RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact of a solution containing a ...mixture of both ion types on RNA folding has remained a challenging problem for decades. By exploiting the large concentration difference between divalent and monovalent ions used in experiments, we develop a theory based on the reference interaction site model (RISM), which allows us to treat divalent cations explicitly while keeping the implicit screening effect due to monovalent ions. Our theory captures both the inner shell and outer shell coordination of divalent cations to phosphate groups, which we demonstrate is crucial for an accurate calculation of RNA folding thermodynamics. The RISM theory for ion–phosphate interactions when combined with simulations based on a transferable coarse-grained model allows us to predict accurately the folding of several RNA molecules in a mixture containing monovalent and divalent ions. The calculated folding free energies and ion-preferential coefficients for RNA molecules (pseudoknots, a fragment of the rRNA, and the aptamer domain of the adenine riboswitch) are in excellent agreement with experiments over a wide range of monovalent and divalent ion concentrations. Because the theory is general, it can be readily used to investigate ion and sequence effects on DNA properties.
Vestigial-like 3 (VGLL3) is a member of the VGLL family, whose members serve as cofactors for TEA domain–containing transcription factors (TEADs). TEADs promote tissue and tumor development together ...with the cofactors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Although VGLL3 is involved in tumor cell proliferation, its relationship with TEADs and YAP/TAZ remains largely unknown. To close this research gap, here we established tumor cells stably expressing VGLL3 and found that they exhibit enhanced proliferation. Notably, YAP and TAZ were inactivated in the VGLL3-expressing cells, coinciding with activation of the Hippo pathway, which suppresses YAP/TAZ activities. VGLL3 in combination with TEADs promoted expression of the Hippo pathway components large tumor suppressor kinase (LATS2) and angiomotin-like 2 (AMOTL2). VGLL3 was highly expressed in malignant breast tumor cells and osteosarcoma cells, and VGLL3 knockdown increased nuclear localization of YAP and TAZ. Knockdown of LATS2 or AMOTL2, as well as VGLL3 knockdown, repressed proliferation of breast tumor cells. Together, these results suggest that VGLL3 together with TEADs promotes cell proliferation by activating the Hippo pathway through LATS2 and AMOTL2, leading to YAP/TAZ inactivation.
Vestigial‐like family member 3 (VGLL3) is a member of the VGLL family that serves as cofactors for TEA‐domain transcription factors. Although VGLL3 is involved in the proliferation of cancer cells, ...the molecular mechanisms underlying VGLL3‐mediated cell proliferation remain largely unknown. In this study, we found that stable expression of VGLL3 in human lung cancer A549 cells affects glutamine metabolism and increases their dependency on de novo nucleotide synthesis for proliferation. Mechanistically, VGLL3 was found to induce the expression of GART, which encodes a trifunctional enzyme that catalyzes de novo purine synthesis from glutamine. GART knockdown and the glycinamide ribonucleotide synthase, aminoimidazole ribonucleotide synthase, and glycinamide ribonucleotide formyltransferase trifunctional protein (GART) inhibitor lometrexol repressed the proliferation and survival of A549 cells stably expressing VGLL3. Mesenchymal breast cancer BT549 cells and MDA‐MB‐231 cells showed high expression of VGLL3, and VGLL3 knockdown was found to reduce GART expression. Lometrexol also repressed the proliferation of these breast cancer cells, whereas addition of inosine monophosphate, an important metabolite downstream of GART, rescued this repression. Taken together, these results suggest that VGLL3 induces GART expression and thereby confers de novo nucleotide‐dependent cell proliferation in cancer cells.
How ions affect RNA folding thermodynamics and kinetics is an important but a vexing problem that remains unsolved. Experiments have shown that the free-energy change, ΔG(c), of RNA upon folding ...varies with the salt concentration (c) as, ΔG(c) = k c ln c + const, where the coefficient k c is proportional to the difference in the ion preferential coefficient, ΔΓ. We performed simulations of a coarse-grained model, by modeling electrostatic interactions implicitly and with explicit representation of ions, to elucidate the molecular underpinnings of the relationship between ΔG and ΔΓ. The simulations quantitatively reproduce the heat capacity for a pseudoknot, thus validating the model. We show that ΔG(c), calculated directly from ΔΓ, varies linearly with ln c (c < 0.2 M), for a hairpin and the pseudoknot, demonstrating a molecular link between the two quantities. Explicit ion simulations also show the linear dependence of ΔG(c) on ln c at all c with k c = 2k B T, except that ΔG(c) values are shifted by ∼2 kcal/mol higher than experiments. The discrepancy is due to an underestimation of Γ for both the folded and unfolded states while giving accurate values for ΔΓ. The predictions for the salt dependence of ΔΓ are amenable to test using single-molecule pulling experiments. The framework provided here can be used to obtain accurate thermodynamics for other RNA molecules as well.
Factors that cause nonuniformity in the luminescence lifetime of pressure-sensitive paints (PSPs) were investigated. The lifetime imaging method of PSP does not theoretically require wind-off ...reference images. Therefore, it can improve measurement accuracy because it can eliminate errors caused by the deformation or movement of the model during the measurement. However, it is reported that the luminescence lifetime of PSP is not uniform on the model, even under uniform conditions of pressure and temperature. Therefore, reference images are used to compensate for the nonuniformity of the luminescence lifetime, which significantly diminishes the advantages of the lifetime imaging method. In particular, fast-responding PSPs show considerable variation in luminescence lifetime compared to conventional polymer-based PSPs. Therefore, this study investigated and discussed the factors causing the nonuniformity of the luminescence lifetime, such as the luminophore solvent, luminophore concentrations, binder thickness, and spraying conditions. The results obtained suggest that the nonuniformity of the luminophore distribution in the binder caused by the various factors mentioned above during the coating process is closely related to the nonuniformity of the luminescence lifetime. For example, when the thickness of the binder became thinner than 8 μm, the fast-responding PSPs showed a tendency to vary significantly in the luminescence lifetime. In addition, it was found that the luminescence lifetime of fast-responding PSP could be changed in the depth direction of the binder depending on the coating conditions. Therefore, it is important to distribute the luminophore uniformly in the binder layer to create PSPs with a more uniform luminescence lifetime distribution.
Colorectal cancer (CRC) cells harboring KRAS or BRAF mutations show a more-malignant phenotype than cells with wild-type KRAS and BRAF. KRAS/BRAF-wild-type CRCs are sensitive to epidermal growth ...factor receptor (EGFR)-targeting agents, whereas KRAS/BRAF-mutant CRCs are resistant due to constitutive activation of the EGFR-downstream KRAS/BRAF signaling pathway. Novel therapeutic strategies to treat KRAS/BRAF mutant CRC cells are thus needed. We recently demonstrated that the telomerase-specific replication-competent oncolytic adenoviruses OBP-301 and p53-armed OBP-702 exhibit therapeutic potential against KRAS-mutant human pancreatic cancer cells. In this study, we evaluated the therapeutic potential of OBP-301 and OBP-702 against human CRC cells with differing KRAS/BRAF status. Human CRC cells with wild-type KRAS/BRAF (SW48, Colo320DM, CACO-2), mutant KRAS (DLD-1, SW620, HCT116), and mutant BRAF (RKO, HT29, COLO205) were used in this study. The antitumor effect of OBP-301 and OBP-702 against CRC cells was analyzed using the XTT assay. Virus-mediated modulation of apoptosis, autophagy, and the EGFR-MEK-ERK and AKT-mTOR signaling pathways was analyzed by Western blotting. Wild-type and KRAS-mutant CRC cells were sensitive to OBP-301 and OBP-702, whereas BRAF-mutant CRC cells were sensitive to OBP-702 but resistant to OBP-301. Western blot analysis demonstrated that OBP-301 induced autophagy and that OBP-702 induced autophagy and apoptosis in human CRC cells. In BRAF-mutant CRC cells, OBP-301 and OBP-702 suppressed the expression of EGFR, MEK, ERK, and AKT proteins, whereas mTOR expression was suppressed only by OBP-702. Our results suggest that p53-armed oncolytic virotherapy is a viable therapeutic option for treating KRAS/BRAF-mutant CRC cells via induction of autophagy and apoptosis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
For simulating proteins at work in millisecond time scale or longer, we develop a coarse-grained (CG) molecular dynamics (MD) method and software, CafeMol. At the resolution of ...one-particle-per-residue, CafeMol equips four structure-based protein models: (1) the off-lattice Go model, (2) the atomic interaction based CG model for native state and folding dynamics, (3) the multiple-basin model for conformational change dynamics, and (4) the elastic network model for quasiharmonic fluctuations around the native structure. Ligands can be treated either explicitly or implicitly. For mimicking functional motions of proteins driven by some external force, CafeMol has various and flexible means to “switch” the energy functions that induce active motions of the proteins. CafeMol can do parallel computation with modest sized PC clusters. We describe CafeMol methods and illustrate it with several examples, such as rotary motions of F1-ATPase and drug exports from a transporter. The CafeMol source code is available at www.cafemol.org.
Co-translational folding (CTF) facilitates correct folding in vivo, but its precise mechanism remains elusive. For the CTF of a three-domain protein SufI, it was reported that the translational ...attenuation is obligatory to acquire the functional state. Here, to gain structural insights on the underlying mechanisms, we performed comparative molecular simulations of SufI that mimic CTF as well as refolding schemes. A CTF scheme that relied on a codon-based prediction of translational rates exhibited folding probability markedly higher than that by the refolding scheme. When the CTF schedule is speeded up, the success rate dropped. These agree with experiments. Structural investigation clarified that misfolding of the middle domain was much more frequent in the refolding scheme than that in the codon-based CTF scheme. The middle domain is less stable and can fold via interactions with the folded N-terminal domain. Folding pathway networks showed the codon-based CTF gives narrower pathways to the native state than the refolding scheme.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
RNA and RNA–protein complexes have recently been intensively studied in experiments, but the corresponding molecular simulation work is much less abundant, primarily due to its large system size and ...the long time scale involved. Here, to overcome these bottlenecks, we develop a coarse-grained (CG) structure-based simulation model for RNA and RNA–protein complexes and test it for several molecular systems. The CG model for RNA contains three particles per nucleotide, each for phosphate, sugar, and a base. Focusing on RNA molecules that fold to well-defined native structures, we employed a structure-based potential, which is similar to the Go-like potential successfully used in CG modeling of proteins. In addition, we tested three means to approximate electrostatic interactions. Many parameters involved in the CG potential were determined via a multiscale method: We matched the native fluctuation of the CG model with that by all-atom simulations for 16 RNA molecules and 10 RNA–protein complexes, from which we derived a generic set of CG parameters. We show that the derived parameters can reproduce native fluctuations well for four RNA and two RNA–protein complexes. For tRNA, the native fluctuation in solution includes large-amplitude motions that reach conformations nearly corresponding to the hybrid state P/E and EF-Tu-bound state A/T seen in the complexes with ribosome. Finally, large-amplitude modes of ribosome are briefly described.
Accurate simulation of urban snow accumulation/melting processes is important to provide reliable information about climate change in snowy urban areas. The Japan Meteorological Agency operates a ...square prism urban canopy (SPUC) model within their regional model to simulate the urban atmosphere. However, presently, this model takes no account of snow processes. Therefore, in this study, we enhanced the SPUC by introducing a snowpack scheme, and assessed the simulated snow over Japanese urban areas by comparing the snow depths from the enhanced SPUC and those from a simple biosphere (iSiB) model with the observations. Snowpack schemes based on two approaches were implemented. The diagnostic approach (sSPUCdgn) uses empirical factors for snow temperature and melting/freezing amounts and the Penman equation for heat fluxes, whereas the prognostic approach (sSPUCprg) calculates snow temperatures using heat fluxes estimated from bulk equations. Both snowpack schemes enabled the model to accurately reproduce the seasonal variations and peaks in snow depth, but it is necessary to use sSPUCprg if we wish to consider the physical processes in the snow layer. Compared to iSiB, sSPUCprg resulted in a good performance for the seasonal variations in snow depth and the error fell to 20 %. While iSiB overestimated the snow depth, a cold bias of over 1°C appeared in the daily mean temperature, which can be attributed to excessive decreases in the snow surface temperature. sSPUCprg reduces the bias by a different calculation method for the snow surface temperature and by including heated building walls without snow; consequently, the simulated snow depth is improved. With an increase in the correlation coefficient, sSPUCprg generated a relationship between the seasonal variations in snowfall and snow depth close to the observed relationship. Therefore, the simulation accuracy of snowfall becomes more crucial for simulating the surface snow processes precisely by using the enhanced SPUC.
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