A Filtered Database Search Algorithm for Endogenous Serum Protein Carbonyl Modifications in a Mouse Model of Inflammation

During inflammation, the resulting oxidative stress can damage surrounding host tissue, forming protein-carbonyls. The SJL mouse is an experimental animal model used to assess in vivo toxicological responses to reactive oxygen and nitrogen species from inflammation. The goals of this study were to identify the major serum proteins modified with a carbonyl functionality and to identify the types of carbonyl adducts. To select for carbonyl-modified proteins, serum proteins were reacted with an aldehyde reactive probe that biotinylated the carbonyl modification. Modified proteins were enriched by avidin affinity and identified by two-dimensional liquid chromatography tandem MS. To identify the carbonyl modification, tryptic peptides from serum proteins were subjected to avidin affinity and the enriched modified peptides were analyzed by liquid chromatography tandem MS. It was noted that the aldehyde reactive probe tag created tag-specific fragment ions and neutral losses, and these extra features in the mass spectra inhibited identification of the modified peptides by database searching. To enhance the identification of carbonyl-modified peptides, a program was written that used the tag-specific fragment ions as a fingerprint (in silico filter program) and filtered the mass spectrometry data to highlight only modified peptides. A de novo-like database search algorithm was written (biotin peptide identification program) to identify the carbonyl-modified peptides. Although written specifically for our experiments, this software can be adapted to other modification and enrichment systems. Using these routines, a number of lipid peroxidation-derived protein carbonyls and direct side-chain oxidation proteins carbonyls were identified in SJL mouse serum.During inflammation, activated phagocytes secrete reactive nitrogen species (RNS) and reactive oxygen species (ROS) that can eliminate infectious agents. If inflammation is chronic, RNS and ROS can also damage surrounding host tissue, resulting in protein modification in the form of protein-carbonyls (1). Total protein carbonylation has been used as a marker of oxidative stress and inflammation and increased levels have been seen in heart disease, lung disease, aging, neurodegenerative disorders, and inflammatory bowel disease (2–7). The carbonylation of proteins can result from the direct oxidation of protein side-chains, forming the glutamate and aminoadipate semialdehydes (Scheme 1) (8, 9), but can also occur from the indirect oxidation of polyunsaturated fatty acids (lipid peroxidation) and carbohydrates, leading to a variety of reactive aldehydes (Scheme 2) (10). These aldehydes covalently modify proteins through conjugate addition (often Michael addition) to nucleophilic amino acid side chains, creating protein-bound carbonyls (10, 11).Open in a separate windowScheme 1.Direct oxidative carbonylation of proteins to form glutamate and aminoadipate semialdehydes.Open in a separate windowScheme 2.Reactive aldehydes, arising from oxidation of polyunsaturated fatty acids and carbohydrates, can indirectly lead to protein carbonylation.In a previous study, DNA oxidative damage products, from tissues from the SJL mouse model of inflammation, were quantitated (12). Only the lipid peroxidation adducts increased in association with inflammation, which suggested an important role of lipids in inflammatory disease progression and established a direct correlation between inflammation and the increased formation of reactive aldehydes from oxidized lipids. Although DNA modification because of inflammation has been the focus of many animal and human studies, it is proteins that are considered most likely to be ubiquitously affected by disease, response, and recovery (13), and the biological consequences include more rapid protein turnover as well as novel signaling (14–16). The overall carbonylation of proteins has been well documented in other inflammatory animal models, which have shown significant increases in protein-carbonyls in the mucosal lining of rat colon (17) and mouse colon (5) whereas increased levels of protein carbonyls were observed in rat serum, along with a higher turnover of proteins from the inflamed tissue (18, 19). Furthermore, increased protein carbonyl modification has been reported in studies of the colon mucosal lining from patients diagnosed with inflammatory bowel disease (20, 21). Taken together, these observations suggest that an increase in carbonylated proteins is likely in the SJL mouse and that the extent and type of protein-carbonyls could potentially be a marker for inflammation and disease.The SJL mouse is an experimental animal model used to assess in vivo toxicological responses to nitric oxide (NO) overproduction from inflammation (22). Injections of RscX lymphoma cells into these mice result in rapid tumor growth as well as host T-cell proliferation in lymph nodes, spleen, and liver, resulting in morbidity within 15 days. The induced macrophages create a 50-fold increase in NO production in spleen and lymph nodes and the post-translational modification 3-nitrotyrosine was highly elevated in spleen tissue.The identification of endogenously formed protein carbonyls in serum is challenging because of their low abundance and the large number of possible modifications (1, 2, 23), some of which are shown in Schemes 1 and 2. We recently identified proteins modified by the carbonyl 9,12-dioxo-10(E)-dodecenoic acid (DODE) in cells treated with the hydroperoxide of linoleic acid (13-HPODE) (24). This work used a technique first demonstrated by Maier and coworkers (25, 26). Protein carbonyls were derivatized with an aldehyde reactive probe (ARP),¹ a biotinylated hydroxylamine that reacts preferentially with aldehyde and keto groups (27), allowing for subsequent enrichment of the modified proteins by avidin affinity. DODE-modified proteins were also identified using an anti-DODE antibody and Western blots. Although a number of DODE modified proteins were identified, we were unable to definitively identify the carbonyl modified peptides by mass spectrometry due both to low abundance and to the interference of ARP-tag-specific fragment ions on database searching.In this current study, SJL mouse serum was screened for the presence of protein carbonyls endogenously formed during inflammation. Carbonyl-modified proteins were then identified using techniques previously established (24); first anti-DODE Western blotting followed by ARP derivatization/enrichment and two-dimensional liquid chromatography tandem MS (2D-LC-MS/MS). These proteins then formed a database of putative carbonyl-modified proteins from SJL mouse serum. To identify the type of carbonyl modification and the modified peptide, the ARP derivatized peptides were enriched and analyzed by mass spectrometry. To minimize the confounding effect of ARP fragmentation, an algorithm (in silico filter) was written that filtered the mass spectrometry data to select only those peptides containing the known ARP pattern of fragmentation. This alone effectively reduced the number of false positives. To further alleviate the interfering effects of ARP fragments on peptide identification by database searching, a de novo searching algorithm (Biotin Peptide Identification program, BPI) was written. Peptides were evaluated against the database of proteins that had been previously identified as potentially carbonyl modified. Because modified peptides were searched against a finite list of proteins and all results were manually evaluated, the BPI program did not calculate a statistical peptide score, which allowed the identification of lower abundant modified peptides that would not be considered significant by standard search engines such as Mascot. The BPI program was also written with the flexibility to evaluate a wide range of known carbonyl-adduct masses and could therefore screen for a large number of carbonyl adducts at one time. This should also allow the program to be used with modification/enrichment systems other than the one used here. The program thus selected a finite number of carbonyl modified peptides, resulting in the identification of a number of proteins that were endogenously carbonylated in serum from the SJL mouse inflammation model.     

Pathogenic VCP/TER94 alleles are dominant actives and contribute to neurodegeneration by altering cellular ATP level in a Drosophila IBMPFD model

Inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) is caused by mutations in Valosin-containing protein (VCP), a hexameric AAA ATPase that participates in a variety of cellular processes such as protein degradation, organelle biogenesis, and cell-cycle regulation. To understand how VCP mutations cause IBMPFD, we have established a Drosophila model by overexpressing TER94 (the sole Drosophila VCP ortholog) carrying mutations analogous to those implicated in IBMPFD. Expression of these TER94 mutants in muscle and nervous systems causes tissue degeneration, recapitulating the pathogenic phenotypes in IBMPFD patients. TER94-induced neurodegenerative defects are enhanced by elevated expression of wild-type TER94, suggesting that the pathogenic alleles are dominant active mutations. This conclusion is further supported by the observation that TER94-induced neurodegenerative defects require the formation of hexamer complex, a prerequisite for a functional AAA ATPase. Surprisingly, while disruptions of the ubiquitin-proteasome system (UPS) and the ER-associated degradation (ERAD) have been implicated as causes for VCP-induced tissue degeneration, these processes are not significantly affected in our fly model. Instead, the neurodegenerative defect of TER94 mutants seems sensitive to the level of cellular ATP. We show that increasing cellular ATP by independent mechanisms could suppress the phenotypes of TER94 mutants. Conversely, decreasing cellular ATP would enhance the TER94 mutant phenotypes. Taken together, our analyses have defined the nature of IBMPFD-causing VCP mutations and made an unexpected link between cellular ATP level and IBMPFD pathogenesis.     

A new drug design targeting the adenosinergic system for Huntington's disease

Background

Huntington''s disease (HD) is a neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The expanded CAG repeats are translated into polyglutamine (polyQ), causing aberrant functions as well as aggregate formation of mutant Htt. Effective treatments for HD are yet to be developed.

Methodology/Principal Findings

Here, we report a novel dual-function compound, N ⁶-(4-hydroxybenzyl)adenine riboside (designated T1-11) which activates the A_2AR and a major adenosine transporter (ENT1). T1-11 was originally isolated from a Chinese medicinal herb. Molecular modeling analyses showed that T1-11 binds to the adenosine pockets of the A_2AR and ENT1. Introduction of T1-11 into the striatum significantly enhanced the level of striatal adenosine as determined by a microdialysis technique, demonstrating that T1-11 inhibited adenosine uptake in vivo. A single intraperitoneal injection of T1-11 in wildtype mice, but not in A_2AR knockout mice, increased cAMP level in the brain. Thus, T1-11 enters the brain and elevates cAMP via activation of the A_2AR in vivo. Most importantly, addition of T1-11 (0.05 mg/ml) to the drinking water of a transgenic mouse model of HD (R6/2) ameliorated the progressive deterioration in motor coordination, reduced the formation of striatal Htt aggregates, elevated proteasome activity, and increased the level of an important neurotrophic factor (brain derived neurotrophic factor) in the brain. These results demonstrate the therapeutic potential of T1-11 for treating HD.

Conclusions/Significance

The dual functions of T1-11 enable T1-11 to effectively activate the adenosinergic system and subsequently delay the progression of HD. This is a novel therapeutic strategy for HD. Similar dual-function drugs aimed at a particular neurotransmitter system as proposed herein may be applicable to other neurotransmitter systems (e.g., the dopamine receptor/dopamine transporter and the serotonin receptor/serotonin transporter) and may facilitate the development of new drugs for other neurodegenerative diseases.     

Curcumin prevents high fat diet induced insulin resistance and obesity via attenuating lipogenesis in liver and inflammatory pathway in adipocytes

Background

Mechanisms underlying the attenuation of body weight gain and insulin resistance in response to high fat diet (HFD) by the curry compound curcumin need to be further explored. Although the attenuation of the inflammatory pathway is an accepted mechanism, a recent study suggested that curcumin stimulates Wnt signaling pathway and hence suppresses adipogenic differentiation. This is in contrast with the known repressive effect of curcumin on Wnt signaling in other cell lineages.

Methodology and Principal Findings

We conducted the examination on low fat diet, or HFD fed C57BL/6J mice with or without curcumin intervention for 28 weeks. Curcumin significantly attenuated the effect of HFD on glucose disposal, body weight/fat gain, as well as the development of insulin resistance. No stimulatory effect on Wnt activation was observed in the mature fat tissue. In addition, curcumin did not stimulate Wnt signaling in vitro in primary rat adipocytes. Furthermore, curcumin inhibited lipogenic gene expression in the liver and blocked the effects of HFD on macrophage infiltration and the inflammatory pathway in the adipose tissue.

Conclusions and Significance

We conclude that the beneficial effect of curcumin during HFD consumption is mediated by attenuating lipogenic gene expression in the liver and the inflammatory response in the adipose tissue, in the absence of stimulation of Wnt signaling in mature adipocytes.     

Decreased circulating endothelial progenitor cell levels and function in patients with nonalcoholic fatty liver disease

Objectives

Nonalcoholic fatty liver disease (NAFLD) is associated with advanced atherosclerosis and a higher risk of cardiovascular disease. Increasing evidence suggests that injured endothelial monolayer is regenerated by circulating bone marrow derived-endothelial progenitor cells (EPCs), and levels of circulating EPCs reflect vascular repair capacity. However, the relation between NAFLD and EPC remains unclear. Here, we tested the hypothesis that patients with nonalcoholic fatty liver disease (NAFLD) might have decreased endothelial progenitor cell (EPC) levels and attenuated EPC function.

Methods and Results

A total of 312 consecutive patients undergoing elective coronary angiography because of suspected coronary artery disease were screened and received examinations of abdominal ultrasonography between July 2009 and November 2010. Finally, 34 patients with an ultrasonographic diagnosis of NAFLD, and 68 age- and sex-matched controls without NAFLD were enrolled. Flow cytometry with quantification of EPC markers (defined as CD34⁺, CD34⁺KDR⁺, and CD34⁺KDR⁺CD133⁺) in peripheral blood samples was used to assess circulating EPC numbers. The adhesive function, and migration, and tube formation capacities of EPCs were also determined in NAFLD patients and controls. Patients with NAFLD had a significantly higher incidence of metabolic syndrome, previous myocardial infarction, hyperuricemia, and higher waist circumference, body mass index, fasting glucose and triglyceride levels. In addition, patients with NAFLD had significantly decreased circulating EPC levels (all P<0.05), attenuated EPC functions, and enhanced systemic inflammation compared to controls. Multivariate logistic regression analysis showed that circulating EPC level (CD34⁺KDR⁺ [cells/10⁵ events]) was an independent reverse predictor of NAFLD (Odds ratio: 0.78; 95% confidence interval: 0.69–0.89, P<0.001).

Conclusions

NAFLD patients have decreased circulating EPC numbers and functions than those without NAFLD, which may be one of the mechanisms to explain atherosclerotic disease progression and enhanced cardiovascular risk in patients with NAFLD.