Journal of Molecular Biology
Crystal Structure of Visfatin/Pre-B Cell Colony-enhancing Factor 1/Nicotinamide Phosphoribosyltransferase, Free and in Complex with the Anti-cancer Agent FK-866
Introduction
The recently identified adipokine visfatin, which is highly enriched in the visceral fat of humans,1 corresponds to pre-B cell colony-enhancing factor 1 (PBEF), which has been described as both a secreted cytokine playing a highly conserved role in innate immunity and an intracellular phosphoribosyltransferase (PRTase). The novel visfatin gene was first isolated from a human peripheral blood lymphocyte cDNA library2 and encodes a secreted 52 kDa cytokine that acts as a growth factor for early stage B cells, even though it lacks a signal peptide. But subsequent experiments revealed additional biological activities that suggested intracellular as well as extracellular localization of visfatin. Interestingly, visfatin mimics insulin signaling by binding to the insulin receptor with an affinity similar to that of insulin (Kd = 3 nM). Visfatin does not compete with insulin, however, suggesting that the two proteins bind to separate sites on the receptor.1
Visfatin is up-regulated in neutrophils and monocytes in response to a variety of inflammatory stimuli and functions as a novel inhibitor of apoptosis. Several lines of evidence suggest the anti-apoptotic activity of visfatin is dependent on its secretion from neutrophils,3 and it appears to be one of the mediators of the later stages of the systemic inflammatory response. Visfatin is also constitutively expressed by the fetal membranes during pregnancy and is up-regulated when the membranes are infected,4 suggesting that it may play a role in infection-induced preterm birth.
Other researchers have found visfatin to be primarily situated in the cell nucleus and cytoplasm. Its possible role in NAD biosynthesis is suggested by the discovery that a bacterial homolog is NadV5 which has the enzymatic activity of nicotinamide phosphoribosyltransferase (NAmPRTase), which catalyzes the reaction between nicotinamide (NAm) and phosphoribosyl pyrophosphate (PRPP) to yield nicotinamide mononucleotide (NMN) (Figure 1).
NAD can also be synthesized from quinolinic acid (QA) and nicotinate (NA) via analogous pathways. Indeed, Rongvaux et al. characterized murine visfatin as an intracellular NAmPRTase and demonstrated that it can rescue a mutant bacterium deficient in this enzymatic activity. Visfatin also was identified as a positive regulator of NAD-dependent protein deacetylase activity, which is essential for maturation of smooth muscle cells.6 Kitani et al. concluded that visfatin is an intracellular protein associated with the cell cycle based on the observation that rat visfatin is localized in the nuclei of confluent cells but in the cytoplasm of proliferating ones.7
Notably, visfatin was also found to be the most highly up-regulated gene in a canine model of acute lung injury, thus making visfatin a potentially novel biomarker in acute lung injury.8 Visfatin also was identified as one of the genes overexpressed in primary colorectal cancer.9 In that regard, FK-866 (Figure 1), which was developed as an anti-cancer agent,10 interferes with NAD biosynthesis, showing a particularly high specificity for NAmPRTase. Inhibition of NAD synthesis using FK-866 induced apoptosis in human leukemia and hepatocarcinoma cells and in various types of tumor xenografts. Moreover, FK-866 has anti-tumoral, anti-metastatic and strong anti-angiogenic activities in RENCA mice,11 and effectively induced delayed apoptotic cell death in HepG2 human liver carcinoma cells with an IC50 of approximately 1 nM.10
To gain insight into the mechanism of action by this multifunctional protein, we have determined the first crystal structure of rat visfatin in three forms: the apo form, the nicotinamide mononucleotide (NMN)-bound form and the FK-866-inhibited form. We found that visfatin forms a homodimer and that FK-866 binds at the interface between the two subunits, partially overlapping the NMN binding site. In addition, we carried out a kinetic study using isothermal titration calorimetry (ITC) to assess FK-866 affinity and enzyme substrate specificity, and structural modeling for the visfatin and insulin receptor complex. Analysis of the structures and biochemical data reveal the enzymatic specificity of visfatin, and suggest the model of visfatin and insulin receptor complex.
Section snippets
Overall structure
To solve the structure of Rattus norvegicus visfatin, we overexpressed the protein in an Escherichia coli expression host and crystallized it as described in Materials and Methods. The crystal structure was solved using the multiwavelength anomalous dispersion (MAD) method with selenomethionine-substituted and K3IrCl6-soaked crystals. Table 1 summarizes the statistics for the crystallographic data collection and refinement. The final models of the visfatin dimer in each crystallographic
Discussion
This study describes the first crystal structure of visfatin, which is a multifunctional protein involved in the differentiation of pre-B cells,2 the NAD biosynthesis and the control of plasma glucose levels through the activation of insulin receptor.1 Interestingly, visfatin contains NAmPRTase, the first enzyme in the salvage pathway enabling the recycling of NAm to NAD.13 The NAD salvage pathway has recently attracted much interest, as it allows restoration of the NAD pool following intensive
Protein expression and purification
The rat visfatin gene was amplified from cDNA by PCR using a GeneAmp PCR system 2400® thermocycler (Perkin Elmer). PCR was run in ThermoPol® buffer (New England Biolabs) using NQ® DNA polymerase (Anygen). The forward and reverse primers (5′-ATCGGATCC-ATGAATGCTGCGGCAGAAGCC-3′ and 5′-TGACTCTCGAGCTAGTGAGGCGCCACA-TCCTGC-3′) were designed based on the visfatin gene sequence in GenBank, accession no. AB081730. The PCR product and the pVFT-His expression vector were digested using BamHI and XhoI,
Acknowledgements
The authors thank Dr John Marshall (University Health Network in Canada) and Dr Hitoshi Fujisawa (Shiga Medical Center Research Institute in Japan) for providing the cDNAs for rat and human visfatin, respectively. We thank the NIMH Chemical Synthesis and Drug Supply Program for providing the FK-866 compound. All data collection was carried out at 6B MX and 4A MXW beamlines of the Pohang Light Source, Korea. This work was supported by a grant from the Center for Functional Analysis of Human
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