Potential Nutraceutical Properties of Leaves from Several Commonly Cultivated Plants.

Chronic dietary ingestion of suitable phytochemicals may assist with limiting or negating neurodegenerative decline. Current therapeutics used to treat Alzheimer disease elicit broad adverse drug reactions, and alternative sources of cholinesterase inhibitors (ChEIs) are required. Herein, we screened methanolic extracts from seven commonly cultivated plants for their nutraceutical potential; ability to inhibit acetylcholinesterase (AChE) and butyryl-cholinesterase (BuChE), and provision of antioxidant activity through their 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging capabilities. Potential neurotoxicity of plant extracts was examined via application to SHSY-5Y neuroblastoma cells and quantitation of cell viability. Methanolic extracts of Citrus limon (Lemon), Bombax ceiba (Red silk-cotton), Lawsonia inermis (Henna), Eucalyptus globulus (Eucalyptus), Ocimum basilicum (Basil), Citrus reticulata (Mandarin orange), and Mentha spicata (Spearmint) all displayed concentration-dependent inhibition of AChE and BuChE. The majority of extracts inhibited AChE and BuChE to near equipotency, with Henna and Eucalyptus extracts the two most potent ChEIs. All plant extracts were able to scavenge free radicals in a concentration-dependent manner, with Eucalyptus the most potent antioxidant. Toxicity of plant extracts to neuronal cells was concentration dependent, with Eucalyptus also the most toxic extract. Fractionation of plant extracts and analysis by mass spectrometry identified a number of plant polyphenols that might have contributed to the cholinesterase inhibition: 3-caffeoylquinic acid, methyl 4-caffeoylquinate, kaempferol-acetyl-glycoside, quercetin 3-rutinoside, quercetin-acetyl-glycoside, kaempferol 3-O-glucoside, and quercetin 3-O-glucoside. In silico molecular modeling of these polyphenols demonstrated their improved AChE and BuChE binding affinities compared to the current FDA-approved dual ChEI, galantamine. Collectively, all the plant extracts contained nutraceutical agents as antioxidants and ChEIs and, therefore, their chronic consumption may prove beneficial to combat the pathological deficits that accrue in Alzheimer disease.

Measurement of the total angiotensinogen and its reduced and oxidised forms in human plasma using targeted LC-MS/MS.

Angiotensinogen (AGT) is a critical protein in the renin-angiotensin-aldosterone system and may have an important role in the pathogenesis of pre-eclampsia. The disulphide linkage between cysteines 18 and 138 has a key role in the redox switch of AGT which modulates the release of angiotensin I with consequential effects on blood pressure. In this paper, we report a quantitative targeted LC-MS/MS method for the reliable measurement of the total AGT and its reduced and oxidised forms in human plasma. AGT was selectively enriched from human plasma using two-dimensional chromatography employing concanavalin A lectin affinity and reversed phase steps and then deglycosylated using PNGase F. A differential alkylation approach was coupled with targeted LC-MS/MS method to identify the two AGT forms in the plasma chymotryptic digest. An additional AGT proteolytic marker peptide was identified and used to measure total AGT levels. The developed MS workflow enabled the reproducible detection of total AGT and its two distinct forms in human plasma with analytical precision of ≤ 15%. The LC-MS/MS assay for total AGT in plasma showed a linear response (R2 = 0.992) with a limit of quantification in the low nanomolar range. The method gave suitable validation characteristics for biomedical application to the quantification of the oxidation level and the total level of AGT in plasma samples collected from normal and pre-eclamptic patients.

Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury.

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo. Increased isoaspartate damage was attenuated by treatment with betaine. To characterize isoaspartate-damaged proteins that accumulate after ethanol administration, rat liver cytosolic proteins were methylated using exogenous PIMT and (3)H-S-adenosylmethionine and proteins resolved by gel electrophoresis. Three major protein bands of ∼ 75-80 kDa, ∼ 95-100 kDa, and ∼ 155-160 kDa were identified by autoradiography. Column chromatography used to enrich isoaspartate-damaged proteins indicated that damaged proteins from ethanol-fed rats were similar to those that accrued in the livers of PIMT knockout (KO) mice. Carbamoyl phosphate synthase-1 (CPS-1) was partially purified and identified as the ∼ 160 kDa protein target of PIMT in ethanol-fed rats and in PIMT KO mice. Analysis of the liver proteome of 4-week ethanol-fed rats and PIMT KO mice demonstrated elevated cytosolic CPS-1 and betaine homocysteine S-methyltransferase-1 when compared to their respective controls, and a significant reduction of carbonic anhydrase-III (CA-III) evident only in ethanol-fed rats. Ethanol feeding of rats for 8 weeks resulted in a larger (∼ 2.3-fold) increase in CPS-1 levels compared to 4-week ethanol feeding indicating that CPS-1 accumulation correlated with the duration of ethanol consumption. Collectively, our results suggest that elevated isoaspartate and CPS-1, and reduced CA-III levels could serve as biomarkers of hepatocellular injury.

Insights into the molecular composition of endogenous unanchored polyubiquitin chains.

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.

Framework for a systems approach to proteomic biomarker profiling in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in females of reproductive age, and its prevalence ranges between 6 and 8%. Associated problems include infertility, menstrual disorders, hirsutism and obesity. In addition, individuals with PCOS may be at increased risk of diabetes, endometrial cancer and, possibly, cardiovascular disease and breast cancer in later life. Biomarkers identified from proteomic analyses may help to improve the clinical management of PCOS, provided that new proteomic data can be integrated with existing knowledge and/or pathways implicated in disease etiology. In this study, a database of identity, descriptions and functions/pathways has been developed from 148 published proteomic biomarkers in PCOS. From analysis of the database, a variety of pathways possibly implicated in PCOS were determined, including those related to fibrinolysis, thrombosis, the antioxidant pathway and the immune system. This database, if developed further, will provide a framework for a systems approach to profiling biomarkers in the future.

Role of Tyr84 in controlling the reactivity of Cys34 of human albumin.

Cys34 in domain I of the three-domain serum protein albumin is the binding site for a wide variety of biologically and clinically important small molecules, provides antioxidant activity, and constitutes the largest portion of free thiol in blood. Analysis of X-ray structures of albumin reveals that the loop containing Tyr84 occurs in multiple conformations. In structures where the loop is well defined, there appears to be an H-bond between the OH of Tyr84 and the sulfur of Cys34. We show that the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with Tyr84Phe mutant albumin is approximately four times faster than with the wild-type protein between pH 6 and pH 8. In contrast, the His39Leu mutant reacts with DTNB more slowly than the wild-type protein at pH < 8, but at a similar rate at pH 8. Above pH 8 there is a dramatic increase in reactivity for the Tyr84Phe mutant. We also report (1)H NMR studies of disulfide interchange reactions with cysteine. The tethering of the two loops containing Tyr84 and Cys34 not only appears to control the redox potential and accessibility of Cys34, but also triggers the transmission of information about the state of Cys34 throughout domain I, and to the domainI/II interface.