Adaptive torsion-angle quasi-statics: a general simulation method with applications to protein structure analysis and design

MOTIVATION: The cost of molecular quasi-statics or dynamics simulations increases with the size of the simulated systems, which is a problem when studying biological phenomena that involve large molecules over long time scales. To address this problem, one has often to either increase the processing power (which might be expensive), or make arbitrary simplifications to the system (which might bias the study). RESULTS: We introduce adaptive torsion-angle quasi-statics, a general simulation method able to rigorously and automatically predict the most mobile regions in a simulated system, under user-defined precision or time constraints. By predicting and simulating only these most important regions, the adaptive method provides the user with complete control on the balance between precision and computational cost, without requiring him or her to perform a priori, arbitrary simplifications. We build on our previous research on adaptive articulated-body simulation and show how, by taking advantage of the partial rigidification of a molecule, we are able to propose novel data structures and algorithms for adaptive update of molecular forces and energies. This results in a globally adaptive molecular quasi-statics simulation method. We demonstrate our approach on several examples and show how adaptive quasi-statics allows a user to interactively design, modify and study potentially complex protein structures.     

Cdc20 hypomorphic mice fail to counteract de novo synthesis of cyclin B1 in mitosis

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome-microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element-binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.     

Overexpression of the E2 ubiquitin–conjugating enzyme UbcH10 causes chromosome missegregation and tumor formation

The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin–conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome. UbcH10 is overexpressed in many human cancer types and is associated with tumor progression. However, whether UbcH10 overexpression causes tumor formation is unknown. To address this central question and to define the molecular and cellular consequences of UbcH10 overexpression, we generated a series of transgenic mice in which UbcH10 was overexpressed in graded fashion. In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy. Importantly, we find that UbcH10 transgenic mice are prone to carcinogen-induced lung tumors and a broad spectrum of spontaneous tumors. Our results identify UbcH10 as a prominent protooncogene that causes whole chromosome instability and tumor formation over a wide gradient of overexpression levels.     

Cdc20 Is Critical for Meiosis I and Fertility of Female Mice

Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.     

Ran-dependent docking of importin-beta to RanBP2/Nup358 filaments is essential for protein import and cell viability

RanBP2/Nup358, the major component of the cytoplasmic filaments of the nuclear pore complex (NPC), is essential for mouse embryogenesis and is implicated in both macromolecular transport and mitosis, but its specific molecular functions are unknown. Using RanBP2 conditional knockout mouse embryonic fibroblasts and a series of mutant constructs, we show that transport, rather than mitotic, functions of RanBP2 are required for cell viability. Cre-mediated RanBP2 inactivation caused cell death with defects in M9- and classical nuclear localization signal (cNLS)-mediated protein import, nuclear export signal-mediated protein export, and messenger ribonucleic acid export but no apparent mitotic failure. A short N-terminal RanBP2 fragment harboring the NPC-binding domain, three phenylalanine-glycine motifs, and one Ran-binding domain (RBD) corrected all transport defects and restored viability. Mutation of the RBD within this fragment caused lethality and perturbed binding to Ran guanosine triphosphate (GTP)-importin-β, accumulation of importin-β at nuclear pores, and cNLS-mediated protein import. These data suggest that a critical function of RanBP2 is to capture recycling RanGTP-importin-β complexes at cytoplasmic fibrils to allow for adequate cNLS-mediated cargo import.     

A novel copper (II) PAmPiCaT complex (cPAmPiCaTc) as a biologically potent candidate: A contraption evidence against methicillin-resistant Staphylococcus aureus (MRSA) and a molecular docking proof

Increasing in the alarm against the resistant bacteria due to the failure of antibiotics, thereby the need of more efficiency/potent molecule to treat infections. In the present investigation, series of piperazine derivatives 5(a-l) compounds were synthesized and they were characterised by different spectral techniques such as ¹H NMR, ¹³C NMR, IR and LCMS. A novel copper complex (cPAmPiCaTc) was developed for the first time by using potent analog 5e and characterized by IR and LCMS. The cPAmPiCaTc evaluated for antibacterial activity and showed excellent antimicrobial effect (12?±?0.08?mm, ZOI) at MIC 20?µg/mL against MRSA compared to standard antibiotics streptomycin and bacitracin at MIC 10?µg/mL. The results show promising anti-staphylococcal action against MRSA which confirmed by membrane damage, bioelectrochemistry, gene regulation (SarA and DHFR), and in silico molecular docking studies. Further, the cPAmPiCaTc also showed excellent blood compatibility and this result pave the way for interesting metallodrug therapeutics in future against MRSA infections.     

Intracellular Endothelin Type B Receptor-driven Ca2+ Signal Elicits Nitric Oxide Production in Endothelial Cells

Endothelin-1 exerts its actions via activation of ET_A and ET_B G_q/11 protein-coupled receptors, located in the plasmalemma, cytoplasm, and nucleus. Although the autocrine/paracrine nature of endothelin-1 signaling has been extensively studied, its intracrine role has been largely attributed to interaction with receptors located on nuclear membranes and the nucleoplasm. Because ET_B receptors have been shown to be targeted to endolysosomes, we used intracellular microinjection and concurrent imaging methods to test their involvement in Ca²⁺ signaling and subsequential NO production. We provide evidence that microinjected endothelin-1 produces a dose-dependent elevation in cytosolic calcium concentration in ET_B-transfected cells and endothelial cells; this response is sensitive to ET_B but not ET_A receptor blockade. In endothelial cells, the endothelin-1-induced Ca²⁺ response is abolished upon endolysosomal but not Golgi disruption. Moreover, the effect is prevented by inhibition of microautophagy and is sensitive to inhibitors of the phospholipase C and inositol 1,4,5-trisphosphate receptor. Furthermore, intracellular endothelin-1 increases nitric oxide via an ET_B-dependent mechanism. Our results indicate for the first time that intracellular endothelin-1 activates endolysosomal ET_B receptors and increase cytosolic Ca²⁺ and nitric oxide production. Endothelin-1 acts in an intracrine fashion on endolysosomal ET_B to induce nitric oxide formation, thus modulating endothelial function.     

Integration of related sequences with protein three-dimensional structural families in an updated version of PALI database

The database of Phylogeny and ALIgnment of homologous protein structures (PALI) contains three-dimensional (3-D) structure-dependent sequence alignments as well as structure-based phylogenetic trees of protein domains in various families. The latest updated version (Release 2.1) comprises of 844 families of homologous proteins involving 3863 protein domain structures with each of these families having at least two members. Each member in a family has been structurally aligned with every other member in the same family using two proteins at a time. In addition, an alignment of multiple structures has also been performed using all the members in a family. Every family with at least three members is associated with two dendrograms, one based on a structural dissimilarity metric and the other based on similarity of topologically equivalenced residues for every pairwise alignment. Apart from these multi-member families, there are 817 single member families in the updated version of PALI. A new feature in the current release of PALI is the integration, with 3-D structural families, of sequences of homologues from the sequence databases. Alignments between homologous proteins of known 3-D structure and those without an experimentally derived structure are also provided for every family in the enhanced version of PALI. The database with several web interfaced utilities can be accessed at: http://pauling.mbu.iisc.ernet.in/~pali.