No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase
Abstract
This study developed an enzymatic method for high-throughput mapping of phosphoproteins on two-dimensional (2-D) polyacrylamide gels. Proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant lambda protein phosphatase and the other was not treated with the enzyme. The two aliquots were then subjected to 2-D electrophoresis. Phosphoproteins could be mapped on the 2-D gel of the nontreated aliquot by comparing the gels of the two aliquots, because the phosphoproteins in the treated aliquot shifted to more basic positions on the gel. This technique revealed that approximately 5% of the detectable proteins were phosphorylated. Fourteen phosphoproteins were identified by mass spectrometry, including proteasome component C8 and small glutamine-rich tetratricopeptide repeat-containing protein. Furthermore, the extent of phosphorylation of two actin modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. The method developed by this study can generally be applied to all biological materials and is useful for high-throughput mapping of phosphoproteins in proteome research.
... It is known that some phosphorylations may lead to the partial depolymerization of the lamina and cause relocation of lamin A/C to the nucleoplasm [24]. As lamin A/C molecule contains over 40 phosphorylation sites [65], and phosphorylation is known to change the isoelectric point (pI) of proteins [66], we tested whether the more acidic lamin A/C forms observed that only bind to NM1 in the presence of PI(4,5)P2 correspond to the phosphorylated A-type lamins, which would also go in line with the nucleoplasmic localization of this complex. In order to test this hypothesis, proteins in HeLa nuclear extracts were dephosphorylated using lambda protein phosphatase (λPP), which has activity towards phosphorylated serine, threonine, and tyrosine residues. ...
Lamins, the nuclear intermediate filaments, are important regulators of nuclear structural integrity as well as nuclear functional processes such as DNA transcription, replication and repair, and epigenetic regulations. A portion of phosphorylated lamin A/C localizes to the nuclear interior in interphase, forming a lamin A/C pool with specific properties and distinct functions. Nucleoplasmic lamin A/C molecular functions are mainly dependent on its binding partners; therefore, revealing new interactions could give us new clues on the lamin A/C mechanism of action. In the present study, we show that lamin A/C interacts with nuclear phosphoinositides (PIPs), and with nuclear myosin I (NM1). Both NM1 and nuclear PIPs have been previously reported as important regulators of gene expression and DNA damage/repair. Furthermore, phosphorylated lamin A/C forms a complex with NM1 in a phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent manner in the nuclear interior. Taken together, our study reveals a previously unidentified interaction between phosphorylated lamin A/C, NM1, and PI(4,5)P2 and suggests new possible ways of nucleoplasmic lamin A/C regulation, function, and importance for the formation of functional nuclear microdomains.
... One interesting phenomenon observed in the present study was that Ybx1 is a basic protein with a calculated pI around pH 10; however, Ybx1 proteins of similar size could be identified across the 2D gel at different pIs with majority of the protein located at the acidic side of the 2D gel, near the region corresponding to pH 3. The significant alteration in pI value suggests that a large proportion of Ybx1 molecules in the oocyte may have been post-translationally modified, most likely by phosphorylation. It has been reported that one phosphorylation on a protein would cause a shift of pI value by 1-2 units on average [52,53]. The shifting of Ybx1 from pH 10 to 3 would suggest phosphorylation at multiple sites of the protein. ...
As in mammals, ovarian folliculogenesis in teleosts also consists of two phases: the primary growth (PG) and secondary growth (SG) phases, which are analogous to the preantral and antral phases respectively in mammals. In this study, we performed a proteomic analysis on zebrafish follicles undergoing the PG-SG transition aiming to identify factors involved in the event. Numerous proteins showed significant changes, and the most prominent one was Y-box binding protein 1 (YB-1; Ybx1/ybx1), a transcription factor and mRNA-binding protein. YB-1 belongs to the Y-box binding protein family, which also includes the gonad-specific YB-2. Interestingly, phylogenetic analysis showed no YB-2 homologue in zebrafish. Although ybx1 mRNA was expressed in various tissues, its protein Ybx1 was primarily produced in the gonads, similar to YB-2 in other species. In the ovary, Ybx1 protein started to appear in early follicles newly emerged from the germ cell cysts, reached the highest level in late PG oocytes, but decreased precipitously when the follicles entered the SG phase. In PG follicles, Ybx1 might function as a key component of the messenger ribonucleoprotein particles (mRNP) in association with other RNA-binding proteins. Similar to mammalian YB-1, zebrafish Ybx1 also contains functional signals that determine its intracellular localization. In conclusion, Ybx1 may play dual roles of YB-1 and YB-2 in zebrafish. In the ovary, Ybx1 binds mRNAs to stabilize them while preventing their translation. At PG-SG transition, Ybx1 is removed to release the masked mRNAs for translation into functional proteins, leading to follicle activation.
... For 2D-Gel, cell pellets were resuspended in 2-D lysis buffer (2 M thiourea, 7 M urea, 4% CHAPS, 1% DTT, protease inhibitors cocktail) and sonicated. Total extracts were treated, where indicated, with Lambda Protein Phosphatase ( -PP) (New England BioLabs) following the protocol described in Yagamata et al., with minor modi cations (31). In short, cells pellets were re-suspended and 20 mM MnCl 2 was added to 150 µg of protein extract in order to obtain a nal concentration of 2 mM -PP buffer and brought to a nal volume of 20 µl with deionized water. ...
Background
Che-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. We have recently demonstrated that Che-1 is able to promote cell proliferation by sustaining global histone acetylation in Multiple Myeloma (MM) cells where it interacts with histone proteins and competes with HDAC class I members for binding.
Methods
Site-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three-serine residue (Ser³¹⁶, Ser³²⁰ and Ser³²¹) in 308–325 AA region. Western blot experiments were conducted to examine the effect of depletion or over-expression of Che-1 and Che-1 3S mutant on histone acetylation, in different human cancer cell lines. Proliferation assays were assessed to estimate the change in cells number when Che-1 was over-expressed or deleted. Immunoprecipitation assays were performed to evaluate Che-1/histone H3 interaction when Ser³¹⁶, Ser³²⁰ and Ser³²¹ were removed. The involvement of CK2 kinase in Che-1 phosphorylation at these residues was analysed by in vitro kinase, 2D gel electrophoresis assays and mass spectrometry analysis.
Results
Here, we confirm that Che-1 depletion reduces cell proliferation with a concomitant general histone deacetylation in several tumor cell lines. Furthermore, we provided evidence that CK2 protein kinase phosphorylates Che-1 at Ser³¹⁶, Ser³²⁰ and Ser³²¹ and that these modifications are required for Che-1/histone H3 binding. These results improve our understanding onto the mechanisms by which Che-1 regulates histone acetylation and cell proliferation.
Conclusions
Che-1 phosphorylation at Ser³¹⁶, Ser³²⁰ and Ser³²¹ by CK2 promotes the interaction with histone H3 and represents an essential requirement for Che-1 pro-proliferative ability.
