Loma Linda University

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Jonathan Neidigh, PhD
Assistant Professor, Basic Sciences
School of Medicine
Publications    Scholarly Journals--Published
  • “The interaction between HPV 16 and FADD is mediated by a novel E6 binding domain” S. S. Tungteakkhun, M. Filippova, J. W. Neidigh, N. Fodor, and P. J. Duerksen-Hughes. Journal of Virology. 82, 9600 - 9614 (2008). ( 11/2008 )
  • “Hypochlorous acid damages histone proteins forming 3-chlorotyrosine and 3,5-dichlorotyrosine” J. I. Kang, Jr. and J. W. Neidigh. Chemical Research in Toxicology. 21, 1028 – 1038 (2008). ( 9/2008 )
  • “The Trp-cage: optimizing the stability of a globular miniprotein” B. Barua, J. C. Lin, V. D. Williams, P. Kummler, J. W. Neidigh, and N. H. Andersen. Protein Engineering Design and Selection. 21, 171 – 185 (2008). ( 8/2008 )
  • “Measurement of the incorporation and repair of exogenous 5-hydroxymethyl-2''''-deoxyuridine in human cells in culture using gas chromatography-negative chemical ionization-mass spectrometry” D. K. Rogstad, A. Darwanto, J. L. Herring, K. N. Rogstad, A. Burdzy, S. R. Hadley, J. W. Neidigh, and L. C. Sowers. Chemical Research in Toxicology. 20, 1787 – 17896 (2008). ( 6/2008 )
  • Holder S, Zemskova, M. Zhang, C, Maryam, T. Bremer, R. Neidigh, JW, Lilly, MB. "Characterization of a Potent and Selective Small-Molecule Inhibitor of the PIM1 Kinase." Molecular Cancer Therapeutics. 6, 162 - 172 (2007) ( 8/2007 )
    The pim-1 kinase is a true oncogene that has been implicated in the development of leukemias, lymphomas, and prostate cancer, and is the target of drug development programs. We have used experimental approaches to identify a selective, cell-permeable small molecule inhibitor of the pim-1 kinase, to foster basic and translational studies of the enzyme. We used an ELISAbased kinase assay to screen a diversity library of potential kinase inhibitors. The flavonol quercetagetin (3, 3?, 4?, 5, 6, 7-hydroxyflavone) was identified as a moderately potent, ATPcompetitive inhibitor (IC50 = 0.34µM). Resolution of the crystal structure of PIM1 in complex with quercetagetin or three other flavonoids revealed a spectrum of binding poses and hydrogen bonding patterns in spite of strong similarity of the ligands. Quercetagetin was a highly selective inhibitor of PIM1 compared to PIM2 and seven other serine-threonine kinases. Quercetagetin was able to inhibit PIM1 activity in intact RWPE2 prostate cancer cells in a dose-dependent manner (ED50 = 5.5µM). RWPE2 cells treated with quercetagetin showed pronounced growth inhibition at inhibitor concentrations that blocked pim-1 kinase activity. Furthermore the ability of quercetagetin to inhibit the growth of other prostate epithelial cell lines varied in proportion to their levels of PIM1 protein. Quercetagetin can function as a moderately potent and selective, cell-permeable inhibitor of the pim-1 kinase, and may be useful for proof-of-concept studies to support the development of clinically useful PIM1 inhibitors.
  • Byrns MC, Vu CC, Neidigh JW, Abad JL, Jones RA, Peterson LA. "Detection of DNA adducts derived from the reactive metabolite of furan, cis-2-butene-1,4-dial.." Chemical Research in Toxicology 19.3 (2006): 414-420. ( 1/2006 ) Link...
    Furan is a toxic and carcinogenic compound used in industry and commonly found in the environment. The mechanism of furan's carcinogenesis is not well-understood and may involve both genotoxic and nongenotoxic pathways. Furan undergoes oxidation by cytochrome P450 to cis-2-butene-1,4-dial, which is thought to mediate furan's toxic effects. Consistently, cis-2-butene-1,4-dial readily reacts with glutathione, amino acids, and nucleosides. To determine the importance of DNA alkylation in furan-induced carcinogenesis, we developed an assay for the detection of cis-2-butene-1,4-dial-derived DNA adducts. DNA samples were treated with O-benzyl-hydroxylamine, which reacts with the aldehyde functionality of the DNA adducts. Enzyme hydrolysates of these samples were then analyzed by capillary electrospray tandem mass spectrometry with selected reaction monitoring. The dCyd and dAdo adducts were detected in digests of DNA treated with nanomolar concentrations of cis-2-butene-1,4-dial. In addition, these adducts were present in DNA isolated from Ames assay strain TA104 treated with mutagenic concentrations of cis-2-butene-1,4-dial. These data support the hypothesis that cis-butene-1,4-dial is a genotoxic metabolite of furan. This method will allow us to explore the role of these adducts in furan-induced carcinogenesis.
  • Valinluck V, Wu W, Liu P, Neidigh JW, Sowers LC. "Impact of cytosine 5-halogens on the interaction of DNA with restriction endonucleases and methyltransferase." Chemical Research in Toxicology 19.4 (2006): 556-562. ( 1/2006 ) Link...
    Growing evidence from both prokaryotes and eukaryotes indicates that pyrimidine 5-methyl groups can have profound biological consequences that are mediated by the affinity of DNA-protein interactions. The presence of the 5-methyl group could potentially create a steric block preventing the binding of some proteins whereas the affinity of many other proteins is substantially increased by pyrimidine methylation. In this paper, we have constructed a series of oligonucleotides containing cytosine and a series of 5-substituted cytosine analogues including all halogens. This set of oligonucleotides has been used to probe the relationship between the size of the substituent and its capacity to modulate cleavage by the methylation-sensitive restriction endonucleases MspI and HpaII. Additionally, we have examined the impact of the halogen substitution on the corresponding bacterial methyltransferase (M.HpaII). We observed that MspI cleavage is only subtly affected by substituted cytosine analogues at the inner position of the CCGG recognition site. In contrast, HpaII cleaves cytosine-containing oligonucleotides completely whereas 5-fluorocytosine-containing oligonucleotides are cleaved at a reduced rate. The presence of the larger halogens Cl, Br, or I as well as a methyl group completely prevents cleavage by HpaII. These data suggest that the steric wall is encountered by HpaII slightly beyond the fluorine substituent, at about 2.65 A from the pyrimidine C5-position. It is known that 5-fluorocytosine in an oligonucleotide can form a covalent irreversible suicide complex with either prokaryotic or eukaryotic methyltransferases. Kinetic data reported here suggest that the 5-fluorocytosine-containing oligonucleotide can also inhibit M.HpaII by formation of a reversible, noncovalent complex. Our results indicate that although a 5-Cl substituent has electronic properties similar to 5-F, 5-chlorocytosine duplexes neither form a complex with M.HpaII nor inhibit enzymatic methylation. Emerging data suggest that halogenation of cytosine can occur in DNA in vivo from inflammation-mediated reactive molecules. The results reported here suggest that the inadvertent halogenation of cytosine residues in DNA could alter the affinity of sequence-specific DNA-binding proteins.
  • N. H. Andersen, Y. Brodsky, J. W. Neidigh, and K. S. Prickett. "Medium-Dependence of the Secondary Structure of Exendin-4 and Glucagon-like-peptide-1." Bioorganic & Medicinal Chemistry 10.1 (2002): 79-85. ( 1/2002 ) Link...
    Exendin-4 is a natural, 39-residue peptide first isolated from the salivary secretions of a Gila Monster (Heloderma suspectum) that has some pharmacological properties similar to glucagon-like-peptide-1 (GLP-1). This paper reports differences in the structural preferences of these two peptides. For GLP-1 in aqueous buffer (pH 3.5 or 5.9), the concentration dependence of circular dichroism spectra suggests that substantial helicity results only as a consequence of helix bundle formation. In contrast, exendin-4 is significantly helical in aqueous buffer even at the lowest concentration examined (2.3 μM). The pH dependence of the helical signal for exendin-4 indicates that helicity is enhanced by a more favorable sequence alignment of oppositely charged sidechains. Both peptides become more helical upon addition of either lipid micelles or fluoroalcohols. The stabilities of the helices were assessed from the thermal gradient of ellipticity (∂[θ]221/∂T values); on this basis, the exendin helix does not melt appreciably until temperatures significantly above ambient. The extent of helix formation for exendin-4 in aqueous buffer (and the thermal stability of the resulting helix) suggests the presence of a stable helix-capping interaction which was localized to the C-terminal segment by NMR studies of NH exchange protection. Solvent effects on the thermal stability of the helix indicate that the C-terminal capping interaction is hydrophobic in nature. The absence of this C-capping interaction and the presence of a flexible, helix-destabilizing glycine at residue 16 in GLP-1 are the likely causes of the greater fragility of the monomeric helical state of GLP-1. The intramolecular hydrophobic clustering in exendin-4 also appears to decrease the extent of helical aggregate formation.
  • J. W. Neidigh, R. M. Fesinmeyer, and N. H. Andersen. "Designing a 20 Residue Protein." Nature Structural Biology 9.6 (2002): 425-430. ( 1/2002 ) Link...
    Truncation and mutation of a poorly folded 39-residue peptide has produced 20-residue constructs that are >95% folded in water at physiological pH. These constructs optimize a novel fold, designated as the 'Trp-cage' motif, and are significantly more stable than any other miniprotein reported to date. Folding is cooperative and hydrophobically driven by the encapsulation of a Trp side chain in a sheath of Pro rings. As the smallest protein-like construct, Trp-cage miniproteins should provide a testing ground for both experimental studies and computational simulations of protein folding and unfolding pathways. Pro Trp interactions may be a particularly effective strategy for the a priori design of self-folding peptides.
  • J. W. Neidigh and N. H. Andersen.. "Peptide Conformational Changes Induced by Trptophan-Phosphocholine Interactions in a Micelle." Biopolymers 65.5 (2002): 354-361. ( 1/2002 ) Link...
    Sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC) micelles are often used to mimic the membrane- or receptor-bound states of peptides in NMR studies. From the present examination of a 26-residue analog of exendin-4 (TrEX4) by NMR and CD in water, aqueous 30% trifluoroethanol (TFE), and bound to both SDS and DPC micelles, it is clear that these two lipid micelles can yield very different peptide structures. The Trp-cage fold (also observed in 30% TFE) is present when TrEX4 is bound to SDS micelles; however, tertiary structure is absent in the presence of DPC micelles. The loss of tertiary structure is attributed to an energetically favorable interaction (estimated as 2-3 kcal/mol) of the tryptophan side chain with the phosphocholine head groups. These dramatic structural differences suggest that care must be taken when using either SDS or DPC to mimic the membrane- or receptor-bound states.
  • J. W. Neidigh, R. M. Fesinmeyer, K. S. Prickett, and N. H. Andersen. "Exendin-4 and Glucagon-like-peptide-1: NMR Structural Comparisons in the Solution and Micelle-Associated States." Biochemistry 40.44 (2001): 13188-13200. ( 1/2001 ) Link...
    Exendin-4, a 39 amino acid peptide originally isolated from the oral secretions of the lizard Heloderma suspectum, has been shown to share certain activities with glucagon-like-peptide-1 (GLP-1), a 30 amino acid peptide. We have determined the structuring preferences of exendin-4 and GLP-1 by NMR in both the solution and dodecylphosphocholine (DPC) micelle-associated states. Based on both chemical shift deviations and the pattern of intermediate range NOEs, both peptides display significant helicity from residue 7 to residue 28 with greater fraying at the N-terminus. Thornton and Gorenstein [(1994) Biochemistry 33, 3532-3539] reported that the presence of a flexible, helix-destabilizing, glycine at residue 16 in GLP-1 was an important feature for membrane and receptor binding. Exendin-4 has a helix-favoring glutamate as residue 16. In the micelle-associated state, NMR data indicate that GLP-1 is less helical than exendin-4 due to the presence of Gly16; chemical shift deviations along the peptide sequence suggest that Gly16 serves as an N-cap for a second, more persistent, helix. In 30 vol-% trifluoroethanol (TFE), a single continuous helix is evident in a significant fraction of the GLP-1 conformers present. Exendin-4 has a more regular and less fluxional helix in both media and displays stable tertiary structure in the solution state. In the micelle-bound state of exendin-4, a single helix (residues 11-27) is observed with residues 31-39 completely disordered and undergoing rapid segmental motion. In aqueous fluoroalcohol or aqueous glycol, the Leu21-Pro38 span of exendin-4 forms a compact tertiary fold (the Trp-cage) which shields the side chain of Trp25 from solvent exposure and produces ring current shifts as large as 3 ppm. This tertiary structure is partially populated in water and fully populated in aqueous TFE. The Leu21-Pro38 segment of exendin-4 may be the smallest protein-like folding unit observed to date. When the Trp-cage forms, fraying of the exendin-4 helix occurs exclusively from the N-terminus; backbone NHs for the C-terminal residues of the helix display H/D exchange protection factors as large as 105 at 9 C. In contrast, no tertiary structure is evident when exendin-4 binds to DPC micelles. An energetically favorable insertion of the tryptophan ring into the DPC micelle is suggested as the basis for this change. With the exception of exendin-4 in media containing fluoro alcohol cosolvents, NMR structure ensembles generated from the NOE data do not fully reflect the conformational averaging present in these systems. Secondary structure definition from chemical shift deviations may be the most appropriate treatment for peptides that lack tertiary structure.
  • N. H. Andersen, R. B. Dyer, R. M. Fesinmeyer, F. Gai, Z. Liu, J. W. Neidigh, and H. Tong.. "Effect of Hexafluoroisopropanol on the Thermodynamics of Peptide Secondary Structure Formation." Journal of the American Chemcal Society 121.42 (1999): 9879-9880. ( 1/1999 ) Link...
  • N. H. Andersen, J. W. Neidigh, S. M. Harris, G. M. Lee, Z. Liu, and H. Tong.. "Extracting Information from the Temperature Gradients of Polypeptide NH Chemical Shifts: I. The Importance of Conformational Averaging." Journal of the American Chemical Society 119.36 (1997): 8547-8561. ( 1/1997 ) Link...
    A detailed analysis of backbone amide NH chemical shift temperature gradients (/T values) for proteins and highly cross-linked peptides reveals that hydrogen-bonded exchange-protected NHs are characterized by /T values of -2.0 1.4 ppb/C while exposed NHs typically display gradients of -6.0 -8.5 ppb/C; however, numerous exceptions to these generalizations occur. For partially folded peptides (rather than proteins), exceptions are more common than concordance with this rule; /T values ranging from -28 to +12 ppb/C have been observed. In the case of the peptide systems for which exchange protection data is available, the common practice of assuming that a /T value less negative than -4 ppb/C indicates that the NH is sequestered from solvent is shown to have zero predictive validity. The analysis of the data for partially folded peptides, protein fragments, and other peptides which are expected to display minimal structuring reveals a significant correlation between /T and the deviation of NH from the random coil reference shift. The analysis was facilitated by plotting NH chemical shift deviations (NH-CSD) versus the /T values. Using such plots, slow-exchanging hydrogen-bonded sites in proteins can be determined with much higher confidence than using the value of the gradient alone. For peptides, the occurrence of large shift deviations and abnormal gradients are diagnostic for partial structuring at lower temperatures which becomes increasingly randomized on warming. A good correlation coefficient (R 0.75) for NH-CSD and /T values indicates that essentially all of the NH shift deviation from reference values is due to the concerted formation of a single structured state on cooling. Correlation coefficients greater than 0.95 were observed for both helix and -hairpin forming peptides. The slope of the correlation plot (parts per thousand/C) is a measure of the decrease in the population of the structured state upon warming. A detailed model which rationalizes the effects of conformational equilibria upon NH shifts is presented. A positive Cp for unfolding is required to rationalize the linearity of NH with temperature that is routinely observed for partially structured peptides. This analysis suggests that ordered states of short peptides achieve significant populations in water only when the hydrophobic effect favors the structured state. This conclusion is pertinent to the current questions concerning the temporal sequence of secondary versus tertiary structure formation during protein folding. Further, it is suggested that the use of NMR parameters (scalar and dipolar couplings) to derive the structural preferences of protein fragments which might serve a "seeding" role in the folding pathway is justified only when the CSD/gradient plot displays both a correlation coefficient greater than 0.70 and significant NH-CSD values (CSD > 0.3).