Characterization of an RNase with two catalytic centers. Human RNase6 catalytic and phosphate-binding site arrangement favors the endonuclease cleavage of polymeric substrates

Background

Human RNase6 is a small cationic antimicrobial protein that belongs to the vertebrate RNaseA superfamily. All members share a common catalytic mechanism, which involves a conserved catalytic triad, constituted by two histidines and a lysine (His15/His122/Lys38 in RNase6 corresponding to His12/His119/Lys41 in RNaseA). Recently, our first crystal structure of human RNase6 identified an additional His pair (His36/His39) and suggested the presence of a secondary active site.

Mechanically stimulated osteocytes reduce the bone-metastatic potential of breast cancer cells in vitro by signaling through endothelial cells

Bone metastases occur in 65% to 75% of patients with advanced breast cancer and significantly worsen their survival and quality of life. We previously showed that conditioned medium (CM) from osteocytes stimulated with oscillatory fluid flow, mimicking bone mechanical loading during routine physical activities, reduced the transendothelial migration of breast cancer cells. Endothelial cells are situated at an ideal location to mediate signals between osteocytes in the bone matrix and metastasizing cancer cells in the blood vessels. In this study, we investigated the specific effects of flow-stimulated osteocytes on the interaction between endothelial cells and breast cancer cells in vitro. We observed that CM from flow-stimulated osteocytes reduced endothelial permeability by 15% and breast cancer cell adhesion onto endothelial monolayers by 18%. The difference in adhesion was abolished with anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing antibodies. Furthermore, CM from endothelial cells conditioned in CM from flow-stimulated osteocytes significantly altered the gene expression in bone-metastatic breast cancer cells, as shown by RNA sequencing. Specifically, breast cancer cell expression of matrix metallopeptidase 9 (MMP-9) was downregulated by 62%, and frizzled-4 (FZD4) by 61%, when the osteocytes were stimulated with flow. The invasion of these breast cancer cells across Matrigel was also reduced by 47%, and this difference was abolished by MMP-9 inhibitors. In conclusion, we demonstrated that flow-stimulated osteocytes downregulate the bone-metastatic potential of breast cancer cells by signaling through endothelial cells. This provides insights into the capability of bone mechanical regulation in preventing bone metastases; and may assist in prescribing exercise or bone-loading regimens to patients with breast cancers.     

Dynamic assembly of the mRNA m6A methyltransferase complex is regulated by METTL3 phase separation

m⁶A methylation is the most abundant and reversible chemical modification on mRNA with approximately one-fourth of eukaryotic mRNAs harboring at least one m⁶A-modified base. The recruitment of the mRNA m⁶A methyltransferase writer complex to phase-separated nuclear speckles is likely to be crucial in its regulation; however, control over the activity of the complex remains unclear. Supported by our observation that a core catalytic subunit of the methyltransferase complex, METTL3, is endogenously colocalized within nuclear speckles as well as in noncolocalized puncta, we tracked the components of the complex with a Cry2-METTL3 fusion construct to disentangle key domains and interactions necessary for the phase separation of METTL3. METTL3 is capable of self-interaction and likely provides the multivalency to drive condensation. Condensates in cells necessarily contain myriad components, each with partition coefficients that establish an entropic barrier that can regulate entry into the condensate. In this regard, we found that, in contrast to the constitutive binding of METTL14 to METTL3 in both the diffuse and the dense phase, WTAP only interacts with METTL3 in dense phase and thereby distinguishes METTL3/METTL14 single complexes in the dilute phase from METTL3/METTL14 multicomponent condensates. Finally, control over METTL3/METTL14 condensation is determined by its small molecule cofactor, S-adenosylmethionine (SAM), which regulates conformations of two gate loops, and some cancer-associated mutations near gate loops can impair METTL3 condensation. Therefore, the link between SAM binding and the control of writer complex phase state suggests that the regulation of its phase state is a potentially critical facet of its functional regulation.

Approximately one-fourth of eukaryotic mRNAs harbor at least one m6A-modified base, but how is this regulated? This study shows that cells can use liquid-liquid phase separation to regulate dynamic assembly of the mRNA m6A methyltransferase complex (METTL3/METTL14/WTAP), with stoichiometries that depend on condensate partitioning in a substrate binding-dependent manner.     

Control of bovine placental progesterone synthesis: roles of cholesterol availability and calcium-activated systems

It was previously reported that dispersed bovine placentome secretes progesterone and that the steroidogenic activity of these cells is stimulated by a calcium-mediated, cyclic nucleotide independent mechanism. In the present study, the influence of substrate availability was explored and the roles of calmodulin and protein kinase C in progestin production examined. Incubation of dispersed fetal cotyledon cells with 25-hydroxycholesterol (25-OH-C), a soluble sterol which readily enters cells and is metabolized to steroid hormones, increased progesterone secretion in a dose-dependent manner. The response to 25-OH-C was dependent on the extracellular calcium concentration. Methyl isobutyl xanthine (MIX) alone also increased pregnenolone as well as progesterone secretion, and the combination of 25-OH-C and MIX stimulated progesterone secretion was inhibited by trifluoperazine. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused no major effects on steroidogenesis but the stimulatory effects of MIX or the ionophore A23187 were enhanced in its presence. These findings suggest that (1) basal progesterone secretion by fetal cotyledon cells is limited by cholesterol availability; (2) MIX increases steroidogenesis in part by increasing the synthesis of pregnenolone, but its actions are expressed independently of cholesterol availability; (3) both calmodulin and protein kinase C may participate in the modulation of bovine placental steroidogenesis.     

New synaptic bouton formation is disrupted by misregulation of microtubule stability in aPKC mutants

The Baz/Par-3-Par-6-aPKC complex is an evolutionarily conserved cassette critical for the development of polarity in epithelial cells, neuroblasts, and oocytes. aPKC is also implicated in long-term synaptic plasticity in mammals and the persistence of memory in flies, suggesting a synaptic function for this cassette. Here we show that at Drosophila glutamatergic synapses, aPKC controls the formation and structure of synapses by regulating microtubule (MT) dynamics. At the presynapse, aPKC regulates the stability of MTs by promoting the association of the MAP1Brelated protein Futsch to MTs. At the postsynapse, aPKC regulates the synaptic cytoskeleton by controlling the extent of Actin-rich and MT-rich areas. In addition, we show that Baz and Par-6 are also expressed at synapses and that their synaptic localization depends on aPKC activity. Our findings establish a novel role for this complex during synapse development and provide a cellular context for understanding the role of aPKC in synaptic plasticity and memory.