... The exact nature of the PTMs in two major SDHB pI fractions needs to be identified in the future. Phosphorylation usually induces an acidic shift in the pI [59] and single phosphorylation may alter pI by 1-2 pH units [60], therefore, multiple phosphorylations may be involved in shifting SDHB pI from basic (pH 9) to an acidic (pH 3-4) pI (Fig. 7EF). Interestingly, high throughput mass spectrometric analysis documented a variety of SDHB PTMs archived in the Phos-phositePlus database [61,62]. ...
Background
The calcium (Ca2+)/calmodulin (CAM)-activated kinase kinase 2 (CAMKK2)-signaling regulates several physiological processes, for example, glucose metabolism and energy homeostasis, underlying the pathogenesis of metabolic diseases. CAMKK2 exerts its biological function through several downstream kinases, therefore, it is expected that depending on the cell-type-specific kinome profile, the metabolic effects of CAMKK2 and its underlying mechanism may differ. Identification of the cell-type-specific differences in CAMKK2-mediated glucose metabolism will lead to unravelling the organ/tissue-specific role of CAMKK2 in energy metabolism. Therefore, the objective of this study was to understand the cell-type-specific regulation of glucose metabolism, specifically, respiration under CAMKK2 deleted conditions in transformed human embryonic kidney-derived HEK293 and hepatoma-derived HepG2 cells.
Methods
Cellular respiration was measured in terms of oxygen consumption rate (OCR). OCR and succinate dehydrogenase (SDH) enzyme activity were measured following the addition of substrates. In addition, transcription and proteomic and analyses of the electron transport system (ETS)-associated proteins, including mitochondrial SDH protein complex (complex-II: CII) subunits, specifically SDH subunit B (SDHB), were performed using standard molecular biology techniques. The metabolic effect of the altered SDHB protein content in the mitochondria was further evaluated by cell-type-specific knockdown or overexpression of SDHB.
Results
CAMKK2 deletion suppressed cellular respiration in both cell types, shifting metabolic phenotype to aerobic glycolysis causing the Warburg effect. However, isolated mitochondria exhibited a cell-type-specific enhancement or dampening of the respiratory kinetics under CAMKK2 deletion conditions. This was mediated in part by the cell-type-specific effect of CAMKK2 loss-of-function on transcription, translation, post-translational modification (PTM), and megacomplex assembly of nuclear-encoded mitochondrial SDH enzyme complex subunits, specifically SDHB. The cell-type-specific increase or decrease in SDHs protein levels, specifically SDHB, under CAMKK2 deletion condition resulted in an increased or decreased enzymatic activity and CII-mediated respiration. This metabolic phenotype was reversed by cell-type-specific knockdown or overexpression of SDHB in respective CAMKK2 deleted cell types. CAMKK2 loss-of-function also affected the overall assembly of mitochondrial supercomplex involving ETS-associated proteins in a cell-type-specific manner, which correlated with differences in mitochondrial bioenergetics.
Conclusion
This study provided novel insight into CAMKK2-mediated cell-type-specific differential regulation of mitochondrial function, facilitated by the differential expression, PTMs, and assembly of SDHs into megacomplex structures.
... For 2D-Gel, cell pellets were resuspended in 2-D lysis buffer (2 M thiourea, 7 M urea, 4% CHAPS, 1% DTT, protease inhibitors cocktail) and sonicated. Total extracts were treated, where indicated, with Lambda Protein Phosphatase ( λ-PP) (New England BioLabs) following the protocol described in Yagamata et al., with minor modifications [32]. In short, cells pellets were resuspended and 20 mM MnCl 2 was added to 150 μg of protein extract in order to obtain a final concentration of 2 mM λ-PP buffer and brought to a final volume of 20 μl with deionized water. ...
Background
Che-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. We have recently demonstrated that Che-1 is able to promote cell proliferation by sustaining global histone acetylation in multiple myeloma (MM) cells where it interacts with histone proteins and competes with HDAC class I members for binding.
Methods
Site-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three serine residues (Ser ³¹⁶ , Ser ³²⁰ and Ser ³²¹ ) in 308–325 aa region. Western blot experiments were conducted to examine the effect of depletion or over-expression of Che-1 and Che-1 3S mutant on histone acetylation, in different human cancer cell lines. Proliferation assays were assessed to estimate the change in cells number when Che-1 was over-expressed or deleted. Immunoprecipitation assays were performed to evaluate Che-1/histone H3 interaction when Ser ³¹⁶ , Ser ³²⁰ and Ser ³²¹ were removed. The involvement of CK2 kinase in Che-1 phosphorylation at these residues was analysed by in vitro kinase, 2D gel electrophoresis assays and mass spectrometry analysis.
Results
Here, we confirmed that Che-1 depletion reduces cell proliferation with a concomitant general histone deacetylation in several tumor cell lines. Furthermore, we provided evidence that CK2 protein kinase phosphorylates Che-1 at Ser ³¹⁶ , Ser ³²⁰ and Ser ³²¹ and that these modifications are required for Che-1/histone H3 binding. These results improve our understanding onto the mechanisms by which Che-1 regulates histone acetylation and cell proliferation.
Conclusions
Che-1 phosphorylation at Ser ³¹⁶ , Ser ³²⁰ and Ser ³²¹ by CK2 promotes the interaction with histone H3 and represents an essential requirement for Che-1 pro-proliferative ability.
... With steadily increasing qualitative information about the identity of PTMs (Grimsrud et al., 2010;Choudhary et al., 2014;Kim et al., 2011;Melo-Braga et al., 2014;Udeshi et al., 2013), tools to investigate the modification quantity, that is the proportion of the protein carrying PTMs, become increasingly important. Thus far, analysis of the PTM extent has mainly focused on quantifying the modified peptides, for example using PTM enrichment methods (Pinkse et al., 2004;Rush et al., 2005;Zhou et al., 2011;Lundby et al., 2012;Lundby et al., 2013), heavy isotope-labeled synthetic peptides (Gerber et al., 2003) or by enzymatically removing (Yamagata et al., 2002;Wu et al., 2011) PTMs. These approaches require prior knowledge of the PTMs and have been primarily applied to the phosphoproteome. ...
Improvements in LC-MS/MS methods and technology have enabled the identification of thousands of modified peptides in a single experiment. However, protein regulation by post-translational modifications (PTMs) is not binary, making methods to quantify the modification extent crucial to understanding the role of PTMs. Here, we introduce FLEXIQuant-LF, a software tool for large-scale identification of differentially modified peptides and quantification of their modification extent without knowledge of the types of modifications involved. We developed FLEXIQuant-LF using label-free quantification of unmodified peptides and robust linear regression to quantify the modification extent of peptides. As proof of concept, we applied FLEXIQuant-LF to data-independent-acquisition (DIA) data of the anaphase promoting complex/cyclosome (APC/C) during mitosis. The unbiased FLEXIQuant-LF approach to assess the modification extent in quantitative proteomics data provides a better understanding of the function and regulation of PTMs. The software is available at https://github.com/SteenOmicsLab/FLEXIQuantLF .
... Recently, phosphoprotein gel stains based on fluorescence-based detection technology were introduced (Schulenberg et al. 2004;. Another method, which does not require any radiolabelling or electroblotting, is the mapping of phosphorylated proteins in 2D gels by the application of a protein phosphatase, as shown by Yamagata et al. (2002). Prior to 2D-PAGE, protein extracts were divided into two aliquots. ...
Os ovos de deferentes espécies de aves possuem em sua composição proteínas de alto valor biológico. Muitas das proteínas localizadas neste complexo fluido permanecem ainda não caracterizadas. Novas proteínas podem ser identificadas apresentando características antimicrobianas ou antivirais, proteínas de transporte, ou fatores de crescimento. Esta investigação exige métodos específicos para separação e caracterização. A proteômica vêm sendo utilizada com êxito para a caracterização das proteínas dos ovos. Através de revisão de literatura, objetivou-se realizar o levantamento das técnicas utilizadas para caracterização das proteínas, conhecimento das proteínas e isoformas identificadas em clara e gema de ovos.
Main conclusion:
Potato eukaryotic elongation factor 1A comprises multiple isoforms, some of which are heat-inducible or heat-upregulated and might be important in alleviating adverse effects of heat stress on plant productivity. Heat stress substantially reduces crop productivity worldwide, and will become more severe due to global warming. Identification of proteins involved in heat stress response may help develop varieties for heat tolerance. Eukaryotic elongation factor 1A (eEF1A) is a cytosolic, multifunctional protein that plays a central role in the elongation phase of translation. Some of the non-canonical eEF1A activities might be important in developing plant heat-stress tolerance. In this study, we investigated effects of heat stress (HS) on eEF1A expression at the protein level in potato, a highly heat vulnerable crop. Our results from both the controlled environment and the field have shown that potato eEF1A is a heat-inducible protein of 49.2-kDa with multiple isoforms (5-8). Increase in eEF1A abundance under HS can be mainly attributed to 2-3 basic polypeptides/isoforms. A significant correlation between eEF1A abundance and the potato productivity in the field was observed in two extremely hot years 2011 and 2012. Genomic Southern blot analysis indicated the existence of multiple genes encoding eEF1A in potato. Identification, isolation and utilization of heat-inducible eEF1A genes might be helpful for the development of the heat-tolerant varieties.
The objective of this study was to investigate changes occurring in μ/m-calpain in post mortem chicken muscles and to determine the origin of the unknown bands found in calpain casein zymography. The unknown bands were reported with slightly greater mobility compared to conventional μ/m-calpain bands in casein zymography. Identification of these bands was accomplished using native polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry and with protein phosphatase treatment. Results showed that the unknown bands were corresponding to μ/m-calpain, and dephosphorylation by protein phosphatase did not change their appearance. The calpain samples were then incubated with various concentrations of Ca(2+) to determine the relationship between changes in μ/m-calpain and the appearance of the unknown bands. The products of μ/m-calpain partial autolysis were found to be consistent with the appearance of the unknown bands. Therefore, the appearance of these bands did not result from phosphorylation of μ/m-calpain as previously hypothesized, but from partial autolysis of μ/m-calpain. Also their presence suggests that μ/m-calpain undergoes partial autolysis during aging which may play certain roles in meat quality improvement.
Phosphoproteome analysis by mass spectrometry is in its infancy, but this research field is developing quickly. Integration of various technologies for phosphoprotein enrichment, phosphopeptide detection and quantitation and phosphopeptide sequencing is clearly needed. These methods have to be coupled with novel bioinformatics tools for analysis and visualization of the protein data that is obtained. Methods are continuously being refined at all these levels, most notably in sample preparation methods, tandem mass spectrometry techniques, and bioinformatics software. The complete characterization of the proteome of a given organism, tissue or organelle must necessarily include identification and detailed structural analysis of the dynamically changing populations of post-translationally modified gene products. Combinations of genetic, biochemical, and immunological techniques with mass spectrometry-based methods result in novel quantitative analytical strategies that may soon allow researchers to study the dynamics of complex cellular processes. Reversible protein phosphorylation is among the best characterized of the hundreds of known post-translational modifications and it plays a pivotal role in cellular development, differentiation, growth and homeostasis. Phosphoproteins are involved in all biochemical processes in the cell, including transcriptional and translational regulation, metabolism, protein degradation, cellular signaling, and communication, and in maintaining the structural integrity of cells. The crucial role of protein phosphorylation and dephosphorylation events is emphasized by the fact that protein kinases, phosphoprotein phosphatases and their respective substrates are implicated in a number of human diseases and ailments, including diabetes and many types of cancer.
Plant kinases are one of the largest protein families in Arabidopsis. There are almost 600 membrane-located receptor kinases and almost 400 soluble kinases with distinct functions in signal transduction. In this minireview we discuss phylogeny and functional context of prominent members from major protein kinase subfamilies in plants.
Peroxisome proliferator (PP)-activated receptor-α (PPARα) agonists exhibit species-specific effects on livers of the rodent and human (h), which has been considered to reside in the difference of PPARα gene structures. However, the contribution of h-hepatocytes (heps) to the species-specificity remains to be clarified. In this study, the effects of fenofibrate were investigated using a hepatocyte-humanized chimeric mouse (m) model whose livers were replaced with h-heps at >70%. Fenofibrate induced hepatocellular hypertrophy, cell proliferation, and peroxisome proliferation in livers of severe combined immunodeficiency (SCID) mice, but not in the h-hep of chimeric mouse livers. Fenofibrate increased the expression of the enzymes of β- and ω-hydroxylation and deoxygenation of lipids at both gene and protein levels in SCID mouse livers, but not in the h-heps of chimeric mouse livers, supporting the studies with h-PPARα-transgenic mice, a hitherto reliable model for studying the regulation of h-PPARα in the h-liver in most respects, except the induction of the peroxisome proliferation. This study indicates the importance of not only h-PPARα gene but also h-heps themselves to correctly predict effects of fibrates on h-livers, and, therefore, suggests that the chimeric mouse is a currently available, consistent, and reliable model to obtain pharmaceutical data concerning the effects of fibrates on h-livers.
Background
Although protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms.
Methods
Phosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry.
Results
A total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases × three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated β-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases.
Conclusions
Protein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations.
Plants adapt to environmental light conditions by photoreceptor-mediated physiological responses, but the mechanism by which photoreceptors perceive and transduce the signals is still unresolved. Here, we used 2-D difference gel electrophoresis (2-D DIGE) and mass spectrometry to characterize early molecular events induced by short blue-light exposures in etiolated Arabidopsis seedlings. We observed the phosphorylation of phototropin 1 (phot1) and accumulation of Weak Chloroplast Movement under Blue Light 1 (WEB1) in the membrane fraction after blue light irradiation. Over 50 spots could be observed for the two rows of phot1 spots in the 2-DE gels, and eight novel phosphorylated Ser/Thr sites were identified in the N-terminus and Hinge 1 region of phot1 in vivo. Blue light caused ubiquitination of phot1, and K526 of phot1 was identified as a putative ubiquitination site. Our study indicates that posttranslational modification of phot1 is more complex than previously reported.
