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At least 703 records · Page 39Linked to original sources
PubMed2005

The murine cardiac 26S proteasome: an organelle awaiting exploration.

Multiprotein complexes have been increasingly recognized as essential functional units for a variety of cellular processes, including the protein degradation system. Selective degradation of proteins in eukaryotes is primarily conducted by the ubiquitin proteasome system. The current knowledge base, pertaining to the proteasome complexes in mammalian cells, relies largely upon information gained in the yeast system, where the 26S proteasome is hypothesized to contain a 20S multiprotein core complex and one or two 19S regulatory complexes. To date, the molecular structure of the proteasome system, the proteomic composition of the entire 26S multiprotein complexes, and the specific designated function of individual components within this essential protein degradation system in the heart remain virtually unknown. A functional proteomic approach, employing multidimensional chromatography purification combined with liquid chromatography tandem mass spectrometry and protein chemistry, was utilized to explore the murine cardiac 26S proteasome system. This article presents an overview on the subject of protein degradation in mammalian cells. In addition, this review shares the limited information that has been garnered thus far pertaining to the molecular composition, function, and regulation of this important organelle in the cardiac cells.

Animals↗
PubMed2005

Expression and function of proteins during development of the basal region in rice seedlings.

A differential display of proteins with a two-dimensional polyacrylamide gel electrophoresis approach was used to analyze protein expression changes during development of the basal region in rice seedlings (Oryza sativa L. cv. Nipponbare). The proteins were detected as 700 Coomassie Brilliant Blue-stained spots with pI values from around 3.5 to 9.0. A proteome reference map was established for the basal region of two-week-old seedlings. The basal region proteome map was used to analyze quantitative variations in the tissue during development from 2-, 4-, 6-, 8-, and 10-week-old seedlings. During development, 31 proteins were up-regulated, and 30 proteins were down-regulated compared with the 2-week-old basal region proteome map. The main functions of these proteins were primary metabolism and protein synthesis or maintenance. Calreticulin precursor, enolase, and voltage-dependent anion channel were identified among the up- and down-regulated proteins. The twin spots of calreticulin precursor and enolase with different pI values are possibly due to post-translational modifications such as phosphorylation. In addition, seven proteins showed developmental stage-specific expression. All of the developmentally regulated proteins of the basal region were clustered by the S-system, a differential equation that fit to time course of cluster and analyzed for cluster relationships. Proteins with unknown functions were tentatively assigned to functional groups based on cluster relationships. Basal region development proteome data will be valuable for resolving questions in functional genomics. In addition, cluster analysis of the basal region proteome during development will be useful for the assessment of functional proteins.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2004

Cell cycle goes global.

The cell division cycle is one of the most intensively studied biological processes, yet, in spite of great effort, many questions remain as to how the cell cycle is controlled by cyclin-dependent kinases and other critical regulators. Recent functional genomic and proteomic approaches have yielded new insights into almost all aspects of cell cycle control, including transcriptional circuits, DNA replication, sister chromatid separation and regulation by environmental signals. Perhaps most notably, systematic analysis has begin to reveal meta-level connections between previously distinct sub-processes. As the interconnections between these huge datasets are beyond intuition, mathematical representation and automated analysis of functional genomic data is an urgent mandate.

Animals↗
PubMed2003

Yeast genomics and proteomics in drug discovery and target validation.

Small, cell permeable, and target-specific chemical ligands are highly valuable, not only as therapeutics but also as research tools. The synthesis, identification and characterization of these compounds is often a difficult task. The straightforward genetics of the budding yeast Saccharomyces cerevisiae, and the high degree of conservation of basic cellular processes between yeast and higher organisms makes yeast an excellent tool for drug development studies, particularly in regards to anticancer and anti-fungal drug discovery. Recent advances in yeast functional genomics and proteomics studies are changing the field of yeast research. Many of these new technologies are readily applicable to drug target identification and other aspects of drug discovery. This review will focus on current genetic, genomic, and proteomic methodologies in S. cerevisiae that have the potential to be useful in drug discovery and target validation.

Animals↗
PubMed2003

Protein and antibody microarray technology.

Following the age of genomics having sequenced the human genome, interest is shifted towards the function of genes. This new age of proteomics brings about a change of methods to study the properties of gene products on a large scale. Protein separation technologies are now applied to allow high-throughput purification and characterisation of proteins. Two-dimensional-gel electrophoresis (2DE) and mass spectrometry (MS) have become widely used tools in the field of proteomics. At the same time, protein and antibody microarrays have been developed as successor of DNA microarrays to soon allow the proteome-wide screening of protein function in parallel. This review is aimed to introduce this new technology and to highlight its current prospects and limitations.

Antibodies↗
PubMed2005

Identification of VCP/p97, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners using membrane-based human proteome arrays.

Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles.

Adenosine Triphosphatases↗
PubMed2005

An emerging role for comprehensive proteome analysis in human pregnancy research.

Elucidation of underlying cellular and molecular mechanisms is pivotal to the comprehension of biological systems. The successful progression of processes such as pregnancy and parturition depends on the complex interactions between numerous biological molecules especially within the uterine microenvironment. The tissue- and stage-specific expression of these bio-molecules is intricately linked to and modulated by several endogenous and exogenous factors. Malfunctions may manifest as pregnancy disorders such as preterm labour, pre-eclampsia and fetal growth restriction that are major contributors to maternal and perinatal morbidity and mortality. Despite the immense amount of information available, our understanding of several aspects of these physiological processes remains incomplete. This translates into significant difficulties in the timely diagnosis and effective treatment of pregnancy-related complications. However, the emergence of powerful mass spectrometry-based proteomic techniques capable of identifying and characterizing multiple proteins simultaneously has added a new dimension to the field of biomedical research. Application of these high throughput methodologies with more conventional techniques in pregnancy-related research has begun to provide a novel perspective on the biochemical blueprint of pregnancy and its related disorders. Further, by enabling the identification of proteins specific to a disease process, proteomics is likely to contribute, not only to the comprehension of the underlying pathophysiologies, but also to the clinical diagnosis of multifactorial pregnancy disorders. Although the application of this technology to pregnancy research is in its infancy, characterization of the cellular proteome, unearthing of functional networks and the identification of disease biomarkers can be expected to significantly improve maternal healthcare in the future.

Female↗
PubMed2006

Neuroproteomics of the synapse and drug addiction.

Proteomic analyses of brain tissues are becoming an integral component of neuroscientific research. In particular, the essential role of the synapse in neurotransmission and plasticity has brought about extensive efforts to identify its protein constituents. Recent studies have used a combination of subcellular fractionation and proteomic techniques to identify proteins associated with different components of the synapse. Thus, a coherent map of the synapse proteome is rapidly emerging, and a timely review of these data is warranted. In the first part of this review, neuroproteomic techniques that have been used to analyze the synapse proteome are described. We then summarize the results from several recent proteomic analyses of mammalian synapses and discuss the similarities and differences in their profiling of synaptic proteins. Important advances in this field of research include the use of proteomics to analyze synaptic function and drug effects on synaptic proteins. This article presents an overview of proteomic analyses of the phosphorylation states of synaptic proteins and recent applications of neuroproteomic techniques to the study of drug addiction. Finally, we discuss the challenges in comparing proteomic studies of drug addiction and the future directions of this field in furthering our understanding of the molecular mechanisms underlying synaptic function and drug addiction.

Animals↗
PubMed2005

Mass spectrometry-based quantitative proteomic profiling.

Quantitative proteomics involves the identification and quantitation of protein components in various biological systems. Stable isotope labelling technology, by both metabolic and chemical methods, has been the most commonly used approach for global proteome-wide profiling. Recently, its capability has been extended from labelled pairs to multiple labels, allowing for the simultaneous quantification of multiplex samples. The ion intensity-based quantitative approach has progressively gained more popularity as mass spectrometry performance has improved significantly. Although some success has been reported, it remains difficult comprehensively to characterise the global proteome, due to its enormous complexity and dynamic range. The use of sub-proteome fractionation techniques permits a simplification of the proteome and provides a practical step towards the ultimate dissection of the entire proteome. Further development of the technology for targeting sub-proteomes on a functional basis - such as selecting proteins with differential expression profiles from mass spectrometric analyses, for further mass spectrometric sequencing in an intelligent manner--is expected in the near future.

Biotin↗
PubMed2005

A human protein atlas for normal and cancer tissues based on antibody proteomics.

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.

Antibodies↗
PubMed2003

Activity-based protein profiling in vivo using a copper(i)-catalyzed azide-alkyne [3 + 2] cycloaddition.

Toward the goal of assigning function to the tens of thousands of protein products encoded by eukaryotic and prokaryotic genomes, the field of proteomics requires new technologies that can functionally characterize proteins within the dynamic environment of the cell, where these biomolecules are subject to myriad posttranslational modifications and the actions of endogenous activators and inhibitors. Here, we report an advanced strategy for activity-based protein profiling (ABPP) that addresses this important need. We show that several enzymes can be labeled in an activity-based manner both in vitro and in vivo by an azido-sulfonate ester probe and that these labeling events can be detected in whole proteomes by copper-catalyzed ligation with a rhodamine-alkyne reagent. This click chemistry-based strategy for ABPP represents a unique and versatile method for functional proteome analysis.

Aldehyde Dehydrogenase↗
PubMed2006

Improvement of domain linker prediction by incorporating loop-length-dependent characteristics.

Protein dissection into structural domains that can fold in isolation is an important issue in both functional and structural proteomics. Here, we analyzed inter- and intradomain loop sequences (respectively named domain linker and nonlinker loops) and computed a domain linker likelihood score, which was used for developing a domain boundary prediction protocol. The analysis confirmed our previous results indicating that the amino acid composition in terms of glycine, proline, aspartic acid, asparagine, lysine, and histidine significantly differs between linker and nonlinker loops. However, a detailed examination revealed that the amino acid composition bias actually depends on the loop length. Indeed, significant frequency deviations were observed for glycine, proline, and aspartic acid in short linker and nonlinker loops, whereas deviations were observed for aspartic acid, proline, asparagine, and lysine in long linker and nonlinker loops. Finally, we incorporated this loop-length-dependent amino acid composition bias in a simple linker prediction protocol, which predicted linkers with a 40.6% specificity and a 36.1% sensitivity. These figures are 4.4 and 2.4% higher than those obtained with our former prediction protocol that does not incorporate loop-length-dependent characteristics. This result should have practical significance for experimental protein dissection, since the probability of obtaining a stably folding structural domain by randomly dissecting a protein sequence is estimated to be 12.6%.

Algorithms↗
PubMed2004

Proteomic investigation of natural variation between Arabidopsis ecotypes.

Two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting were used to investigate the natural variation in the proteome among 8 Arabidopsis thaliana ecotypes, of which 3 were previously shown to display atypical responses to environmental stress. Comparison of 2-D maps demonstrated that only one-quarter of spots was shared by all accessions. On the other hand, only 15% of the 25 majors spots accounting for half the total protein amount could be classified as major spots in all ecotypes. Identification of these major spots demonstrated large differences between the major functions detected. Accordingly, the proteomes appeared to reveal important variations in terms of function between ecotypes. Hierarchical clustering of proteomes according to either the amount of all anonymous spots, that of the 25 major spots or the functions of these major spots identified the same classes of ecotypes, and grouped the three atypical ecotypes. It is proposed that proteome comparison has the capacity to evidence differences in the physiological status of ecotypes. Results are discussed with respect to the possibility to infer such differences from limited comparisons of major proteins. It is concluded that classical proteomics could constitute a powerful tool to mine the biodiversity between ecotypes of a single plant species.

Arabidopsis↗
PubMed2006

A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability.

A detailed understanding of the molecular basis of protein folding and stability determinants partly relies on the study of proteins with enhanced conformational stability properties, such as those from thermophilic organisms. In this study, we set up a methodology aiming at identifying the subset of cytosolic hyperstable proteins using Sulfurispharea sp., a hyperthermophilic archaeon, able to grow between 70 and 97 degrees C, as a model organism. We have thermally and chemically perturbed the cytosolic proteome as a function of time (up to 96 h incubation at 90 degrees C), and proceeded with analysis of the remaining proteins by combining one- and two-dimensional gel electrophoresis, liquid chromatography fractionation, and protein identification by N-terminal sequencing and mass spectrometry methods. In total, 14 proteins with enhanced stabilities which are involved in key cellular processes such as detoxification, nucleic acid processing, and energy metabolism were identified including a superoxide dismutase, a peroxiredoxin, and a ferredoxin. We demonstrate that these proteins are biologically active after extensive thermal treatment of the proteome. The relevance of these and other targets is discussed in terms of the organism's ecology. This work thus illustrates an experimental approach aimed at mining a proteome for hyperstable proteins, a valuable tool for target selection in protein stability and structural studies.

Amino Acid Sequence↗
PubMed2001

Effect of antibodies to sperm-specific recombinant contraceptive vaccinogen (rCV) on murine fertilization: search for an animal model to examine its contraceptive potential.

Recently, we cloned and sequenced a sperm-specific antigen, designated as Contraceptive Vaccinogen (rCV), from human testis (Naz et al., 2001). The present study was conducted to examine its proteomic homologue and function in murine sperm, in order to find out whether or not the mouse can provide a suitable model for examining its immunocontraceptive effects. This was examined by using purified antibodies (Ab) raised against the recombinant (r) human CV antigen of approximately 44 kD. In the Western blot procedure, rCV antibodies recognized a specific protein band of approximately 64 +/- 5 kD in murine testis and murine sperm extracts, the band similar to that found in human testis and human sperm. In the immunoprecipitation procedure, rCV Ab immunoprecipitated a protein band of similar size from murine sperm and murine testis extracts. The immunocytochemical (ICT), immunoscanning electronmicroscopic (ISEM) and the immunobead binding technique (IBT) revealed the subcellular localization of CV antigen on the surface of acrosome and tail regions of the noncapacitated and capacitated murine sperm cell. In functional bioassays, rCV Ab inhibited the acrosome reaction as well as sperm-egg binding in vitro. These data indicate that the CV antigen is expressed in murine sperm and has a biological role in sperm function and sperm-egg binding. In vitro inhibition of capacitation/acrosome reaction and sperm-zona binding suggest that the mouse can provide a suitable model to examine the immunocontraceptive effects of CV antigen in actively-immunized animals.

Animals↗
PubMed2006

Chemical and structural diversity of siRNA molecules.

Short interfering RNAs (siRNAs) are 21-23 nt long double-stranded oligoribonucleotides which in mammalian cells exhibit a potency for sequence-specific gene silencing via an RNA interference (RNAi) pathway. It has been already proven that exogenous, chemically synthesized siRNA molecules are effective inhibitors of gene expression and are widely applied for analysis of protein function and proteomics-based target identification. Moreover, since their discovery siRNA molecules have been implemented as potential candidates for therapeutic applications. Variously modified siRNA molecules containing sugar modifications (2'-OMe, -F, -O-allyl, -amino, orthoesters and LNA analogues), internucleotide phospodiester bond modifications (phosphorothioates, boranophosphates), base modifications (s(2)U) as well as 3'-terminal cholesterol-conjugated constructs were investigated as potential candidates for effective inhibition of gene expression. This chapter reviews an impact of chemical and structural modifications of siRNA molecules on their serum and thermal stability, cellular and in vivo activity, cellular uptake, biodistribution and cytotoxicity. Functional analysis of chemically modified siRNA molecules allows for better understanding of the mechanism of the RNA interference process as well as demonstrates immense efforts in optimizing in vivo potency of siRNA molecules for RNAi-based drug design.

Animals↗
PubMed2005

A human protein-protein interaction network: a resource for annotating the proteome.

Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

Axin Protein↗
PubMed2003

[Studies on the parasite Leishmania in the post-genomic era].

Leishmania is a protozoan parasite responsible for considerable morbidity worldwide. The pathologies caused by Leishmania infections are varying with the species. The ongoing determination of the Leishmania major genome sequence represents a milestone for Leishmania research. We discuss here the use of transcriptomics and proteomics to accelerate our understanding of key processes related to Leishmania biology. These two techniques should be useful to find genes and proteins that are expressed in a stage-specific manner and examples of the use of such techniques are provided. Both approaches will complement each others. Indeed, while a number of stage-specific genes have increased stable RNA levels, an even larger subset of the Leishmania amastigote genes are regulated at the level of translation. The availability of the Leishmania genome should also permit important advances in finding species-specific genes that could explain different pathologies. Functional genomic and proteomic approaches should also be useful for understanding the mechanisms of drug resistance in the parasite. The availability of both the Leishmania genome and of its human host or of the mouse animal model will facilitate large scale studies and increase our understanding of host-pathogen interactions.

Animals↗