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1.
Chem Sci ; 12(19): 6719-6725, 2021 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-34040747

RESUMEN

One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.

2.
Chemistry ; 25(41): 9592-9596, 2019 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-31111975

RESUMEN

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580-650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.


Asunto(s)
Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/química , Rodanina/análogos & derivados , Núcleo Celular/ultraestructura , Fluorescencia , Células HEK293 , Células HeLa , Humanos , Microscopía Confocal , Microscopía Fluorescente , Imagen Óptica
3.
Sci Rep ; 8(1): 16759, 2018 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-30425281

RESUMEN

Silicateins play a key role in biosynthesis of spicules in marine sponges; they are also capable to catalyze formation of amorphous silica in vitro. Silicateins are highly homologous to cathepsins L - a family of cysteine proteases. Molecular mechanisms of silicatein activity remain controversial. Here site-directed mutagenesis was used to clarify significance of selected residues in silica polymerization. A number of mutations were introduced into two sponge proteins - silicatein A1 and cathepsin L from Latrunculia oparinae, as well as into human cathepsin L. First direction was alanine scanning of the proposed catalytic residues. Also, reciprocal mutations were introduced at selected positions that differ between cathepsins L and silicateins. Surprisingly, all the wild type and mutant proteins were capable to catalyze amorphous silica formation with a water-soluble silica precursor tetra(glycerol)orthosilicate. Some mutants possessed several-fold enhanced silica-forming activity and can potentially be useful for nanomaterial synthesis applications. Our findings contradict to the previously suggested mechanisms of silicatein action via a catalytic triad analogous to that in cathepsins L. Instead, a surface-templated biosilification by silicateins and related proteins can be proposed.


Asunto(s)
Catepsina L/metabolismo , Catepsinas/metabolismo , Glicerol/química , Glicerol/metabolismo , Silicatos/química , Dióxido de Silicio/metabolismo , Secuencia de Aminoácidos , Animales , Catepsina L/química , Catepsina L/genética , Catepsinas/química , Catepsinas/genética , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Polimerizacion , Poríferos/genética , Dióxido de Silicio/química
4.
Biochem Biophys Res Commun ; 495(2): 2066-2070, 2018 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-29253563

RESUMEN

Silicateins, the spicule-forming proteins from marine demosponges capable to polymerize silica, are popular objects of biomineralization studies due to their ability to form particles varied in shape and composition under physiological conditions. Despite the occurrence of the many approaches to nanomaterial synthesis using silicateins, biochemical properties of this protein family are poorly characterized. The main reason for this is that tetraethyl orthosilicate (TEOS), the commonly used silica acid precursor, is almost insoluble in water and thus is poorly available for the protein. To solve this problem, we synthesized new water-soluble silica precursor, tetra(glycerol)orthosilicate (TGS), and characterized biochemical properties of the silicatein A1 from marine sponge Latrunculia oparinae. Compared to TEOS, TGS ensured much greater activity of silicatein and was less toxic for the mammalian cell culture. We evaluated optimum conditions for the enzyme - pH range, temperature and TGS concentration. We concluded that TGS is a useful silica acid precursor that can be used for silica particles synthesis and in vivo applications.


Asunto(s)
Materiales Biomiméticos/síntesis química , Catepsinas/química , Polímeros/síntesis química , Poríferos/química , Dióxido de Silicio/síntesis química , Agua/química , Animales , Ensayo de Materiales , Solubilidad
5.
Int J Mol Sci ; 18(7)2017 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-28704934

RESUMEN

Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed "trio-FPs" consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression.


Asunto(s)
Antozoos/metabolismo , Animales , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/metabolismo , Microscopía Confocal
6.
J Virol ; 86(10): 5574-83, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22438537

RESUMEN

Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.


Asunto(s)
Infecciones por Cardiovirus/virología , Efecto Citopatogénico Viral , Regulación hacia Abajo , Interacciones Huésped-Patógeno , Mengovirus/fisiología , Replicación Viral , Infecciones por Cardiovirus/inmunología , Infecciones por Cardiovirus/patología , Células HeLa , Humanos , Mengovirus/genética , Mengovirus/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología
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