Identification of lactate dehydrogenase as a mammalian pyrroloquinoline quinone (PQQ)-binding protein

Pyrroloquinoline quinone (PQQ), a redox-active o-quinone, is an important nutrient involved in numerous physiological and biochemical processes in mammals. Despite such beneficial functions, the underlying molecular mechanisms remain to be established. In the present study, using PQQ-immobilized Sepharose beads as a probe, we examined the presence of protein(s) that are capable of binding PQQ in mouse NIH/3T3 fibroblasts and identified five cellular proteins, including l-lactate dehydrogenase (LDH) A chain, as potential mammalian PQQ-binding proteins. In vitro studies using a purified rabbit muscle LDH show that PQQ inhibits the formation of lactate from pyruvate in the presence of NADH (forward reaction), whereas it enhances the conversion of lactate to pyruvate in the presence of NAD+ (reverse reaction). The molecular mechanism underlying PQQ-mediated regulation of LDH activity is attributed to the oxidation of NADH to NAD+ by PQQ. Indeed, the PQQ-bound LDH oxidizes NADH, generating NAD+, and significantly catalyzes the conversion of lactate to pyruvate. Furthermore, PQQ attenuates cellular lactate release and increases intracellular ATP levels in the NIH/3T3 fibroblasts. Our results suggest that PQQ, modulating LDH activity to facilitate pyruvate formation through its redox-cycling activity, may be involved in the enhanced energy production via mitochondrial TCA cycle and oxidative phosphorylation

Phenethyl iosothiocyanate activates leptin signaling

Obesity, a principal risk factor for the development of diabetes mellitus, heart disease, and hypertension, is a growing and serious health problem all over the world. Leptin is a weight-reducing hormone produced by adipose tissue, which decreases food intake via hypothalamic leptin receptors (Ob-Rb) and the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway. Protein tyrosine phosphatase 1B (PTP1B) negatively regulates leptin signaling by dephosphorylating JAK2, and the increased activity of PTP1B is implicated in the pathogenesis of obesity. Hence, inhibition of PTP1B may help prevent and reduce obesity. In this study, we revealed that phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate in certain cruciferous vegetables, potently inhibits recombinant PTP1B by binding to the reactive cysteinyl thiol. Moreover, we found that PEITC causes the ligand-independent phosphorylation of Ob-Rb, JAK2, and STAT3 by inhibiting cellular PTP1B in differentiated human SH-SY5Y neuronal cells. PEITC treatment also induced nuclear accumulation of phosphorylated STAT3, resulting in enhanced anorexigenic POMC expression and suppressed orexigenic NPY/AGRP expression. We demonstrated that oral administration of PEITC to mice significantly reduces food intake, and stimulates hypothalamic leptin signaling. Our results suggest that PEITC might help prevent and improve obesity

Oxidative Deamination Activity of EGCG

(-)-Epigallocatechin-3-O-gallate (EGCG), the most abundant polyphenol in green tea, mediates the oxidative modification of proteins, generating protein carbonyls. However, the underlying molecular mechanism remains unclear. Here we analyzed the EGCG-derived intermediates generated upon incubation with the human serum albumin (HSA) and established that EGCG selectively oxidized the lysine residues via its oxidative deamination activity. In addition, we characterized the EGCG-oxidized proteins and discovered that the EGCG could be an endogenous source of the electrically-transformed proteins that could be recognized by the natural antibodies. When HSA was incubated with EGCG in the phosphate-buffered saline (pH 7.4) at 37°C, the protein carbonylation was associated with the formation of EGCG-derived products, such as the protein-bound EGCG, oxidized EGCG, and aminated EGCG. The aminated EGCG was also detected in the sera from the mice treated with EGCG in vivo. EGCG selectively oxidized lysine residues at the EGCG-binding domains in HSA to generate an oxidatively deaminated product, aminoadipic semialdehyde. In addition, EGCG treatment results in the increased negative charge of the protein due to the oxidative deamination of the lysine residues. More strikingly, the formation of protein carbonyls by EGCG markedly increased its cross-reactivity with the natural IgM antibodies. These findings suggest that many of the beneficial effects of EGCG may be partly attributed to its oxidative deamination activity, generating the oxidized proteins as a target of natural antibodies

The effects of PQQ on brain function

Pyrroloquinoline quinone disodium salt (PQQ) is a red trihydrate crystal that was approved as a new food ingredient by FDA in 2008. Now, it is approved as a food in Japan and the EU. PQQ has redox properties and exerts antioxidant, neuroprotective, and mitochondrial biogenesis effects. The baseline intake level of PQQ is considered to be 20 mg / day. PQQ ingestion lowers blood lipid peroxide levels in humans, suggesting antioxidant activity. In the field of cognitive function, double-blind, placebo-controlled trials have been conducted. Various improvements have been reported regarding general memory, verbal memory, working memory, and attention. Furthermore, a stratified analysis of a population with a wide range of ages revealed unique effects in young people (20–40 years old) that were not observed in older adults (41–65 years old). Specifically, cognitive flexibility and executive speed improved more rapidly in young people at 8 weeks. Co-administration of PQQ and coenzyme Q10 further enhanced these effects. In an open-label trial, PQQ was shown to improve sleep and mood. Additionally, PQQ was found to suppress skin moisture loss and increase PGC-1α expression. Overall, PQQ is a food with various functions, including brain health benefits

Phenethyl isothiocyanate activates leptin signaling and decreases food intake.

Obesity, a principal risk factor for the development of diabetes mellitus, heart disease, and hypertension, is a growing and serious health problem all over the world. Leptin is a weight-reducing hormone produced by adipose tissue, which decreases food intake via hypothalamic leptin receptors (Ob-Rb) and the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway. Protein tyrosine phosphatase 1B (PTP1B) negatively regulates leptin signaling by dephosphorylating JAK2, and the increased activity of PTP1B is implicated in the pathogenesis of obesity. Hence, inhibition of PTP1B may help prevent and reduce obesity. In this study, we revealed that phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate in certain cruciferous vegetables, potently inhibits recombinant PTP1B by binding to the reactive cysteinyl thiol. Moreover, we found that PEITC causes the ligand-independent phosphorylation of Ob-Rb, JAK2, and STAT3 by inhibiting cellular PTP1B in differentiated human SH-SY5Y neuronal cells. PEITC treatment also induced nuclear accumulation of phosphorylated STAT3, resulting in enhanced anorexigenic POMC expression and suppressed orexigenic NPY/AGRP expression. We demonstrated that oral administration of PEITC to mice significantly reduces food intake, and stimulates hypothalamic leptin signaling. Our results suggest that PEITC might help prevent and improve obesity

Pyrroloquinoline Quinone, a Redox-Active <i>o</i>‑Quinone, Stimulates Mitochondrial Biogenesis by Activating the SIRT1/PGC-1α Signaling Pathway

Pyrroloquinoline quinone (PQQ), a redox-active <i>o</i>-quinone found in various foods and mammalian tissues, has received an increasing amount of attention because of a number of health benefits that can be attributed to its ability to enhance mitochondrial biogenesis. However, its underlying molecular mechanism remains incompletely understood. We have now established that the exposure of mouse NIH/3T3 fibroblasts to a physiologically relevant concentration of PQQ significantly stimulates mitochondrial biogenesis. The exposure of NIH/3T3 cells to 10–100 nM PQQ for 48 h resulted in increased levels of Mitotracker staining, mitochondrial DNA content, and mitochondrially encoded cytochrome <i>c</i> oxidase subunit 1 (MTCO1) protein. Moreover, we observed that PQQ treatment induces deacetylation of the peroxisome proliferator-activated receptor-γ-coactivator 1α (PGC-1α) and facilitates its nuclear translocation and target gene expression but does not affect its protein levels, implying increased activity of the NAD⁺-dependent protein deacetylase sirtuin 1 (SIRT1). Indeed, treatment with a SIRT1 selective inhibitor, EX-527, hampered the ability of PQQ to stimulate PGC-1α-mediated mitochondrial biogenesis. We also found that the PQQ treatment caused a concentration-dependent increase in the cellular NAD⁺ levels, but not the total NAD⁺ and NADH levels. Our results suggest that PQQ-inducible mitochondrial biogenesis can be attributed to activation of the SIRT1/PGC-1α signaling pathway by enhancing cellular NAD⁺ formation

Oxidative deamination of lysine residues by polyphenols generates an equilibrium of aldehyde and 2-piperidinol products

peer reviewedPolyphenols, especially catechol-type polyphenols, exhibit lysyl oxidase-like activity and mediate oxidative deamination of lysine residues in proteins. Previous studies have shown that polyphenol-mediated oxidative deamination of lysine residues can be associated with altered electrical properties of proteins and increased cross-reactivity with natural IgM antibodies. This interaction suggested that oxidized proteins could act as innate antigens and elicit an innate immune response. However, the structural basis for oxidatively deaminated lysine residues remains unclear. In the present study, to establish the chemistry of lysine oxidation, we characterized oxidation products obtained via incubation of the lysine analog N-biotinyl-5-aminopentylamine (Bt-APA) with eggshell membranes containing lysyl oxidase and identified a unique six-membered ring 2-piperidinol derivative equilibrated with a ring-open product (aldehyde) as the major product. By monitoring these aldehyde/2-piperidinol products, we evaluated the lysyl oxidase-like activity of polyphenols. We also observed that this reaction was mediated by some polyphenols, especially o-diphenolic-type polyphenols, in the presence of copper ions. Interestingly, the natural IgM monoclonal antibody recognized these aldehyde/2-piperidinol products as an innate epitope. These findings establish the existence of a dynamic equilibrium of oxidized lysine and provide important insights into the chemopreventive function of dietary polyphenols for chronic diseases