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PeptidePicker: A scientific workflow with web interface for selecting appropriate peptides for targeted proteomics experiments.
Get PDFOne challenge in Multiple Reaction Monitoring (MRM)-based proteomics is to select the most appropriate surrogate peptides to represent a target protein. We present here a software package to automatically generate these most appropriate surrogate peptides for an LC/MRM-MS analysis. Our method integrates information about the proteins, their tryptic peptides, and the suitability of these peptides for MRM which is available online in UniProtKB, NCBI's dbSNP, ExPASy, PeptideAtlas, PRIDE, and GPMDB. The scoring algorithm reflects our knowledge in choosing the best candidate peptides for MRM, based on the uniqueness of the peptide in the targeted proteome, its physiochemical properties, and whether it previously has been observed. The modularity of the workflow allows further extension and additional selection criteria to be incorporated. We have developed a simple Web interface where the researcher provides the protein accession number, the subject organism, and peptide-specific options. Currently, the software is designed for human and mouse proteomes, but additional species can be easily be added. Our software improved the peptide selection by eliminating human error, considering multiple data sources and all of the isoforms of the protein, and resulted in faster peptide selection - approximately 50 proteins per hour compared to 8 per day
Genetic diversity ofSCN5A gene and its possible association with the concealed form 2 of Brugada Syndrome development in Polish group of patients
Get PDFBrugada Syndrome (BS) is an inherited channelopathy associated with a high incidence of sudden cardiac death. The article presents the discovery of new genetic variants at SCN5A gene which might be associated with the development of concealed form of Brugada Syndrome. The study involved a group of 59 patients (37 men) with suspected concealed form of Brugada Syndrome. Pharmacological provocation with intravenous ajmaline administration was performed. Six patients with positive test result were subjected to molecular analysis of SCN5A gene with MSSCP method. Additionally, MSSCP genotyping was performed for samples obtained from the family members with Brugada Syndrome, despite they had negative ajmaline challenge test results.Genetic examinations of the SCN5A gene at 6 positive patients showed 6 known polymorphisms, 8 new single nucleotide point (SNP) variants located at exons and 12 new single nucleotide point variants at introns. Among detected in exons SNP, two of them were synonymous SNPs (no change in the coded amino acids), whereas 3 represented non-synonymous SNPs and affected the protein sequences
Transferrin as a drug carrier: Cytotoxicity, cellular uptake and transport kinetics of doxorubicin transferrin conjugate in the human leukemia cells.
Get PDFLeukemias are one of most common malignancies worldwide. There is a substantial need for new chemotherapeutic drugs effective against this cancer. Doxorubicin (DOX), used for treatment of leukemias and solid tumors, is poorly efficacious when it is administered systemically at conventional doses. Therefore, several strategies have been developed to reduce the side effects of this anthracycline treatment. In this study we compared the effect of DOX and doxorubicin-transferrin conjugate (DOX-TRF) on human leukemia cell lines: chronic erythromyeloblastoid leukemia (K562), sensitive and resistant (K562/DOX) to doxorubicin, and acute lymphoblastic leukemia (CCRF-CEM). Experiments were also carried out on normal cells, peripheral blood mononuclear cells (PBMC). We analyzed the chemical structure of DOX-TRF conjugate by using mass spectroscopy. The in vitro growth-inhibition assay XTT, indicated that DOX-TRF is more cytotoxic for leukemia cells sensitive and resistant to doxorubicin and significantly less sensitive to normal cells compared to DOX alone. During the assessment of intracellular DOX-TRF accumulation it was confirmed that the tested malignant cells were able to retain the examined conjugate for longer periods of time than normal lymphocytes. Comparison of kinetic parameters showed that the rate of DOX-TRF efflux was also slower in the tested cells than free DOX. The results presented here should contribute to the understanding of the differences in antitumor activities of the DOX-TRF conjugate and free drug
Effects of liposomes with polyisoprenoids, potential drug carriers,on the cardiovascular and excretory system in rats
Get PDFBackground: The unpredictable side effects of a majority currently used drugs are the substantial issue, in
which patients and physicians are forced to deal with. Augmenting the therapeutic efficacy of drugsmay
prove more fruitful than searching for the new ones. Since recent studies show that new cationic
derivatives of polyisoprenoid alcohols (APrens) might exhibit augmenting properties, we intend to use
them as a component of liposomal drug carriers. In this study we investigate if these compounds do not
per se cause untoward effects on the living organism.
Methods: Male Sprague–Dawley rats received for four weeks daily injections (0.5 ml sc) of liposomes
built of dioleoyl phosphatidylethanolamine (DOPE), liposomes built of DOPE and APren-7 (ratio 10:1) or
water solvent. Weekly, rats were observed in metabolic cages (24 h); blood and urine were sampled for
analysis; body weight (BW) and systolic blood pressure (SBP) were determined. After chronic
experiment, kidneys and heart were harvested for histological and morphometric analysis.
Results: The 4-week BW increments were in the range of 97 ± 4 to 102 ± 4%, intergroup differences were not significant. Microalbuminuria was the lowest in the group receiving liposomes with APren-7 (0.22 ± 0.03 mg/day). Water and food intake, plasma and urine parameters were similar in all groups.
Conclusions: Newly designed liposomes containing APren-7 did not affect functions of the excretory and cardiovascular systems, and renal morphology; therefore we find them suitable as a component of liposomal drug carriers
The Diversity of Bacteria Isolated from Antarctic Freshwater Reservoirs Possessing the Ability to Produce Polyhydroxyalkanoates
No full textThe diversity of polyhydroxyalkanoates-pro-
ducing bacteria in freshwater reservoirs in the EcologyGlacier foreland, Antarctica, was examined by a cultiva-
tion-dependent method. Isolated strains were analyzed
phylogenetically by 16S rRNA gene sequencing, and clas-
sified as members of Alpha-, Beta-, or Gammaproteobac-
teria classes. Polymerase chain reaction was used to detect
PHA synthase genes. Potential polyhydroxyalkanoates
(PHAs) producers belonging mainly to Pseudomonas sp.,
and Janthinobacterium sp. were isolated from all five
sampling sites, suggesting that PHA synthesis is a common
bacterial feature at pioneer sites. All Pseudomonas strains
had the genetic potential to synthesize medium-chain-
length PHAs, whereas some isolated Janthinobacterium
strains might produce short-chain-length PHAs or medium-
chain-length PHAs. It is the first report revealing that
Janthinobacterium species could have the potential to
produce medium-chain-length PHAs
Identification of proteins involved in starch and polygalacturonic acid degradation using LC/MS
Get PDFPlant biomass in the form of cheap wastes, such as straw, corn stalks, wood chips, sawdust, bagasse, pomace, etc.,
is abundant throughout the world. To convert these wastes into the useful value-added compounds microbial enzymes are the
preferred choice. In this paper, we identify enzymes involved in the degradation of starch and polygalacturonic acid using liquid
chromatography/mass spectrometry based analysis. We analysed total protein from soil and compost samples. Extracellular
proteins from enrichment cultures were analysed in parallel and used as controls in the sample preparation and identification of
proteins. In general, both protein sequence coverage and the number of identified peptides were higher in the samples obtained
from the enrichment cultures than from the total protein from soil and compost. The influence of the nature of gel (zymography
vs. SDS/polyacrylamide) was negligible. Thus, starch and polygalacturonic acid degradation associated proteins can be directly
excised from the zymograms without the need to align zymograms with the SDS/polyacrylamide gels. A range of starch and
polygalacturonic acid degradation associated enzymes were identified in both total protein samples and extracellular proteins from
the enrichment cultures. Our results show that proteins involved in starch and polygalacturonic acid degradation can be identified by
liquid chromatography/mass spectrometry from the complex protein mixtures both with and without cultivation of microorganisms
Mitochondrial protein sorting as a therapeutic target for ATP synthase disorders
Get PDFMitochondrial diseases are systemic, prevalent and often fatal; yet treatments remain scarce. Identifying molecular intervention points that can be therapeutically targeted remains a major challenge, which we confronted via a screening assay we developed. Using yeast models of mitochondrial ATP synthase disorders, we screened a drug repurposing library, and applied genomic and biochemical techniques to identify pathways of interest. Here we demonstrate that modulating the sorting of nuclear-encoded proteins into mitochondria, mediated by the TIM23 complex, proves therapeutic in both yeast and patient-derived cells exhibiting ATP synthase deficiency. Targeting TIM23-dependent protein sorting improves an array of phenotypes associated with ATP synthase disorders, including biogenesis and activity of the oxidative phosphorylation machinery. Our study establishes mitochondrial protein sorting as an intervention point for ATP synthase disorders, and because of the central role of this pathway in mitochondrial biogenesis, it holds broad value for the treatment of mitochondrial diseases
Human Annexins A1, A2, and A8 as Potential Molecular Targets for Ni(II) Ions
No full textNickel is harmful for humans, but molecular mechanisms of its toxicity are far from being fully elucidated. One of such mechanisms may be associated with the Ni(II)-dependent peptide bond hydrolysis, which occurs before Ser/Thr in Ser/Thr-Xaa-His sequences. Human annexins A1, A2, and A8, proteins modulating the immune system, contain several such sequences. To test if these proteins are potential molecular targets for nickel toxicity we characterized the binding of Ni(II) ions and hydrolysis of peptides Ac-KALTGHLEE-am (A1-1), Ac-TKYSKHDMN-am (A1-2), and Ac-GVGTRHKAL-am (A1-3), from annexin A1, Ac-KMSTVHEIL-am (A2-1) and Ac-SALSGHLET-am (A2-2), from annexin A2, and Ac-VKSSSHFNP-am (A8-1), from annexin A8, using UV-vis and circular dichroism (CD) spectroscopies, potentiometry, isothermal titration calorimetry, high-performance liquid chromatography (HPLC), and electrospray ionization mass spectrometry (ESI-MS). We found that at physiological conditions (pH 7.4 and 37 °C) peptides A1-2, A1-3, A8-1, and to some extent A2-2 bind Ni(II) ions sufficiently strongly in 4N complexes and are hydrolyzed at sufficiently high rates to justify the notion that these annexins can undergo nickel hydrolysis in vivo. These results are discussed in the context of specific biochemical interactions of respective proteins. Our results also expand the knowledge about Ni(II) binding to histidine peptides by determination of thermodynamic parameters of this process and spectroscopic characterization of 3N complexes. Altogether, our results indicate that human annexins A1, A2, and A8 are potential molecular targets for nickel toxicity and help design appropriate cellular studies
Carboxy terminus of GATA4 transcription factor is required for its cardiogenic activity and interaction with CDK4.
Get PDFGATA4-6 transcription factors regulate numerous aspects of development and homeostasis in multiple tissues of mesodermal and endodermal origin. In the heart, the best studied of these factors, GATA4, has multiple distinct roles in cardiac specification, differentiation, morphogenesis, hypertrophy and survival. To improve understanding of how GATA4 achieves its numerous roles in the heart, here we have focused on the carboxy-terminal domain and the residues required for interaction with cofactors FOG2 and Tbx5. We present evidence that the carboxy terminal region composed of amino acids 362-400 is essential for mediating cardiogenesis in Xenopus pluripotent explants and embryos. In contrast, the same region is not required for endoderm-inducing activity of GATA4. Further evidence is presented that the carboxy terminal cardiogenic region of GATA4 does not operate as a generic transcriptional activator. Potential mechanism of action of the carboxy terminal end of GATA4 is provided by the results showing physical and functional interaction with CDK4, including the enhancement of cardiogenic activity of GATA4 by CDK4. These results establish CDK4 as a GATA4 partner in cardiogenesis. The interactions of GATA4 with its other well described cofactors Tbx5 and FOG2 are known to be involved in heart morphogenesis, but their requirement for cardiac differentiation is unknown. We report that the mutations that disrupt interactions of GATA4 with Tbx5 and FOG2, G295S and V217G, respectively, do not impair cardiogenic activity of GATA4. These findings add support to the view that distinct roles of GATA4 in the heart are mediated by different determinants of the protein. Finally, we show that the rat GATA4 likely induces cardiogenesis cell autonomously or directly as it does not require activity of endodermal transcription factor Sox17, a GATA4 target gene that induces cardiogenesis non-cell autonomously
Urine proteomes of healthy aging humans reveal extracellular matrix (ECM) alterations and immune system dysfunction.
Get PDFAging is a complex physiological process that poses considerable conundrums to rapidly aging societies. For example, the risk of dying from cardiovascular diseases and/or cancer steadily declines for people after their 60s and other causes of death predominate for seniors older than 80 years of age. Thus, physiological aging presents numerous unanswered questions, particularly with regard to changing metabolic patterns. Urine proteomics analysis is becoming a non-invasive, and reproducible diagnostic method. We investigated the urine proteomes in healthy elderly people to determine which metabolic processes were weakened or strengthened in aging humans. Urine samples from 37 healthy volunteers aged 19-90 years (19 men; 18 women) were analyzed for protein expression by LC-MS/MS. This generated a list of 19 proteins that were differentially expressed in different age groups (young, intermediate, and old age). In particular, the oldest group showed protein changes reflective of altered extracellular matrix turnover and declining immune function, which changes corresponded to reported changes in cardiovascular tissue remodeling and immune disorders in the elderly. Thus, urinary proteomes changes in the elderly appear to reflect the physiological processes of aging and are particularly clearly represented in the circulatory and immune systems. Detailed identification of “protein trails” creates a more global picture of metabolic changes that occur in the elderly