The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major pest of potato plants, and its digestive system is a promising target for development of pest control strategies. This work focuses on functional proteomic analysis of the digestive proteolytic enzymes expressed in the CPB gut. We identified a set of peptidases using imaging with specific activity-based probes and activity profiling with selective substrates and inhibitors. The secreted luminal peptidases were classified as: (i) endopeptidases of cathepsin D, cathepsin L, and trypsin types and (ii) exopeptidases with aminopeptidase (cathepsin H), carboxypeptidase (serine carboxypeptidase, prolyl carboxypeptidase), and carboxydipeptidase (cathepsin B) activities. The proteolytic arsenal also includes non-luminal peptidases with prolyl oligopeptidase and metalloaminopeptidase activities. Our results indicate that the CPB gut employs a multienzyme network of peptidases with complementary specificities to efficiently degrade ingested proteins. This proteolytic system functions in both CPB larvae and adults and is controlled mainly by cysteine and aspartic peptidases and supported by serine and metallopeptidases. The component enzymes identified here are potential targets for inhibitors with tailored specificities that could be engineered into potato plants to confer resistance to CPB.

• MeSH
• brouci enzymologie   genetika   růst a vývoj   metabolismus   MeSH
• fyziologie výživy zvířat MeSH
• gastrointestinální trakt enzymologie   MeSH
• hmyzí proteiny genetika   metabolismus   MeSH
• larva genetika   růst a vývoj   metabolismus   MeSH
• proteasy genetika   metabolismus   MeSH
• proteolýza MeSH
• proteomika MeSH
• rostlinné proteiny metabolismus   MeSH
• trávení MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Although activity-based protein profiling (ABPP) has been used to study a variety of enzyme classes, its application to intramembrane proteases is still in its infancy. Intramembrane proteolysis is an important biochemical mechanism for activating proteins residing within the membrane in a dormant state. Rhomboid proteases (intramembrane serine proteases) are embedded in the lipid bilayers of membranes and occur in all phylogenetic domains. The study of purified rhomboid proteases has mainly been performed in detergent micelle environments. Here we report on the reconstitution of rhomboids in liposomes. Using ABPP, we have been able to detect active rhomboids in large and giant unilamellar vesicles. We have found that the inhibitor profiles of rhomboids in micelles and liposomes are similar, thus validating previous inhibitor screenings. Moreover, fluorescence microscopy experiments on the liposomes constitute the first steps towards activity-based imaging of rhomboid proteases in membrane environments.

• MeSH
• enzymatické testy metody   MeSH
• inhibitory proteas farmakologie   MeSH
• micely MeSH
• proteasy metabolismus   MeSH
• unilamelární lipozómy chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Intrinsically disordered regions (IDRs) are protein regions that lack persistent secondary or tertiary structure under native conditions. IDRs represent >40% of the eukaryotic proteome and play a crucial role in protein-protein interactions. The classical approach for identification of these interaction interfaces is based on mutagenesis combined with biochemical techniques such as coimmunoprecipitation or yeast two-hybrid screening. This approach either provides information of low resolution (large deletions) or very laboriously tries to precisely define the binding epitope via single amino acid substitutions. Here, we report the use of a peptide microarray based on the human scaffold protein AXIN1 for high-throughput and -resolution mapping of binding sites for several AXIN1 interaction partners in vitro For each of the AXIN1-binding partners tested, i.e. casein kinase 1 ϵ (CK1ϵ); c-Myc; peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1); and p53, we found at least three different epitopes, predominantly in the central IDR of AXIN1. We functionally validated the specific AXIN1-CK1ϵ interaction identified here with epitope-mimicking peptides and with AXIN1 variants having deletions of short binding epitopes. On the basis of these results, we propose a model in which AXIN1 competes with dishevelled (DVL) for CK1ϵ and regulates CK1ϵ-induced phosphorylation of DVL and activation of Wnt/β-catenin signaling.

• MeSH
• axin protein metabolismus   MeSH
• beta-katenin metabolismus   MeSH
• čipová analýza proteinů metody   MeSH
• fosforylace MeSH
• interakční proteinové domény a motivy * MeSH
• kaseinkinasa Iepsilon metabolismus   MeSH
• kompetitivní vazba MeSH
• lidé MeSH
• peptidy metabolismus   MeSH
• protein dishevelled metabolismus   MeSH
• proteiny Wnt metabolismus   MeSH
• signální dráha Wnt MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

INTRODUCTION: Microarray-based gene expression profiling represents a major breakthrough for understanding the molecular complexity of breast cancer. cDNA expression profiles cannot detect changes in activities that arise from post-translational modifications, however, and therefore do not provide a complete picture of all biologically important changes that occur in tumors. Additional opportunities to identify and/or validate molecular signatures of breast carcinomas are provided by proteomic approaches. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) offers high-throughput protein profiling, leading to extraction of protein array data, calling for effective and appropriate use of bioinformatics and statistical tools. METHODS: Whole tissue lysates of 105 breast carcinomas were analyzed on IMAC 30 ProteinChip Arrays (Bio-Rad, Hercules, CA, USA) using the ProteinChip Reader Model PBS IIc (Bio-Rad) and Ciphergen ProteinChip software (Bio-Rad, Hercules, CA, USA). Cluster analysis of protein spectra was performed to identify protein patterns potentially related to established clinicopathological variables and/or tumor markers. RESULTS: Unsupervised hierarchical clustering of 130 peaks detected in spectra from breast cancer tissue lysates provided six clusters of peaks and five groups of patients differing significantly in tumor type, nuclear grade, presence of hormonal receptors, mucin 1 and cytokeratin 5/6 or cytokeratin 14. These tumor groups resembled closely luminal types A and B, basal and HER2-like carcinomas. CONCLUSION: Our results show similar clustering of tumors to those provided by cDNA expression profiles of breast carcinomas. This fact testifies the validity of the SELDI-TOF MS proteomic approach in such a type of study. As SELDI-TOF MS provides different information from cDNA expression profiles, the results suggest the technique's potential to supplement and expand our knowledge of breast cancer, to identify novel biomarkers and to produce clinically useful classifications of breast carcinomas.

• MeSH
• biologické modely MeSH
• čipová analýza proteinů metody   MeSH
• diagnostické techniky molekulární MeSH
• financování organizované MeSH
• komplementární DNA metabolismus   MeSH
• lidé MeSH
• nádorové biomarkery MeSH
• nádory prsu genetika   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• proteomika metody   MeSH
• regulace genové exprese MeSH
• shluková analýza MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• stanovení celkové genové exprese MeSH
• výpočetní biologie metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH

Osteoblastic differentiation is a multistep process characterized by osteogenic induction of mesenchymal stem cells, which then differentiate into proliferative pre-osteoblasts that produce copious amounts of extracellular matrix, followed by stiffening of the extracellular matrix, and matrix mineralization by hydroxylapatite deposition. Although these processes have been well characterized biologically, a detailed transcriptional analysis of murine primary calvaria osteoblast differentiation based on RNA sequencing (RNA-seq) analyses has not previously been reported. Here, we used RNA-seq to obtain expression values of 29,148 genes at four time points as murine primary calvaria osteoblasts differentiate in vitro until onset of mineralization was clearly detectable by microscopic inspection. Expression of marker genes confirmed osteogenic differentiation. We explored differential expression of 1386 protein-coding genes using unsupervised clustering and GO analyses. 100 differentially expressed lncRNAs were investigated by co-expression with protein-coding genes that are localized within the same topologically associated domain. Additionally, we monitored expression of 237 genes that are silent or active at distinct time points and compared differential exon usage. Our data represent an in-depth profiling of murine primary calvaria osteoblast differentiation by RNA-seq and contribute to our understanding of genetic regulation of this key process in osteoblast biology.

• MeSH
• alternativní sestřih MeSH
• buněčná diferenciace genetika   MeSH
• kultivované buňky MeSH
• lebka fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• osteoblasty fyziologie   MeSH
• osteogeneze genetika   MeSH
• RNA analýza   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies.

• MeSH
• Arabidopsis genetika   MeSH
• fylogeneze MeSH
• konformace proteinů MeSH
• kyseliny indoloctové metabolismus   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• multigenová rodina genetika   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Goeckerman's therapy (GT) of psoriasis is based on daily application of pharmacy grade coal tar on affected skin with subsequent exposure to UV light. The aim of this study was to evaluate the influence of Goeckerman's therapy of psoriasis on the levels of proangiogenic chemokines ENA-78 (CXCL5, Epithelial Cell Derived Neutrophil Attractant- 78), GRO alpha (CXCL1, Growth-Related Oncogene), IL-8 (CXCL8, Interleukin-8), MCP-1 (CCL2, Monocyte Chemotactic (Chemoattractant) Protein 1) and RANTES (CCL5, Regulated on Activation of Normal T Cell Expressed and Secreted) in peripheral blood of 22 children's patients with psoriasis. 22 otherwise healthy children serve as a control group. The serum levels of chemokines were determined by commercial membrane protein array technique (RayBiotech, USA). Efficacy of Goeckerman's therapy was delineated by PASI score. Disease activity was significantly diminished by Goeckerman's therapy (p<0.001). Serum levels of GRO alpha and MCP-1 in patients before GT were significantly higher than those measured in healthy blood donors (GRO alpha: p=0.0128 and MCP-1: p=0.0003). Serum levels of GRO alpha, MCP-1 and RANTES were significantly diminished by GT (GRO alpha: p=0.002, MCP-1: p=0.048 and RANTES: p=0.0131). Compared to the healthy controls, serum level of MCP-1 remained significantly increased in psoriasis patients after GT (p<0.0001). In conclusion, we found that the GT of psoriasis influenced the serum levels of proinflammatory and proangiogenic chemokines, especially GRO alpha, MCP-1 and RANTES. It could be the cause for decreased proangiogenic activity which is described after GT of psoriasis.

• MeSH
• chemokiny CC krev   MeSH
• chemokiny CXC krev   MeSH
• čipová analýza proteinů MeSH
• dehet uhelný aplikace a dávkování   MeSH
• dítě MeSH
• financování organizované MeSH
• keratolytika aplikace a dávkování   MeSH
• lidé MeSH
• mladiství MeSH
• psoriáza krev   terapie   MeSH
• terapie ultrafialovými paprsky MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH

While targeted therapy based on the idea of attenuating the activity of a preselected, therapeutically relevant protein has become one of the major trends in modern cancer therapy, no truly specific targeted drug has been developed and most clinical agents have displayed a degree of polypharmacology. Therefore, the specificity of anticancer therapeutics has emerged as a highly important but severely underestimated issue. Chemical proteomics is a powerful technique combining postgenomic drug-affinity chromatography with high-end mass spectrometry analysis and bioinformatic data processing to assemble a target profile of a desired therapeutic molecule. Due to high demands on the starting material, however, chemical proteomic studies have been mostly limited to cancer cell lines. Herein, we report a down-scaling of the technique to enable the analysis of very low abundance samples, as those obtained from needle biopsies. By a systematic investigation of several important parameters in pull-downs with the multikinase inhibitor bosutinib, the standard experimental protocol was optimized to 100 μg protein input. At this level, more than 30 well-known targets were detected per single pull-down replicate with high reproducibility. Moreover, as presented by the comprehensive target profile obtained from miniaturized pull-downs with another clinical drug, dasatinib, the optimized protocol seems to be extendable to other drugs of interest. Sixty distinct human and murine targets were finally identified for bosutinib and dasatinib in chemical proteomic experiments utilizing core needle biopsy samples from xenotransplants derived from patient tumor tissue. Altogether, the developed methodology proves robust and generic and holds many promises for the field of personalized health care.

• MeSH
• aniliny chemie   MeSH
• anotace sekvence MeSH
• biopsie MeSH
• buňky K562 MeSH
• chinoliny chemie   MeSH
• chromatografie afinitní MeSH
• cílená molekulární terapie MeSH
• inhibitory proteinkinas chemie   MeSH
• lidé MeSH
• mapy interakcí proteinů MeSH
• myši inbrední NOD MeSH
• myši SCID MeSH
• myši MeSH
• nádory plic farmakoterapie   enzymologie   patologie   MeSH
• nemalobuněčný karcinom plic farmakoterapie   enzymologie   patologie   MeSH
• nitrily chemie   MeSH
• proteomika MeSH
• pyrimidiny chemie   MeSH
• thiazoly chemie   MeSH
• transplantace nádorů MeSH
• tyrosinkinasy izolace a purifikace   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

INTRODUCTION: Studies have correlated living close to major roads with Alzheimer's disease (AD) risk. However, the mechanisms responsible for this link remain unclear. METHODS: We exposed olfactory mucosa (OM) cells of healthy individuals and AD patients to diesel emissions (DE). Cytotoxicity of exposure was assessed, mRNA, miRNA expression, and DNA methylation analyses were performed. The discovered altered pathways were validated using data from the human population-based Rotterdam Study. RESULTS: DE exposure resulted in an almost four-fold higher response in AD OM cells, indicating increased susceptibility to DE effects. Methylation analysis detected different DNA methylation patterns, revealing new exposure targets. Findings were validated by analyzing data from the Rotterdam Study cohort and demonstrated a key role of nuclear factor erythroid 2-related factor 2 signaling in responses to air pollutants. DISCUSSION: This study identifies air pollution exposure biomarkers and pinpoints key pathways activated by exposure. The data suggest that AD individuals may face heightened risks due to impaired cellular defenses. HIGHLIGHTS: Healthy and AD olfactory cells respond differently to DE exposure. AD cells are highly susceptible to DE exposure. The NRF2 oxidative stress response is highly activated upon air pollution exposure. DE-exposed AD cells activate the unfolded protein response pathway. Key findings are also confirmed in a population-based study.

• MeSH
• Alzheimerova nemoc * genetika   metabolismus   MeSH
• čichová sliznice metabolismus   MeSH
• epigenomika MeSH
• faktor 2 související s NF-E2 genetika   metabolismus   MeSH
• látky znečišťující vzduch škodlivé účinky   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA * MeSH
• mikro RNA metabolismus   genetika   MeSH
• senioři MeSH
• stanovení celkové genové exprese MeSH
• transkriptom MeSH
• výfukové emise vozidel * toxicita   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Numerous cellular functions including respiration require iron. Plants and phytoplankton must also maintain the iron-rich photosynthetic electron transport chain, which most likely evolved in the iron-replete reducing environments of the Proterozoic ocean [1]. Iron bioavailability has drastically decreased in the contemporary ocean [1], most likely selecting for the evolution of efficient iron acquisition mechanisms among modern phytoplankton. Mesoscale iron fertilization experiments often result in blooms dominated by diatoms [2], indicating that diatoms have adaptations that allow survival in iron-limited waters and rapid multiplication when iron becomes available. Yet the genetic and molecular bases are unclear, as very few iron uptake genes have been functionally characterized from marine eukaryotic phytoplankton, and large portions of diatom iron starvation transcriptomes are genes encoding unknown functions [3-5]. Here we show that the marine diatom Phaeodactylum tricornutum utilizes ISIP2a to concentrate Fe(III) at the cell surface as part of a novel, copper-independent and thermodynamically controlled iron uptake system. ISIP2a is expressed in response to iron limitation several days prior to the induction of ferrireductase activity, and it facilitates significant Fe(III) uptake during the initial response to Fe limitation. ISIP2a is able to directly bind Fe(III) and increase iron uptake when heterologously expressed, whereas knockdown of ISIP2a in P. tricornutum decreases iron uptake, resulting in impaired growth and chlorosis during iron limitation. ISIP2a is expressed by diverse marine phytoplankton, indicating that it is an ecologically significant adaptation to the unique nutrient composition of marine environments.

• MeSH
• druhová specificita MeSH
• fytoplankton metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• mořská biologie MeSH
• mořská voda chemie   MeSH
• rozsivky metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• terciární struktura proteinů MeSH
• železo metabolismus   farmakokinetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH