Quantitative Analysis of IGF1R/AKT/mTOR Pathway using Multiplex-Immunoprecipi...Thermo Fisher Scientific
Determine the efficacy of multiplex IP to targeted MS (mIP-tMS) technique for measurement of the total and phosphorylated AKT-mTOR pathway targets and to evaluate whether mIP-tMS assays are as effective as the current singleplex immunoassay (Western Blot (WB) and ELISA) and multiplex Luminex assays.
Quantitative Analysis of IGF1R/AKT/mTOR Pathway using Multiplex-Immunoprecipi...Thermo Fisher Scientific
Determine the efficacy of multiplex IP to targeted MS (mIP-tMS) technique for measurement of the total and phosphorylated AKT-mTOR pathway targets and to evaluate whether mIP-tMS assays are as effective as the current singleplex immunoassay (Western Blot (WB) and ELISA) and multiplex Luminex assays.
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.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
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
System response of metabolic networks in Chlamydomonas reinhardtii during Nit...Nishikant Wase
Nitrogen starvation induces a global stress response in microalga that results in the accumulation of triglycerides in lipid bodies. To identify components and mechanisms leading to lipid accumulation during nitrogen stress, we used GC-MS based metabolite profiling and iTRAQ based quantitative proteomics to examine Chlamydomonas reinhardtii cultured up to 144 hours without nitrogen. When nitrogen is limiting, starch and lipid accumulated rapidly, with lipid becoming the major storage compound by 144 hours. Our FAMEs data showed that the percentage of highly unsaturated fatty acids was reduced and the percentage of saturated and monounsaturated fatty acids were increased. Using information from the GC-MS based metabolite profiling; a Partial Least Squares Discriminant Analysis model was created to evaluate the role of different intracellular metabolites during lipid accumulation. We observed decreased abundance of key amino acids whereas some important metabolites including citric acid, trehalose, triethanolamine, nicotinamine, methnionine, citramalic acid and sorbitol were increased in abundance. Addition of citric acid (from 4 mM to 6 mM) to the growth media significantly improves the lipid yield in Chlamydomonas reinhardtii while growing in TAP media containing nitrogen. Examination of differentially expressed proteins revealed that 100 of 793 identified proteins were induced after 144 hours, while 77 proteins were reduced in abundance. Proteins involved in nitrogen assimilation, oxidative phosphorylation, the glycolytic pathway, TCA cycle, starch, and lipid metabolism were found to be higher in abundance than in non-stressed cultures. Another effect of nitrogen starvation was reduction of proteins of the photosynthetic apparatus (including PS-I and PS-II) and light harvesting complex proteins. We conclude that during nitrogen starvation, carbon availability is the most important factor controlling oil biosynthesis and that there is carbon partitioning between starch and oil synthesis.
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.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
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
System response of metabolic networks in Chlamydomonas reinhardtii during Nit...Nishikant Wase
Nitrogen starvation induces a global stress response in microalga that results in the accumulation of triglycerides in lipid bodies. To identify components and mechanisms leading to lipid accumulation during nitrogen stress, we used GC-MS based metabolite profiling and iTRAQ based quantitative proteomics to examine Chlamydomonas reinhardtii cultured up to 144 hours without nitrogen. When nitrogen is limiting, starch and lipid accumulated rapidly, with lipid becoming the major storage compound by 144 hours. Our FAMEs data showed that the percentage of highly unsaturated fatty acids was reduced and the percentage of saturated and monounsaturated fatty acids were increased. Using information from the GC-MS based metabolite profiling; a Partial Least Squares Discriminant Analysis model was created to evaluate the role of different intracellular metabolites during lipid accumulation. We observed decreased abundance of key amino acids whereas some important metabolites including citric acid, trehalose, triethanolamine, nicotinamine, methnionine, citramalic acid and sorbitol were increased in abundance. Addition of citric acid (from 4 mM to 6 mM) to the growth media significantly improves the lipid yield in Chlamydomonas reinhardtii while growing in TAP media containing nitrogen. Examination of differentially expressed proteins revealed that 100 of 793 identified proteins were induced after 144 hours, while 77 proteins were reduced in abundance. Proteins involved in nitrogen assimilation, oxidative phosphorylation, the glycolytic pathway, TCA cycle, starch, and lipid metabolism were found to be higher in abundance than in non-stressed cultures. Another effect of nitrogen starvation was reduction of proteins of the photosynthetic apparatus (including PS-I and PS-II) and light harvesting complex proteins. We conclude that during nitrogen starvation, carbon availability is the most important factor controlling oil biosynthesis and that there is carbon partitioning between starch and oil synthesis.
Aptamers provide opportunities for structure-based drug design strategies relevant to therapeutic intervention. Recent advances in the chemical modifications of nucleic acids suggest that one of the major barriers to use, stability, can be overcome. The high affinity and specificity of aptamers rival antibodies and make them a promising tool in diagnostic and therapeutic application. We should expect more aptamers to be isolated in the near future against an ever increasing repertoire of targets, using these different SELEX approaches with increased speed and efficiency. Aptamers are poised to successfully compete with monoclonal Abs in therapeutics and drug development within the next few decades.
Personalized nanomedicine for the treatment of vascular hypertensionSusanta Kumar Rout
This study includes designing a nanomedical device for the treatment of vascular hypertension in polycystic kidney diseases (PKD) model through cilia targeting.
They generated and compared two different metal and polymer cilia-targeted nanoparticle drug delivery systems (DDS), i.e. gold (Au) and poly-lactic-co-glycolic acid (PLGA) nanoparticles (NPs)
The target is Dopamine-receptor type-5 (DRS) on primary cilia.
The drug-loaded is Fenoldopam (FD).
Synthesis, Anti-HIV activity and Cytotoxicity of N-Substituted Phthalimide de...
Activity Studies of Nampt Summer 2012
1. Acknowledgements:
This research is based in part upon work conducted using the Rhode Island Genomics and Sequencing Center which is supported in part by the National Science Foundation (MRI Grant No. DBI-0215393 and EPSCoR Grant Nos.
0554548 & EPS-1004057), the US Department of Agriculture (Grant Nos. 2002-34438-12688 and 2003-34438-13111), and the University of Rhode Island. The project described was supported by grants from the National Center for
Research Resources (5P20RR016457-11) and the National Institute for General Medical Science (8 P20 GM103430-11), components of the National Institutes of Health (NIH), and EPSCoR grants (Nos. 0554548 & EPS-1004057). Its
contents are solely the responsibility of the authors and do not necessarily represent the official views of the NSF, NIGMS, or the NIH.
Introduction
Background
Nicotinamide adenine dinucleotide (NAD+) is of paramount importance as a cellular me-
tabolite, and is a cofactor in more than 200 oxidation reduction reactions. NAD+ serves as a
substrate for post-translational modification proteins such as PARP1, among many others.
Some cancer cells are thought to be especially sensitive to NAD+ levels. Nicotinamide
phosphoribosyltransferase (Nampt) is the rate-limiting enzyme in the NAD+ salvage path-
way and its expression and activity is correlated to the concentration of NAD+ synthesized
in the body, making it a potential target for regulation by inhibition.
Nampt is a 55 kDa protein that forms a homodimer whose active site is located along the di-
merization plane. Nampt has several post-translation modification and phosphorylation
sites. The 188 Tyrosine site, located within the substrate channel outside of the active site,
was mutated to mimic phosphorylation Y-188D and mimic the non-phosphorylated state
Y-188F.
Nampt activity was monitored via conversion of its enzymatic product, Nicotinamide mono-
nucleoside NMN, to a fluorescence derivative. A linear relationship between fluorescence
intensity and NMN concentration will be presented along with experiments investigating
enzyme kinetics (including optimization of time course and preliminary Michaelis–Menten
kinetics). These analyses will be instrumental in evaluating Nampt kinetics, by matching cal-
culated Nampt Km and Kcat to literature values and comparing generated mutants and po-
tential inhibitors with this framework.
Kinetics Assay Calibration
Activity Studies of Nicotinamide Phosphoribosyltransferase (Nampt)
Christopher Funk, Cailyn Mather, Katelyn Pina, and Karen H. Almeida, Ph.D.
Physical Sciences Department, Rhode Island College, Providence, RI
PARP-1 is an essential protein in the detection of metabolic, chemical or radiation-induced DNA
strand breaks. Upon binding, PARP catalyzes the production of long poly(ADP-ribose)polymers
called PAR. PAR chains act as a signal to recruit other DNA damage repair proteins. After suc-
cessful repair, PAR chains are degraded by PAR glycohydrolase (PARG) to allow for further
damage detection (right panel). ADP-ribose monomers are transferred from NAD+, yielding nico-
tinamide that is recycled into NAD+ via the NAD+ salvage pathway (left panel). Excessive DNA
damage can lead to hyper-activation of PARP and via the connection of these two processes, po-
tential depletion of NAD+ cellular levels. Nampt is the rate-limiting enzyme in the NAD+ salvage
pathway and therefore critical in maintaining sufficient cellular energy.
References
Role of Nicotinamide in DNA Damage, Mutagensis and DNA Repair. (2010) Journal of Nucleic
Acids. 2010: 157591. PMID: 20725615
Molecular basis for the inhibition of human NMPRTase, a novel target for anticancer agents.
(2006) Nat.Struct.Mol.Biol. 13: 582-588. PMID: 16826227
A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransfer-
ase. (2011) Anal. Biochem. 412:18-25. PMID: 21211508
Development of Fluorescent assay for NMN detection. Chemical transformation of nicotinamide
(NAM) to NAD+ via the NAD+ salvage pathway. The intermediate, nicotinamide mononucleotide
(NMN) is converted to a fluorescent derivative for quantitative detection, excitation 375 nm and
emission from 410nm - 460nm.
Left Structure: X-ray structure of human Nampt complexed with FK866 inhibitor. Active site resi-
dues are highlighted in orange and yellow, FK866 in magenta and tyrosine188 is highlighted in red.
PDB ID: 2GVJ. Right Structure: Chemical structure of FK866 (APO-866).
Kinetics Tables
Inhibition of Nampt Wild Type with known inhibitor FK866.Panel A:Activity of Nampt Wild Type
(31ug/mL) with varying concentrations of FK866.Panel B:Extrapolated results from panel A using
theoretical controls showing the percent activity of Nampt Wild Type versus [FK866].The IC50 value was
determined to be 1.204 uM. The Zhang paper reported an IC50 value at 0.9+ or - 0.1nM.
Conclusions/Future Work
Assay standardization using Nampt Wild Type. Panel A : Maintaining Nampt at a constant
25ug/mL, while varying NAM concentrations to determine the appropriate [NAM] range for ki-
netics calculations. Panel B: Standard curve of fluorescence versus NMN derivative concentra-
tion. Panel C: Nampt Wild Type Assay (Michalelis-Menten Curve) with a R-squared value of
0.97 using calibrated assay parameters with a 15-minute incubation time at 37 degrees C.
FK866 Inhibition
Average Kcat/Km value is around 5,377 (M-1 sec-1) for Nampt Wild Type.
Preliminary data suggests no evidence in changing the tyrosine Y188D.
Y188F has no activity, Y188 residue is important for the activity of Nampt.
For FK866 the IC50 value is 1.204 uM. This preliminary data confirms that we can inhibit Nampt activity in our lab.
Previous fluorescence assay protocols were revised for incubation and [NAM] for improved kinetic data collection.
Calculated Km and Kcat values are specific to concentration and to protein used in each experiments. Establish a
range within our assays.
Preliminary data on Y188F activity suggests inhibited activity as previously hypothesized. Future assays will hope-
fully confirm these results.
A decrease in fluorescence was noted with the addition of FK866 inhibitor to Nampt Wild Type. Future work will in-
clude additional controls to confirm the theoretical percent activity. Include additional controls to confirm the theo-
retical percent activity.
A
B C
NAMPT WT
0 20 40
0.0
0.1
0.2
0.3
[NAM] uM
[NMN]generated
uM/min
NAMPT Y188D
0 20 40
0.0
0.1
0.2
0.3
[NAM] uM
[NMN]generated
uM/min
NAMPT Y188F
0 20 40
0.0
0.1
0.2
0.3
[NAM] uM
[NMN]generated
uM/min
A B C
Nampt Wild Type and Y188 mutant activity using 100% pure protein (coomassie stain
validation). Panel A: [NMN] generated per minute versus [NAM] using 25ug/mL con-
stant Nampt Wild Type with a 15 minute incubation time at 37 degrees C, with a R-
squared value of 0.4561. Panel B: [NMN] generated per minute versus [NAM] using
41ug/mL constant Nampt Y188D with a 15 minute incubation time at 37 degrees C,
and a R-squared value of 0.38. Panel C: [NMN] generated per minute versus [NAM]
using 25ug/mL constant Nampt Y188F with a 15 minute incubation time at 37 degrees
C, and a R-squared value of 0.267.
CM 70612
0 20 40 60 80 100
0.0
0.2
0.4
0.6
0.8
1.0
[NAM] uM
[NMN]generated
uM/min
kcat,
(min-1)
Km,
(uM)
Kcat/Km,
(uM-1 min-
1)
Kcat/Km, (M-
1 sec-1) Reference R-squared time
Burgos and
Schramm 0.46 0.01 92.0000 1,533,333 Biochem 2008
Revollo 6.3 1.8 3.5000 58,333 Cell Metab 2007
Revollo 1.2 0.92 1.3043 21,739 JBC 2004
KHA 3/13 0.298 1.84 0.1620 2,699 0.99 60
CF 7/2 0.7314 6.45 0.1134 1,890 0.93 20
CF 7/3 0.5211 1.03 0.5059 8,432 0.75 20
CF 7/5 1.947 8.189 0.2378 3,963 0.98 15
KP 7/5 0.8008 2.18 0.3670 6,117 0.61 15
KP 7/3 0.6867 3.25 0.2113 3,522 0.79 20
CM 7/6 1.992 6.82 0.2923 4,872 0.97 15
CF 7/12 0.5676 0.821 0.6914 11,524 0.4561 15
NAMPT Y188D
kcat,
(min-1)
Km,
(uM)
Kcat/Km,
(uM-1 min-
1)
Kcat/Km, (M-
1 sec-1) Reference R-squared time
CF 71212 0.2097 0.275 0.7625 12,709 0.38 15
NAMPT Y188F
kcat,
(min-1)
Km,
(uM)
Kcat/Km,
(uM-1 min-
1)
Kcat/Km, (M-
1 sec-1) Reference R-squared time
CF 71212 0.1145 -0.331 -0.3459 -5,765 0.267 15
Calculated Kcat and Km values from fluorescence assays of Nampt Wild Type, Nampt Y188D,
and Nampt Y188F. Panel A: Collection of Kcat and Km values from literature and experimen-
tal data of Nampt Wild Type. The average Kcat/Km (M-1 sec-1) is 5,377 for Nampt Wild Type.
Panel B: Experimental values of Kcat and Km from Nampt mutants.
A
B
0 2 4 6
0
50
100
[FK866] uM
%Activity
B
Fluorescence Assay Mechanism
Nampt Mutations Kinetics
NAD+
N
O
NH2
N
NN
N
NH2
O
HOH
OP
O-
O
O
O
OHOH
OPO
O-
O
N+
O
NH2
O
OHOH
OP-
O
O-
O
PO
O-
O
O-
PO
O-
O
PRPP
Nicotinamide
O
OHOH
OP-
O
O-
O
N+
O
NH2
NMN
NAMPT
PPi
Nmnat
ATP PPi
O
OHOH
OP-
O
O-
O
N
NH
O
H
O
OH
O
KOH
Fluorescent
NMN derivative
+
NMN
PARP-1
Nicotinamide
NAD+
Nampt
Nmnat
Redox Reactions
NADH
ADP/ATP
DNA strand breaks
Poly-ADP-ribosylation
Reactions
Poly(ADP-ribose) polymers
PARG
Free ADP-ribose
PARP-1 (modified)
O
Structures
Activity %= F-Fco/ F100%-Fo
F= Sample Reactions containing buffer,NAM,Fk866
Fco= Buferr,no NAM,Fk866
Fo= Containing only buffer
F100%= Containing buffer and NAM
!
A
1.0
10-07
1.0
10-06
1.0
10-05
1.0
10-04
1.0
10-03
1.0
10-02
1.0
10-01
1.0
1000
1.0
1001
80
100
120
140
160
[FK866], uM
FluorescenceUnits