Protein glycation is a universal, non-enzymatic modification that occurs when a sugar covalently attaches to a primary amine. These spontaneous modifications may have deleterious or regulatory effects on protein function, and their removal is mediated by the conserved metabolic kinase fructosamine-3-kinase (FN3K). Despite its crucial role in protein repair, we currently have a poor understanding of how FN3K engages or phosphorylates its substrates. By integrating structural biology and biochemistry, we elucidated the catalytic mechanism for FN3K-mediated protein deglycation. Our work identifies key amino acids required for binding and phosphorylating glycated substrates and reveals the molecular basis of an evolutionarily conserved protein repair pathway. Additional structural-functional studies revealed unique structural features of human FN3K as well as differences in the dimerization behavior and regulation of FN3K family members. Our findings improve our understanding of the structure of FN3K and its catalytic mechanism, which opens new avenues for therapeutically targeting FN3K.

Glycation reactions in food lead to the formation of the Amadori rearrangement product (ARP) N-ε-fructosyllysine (fructoselysine, FL), which is taken up with the daily diet and comes into contact with the gut microbiota during digestion. In the present study, nine commercially available probiotic preparations as well as single pure strains thereof were investigated for their FL-degrading capability under anaerobic conditions. One of the commercial preparations as well as three single pure strains thereof was able to completely degrade 0.25 mM FL within 72 h. Three new deglycating lactic acid bacteria species, namely, Lactobacillus buchneri DSM 20057, Lactobacillus jensenii DSM 20557, and Pediococcus acidilactici DSM 25404, could be identified. Quantitative experiments showed that FL was completely deglycated to lysine. Using 13C6-labeled FL as the substrate, it could be proven that the sugar moiety of the Amadori product is degraded to lactic acid, showing for the first time that certain lactic acid bacteria can utilize the sugar moiety as a substrate for lactic acid fermentation.

After preliminary ab initio calculations, 3-phenacyl substituted thiazolium salts, analogs of Alagebrium, were synthesized and investigated in vitro as glycation reaction inhibitors. The most part of investigations focused on the potential of the title compounds to attenuate the formation of fluorescent AGEs as well on their ability to disrupt the cross-linking formation among glycated proteins. Additionally, the capability of thiazolium salts to deglycate in the reaction of early glycation products with nitroblue tetrazolium was determined. Cytotoxicological properties of the title compounds were evaluated using LDH and MTT assays. The leader compound (3-[2-(biphenyl-4-yl)-2-oxoethyl]-1,3-thiazol-3-ium bromide) in a 50 mg/kg dose (p.o. 14 days) was further tested within an in vivo carbonyl stress model (rats, methylglyoxal 86.25 mg/kg/d, i.p., 14 days). As a result, the leader-molecule revealed a high effectiveness against all three examined mechanisms of glycation reaction inhibition in in vitro tests and was able to suppress capacity of methylglyoxal to form AGEs in vivo.

Diabetes is a multifactorial disorder that increases mortality and disability due to its complications. A key driver of these complications is nonenzymatic glycation, which generates advanced glycation end-products (AGEs) that impair tissue function. Therefore, effective nonenzymatic glycation prevention and control strategies are urgently needed. This review comprehensively describes the molecular mechanisms and pathological consequences of nonenzymatic glycation in diabetes and outlines various anti-glycation strategies, such as lowering plasma glucose, interfering with the glycation reaction, and degrading early and late glycation products. Diet, exercise, and hypoglycemic medications can reduce the onset of high glucose at the source. Glucose or amino acid analogs such as flavonoids, lysine and aminoguanidine competitively bind to proteins or glucose to block the initial nonenzymatic glycation reaction. In addition, deglycation enzymes such as amadoriase, fructosamine-3-kinase, parkinson\'s disease protein, glutamine amidotransferase-like class 1 domain-containing 3A and terminal FraB deglycase can eliminate existing nonenzymatic glycation products. These strategies involve nutritional, pharmacological, and enzymatic interventions that target different stages of nonenzymatic glycation. This review also emphasizes the therapeutic potential of anti-glycation drugs for preventing and treating diabetes complications.

Non-enzymatic glycation (a.k.a. Maillard reaction) is a series of random spontaneous reactions between reducing sugars and amines, resulting in the formation of irreversible advanced glycation endproducts (AGE\'s). In food chemistry, this process is beneficial by contributing to the flavor, aroma, texture, and appearance of cooked foods. In vivo, however, Maillard reaction is deleterious because uncontrolled modification and crosslinking of biological macromolecules impairs their function. Consequently, chronic hyperglycemia of diabetes mellitus, for instance, leads to increased non-enzymatic glycation and diverse, multi-organ pathologies of diabetic complications. Based on the fact that toxic compounds, such as free radicals, are detoxified in vivo by specific defense mechanisms, one would expect to find mechanisms to control glucose toxicity as well. Thus far, only one such enzyme, fructosamine-3-kinase (FN3K), has been characterized. It operates intracellularly by catalyzing ATP-dependent removal of Maillard adducts, D-fructoselysines, from proteins, thereby reducing the Maillard reaction flux from glucose to AGE\'s. When FN3K was isolated, a closely related but distinct protein copurified with it. Unlike FN3K, however, this enzyme, fructosamine-3-kinase-related protein (FN3KRP), does not phosphorylate D-fructoselysines but it does phosphorylate several other (non-physiological) substrates. Interestingly, the distribution of FN3KRP in nature appears to be nearly universal whereas that of FN3K is limited to endotherms. In this article, it is suggested that the function of FN3KRP is deglycation of Maillard adducts downstream from fructoselysines. Such a mechanism, if proven correct, would be valuable given reports on apparent correlations between FN3KRP and some chronic conditions and/or diseases, such as a recent publication which proposes that the FN3KRP gene may be a longevity gene.

Cataracts are the major cause of blindness worldwide, largely resulting from aging and diabetes mellitus. Advanced glycation end products (AGEs) have been identified as major contributors in cataract formation because they alter lens protein structure and stability and induce covalent cross-linking, aggregation, and insolubilization of lens crystallins. We investigated the potential of the deglycating enzyme fructosamine-3-kinase (FN3K) in the disruption of AGEs in cataractous lenses. Macroscopic changes of equine lenses were evaluated after ex vivo intravitreal FN3K injection. The mechanical properties of an equine lens pair were evaluated after treatment with saline and FN3K. AGE-type autofluorescence (AF) was measured to assess the time-dependent effects of FN3K on glycolaldehyde-induced AGE-modified porcine lens fragments and to evaluate its actions on intact lenses after in vivo intravitreal FN3K injection of murine eyes. A potential immune response after injection was evaluated by analysis of IL-2, TNFα, and IFNγ using an ELISA kit. Dose- and time-dependent AF kinetics were analyzed on pooled human lens fragments. Furthermore, AF measurements and a time-lapse of macroscopic changes were performed on intact cataractous human eye lenses after incubation with an FN3K solution. At last, AF measurements were performed on cataractous human eyes after crossover topical treatment with either saline- or FN3K-containing drops. While the lenses of the equine FN3K-treated eyes appeared to be clear, the saline-treated lenses had a yellowish-brown color. Following FN3K treatment, color restoration could be observed within 30 min. The extension rate of the equine FN3K-treated lens was more than twice the extension rate of the saline-treated lens. FN3K treatment induced significant time-dependent decreases in AGE-related AF values in the AGE-modified porcine lens fragments. Furthermore, in vivo intravitreal FN3K injection of murine eyes significantly reduced AF values of the lenses. Treatment did not provoke a systemic immune response in mice. AF kinetics of FN3K-treated cataractous human lens suspensions revealed dose- and time-dependent decreases. Incubation of cataractous human eye lenses with FN3K resulted in a macroscopic lighter color of the cortex and a decrease in AF values. At last, crossover topical treatment of intact human eyes revealed a decrease in AF values during FN3K treatment, while showing no notable changes with saline. Our study suggests, for the first time, a potential additional role of FN3K as an alternative treatment for AGE-related cataracts.

Glycated stress is mediated by the advanced glycation end products (AGE) and the binding of AGEs to the receptors for advanced glycation end products (RAGEs) in cancer cells. RAGEs are involved in mediating tumorigenesis of multiple cancers through the modulation of several downstream signaling cascades. Glycated stress modulates various signaling pathways that include p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor kappa-B (NF-κB), tumor necrosis factor (TNF)-α, etc., which further foster the uncontrolled proliferation, growth, metastasis, angiogenesis, drug resistance, and evasion of apoptosis in several cancers. In this review, a balanced overview on the role of glycation and deglycation in modulating several signaling cascades that are involved in the progression of cancers was discussed. Further, we have highlighted the functional role of deglycating enzyme fructosamine-3-kinase (FN3K) on Nrf2-driven cancers. The activity of FN3K is attributed to its ability to deglycate Nrf2, a master regulator of oxidative stress in cells. FN3K is a unique protein that mediates deglycation by phosphorylating basic amino acids lysine and arginine in various proteins such as Nrf2. Deglycated Nrf2 is stable and binds to small musculoaponeurotic fibrosarcoma (sMAF) proteins, thereby activating cellular antioxidant mechanisms to protect cells from oxidative stress. This cellular protection offered by Nrf2 activation, in one way, prevents the transformation of a normal cell into a cancer cell; however, in the other way, it helps a cancer cell not only to survive under hypoxic conditions but also, to stay protected from various chemo- and radio-therapeutic treatments. Therefore, the activation of Nrf2 is similar to a double-edged sword and, if not controlled properly, can lead to the development of many solid tumors. Hence, there is a need to develop novel small molecule modulators/phytochemicals that can regulate FN3K activity, thereby maintaining Nrf2 in a controlled activation state.

DJ-1 is a multifunctional protein associated with cancers and autosomal early-onset Parkinson disease. Besides the well-documented antioxidative stress activity, recent studies show that DJ-1 has deglycation enzymatic activity and anti-ferroptosis effect. It has been shown that DJ-1 forms the homodimerization, which dictates its antioxidative stress activity. In this study, we investigated the relationship between the dimeric structure of DJ-1 and its newly reported activities. In HEK293T cells with Flag-tagged and Myc-tagged DJ-1 overexpression, we performed deletion mutations and point mutations, narrowed down the most critical motif at the C terminus. We found that the deletion mutation of the last three amino acids at the C terminus of DJ-1 (DJ-1 ΔC3) disrupted its homodimerization with the hydrophobic L187 residue being of great importance for DJ-1 homodimerization. In addition, the ability in methylglyoxal (MGO) detoxification and deglycation was almost abolished in the mutation of DJ-1 ΔC3 and point mutant L187E compared with wild-type DJ-1 (DJ-1 WT). We also showed the suppression of erastin-triggered ferroptosis in DJ-1-/- mouse embryonic fibroblast cells was abolished by ΔC3 and L187E, but partially diminished by V51C. Thus, our results demonstrate that the C terminus of DJ-1 is crucial for its homodimerization, deglycation activity, and suppression of ferroptosis.

Glycation is a common non-enzymatic reaction between proteins and sugars, which gives rise in the human body to the formation of advanced glycation end products (AGEs). These modifications impacts both extra and intracellular proteins, leading to cells and tissues dysfunctions. In the skin, accumulation of AGEs leads to aesthetic consequences, wrinkles, dark spots and yellowish skin tone, as it can be seen in diabetic patients. Consequently, there is a growing dermatological interest to find compounds able to eliminate AGEs accumulated in skin. In this context, a method has been developed to detect and quantify intracellular glycation in human dermal fibroblasts. After cultivation of fibroblasts, cell lysates were injected in an HPLC system coupled with a fluorescence detector in by-pass mode. The system allows the simultaneous measurement of global AGEs and particular pentosidine amounts using two sets of wavelengths in a single run of 1 min. The immunocytochemistry approach was used to valid the HPLC analysis data. The method developed was able to quantify changes in global AGEs and pentosidine content in cells in response to glyoxal treatment. Fibroblasts treated with 500 μM of glyoxal for 48 h showed a significant 2.3-fold and 2.6-fold increase in the content of AGEs and pentosidine respectively compared to control cells. As an application, a screening of natural extracts have been done and the method allowed identifying extracts able to significantly reduce the amount of pentosidine in fibroblasts (-32%). These extracts act as deglycation agents of interest in the field of dermatology and cosmetology.