Despite the progress in performance of MS-driven analytical methods during the last decade with regard to sensitivity and selectivity, the identification of phosphorylation sites is still not a trivial task. Thus, today no single method can reliably detect and characterize all modified residues in a phosphoprotein and far most successful analysis strategies for phosphoprotein and phosphoproteome, including multiple levels of enrichment and separation methods as well as biological follow-up analysis. However, recent improvements in MS have spawned improved and far more robust analytical strategies. An improved effiency of enrichment and separation techniqes on both peptide and protein level, the improved data quality by ECD or ETD peptide fragmentation and the improved confidence in phosphopeptide detection by MS 3 phosphopeptide sequencing using high mass accuracy FTICR-based mass spectrometers have enabled multiple comprehensive studies of protein phsophorylation. Last, but not least, multiple complementary MS-driven strategies for relative and absolute quantitation of protein phosphorylation will ease rapid investigations of signal transduction systems and, thus, provide the basis for great advances.
Small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) is a steroid receptor molecular co-chaperone that may substantially influence hormone action and, consequently, hormone-mediated carcinogenesis. To date, published studies describe SGTA as a protein that is potentially critical in a range of biological processes, including viral infection, cell division, mitosis, and cell cycle checkpoint activation. SGTA interacts with the molecular chaperones, heat shock protein 70 (HSP70) and HSP90, and with steroid receptor complexes, including those containing the androgen receptor. Steroid receptors are critical for maintaining cell growth and differentiation in hormonally regulated tissues, such as male and female reproductive tissues, and also play a role in disease states involving these tissues. There is growing evidence that, through its interactions with chaperones and steroid receptors, SGTA may be a key player in the pathogenesis of hormonally influenced disease states, including prostate cancer and polycystic ovary syndrome. Research into the function of SGTA has been conducted in several model organisms and cell types, with these studies showing that SGTA functionality is cell-specific and tissue-specific. However, very few studies have been replicated in multiple cell types or experimental systems. Although a broad range of functions have been attributed to SGTA, there is a serious lack of mechanistic information to describe how SGTA acts. In this review, published evidence linking SGTA with hormonally regulated disease states is summarized and discussed, highlighting the need for future research to more clearly define the biological function(s) of this potentially important co-chaperone.
Neutrophils are essential in host defense against periodontopathic bacteria. Immunoglobulin G Fc receptor IIIb (FcγRIIIb) is a neutrophil-specific receptor for immunoglobulin G and bears the functional NA1-NA2 polymorphism. Accumulating evidence suggests a significant association between FcγRIIIb gene polymorphism and periodontitis. In this study, we employed a proteomic approach to evaluate the relevance of FcγRIIIb polymorphism to protein expression profiles of neutrophils.
Neutrophils were collected from ten healthy subjects whose FcγRIIIb genotypes were determined by allele-specific PCRs. Expressions of proteins induced by interaction via FcγRIIIb were examined between the FcγRIIIb genotypes with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins that were significantly different in expression levels between the FcγRIIIb genotypes were determined with computer image analysis, and identified with mass spectrometry and protein databases.
A total of 757 protein spots were observed in the two-dimensional electrophoretograms of neutrophils from five FcγRIIIb-NA1/NA1 and five FcγRIIIb-NA2/NA2 donors. A statistical analysis revealed that the expression levels of five proteins were significantly different between the FcγRIIIb genotypes (p < 0.05). The FcγRIIIb-NA1/NA1 neutrophils exhibited two spots that were significantly underexpressed (protein-arginine deiminase type-4 and annexin VI) and three spots that were significantly overexpressed (Cdc42hs-Gdp complex, myosin light chain 12A and coactosin-like 1) when compared with FcγRIIIb-NA2/NA2 neutrophils. The same expression profiles of protein-arginine deiminase type-4 were obtained by ELISA.
Differential protein expression profiles were observed in neutrophils between FcγRIIIb genotypes.
Reversible protein phosphorylation is an essential mechanism in the regulation of diverse biological processes, nonetheless is frequently altered in disease. As most phosphoproteome studies are based on optimized in-vitro cell culture studies new methods are in need to improve de novo identification and characterization of phosphoproteins in extracts from tissues. Here, we describe a rapid and reliable method for the detection of phosphoproteins in tissue extract based on an experimental strategy that employs 1D and 2D SDS PAGE, Western immunoblotting of phosphoproteins, in-gel protease digestion and enrichment of phosphorpeptides using metal oxide affinity chromatography (MOAC). Subsequently, phosphoproteins are identified by MALDI-TOF-MS/MS with the CHCA-TL or DHB ML sample matrix preparation method and further characterized by various bioinformatic software tools to search for candidate kinases and phosphorylation-dependent binding motifs. The method was applied to mouse lung tissue extracts and resulted in an identification of 160 unique phosphoproteins. Notably, TiO(2) enrichment of pulmonary protein extracts resulted in an identification of additional 17 phosphoproteins and 20 phosphorylation sites. By use of MOAC, new phosphorylation sites were identified as evidenced for the advanced glycosylation end product-specific receptor. So far this protein was unknown to be phosphorylated in lung tissue of mice. Overall the developed methodology allowed efficient and rapid screening of phosphorylated proteins and can be employed as a general experimental strategy for an identification of phosphoproteins in tissue extracts.
In order to keep subscribers up-to-date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly-published material on comparative and functional genomics. Each bibliography is divided into 16 sections. 1 Reviews & symposia; 2 General; 3 Large-scale sequencing and mapping; 4 Genome evolution; 5 Comparative genomics; 6 Gene families and regulons; 7 Pharmacogenomics; 8 Large-scale mutagenesis programmes; 9 Functional complementation; 10 Transcriptomics; 11 Proteomics; 12 Protein structural genomics; 13 Metabolomics; 14 Genomic approaches to development; 15 Technological advances; 16 Bioinformatics. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted
The reversible change of the phosphorylation state of proteins regulates key cellular processes. In the present study three different gel-based approaches were compared with regard to their applicability to quantitatively analyse the phosphoproteome of scarce biological material obtained ex vivo. Our results show that the phosphoproteome characterization of oviductal epithelial cells isolated from the female reproductive tract requires affinity enrichment and pre - electrophoretic labelling using fluorescence dyes. Using this approach 30 μg of enriched phosphoproteins proved to be sufficient for the phosphoproteome characterization. In contrast, sequential fluorescence staining of two-dimensional separated total cell lysates as well as sequential staining in conjunction with a pre - enrichment step led to detection discrepancies and excluded further analysis steps. Information gained from this study provides a successful approach for the phosphoproteome analysis of scarce samples. In addition, the cellular processes taking place in the female reproductive tract can be monitored ex vivo.
A peptide-based 2-D liquid phase fractionation (PF2D) system was used in a quantitative proteomic analysis of hepatocellular carcinoma. 2-D liquid maps of peptide specimens showed better resolution than those of proteins, leading to the identification of differentially expressed proteins. Peptide-based PF2D gave well-matched theoretical and experimental pI values and was proven to be a very efficient and versatile analytical tool for both large-scale profiling and quantification of phosphoproteins in disease biomarker discovery.
This study reports a novel class of antifungal protein derived from bacterial origin. Bacillus thuringiensis SF361, the strain also responsible for producing the novel bacteriocin thurincin H, exhibits broad antifungal activity against
select members of several fungal genera, including Aspergillus, Byssochlamys, and Penicillium, as well as the pathogenic yeast Candida albicans. Optimal antifungal production and secretion were observed after-log phase growth when incubated at 37°C in a carbohydrate-free
growth medium. High-performance liquid chromatography purification was performed after pH-selective ammonium sulfate precipitation
and size-exclusion chromatography. Intact mass analysis and peptide mass fingerprinting identified the 13,484-Da protein to
be a mass homolog to the YvgO protein construct sequenced from Bacillus cereus AH 1134. Further analysis via amino-terminal sequencing also revealed the existence of four distinct yet equally efficacious
YvgO variants differing only within the first four N-terminal residues. YvgO was found to be remarkably stable, maintaining
its antifungal activity under a wide pH and temperature range. When assayed against the toxigenic species Byssochlamys fulva H25, the selected primary filamentous fungal indicator, the MIC was estimated to be 1.5 ppm. Candida albicans 3153 was more resistant, exhibiting MICs between 25 and 800 ppm, depending on growth conditions. YvgO is unique among antifungals,
showing no known sequential or functional homology to the typical classes of antifungal proteins, including common membrane-acting
agents such as cellulases and glucanases. Due to its activity against an array of pathogenic and spoilage fungi, the potentials
for clinical, agricultural, and food-processing applications are encouraging.
The organophosphorus pesticide dimethoate (DM) has been widely used in agriculture, and its extensive use could still have left many environmental problems.
In the present study, the oyster (Saccostrea cucullata) was subjected to acute DM toxicity (2 mg/L), and gas chromatographic analysis revealed and quantified residues of DM in the oyster gonad.
Two-dimensional gel electrophoresis showed 12 differentially expressed proteins in the DM-exposed oyster gonad in comparison to the control. Among these 12 protein spots, nine were down-regulated, and three were up-regulated. Both matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry and database searching were utilized to identify these differential proteins, and revealed five proteins previously described as being related to DM toxicity. In addition, the levels of mRNA expression corresponding to these differential proteins were further proved in part by real-time PCR. The functions of these proteins were summarized as: carrying out energy metabolism, DNA repair, DNA transcriptional regulation, and oxidative protection. The remaining seven protein spots were of particular interest in terms of their responses to DM, which have seldom been reported.
These data might point to a number of novel and significant biomarkers for evaluating the contamination levels of DM and provide useful insight into the mechanisms of DM toxicity in vivo.
Previous studies have shown that the Epstein-Barr virus-encoded latent membrane protein1 (LMP1) could activate nuclear factor kappa B, activator protein-1, and Janus kinases/signal transducer and activation of transcription factors pathways. However, many signaling molecules and downstream target proteins triggered by LMP1 have not been identified. To determine the functional components in signaling pathways triggered by LMP1, we combined the novel strategy of phosphoprotein enrichment with proteomics technology to elucidate the signaling cascade activated by LMP1. We found that LMP1 could increase the quantity of total phosphoproteins by 18.03%, and 43 proteins showed significant changes in the degree of phosphorylation when LMP1 was expressed. Twenty-five signaling molecules or downstream targets of signaling pathways triggered by LMP1 were identified, several of which had previously been implicated in LMP1 signal pathways. The other proteins, including annexin A2, heat shock protein 27, stathmin, annexin I, basic transcription factor 3, and porin, were novel signaling molecules or targets with no previously known function in LMP1 signal transduction. The method used here has proven to be suitable for the identification of molecules involved in various signaling pathways.
2-DE proved to be a key technology in protein science since the two orthogonal separation dimensions are capable of protein isoform separation. Recently, Agilent introduced the OFFGEL 3100 fractionator for in solution IEF (off-gel) of proteins with the help of a 12- or 24-well frame. With this instrument also conventional focusing in IPG strips after passive in-tray rehydration can be performed. In this study, two novel IEF applications using the OFFGEL electrophoresis were developed. First, a sample cup was built and a cup-loading method for the OFFGEL device was implemented. Applying proteins via cup resulted in higher reproducibility and less protein loss compared with conventional in-tray rehydration loading. Especially, the recovery of basic and high-molecular-mass proteins seems to be favored by cup loading. These effects are more pronounced with low microgram sample amounts. Second, a 48-well OFFGEL frame was developed, which doubles the resolution of the commercially available 24-well frame. It is capable of separating proteins with small pI differences and shows potential for isoform/PTM separation.
Peach softening is usually attributed to the dismantling of the cell wall in which endo-polygalacturonase (endo-PG)-catalysed depolymerization of pectins plays a central role. In this study, the hypothesis that the function of endo-PG is critical for achieving a melting flesh fruit texture but not for reducing fruit firmness was tested by comparing pericarp morphology and endo-PG expression and localization in melting (MF) and non-melting flesh (NMF) fruit at successive stages of ripening. MF Bolero, Springbelle, and Springcrest, and NMF Oro-A and Jonia cultivars were analysed. Both MF and NMF fruit were left to ripen on the tree and reached a firmness of <10 Newtons (N). The image analysis of pericarp tissues revealed that during softening the loss of cell turgidity was a process common to mesocarp cells of all MF and NMF fruit and was clearly visible in peaches with a firmness of less than ∼20 N. In contrast, the loss of cell adhesion was a feature exclusively observed in ripe MF fruit pericarp. In this ripe fruit, large numbers of endo-PG isoforms were highly expressed and the enzyme localization corresponded to the middle lamella. As a consequence, wide apoplastic spaces characterized the pericarp of ripe MF peaches. In contrast, no loss of cell adhesion was observed in any NMF fruit or in unripe MF peaches. Accordingly, no endo-PG was detected in unripe NMF fruit, whereas few and poorly expressed enzyme isoforms were revealed in ripe NMF and in unripe MF peaches. In this fruit, the poorly expressed endo-PG localized mainly in vesicles within the cytoplasm and inner primary cell wall. On the whole the results suggested that endo-PG function was needed to achieve melting flesh texture, which was characterized by wide apoplastic spaces and partially deflated mesocarp cells. Conversely, endo-PG activity had no critical influence on the reduction of fruit firmness given the capacity of NMF peaches to soften, reaching values of 5-10 N. As in tomato, the change of symplast/apoplast water status seems to be the main process through which peach fruit regulates its firmness.
hMena and the epithelial specific isoform hMena(11a) are actin cytoskeleton regulatory proteins belonging to the Ena/VASP family. EGF treatment of breast cancer cell lines upregulates hMena/hMena(11a) expression and phosphorylates hMena(11a), suggesting cross-talk between the ErbB receptor family and hMena/hMena(11a) in breast cancer. The aim of this study was to determine whether the hMena/hMena(11a) overexpression cooperates with HER-2 signalling, thereby affecting the HER2 mitogenic activity in breast cancer. In a cohort of breast cancer tissue samples a significant correlation among hMena, HER2 overexpression, the proliferation index (high Ki67), and phosphorylated MAPK and AKT was found and among the molecular subtypes the highest frequency of hMena overexpressing tumors was found in the HER2 subtype. From a clinical viewpoint, concomitant overexpression of HER2 and hMena identifies a subgroup of breast cancer patients showing the worst prognosis, indicating that hMena overexpression adds prognostic information to HER2 overexpressing tumors. To identify a functional link between HER2 and hMena, we show here that HER2 transfection in MCF7 cells increased hMena/hMena(11a) expression and hMena(11a) phosphorylation. On the other hand, hMena/hMena(11a) knock-down reduced HER3, AKT and p44/42 MAPK phosphorylation and inhibited the EGF and NRG1-dependent HER2 phosphorylation and cell proliferation. Of functional significance, hMena/hMena(11a) knock-down reduced the mitogenic activity of EGF and NRG1. Collectively these data provide new insights into the relevance of hMena and hMena(11a) as downstream effectors of the ErbB receptor family which may represent a novel prognostic indicator in breast cancer progression, helping to stratify patients.
Quantitative proteomics based on 2D electrophoresis (2-DE) coupled with peptide mass fingerprinting is still one of the most widely used quantitative proteomics approaches in microbiology research. Our view on the exploitation of this global expression analysis technique and its contribution and potential to push forward the field of molecular microbial physiology towards a molecular systems microbiology perspective is discussed in this article. The advances registered in 2-DE-based quantitative proteomic analysis leading to increased protein resolution, sensitivity and accuracy, and the promising use of 2-DE to gain insights into post-translational modifications at a proteome-wide level (considering all the proteins/protein forms expressed by the genome) are focused on. Given the progress made in this field, it is foreseen that the 2-DE-based approach to quantitative proteomics will continue to be a fundamental tool for microbiologists working at a genome-wide scale. Guidelines are also provided for the exploitation of expression proteomics data, based on useful computational tools, and for the integration of these data with other genome-wide expression information. The advantages and limitations of a complete 2-DE-based expression proteomics analysis, envisaging the quantification of the global changes occurring in the proteome of a given cell depending on environmental or genetic manipulations, are discussed from the microbiologist's perspective. Particular focus is given to the emerging field of toxicoproteomics, a new systems toxicity approach that offers a powerful tool to directly monitor the earliest stages of the toxicological response by identifying critical proteins and pathways that are affected by, and respond to, a chemical stress. The experimental design and the bioinformatics analysis of data used in our laboratory to gain mechanistic insights through expression proteomics into the responses of the eukaryotic model Saccharomyces cerevisiae or of Pseudomonas strains to environmental toxicants are presented as case studies.
We identified the antibody against mitochondrial heat shock protein 70 (mtHSP70) in serum from multiple sclerosis (MS) patients by proteomics-based analysis. The prevalence of the anti-mtHSP70 antibody is significantly higher in serum from MS patients than in serum from Parkinson disease patients, multiple cerebral infarction patients, infectious meningoencephalitis patients, and healthy controls (HCs) (68% sensitivity; 74% specificity). We studied the clinical features and magnetic resonance imaging findings of MS patients with the anti-mtHSP70 antibody. As a result, there were no significant differences between the anti-mtHSP70-antibody-positive and -negative MS patients. Additionally, in our comprehensive analysis of the prevalence of both the anti-mtHSP70 antibody and the anti-phosphoglycerate mutase 1 (PGAM1) antibody, which was previously reported by us to also show a higher prevalence in serum from MS patients, the positivity rates of both these antibodies were significantly higher in serum from MS patients than in serum from patients with other neurological diseases and from HCs; moreover, the specificity of this combination assay was higher than that of the assay of only one antibody (57% sensitivity; 93% specificity). Results of our study suggest that not only the anti-PGAM1 antibody but also the anti-mtHSP70 antibody is good diagnostic markers of MS and the combination of both these antibodies is useful for a more specific diagnosis of MS.
Post-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins, hepatitis C virus (HCV) can orchestrate its complete viral life cycle. HCV non-structural protein 3 (NS3) has many functions. It has protease and helicase activities, interacts with several host-cell proteins and plays a role in translation, replication and virus-particle formation. Organization of all these functions is necessary and could be regulated by PTMs. We therefore searched for modifications of the NS3 protein in the subgenomic HCV replicon. When performing a tag-capture approach coupled with two-dimensional gel electrophoresis analyses, we observed that isolated His6-NS3 yielded multiple spots. Individual protein spots were digested in gel and analysed by mass spectrometry. Differences observed between the individual peptide mass fingerprints suggested the presence of modified peptides and allowed us to identify N-terminal acetylation and an adaptive mutation of NS3 (Q1067R). Further analysis of other NS3 variants revealed phosphorylation of NS3.
Reactive oxygen species (ROS) are known to participate in neurodegeneration after ischemia-reperfusion. With the aid of ROS, the calpain-induced lysosomal rupture provokes ischemic neuronal death in the cornu Ammonis (CA) 1 of the hippocampus; however, the target proteins of ROS still remain unknown. Here a proteomic analysis was done to identify and characterize ROS-induced carbonyl modification of proteins in the CA1 of the macaque monkey after transient whole-brain ischemia followed by reperfusion. We found that carbonyl modification of heat shock 70-kDa protein 1 (Hsp70-1), a major stress-inducible member of the Hsp70 family, was extensively increased before the neuronal death in the CA1 sector, and the carbonylation site was identified to be Arg469 of Hsp70-1. The CA1 neuronal death conceivably occurs by calpain-mediated cleavage of carbonylated Hsp70 that becomes prone to proteolysis with the resultant lysosomal rupture. In addition, the carbonyl levels of dihydropyrimidinase-like 2 isoform 2, glial fibrillary acidic protein, and beta-actin were remarkably increased in the postischemic CA1. Therefore, ischemia-reperfusion-induced oxidative damage to these proteins in the CA1 may lead to loss of the neuroprotective function, which contributes to neuronal death.
Our recent clinical studies have demonstrated that autologous implantation of human cultured periosteal (hCP) sheets in combination with porous hydroxylapatite (HAp) particles at the site of periodontal bone defects strikingly facilitates tissue regeneration. To better understand how the hCP sheet functions at the implantation site, we have now examined its biochemical and morphological characteristics in vitro and its ectopic osteoinductivity in nude mice. Cultured human periosteal tissue segments produced periosteal cells that migrated out from the central region within 4-8 days and grew more rapidly with longer culture times. Alkaline phosphatase activity increased in parallel with actual osteoblastic induction. Cytokine array assays demonstrated that osteoblastic induction downregulated IL-6 and thrombopoietin, but upregulated IL-8, IL-13, IGF-I and IGFBP-2 in hCP sheets. When differentiated hCP sheets were implanted alone, areas of osteoid and mineralized tissue were formed within 2 weeks, but non-induced, immature hCP sheets did not produce much mineralization. These findings suggest that mature hCP sheets potentially function not only as seeds of ectopic bone formation without the need of synthetic tissue scaffolds, but also as living drug-delivery systems, to influence cells near implantation sites by producing several important cytokines. These two major characteristics indicate that a mature hCP sheet is a promising osteoinductive biomaterial, even without conventional scaffolds for periodontal regenerative therapy.
