Pentosinane, a Post-Translational Modification of Human Proteins with Underappreciated Stability.

Pentosinane is a structurally complex nonenzymatic post-translational modification of proteins believed to be present in all living things. It falls into the category of advanced glycation end products (AGEs) and is structurally related to the other AGEs pentosidine and glucosepane. Although pentosidine and glucosepane have been widely studied for their role in wide-ranging conditions (e.g., diabetes mellitus, Alzheimer's disease, and human aging), relatively little is known about pentosinane. Interestingly, previous reports have suggested that pentosidine may derive from pentosinane. The (patho)physiological significance of pentosinane in humans is largely unexplored. As a first step to address this knowledge gap, we report herein the first total synthesis of pentosinane. Our synthesis is high yielding (1.7% over seven steps), concise, and enantioselective, and it leverages a strategy for synthesizing 2,5-diaminoimidazoles previously developed by our lab. Access to synthetic pentosinane has allowed us to perform additional studies showing that its oxidation to pentosidine is both pH and oxygen dependent and is substantially slower under physiological conditions than previously believed. Additionally, pentosinane rapidly decomposes under harshly acidic conditions typically employed for pentosidine isolation. Taken together, these results suggest that pentosinane is likely to be more abundant in vivo than previously appreciated. We believe these results represent a critical step toward illuminating the role(s) of pentosinane in human biology.

Methylglyoxal-derived posttranslational arginine modifications are abundant histone marks.

Histone posttranslational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. Herein we describe the existence of Lys and Arg modifications on histones by a glycolytic by-product, methylglyoxal (MGO). Our data demonstrate that adduction of histones by MGO is an abundant modification, present at the same order of magnitude as Arg methylation. These modifications were detected on all four core histones at critical residues involved in both nucleosome stability and reader domain binding. In addition, MGO treatment of cells lacking the major detoxifying enzyme, glyoxalase 1, results in marked disruption of H2B acetylation and ubiquitylation without affecting H2A, H3, and H4 modifications. Using RNA sequencing, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Finally, we show that the deglycase DJ-1 protects histones from adduction by MGO. Collectively, our findings demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far-reaching implications for the control of gene expression and protein transcription linked to metabolism.

One-Step Synthesis of 2,5-Diaminoimidazoles and Total Synthesis of Methylglyoxal-Derived Imidazolium Crosslink (MODIC).

Here we describe a general method for the synthesis of 2,5-diaminoimidazoles, which involves a thermal reaction between α-aminoketones and substituted guanylhydrazines without the need for additives. As one of the few known ways to access the 2,5-diaminoimidazole motif, our method greatly expands the number of reported diaminoimidazoles and further supports our previous observations that these compounds spontaneously adopt the non-aromatic 4(H) tautomer. The reaction works successfully on both cyclic and acyclic amino ketone starting materials, as well as a range of substituted guanylhydrazines. Following optimization, the method was applied to the efficient synthesis of the advanced glycation end product (AGE) methylglyoxal-derived imidazolium crosslink (MODIC). We expect that this method will enable rapid access to a variety of biologically important 2,5-diaminoimidazole-containing products.

Generation and characterization of antibodies against arginine-derived advanced glycation endproducts.

Although antibodies reagents have been widely employed for studying advanced glycation end-products (AGEs), these materials have been produced using complex mixtures of immunogens. Consequently, their epitope specificity remains unknown. Here we have generated the first antibodies capable of recognizing each of the three isomers of the methylglyoxal hydroimidazolones (MG-Hs) by using chemical synthesis to create homogenous immunogens. Furthermore, we have thoroughly characterized the epitope specificity of both our antibodies and that of two existing monoclonals by implementing a direct ELISA protocol employing synthetic MG-H antigens. Finally, we employed the reported anti-MG-H antibodies to the detection of MG-Hs in cellular systems using immunofluorescence microscopy. These studies have demonstrated that anti-MG-H1 and anti-MG-H3 staining is concentrated within the nucleus, while anti-MG-H2 affords only minimal signal. These observations are consistent with reported formation preferences for MG-Hs, and may suggest novel nuclear targets for non-enzymatic posttranslational modification. The antibody reagents reported herein, as well as the strategy employed for their creation, are likely to prove useful for the immunochemical study of AGEs in biological systems.

A three-step process to facilitate the annulation of polycyclic aromatic hydrocarbons.

A new efficient three-step process to annulate polycyclic aromatic hydrocarbons (PAHs) has been developed, providing access to PAHs with saturated rings that under current chemical methods would be difficult to produce in an efficient manner. This method relies on a palladium-catalyzed cross-coupling reaction of various brominated PAHs with cyclohexanone to yield α-arylated ketones, which are converted to regiospecific vinyl triflates and cyclized by a palladium-catalyzed intramolecular arene-vinyl triflate coupling to produce PAHs with incorporated saturated rings or "tetrahydroindeno-annulated" PAHs.

Targeting the Expanded TCF4/Fuchs' Endothelial Corneal Dystrophy CUG Repeat with Morpholino Peptide Conjugates.

Fuchs' corneal endothelial dystrophy (FECD) is a major cause of vision loss. Corneal transplantation is the only effective curative treatment, but this surgery has limitations. A pharmacological intervention would complement surgery and be beneficial for many patients. FECD is caused by an expanded CUG repeat within intron 2 of the TCF4 RNA. Agents that recognize the expanded repeat can reverse the splicing defects associated with the disease. Successful drug development will require diverse strategies for optimizing the efficacy of anti-CUG oligomers. In this study, we evaluate anti-CUG morpholinos conjugated to cyclic cell penetrating peptides. The morpholino domain of the conjugate is complementary to the repeat, while the peptide has been optimized for import across cell membranes. We show that morpholino conjugates can enter corneal endothelial cells and block the CUG RNA foci associated with the disease. These experiments support morpholino peptide conjugates as an approach for developing anti-CUG therapies for FECD.

Generation and Characterization of Anti-Glucosepane Antibodies Enabling Direct Detection of Glucosepane in Retinal Tissue.

Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.

A low glycemic diet protects disease-prone Nrf2-deficient mice against age-related macular degeneration.

Age-related macular degeneration (AMD) is a major blinding disease, affecting over 14% of the elderly. Risk for AMD is related to age, diet, environment, and genetics. Dietary modulation of AMD risk is a promising treatment modality, but requires appropriate animal models to demonstrate advantages of diet. Mice lacking the antioxidant transcription factor Nrf2 (Nfe2l2) develop age-related retinopathy relevant to human AMD. Here we evaluated the effect of consuming high glycemic (HG) or low glycemic (LG) diets until 18-months of age on development of features relevant to AMD in Nrf2-null mice. Nrf2-null mice that consumed HG diets developed atrophic AMD, characterized by photoreceptor degeneration, retinal pigment epithelium (RPE) atrophy and pigmentary abnormalities, basal laminar deposits, and loss of the choriocapillaris. In contrast, Nrf2-null-mice that consumed LG diets did not develop retinal disease phenotypes. Consumption of HG diets was associated with accumulation of advanced glycation end-products in the RPE and systemically, whereas consumption of the LG diet was associated with increased levels of anti-glycative and anti-oxidative detoxification machinery. Together our data indicate that the Nrf2-null HG mouse is a good model for atrophic AMD studies and that the LG diet can activate protective pathways to prevent AMD, even in a genetically predisposed animal.

Non-enzymatic Lysine Lactoylation of Glycolytic Enzymes.

Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feedforward mechanisms of regulation. We have identified a PTM that is derived from the glycolytic by-product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D-lactate. We have identified the non-enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a "LactoylLys" modification on proteins. GLO2 knockout cells have elevated LGSH and a consequent marked increase in LactoylLys. Using an alkyne-tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg-like conditions.