Prioritizing targets for structural biology through the lens of proteomics: The archaeal protein TGAM_1934 from Thermococcus gammatolerans

ORFans are hypothetical proteins lacking any significant sequence similarity with other proteins. Here, we highlighted by quantitative proteomics the TGAM_1934 ORFan from the hyperradioresistant Thermococcus gammatolerans archaeon as one of the most abundant hypothetical proteins. This protein has been selected as a priority target for structure determination on the basis of its abundance in three cellular conditions. Its solution structure has been determined using multidimensional heteronuclear NMR spectroscopy. TGAM_1934 displays an original fold, although sharing some similarities with the 3D structure of the bacterial ortholog of frataxin, CyaY, a protein conserved in bacteria and eukaryotes and involved in iron–sulfur cluster biogenesis. These results highlight the potential of structural proteomics in prioritizing ORFan targets for structure determination based on quantitative proteomics data. The proteomic data and structure coordinates have been deposited to the ProteomeXchange with identifier PXD000402 ( http://proteomecentral.proteomexchange.org/dataset/PXD000402 ) and Protein Data Bank under the accession number 2mcf, respectively.     

Separate physiological roles for two isozymes of pyridine nucleotide-linked glycerol-3-phosphate dehydrogenase in chicken

Summary Two isozymes of diphosphopyridine nucleotide-linked glycerol-3-P dehydrogenase (E.C. 1.1.1.8) were studied with respect to their tissue distribution in chicken, their ontogeny in chicken liver, and their avian taxonomic distribution. These isozymes in chicken are designated liver type and muscle type based on the tissue of highest concentration.Electrophoretic analysis shows that the liver type is found in small amounts or is undetectable in all tissues studied exept liver. The muscle type is found in skeletal muscles and kidney. Presumptive hybrid enzymes occur at low levels in chicken liver and kidney.The tissue distribution of glycerol-3-P dehydrogenase in several birds capable of sustained flight is different than in chicken. A single electrophoretic form is predominant in both liver and breast muscle with the activity in liver about ten times greater than in breast muscle. Chicken liver and breast muscle extracts have similar levels of glycerol-3-P dehydrogenase activity.Glycerol-3-P dehydrogenase, solely of the liver type, appears simultaneously with liver formation in the chicken embryo and reaches peak concentrations in the liver between the 10th and 14th day of embryonic development. This embryonic pattern is quite different from the muscle type in breast muscle where the enzyme does not appear until after hatching.These observations, when correlated with the metabolism of lipids and carbohydrates in liver and breast muscle of chicken and birds capable of sustained flight, lend support to a hypothesis that the two isozymes have distinct physiological roles. The liver type is associated with the metabolism of the glycerol moiety of triglycerides and phospholipids whereas the muscle type operates in concert with muscle type lactate dehydrogenase to oxidize DPNH during anaerobic muscle glycolysis. The role of glycerol-3-P dehydrogenase in muscle appears to be essential for prolonging anaerobic glycolysis. It is proposed that the isozymes of glycerol-3-P dehydrogenase originated by gene duplication and then diverged via selective evolution to fulfill the metabolic roles proposed.This research was supported in part by Grant NSG-374 from the National Aeronautics and Space Administration, by Grant CA-03611 from the National Institute of Health, and by Grant P77 J from the American Cancer Society. Publication 810 from the Graduate Department of Biochemistry, Brandeis University.Recipient of Training Grant GM-0212 from the National Institute of General Medical Sciences.     

Investigation on glycosylation patterns of proteins from human liver cancer cell lines based on the multiplexed proteomics technology

Glycosylation, a very important post-translational modification of proteins, is increasingly coming into notice. However, large-scale, throughput investigations on glycosylated proteins are few. We applied a sensitive and fast fluorescence-based multiplexed proteomics (MP) technology which included two-dimensional gel electrophoresis (2-DE) followed by the fluorescence staining of glycoprotein and mass spectrometry identification for the purpose of constructing glycoprotein databases of the typical human hepatocellular carcinoma cell lines including Hep3B cell line without metastasis and MHCC97H with highly metastatic potential as well as the control non-tumor Chang liver cell. 74+/-2 (n=3), 78+/-3 (n=3) and 72+/-5 (n=3) glycoprotein spots were detected on 2-DE gels from Chang liver, Hep3B and MHCC97H cell sample using this MP technique, respectively. In all, 80 glycoproteins from three cell lines were successfully identified via peptide mass profiling using MALDI-TOF-MS/MS and the identified glycoproteins were annotated to our databases. In addition, we also found the glycosylation pattern differences among these three cell lines. The protein glycosylation alteration would be have great significance for the diagnosis of HCC and prediction of its metastasis. This study described the construction of glycosylation patterns of proteins and glycoproteome databases of human liver cells by the novel technological platform. The glycoproteome databases also provide essential basis for following study.     

