Strong evidence indicates that regulated mRNA translation in neuronal dendrites underlies synaptic plasticity and brain development. The fragile X mental retardation protein (FMRP) is involved in this process; here, we show that it acts by inhibiting translation initiation. A binding partner of FMRP, CYFIP1/Sra1, directly binds the translation initiation factor eIF4E through a domain that is structurally related to those present in 4E-BP translational inhibitors. Brain cytoplasmic RNA 1 (BC1), another FMRP binding partner, increases the affinity of FMRP for the CYFIP1-eIF4E complex in the brain. Levels of proteins encoded by known FMRP target mRNAs are increased upon reduction of CYFIP1 in neurons. Translational repression is regulated in an activity-dependent manner because BDNF or DHPG stimulation of neurons causes CYFIP1 to dissociate from eIF4E at synapses, thereby resulting in protein synthesis. Thus, the translational repression activity of FMRP in the brain is mediated, at least in part, by CYFIP1.

We demonstrate temperature-controlled encapsulation and release of the enzyme horseradish peroxidase using a preassembled and covalently closed three-dimensional DNA cage structure as a controllable encapsulation device. The utilized cage structure was covalently closed and composed of 12 double-stranded B-DNA helices that constituted the edges of the structure. The double stranded helices were interrupted by short single-stranded thymidine linkers constituting the cage corners except for one, which was composed by four 32 nucleotide long stretches of DNA with a sequence that allowed them to fold into hairpin structures. As demonstrated by gel-electrophoretic and fluorophore-quenching experiments this design imposed a temperature-controlled conformational transition capability to the structure, which allowed entrance or release of an enzyme cargo at 37 °C while ensuring retainment of the cargo in the central cavity of the cage at 4 °C. The entrapped enzyme was catalytically active inside the DNA cage and was able to convert substrate molecules penetrating the apertures in the DNA lattice that surrounded the central cavity of the cage.

A system containing the globular protein azurin and 3,658 water molecules has been simulated to investigate the influence on water dynamics exerted by a protein surface. Evaluation of water mean residence time for elements having different secondary structure did not show any correlation. Identically, comparison of solvent residence time for atoms having different charge and polarity did not show any clear trend. The main factor influencing water residence time in proximity to a specific site was found to be its solvent accessibility. In detail for atoms belonging to lateral chains and having solvent-accessible surface lower than approximately 16 A(2)a relation is found for which charged and polar atoms are surrounded by water molecules characterized by residence times longer than the non polar ones. The involvement of the low accessible protein atom in an intraprotein hydrogen bond further modulates the length of the water residence time. On the other hand for surfaces having high solvent accessibility, all atoms, independently of their character, are surrounded by water molecules which rapidly exchange with the bulk solvent. Proteins 2000;39:56-67.

DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance. DNA topoisomerase I (Top1p)1 catalyzes the relaxation of supercoiled DNA through the transient cleavage of one strand of a DNA duplex and is fundamental to processes such as replication, recombination, and transcription (1-4). The threedimensional structure of reconstituted and N-terminal truncated versions of human topoisomerase I (hTop1p) in complex with a 22-bp DNA molecule have shown that the enzyme is organized in multiple domains that "clamp" around the DNA molecule (5, 6). Changes in DNA topology are achieved by introducing a transient break in the phosphodiester bond of one strand in the duplex DNA. The phosphodiester bond energy is preserved during catalysis through the formation of a transient covalent phosphotyrosine bond between the catalytic Tyr-723 and the 3Ј end of the broken DNA strand. DNA relaxation has been proposed to proceed via "controlled rotation" in which the covalently bound enzyme holds one end of the DNA duplex and allows the end downstream of the cleavage site to rotate around the remaining phosphodiester bond (6). Eukaryotic Top1p is the cellular target of the anti-tumor drug camptothecin (CPT), which reversibly stabilizes the cleavable intermediate complex formed in the catalytic cycle of the enzyme (1, 2, 7).Recently Staker et al. (8) have solved the x-ray crystal structure of hTop1p covalently joined to double-stranded DNA and bound to the CPT analog Topotecan (8). This structure revealed that the drug molecule intercalates between upstream (Ϫ1) and downstream (ϩ1) bp, displacing the downstream DNA and thus preventing the religation of the cleaved strand. The structure helps to clarify the role of several, but not all, mutant residues that produce a CPT-resistant enzyme. In particular the structure permits to explain the CPT resistance for mutations involving residues that interact directly with the drug or that would alter the interaction with DNA...

Lactoferrin (Lf) is a cationic glycoprotein synthetized by exocrine glands and is present in all human secretions. It is also secreted by neutrophils in infection and inflammation sites. This glycoprotein possesses antimicrobial activity due to its capability to chelate two ferric ions per molecule, as well as to interact with bacterial and viral anionic surface components. The cationic features of Lf bind to cells, protecting the host from bacterial and viral injuries. Its anti-inflammatory activity is mediated by the ability to enter inside the nucleus of host cells, thus inhibiting the synthesis of proinflammatory cytokine genes. In particular, Lf down-regulates the synthesis of IL-6, which is involved in iron homeostasis disorders and leads to intracellular iron overload, favoring viral replication and infection. The well-known antiviral activity of Lf has been demonstrated against DNA, RNA, and enveloped and naked viruses and, therefore, Lf could be efficient in counteracting also SARS-CoV-2 infection. For this purpose, we performed in vitro assays, proving that Lf exerts an antiviral activity against SARS-COV-2 through direct attachment to both SARS-CoV-2 and cell surface components. This activity varied according to concentration (100/500 μg/ml), multiplicity of infection (0.1/0.01), and cell type (Vero E6/Caco-2 cells). Interestingly, the in silico results strongly supported the hypothesis of a direct recognition between Lf and the spike S glycoprotein, which can thus hinder viral entry into the cells. These in vitro observations led us to speculate a potential supplementary role of Lf in the management of COVID-19 patients.