Regulation of human 4-hydroxy-2-oxoglutarate aldolase by pyruvate and α-ketoglutarate: implications for primary hyperoxaluria type-3.

4-hydroxy-2-oxoglutarate aldolase (HOGA1) is a mitochondrial enzyme that plays a gatekeeper role in hydroxyproline metabolism. Its loss of function in humans causes primary hyperoxaluria type 3 (PH3), a rare condition characterised by excessive production of oxalate. In this study, we investigated the significance of the associated oxaloacetate decarboxylase activity which is also catalysed by HOGA1. Kinetic studies using the recombinant human enzyme (hHOGA1) and active site mutants showed both these dual activities utilise the same catalytic machinery with micromolar substrate affinities suggesting that both are operative in vivo. Biophysical and structural studies showed that pyruvate was a competitive inhibitor with an inhibition constant in the micromolar range. By comparison α-ketoglutarate was a weak inhibitor with an inhibition constant in the millimolar range and could only be isolated as an adduct with the active site Lys196 in the presence of sodium borohydride. These studies suggest that pyruvate inhibits HOGA1 activity during gluconeogenesis. We also propose that loss of HOGA1 function could increase oxalate production in PH3 by decreasing pyruvate availability and metabolic flux through the Krebs cycle.

Synthesis of isotopically labelled αCGRP8-37 and its lipidated analogue.

α-Calcitonin gene related peptide (αCGRP) inhibitors are important medicinal targets due to their ability to produce antimigraine effects, thus, the discovery of long-acting αCGRP inhibitors is of significant interest. Herein we report the synthesis of an isotopically labelled version of the well-known CGRP receptor antagonist, αCGRP8-37 , as well as lipidated αCGRP8-37 with comparable antagonistic activity. These isotopically labelled peptides can be employed in assays to determine the metabolic stability of the lipidated αCGRP8-37 and compare this with the stability of known αCGRP8-37 .

Solid-Phase Thiol-Ene Lipidation of Peptides for the Synthesis of a Potent CGRP Receptor Antagonist.

We report a new method herein coined SP-CLipPA (solid-phase cysteine lipidation of a peptide or amino acid) for the synthesis of mono-S-lipidated peptides. This technique utilizes thiol-ene chemistry for conjugation of a vinyl ester to a free thiol of a semiprotected, resin-bound peptide. Advantages of SP-CLipPA include: ease of handling, conversions of up to 91 %, by-product removal by simple filtration, and a single purification step. Additionally, the desired lipidated products show high chromatographic separation from impurities, thus facilitating RP-HPLC purification. To showcase the utility of SP-CLipPA, we synthesized a potent calcitonin gene-related peptide (CGRP) receptor antagonist peptide in excellent yield and purity. This peptide, selected from a series of lipidated analogues of CGRP8-37 and CGRP7-37 , has potential for the treatment of migraine.

Rutin suppresses human-amylin/hIAPP misfolding and oligomer formation in-vitro, and ameliorates diabetes and its impacts in human-amylin/hIAPP transgenic mice.

Pancreatic islet ß-cells secrete the hormones insulin and amylin, and defective ß-cell function plays a central role in the pathogenesis of type-2 diabetes (T2D). Human amylin (hA, also termed hIAPP) misfolds and forms amyloid aggregates whereas orthologous mouse amylin does neither. Furthermore, hA elicits apoptosis in cultured ß-cells and ß-cell death in ex-vivo islets. In addition, hA-transgenic mice that selectively express hA in their ß-cells, manifest ß-cell apoptosis and progressive islet damage that leads to diabetes closely resembling that in patients with T2D. Aggregation of hA is thus linked to the causation of diabetes. We employed time-dependent thioflavin-T spectroscopy and ion-mobility mass spectrometry to screen potential suppressors of hA misfolding for anti-diabetic activity. We identified the dietary flavonol rutin as an inhibitor of hA-misfolding and measured its anti-diabetic efficacy in hA-transgenic mice. In vitro, rutin bound hA, suppressed misfolding, disaggregated oligomers and reverted hA-conformation towards the physiological. In hA-transgenic mice, measurements of glucose, fluid-intake, and body-weight showed that rutin-treatment slowed diabetes-progression by lowering of rates of elevation in blood glucose (P = 0.030), retarding deterioration from symptomatic diabetes to death (P = 0.014) and stabilizing body-weight (P < 0.0001). In conclusion, rutin treatment suppressed hA-aggregation in vitro and doubled the lifespan of diabetic mice (P = 0.011) by a median of 69 days compared with vehicle-treated control-diabetic hA-transgenic mice.

Structure and function of human xylulokinase, an enzyme with important roles in carbohydrate metabolism.

D-Xylulokinase (XK; EC 2.7.1.17) catalyzes the ATP-dependent phosphorylation of d-xylulose (Xu) to produce xylulose 5-phosphate (Xu5P). In mammals, XK is the last enzyme in the glucuronate-xylulose pathway, active in the liver and kidneys, and is linked through its product Xu5P to the pentose-phosphate pathway. XK may play an important role in metabolic disease, given that Xu5P is a key regulator of glucose metabolism and lipogenesis. We have expressed the product of a putative human XK gene and identified it as the authentic human d-xylulokinase (hXK). NMR studies with a variety of sugars showed that hXK acts only on d-xylulose, and a coupled photometric assay established its key kinetic parameters as K(m)(Xu) = 24 ± 3 µm and k(cat) = 35 ± 5 s(-1). Crystal structures were determined for hXK, on its own and in complexes with Xu, ADP, and a fluorinated inhibitor. These reveal that hXK has a two-domain fold characteristic of the sugar kinase/hsp70/actin superfamily, with glycerol kinase as its closest relative. Xu binds to domain-I and ADP to domain-II, but in this open form of hXK they are 10 Å apart, implying that a large scale conformational change is required for catalysis. Xu binds in its linear keto-form, sandwiched between a Trp side chain and polar side chains that provide exquisite hydrogen bonding recognition. The hXK structure provides a basis for the design of specific inhibitors with which to probe its roles in sugar metabolism and metabolic disease.

NAD+ dependent UPRmt activation underlies intestinal aging caused by mitochondrial DNA mutations.

Aging in mammals is accompanied by an imbalance of intestinal homeostasis and accumulation of mitochondrial DNA (mtDNA) mutations. However, little is known about how accumulated mtDNA mutations modulate intestinal homeostasis. We observe the accumulation of mtDNA mutations in the small intestine of aged male mice, suggesting an association with physiological intestinal aging. Using polymerase gamma (POLG) mutator mice and wild-type mice, we generate male mice with progressive mtDNA mutation burdens. Investigation utilizing organoid technology and in vivo intestinal stem cell labeling reveals decreased colony formation efficiency of intestinal crypts and LGR5-expressing intestinal stem cells in response to a threshold mtDNA mutation burden. Mechanistically, increased mtDNA mutation burden exacerbates the aging phenotype of the small intestine through ATF5 dependent mitochondrial unfolded protein response (UPRmt) activation. This aging phenotype is reversed by supplementation with the NAD+ precursor, NMN. Thus, we uncover a NAD+ dependent UPRmt triggered by mtDNA mutations that regulates the intestinal aging.

Proteomic Analysis of Honey: Peptide Profiling as a Novel Approach for New Zealand Manuka (Leptospermum scoparium) Honey Authentication.

New Zealand manuka (Leptospermum scoparium) honey is a premium food product. Unfortunately, its high demand has led to "not true to label" marketed manuka honey. Robust methods are therefore required to determine authenticity. We previously identified three unique nectar-derived proteins in manuka honey, detected as twelve tryptic peptide markers, and hypothesized these could be used to determine authenticity. We invoked a targeted proteomic approach based on parallel reaction-monitoring (PRM) to selectively monitor relative abundance of these peptides in sixteen manuka and twenty six non-manuka honey samples of various floral origin. We included six tryptic peptide markers derived from three bee-derived major royal jelly proteins as potential internal standards. The twelve manuka-specific tryptic peptide markers were present in all manuka honeys with minor regional variation. By comparison, they had negligible presence in non-manuka honeys. Bee-derived peptides were detected in all honeys with similar relative abundance but with sufficient variation precluding their utility as internal standards. Manuka honeys displayed an inverse relationship between total protein content and the ratio between nectar- to bee-derived peptide abundance. This trend reveals an association between protein content on possible nectar processing time by bees. Overall, these findings demonstrate the first successful application of peptide profiling as an alternative and potentially more robust approach for manuka honey authentication.

The Leptospermum scoparium (Manuka)-Specific Nectar and Honey Compound 3,6,7-Trimethyllumazine (LepteridineTM) That Inhibits Matrix Metalloproteinase 9 (MMP-9) Activity.

3,6,7-trimethyllumazine (Lepteridine™) is a newly discovered natural pteridine derivative unique to Manuka (Leptospermum scoparium) nectar and honey, with no previously reported biological activity. Pteridine derivative-based medicines, such as methotrexate, are used to treat auto-immune and inflammatory diseases, and Manuka honey reportedly possesses anti-inflammatory properties and is used topically as a wound dressing. MMP-9 is a potential candidate protein target as it is upregulated in recalcitrant wounds and intestinal inflammation. Using gelatin zymography, 40 µg/mL LepteridineTM inhibited the gelatinase activities of both pro- (22%, p < 0.0001) and activated (59%, p < 0.01) MMP-9 forms. By comparison, LepteridineTM exerted modest (~10%) inhibition against a chromogenic peptide substrate and no effect against a fluorogenic peptide substrate. These findings suggest that LepteridineTM may not interact within the catalytic domain of MMP-9 and exerts a negligible effect on the active site hydrolysis of small soluble peptide substrates. Instead, the findings implicate fibronectin II domain interactions by LepteridineTM which impair gelatinase activity, possibly through perturbed tethering of MMP-9 to the gelatin matrix. Molecular modelling analyses were equivocal over interactions at the S1' pocket versus the fibronectin II domain, while molecular dynamic calculations indicated rapid exchange kinetics. No significant degradation of synthetic or natural LepteridineTM in Manuka honey occurred during simulated gastrointestinal digestion. MMP-9 regulates skin and gastrointestinal inflammatory responses and extracellular matrix remodelling. These results potentially implicate LepteridineTM bioactivity in Manuka honey's reported beneficial effects on wound healing via topical application and anti-inflammatory actions in gastrointestinal disorder models via oral consumption.

Purification, crystallization and preliminary crystallographic analysis of human dihydrodipicolinate synthase-like protein (DHDPSL).

Human dihydrodipicolinate synthase-like protein (DHDPSL) is a gene product of unknown function. It is homologous to bacterial pyruvate-dependent aldolases such as dihydrodipicolinate synthase (DHDPS), which functions in lysine biosynthesis. However, it cannot have this function and instead is implicated in a genetic disorder that leads to excessive production of oxalate and kidney-stone formation. In order to better understand its function, DHDPSL was expressed as an MBP-fusion protein and crystallized using an in situ proteolysis protocol. Two crystal forms were obtained, both of which diffracted X-rays to approximately 2.0 Å resolution. One of these, belonging to space group P6(2)22 or P6(4)22 with unit-cell parameters a = b = 142.9, c = 109.8 Å, α = ß = 90, γ = 120°, was highly reproducible and suitable for structure determination by X-ray crystallography.

Use of a repetitive seeding protocol to obtain diffraction-quality crystals of a putative human D-xylulokinase.

In mammals, the enzyme D-xylulokinase (XK; EC 2.7.1.17) catalyses the last step of the glucuronate-xylulose pathway, in which the ketopentose sugar D-xylulose is phosphorylated to yield D-xylulose 5-phosphate (Xu5P). Xu5P is also a metabolite of the pentose phosphate pathway and acts as a signalling molecule that regulates lipogenesis and glycolysis in the liver. To date, no eukaryotic XK has been structurally characterized. A putative human XK was expressed in Escherichia coli aided by molecular chaperones, purified and crystallized. A seeding procedure involving repeated rounds of seeding was developed and proved to be essential for obtaining diffraction-quality crystals. Preliminary X-ray diffraction analysis was performed using synchrotron radiation. This resulted in the collection of a complete diffraction data set to 2.7 Šresolution from a crystal belonging to the trigonal space group P3(1) or P3(2) with unit-cell parameters a = b = 101.87, c = 158.85 Å.

Structure-Based Discovery and Optimization of Furo[3,2-c]pyridin-4(5H)-one Derivatives as Potent and Second Bromodomain (BD2)-Selective Bromo and Extra Terminal Domain (BET) Inhibitors.

Pan-bromodomain and extra terminal (Pan-BET) inhibitors show profound efficacy but exhibit pharmacology-driven toxicities in clinical trials. The development of domain-selective BET inhibitors to separate efficacy and toxicity is urgently needed. Herein, we report a series of furo[3,2-c]pyridin-4(5H)-one derivatives as novel BD2-selective BET inhibitors. The representative compound 8l (XY153) potently bound to BRD4 BD2 with an half-maximum inhibitory concentration (IC50) value of 0.79 nM and displayed 354-fold selectivity over BRD4 BD1. Besides, 8l exhibited 6-fold BRD4 BD2 domain selectivity over other BET BD2 domains. Compound 8l displayed potent antiproliferative activity against multiple tumor cell lines, especially MV4-11 (IC50 = 0.55 nM), while showing weak cytotoxicity against the normal lung fibroblast cell line. It highlights the safety profile of this series of BD2 inhibitors. 8l also demonstrated good metabolic stability in vitro. These data indicate that 8l may serve as a new and valuable lead compound for the development of potential therapeutics against acute myeloid leukemia (AML).

Lipidated Calcitonin Gene-Related Peptide (CGRP) Peptide Antagonists Retain CGRP Receptor Activity and Attenuate CGRP Action In Vivo.

Signaling through calcitonin gene-related peptide (CGRP) receptors is associated with pain, migraine, and energy expenditure. Small molecule and monoclonal antibody CGRP receptor antagonists that block endogenous CGRP action are in clinical use as anti-migraine therapies. By comparison, the potential utility of peptide antagonists has received less attention due to suboptimal pharmacokinetic properties. Lipidation is an established strategy to increase peptide half-life in vivo. This study aimed to explore the feasibility of developing lipidated CGRP peptide antagonists that retain receptor antagonist activity in vitro and attenuate endogenous CGRP action in vivo. CGRP peptide analogues based on the archetypal CGRP receptor antagonist, CGRP8-37, were palmitoylated at the N-terminus, position 24, and near the C-terminus at position 35. The antagonist activities of the lipidated peptide analogues were tested in vitro using transfected Cos-7 cells expressing either the human or mouse CGRP receptor, amylin subtype 1 (AMY1) receptor, adrenomedullin (AM) receptors, or calcitonin receptor. Antagonist activities were also evaluated in SK-N-MC cells that endogenously express the human CGRP receptor. Lipidated peptides were then tested for their ability to antagonize endogenous CGRP action in vivo using a capsaicin-induced dermal vasodilation (CIDV) model in C57/BL6J mice. All lipidated peptides except for the C-terminally modified analogue retained potent antagonist activity compared to CGRP8-37 towards the CGRP receptor. The lipidated peptides also retained, and sometimes gained, antagonist activities at AMY1, AM1 and AM2 receptors. Several lipidated peptides produced robust inhibition of CIDV in mice. This study demonstrates that selected lipidated peptide antagonists based on αCGRP8-37 retain potent antagonist activity at the CGRP receptor and are capable of inhibition of endogenous CGRP action in vivo. These findings suggest that lipidation can be applied to peptide antagonists, such as αCGRP8-37 and are a potential strategy for antagonizing CGRP action.

The chaperone proteins HSP70, HSP40/DnaJ and GRP78/BiP suppress misfolding and formation of ß-sheet-containing aggregates by human amylin: a potential role for defective chaperone biology in Type 2 diabetes.

Misfolding of the islet ß-cell peptide hA (human amylin) into ß-sheet-containing oligomers is linked to ß-cell apoptosis and the pathogenesis of T2DM (Type 2 diabetes mellitus). In the present study, we have investigated the possible effects on hA misfolding of the chaperones HSP (heat-shock protein) 70, GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) and HSP40/DnaJ. We demonstrate that hA underwent spontaneous time-dependent ß-sheet formation and aggregation by thioflavin-T fluorescence in solution, whereas rA (rat amylin) did not. HSP70, GRP78/BiP and HSP40/DnaJ each independently suppressed hA misfolding. Maximal molar protein/hA ratios at which chaperone activity was detected were 1:200 (HSP70, HSP40/DnaJ and GRP78/BiP). By contrast, none of the chaperones modified the secondary structure of rA. hA, but not rA, was co-precipitated independently with HSP70 and GRP78/BiP by anti-amylin antibodies. As these effects occur at molar ratios consistent with chaperone binding to relatively rare misfolded hA species, we conclude that HSP70 and GRP78/BiP can detect and bind misfolded hA oligomers, thereby effectively protecting hA against bulk misfolding and irreversible aggregation. Defective ß-cell chaperone biology could contribute to hA misfolding and initiation of apoptosis in T2DM.

Proteomic analysis of honey. Identification of unique peptide markers for authentication of NZ manuka (Leptospermum scoparium) honey.

Proteomics is an emerging tool in food authentication that has not been optimised for honey analysis. In this study, we present a qualitative proteomic analysis of New Zealand manuka (Leptospermum scoparium) honey. A total of fifty bee-derived proteins were identified in the honey, the most predominant being major royal jelly proteins (MRJPs). We also demonstrate for the first time the presence of unique nectar-derived proteins in manuka honey. A total of 17 manuka plant proteins were identified, a-third of which were putative pathogenesis-related proteins. Two proteins involved in drought tolerance were also identified. Twelve candidate peptides were selected as potential authentication markers based on their uniqueness to manuka honey. Nectar analyses confirmed the origin and specificity of these peptides to L. scoparium nectar, thus presenting peptide profiling as a viable and novel approach for manuka honey authentication. Raw data are available via ProteomeXchange with identifier PXD021730.

Utility of the Leptospermum scoparium Compound Lepteridine as a Chemical Marker for Manuka Honey Authenticity.

Manuka honey is a premium food product with unique antimicrobial bioactivity. Concerns with mislabeled manuka honey require robust assays to determine authenticity. Lepteridine is a Leptospermum-specific fluorescent molecule with potential as an authenticity marker. We describe a mass spectrometry-based assay to measure lepteridine based on an isotopically labeled lepteridine standard. Using this assay, lepteridine concentrations in manuka honey samples strongly correlated with concentrations quantitated by either high-performance liquid chromatography-ultraviolet (HPLC-UV) or fluorescence. A derived minimum lepteridine threshold concentration was compared with the New Zealand regulatory definition for manuka honey to determine "manuka honey" authenticity on a set of commercial samples. Both methods effectively distinguished manuka honey from non-manuka honeys. The regulatory definition excludes lepteridine but otherwise includes the quantification of multiple floral markers together with pollen analysis. Our findings suggest that the quantification of lepteridine alone or in combination with leptosperin could be implemented as an effective screening method to identify manuka honey, likely to achieve an outcome similar to the regulatory definition.

Mapping of the ATP-binding domain of human fructosamine 3-kinase-related protein by affinity labelling with 5'-[p-(fluorosulfonyl)benzoyl]adenosine.

The modification of proteins by reducing sugars through the process of non-enzymatic glycation is one of the principal mechanisms by which hyperglycaemia may precipitate the development of diabetic complications. Fn3K (fructosamine 3-kinase) and Fn3KRP (Fn3K-related protein) are two recently discovered enzymes that may play roles in metabolizing early glycation products. However, although the activity of these enzymes towards various glycated substrates has been established, very little is known about their structure-function relationships or their respective mechanisms of action. Furthermore, their only structural similarities noted to date with members of other kinase families has been with the bacterial aminoglycoside kinases. In the present study, we employed affinity labelling with the ATP analogue FSBA {5'-p-[(fluorosulfonyl)benzoyl]adenosine} to probe the active-site topology of Fn3KRP as an example of this enigmatic family of kinases. FSBA was found to modify Fn3KRP at five distinct sites; four of these were predicted to be localized in close proximity to its ATP-binding site, based on alignments with the aminoglycoside kinase APH(3')-IIIa, and examination of its published tertiary structure. The results of the present studies provide evidence that Fn3KRP possesses an ATP-binding domain that is structurally related to that of both the aminoglycoside kinases and eukaryotic protein kinases.

Evidence that alpha-calcitonin gene-related peptide is a neurohormone that controls systemic lipid availability and utilization.

Alpha-calcitonin gene-related peptide (alphaCGRP) is released mainly from sensory and motor nerves in response to physiological stimuli. Despite well-documented pharmacological effects, its primary physiological role has thus far remained obscure. Increased lipid content, particularly in skeletal muscle and liver, is strongly implicated in the pathogenesis of insulin resistance, but the physiological regulation of organ lipid is imperfectly understood. Here we report our systematic investigations of the effects of alphaCGRP on in vitro and in vivo indices of lipid metabolism. In rodents, levels of alphaCGRP similar to those in the blood markedly stimulated fatty acid beta-oxidation and evoked concomitant mobilization of muscle lipid via receptor-mediated activation of muscle lipolysis. alphaCGRP exerted potent in vivo effects on lipid metabolism in muscle, liver, and the blood via receptor-mediated pathways. Studies with receptor antagonists were consistent with tonic regulation of lipid metabolism by an endogenous CGRP agonist. These data reveal that alphaCGRP is a newly recognized regulator of lipid availability and utilization in key tissues and that it may elevate the availability of intramyocellular free fatty acids to meet muscle energy requirements generated by contraction by evoking their release from endogenous triglyceride.

New approach: Chemical and fluorescence profiling of NZ honeys.

New Zealand manuka (Leptospermum scoparium) and kanuka (Kunzea ericoides) honeys contain a unique array of chemical markers useful for chemical fingerprinting. We investigated the presence of 13 potential marker compounds in nectars of the major honey crop species. We confirmed that leptosperin, lepteridine, 2'-methoxyacetophenone, and 2-methoxybenzoic acid are exclusive to manuka nectar whereas lumichrome is unique to kanuka nectar. 3-Phenyllactic acid and 4-hydroxyphenyllactic acid are present in manuka and kanuka nectars. Leptosperin, lepteridine, 3-phenyllactic acid, and 4-hydroxyphenyllactic acid are chemically stable over prolonged storage, but not 2-methoxybenzoic acid and 2'-methoxyacetophenone. Accordingly, leptosperin and lepteridine are definitive chemical markers for authentication of manuka honey. An optimal concentration cut-off was established for the floral source-specific markers: leptosperin (94mg/kg), lepteridine (2.1mg/kg), 2'-methoxyacetophenone (2.0mg/kg) for manuka honey, and lumichrome (4.5mg/kg) for kanuka honey. The use of leptosperin and lepteridine as fluorescence markers for manuka honey authentication is reinforced.

Lepteridine as a unique fluorescent marker for the authentication of manuka honey.

The recent discovery of two unique manuka marker fluorescence wavelengths (MM1 and MM2) potentially offers a rapid and cost-effective approach for manuka honey authentication using spectroscopy. The fluorophore responsible for the MM1 marker has been identified as leptosperin. We investigated whether lepteridine may be responsible for the MM2 fluorescence. We quantified the lepteridine in manuka honey and manuka nectar, which ranged between 5-52mg/kg and 80-205mg/kg, respectively. Notably, the fluorescent spectrum of synthetic lepteridine matched the MM2 fluorescence signature. Fluorescence quenching was observed in the honey matrix but otherwise, lepteridine was stable over prolonged storage at 37°C. Lepteridine was also found in Australian Leptospermum honeys and nectars. Lepteridine concentration was positively correlated with concentrations of the MM1 fluorescence marker leptosperin in honeys. These findings identify lepteridine as the principle compound responsible for MM2 fluorescence, and support the utility as a marker compound for manuka honey authentication.

Leptosperin is a distinct and detectable fluorophore in Leptospermum honeys.

New Zealand manuka (Leptospermum scoparium) honey exhibits two unique fluorescence signatures that distinguish it from other honey types. One of these is the MM1 fluorescence marker (270-365nm excitation-emission) which we show is due to a Leptospermum nectar-derived compound, leptosperin. Synthetic or honey-purified leptosperin not only displayed an identical fluorescence spectrum, but supplementation of leptosperin into clover or artificial honeys generated the MM1 fluorescence signature. There was a quenching effect of the honey matrix on leptosperin fluorescence but otherwise leptosperin was chemically stable over prolonged storage at 37°C. Leptosperin was also present in the woody-fruited Australian Leptospermum species at elevated concentrations but virtually absent in Leptospermum subtenue suggesting its elevated expression developed following the mid-Miocene separation of the genus. These findings suggest that fluorescence spectroscopy could offer a rapid and high-throughput screening method for identification of Leptospermum honeys using the MM1 fluorescence marker.