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.
Ubiquitin & Proteasome: Role in Transcription RegulationAnjali Dahiya
Eukaryotic gene transcription is a phenomenally complex process. Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression can lead to cellular catastrophe and disease. One of the ways, cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin proteasome system (UPS)
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.
Ubiquitin & Proteasome: Role in Transcription RegulationAnjali Dahiya
Eukaryotic gene transcription is a phenomenally complex process. Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression can lead to cellular catastrophe and disease. One of the ways, cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin proteasome system (UPS)
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
Crimson Publishers- CPG Methylation in G-Quadruplex and IMotif DNA StructuresCrimsonPublishers-SBB
Abberant hypomethylation in DNA regions with noncanonical folding potential (ncDNA motifs) is believed to predetermine tumor development - presumably, by facilitating G-quadruplex (G4) and/or i-motif (IM) formation via altering nucleosome positioning (stable G4s induce subsequent genomic rearrangements). We questioned whether CpG methylation per se affects the dsDNA-ncDNA equilibrium. Thermodynamic studies of genomic and model oligonucleotides with methylated CpG sites at different positions are reported. The genomic oligonucleotides analyzed in this work are DNA fragments with reportedly different methylation statuses in colorectal cancer and normal cells. Free energies of duplex, ncDNA formation from single strands were calculated based on melting curve analyses. Polyethylenglycole was used to imitate crowding effect. Our results suggest that CpG methylation may alter the energetic barrier for dsDNA-IM transitions.
Melanoma and Parkinson disease & Link between them.SAKEEL AHMED
Parkinson disease is the progressive neurodegenerative disorder in which mainly dopaminergic neuron in the substantia nigra pars compacta.
Melanoma is a type of skin cancer.
Sima lev: Lipid Transfer Proteins and Membrane Contact Sites in Human CancerSima Lev
Lipid-transfer proteins (LTPs) were initially discovered as cytosolic factors that facilitate lipid transport between membrane bilayers in vitro. Since then, many LTPs have been isolated from bacteria, plants, yeast, and mammals, and extensively studied in cell-free systems and intact cells. A major advance in the LTP field was associated with the discovery of intracellular membrane contact sites (MCSs), small cytosolic gaps between the endoplasmic reticulum (ER) and other cellular membranes, which accelerate lipid transfer by LTPs. As LTPs modulate the distribution of lipids within cellular membranes, and many lipid species function as second messengers in key signaling pathways that control cell survival, proliferation, and migration, LTPs have been implicated in cancer-associated signal transduction cascades. Increasing evidence suggests that LTPs play an important role in cancer progression and metastasis. This review by Sima Lev describes how different LTPs as well as MCSs can contribute to cell transformation and malignant phenotype, and discusses how “aberrant” MCSs are associated with tumorigenesis in human.
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
1. Pyruvate Kinase and the Second Secret of
Life
Pyruvate Kinase and the Second Secret ofPyruvate Kinase and the Second Secret of
LifeLife
‘The second secret of life’ (Monod, 1963)
Allostery
the comparison of how one ligand binds in the absence,
versus the presence, of a second ligand
The first secret of life
‘The structure of DNA’ (Perutz)
2. Monod Wyman, Changeux (MWC) model for allostery:
proteins adopt various conformations in thermal equilibrium and allosteric
regulators stabilize selected conformations
Allostery: the basis of protein communication
Signal Transduction MetabolismTranscription
p53 CDK pyruvate kinase
Mdm2 cyclin/phosphate fructose bisphosphate
DNA ATP/protein substrate ADP/phosphenolpyruvate
4. LeishmaniaLeishmania mexicanamexicana
PYKPYK
regulated by F26BPregulated by F26BP
M2 PYK (embryonicM2 PYK (embryonic
or cancer cells)or cancer cells)
regulated by F16BPregulated by F16BP
M1 PYK (adult tissue)M1 PYK (adult tissue)
constitutively activeconstitutively active
O
O
O
P
O
O
O
O O
P
O
O
O
O
6
2
O
O
O
P
O
P O
O
O
O
O
O
O O
6 1
Phosphoenolpyruvate Pyruvate
ADP + + ATP
O
O
O
P
O
O
O
O
O
O
5. T to R Conformational transition of Leishmania
Pyruvate Kinase (LmPYK)
O
O
O
P
O
O
O
Phosphoenolpyruvate Pyruvate
PyK
ADP
ATP
Active
Site
Effector
Site
Pyruvate kinase monomer
6. Active site of ATP-bound LmPYK
Phosphoenolpyruvate Pyruvate
ADP
ATP
O
O
O
P
O
O
O
O
O
O
7. F-2,6-BP binding stabilises effector loop and rigidifies tetramer
• Forms 4 salt bridges (K484….E498 & R493….D482)
• effector loop pushed towards adjacent chain in tetramer
cc_ribbon.png
8. T to R Conformational transition of
Leishmania Pyruvate Kinase (LmPYK)
consistent with Monod, Wyman,
and Changeux allostery model.
