Search

Breast cancer is the most common type of cancer in women in many areas and is increasing found in developing countries, where the majority of cases are diagnosed in late stages. Retinoic acids, through their associated nuclear receptors, exert intoxicating effects on cell growth, differentiation and apoptosis, and hold significant promise in relation to cancer therapy and chemoprevention. To enhance our understanding of the molecular mechanisms associated with retinoic acids in the breast cancer cell line MCF-7 in a time-dependent manner, we conducted a proteomic analysis of MCF-7 cells using the 2-DE couple with high-throughput mass spectrometry and bioinformatics tools. In the 2-DE patterns of MCF-7 cells treated with retinoic acid in a time-dependent manner, 35 protein spots were found to be differentially expressed. These were 17 increased, 4 decreased, and 14 unevenly expressed protein spots, all of which were analyzed using LTQ-FTICR mass spectrometry. Furthermore, five candidate proteins, up-regulated, were validated by western blotting. These were nucleoredoxin, latexin, aminomethyltransferase, translationally controlled one tumor protein, and rab GDP dissociation inhibitor β. These observations represent novel findings leading to new insight into the exact mechanism behind the effect of retinoic acids in MCF-7 cells while also identifying possible therapeutic targets for breast cancer diagnosis and novel drug development paths for the treatment of this disease.

Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n = 1,001) were identified from cells cultured either with (n = 857) or without (n = 667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.

We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat.

The dermal papilla cells (DPCs) of hair follicles are known to secrete paracrine factors for follicular cells. Shotgun proteomic analysis was performed to compare the expression profiles of the secretomes of human DPCs and dermal fibroblasts (DFs). In this study, the proteins secreted by DPCs and matched DFs were analyzed by 1DE/LTQ FTICR MS/MS, semi-quantitatively determined using emPAI mole percent values and then characterized using protein interaction network analysis. Among the 1,271 and 1,188 proteins identified in DFs and DPCs, respectively, 1,529 were further analyzed using the Ingenuity Pathway Analysis tool. We identified 28 DPC-specific extracellular matrix proteins including transporters (ECM1, A2M), enzymes (LOX, PON2), and peptidases (C3, C1R). The biochemically- validated DPC-specific proteins included thrombospondin 1 (THBS1), an insulin-like growth factor binding protein3 (IGFBP3), and, of particular interest, an integrin beta1 subunit (ITGB1) as a key network core protein. Using the shotgun proteomic technique and network analysis, we selected ITGB1, IGFBP3, and THBS1 as being possible hair-growth modulating protein biomarkers.

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi-lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2-DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin-avidin affinity column were separated by 2-DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole-time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF-induced morphological change of MSCs.

The proteome of a HUPO human serum reference sample was analyzed using multidimensional separation techniques at both the protein and the peptide levels. To eliminate false-positive identifications from the search results, we employed a data filtering method using molecular weight (MW) correlations derived from denaturing 1-DE. First, the six most abundant serum proteins were removed from the sample using immunoaffinity chromatography. 1-DE was then used to fractionate the remaining serum proteins according to the MW. Gel bands were isolated and in-gel digested with trypsin, and the resulting peptides were analyzed by 2-D LC/ESI-MS/MS. A SEQUEST search using the MS/MS results identified 494 proteins. Of these, 202 were excluded formally using protein data filtering as they were single-assignment proteins and their theoretical and electrophoretically-derived MWs did not correlate at high confidence. To evaluate this method, the results were compared with those of 1-D LC/MALDI-TOF/TOF and HUPO Plasma Proteome Project analyses. Our data filtering approach proved valuable in analysis of complex, large-scale proteomes such as human serum.

Sarcopenia, characterized by reduced muscle mass and fiber number leading to muscular atrophy, has been associated with serious socioeconomic challenges among the elderly in developed countries. Therefore, preventing sarcopenia could be a promising strategy for achieving a healthy aging society. Nutmeg (Myristica fragrans) has been used as a spice to increase flavor and prevent putrefaction of food. Nutmeg contains various bioactive components that improve muscle activity. To determine the potential effect of lignan-enriched nutmeg extract (LNX) on sarcopenia, LNX (100 mg/kg body weight)-fed aged mice were subjected to forced exercise. Herein, aged (22-month-old) mice fed LNX for three weeks exhibited a shortened and thickened soleus muscle. The ratio of the soleus muscle mass (%) to body weight was significantly increased in LNX-fed aged mice. The relative increase in muscle mass in LNX-fed aged mice improved exercise activities, including rotarod, swimming, and grip strength test results. Proteome profiles of the soleus muscle of LNX-fed mice were used to analyze protein–protein interaction network. Several myosin heavy chain isoforms were found to interact with actin, ACTA1, which functions as a hub protein. Furthermore, the expression of myogenic proteins, such as MYH1, MYH4, and ACTA1, was dose-dependently increased in vivo. In result, our functional proteomic analysis revealed that feeding LNX restored muscle proteins in aged mice. [ABSTRACT FROM AUTHOR]

Summary: The omics‐based studies are important for identifying characteristic proteins in plants to elucidate the mechanism of ACC deaminase producing bacteria‐mediated salt tolerance. This study evaluates the changes in the proteome of rice inoculated with ACC deaminase producing bacteria under salt‐stress conditions. Salt stress resulted in a significant decrease in photosynthetic pigments, whereas inoculation of Methylobacterium oryzae CBMB20 had significantly increased pigment contents under normal and salt‐stress conditions. A total of 76, 51 and 33 differentially abundant proteins (DAPs) were identified in non‐inoculated salt‐stressed plants, bacteria‐inoculated plants under normal and salt stress conditions respectively. The abundances of proteins responsible for ethylene emission and programmed cell death were increased, and that of photosynthesis‐related proteins were decreased in non‐inoculated plants under salt stress. However, bacteria‐inoculated plants had shown higher abundance of antioxidant proteins, RuBisCo and ribosomal proteins that are important for enhancing stress tolerance and improving plant physiological traits. Collectively, salt stress might affect plant physiological traits by impairing photosynthetic machinery and accelerating apoptosis leading to a decline in biomass. However, inoculation of plants with bacteria can assist in enhancing photosynthetic activity, antioxidant activities and ethylene regulation related proteins for attenuating salt‐induced apoptosis and sustaining growth and development. [ABSTRACT FROM AUTHOR]

The spiny mouse, Acomys cahirinus displays a unique wound healing ability with regeneration of all skin components in a scar-free manner. To identify orchestrators of this regenerative response we have performed proteomic analyses of skin from Acomys and Mus musculus before and after wounding. Of the ~2000 proteins identified many are expressed at similar levels in Acomys and Mus, but there are significant differences. Following wounding in Mus the complement and coagulation cascades, PPAR signaling pathway and ECM-receptor interactions predominate. In Acomys, other pathways predominate including the Wnt, MAPK, the ribosome, proteasome, endocytosis and tight junction pathways. Notable among Acomys specific proteins are several ubiquitin-associated enzymes and kinases, whereas in Mus immuno-modulation proteins characteristic of inflammatory response are unique or more prominent. ECM proteins such as collagens are more highly expressed in Mus, but likely more important is the higher expression of matrix remodeling proteases in Acomys. Another distinctive difference between Acomys and Mus lies in the macrophage-produced arginase 1 is found in Mus whereas arginase 2 is found in Acomys. Thus, we have identified several avenues for experimental approaches whose aim is to reduce the fibrotic response that the typical mammal displays in response to wounding. [ABSTRACT FROM AUTHOR]

The objective of this study is to demonstrate characteristics of a y-type high molecular weight glutenin subunit (D1y HMW-GS) at Glu-D1 found in IT212991, a North Korean landrace wheat compared to Dy12 and Dy12.K as a novel HMW-GS in JB20, a Korean wheat line onto molecular analyses as PCR, cloning, DNA sequencing, and RP-HPLC and proteomic analyses as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional electrophoresis (2-DE), Fourier-transform mass spectrometry (LTQ-FT-MS). The D1y of IT212991 was identified to have faster electrophoretic mobility than that of Dy12 by SDS-PAGE. HMW-GS components of IT212991 were identified to be different from Chinese Spring (CS) and JB20, a Korean wheat line by RP-HPLC. The result of mass spectrometric analysis, the D1y of IT212991 (68510.8 Da) was similar to that of Dy12.K of JB20 (68514.4 Da), and lower than Dy12 of CS (69151.2 Da). The result of LTQ-FT-MS based on 2-DE, the D1y of IT212991 was identified to be similar with Dy12 corresponding to the protein function as ‘Glutenin, high molecular weight subunit 12’. The D1y encoding the D1y of IT212991 was identified to consist of 652 amino acid sequences corresponding to 1962 bp according to DNA sequencing. The gene was identified to have a insertion and deletion (InDel) corresponding to 18 bp sequences ‘AACAGGACAAGGGCAACA’ compared to ordinary Dy12 gene. It was demonstrated that the D1y of IT212991 is the same as Dy12.K. [ABSTRACT FROM AUTHOR]

Acinetobacter lwoffii K24 known as an aniline degrading bacterium has also been found to utilize p-hydroxybenzoate as a sole carbon source. In this study, 2-DE using Q-Sepharose column separation was attempted for fast screening of protocatechuate 3,4-dioxygenase for catabolism of p-hydroxybenzoate in A. lwoffii K24. Two protocatechuate 3,4-dioxygenase subunits, pcaG and pcaH were detected and identified with N-terminal and internal sequencing, suggesting proteomics using a column separation may be helpful for the identification of specific protein spots and maximizing the detectable protein spots on the 2-DE gel. The PCR process using degenerate primers for protocatechuate 3,4-dioxygenase and sequence analyses of the PCR products revealed the existence of pcaH and pcaG in A. lwoffii K24. These two subunits were found to be closely located and share extensive homology with pcaH and pcaG of Pseudomonas marginata or Pseudomonas cepacia, providing the evidence that A. lwoffi K24 has the protocatechuate branches as well as catechol branches of β-ketoadipate pathway. [Copyright &y& Elsevier]

Citric acid (CA), as an organic chelator, plays a vital role in alleviating copper (Cu) stress-mediated oxidative damage, wherein a number of molecular mechanisms alter in plants. However, it remains largely unknown how CA regulates differentially abundant proteins (DAPs) in response to Cu stress in Brassica napus L. In the present study, we aimed to investigate the proteome changes in the leaves of B. L. seedlings in response to CA-mediated alleviation of Cu stress. Exposure of 21-day-old seedlings to Cu (25 and 50 μM) and CA (1.0 mM) for 7 days exhibited a dramatic inhibition of overall growth and considerable increase in the enzymatic activities (POD, SOD, CAT). Using a label-free proteome approach, a total of 6345 proteins were identified in differentially treated leaves, from which 426 proteins were differentially expressed among the treatment groups. Gene ontology (GO) and KEGG pathways analysis revealed that most of the differential abundance proteins were found to be involved in energy and carbohydrate metabolism, photosynthesis, protein metabolism, stress and defense, metal detoxification, and cell wall reorganization. Our results suggest that the downregulation of chlorophyll biosynthetic proteins involved in photosynthesis were consistent with reduced chlorophyll content. The increased abundance of proteins involved in stress and defense indicates that these DAPs might provide significant insights into the adaptation of Brassica seedlings to Cu stress. The abundances of key proteins were further verified by monitoring the mRNA expression level of the respective transcripts. Taken together, these findings provide a potential molecular mechanism towards Cu stress tolerance and open a new route in accelerating the phytoextraction of Cu through exogenous application of CA in B. napus. [ABSTRACT FROM AUTHOR]

Abstract: Brown adipocytes oxidize fatty acids to produce heat in response to cold or caloric overfeeding. The motivation and function of the development of brown fat may thus counteract obesity, though this remains uncertain. We investigated the brown adipocyte proteome by two-dimensional gel electrophoresis followed by mass spectrometry. Comparative analyses of proteins focused on total protein spots to filter differentially expressed proteins during the differentiation of mouse primary brown preadipocytes. A Western blot analysis was performed to verify the target proteins. The results indicated that 10 protein spots were differentially expressed with significant changes, including the three up-regulated proteins of prohibitin, hypoxanthine–guanine phosphoribosyltransferase, and enoyl-CoA hydratase protein; the 5 down-regulated proteins of triosephosphate isomerase, elongation factor 2, α-tropomyosin slow, endophilin-B1, and cofilin-1 (CFL1); and the two unequivocally expressed proteins of peroxiredoxin-1 and collagen α-1(i) chain precursor. We found that during brown adipogenesis, CFL1 has an inhibitory effect on brown adipocyte differentiation. The overexpression of CFL1 inhibited the brown fat deposition and repressed the brown marker genes UCP1, PRDM16, PGC-1α and PPARγ via actin dynamics and polymerization. These observations may be novel findings that bring new insight into the detailed mechanisms of brown adipogenesis and identify possible therapeutic targets for anti-obesity. Biological significance: We use 2-DE to compare the proteomes of adipocytes during the brown adipogenesis of primary mouse preadipocytes. We identified 10 proteins that are differentially expressed. Among these, we found that the actin binding protein CFL1 inhibits the differentiation of brown preadipocytes. CFL1 overexpressing cells showed lower deposition of brown fat droplets, and the brown marker genes of UCP1, PRDM16, PGC-1α and PPARγ were decreased through actin dynamics and polymerization. [Copyright &y& Elsevier]

Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are typically asymptomatic and slow-progressing but potentially fatal diseases that are common causes of liver cirrhosis and related complications. Exosomes are nano-sized extracellular vesicles that have been linked to various intercellular communication processes and can carry biological materials reflecting the state and severity of disease. In this study, shotgun proteomic analysis of the protein expression profiles of extracellular vesicles, including exosomes and microvesicles, enriched from human serum samples of 24 patients diagnosed with various fatty liver diseases was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS) followed by protein identification and label-free quantification using the MaxQuant platform. A total of 329 proteins, including 190 previously reported exosome-specific proteins, were identified from four types of liver disease, where significant differences in protein expression were found in apolipoproteins, immunoglobulins, and other previously reported markers of liver disease. Principal component analysis of 61 proteins identified from MaxQuant analysis of the LC-MS/MS data provided a confident differentiation between ALD and NAFLD. The current investigation revealed the difference among various types of liver disease using LC-MS/MS of exosomes enriched from human serum samples of 24 patients where the most significantly up-regulation proteins were alpha-2-macroglobulin for alcoholic hepatitis and apolipoprotein C3 for nonalcoholic fatty liver disease. [Display omitted] • Exosomes were enriched from human serum samples with fatty liver diseases. • LC-MS/MS analyses of digested exosomal proteins were performed in triplicate. • A total of 329 proteins were identified from the enriched exosomes. • Confident differentiation was obtained between alcoholic liver disease and nonalcoholic fatty liver disease. [ABSTRACT FROM AUTHOR]