Nejvíce citovaný článek - PubMed ID 25799115
The use of human growth hormone (hGH) by athletes remains widespread despite being banned by the World Anti-Doping Agency (WADA). Current commercial ELISA kits for the determination of hGH are predominantly based on the sandwich format of this method, which allows the analysis of the whole hGH molecules, but not their fragments. The abuse of these fragments by athletes is the current trend in doping. Therefore, a competitive ELISA format is needed; however, only a limited number of kits on the market are based on this principle. Furthermore, these kits are designed to detect and quantify hGH only in blood plasma, serum or tissue cultures. When analysing a nutritional supplement containing hGH using one of these ELISA kits, a false negative result was observed. In the present work, two competitive ELISA methods for the detection of hGH in illegal nutritional supplements were developed. The development of the methods involved the selection of suitable immunoreagents and their combinations with subsequent optimisation of the conditions for their use. The methods were then characterised (with LOD 0.4 and 18.4 ng/mL, according to the primary antibody) and applied to analyse real nutritional supplements. In all 34 samples suspected of containing hGH, the hormone was detected using both ELISA methods and subsequently confirmed by LC-UV and LC-MS. Therefore, the newly developed ELISA methods could become a valuable tool for the police in identifying illegal preparations.
- Klíčová slova
- black market, competitive ELISA, doping, growth hormone, nutritional supplements,
- Publikační typ
- časopisecké články MeSH
Protein-RNA interactions play important biological roles and hence reactive RNA probes for cross-linking with proteins are important tools in their identification and study. To this end, we designed and synthesized 5'-O-triphosphates bearing a reactive squaramate group attached to position 5 of cytidine or position 7 of 7-deazaadenosine and used them as substrates for polymerase synthesis of modified RNA. In vitro transcription with T7 RNA polymerase or primer extension using TGK polymerase was used for synthesis of squaramate-modified RNA probes which underwent covalent bioconjugations with amine-linked fluorophore and lysine-containing peptides and proteins including several viral RNA polymerases or HIV reverse transcriptase. Inhibition of RNA-depending RNA polymerases from Japanese Encephalitis virus was observed through formation of covalent cross-link which was partially identified by MS/MS analysis. Thus, the squaramate-linked NTP analogs are useful building blocks for the synthesis of reactive RNA probes for bioconjugations with primary amines and cross-linking with lysine residues.
- Publikační typ
- časopisecké články MeSH
DNA modifications on pyrimidine nucleobases play diverse roles in biology such as protection of bacteriophage DNA from enzymatic cleavage, however, their role in the regulation of transcription is underexplored. We have designed and synthesized a series of uracil 2'-deoxyribonucleosides and 5'-O-triphosphates (dNTPs) bearing diverse modifications at position 5 of nucleobase, including natural nucleotides occurring in bacteriophages, α-putrescinylthymine, α-glutaminylthymine, 5-dihydroxypentyluracil, and methylated or non-methylated 5-aminomethyluracil, and non-natural 5-sulfanylmethyl- and 5-cyanomethyluracil. The dNTPs bearing basic substituents were moderate to poor substrates for DNA polymerases, but still useful in primer extension synthesis of modified DNA. Together with previously reported epigenetic pyrimidine nucleotides, they were used for the synthesis of diverse DNA templates containing a T7 promoter modified in the sense, antisense or in both strands. A systematic study of the in vitro transcription with T7 RNA polymerase showed a moderate positive effect of most of the uracil modifications in the non-template strand and some either positive or negative influence of modifications in the template strand. The most interesting modification was the non-natural 5-cyanomethyluracil which showed significant positive effect in transcription.
- Publikační typ
- časopisecké články MeSH
UNLABELLED: Adenovirus (AdV) infection of the respiratory epithelium is common but poorly understood. Human AdV species C types, such as HAdV-C5, utilize the Coxsackie-adenovirus receptor (CAR) for attachment and subsequently integrins for entry. CAR and integrins are however located deep within the tight junctions in the mucosa where they would not be easily accessible. Recently, a model for CAR-independent AdV entry was proposed. In this model, human lactoferrin (hLF), an innate immune protein, aids the viral uptake into epithelial cells by mediating interactions between the major capsid protein, hexon, and yet unknown host cellular receptor(s). However, a detailed understanding of the molecular interactions driving this mechanism is lacking. Here, we present a new cryo-EM structure of HAdV-5C hexon at high resolution alongside a hybrid structure of HAdV-5C hexon complexed with human lactoferrin (hLF). These structures reveal the molecular determinants of the interaction between hLF and HAdV-C5 hexon. hLF engages hexon primarily via its N-terminal lactoferricin (Lfcin) region, interacting with hexon's hypervariable region 1 (HVR-1). Mutational analyses pinpoint critical Lfcin contacts and also identify additional regions within hLF that critically contribute to hexon binding. Our study sheds more light on the intricate mechanism by which HAdV-C5 utilizes soluble hLF/Lfcin for cellular entry. These findings hold promise for advancing gene therapy applications and inform vaccine development. IMPORTANCE: Our study delves into the structural aspects of adenovirus (AdV) infections, specifically HAdV-C5 in the respiratory epithelium. It uncovers the molecular details of a novel pathway where human lactoferrin (hLF) interacts with the major capsid protein, hexon, facilitating viral entry, and bypassing traditional receptors such as CAR and integrins. The study's cryo-EM structures reveal how hLF engages hexon, primarily through its N-terminal lactoferricin (Lfcin) region and hexon's hypervariable region 1 (HVR-1). Mutational analyses identify critical Lfcin contacts and other regions within hLF vital for hexon binding. This structural insight sheds light on HAdV-C5's mechanism of utilizing soluble hLF/Lfcin for cellular entry, holding promise for gene therapy and vaccine development advancements in adenovirus research.
- Klíčová slova
- adenovirus, cryo-EM, immune evasion, lactoferrin, viral entry,
- MeSH
- adenovirové infekce lidí metabolismus virologie MeSH
- biologické modely MeSH
- elektronová kryomikroskopie MeSH
- internalizace viru * MeSH
- laktoferrin * chemie genetika metabolismus ultrastruktura MeSH
- lidé MeSH
- lidské adenoviry * chemie genetika metabolismus ultrastruktura MeSH
- mutace MeSH
- respirační sliznice cytologie metabolismus virologie MeSH
- rozpustnost MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- virové plášťové proteiny * chemie genetika metabolismus ultrastruktura MeSH
- virové receptory * chemie genetika metabolismus ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- hexon capsid protein, Adenovirus MeSH Prohlížeč
- laktoferrin * MeSH
- virové plášťové proteiny * MeSH
- virové receptory * MeSH
Human interleukin 24 (IL-24) is a multifunctional cytokine that represents an important target for autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers exciting possibilities in basic research and may have applications in therapy. As a proof-of-concept, we developed a strategy based on recombinant soluble protein variants and genetic code expansion technology to photocontrol the binding between IL-24 and one of its receptors, IL-20R2. Screening of non-canonical ortho-nitrobenzyl-tyrosine (NBY) residues introduced at several positions in both partners was done by a combination of biophysical and cell signaling assays. We identified one position for installing NBY, tyrosine70 of IL-20R2, which results in clear impairment of heterocomplex assembly in the dark. Irradiation with 365-nm light leads to decaging and reconstitutes the native tyrosine of the receptor that can then associate with IL-24. Photocaged IL-20R2 may be useful for the spatiotemporal control of the JAK/STAT phosphorylation cascade.
Identification of metal-binding sites in proteins and understanding metal-coupled protein folding mechanisms are aspects of high importance for the structure-to-function relationship. Mass spectrometry (MS) has brought a powerful adjunct perspective to structural biology, obtaining from metal-to-protein stoichiometry to quaternary structure information. Currently, the different experimental and/or instrumental setups usually require the use of multiple data analysis software, and in some cases, they lack some of the main data analysis steps (MS processing, scoring, identification). Here, we present a comprehensive data analysis pipeline that addresses charge-state deconvolution, statistical scoring, and mass assignment for native MS, bottom-up, and native top-down with emphasis on metal-protein complexes. We have evaluated all of the approaches using assemblies of increasing complexity, including free and chemically labeled proteins, from low- to high-resolution MS. In all cases, the results have been compared with common software and proved how MetaOdysseus outperformed them.
- Klíčová slova
- Cys-rich, R package, mass spectrometry, metalloprotein, zinc,
- MeSH
- cystein * MeSH
- hmotnostní spektrometrie MeSH
- proteiny * MeSH
- software MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cystein * MeSH
- proteiny * MeSH