9. The active conformer
(R-state) of LmPYK is
stabilised by the F26BP
effector molecule
Increased activity correlates with increased thermal stability
11. Using EDULISS to find hits for the active site
Look for ligands to mimic the γ-phosphate of
ATP and up to 3 coordinating water molecules
Sample hits
Sulphonate mimics of ATP are reminiscent of
trypan blue and suramin
12. SURAMIN binds to LmPYK (IC50 = 7μM) and overlaps
with the ATP binding pocket
N
H
N
H
O
NHO
N
H
O
S
S
O
O
O
S
O
O
O
ONH
O
N
H
S
SS
O
O
O
O
O
O
O
O
O
O
O
O
Used since 1920’s as antihelminthic
and against trypanosomiasis
13. HTS screen for LmPYK inhibitors using 300,000 compound library yields
70 hits with IC 50 values between 1 to 50 μM
(NIH, Doug Auld)
14. M2 PYK (embryonicM2 PYK (embryonic
or cancer cells)or cancer cells)
regulated by F16BPregulated by F16BP
M1 PYK (adult tissue)M1 PYK (adult tissue)
constitutively activeconstitutively active
pyruvate
glucose
mitochondrionpyruvate
glucose
X
Amino acid synthesis
cell division
Pyruvate Kinase and Cancer
Warburg Effect: increased uptake of glucose but low oxidative phosphorylation
caused by replacing M1 PYK isoform by allosterically controlled M2 isoform
16. M2 PYK is a splice variant of M1 PYK and differs by 22 AA over a 45 AA stretch
M1 (constitutively active) forms tetramers
M2 (allosterically activated) forms dimers
17. Structure of human M1 PYK
The 45 amino acid splice variant defines the C-C interface
18. 12 of the 22 residues that differ between M1 and M2 (black) are in
a loop that clamps K142 and stabilises the dimer interface
19. 300 Å
M2 PYK negative staining EM images show
tetramers (black circles) and dimers (red circles)
Images from Crick Wang and Laura Spagnolo
20. buffer 6 new PYK pro001:10_UV f16 only001:10_UV buffer 6 new PYK pro001:10_Fractions
0
20
40
60
80
100
120
mAU
0.0 5.0 10.0 15.0 20.0 25.0 30.0 ml
F2 1A2 1A4 1A6 1A8 1A10 1A12 1B2 1B4 1B6 1B8 1B10 1B12 1C2 1C4 1C6 1C8 1C10 1C12 1D2 1D4 1D6 1D8 1D10 1D12 1E2 1E4 1E6 1E8 1E10 1E12 1F2 1F4 1F6 1F8 1F10 Wast
Addition of effector (F16BP) pushes M2 to the tetrameric state
Gel filtration of M2 PYK (12 mg/ml)
Red: addition of F16BP (1mM)
M2 is tetrameric
Blue: no F16BP added
M2 is a mixture of tetramer and dimer
tetramer
dimer
21. M2 (apo) M2 + F16BP
M2 in 100mM KCl M2 in 10mM MgCl2
10nm 20nm
Dynamic Light Scattering results for M2 PYK are consistent with
a dimer- tetramer equilibrium.
Tetramers are favoured by addition of F16BP (and / or Mg ions)
10nm 20nm
23. • B-domain movement
• effector loop movement
• slight rotation of C domains
away from the central cavity
M2: Inactive to active transition (binding F16BP)
24. • T408M may repulse Q489 and
reintroduce E409 interactions in M2.
Comparison of C-C interface between M1(blue) and M2 (green) PYK
25. •The addition of the effector F16BP greatly reduces overall thermal
motion (B-factor) and increases Tm
M2 PYK (apo) Tm = 420C
M2 PYK + F16BPTm = 520C
M2 Pyruvate kinase X-ray structures with and without effector molecule
Red = hot, Blue = cold
26. M2 PYK is prevented from forming tetramers by:
- allosteric inhibitors (small molecules, phosphorylated proteins)
- phosphorylation
- viral oncoproteins
- lack of effector (F16BP)
oncoprotein
Inactive M2 PYK Active M2 PYK
M2 PYK activation provides a potential cancer therapy
27. Evolution of Allosteric Control in Pyruvate Kinase
No effector bound
(F16BP or F26BP)
-Inactive T-state
(flexible)
Effector bound
(F16BP or F26BP)
-Active R-state
(rigid)
clamp rock and lock dissociation
28. SUPPORT
MRC, Wellcome, BBSRC, EC, NIH
Glycolytic Enzymes
Hugh Morgan
Iain McNae
Matt Nowicki
Liam Worrall
Lindsay Tulloch
Linda Gilmore
Paul Michels
LIDAEUS/ EDULISS
Paul Taylor
Kun Yi Hsin
Steven Shave
CTCB Facilities
Martin Wear
Liz Blackburn
Janice Bramham
Sandra Bruce
Conny Ludwig