Prostaglandin E(1) (PGE(1)) lowers dermal interstitial fluid pressure (IFP) in vivo and inhibits fibroblast-mediated collagen gel contraction in vitro. PDGF-BB, in contrast, stimulates contraction and normalizes IFP lowered as a result of anaphylaxis. Human diploid AG1518 fibroblasts expressed EP2, EP3 and IP prostaglandin receptors. The inhibitory effect of PGE(1) on contraction depended on cAMP. Short-term stimulation with PDGF-BB transiently induced formation of actin-containing membrane and circular ruffles and breakdown of stress fibers. PGE(1) had no effect on stress fibers nor did it modulate the effects of PDGF-BB. PGE(1) alone or in combination with PDGF-BB inhibited initial adhesion and spreading to collagen. PDGF-BB had no effect on adhesion but stimulated cell spreading. Two-dimensional gel electrophoresis and MALDI TOF analyses of SDS/Triton X-100-soluble proteins revealed changes in migration pattern of actin-binding proteins. Interestingly, PDGF-BB and PGE(1) affected both similar and different sets of actin-binding proteins. PDGF-BB and PGE(1) did not trans-modulate their respective effects on actin-binding proteins, cytoskeletal organization or initial adhesion. Our data show that PDGF-BB stimulates actin cytoskeleton dynamics, whereas PGE(1) inhibits processes dependent on cytoskeletal motor functions. We suggest that these different activities may partly explain the contrasting effects of PGE(1) and PDGF-BB on contraction and IFP.
Genomic studies have shown that there are four abundant type I and type II intermediate filament proteins (IFPs) in wool. When separated using 2D-PAGE, the type I IFPs separated into four clearly defined major rows. The type II IFPs separated into two distinct staggered rows. The large number of spots seen by 2D-PAGE has previously been attributed to charge heterogeneity caused by post-translational modification of the protein. However, analysis of wool IFPs by 2D-PAGE techniques and mass spectrometry suggested an absence of phosphorylation or glycosylation modifications. Investigations with both the type I and type II IFPs showed that when single protein spots from a 2D-PAGE separation are eluted, re-focused and re-electrophoresed, several spots are formed on both the acidic and basic side of the original spot. Amino acid analysis, mass spectrometry and Ellman's assay support the hypothesis that the proteins have the same sequence but vary in isoelectric charge, due to differences in exposure of charged residues on the molecular surface. The cause of IFP charge heterogeneity is thus proposed to be a conformational equilibrium between several different forms of the same protein in the rehydration solution used for the first dimension.
Post-translational modifications modulate the activity of most eukaryote proteins. Analysis of these modifications presents formidable challenges but their determination generates indispensable insight into biological function. Strategies developed to characterize individual proteins are now systematically applied to protein populations. The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.
Eukaryotic organisms from yeast to human harbor genes encoding the small glutamine-rich tetratricopeptide repeat-containing (SGT) protein. Work presented here demonstrated the presence of human SGT (hSGT) protein in a panel of human cell lines and throughout the cell cycle. To identify cellular processes in which hSGT is involved, knock down populations were analyzed which were generated through transfection of hsgt-specific small interfering RNA. Most strikingly, depletion of hSGT led to reduced proliferation of the affected cell populations while the mitotic index was increased. Time-lapse video microscopy revealed that cells from hSGT-depleted populations were unable to complete cell division due to mitotic arrest which was frequently followed by cell death. Further evidence for a role in cell division was given by the accumulation of hSGT in the midzone and the midbody, and by a mitosis-specific migration pattern of hSGT as detected by Western blotting after SDS-PAGE or two-dimensional gel electrophoresis. In conclusion, results obtained in this study demonstrate that hSGT protein is a constitutive component of all human cell lines tested and that this protein is essential for successful completion of cell division.
Post-translational modifications generate tremendous diversity, complexity and heterogeneity of gene products, and their determination is one of the main challenges in proteomics research. Recent developments in mass spectrometry based approaches for systematic, qualitative and quantitative determination of modified proteins promise to bring new insights on the dynamics and spatio-temporal control of protein activities by post-translational modifications, and reveal their roles in biological processes and pathogenic conditions. Combinations of affinity-based enrichment and extraction methods, multidimensional separation technologies and mass spectrometry are particularly attractive for systematic investigation of post-translationally modified proteins in proteomics.
The 26S proteasome complex, which consists of a 20S proteasome and a pair of 19S regulatory particles, plays important roles in the degradation of ubiquitinated proteins in eukaryotic cells. The alpha7 subunit of the budding yeast 20S proteasome is a major phosphorylatable subunit; serine residue(s) in its C-terminal region are phosphorylated in vitro by CKII. However, the exact in vivo phosphorylation sites have not been identified. In this study, using electrospray ionization quadrupole time-of-flight mass spectrometry analysis, we detected a mixture of singly, doubly, and triply phosphorylated C-terminal peptides isolated from a His-tagged construct of the alpha7 subunit by nickel-immobilized metal affinity chromatography. In addition, we identified three phosphorylation sites in the C-terminal region using MS/MS analysis and site-directed mutagenesis: Ser258, Ser263, and Ser264 residues. The MS/MS analysis of singly phosphorylated peptides showed that phosphorylation at these sites did not occur successively.
Proteomics, the analysis of expressed proteins, has been an important developing area of research for the past two decades [Anderson, NG, Anderson, NL. Twenty years of two-dimensional electrophoresis: past, present and future. Electrophoresis 1996;17:443-453]. Advances in technology have led to a rapid increase in applications to a wide range of samples; from initial experiments using cell lines, more complex tissues and biological fluids are now being assessed to establish changes in protein expression. A primary aim of clinical proteomics is the identification of biomarkers for diagnosis and therapeutic intervention of disease, by comparing the proteomic profiles of control and disease, and differing physiological states. This expansion into clinical samples has not been without difficulties owing to the complexity and dynamic range in plasma and human tissues including tissue biopsies. The most widely used techniques for analysis of clinical samples are surface-enhanced laser desorption/ionisation mass spectrometry (SELDI-MS) and 2-dimensional gel electrophoresis (2-DE) coupled to matrix-assisted laser desorption ionisation [Person, MD, Monks, TJ, Lau, SS. An integrated approach to identifying chemically induced posttranslational modifications using comparative MALDI-MS and targeted HPLC-ESI-MS/MS. Chem. Res. Toxicol. 2003;16:598-608]-mass spectroscopy (MALDI-MS). This review aims to summarise the findings of studies that have used proteomic research methods to analyse samples from clinical studies and to assess the impact that proteomic techniques have had in assessing clinical samples.
In order to investigate the cellular system of the freshwater sponge, Ephydatia fluviatilis, we isolated a molecular marker for the most prominent cell type, the choanocyte. After feeding sponge with fluorescent beads, fluorescent-labeled choanocytes were collected by fluorescence activated cell sorting (FACS). By protein profiling choanocyte and archeocyte (stem cell)-rich fractions, proteins characteristic of choanocyte were identified. The partial amino-acid sequence of one of the proteins characteristic of choanocyte matches the deduced amino-acid sequence of sponge expression tag (EST) clones and mouse annexin VII. These EST clones overlap and encode a protein, designated Ef annexin, which includes four annexin domains. Whole mount in situ hybridization shows Ef annexin expression in chamber-forming choanocytes in 7-day-old sponge, leading us to conclude that Ef annexin can be used as a choanocyte marker. In the early development stage, Ef annexin expression can be detected in both large single cells, characteristic of archeocytes, and cells forming 2-, 4- and multiple-cell clusters. These results indicate that Ef annexin is initially expressed in the choanocyte-committed archeocyte which then undergoes several mitotic cell divisions to form a choanocyte chamber. This suggests that the single choanocyte chamber essentially originates from a single archeocyte.
