Application of quantitative proteomics to the integrated analysis of the ubiquitylated and global proteomes of xenograft tumor tissues

Background

Post-translational modification by ubiquitin is a fundamental regulatory mechanism that is implicated in many cellular processes including the cell cycle, apoptosis, cell adhesion, angiogenesis, and tumor growth. The low stoichiometry of ubiquitylation presents an analytical challenge for the detection of endogenously modified proteins in the absence of enrichment strategies. The recent availability of antibodies recognizing peptides with Lys residues containing a di-Gly ubiquitin remnant (K-ε-GG) has greatly improved the ability to enrich and identify ubiquitylation sites from complex protein lysates via mass spectrometry. To date, there have not been any published studies that quantitatively assess the changes in endogenous ubiquitin-modification protein stoichiometry status at the proteome level from different tissues.

Results

In this study, we applied an integrated quantitative mass spectrometry based approach using isobaric tags for relative and absolute quantitation (iTRAQ) to interrogate the ubiquitin-modified proteome and the cognate global proteome levels from luminal and basal breast cancer patient-derived xenograft tissues. Among the proteins with quantitative global and ubiquitylation data, 91 % had unchanged levels of total protein relative abundance, and less than 5 % of these proteins had up- or down-regulated ubiquitylation levels. Of particular note, greater than half of the proteins with observed changes in their total protein level also had up- or down-regulated changes in their ubiquitylation level.

Conclusions

This is the first report of the application of iTRAQ-based quantification to the integrated analysis of the ubiquitylated and global proteomes at the tissue level. Our results underscore the importance of conducting integrated analyses of the global and ubiquitylated proteomes toward elucidating the specific functional significance of ubiquitylation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9086-5) contains supplementary material, which is available to authorized users.     

Enzymes that hydrolyze adenine nucleotides in a model of hypercholesterolemia induced by Triton WR-1339: protective effects of β-caryophyllene

In waterfowls, overfeeding leads to a hepatic steatosis, also called “foie gras.” We decided to investigate the role of glucose metabolism in steatosis emergence. For this, we measured the expression of genes during the 12 h following the last meal of the overfeeding period. As expected, it showed that the expression of glucose transporter is more precocious in jejunal mucosa, especially for SGLT1, known to be the major transporter at the apical surface. In the liver, GLUT2 and HK1 are upregulated at the same time and seem to work together to import glucose. In peripherals tissues, such as muscle and subcutaneous adipose tissue (SAT), expression of genes of interest occurs later than the one in jejunum and liver. These results are in accordance with the evolution of glycemia. This study allows us to better understand the kinetic treatment of glucose after a meal in overfed ducks. It also will allow researchers to better target their sampling time knowing the optimal point of expression of each gene.     

Studies on synthesis and degradation rates and some molecular properties of guinea-pig muscle acylphosphatase.

Acylphosphatase (acylphosphate phosphohydrolase, EC 3.6.1.7) was purified from guinea-pig muscle by a procedure involving immuno-affinity chromatography and a subsequent ion-exchange chromatography. This purification technique gave an overall yield of about 60% and permitted the isolation of three molecular forms with acylphosphatase activity, with a distribution greatly resembling those found in horse and turkey muscle. The main form appears to be very similar to the corresponding form in horse and turkey muscle, as indicated by amino acid composition, end-group analysis, the presence of glutathione as a mixed disulphide in almost the same stoichiometric ratio and kinetic analysis. From turnover data, the main form of acylphosphatase in guinea-pig muscle exhibits a degradation constant of 0.10 day-1, corresponding to a half-life of 6.8 days. These values are very close to those found for muscle total soluble proteins.     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

L78部分氨基酸残基对SERCA1a钙转运功能的影响

跨肌浆网膜的钙离子ATP酶SERCA1a可在水解ATP的同时逆浓度梯度从胞浆转运钙离子入肌浆网,引发收缩的骨骼肌细胞舒张.目前对SERCA1a结构与功能的研究,要领先于对其他P型离子转运ATP酶的研究.为了解SERCA1a跨膜螺旋M7与M8膜内侧连接部Linker78(L78)的功能,我们评估了L78部分氨基酸残基突变对SERCA1a反应循环的影响,发现:除G864A之外的所有突变体均可不同程度地降低钙离子转运速率;G862或P863氨基酸残基的突变导致SERCA1a的ATP酶活性显著降低或丧失,并引起由ATP或无机磷酸Pi生成的磷酸化酶中间体EP量显著减少;A893被P取代对SERCA1a的影响与G862、P863突变相似;与野生型SERCA1a相比,G864A与FMQ873-875单突变体ATP酶活性及EP生成量无明显差别.上述结果表明,M7、M8膜内侧连接部L78不仅与跨膜区钙离子转运过程密切相关,而且L78在GPG862-864处及A893附近的正确转折对胞浆结构域P的磷酸化也具有关键的远距离调控作用,提示L78的结构及柔度对SERCA1a构象正常的周期性变化至关重要.     

