Pathological secondary modification of proteins

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2013Cataloged from PDF version of thesis.Includes bibliographical references.There is increasing recognition that aberrant protein modifications play an important role in the pathophysiology of inflammation and oxidative stress in cells. We recently discovered that N⁶-formylation of lysine is an abundant endogenous modification of histone and chromatin proteins. The high abundance of N⁶-formyllysine in histone proteins and its chemical similarity to the biologically important N⁶- acetyllysine has raised questions about its mechanisms of formation and biological consequences. Using novel ultrasensitive and specific liquid chromatography-coupled tandem mass spectrometry methods (LC-MS/MS) to quantify N⁶-formyllysine lesions in proteins, we aimed to investigate the sources as well as the fate of this abundant endogenous protein modification. We present evidence that endogenous formaldehyde is a major source of N⁶-formyllysine and that this adduct is widespread among proteins in all cellular compartments. We observed in vitro as well as in vivo that formaldehyde exposure leads to a dose-dependent increase in N⁶-formyllysine protein adducts, with the use of isotopically-labeled formaldehyde to dissect endogenous from exogenous formaldehyde as sources of the adduct. Further, other isotope labeling studies revealed that lysine demethylation in histone proteins is not a source of N⁶-formyllysine. With regard to N⁶-formyllysine persistence in cells, our investigation of histone deacetylases revealed that despite chemical similarity of N⁶-formyllysine to N⁶-acetyllysine, the former is refractory to removal by histone deacetylases, which suggests that they will persist throughout the life of individual histone proteins. If not repaired, lysine formylation could accumulate to significant levels. The resemblance of N⁶-formyllysine to N⁶-acetyllysine, together with recent studies that mapped its location on many conserved lysine acetylation and methylation sites along histone proteins, support the idea that this abundant protein modification could interfere with normal regulation of gene expression, potentially leading to an epigenetic mechanism of disruption of cell function.by Bahar Edrissi.Ph.D

N 6 -Formyllysine as a Biomarker of Formaldehyde Exposure: Formation and Loss of N 6 -Formyllysine in Nasal Epithelium in Long-Term, Low-Dose Inhalation Studies in Rats

Exposure to both endogenous and exogenous formaldehyde has been established to be carcinogenic, likely by virtue of forming nucleic acid and proteins adducts such as N6-formyllysine. To better assess N6-formyllysine as a biomarker of formaldehyde exposure, we studied accumulation of N6-formyllysine adducts in tissues of rats exposed by inhalation to 2 ppm [13C2H2]-formaldehyde for 7, 14, 21, and 28 days (6 h/day) and investigated adduct loss over a 7-day postexposure period using liquid chromatography-coupled tandem mass spectrometry. Our results showed formation of exogenous adducts in nasal epithelium and to some extent in trachea but not in distant tissues of lung, bone marrow, or white blood cells, with a 2-fold increase over endogenous N6-formyllysine over a 3-week exposure period. Postexposure analyses indicated a biexponential decay of N6-formyllysine in proteins extracted from different cellular compartments, with half-lives of ∼25 and ∼182 h for the fast and slow phases, respectively, in cytoplasmic proteins. These results parallel the behavior of DNA adducts and DNA-protein cross-links, with protein adducts cleared faster than DNA-protein cross-links, and point to the potential utility of N6-formyllysine protein adducts as biomarkers of formaldehyde

Dosimetry of N 6 -Formyllysine Adducts Following [ 13 C 2 H 2 ]-Formaldehyde Exposures in Rats

With formaldehyde as the major source of endogenous N6-formyllysine protein adducts, we quantified endogenous and exogenous N6-formyllysine in the nasal epithelium of rats exposed by inhalation to 0.7, 2, 5.8, and 9.1 ppm [13C2H2]-formaldehyde using liquid chromatography-coupled tandem mass spectrometry. Exogenous N6-formyllysine was detected in the nasal epithelium, with concentration-dependent formation in total as well as fractionated (cytoplasmic, membrane, nuclear) proteins, but was not detected in the lung, liver, or bone marrow. Endogenous adducts dominated at all exposure conditions, with a 6 h 9.1 ppm formaldehyde exposure resulting in one-third of the total load of N6-formyllysine being derived from exogenous sources. The results parallel previous studies of formaldehyde-induced DNA adducts

Quantitative Analysis of Histone Modifications: Formaldehyde Is a Source of Pathological N6-Formyllysine That Is Refractory to Histone Deacetylases

Aberrant protein modifications play an important role in the pathophysiology of many human diseases, in terms of both dysfunction of physiological modifications and the formation of pathological modifications by reaction of proteins with endogenous electrophiles. Recent studies have identified a chemical homolog of lysine acetylation, N[superscript 6]-formyllysine, as an abundant modification of histone and chromatin proteins, one possible source of which is the reaction of lysine with 3′-formylphosphate residues from DNA oxidation. Using a new liquid chromatography-coupled to tandem mass spectrometry method to quantify all N[superscript 6]-methyl-, -acetyl- and -formyl-lysine modifications, we now report that endogenous formaldehyde is a major source of N[superscript 6]-formyllysine and that this adduct is widespread among cellular proteins in all compartments. N[superscript 6]-formyllysine was evenly distributed among different classes of histone proteins from human TK6 cells at 1–4 modifications per 10[superscript 4] lysines, which contrasted strongly with lysine acetylation and mono-, di-, and tri-methylation levels of 1.5-380, 5-870, 0-1400, and 0-390 per 10[superscript 4] lysines, respectively. While isotope labeling studies revealed that lysine demethylation is not a source of N[superscript 6]-formyllysine in histones, formaldehyde exposure was observed to cause a dose-dependent increase in N[superscript 6]-formyllysine, with use of [[superscript 13]C,[superscript 2]H[subscript 2]]-formaldehyde revealing unchanged levels of adducts derived from endogenous sources. Inhibitors of class I and class II histone deacetylases did not affect the levels of N[superscript 6]-formyllysine in TK6 cells, and the class III histone deacetylase, SIRT1, had minimal activity (<10%) with a peptide substrate containing the formyl adduct. These data suggest that N[superscript 6]-formyllysine is refractory to removal by histone deacetylases, which supports the idea that this abundant protein modification could interfere with normal regulation of gene expression if it arises at conserved sites of physiological protein secondary modification

Different lysine species detected in purified histone H4 from TK6 cells.

<p>Lysine adducts were monitored by tandem mass spectrometry, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003328#s4" target="_blank"><i>Materials and Methods</i></a>. Abbreviations: FK, N⁶-formyllysine; AK, N⁶-acetyllysine; K, lysine; MK, N⁶-mono-methyllysine; M₂K, N⁶-di-methyllysine; M₃K, N⁶-tri-methyllysine.</p

Addition of [¹³C,²H₂]-formaldehyde to TK6 cells distinguishes exogenous from endogenous sources of N⁶-formyllysine.

<p>(A) LC-MS/MS analysis showing signals for the three isotopomeric N⁶-formyllysine species, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003328#s4" target="_blank"><i>Materials and Methods</i></a>. (B) Plot of N⁶-formyllysine levels as a function of exposure to [¹³C,²H₂]-formaldehyde. Data represent mean ± SD for N = 3.</p

Formaldehyde is a source of N⁶-formyllysine.

<p>Formation of N⁶-formyllysine in (A) <i>in vitro</i> reactions of 1 mM L-lysine with formaldehyde for 2 h at 37°C, and in (B) TK6 cells exposed to formaldehyde, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003328#s4" target="_blank"><i>Materials and Methods</i></a>. Data represent mean ± SD for N = 3, with asterisks denoting statistically significant differences by Student's t-test (p<0.05).</p

Summary of mass spectrometry parameters for each species.

<p>Summary of mass spectrometry parameters for each species.</p