We used a proteomic approach for identifying molecules involved in the pathogenesis of chronic lymphocytic leukemia (CLL). We investigated 14 patients who were completely concordant for IgV(H) mutational status (unmutated vs. mutated), CD38 expression (positive vs. negative), and clinical behavior (progressive vs. stable); these patients were characterized as having either poor or good prognoses. The 2 patient subsets differed in the expression of hematopoietic lineage cell-specific protein 1 (HS1). In patients with poor prognoses, most HS1 protein was constitutively phosphorylated, whereas only a fraction was phosphorylated in patients with good prognoses. This difference was investigated in a larger cohort of 26 unselected patients. The survival curve of all 40 patients analyzed revealed that patients with predominately phosphorylated HS1 experience a significantly shorter median survival time. As HS1 is a protein pivotal in the signal cascade triggered by B cell receptor (BCR) stimulation, we studied its pattern of expression following BCR engagement. Normal mature B cells stimulated by anti-IgM shifted the non- or less-phosphorylated form of HS1 toward the more phosphorylated form. Naive B cells showed both HS1 forms while memory B cells expressed mainly the phosphorylated fraction. These data indicate a central role for antigen stimulation in CLL and suggest a new therapeutic target for patients with aggressive disease.
Substantial advances have been made in the fundamental understanding of human biology, ranging from DNA structure to identification of diseases associated with genetic abnormalities. Genome sequence information is becoming available in unprecedented amounts. The absence of a direct functional correlation between gene transcripts and their corresponding proteins, however, represents a significant roadblock for improving the efficiency of biological discoveries. The success of proteomics depends on the ability to identify and analyze protein products in a cell or tissue and, this is reliant on the application of several key technologies. Proteomics is in its exponential growth phase. Two-dimensional electrophoresis complemented with mass spectrometry provides a global view of the state of the proteins from the sample. Proteins identification is a requirement to understand their functional diversity. Subtle difference in protein structure and function can contribute to complexity and diversity of life. This review focuses on the progress and the applications of proteomics science with special reference to integration of the evolving technologies involved to address biological questions.
We constructed a novel database of the proteome of DLD-1 colon cancer cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of fluorescence-labeled proteins followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. The database consists of 258 functionally categorized proteins corresponding to 314 protein spots. The majority of the proteins are oxidoreductases, cytoskeletal proteins and nucleic acid binding proteins. Phosphatase treatment showed that 28% of the protein spots on the gel are phosphorylated, and mass spectrometric analysis identified 21 of them. Proteins of DLD-1 cells and of laser-microdissected colon cancer tissues showed similar distribution on 2D gels, suggesting the utility of our database for clinical proteomics.
Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult task especially if quantitative comparisons have to be made. Several different experimental strategies can be employed to explore phosphoproteomes and this review will cover the most important ones such as incorporation of radiolabeled phosphate into proteins, application of specific antibodies against phosphorylated residues and direct staining of phosphorylated proteins in polyacrylamide gels. Moreover, methods to enrich phosphorylated proteins such as affinity chromatography (IMAC) and immunoprecipitation as well as mass spectrometry for identification of phosphorylated peptides and phosphorylation sites are also described.
Although protein phosphorylation is probably the most studied post-translational modification occurring in cells, the number of proteins, which are the target of this modification, is still largely unknown. Increasing the coverage of the phosphoproteome as well as the detection of variation at the phosphorylation level would be very helpful for understanding the mechanisms of cell life and the modifications of the cell state leading to pathological conditions such as neurodegeneration. In order to further investigate variations occurring at the phosphorylation level, we have initiated the creation of a reference map of phosphorylated proteins in rat cortical neurons, employing a combination of phosphatase treatment and 2-DE/differential in gel electrophoresis technology. About 131 spots were recognized as phosphorylated proteins as they showed different migration behaviour after phosphatase treatment. The analysis of 42 selected spots was carried out by LC/MS/MS technology resulting in the identification of two new phosphoproteins.
Most cyanobacteria take up nitrate or nitrite through a multisubunit ABC transporter (ATP-binding cassette) located in the cytoplasmic membrane. Nitrate and nitrite transport activity is instantaneously blocked by the presence of ammonium in the medium. Previous biochemical studies reported the existence of phosphorylation/dephosphorylation events of the nitrate transporter (NRT) related to the presence of ammonium-sensitive kinase/phosphatase activities in plasma membranes of the cyanobacterium Synechococcus elongatus PCC 6301. In this work, we have analyzed the biochemical properties of the periplasmic nitrate/nitrite-binding subunit (NrtA) of NRT from the thermophilic nondiazotrophic cyanobacterium Phormidium laminosum. Our results show that cyanobacterial NrtA is phosphorylated in vivo. However, substrate binding activity in vitro is not affected by the phosphorylation state of the protein, ruling out the possibility that phosphorylation/dephosphorylation of NrtA is involved in the regulation of the nitrate/nitrite uptake by NRT transporter. Moreover, NrtA is present as multiple isoforms showing the same molecular mass but different isoelectric points ranging from pI 5 to 6. Mass spectrometric characterization of NrtA isoforms shows that the protein is phosphorylated at residue Tyr203, and contains several methionine sulphoxide residues which account for the observed isoforms. Both phosphorylated and non-phosphorylated forms of NrtA are active in vitro, showing comparable binding affinity for nitrate and nitrite. Both substrates behave as pure competitive inhibitors with a binding stoichiometry of one molecule of anion per NrtA monomer.
Mps-one-binder (Mob) proteins play a crucial role in yeast cytokinesis. After cloning two Mob1-like genes, MsMob1-A and MsMob1-B from alfalfa (Medicago sativa L.) we show that, although they are constitutively expressed in roots, stems, leaves, flowers and pods, their transcripts and proteins are mostly produced in actively proliferating tissues. A polyclonal antibody specifically raised against MsMob1 proteins was used for immunolocalization studies in synchronized root tip cells. The subcellular localization of MsMob1-like proteins is demonstrated to be cell cycle-regulated. Cytoplasmic localization is faint and diffused during G1 and S. It becomes concentrated in punctuate and fibrillar structures in G2 as well as M phase. At the stage of cytokinesis, the protein is found at the emerging cell plate marking the progressive formation of the septum. Mob1 proteins partially co-localize with microtubules structures functionally related to the spindles and important for cytokinesis in eukaryotic cells. The MsMob1 expression cannot rescue the lethality of the yeast mob1 mutant, suggesting that interaction of Mob1 proteins with their effectors may be species-specific. Localization of Mob1 proteins in the inner layer of the root cap indicates an additional function for this class of proteins in plants, which is likely related to the onset of programmed cell death.
ResearchGate has not been able to resolve any references for this publication.