Interferon-induced tetherin restricts vesicular stomatitis virus release in neurons

Tetherin, a recently identified interferon (IFN)-inducible, type 2 transmembrane protein, has been shown to be a cellular antiviral restriction factor that retains newly formed virions in infected cells. Thus, tetherin plays an important role in the innate cell-autonomous immune response. The aim of this study was to examine the antiviral activities of tetherin in vesicular stomatitis virus infections of murine neuronal cells. Both IFN-β and IFN-γ induce the expression of tetherin mRNA and protein. Tetherin knockdown experiments were carried out by transfection of tethrin shRNA into murine neuroblastoma cells using a vector containing the pCMV-driven tGFP gene. The efficiency of transfection was monitored through GFP expression by the transfected cells. Selected transfected cells were used for further mRNA and protein analysis, fluorescent immunocytolocalization, and viral infection to study the impact of tetherin knockdown. Our research indicates that tetherin is expressed on the outer face of the plasma membrane of murine neuroblastoma cells, its expression can be induced with both IFN-γ and IFN-β, and tetherin restricts progeny virus release up to 100-fold in mammalian neurons, thus contributing to a potent antiviral state within the host cell.     

Characterization of an extracellular alkaline serine protease from marine <Emphasis Type="Italic">Engyodontium album</Emphasis> BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme. K _m, V _max, and K _cat of the enzyme were 4.727 × 10⁻² mg/ml, 394.68 U, and 4.2175 × 10⁻² s⁻¹, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65°C), with maximum activity at pH 11 and 60°C. CaCl₂ (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65°C. Hg²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Cd⁺, and Al³⁺ inhibited enzyme activity, while 1 mM Co²⁺ enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.     

Estradiol-mediated internalisation of the non-activated estrogen receptor from the goat uterine plasma membrane: Identification of the proteins involved

An indirect approach has been made to study the molecular details associated with the estradiol-induced internalisation of the non-activated estrogen receptor (naER) from the goat uterine plasma membrane. The internalisation of naER appears to be an energy dependent process. Exposure of the plasma membrane to estradiol results in the activation of a Mg2+ dependent ATPase associated with the membrane fraction. Presence of quercetin in the medium prevented the activation of the Mg2+ ATPase as well as the dissociation of naER from the plasma membrane. Using isolated plasma membrane preparations it has been possible to identify the proteins which interact with naER during various stages of its internalisation. The main proteins identified are: (1) a 58 kDa protein, p58, which apparently recognizes the nuclear localization signals on the naER and transports it to the nucleus: (2) hsp70: (3) hsp90, the functional roles of which remain unknown at this stage; (4) a 50 kDa protein associated with the clathrin coated vesicles, presumed to be involved in recognizing the tyrosine based internalisation signals on the naER; (5) actin which mediates the plasma membrane-to-nucleus movement of the naER-p58 complex.     

Import and export of nuclear proteins: Focus on the nucleocytoplasmic movements of two different species of mammalian estrogen receptor

There is a wealth of information regarding the import and export of nuclear proteins in general. Nevertheless, the available data that deals with the nucleocytoplasmic movement of steroid hormone receptors remains highly limited. Some research findings reported during the past five years have succeeded in identifying proteins related to the movement of estrogen receptor from the cytoplasm to the nucleus. What is striking in these findings is the facilitatory role of estradiol in the transport process. A similar conclusion has been drawn from the studies on the plasma membrane-to nucleus movement of the alternative form of estrogen receptor, the non-activated estrogen receptor (naER). The internalization of naER from the plasma membrane takes place only in the presence of estradiol. While the gene regulatory functions of ER appear to get terminated following its ubiquitinization within the nucleus, the naER, through its deglycosylated form, the nuclear estrogen receptor II (nER II) continues to remain functional even beyond its existence within the nucleus. Recent studies have indicated the possibility that the estrogen receptor that regulates the nucleo cytoplasmic transport of m RNP is the nERII. This appears to be the result of the interaction between nERII and three proteins belonging to a group of small nuclear ribonucleo proteins (snRNP). The interaction of nERII with two of this protein appears to activate the inherent Mg²⁺ ATPase activity of the complex, which leads to the exit of the RNP through the nuclear pore complex.     

Cholesterol inhibits the nuclear entry of estrogen receptor activation factor (E-RAF) and its dimerization with the nonactivated estrogen receptor (naER) in goat uterus

An alternative form of estrogen receptor isolated from goat uterus, the nonactivated estrogen receptor (naER), has no DNA-binding function, although it is closely similar to the classical estrogen receptor (ER) in its hormone binding affinity and specificity. The naER dimerizes with a DNA binding protein, estrogen receptor activation factor (E-RAF). The heterodimer binds to the DNA. Assays carried out during the purification of E-RAF showed that an endogenous inhibitor that is heat stable and dialyzable bound to the E-RAF and prevented the formation of the heterodimer. The inhibitor has been isolated and purified. GC-MS analysis identifies this molecule to be cholesterol. Circular dichroism measurement has shown that the high-affinity binding of cholesterol to E-RAF results in subtle changes in the secondary and the tertiary structure of the protein. The E-RAF with altered conformation fails to dimerize with the naER. Instead of facilitating E-RAF entry into the nucleus, dimerization with the naER prevents it. Similarly, cholesterol binding blocks the nuclear entry of the protein, showing that E-RAF with altered conformation is incapable of interaction with the nuclear pore complex/membrane proteins. The naER-E-RAF heterodimer remains at the nuclear periphery, incapable of further transport. These results indicate the possibility that the dimerization between naER and the E-RAF takes place only within the nuclear compartment. The observation that cholesterol binding prevents nuclear entry of the E-RAF reflects the similarity of E-RAF with the sterol regulatory element (SRE) binding protein that enters the nucleus and binds to SRE only when the intracellular level of cholesterol remains low.     

TOP1 inhibition therapy protects against SARS-CoV-2-induced lethal inflammation

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IRF1 governs the differential interferon-stimulated gene responses in human monocytes and macrophages by regulating chromatin accessibility

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