and not
degradation.
STEP levels are elevated in Fragile X mice.
Blocking STEP activity with TC-2153 leads to increase of phosphorylation of
its substrates pGluN2B, pPyk and pERK.
TC-2153 can be used as a potential drug treatment of Fragile X Syndrome
Abstract
Methods
Figure 2: Structure of 8-(trifluoromethyl)-
1,2, 4,5-benzopentathiepin-6-amine or
TC-2153. TC-2153 is a potent and
specific inhitor to the isoforms of STEP:
STEP61 and STEP46 (Xu et. al, 2014).
Wild Type Vehicle Wild TC-2153 Treated
STEP
STEP
pNR2B
pNR2B
Pictorial representation of hypothesis that
inhibition of STEP results in reduced
dephosphorylation activity and thus increase in
phosphorylation and activation of STEP
substrates.
References
Fitzpatrick CJ and Lomproso PJ (2011). The role of striatal-enriched protein tyrosine
phosphatase (STEP) in cognition. Front Neuroanat. 5:47. dio:10.3389/fnana.2011.00047.
Xu J, Chatterjee M, Baguley TD, Brouillette J, Kurup P, Ghosh D, et al. (2014) Inhibitor of the
Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's
Disease. PLoS Biol 12(8): e1001923. doi:10.1371/journal.pbio.1001923
Goebel-Goody SM, Wilson-Wallis ED, Royston S, Tagliatela SM, Naegele JR, Lombroso PJ
(2012). Genetic manipulation of STEP reverses behavioral abnormalities in a fragile X
syndrome mouse model. Genes, brain, and behavior. 11(5):586-600. doi:10.1111/j.1601-
183X/2012/00781.x
Reduction of STEP Levels in WT and Fmr1KO Mice
upon TC-2153 Treatment
STEP
Increase in NR2B Phosphorylation in Fmr1KO Mice
upon TC-2153 Treatment
P2 fractionation and Western blot of
hippocampus brain region of Fmr1KO mice
three hours post vehicle or 10mg/kg TC-
2153 treatment. Tyr phosphorylation of
STEP substrate NR2B was determined as
a function of immunoreactivity normalized
to total protein signal. Increased Tyr
phosphorylation of STEP substrate NR2B
upon TC-2153 treatment was indicative of
effective STEP inhibition in the disease
model.
pNR2B