Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

• Klíčová slova
• PAS domain, basic helix–loop–helix/transcription factor, hormone receptor, insect, juvenile hormone, ligand-binding protein, methoprene, nuclear translocation, protein phosphorylation, protein purification,
• MeSH
• Aedes genetika   metabolismus   MeSH
• fosforylace MeSH
• hmyzí proteiny genetika   metabolismus   MeSH
• juvenilní hormony metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• receptory buněčného povrchu genetika   metabolismus   MeSH
• Sf9 buňky MeSH
• Spodoptera MeSH
• Tribolium genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hmyzí proteiny MeSH
• juvenilní hormony MeSH
• receptory buněčného povrchu MeSH

Human protoporphyrinogen oxidase IX (hPPO) is an oxygen-dependent enzyme catalyzing the penultimate step in the heme biosynthesis pathway. Mutations in the enzyme are linked to variegate porphyria, an autosomal dominant metabolic disease. Here we investigated eukaryotic cells as alternative systems for heterologous expression of hPPO, as the use of a traditional bacterial-based system failed to produce several clinically relevant hPPO variants. Using bacterially-produced hPPO, we first analyzed the impact of N-terminal tags and various detergent on hPPO yield, and specific activity. Next, the established protocol was used to compare hPPO constructs heterologously expressed in mammalian HEK293T17 and insect Hi5 cells with prokaryotic overexpression. By attaching various fusion partners at the N- and C-termini of hPPO we also evaluated the influence of the size and positioning of fusion partners on expression levels, specific activity, and intracellular targeting of hPPO fusions in mammalian cells. Overall, our results suggest that while enzymatically active hPPO can be heterologously produced in eukaryotic systems, the limited availability of the intracellular FAD co-factor likely negatively influences yields of a correctly folded protein making thus the E.coli a system of choice for recombinant hPPO overproduction. At the same time, PPO overexpression in eukaryotic cells might be preferrable in cases when the effects of post-translational modifications (absent in bacteria) on target protein functions are studied.

• MeSH
• buněčné linie MeSH
• Escherichia coli genetika   MeSH
• flavoproteiny biosyntéza   genetika   izolace a purifikace   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mitochondriální proteiny biosyntéza   genetika   izolace a purifikace   MeSH
• protoporfyrinogenoxidasa biosyntéza   genetika   izolace a purifikace   MeSH
• rekombinantní fúzní proteiny biosyntéza   genetika   izolace a purifikace   MeSH
• Sf9 buňky MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• flavoproteiny MeSH
• mitochondriální proteiny MeSH
• PPOX protein, human MeSH Prohlížeč
• protoporfyrinogenoxidasa MeSH
• rekombinantní fúzní proteiny MeSH

Although the modulation of host physiology has been interpreted as an essential process supporting baculovirus propagation, the requirement of energy supply for host antivirus reactions could not be ruled out. Our present study showed that metabolic induction upon AcMNPV (budded virus) infection of Bombyx mori stimulated virus clearance and production of the antivirus protein, gloverin. In addition, we demonstrated that adenosine receptor signaling (AdoR) played an important role in regulating such metabolic reprogramming upon baculovirus infection. By using a second lepidopteran model, Spodoptera frugiperda Sf-21 cells, we demonstrated that the glycolytic induction regulated by adenosine signaling was a conservative mechanism modulating the permissiveness of baculovirus infection. Another interesting finding in our present study is that both BmNPV and AcMNPV infection cause metabolic activation, but it appears that BmNPV infection moderates the level of ATP production, which is in contrast to a dramatic increase upon AcMNPV infection. We identified potential AdoR miRNAs induced by BmNPV infection and concluded that BmNPV may attempt to minimize metabolic activation by suppressing adenosine signaling and further decreasing the host's anti-baculovirus response. Our present study shows that activation of energy synthesis by adenosine signaling upon baculovirus infection is a host physiological response that is essential for supporting the innate immune response against infection.

• Klíčová slova
• Bombyx mori, Spodoptera frugiperda, adenosine signaling, baculovirus, gloverin, glycolysis,
• MeSH
• adenosin metabolismus   MeSH
• adenosintrifosfát biosyntéza   MeSH
• bourec metabolismus   virologie   MeSH
• deoxyglukosa farmakologie   MeSH
• energetický metabolismus MeSH
• glykolýza účinky léků   genetika   MeSH
• hmyzí proteiny metabolismus   MeSH
• infekce DNA virem metabolismus   virologie   MeSH
• interakce hostitele a patogenu imunologie   MeSH
• mezibuněčné signální peptidy a proteiny metabolismus   MeSH
• nukleopolyhedroviry fyziologie   MeSH
• purinergní receptory P1 genetika   metabolismus   MeSH
• replikace viru účinky léků   MeSH
• Sf9 buňky MeSH
• Spodoptera MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• adenosintrifosfát MeSH
• deoxyglukosa MeSH
• gloverin MeSH Prohlížeč
• hmyzí proteiny MeSH
• mezibuněčné signální peptidy a proteiny MeSH
• purinergní receptory P1 MeSH

We recently reported on a successful vaccine for carp against SVCV based on the intramuscular injection of a DNA plasmid encoding the SVCV glycoprotein (SVCV-G). This shows that the intramuscular (i.m.) route of vaccination is suitable to trigger protective responses against SVCV, and that the SVCV G-protein is a suitable vaccine antigen. Yet, despite the general success of DNA vaccines, especially against fish rhabdoviruses, their practical implementation still faces legislative as well as consumer's acceptance concerns. Furthermore, the i.m. route of plasmid administration is not easily combined with most of the current vaccination regimes largely based on intraperitoneal or immersion vaccination. For this reason, in the current study we evaluated possible alternatives to a DNA-based i.m. injectable vaccine using the SVCV-G protein as the vaccine antigen. To this end, we tested two parallel approaches: the first based on the optimization of an alginate encapsulation method for oral delivery of DNA and protein antigens; the second based on the baculovirus recombinant expression of transmembrane SVCV-G protein in insect cells, administered as whole-cell subunit vaccine through the oral and injection route. In addition, in the case of the oral DNA vaccine, we also investigated the potential benefits of the mucosal adjuvants Escherichia coli lymphotoxin subunit B (LTB). Despite the use of various vaccine types, doses, regimes, and administration routes, no protection was observed, contrary to the full protection obtained with our reference i.m. DNA vaccine. The limited protection observed under the various conditions used in this study, the nature of the host, of the pathogen, the type of vaccine and encapsulation method, will therefore be discussed in details to provide an outlook for future vaccination strategies against SVCV.

• Klíčová slova
• Alginate encapsulation, Baculovirus, DNA vaccine, Insect cells, SVCV glycoprotein,
• MeSH
• DNA vakcíny aplikace a dávkování   klasifikace   farmakologie   MeSH
• infekce viry z čeledi Rhabdoviridae imunologie   prevence a kontrola   veterinární   virologie   MeSH
• kapři * MeSH
• nemoci ryb imunologie   prevence a kontrola   virologie   MeSH
• Rhabdoviridae imunologie   MeSH
• Sf9 buňky MeSH
• Spodoptera MeSH
• subjednotkové vakcíny aplikace a dávkování   klasifikace   farmakologie   MeSH
• vakcinace veterinární   MeSH
• virové vakcíny aplikace a dávkování   klasifikace   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vakcíny MeSH
• subjednotkové vakcíny MeSH
• virové vakcíny MeSH

Many enzymes from the short-chain dehydrogenase/reductase superfamily (SDR) have already been well characterized, particularly those that participate in crucial biochemical reactions in the human body (e.g. 11β-hydroxysteroid dehydrogenase 1, 17β-hydroxysteroid dehydrogenase 1 or carbonyl reductase 1). Several other SDR enzymes are completely or almost completely uncharacterized, such as DHRS1 (also known as SDR19C1). Based on our in silico and experimental approaches, DHRS1 is described as a likely monotopic protein that interacts with the membrane of the endoplasmic reticulum. The highest expression level of DHRS1 protein was observed in human liver and adrenals. The recombinant form of DHRS1 was purified using the detergent n-dodecyl-β-D-maltoside, and DHRS1 was proven to be an NADPH-dependent reductase that is able to catalyse the in vitro reductive conversion of some steroids (estrone, androstene-3,17-dione and cortisone), as well as other endogenous substances and xenobiotics. The expression pattern and enzyme activities fit to a role in steroid and/or xenobiotic metabolism; however, more research is needed to fully clarify the exact biological function of DHRS1.

• Klíčová slova
• DHRS1, SDR superfamily, SDR19C1, Steroid hormones, Xenobiotics,
• MeSH
• dehydrogenasy/reduktasy s krátkým řetězcem metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• estron metabolismus   MeSH
• HeLa buňky MeSH
• játra metabolismus   MeSH
• kortison metabolismus   MeSH
• lidé MeSH
• nadledviny metabolismus   MeSH
• nádorové buněčné linie MeSH
• oxidoreduktasy genetika   metabolismus   MeSH
• rekombinantní proteiny biosyntéza   genetika   MeSH
• sekvence aminokyselin MeSH
• Sf9 buňky MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dehydrogenasy/reduktasy s krátkým řetězcem MeSH
• DHRS1 protein, human MeSH Prohlížeč
• estron MeSH
• kortison MeSH
• oxidoreduktasy MeSH
• rekombinantní proteiny MeSH

Heliothis zea nudivirus-1 (HzNV-1) is an insect virus that can induce both lytic and latent infections in various insect cell lines. During latent infection, several microRNAs (miRNAs) are produced from persistency-associated gene 1 (pag1) as the only detectable HzNV-1 transcript. Previous studies have shown that the pag1 gene suppresses the immediate-early gene hhi1 and promotes host switching into a latent infection via miRNAs derived from pag1. Although other functions of the miRNAs derived from pag1 have not yet been elucidated, several studies have suggested that miRNAs encoded from latency-associated genes can regulate histone-associated enzymes. Because pag1 is a noncoding transcript, it potentially regulates host chromatin structure through miRNAs upon infection. Nevertheless, the exact mechanism by which pag1 alters viral infections remains unknown. In this study, we found that the pag1-encoded miRNA miR-420 suppresses expression of the histone modification-associated enzyme su(var)3-9. Therefore, this miRNA causes histone modification to promote HzNV-1 infection. These results suggest that HzNV-1 may directly influence epigenetic regulation in host cells through interactions with pag1 miRNAs to promote lytic infection. This study provides us with a better understanding of both the HzNV-1 infection pathway and the relationship between viral miRNAs and epigenetic regulation.

• MeSH
• epigeneze genetická * MeSH
• histony metabolismus   MeSH
• hmyzí proteiny metabolismus   MeSH
• metylace MeSH
• mikro RNA biosyntéza   MeSH
• nukleopolyhedroviry fyziologie   MeSH
• regulace exprese virových genů * MeSH
• RNA virová biosyntéza   MeSH
• Sf9 buňky MeSH
• Spodoptera * metabolismus   virologie   MeSH
• virové proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histony MeSH
• hmyzí proteiny MeSH
• mikro RNA MeSH
• Pag1 protein, Heliothis zea virus 1 MeSH Prohlížeč
• RNA virová MeSH
• virové proteiny MeSH

The aim of this study was to develop a suitable vaccine antigen against porcine circovirus 2 (PCV2), the causative agent of post-weaning multi-systemic wasting syndrome, which causes significant economic losses in swine breeding. Chimeric antigens containing PCV2b Cap protein sequences based on the mouse polyomavirus (MPyV) nanostructures were developed. First, universal vectors for baculovirus-directed production of chimeric MPyV VLPs or pentamers of the major capsid protein, VP1, were designed for their exploitation as vaccines against other pathogens. Various strategies were employed based on: A) exposure of selected immunogenic epitopes on the surface of MPyV VLPs by insertion into a surface loop of the VP1 protein, B) insertion of foreign protein molecules inside the VLPs, or C) fusion of a foreign protein or its part with the C-terminus of VP1 protein, to form giant pentamers of a chimeric protein. We evaluated these strategies by developing a recombinant vaccine against porcine circovirus 2. All candidate vaccines induced the production of antibodies against the capsid protein of porcine circovirus after immunization of mice. The candidate vaccine, Var C, based on fusion of mouse polyomavirus and porcine circovirus capsid proteins, could induce the production of antibodies with the highest PCV2 neutralizing capacity. Its ability to induce the production of neutralization antibodies was verified after immunization of pigs. The advantage of this vaccine, apart from its efficient production in insect cells and easy purification, is that it represents a DIVA (differentiating infected from vaccinated animals) vaccine, which also induces an immune response against the mouse polyoma VP1 protein and is thus able to distinguish between vaccinated and naturally infected animals.

• MeSH
• Circovirus * genetika   imunologie   MeSH
• myši MeSH
• nanostruktury * MeSH
• Polyomavirus * genetika   imunologie   MeSH
• prasata MeSH
• rekombinantní fúzní proteiny * genetika   imunologie   MeSH
• Sf9 buňky MeSH
• Spodoptera MeSH
• virové plášťové proteiny * genetika   imunologie   MeSH
• virové vakcíny * genetika   imunologie   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• rekombinantní fúzní proteiny * MeSH
• virové plášťové proteiny * MeSH
• virové vakcíny * MeSH
• VP1 protein, Poliovirus MeSH Prohlížeč

Dehydrogenase/reductase (SDR family) member 3 (DHRS3), also known as retinal short-chain dehydrogenase/reductase (retSDR1) is a member of SDR16C family. This family is thought to be NADP(H) dependent and to have multiple substrates; however, to date, only all-trans-retinal has been identified as a DHRS3 substrate. The reductive reaction catalysed by DHRS3 seems to be physiological, and recent studies proved the importance of DHRS3 for maintaining suitable retinoic acid levels during embryonic development in vivo. Although it seems that DHRS3 is an important protein, knowledge of the protein and its properties is quite limited, with the majority of information being more than 15 years old. This study aimed to generate a more comprehensive characterisation of the DHRS3 protein. Recombinant enzyme was prepared and demonstrated to be a microsomal, integral-membrane protein with the C-terminus oriented towards the cytosol, consistent with its preference of NADPH as a cofactor. It was determined that DHRS3 also participates in the metabolism of other endogenous compounds, such as androstenedione, estrone, and DL-glyceraldehyde, and in the biotransformation of xenobiotics (e.g., NNK and acetohexamide) in addition to all-trans-retinal. Purified and reconstituted enzyme was prepared for the first time and will be used for further studies. Expression of DHRS3 was shown at the level of both mRNA and protein in the human liver, testis and small intestine. This new information could open other areas of DHRS3 protein research.

• Klíčová slova
• DHRS3, Expression, Membrane topology, Reductase activity, retSDR1,
• MeSH
• alkoholoxidoreduktasy metabolismus   MeSH
• cytosol metabolismus   MeSH
• jaterní mikrozomy enzymologie   metabolismus   MeSH
• játra enzymologie   metabolismus   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• NADH, NADPH oxidoreduktasy metabolismus   MeSH
• NADP metabolismus   MeSH
• Sf9 buňky MeSH
• Spodoptera metabolismus   MeSH
• syntázy mastných kyselin metabolismus   MeSH
• tenké střevo enzymologie   metabolismus   MeSH
• testis enzymologie   metabolismus   MeSH
• tretinoin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkoholoxidoreduktasy MeSH
• membránové proteiny MeSH
• NADH, NADPH oxidoreduktasy MeSH
• NADP MeSH
• short chain trans-2-enoyl-CoA reductase MeSH Prohlížeč
• syntázy mastných kyselin MeSH
• tretinoin MeSH

Regulated protein synthesis via changes in mRNA structures forms an important part of how prokaryotic cells adapt protein expression in response to changes in the environment. Little is known regarding how this concept has adapted to regulate mRNA translation via signaling pathways in mammalian cells. Here, we show that following phosphorylation by the ataxia telangiectasia mutated (ATM) kinase at serine 403, the C-terminal RING domain of HDMX binds the nascent p53 mRNA to promote a conformation that supports the p53 mRNA-HDM2 interaction and the induction of p53 synthesis. HDMX and its homolog HDM2 bind the same p53 internal ribosome entry sequences (IRES) structure but with different specificity and function. The results show how HDMX and HDM2 act as nonredundant IRES trans-acting factors (ITAFs) to bring a positive synergistic effect on p53 expression during genotoxic stress by first altering the structure of the newly synthesized p53 mRNA followed by stimulation of translation.

• MeSH
• ATM protein metabolismus   MeSH
• fosforylace MeSH
• jaderné proteiny chemie   fyziologie   MeSH
• lidé MeSH
• messenger RNA chemie   genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• obrácené repetice MeSH
• poškození DNA MeSH
• posttranskripční úpravy RNA MeSH
• posttranslační úpravy proteinů MeSH
• proteiny buněčného cyklu MeSH
• proteosyntéza MeSH
• protoonkogenní proteiny c-mdm2 metabolismus   MeSH
• protoonkogenní proteiny chemie   fyziologie   MeSH
• regulace genové exprese MeSH
• sbalování RNA MeSH
• sekvence nukleotidů MeSH
• Sf9 buňky MeSH
• Spodoptera MeSH
• substrátová specifita MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATM protein, human MeSH Prohlížeč
• ATM protein MeSH
• jaderné proteiny MeSH
• MDM2 protein, human MeSH Prohlížeč
• MDM4 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• nádorový supresorový protein p53 MeSH
• proteiny buněčného cyklu MeSH
• protoonkogenní proteiny c-mdm2 MeSH
• protoonkogenní proteiny MeSH
• TP53 protein, human MeSH Prohlížeč

Dehydrogenase/reductase SDR family member 7 (DHRS7, SDR34C1, retSDR4) is one of the many endoplasmic reticulum bound members of the SDR superfamily. Preliminary results indicate its potential significance in human metabolism. DHRS7 containing TEV-cleavable His10 and FLAG-tag expressed in the Sf9 cell line was solubilised, purified, and reconstituted into liposomes to enable the improved characterisation of this enzyme in the future. Igepal CA-630 was determined to be the best detergent for the solubilisation process. The solubilised DHRS7 was purified using affinity chromatography, and the purified enzyme was subjected to TEV cleavage of the affinity tags and then repurified using subtractive Ni-IMAC. The cleaved and uncleaved versions of DHRS7 were successfully reconstituted into liposomes. In addition, using tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as the substrate, the cleaved liposomal DHRS7 was found to be inactive, whereas the pure and uncleaved liposomal DHRS7 were confirmed as enzymes, which reduce carbonyl group of the substrates.

• Klíčová slova
• Carbonyl reductase activity, DHRS7, Purification, Reconstitution, Short-chain dehydrogenases/reductases, Solubilisation,
• MeSH
• buněčná membrána MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• oxidoreduktasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Sf9 buňky MeSH
• Spodoptera MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• oxidoreduktasy MeSH
• rekombinantní proteiny MeSH