Poly(ADP-Ribose) Polymerases in Host-Pathogen Interactions, Inflammation, and Immunity.

The literature review presented here details recent research involving members of the poly(ADP-ribose) polymerase (PARP) family of proteins. Among the 17 recognized members of the family, the human enzyme PARP1 is the most extensively studied, resulting in a number of known biological and metabolic roles. This review is focused on the roles played by PARP enzymes in host-pathogen interactions and in diseases with an associated inflammatory response. In mammalian cells, several PARPs have specific roles in the antiviral response; this is perhaps best illustrated by PARP13, also termed the zinc finger antiviral protein (ZAP). Plant stress responses and immunity are also regulated by poly(ADP-ribosyl)ation. PARPs promote inflammatory responses by stimulating proinflammatory signal transduction pathways that lead to the expression of cytokines and cell adhesion molecules. Hence, PARP inhibitors show promise in the treatment of inflammatory disorders and conditions with an inflammatory component, such as diabetes, arthritis, and stroke. These functions are correlated with the biophysical characteristics of PARP family enzymes. This work is important in providing a comprehensive understanding of the molecular basis of pathogenesis and host responses, as well as in the identification of inhibitors. This is important because the identification of inhibitors has been shown to be effective in arresting the progression of disease.

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights.

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.

Methods to identify the NMR resonances of the ¹³C-dimethyl N-terminal amine on reductively methylated proteins.

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive (13)C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive (13)C-methylation is a chemical reaction performed under mild conditions that modifies a protein's primary amino groups (lysine ε-amino groups and the N-terminal α-amino group) to (13)C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive (13)C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein (13)C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of (13)C-labeling at each primary amino group. One limitation of the method arises when the protein's N-terminal residue is a lysine because the α- and ε-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the (13)C-dimethylamines associated with both the N-terminal α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme, Lys1, are identified in (1)H-(13)C heteronuclear single-quantum coherence spectra.

Multicomponent analyses of chiral samples by use of regression analysis of UV-visible spectra of cyclodextrin guest-host complexes.

We report the first combined use of analytical spectroscopy, guest-host chemistry, and multivariate regression analysis for determination of enantiometric composition of multicomponent samples of chiral analytes. Sample solutions containing multicomponent analytes of ephedrine, tryptophan, propranolol, and proline of varying enantiomeric composition with beta-cyclodextrin (BCD) or methyl-beta-cyclodextrin (Me-BCD) as chiral host molecules were investigated using ultraviolet (UV)-visible spectroscopy. The interactions of enantiomers of chiral analytes with chiral hosts resulted in the formation of transient diastereomeric inclusion complexes with varying spectral properties. Multivariate analysis using partial-least-square (PLS) regression was used to correlate subtle changes in the UV-visible spectra of the guest-host complexes with the enantiomeric composition of the calibration samples. These PLS regressions were carefully optimized and then used to predict the enantiomeric composition of multicomponent chiral analytes of validation samples. The results of these validation studies demonstrate the predictive ability of the regression models for determination of future enantiomeric composition of samples. The accuracy of the models to correctly predict the enantiomeric composition of samples, evaluated by use of the root mean square percent relative error (RMS%RE) was analyte and chiral host dependent. In general, better prediction of enantiomeric composition of samples and low RMS%RE values were obtained when Me-BCD was used as the chiral host. The analyses procedure reported here is simple, rapid, and inexpensive. In addition, this approach does not require prior separation of chiral analytes, thus reducing analysis time and eliminating the need for expensive chiral columns.