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 Resou -

rces (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 re-

sponsibility of the authors and do not necessarily represent the official views of the NSF, NIGMS, or the NIH.

Introduction

Background

NAD+/NADH are customarily known for their role in oxidation/reduction reactions in biological systems. Recently it has been established that NAD+ is consumed during times of stress. In such situations, in-creased activity of poly (ADP-ribose) polymerase has been noted resulting in an increase in DNA damage repair. Stressful situations also cause heightened activity of Sirtuin proteins and cellular sur-vival. The increased activity of these protein families results in the conversion of NAD+ to nicotinamide (NAM). As NAD+ levels decrease, the NAD+ salvage pathway is activated in an attempt to replenish the stock. In the two-step process, NAM is recycled back to NAD+. In the first step, nicotinamide phosphori-bosyltransferase (NAMPT) converts NAM to nicotinamide mononuculeotide (NMN). This step is the rate-determining step and has been linked to diseases such as cancer, diabetes, and Alzheimer’s dis-ease. NAD+ is then regenerated by the adenylation of NMN.

NAMPT is a 55-kDa enzyme existing as a homodimer with two active sites. Currently there are 19 known post-translational modification (PTM) sites reported for NAMPT. The modifications can be the phosphorylation of serine or tyrosine or the ubiquitination of lysine. Our hypothesis states that the PTMs of NAMPT regulate the overall activity of the protein. The PTM will be modified in a series of sys-tematic mutations. Each PTM involving serine will be changed to alanine or glutamate to represent the unphosphorylated or phosphorylated states respectively. Sites involving tyrosine will be mutated to phe-nylalanine or glutamate to represent the unphosphorylated or phosphorylated states respectively. The ubiquitination sites will be changed to alanine. Mutations studied will be S199D, S200D, H247A, K228A, Y188D, Y188F and K389A. It is the aim of this experiment to gain a more comprehensive understand-ing of the regeneration of NAD+ while furthering our knowledge on the role of NAMPT in the restoration of NAD+.

Nampt cDNA was PCR amplified and cloned into Gateway entry vectors, sequenced and recombined into destination vectors specific for bacterial protein expression with an N-terminal 6X Histidine epitope tag. Recombinant Nampt expression was verified by immunoblot analysis and purified on Nickel NTA resin. Protein purity was determined by SDS-PAGE gel electrophoresis followed by Coomassie stain-ing, supplemented by concentration determination by A280 analysis..

Nicotinamide Phosphoribosyltransferase (Nampt) Point Mutation Generation and Purification

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

Nampt structure indicates post translational mutations (yellow and red highlights) to be studied.The red sites are phosphorylation sites while the yellow sites are the ubiquitination sites. Y188 is highlighted in red on the far right.

Purification/Concentration

Gravity drip purification on Ni-NTA column. Panel A: Post purification coomassie stain shows usable con-centrations of Nampt in the 75, 100, 125, 150 mM Imidazole elutions. Panel B: After overnight dialysis in Nampt Buffer (20 mM Tris HCl pH 7.5, 300 mM NaCl, and 10% Glycerol) 125 mM (1a) and 150 mM (2a) Im-idazole elutions were concentrated (1b,2b) and then 15uL of each sample was run down a gel for coo-massie stain analysis. Panel C: Same procedure as Panel B, but with a 1ug calculated (from A280) protein load to normalize each sample. Impurities seen in previous coomassie stains no longer detectable. Panel D: Western Blot Verification of induction bypass on pENTR Nampt Y188F1 purified elutions of 75, 100, 125, 150 mM Imidazole and flow through.

Conclusions/Future Work

Target PTM’s

6 hour inductions are not necessary to produce viable quantity of protein and will be removed from protocols.

Gravity column purification through Ni-NTA is extremely efficient requiring only one binding and 75, 100, 125, and 150 mM Imidazole elutions contain highest quantitiy of proteins with minimal impuri-ties.

Using A280 to quanitify protein and calculate 1ug per load for coomassie stain to normalize coo-massie analysis is the new standard protocol. This gives best representatioin of purity for each elu-tion.

Each mutation made needs to be recombined into the pDEST17 vector for expression in BL21(DE3) pLysS bacteria to generate a 6X HIS epitope tagged protein for further analysis.

Additional mutations are being sequenced and in the process of being made to further analyze. These mutations include H247A, Y64A, Y69A, and Y240 at sequencing.

Western Blot analysis of induction (30 second exposure) of Nampt Wild Type, Y188D1(2), and Y188F1(2). Induced with 1.5 mM IPTG for 6 hours at 37 degrees Celsius in shaker. Lane 1: Nampt Wild Type T=0. Lane 2: Nampt Wild Type T=6. Lane 3: Y188D1 T=0. Lane 4: Y188D1 T=6. Lane 5: Y188D2 T=0. Lane 6: Y188D2 T=6. Lane 7: Y188F1 T=0. Lane 8: Y188F1 T=6. Lane 9: Y188F2 T=0. Lane 10: Y188F2 T=6. Analysis indicates that induction is unnecessary due to indiscernible differences in Nampt protein generated between the T=0 and T=6. Protein generated via overnight cultures and purified using Ni-NTA column gravity purification.

Mutagenesis

Induction Bypass

NMN

PARP-1

Nicotinamide

NAD+

Nampt

Nmnat

Redox Reactions

NADH

ADP/ATP

DNA strand breaks

Poly-ADP-ribosylationReactions

Poly(ADP-ribose) polymers

PARG

Free ADP-ribose

PARP-1 (modified)

Panel A: Nampt cDNA was engineered to contain the mutations shown. These muta-tions include Y188D, Y188F, S200D, K228A, and K389A. Panel B: Transformation plates containing 5ng of pENTR Nampt DNA mutated into pENTR Nampt S200D. Panel C: Verification that the DNA was full length using Not1 for the restriction enzyme digest at 4074 kb.

A

B

C

A

B C

D

4074 kb

~55kDa

1 2 3 4 5 6 7 8 9 10

Indicates ~55 kDa

75 75 100 100 (mM Imidazole) 125 125 150 150 (mM Imidazole)

1a 1b 2a 2b 1a 1b 2a 2b

FT 75 100 125 150 (mM Imidazole)

NAMPT Mutations

WT 552 GGTCTGGAATACAAGTTACATGAT 575

G L E Y K L H D

Y188D 552 GCTCTGGAAGACAAGTTACATGAT 575

G L E D K L H D

Y188F 552 GCTCTGGAATTCAAGTTACATGAT 575

G L E F K L H D

WT 588 GGAGTCTCTTCCCAAGAGACTGTC 611

G V S S Q E T A

S200D 588 GGAGTCTCTGACCAAGAGACTGCT 611

G V S D Q E T A

WT 672 GCTCTAATTAAAAAATATTATGGA 695

A L I K K Y Y G

K228A 672 GCTCTAATTGCAAAATATTATGGA 695

A L I A K Y Y G

WT 1155 TTGCTACAGAAGTTGACAAGAGAT 1178

L L Q K L T R D

K389A 1155 TTGCTACAGGCGTTGACAAGAGAT 1178

L L Q A L T R D