Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.

• MeSH
• antivirové látky farmakologie   chemie   metabolismus   MeSH
• diferenciační antigeny * metabolismus   chemie   MeSH
• konformace proteinů * MeSH
• lidé MeSH
• rekombinantní proteiny chemie   izolace a purifikace   metabolismus   biosyntéza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitochondria and oxidative phosphorylation (OXPHOS) are emerging as intriguing targets for the efficient elimination of cancer cells. The specificity of this approach is aided by the capacity of non-proliferating non-cancerous cells to withstand oxidative insult induced by OXPHOS inhibition. Recently we discovered that mitochondrial targeting can also be employed to eliminate senescent cells, where it breaks the interplay between OXPHOS and ATP transporters that appear important for the maintenance of mitochondrial morphology and viability in the senescent setting. Hence, mitochondria/OXPHOS directed pharmacological interventions show promise in several clinically-relevant scenarios that call for selective removal of cancer and senescent cells.

• MeSH
• adenosindifosfát metabolismus   MeSH
• adenosintrifosfát metabolismus   MeSH
• biologický transport MeSH
• buněčná smrt MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• nádory metabolismus   patologie   MeSH
• oxidativní fosforylace MeSH
• proliferace buněk MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• stárnutí buněk * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Kallikrein-related proteases (KLKs) play a critical role in epidermis physiology and have been implicated in skin pathologies such as Netherton syndrome. The contribution of individual KLKs to skin proteolysis is poorly understood. Monitoring of their activities in skin proteome is hampered by overlapping substrate specificities, and there is a need for novel assays. Here, we present a platform of selective and sensitive fluorogenic substrates and inhibitors for profiling KLK5, KLK7 and KLK14. These chemical tools were evaluated using recombinant KLKs and tissue from a unique set of mice deficient in eight combinations of KLKs and their natural regulator LEKTI.

• MeSH
• kalikreiny nedostatek   genetika   metabolismus   MeSH
• kůže metabolismus   MeSH
• lidé MeSH
• modely nemocí na zvířatech * MeSH
• myši knockoutované MeSH
• myši MeSH
• proteolýza * MeSH
• stanovení celkové genové exprese MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Every year, influenza A virus (IAV) affects and kills many people worldwide. The viral hemagglutinin (HA) is a critical actor in influenza virus infectivity which needs to be cleaved by host serine proteases to exert its activity. KLK5 has been identified as an activating protease in humans with a preference for the H3N2 IAV subtype. We investigated the origin of this preference using influenza A/Puerto Rico/8/34 (PR8, H1N1) and A/Scotland/20/74 (Scotland, H3N2) viruses. Pretreatment of noninfectious virions with human KLK5 increased infectivity of Scotland IAV in MDCK cells and triggered influenza pneumonia in mice. These effects were not observed with the PR8 IAV. Molecular modeling and in vitro enzymatic studies of peptide substrates and recombinant HAs revealed that the sequences around the cleavage site do not represent the sole determinant of the KLK5 preference for the H3N2 subtype. Using mouse Klk5 and Klk5-deficient mice, we demonstrated in vitro and in vivo that the mouse ortholog protease is not an IAV activating enzyme. This may be explained by unfavorable interactions between H3 HA and mKlk5. Our data highlight the limitations of some approaches used to identify IAV-activating proteases.

• MeSH
• buňky MDCK MeSH
• kalikreiny nedostatek   metabolismus   MeSH
• lidé MeSH
• modely nemocí na zvířatech * MeSH
• molekulární modely MeSH
• myši knockoutované MeSH
• myši MeSH
• psi MeSH
• roční období MeSH
• serinové proteasy metabolismus   MeSH
• virus chřipky A metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We show that the plant quaternary benzo[c]phenanthridine alkaloid sanguilutine (SL) is a strong inducer of caspase-independent non-apoptotic death in human melanoma cells. Necrostatin-1, a specific inhibitor of necroptosis, completely reversed the cytotoxic effect of SL, suggesting that necroptosis was a predominant type of cell death induced by SL in these cells. In addition, we showed that SL can trigger an autophagic response, as confirmed by GFP-LC3 puncta formation and LC3-II accumulation. Interestingly, we observed a significant decrease in the viability of melanoma cells treated with combination of autophagy inhibitors (3-methyladenine, bafilomycin-A1 and LY294002) and SL. Our results further indicated that autophagy may serve as a pro-survival mechanism, delaying the induction of necroptosis in melanoma cells. The ability of SL to induce caspase-independent non-apoptotic cell death (necroptosis) suggests its possible therapeutic potential in the treatment of apoptosis-resistant melanoma tumours. Furthermore, SL might serve as a useful tool for studying the mechanisms of necroptosis and autophagy induction and the interplay between these two processes.

• MeSH
• adenin analogy a deriváty   farmakologie   MeSH
• antitumorózní látky antagonisté a inhibitory   farmakologie   MeSH
• autofagie účinky léků   MeSH
• benzofenantridiny antagonisté a inhibitory   farmakologie   MeSH
• imidazoly farmakologie   MeSH
• indoly farmakologie   MeSH
• kaspasy metabolismus   MeSH
• lidé MeSH
• melanom patologie   MeSH
• nádorové buněčné linie MeSH
• nekróza chemicky indukované   MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• protoonkogenní proteiny c-bcl-2 metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Viroid-caused pathogenesis is a specific process dependent on viroid and host genotype(s), and may involve viroid-specific small RNAs (vsRNAs). We describe a new PSTVd variant C3, evolved through sequence adaptation to the host chamomile (Matricaria chamomilla) after biolistic inoculation with PSTVd-KF440-2, which causes extraordinary strong ('lethal') symptoms. The deletion of a single adenine A in the oligoA stretch of the pathogenicity (P) domain appears characteristic of PSTVd-C3. The pathogenicity and the vsRNA pool of PSTVd-C3 were compared to those of lethal variant PSTVd-AS1, from which PSTVd-C3 differs by five mutations located in the P domain. Both lethal viroid variants showed higher stability and lower variation in analyzed vsRNA pools than the mild PSTVd-QFA. PSTVd-C3 and -AS1 caused similar symptoms on chamomile, tomato, and Nicotiana benthamiana, and exhibited similar but species-specific distributions of selected vsRNAs as quantified using TaqMan probes. Both lethal PSTVd variants block biosynthesis of lignin in roots of cultured chamomile and tomato. Four 'expression markers' (TCP3, CIPK, VSF-1, and VPE) were selected from a tomato EST library to quantify their expression upon viroid infection; these markers were strongly downregulated in tomato leaf blades infected by PSTVd-C3- and -AS1 but not by PSTVd-QFA.

• MeSH
• fyziologická adaptace * MeSH
• genetické markery genetika   MeSH
• interakce hostitele a patogenu MeSH
• lignin metabolismus   MeSH
• malá nekódující RNA genetika   MeSH
• Matricaria virologie   MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární evoluce * MeSH
• molekulární sekvence - údaje MeSH
• RNA virová genetika   MeSH
• sekvence nukleotidů MeSH
• Solanum lycopersicum virologie   MeSH
• Solanum tuberosum metabolismus   virologie   MeSH
• termodynamika MeSH
• viroidy genetika   patogenita   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Sequence-specific DNA binding is the key function through which tumor suppressor p53 exerts transactivation of the downstream target genes, often being impaired in cancer cells by mutations in the TP53 gene. Functional protein microarray technology enables a high-throughput parallel analysis of protein properties within one experiment under the same conditions. Using an array approach, we analyzed the DNA binding activity of wild type p53 protein and of 49 variants. Our results show significant differences in the binding properties between the p53 mutants. The C-terminal mutant R337C displayed the highest DNA binding activity on the array. However, the same mutant showed only a partial activation in the reporter gene assay and almost no activation of downstream target genes after transfection of expression vector into cells lacking endogenous p53. These observations demonstrate that DNA binding itself is not sufficient for activating the p53 target genes in at least some of the p53 mutants and, therefore, in vitro studies might not always reflect in vivo conditions.

