Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.

• MeSH
• adenosin analogy a deriváty   metabolismus   farmakologie   MeSH
• Betacoronavirus enzymologie   MeSH
• chemické modely MeSH
• COVID-19 MeSH
• inhibitory enzymů metabolismus   farmakologie   MeSH
• katalytická doména MeSH
• koronavirové infekce virologie   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• methyltransferasy chemie   metabolismus   MeSH
• molekulární modely MeSH
• pandemie MeSH
• RNA čepičky MeSH
• RNA virová metabolismus   MeSH
• SARS-CoV-2 MeSH
• stabilita RNA MeSH
• virová pneumonie virologie   MeSH
• virové nestrukturální proteiny chemie   metabolismus   MeSH
• virové regulační a přídatné proteiny chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• inhibitory enzymů MeSH
• methyltransferasy MeSH
• NSP10 protein, SARS-CoV-2 MeSH Prohlížeč
• NSP16 protein, SARS-CoV-2 MeSH Prohlížeč
• RNA 2'-O-methyltransferase MeSH Prohlížeč
• RNA čepičky MeSH
• RNA virová MeSH
• sinefungin MeSH Prohlížeč
• virové nestrukturální proteiny MeSH
• virové regulační a přídatné proteiny MeSH

Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biosenzitivní techniky MeSH
• buněčná membrána metabolismus   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• fosfatidylinositoly metabolismus   MeSH
• fosfolipasy typu C genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• intracelulární membrány metabolismus   MeSH
• kinetika MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• systémy druhého messengeru MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fosfatidylinositolfosfáty MeSH
• fosfatidylinositoly MeSH
• fosfolipasy typu C MeSH
• luminescentní proteiny MeSH
• rekombinantní fúzní proteiny MeSH

Many picornaviruses hijack the Golgi resident Acyl-coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4-kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4-phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small-angle X-ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.

• Klíčová slova
• ACBD3, RNA virus, coarse-grained simulations, host factor, intrinsically disordered regions, picornavirus, small-angle X-ray scattering (SAXS),
• MeSH
• acylkoenzym A chemie   metabolismus   MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• lidé MeSH
• membránové proteiny chemie   metabolismus   MeSH
• Picornaviridae chemie   metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ACBD3 protein, human MeSH Prohlížeč
• acylkoenzym A MeSH
• adaptorové proteiny signální transdukční MeSH
• membránové proteiny MeSH

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

• Klíčová slova
• endosome, phosphoinositide, vesicular traffic,
• MeSH
• 1-fosfatidylinositol-4-kinasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• biologický transport MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• endozomy účinky léků   metabolismus   MeSH
• Golgiho aparát účinky léků   metabolismus   MeSH
• HEK293 buňky MeSH
• inhibitory enzymů metabolismus   farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• preklinické hodnocení léčiv MeSH
• rychlé screeningové testy * MeSH
• simulace molekulového dockingu MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• 1-fosfatidylinositol-4-kinasa MeSH
• inhibitory enzymů MeSH

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a key enzyme of the Golgi system because it produces its lipid hallmark - the phosphatidylinositol 4-phosphate (PI4P). It is recruited to Golgi by the Golgi resident ACBD3 protein, regulated by 14-3-3 proteins and it also serves as an adaptor because it recruits the small GTPase Rab11. Here, we analyzed the protein complexes formed by PI4KB in vitro using small angle x-ray scattering (SAXS) and we discovered that these protein complexes are highly flexible. The 14-3-3:PI4KB:Rab11 protein complex has 2:1:1 stoichiometry and its different conformations are rather compact, however, the ACBD3:PI4KB protein complex has both, very compact and very extended conformations. Furthermore, in vitro reconstitution revealed that the membrane is necessary for the formation of ACBD3:PI4KB:Rab11 protein complex at physiological (nanomolar) concentrations.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus   MeSH
• intracelulární membrány metabolismus   MeSH
• maloúhlový rozptyl MeSH
• membránové proteiny metabolismus   MeSH
• multimerizace proteinu * MeSH
• proteiny 14-3-3 metabolismus   MeSH
• Rab proteiny vázající GTP metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ACBD3 protein, human MeSH Prohlížeč
• adaptorové proteiny signální transdukční MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• membránové proteiny MeSH
• phosphatidylinositol 4-kinase IIIbeta, human MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• Rab proteiny vázající GTP MeSH
• rab11 protein MeSH Prohlížeč
• rekombinantní proteiny MeSH

Zika virus is a global health threat due to significantly elevated risk of fetus malformations in infected pregnant women. Currently, neither an effective therapy nor a prophylactic vaccination is available for clinical use, desperately necessitating novel therapeutics and approaches to obtain them. Here, we present a structural model of the Zika virus RNA-dependent RNA polymerase (ZIKV RdRp) in complex with template and nascent RNAs, Mg2+ ions and accessing nucleoside triphosphate. The model allowed for docking studies aimed at effective pre-screening of potential inhibitors of ZIKV RdRp. Applicability of the structural model for docking studies was illustrated with the NITD008 artificial nucleotide that is known to effectively inhibit the function of the ZIKV RdRp. The ZIKV RdRp - RNA structural model is provided for all possible variations of the nascent RNA bases pairs to enhance its general utility in docking and modelling experiments. The developed model makes the rational design of novel nucleosides and nucleotide analogues feasible and thus provides a solid platform for the development of advanced antiviral therapy.

• MeSH
• adenosin analogy a deriváty   chemie   farmakologie   MeSH
• hořčík chemie   MeSH
• infekce virem zika genetika   virologie   MeSH
• konformace proteinů účinky léků   MeSH
• lidé MeSH
• molekulární modely MeSH
• nukleosidy chemie   MeSH
• nukleotidy chemie   MeSH
• polyfosfáty chemie   MeSH
• replikace viru genetika   MeSH
• RNA-dependentní RNA-polymerasa chemie   genetika   MeSH
• RNA chemie   genetika   MeSH
• simulace molekulového dockingu MeSH
• virové nestrukturální proteiny chemie   genetika   MeSH
• virus zika chemie   genetika   patogenita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• hořčík MeSH
• NITD008 MeSH Prohlížeč
• nukleosidy MeSH
• nukleotidy MeSH
• polyfosfáty MeSH
• RNA-dependentní RNA-polymerasa MeSH
• RNA MeSH
• triphosphoric acid MeSH Prohlížeč
• virové nestrukturální proteiny MeSH

Most single stranded plus RNA viruses hijack phosphatidylinositol 4-kinases (PI4Ks) to generate membranes highly enriched in phosphatidylinositol 4-phosphate (PI4P). These membranous compartments known as webs, replication factories or replication organelles are essential for viral replication because they provide protection from the innate intracellular immune response while serving as platforms for viral replication. Using purified recombinant proteins and biomimetic model membranes we show that the nonstructural viral 3A protein is sufficient to promote membrane hyper-phosphorylation given the proper intracellular cofactors (PI4KB and ACBD3). However, our bio-mimetic in vitro reconstitution assay revealed that rather than the presence of PI4P specifically, negative charge alone is sufficient for the recruitment of 3Dpol enzymes to the surface of the lipid bilayer. Additionally, we show that membrane tethered viral 3B protein (also known as Vpg) works in combination with the negative charge to increase the efficiency of membrane recruitment of 3Dpol.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem genetika   metabolismus   MeSH
• Kobuvirus enzymologie   MeSH
• lidé MeSH
• membránové proteiny genetika   metabolismus   MeSH
• pikornavirové infekce metabolismus   virologie   MeSH
• virové nestrukturální proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ACBD3 protein, human MeSH Prohlížeč
• adaptorové proteiny signální transdukční MeSH
• fosfatidylinositolfosfáty MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• membránové proteiny MeSH
• phosphatidylinositol 4-kinase IIIbeta, human MeSH Prohlížeč
• phosphatidylinositol 4-phosphate MeSH Prohlížeč
• virové nestrukturální proteiny MeSH

Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein-coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions-binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized-metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X-ray crystallography.

• Klíčová slova
• crystal structure, endolysin, histidine tag, lysozyme, phage T4, protein purification,
• MeSH
• bakteriofág T4 * enzymologie   genetika   MeSH
• chromatografie afinitní metody   MeSH
• krystalografie rentgenová metody   MeSH
• muramidasa * chemie   genetika   izolace a purifikace   MeSH
• mutace * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• muramidasa * MeSH

14-3-3 proteins bind phosphorylated binding partners to regulate several of their properties, including enzymatic activity, stability and subcellular localization. Here, two crystal structures are presented: the crystal structures of the 14-3-3 protein (also known as Bmh1) from the yeast Lachancea thermotolerans in the unliganded form and bound to a phosphopeptide derived from human PI4KB (phosphatidylinositol 4-kinase B). The structures demonstrate the high evolutionary conservation of ligand recognition by 14-3-3 proteins. The structural analysis suggests that ligand recognition by 14-3-3 proteins evolved very early in the evolution of eukaryotes and remained conserved, underlying the importance of 14-3-3 proteins in physiology.

• Klíčová slova
• 14-3-3 proteins, Bmh1, Bmh2, Lachancea thermotolerans, PI4KB, crystal structure, phosphopeptide,
• MeSH
• 1-fosfatidylinositol-4-kinasa chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• fosfoproteiny chemie   genetika   metabolismus   MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• klonování DNA MeSH
• konformace proteinů, alfa-helix MeSH
• konzervovaná sekvence MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• ligandy MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• plazmidy chemie   metabolismus   MeSH
• protein - isoformy chemie   genetika   metabolismus   MeSH
• proteiny 14-3-3 chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• Saccharomycetales chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• strukturní homologie proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 1-fosfatidylinositol-4-kinasa MeSH
• fosfoproteiny MeSH
• fungální proteiny MeSH
• ligandy MeSH
• protein - isoformy MeSH
• proteiny 14-3-3 MeSH
• rekombinantní proteiny MeSH

We report on an extensive structure-activity relationship study of novel PI4K IIIβ inhibitors. The purine derivative of the potent screening hit T-00127-HEV1 has served as a suitable starting point for a thorough investigation of positions 8 and 2. While position 8 of the purine scaffold can only bear a small substituent to maintain the inhibitory activity, position 2 is opened for extensive modification and can accommodate even substituted phenyl rings without the loss of PI4K IIIβ inhibitory activity. These empirical observations nicely correlate with the results of our docking study, which suggests that position 2 directs towards solution and can provide the necessary space for the interaction with remote residues of the enzyme, whereas the cavity around position 8 is strictly limited. The obtained compounds have also been subjected to antiviral screening against a panel of (+)ssRNA viruses.

• Klíčová slova
• Antiviral agent, Hepatitis C virus, PI4K IIIβ, Phosphatidylinositol 4-kinase, Purine,
• MeSH
• antivirové látky chemická syntéza   chemie   farmakologie   MeSH
• enterovirus B lidský účinky léků   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem antagonisté a inhibitory   metabolismus   MeSH
• HeLa buňky MeSH
• Hepacivirus účinky léků   MeSH
• inhibitory proteinkinas chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• puriny chemická syntéza   chemie   farmakologie   MeSH
• Rhinovirus účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antivirové látky MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• inhibitory proteinkinas MeSH
• phosphatidylinositol 4-kinase IIIbeta, human MeSH Prohlížeč
• purine MeSH Prohlížeč
• puriny MeSH