Initial denaturing conditions influence the slow folding phase of acylphosphatase associated with proline isomerization

The folding kinetics of human common-type acylphosphatase (cAcP) from its urea- and TFE-denatured states have been determined by stopped-flow fluorescence techniques. The refolding reaction from the highly unfolded state formed in urea is characterized by double exponential behavior that includes a slow phase associated with isomerism of the Gly53-Pro54 peptide bond. However, this slow phase is absent when refolding is initiated by dilution of the highly a-helical denatured state formed in the presence of 40% trifluoroethanol (TFE). NMR studies of a peptide fragment corresponding to residues Gly53-Gly69 of cAcP indicate that only the native-like trans isomer of the Gly-Pro peptide bond is significantly populated in the presence of TFE, whereas both the cis and trans isomers are found in an approximately 1:9 ratio for the peptide bond in aqueous solution. Molecular modeling studies in conjunction with NMR experiments suggest that the trans isomer of the Gly53-Pro54 peptide bond is stabilized in TFE by the formation of a nonnative-like hydrogen bond between the CO group of Gly53 and the NH group of Lys57. These results therefore reveal that a specific nonnative interaction in the denatured state can increase significantly the overall efficiency of refolding.     

Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress

DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4–5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic‐membrane‐located inhibitor of proton‐driven F₁F₀ ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin‐resistant (Nov^R) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with Nov^R gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug‐treated bacteria. The Salmonella cytosol reaches pH 5–6 in response to an external pH of 4–5: the ATP‐dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP‐dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid‐mediated impairment of the negative supercoiling activity of gyrase.     

Interim Response of Wading Birds (Pelecaniformes and Ciconiiformes) and Waterfowl (Anseriformes) to the Kissimmee River Restoration Project,Florida, U.S.A

Success of the Kissimmee River Restoration Project will be evaluated in part by monitoring populations of wading birds (Pelecaniformes and Ciconiiformes) and waterfowl (Anseriformes). These two waterbird guilds were integral components of the pre‐channelization river–floodplain ecosystem, and both declined substantially following channelization. Restoration is expected to attract wading birds and waterfowl by reintroducing naturally fluctuating water levels, seasonal hydroperiods, and historic vegetation communities. Post‐construction aerial surveys (November 2001 to May 2008) within the Phase I restoration area indicate that the abundance and species richness of both wading birds and waterfowl have shown a positive restoration response thus far. Dry season abundance of aquatic wading birds and waterfowl has exceeded restoration expectations (≥30.6 birds/km² and ≥3.9 birds/km², respectively) each year since the completion of restoration Phase I in 2001. While there has been a significant positive restoration effect on waterfowl abundance, waterfowl species richness (n = 6) has not yet reached the restoration expectation of ≥13 species. Abundance of the terrestrial cattle egret (Bubulcus ibis), which increased dramatically after the majority of floodplain wetlands were converted to cattle pastures in the channelized system, has shown a significant negative response to restoration. It is anticipated that completion of the remaining phases of restoration (II/III), and implementation of the Kissimmee River Headwaters Revitalization water regulation schedule by 2019, will further increase and improve habitat for wading birds and waterfowl by reestablishing floodplain hydrology that more closely mimics historical conditions.     

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).     

Blood Dendritic Cell Frequency Declines in Idiopathic Parkinson’s Disease and Is Associated with Motor Symptom Severity

The role of inflammation in Parkinson’s Disease (PD) is well appreciated, but its underlying mechanisms are still unclear. Our objective was to determine whether dendritic cells (DC), a unique type of migratory immune cells that regulate immunological response and inflammation have an impact on PD. In a case-control study including 80 PD patients and 80 age- and gender-matched healthy control subjects, the two main blood subsets of plasmacytoid and myeloid DC were defined by flow cytometry analysis. Clinical evaluation of subjects consisting of cognition and depression assessment was performed using the Mini Mental State Examination and the Beck Depression Inventory. The severity of motor symptoms was measured using the Unified Parkinson’s Disease Rating Scale-Part III. Comparison between patient and control DC measures and their relationships with clinical assessments were evaluated.The following main results were obtained: 1) the level of circulating DC (mainly the myeloid subset) was significantly reduced in PD patients in comparison with healthy controls; 2) after controlling for depressive and cognitive characteristics, the frequency of myeloid DC was confirmed as one of the independent determinants of PD; 3) the number of both myeloid and plasmacytoid DC was negatively associated with motor symptom severity. Overall, the decline of blood DC, perhaps due to the recruitment of immune cells to the site of disease-specific lesions, can be considered a clue of the immune alteration that characterizes PD, suggesting innovative exploitations of DC monitoring as a clinically significant tool for PD treatment. Indeed, this study suggests that reduced peripheral blood DC are a pathologically-relevant factor of PD and also displays the urgency to better understand DC role in PD for unraveling the immune system contribution to disease progression and thus favoring the development of innovative therapies ideally based on immunomodulation.