• MeSH
• aktivace transkripce MeSH
• čipová analýza proteinů MeSH
• DNA metabolismus   MeSH
• genetická transkripce genetika   MeSH
• genetická variace genetika   MeSH
• lidé MeSH
• molekulární modely MeSH
• mutace MeSH
• mutantní proteiny genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Bcl-2 protein is one of the key components of biochemical pathways controlling programmed cell death. The function of this protein can be regulated by posttranslational modifications. Phosphorylation of Bcl-2 has been considered to be significantly associated with cell cycle arrest in the G2/M phase of the cell cycle, and with cell death caused by defects of microtubule dynamics. This study shows that phosphorylation of Bcl-2 can be induced by heavy metals due to activation of the Jun N-terminal kinase pathway that is not linked to the G2/M cell cycle arrest. Furthermore, we demonstrate that hyperphosphorylated Bcl-2 protein is a more potent inhibitor of zinc-induced cell death than its hypophosphorylated mutant form. These data suggest that regulation of Bcl-2 protein function by phosphorylation is an important part of cell responses to stress.

• MeSH
• apoptóza účinky léků   MeSH
• elektroforéza MeSH
• financování organizované MeSH
• fosforylace účinky léků   MeSH
• fyziologický stres účinky léků   MeSH
• JNK mitogenem aktivované proteinkinasy metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• posttranslační úpravy proteinů účinky léků   MeSH
• protoonkogenní proteiny c-bcl-2 metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• těžké kovy farmakologie   MeSH
• zinek farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

Similar to growing and metabolically active tissues, tumors require a dense vasculature to gain access to oxygen and nutrients. However, blood vessels in tumors differ from vessels in normal tissues in many respects. In particular, the tumor vasculature is in an active state of angiogenesis or vasculogenesis, and it is immature and leaky. Blood vessels are multicellular tubes formed by polarized endothelial cells, which face the patent vascular lumen with their apical cell surface, whereas their basal cell surface faces extracellular matrix on the outside of the vessels. The same cell polarity can be found in other tubular structures, such as in the bronchial tubes of the lung or the kidney tubules. In contrast, blood vessels in invertebrates often have a vascular lumen lined by basal cell surfaces. These vessels are often formed by a process named 'ancestral vascular tube formation'. Here, we discuss the hypothesis that the supply of tumors with blood can be achieved by both endothelial cell-lined tubes as well as tubes formed by the tumor cells themselves using the ancestral vascular tube formation mechanism. We discuss this hypothesis with a particular focus on gastrointestinal tumors.

• MeSH
• cévní endotel cytologie   metabolismus   MeSH
• extracelulární matrix metabolismus   MeSH
• fyziologická neovaskularizace MeSH
• gastrointestinální nádory krevní zásobení   metabolismus   patofyziologie   MeSH
• lidé MeSH
• nádory krevní zásobení   metabolismus   patofyziologie   MeSH
• patologická angiogeneze MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

Secreted aspartic proteases (Saps) of pathogenic Candida spp. represent a specific target for antifungal drug development. We synthesized a series of peptidomimetic inhibitors with different isosteric groups and modifications at individual positions and tested them with purified Saps from C. albicans (Sap2p), C. tropicalis (Sapt1p), and C. parapsilosis (Sapp1p). The kinetic parameters indicated that all three proteases prefer binding of inhibitors containing bulky hydrophobic residues between positions P3 and P3'. The most divergent specificity was found for Sapp1p. The sequence alignment of Sap2p, Sapt1p, and Sapp1p, and homology modeling of Sapp1p with the crystal structure of Sapt1p and the complex of Sap2p with a peptidomimetic inhibitor showed that the overall folds of Sap2p, Sapt1p, and Sapp1p are similar. However, the N- and C-terminal loops formed by disulfide bonds between residues 47-53 and 258-292 are significantly shorter in Sapp1p, and a unique insertion following Tyr 129 in Sapp1p results in the formation of a loop that can interact with inhibitor residues. These Sapp1p structural differences might lead to its altered susceptibility to inhibition.

• MeSH
• aspartátové endopeptidasy chemie   účinky léků   MeSH
• Candida enzymologie   MeSH
• financování organizované MeSH
• inhibitory proteas farmakologie   chemická syntéza   chemie   MeSH
• izoenzymy chemie   účinky léků   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• stereoizomerie MeSH
• substrátová specifita MeSH
• terciární struktura proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH