A series of organometallic ruthenium(II) complexes containing iminophosphorane ligands have been synthesized and characterized. Cationic compounds with chloride as counterion are soluble in water (70–100 mg/mL). Most compounds (especially highly water-soluble 2) are more cytotoxic to a number of human cancer cell lines than cisplatin. Initial mechanistic studies indicate that the cell death type for these compounds is mainly through canonical or caspase-dependent apoptosis, nondependent on p53, and that the compounds do not interact with DNA or inhibit protease cathepsin B. In vivo experiments of 2 on MDA-MB-231 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (shrinkage) of 56% after 28 days of treatment (14 doses of 5 mg/kg every other day) with low systemic toxicity. Pharmacokinetic studies showed a quick absorption of 2 in plasma with preferential accumulation in the breast tumor tissues when compared to kidney and liver, which may explain its high efficacy in vivo.

The cytoplasmic polyadenylation element-binding protein 3 (CPEB3), is an RNA-binding protein which in its soluble state is localized in membraneless neuronal RNA granules keeping target mRNAs in a repressed state. The stimulus-dependent aggregation of CPEB3 activates target mRNAs translation, a central event for the maintenance of long-term memory-related synaptic plasticity in mammals. To date, the molecular determinants that govern both connected events remain unclear. Here, to gain insight into these processes, the biophysical properties of the human CPEB3 (hCPEB3) are characterized. We found that hCPEB3 homotypic condensation is mainly driven by hydrophobic interactions and occurs under physiological conditions. Moreover, hCPEB3 biomolecular condensates are dynamic inside living cells, whose localization and stabilization are mediated by its RNA-recognition domains.In contrast, the hCPEB3 polar N-terminal region is crucial for hCPEB3 amyloid-like aggregation in vitro, which is disrupted by the polyglutamine binding peptide 1 (QBP1), Aβ42 seeds and Hsp70, highlighting the importance of the Q4RQ4 tract as well as the hydrophobic residues for hCPEB3 functional aggregation. Based on these findings, we postulate a model for hCPEB3's role in memory persistence that advances a rather sophisticated control for hCPEB3 condensate dissociation and amyloid-like formation to achieve its physiological function.hCPEB3's demixing and amyloidogenesis Highlights • hCPEB3 forms toxic intermediates that persist longer than in other functional amyloids.• RNA-recognition domains stabilize hCPEB3 granule formation and dynamics.• Different segments within hCPEB3 promote amyloidogenesis and liquid demixing.• hCPEB3 amyloid formation requires both hydrophobic and polyQ segments. Graphical AbstracthCPEB3's demixing and amyloidogenesis

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Background Current understanding of the molecular basis of memory consolidation points to an important function of amyloid formation by neuronal-specific isoforms of the cytoplasmic polyadenylation element binding (CPEB) protein family. In particular, CPEB is thought to promote memory persistence through formation of self-sustaining prion-like amyloid assemblies at synapses, mediated by its intrinsically disordered region (IDR) and leading to permanent physical alterations at the basis of memory persistence. Although the molecular mechanisms by which amyloid formation takes place in CPEB have been described in invertebrates, the way amyloid formation occurs in the human homolog CPEB3 (hCPEB3) remains unclear. Here, we characterize by NMR spectroscopy the atomic level conformation and ps-ms dynamics of the 426-residue IDR of hCPEB3, which has been associated with episodic memory in humans. Results We show that the 426-residue N-terminal region of hCPEB3 is a dynamic, intrinsically disordered region (IDR) which lacks stable folded structures. The first 29 residues, M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical + disordered motif, and residues 86–93: P83QQPPPP93, and 166–175: P166PPPAPAPQP175 form polyproline II (PPII) helices. The (VG)5 repeat motif is completely disordered, and residues 200–250 adopt three partially populated α-helices. Residues 345–355, which comprise the nuclear localization signal (NLS), form a modestly populated α-helix which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids. Conclusion Our NMR spectroscopic analysis of hCPEB3 provides insights into the first structural transitions involved in protein–protein and protein-mRNA interactions. The atomic level understanding of these structural transitions involved in hCPEB3 aggregation is a key first step toward understanding memory persistence in humans, as well as sequence features that differentiate beneficial amyloids from pathological ones. Areas Biophysics, Structural Biology, Biochemistry & Neurosciences.

Highlights:· Human CPEB3's aggregation-prone disordered region is studied by NMR. · In the monomeric state, the polyQ stretch is disordered and flexible. · 5 a-helices, 2 PPII helices and a rigid nonpolar stretch are identified. · Association of the first 4 a-helices could promote functional amyloid formation. · The PPII helices could negatively regulate this process. · The last a-helix forms the NES and putatively regulates nucleocytoplasmic transport via STAT5B. AbstractAmyloids play key pathological roles in a score of neurodegenerative diseases, but they are also essential to several physiological processes. Perhaps the most fascinating functional amyloid is formed by the Cytoplasmic Polyadenylation Element Binding protein (CPEB) protein family as its structural conversion is essential for memory consolidation in different animals. In vitro, CPEB amyloid formation is driven by the intrinsically disordered region (IDR) present in neuronal-specific isoforms. However, the underlying conformational transitions from the monomeric state to the amyloid state remain poorly understood. Here, we characterize the residue level conformational preferences and ps-ns dynamics for human CPEB3 (hCPEB3) by high field, heteronuclear NMR spectroscopy. At 426 residues, this is the second longest IDR characterized to date in such detail.We find that the residues 1-29: M1QDDLLMDKSKTQPQPQQQQRQQQQQPQP29, adopt an a-helical+disordered Qrich motif. Similar helix + Q/N-rich motifs are observed in CPEB homologs as well as other RNA-binding proteins like TDP-43 that form pathological amyloids. Residues 86-93: P83QQPPPP93, and residues 166-175: P166PPPAPAPQP175 form polyproline-II (PPII) helices, and we propose NMR chemical shift-based criteria for identifying this conformation. We advance that the presence of helix and amyloid breaker residues, such as proline, in hCPEB3 and its absence in TDP-43 may be a key difference between the functional and pathological amyloids in higher eukaryotes. While the (VG)5 repeat motif (residues 272-282) appears to be completely disordered, residues S221-A235 form a highly populated, rather rigid a-helix and two nearby segments adopt partially populated a-helices. Residues 345-355 also form a partially populated a-helix. These residues comprise the nuclear export signal and border a putative phosphoTyr site which may mediate STAT5B binding. Thus, nascent hCPEB3 protein is not fully disordered like a blank sheet of paper; instead, it contains creases which guide the engraving of our memories. Based on these findings and previous results, a working model for hCPEB3 structural transitions in human memory consolidation is advanced.