Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry

Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.     

Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP

The E3 ubiquitin ligase CHIP is involved in protein triage, serving as a co-chaperone for refolding as well as catalyzing ubiquitination of substrates. CHIP functions with both the stress induced Hsp70 and constitutive Hsc70 chaperones, and also plays a role in maintaining their balance in the cell. When the chaperones carry no client proteins, CHIP catalyzes their polyubiquitination and subsequent proteasomal degradation. Although Hsp70 and Hsc70 are highly homologous in sequence and similar in structure, CHIP mediated ubiquitination promotes degradation of Hsp70 with a higher efficiency than for Hsc70. Here we report a detailed and systematic investigation to characterize if there are significant differences in the CHIP in vitro ubiquitination of human Hsp70 and Hsc70. Proteomic analysis by mass spectrometry revealed that only 12 of 39 detectable lysine residues were ubiquitinated by UbcH5a in Hsp70 and only 16 of 45 in Hsc70. The only conserved lysine identified as ubiquitinated in one but not the other heat shock protein was K159 in Hsc70. Ubiquitination assays with K-R ubiquitin mutants showed that multiple Ub chain types are formed and that the distribution is different for Hsp70 versus Hsc70. CHIP ubiquitination with the E2 enzyme Ube2W is predominantly directed to the N-terminal amine of the substrate; however, some internal lysine modifications were also detected. Together, our results provide a detailed view of the differences in CHIP ubiquitination of these two very similar proteins, and show a clear example where substantial differences in ubiquitination can be generated by a single E3 ligase in response to not only different E2 enzymes but subtle differences in the substrate.     

Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress

Genetic deletion of the mitochondrial deacetylase sirtuin-3 (Sirt3) results in increased mitochondrial superoxide, a tumor-permissive environment, and mammary tumor development. MnSOD contains a nutrient- and ionizing radiation (IR)-dependent reversible acetyl-lysine that is hyperacetylated in Sirt3?/? livers at 3 months of age. Livers of Sirt3?/? mice exhibit decreased MnSOD activity, but not immunoreactive protein, relative to wild-type livers. Reintroduction of wild-type but not deacetylation null Sirt3 into Sirt3?/? MEFs deacetylated lysine and restored MnSOD activity. Site-directed mutagenesis of MnSOD lysine 122 to an arginine, mimicking deacetylation (lenti-MnSOD(K122-R)), increased MnSOD activity when expressed in MnSOD?/? MEFs, suggesting acetylation directly regulates function. Furthermore, infection of Sirt3?/? MEFs with lenti-MnSOD(K122-R) inhibited in vitro immortalization by an oncogene (Ras), inhibited IR-induced genomic instability, and decreased mitochondrial superoxide. Finally, IR was unable to induce MnSOD deacetylation or activity in Sirt3?/? livers, and these irradiated livers displayed significant IR-induced cell damage and microvacuolization in their hepatocytes.     

Yeast Asc1p and mammalian RACK1 are functionally orthologous core 40S ribosomal proteins that repress gene expression

Translation of mRNA into protein is a fundamental step in eukaryotic gene expression requiring the large (60S) and small (40S) ribosome subunits and associated proteins. By modern proteomic approaches, we previously identified a novel 40S-associated protein named Asc1p in budding yeast and RACK1 in mammals. The goals of this study were to establish Asc1p or RACK1 as a core conserved eukaryotic ribosomal protein and to determine the role of Asc1p or RACK1 in translational control. We provide biochemical, evolutionary, genetic, and functional evidence showing that Asc1p or RACK1 is indeed a conserved core component of the eukaryotic ribosome. We also show that purified Asc1p-deficient ribosomes have increased translational activity compared to that of wild-type yeast ribosomes. Further, we demonstrate that asc1Delta null strains have increased levels of specific proteins in vivo and that this molecular phenotype is complemented by either Asc1p or RACK1. Our data suggest that one of Asc1p's or RACK1's functions is to repress gene expression.     

Differential antioxidant properties of red wine in water soluble and lipid soluble peroxyl radical generating systems

Red wine and its components have been shown to possess cardioprotective and anti-atherogenic effects. Additionally, red wine and many of its components like catechin, epicatechin, rutin, transresveratrol and quercetin possess antioxidant properties. Oxidized low density lipoprotein (LDL) is involved in the development of an atherosclerotic lesion. Red wine, therefore, may be anti-atherogenic because of its antioxidant effects on LDL modification. This study examined the antioxidant effects of catechin, epicatechin, rutin, transresveratrol, quercetin and Merlot wines on LDL oxidation. Merlot was chosen because although other red wines have been tested, limited information exists for this variety. Oxidation was carried out with AAPH (2,2-Azo-bis(2-amidinopropane) dihydrochloride) and AMVN (2,2-Azo-bis(2,4-dimethylvaleronitrile)), as water and lipid soluble peroxyl radical generating systems (FRGS), respectively. This allowed us to determine the lipophilic antioxidant characteristics of the wine and its components. Conjugated diene assays were used to measure LDL oxidation over 6 hrs. In an AAPH system, all polyphenolic compounds except transresveratrol displayed an antioxidant effect. LDL oxidation by AAPH was also inhibited by aliquots of Merlot wine. No antioxidant effects were observed in an AMVN environment except for a mild antioxidant effect by quercetin. Surprisingly, incubation of LDL with Merlot wine strongly protected against oxidation by AMVN. In summary, the five phenolic compounds displayed antioxidant effects in a water soluble free radical generating system, but only quercetin showed this in a lipid soluble one. However, red wine inhibited LDL oxidation by both water and lipid soluble free radical generating systems. Our data suggest, therefore, that red wines contain unidentified antioxidants that provide protection against LDL oxidation within a lipid soluble environment. (Mol Cell Biochem 263: 211–215, 2004)     

Helicobacter pylori exploits a unique repertoire of type IV secretion system components for pilus assembly at the bacteria-host cell interface

Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS.     

The importance of lipid solubility in antioxidants and free radical generating systems for determining lipoprotein proxidation.

The oxidative modification of low-density lipoprotein (LDL) plays an important role in atherosclerosis. Protecting LDL from oxidation has been shown to reduce the risk of coronary heart disease. In this study, we compared the protective effects of two lipophilic antioxidants (vitamin E and lazaroid) with two hydrophilic antioxidants (trolox and vitamin C) in the presence of several different free radical generating systems. Vitamin E (IC50 = 5.9 microM) and lazaroid (IC50 = 5.0 microM) were more effective in inhibiting lipid peroxidation caused by a Fe-ADP free radical generating system than vitamin C (IC50 = 5.2 x 10(3) microM) and trolox (IC5 = 1.2 x 10(3) microM). Preincubation of lipoproteins with a lipophilic antioxidant increased the protective effect against various free radicals. Preincubation with hydrophilic antioxidants did not have an effect. We also tested the efficacy of the antioxidants when the free radicals were generated within the lipid or the aqueous environment surrounding the LDL. For this purpose, we used the peroxyl generating azo-compounds AMVN (2,2'-azobis(2,4-dimethylvaleronitrile)) and AAPH (2,2'azobis(2-amidinopropane) dihydrochloride). All of the antioxidants tested were more effective against free radicals generated in a water soluble medium than they were against free radicals generated in a lipid environment. In conclusion, our data demonstrate that lipid solubility is an important factor for both the antioxidant and the free radical generating systems in determining the extent of lipid peroxidation in LDL. Our data also demonstrate that antioxidant efficacy in one set of experimental conditions may not necessarily translate into a similar degree of protection in another set of conditions where lipophilicity is a variable.     

Obesity and altered glucose metabolism impact HDL composition in CETP transgenic mice: a role for ovarian hormones

Mechanisms underlying changes in HDL composition caused by obesity are poorly defined, partly because mice lack expression of cholesteryl ester transfer protein (CETP), which shuttles triglyceride and cholesteryl ester between lipoproteins. Because menopause is associated with weight gain, altered glucose metabolism, and changes in HDL, we tested the effect of feeding a high-fat diet (HFD) and ovariectomy (OVX) on glucose metabolism and HDL composition in CETP transgenic mice. After OVX, female CETP-expressing mice had accelerated weight gain with HFD-feeding and impaired glucose tolerance by hyperglycemic clamp techniques, compared with OVX mice fed a low-fat diet (LFD). Sham-operated mice (SHAM) did not show HFD-induced weight gain and had less glucose intolerance than OVX mice. Using shotgun HDL proteomics, HFD-feeding in OVX mice had a large effect on HDL composition, including increased levels of apoA2, apoA4, apoC2, and apoC3, proteins involved in TG metabolism. These changes were associated with decreased hepatic expression of SR-B1, ABCA1, and LDL receptor, proteins involved in modulating the lipid content of HDL. In SHAM mice, there were minimal changes in HDL composition with HFD feeding. These studies suggest that the absence of ovarian hormones negatively influences the response to high-fat feeding in terms of glucose tolerance and HDL composition. CETP-expressing mice may represent a useful model to define how metabolic changes affect HDL composition and function.     

The importance of lipid solubility in antioxidants and free radical generating systems for determining lipoprotein peroxidation

The oxidative modification of low-density lipoprotein (LDL) plays an important role in atherosclerosis. Protecting LDL from oxidation has been shown to reduce the risk of coronary heart disease. In this study, we compared the protective effects of two lipophilic antioxidants (vitamin E and lazaroid) with two hydrophilic antioxidants (trolox and vitamin C) in the presence of several different free radical generating systems. Vitamin E (IC₅₀ = 5.9 μM) and lazaroid (IC₅₀ = 5.0 μM) were more effective in inhibiting lipid peroxidation caused by a Fe-ADP free radical generating system than vitamin C (IC₅₀ = 5.2 × 10³ μM) and trolox (IC₅ = 1.2 × 10³ μM). Preincubation of lipoproteins with a lipophilic antioxidant increased the protective effect against various free radicals. Preincubation with hydrophilic antioxidants did not have an effect. We also tested the efficacy of the antioxidants when the free radicals were generated within the lipid or the aqueous environment surrounding the LDL. For this purpose, we used the peroxyl generating azo-compounds AMVN (2,2′-azobis(2,4-dimethylvaleronitrile)) and AAPH (2,2′azobis (2-amidinopropane) dihydrochloride). All of the antioxidants tested were more effective against free radicals generated in a water soluble medium than they were against free radicals generated in a lipid environment. In conclusion, our data demonstrate that lipid solubility is an important factor for both the antioxidant and the free radical generating systems in determining the extent of lipid peroxidation in LDL. Our data also demonstrate that antioxidant efficacy in one set of experimental conditions may not necessarily translate into a similar degree of protection in another set of conditions where lipophilicity is a variable.     

Global impact of oncogenic Src on a phosphotyrosine proteome

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype.