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Summary: The status of spindle checkpoint signaling depends on the balance of two opposing dynamic processes that regulate the highly unusual two-state behavior of Mad2. In mitosis, a Mad1-Mad2 core complex recruits cytosolic Mad2 to kinetochores through Mad2 dimerization and converts Mad2 to a conformer amenable to Cdc20 binding, thereby facilitating checkpoint activation. p31comet inactivates the checkpoint through binding to Mad1- or Cdc20-bound Mad2, thereby preventing Mad2 activation and promoting the dissociation of the Mad2-Cdc20 complex. Here, we report the crystal structure of the Mad2-p31comet complex. The C-terminal region of Mad2 that undergoes rearrangement in different Mad2 conformers is a major structural determinant for p31comet binding, explaining the specificity of p31comet toward Mad1- or Cdc20-bound Mad2. p31comet adopts a fold strikingly similar to that of Mad2 and binds at the dimerization interface of Mad2. Thus, p31comet exploits the two-state behavior of Mad2 to block its activation by acting as an “anti-Mad2.” [Copyright &y& Elsevier]

In this study, we discuss the tunability of valley splitting using first-principles calculations with a monolayer MoTe 2 and layered ferromagnetic MnS 2 heterostructure as an example. We observe that, due to the magnetic proximity effect (MPE) at the interface, a monolayer of MoTe 2 can exhibit a significant valley splitting of 55.2 meV. The production of the interlayer dipoles with spin-adapted configuration could be the origin of MPE at the interface. Furthermore, the valley splitting can be regulated continuously by the perpendicular electric field and biaxial strain. Interestingly, the valley splitting increases with the increasing induced magnetic moments in MoTe 2 by applying an electric field while the inverse laws are presented by applying biaxial strains, which indicates that the mechanisms of valley splitting manipulating in these two ways are quite different. The calculation results suggest that the electric field influences the electric dipole distributions at the interface, which determines the induced magnetic moments in monolayer MoTe 2 , and results in valley splitting variations. However, biaxial strains not only affect MPE at the interface but also the intrinsic spin splitting caused by spin-orbital coupling (SOC) effects of monolayer MoTe 2 itself and the latter is even the dominating mechanism of valley splitting variations., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Increased expression of TGFB regulatory factors DNMT3A and DNMT3B in non-neoplastic liver tissues of HCC patients is the goal of this study. Furthermore, we demonstrate that TGF- is capable of elevating the percentage of CD133+ cells present in liver cancer cell lines in a manner that is both consistent and long-lasting over several cell divisions. This process is linked to stable alterations in DNA methylation that occur over the whole of the genome and continue even after cell division. In addition, the silencing of de novo DNA methyl-transferases with siRNA is able to inhibit the phenotypic changes that are induced by TGF-. According to the findings of our research, there is a self-sustaining interaction between the DNA methylation machinery and the TGF- signaling pathway, which may be significant in the development of cellular phenotypes. CD133 positive and negative fractions expand within liver cancer cell lines in proportions that remain stable throughout time. In contrast to their CD133- counterparts, MACS-sorted CD133+ Huh7cells demonstrated the ability to shape themselves into spheres when grown under non-attachment circumstances. This study also found that the TGF- is responsible for the de novo induction of CD133, which is linked to an increase in the expression of DNMT3 genes and there is a correlation between the TGF-induced transition in the cell subpopulation and a distinct DNA methylome. TGF- has the potential to generate genome-wide alterations in DNA methylation, which ultimately leads to a persistent shift in the fraction of liver cancer cell subpopulations.

The Hippo pathway controls organ size and maintains tissue homeostasis through a central MST-LATS kinase cascade. When Hippo signaling is on, activated MST1/2 partner with SAV1 to phosphorylate and activate the LATS1/2-MOB1 complexes, which in turn phosphorylate and inactivate YAP/TAZ transcription co-activators. This process halts the expression of Hippo-responsive genes, thereby inhibiting cell proliferation and promoting apoptosis. Our studies have shown that two core adaptor proteins MOB1 and SAV1 use distinctive mechanisms to enhance Hippo signaling. MOB1 promotes MST-dependent LATS activation through dynamic scaffolding and allosteric regulation. SAV1 promotes MST activation by antagonizing the PP2A phosphatase activity. Here we describe the detailed methods for the purification and crystallization of the MST2-SAV1 and pMOB1-LATS1 complexes, for assaying the SAV1-dependent inhibition of PP2A, and for analyzing LATS1 kinase activation using in vitro reconstitution.

Classical swine fever virus (CSFV) is capable of counteracting innate cellular antiviral responses by inhibiting type I interferon (IFN)-alpha/beta induction. A function associated with CSFV N(pro), with respect to the inhibition of IFN-beta production, has been clearly elucidated. In this study, we explored the role of CSFV E(rns) in IFN-beta induction by exogenous double-stranded (ds) RNA. Synthetic dsRNA (poly (IC)) was used as an exogenous stimulus to trigger IFN-beta induction. CSFV E(rns) inhibited IFN-beta promoter-driven luciferase activity induced by poly (IC) in different cell lines, and the inhibitory effect was dose-dependent. Moreover, E(rns) reduced IFN-beta mRNA synthesis and blocked IFN-alpha/beta production induced by poly (IC), suggesting that this inhibition occurs at the transcriptional level. Furthermore, E(rns) counteracted poly (IC)-mediated IFN-beta induction independent of its ribonuclease activity. In conclusion, CSFV E(rns) antagonizes extracellular dsRNA-mediated IFN-beta expression. These findings contribute to our understanding of the pathogenesis of CSFV.

To maintain the fidelity of chromosome inheritance, cells utilize a surveillance mechanism called the spindle checkpoint to sense improper attachment of sister chromatids to the mitotic spindle prior to chromosome segregation. The target of the spindle checkpoint is a ubiquitin ligase called the anaphase-promoting complex or cyclosome (APC/C). The spindle checkpoint protein Mad2 inhibits the activity of APC/C through direct binding to its activator Cdc20. Studies have shown that Mad2 has two distinct natively folded conformations and that the unusual two-state behavior of Mad2 plays a crucial role in checkpoint signaling. This article describes methods for the purification of the two Mad2 conformers and for the analysis of their activities in APC/C inhibition in Xenopus egg extracts.

To better understand origin recognition and initiation of DNA replication, we have examined by NMR complexes formed between the origin-binding domain of SV40 T antigen (T-ag-obd), the initiator protein of the SV40 virus, and cognate and noncognate DNA oligomers. The results reveal two structural effects associated with "origin-specific" binding that are absent in nonspecific DNA binding. The first is the formation of a hydrogen bond (H-bond) involving His 203, a residue that genetic studies have previously identified as crucial to both specific and nonspecific DNA binding in full-length T antigen. In free T-ag-obd, the side chain of His 203 has a pK(a) value of approximately 5, titrating to the N(epsilon)(1)H tautomer at neutral pH (Sudmeier, J. L., et al. (1996) J. Magn. Reson., Ser. B 113, 236-247). In complexes with origin DNA, His 203 N(delta)(1) becomes protonated and remains nontitrating as the imidazolium cation at all pH values from 4 to 8. The H-bonded N(delta1)H resonates at 15.9 ppm, an unusually large N-H proton chemical shift, of a magnitude previously observed only in the catalytic triad of serine proteases at low pH. The formation of this H-bond requires the middle G/C base pair of the recognition pentanucleotide, GAGGC. The second structural effect is a selective distortion of the A/T base pair characterized by a large (0.6 ppm) upfield chemical-shift change of its Watson-Crick proton, while nearby H-bonded protons remain relatively unaffected. The results indicate that T antigen, like many other DNA-binding proteins, may employ "catalytic" or "transition-state-like" interactions in binding its cognate DNA (Jen-Jacobson, L. (1997) Biopolymers 44, 153-180), which may be the solution to the well-known paradox between the relatively modest DNA-binding specificity exhibited by initiator proteins and the high specificity of initiation.