稻米品质形成的生理基础研究进展

综述了水稻(Oryza sativa L)米质形成的生理基础的研究进展,主要包括稻米品质形成期的源库特征、淀粉合成的关键酶以及淀粉粒的形态、结构及其糊化特性等几个内容,从植物生理学角度来揭示稻米品质形成的规律有着重要的理论意义.     

Proteomic identification of nitrated brain proteins in amnestic mild cognitive impairment: a regional study

Oxidative stress is an imbalance between the level of antioxidants and oxidants in a cell. Oxidative stress has been shown in brain of subjects with mild cognitive impairment (MCI) as well Alzheimer's disease (AD). MCI is considered as a transition phase between control and AD. The focus of the current study was to identify nitrated proteins in the hippocampus and inferior parietal lobule (IPL) brain regions of subjects with amnestic MCI using proteomics. The identified nitrated proteins in MCI brain were compared to those previously reported to be nitrated and oxidatively modified in AD brain, a comparison that might provide an invaluable insight into the progression of the disease.     

小菜蛾溴氰菊酯敏感和抗性品系蛋白质表达谱的比较分析

杀虫剂抗性是害虫防治中的一个主要挑战。为有效治理抗性, 我们必须了解杀虫剂诱导的害虫生理生化变化。目前害虫抗药性的一些机理已经清楚, 但更多的相关机制还有待探究。本研究通过蛋白质组学方法检测了小菜蛾Plutella xylostella溴氰菊酯敏感和抗性品系间蛋白质组的表达差异。结果显示： SDS-PAGE胶上有大约300个蛋白差异点, 其中23个蛋白点具2.5倍以上的表达差异, 通过MALDI-TOF-MS, 我们成功鉴定出8个蛋白, 其中包括化感蛋白CSP2、 铜锌超氧化物歧化酶和peroxiredoxin样蛋白。通过实时定量PCR（real-time quantitative PCR, qPCR）分析了其中5个蛋白的mRNA 表达水平, 结果表明mRNA 表达水平不能真实反映蛋白的表达水平。免疫印迹验证了双向电泳中SOD1的表达差异。本研究有力地证明溴氰菊酯诱导小菜蛾成虫蛋白质组表达变化, 这为进一步筛选抗性靶标提供很大的帮助。     

Circadian rhythms and feeding time in fishes

Synopsis Although several studies have described effects of meal frequency and timing of meals on growth performance and body composition of different species of fishes, the mechanisms by which such variables influence the energy partitioning processes is not known. They may interact with the natural feeding rhythm of the fish, or with various behavioural and physiological parameters that exhibit circadian-like patterns; however, in most cases, the endogenous character of such rhythms is not clear. The time of feeding, perse, can act as a Zeitgeber and override the effect of the light/dark alternation. Fish that are fed always at the same time of day show typical pre-feeding activity. Blood levels of some nutrients and hormones also show peaks or troughs at the time of feeding, but whether these are pre-feeding or post-prandial events is not clear. These results from fish are compared with similar studies with mammals. The existence and location of an endogenous multi-oscillator system is also discussed.     

Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC)

We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.     

Motor pattern selection by combinatorial code of interneuronal pathways

We use a modeling approach to examine ideas derived from physiological network analyses, pertaining to the switch of a motor control network between two opposite control modes. We studied the femur–tibia joint control system of the insect leg, and its switch between resistance reflex in posture control and “active reaction” in walking, both elicited by the same sensory input. The femur–tibia network was modeled by fitting the responses of model neurons to those obtained in animals. The strengths of 16 interneuronal pathways that integrate sensory input were then assigned three different values and varied independently, generating a database of more than 43 million network variants. We demonstrate that the same neural network can produce the two different behaviors, depending on the combinatorial code of interneuronal pathways. That is, a switch between behaviors, such as standing to walking, can be brought about by altering the strengths of selected sensory integration pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.