LK-3, an amphipathic dimeric peptide linked by two disulfide bonds, and related isomeric bundles were synthesized, and their cell penetrating abilities were investigated. The measurements using size exclusion chromatography and dynamic light scattering show that LK-3 and its isomers form cell penetrating oligomers. Calculations, performed for various types of peptide isomers, elucidate a strong correlation between the amphipathic character of dimers and cell penetration ability. The results suggest that the amphipathicities of LK-3 and related bundle dimers are responsible for their oligomerization propensities which in turn determine their cell penetrating abilities. The observations made in this study provide detailed information about the mechanism of cell uptake of LK-3 and suggest a plausible insight of the early stage of nanoparticle formation of the cell penetrating amphipathic peptides.

Human ribosomal protein S3 (hRpS3) is a component of the 40S ribosomal subunit that associated in protein synthesis. hRpS3 has additional ribosomal functions such as DNA repair, transcription, metastasis, and apoptosis via interaction with numerous signaling molecules and has different modifications. Cyclin-dependent kinases (CDKs) are heterodimeric serine/threonine protein kinases that regulate cell cycle progression. Among its members, the Cdk1-cyclin B complex is known to control cell progression in the G2/M phase, while Cdk2-cyclin E/A complexes function in G1/S and S/G2 transition. In our previous study, we observed interaction between hRpS3 and Cdk1. The present study investigated the interaction between hRpS3 and Cdk2. Cdk2 phosphorylated hRps3 at amino acid residues S6 and T221 during the S-phase. Furthermore, hRpS3 knockdown delayed cell cycle progression by modulating the expression of cell cycle-related proteins, including cyclin B1 and cyclin E1. These findings suggest that hRpS3 is involved in Cdk2-mediated cell cycle regulation.

Crosstalk between G-protein signaling and glutamatergic transmission within the brain reward circuits is critical for long-term emotional effects (depression and anxiety), cravings, and negative withdrawal symptoms associated with opioid addiction. A previous study showed that Regulator of G-protein signaling 4 (RGS4) may be implicated in opiate action in the nucleus accumbens (NAc). However, the mechanism of the NAc-specific RGS4 actions that induce the behavioral responses to opiates remains largely unknown. The present study used a short hairpin RNA (shRNA)-mediated knock-down of RGS4 in the NAc of the mouse brain to investigate the relationship between the activation of ionotropic glutamate receptors and RGS4 in the NAc during morphine reward. Additionally, the shRNA-mediated RGS4 knock-down was implemented in NAc/striatal primary-cultured neurons to investigate the role that striatal neurons have in the morphine-induced activation of ionotropic glutamate receptors. The results of this study show that the NAc-specific knockdown of RGS4 significantly increased the behaviors associated with morphine and did so by phosphorylation of the GluR1 (Ser831) and NR2A (Tyr1325) glutamate receptors in the NAc. Furthermore, the knock-down of RGS4 enhanced the phosphorylation of the GluR1 and NR2A glutamate receptors in the primary NAc/striatal neurons during spontaneous morphine withdrawal. These findings show a novel molecular mechanism of RGS4 in glutamatergic transmission that underlies the negative symptoms associated with morphine administration.

Absence seizures are caused by abnormal synchronized oscillations in the thalamocortical (TC) circuit, which result in widespread spike-and-wave discharges (SWDs) on electroencephalography (EEG) as well as impairment of consciousness. Thalamic reticular nucleus (TRN) and TC neurons are known to interact dynamically to generate TC circuitry oscillations during SWDs. Clinical studies have suggested the association of Plcβ1 with early-onset epilepsy, including absence seizures. However, the brain regions and circuit mechanisms related to the generation of absence seizures with Plcβ1 deficiency are unknown. In this study, we found that loss of Plcβ1 in mice caused spontaneous complex-type seizures, including convulsive and absence seizures. Importantly, TRN-specific deletion of Plcβ1 led to the development of only spontaneous SWDs, and no other types of seizures were observed. Ex vivo slice patch recording demonstrated that the number of spikes, an intrinsic TRN neuronal property, was significantly reduced in both tonic and burst firing modes in the absence of Plcβ1 . We conclude that the loss of Plcβ1 in the TRN leads to decreased excitability and impairs normal inhibitory neuronal function, thereby disrupting feedforward inhibition of the TC circuitry, which is sufficient to cause hypersynchrony of the TC system and eventually leads to spontaneous absence seizures. Our study not only provides a novel mechanism for the induction of SWDs in Plcβ1 -deficient patients but also offers guidance for the development of diagnostic and therapeutic tools for absence epilepsy.

Human prostaglandin E2 receptor 4 (EP4) is one of the four subtypes of prostaglandin E2 (PGE2) receptors and belongs to the rhodopsin-type G protein-coupled receptor (GPCR) family. Particularly, EP4 is expressed in various cancer cells and is involved in cancer-cell proliferation by a G protein signaling cascade. To prepare an active form of EP4 for biochemical characterization and pharmaceutical application, this study designed a recombinant protein comprising human EP4 fused to the P9 protein (a major envelope protein of phi6 phage) and overexpressed the P9-EP4 fusion protein in the membrane fraction of E. coli. The solubilized P9-EP4 with sarkosyl (a strong anionic detergent) was purified by affinity chromatography. The purified protein was stabilized with amphiphilic polymers derived from poly-γ-glutamate. The polymer-stabilized P9-EP4 showed specific interaction with the alpha subunits of Gs or Gi proteins, and a high content of α-helical structure by a circular dichroism spectroscopy. Furthermore, the polymer-stabilized P9-EP4 showed strong heat resistance compared with P9-EP4 in detergents. The functional preparation of EP4 and its stabilization with amphiphilic polymers could facilitate both the biochemical characterization and pharmacological applications targeting EP4.

Established fear memory becomes vulnerable to disruption after memory retrieval and extinction; this labile state is critical for inhibiting the return of fear memory. However, the labile state has a very narrow time window after retrieval, and underlying molecular mechanisms are not well known. To that end, we isolated the hippocampus immediately after fear memory retrieval and performed proteomics. We identified Neurobeachin (NBEA), an autism-related regulator of synaptic protein trafficking, to be upregulated after contextual fear memory retrieval. NBEA protein expression was rapid and transient after fear memory retrieval at the synapse. Nbea mRNA was enriched at the synapses, and the rapid induction of NBEA expression was blocked by inhibition of the mammalian target of rapamycin (mTOR)-dependent signaling pathway. Mice with cornu ammonis 1 (CA1)-specific Nbea shRNA knockdown showed normal fear acquisition and contextual fear memory but impaired extinction, suggesting an important role of Nbea in fear memory extinction processes. Consistently, Nbea heterozygotes showed normal fear acquisition and fear memory recall but showed impairment in extinction. Our data suggest that NBEA is necessary either for induction of memory lability or for the physiological process of memory extinction.

Human DNA topoisomerase II-binding protein 1 (hTopBP1) plays an important role in DNA replication and the DNA damage checkpoint pathway. The human mutY homolog (hMYH) is a base excision repair DNA glycosylase that excises adenines or 2-hydroxyadenines that are mispaired with guanine or 7,8-dihydro-8-oxoguanine (8-oxoG). hTopBP1 and hMYH were involved in ATR-mediated Chk1 activation, moreover, both of them were associated with ATR and hRad9 which known as checkpoint-involved proteins. Therefore, we investigated whether hTopBP1 interacted with hMYH, and what the function of their interaction is.

Adenylate kinase 2 (AK2), which balances adenine nucleotide pool, is a multi-functional protein. Here we show that AK2 negatively regulates tumour cell growth. AK2 forms a complex with dual-specificity phosphatase 26 (DUSP26) phosphatase and stimulates DUSP26 activity independently of its AK activity. AK2/DUSP26 phosphatase protein complex dephosphorylates fas-associated protein with death domain (FADD) and regulates cell growth. AK2 deficiency enhances cell proliferation and induces tumour formation in a xenograft assay. This anti-growth function of AK2 is associated with its DUSP26-stimulating activity. Downregulation of AK2 is frequently found in tumour cells and human cancer tissues showing high levels of phospho-FADD(Ser194). Moreover, reconstitution of AK2 in AK2-deficient tumour cells retards both cell proliferation and tumourigenesis. Consistent with this, AK2(+/-) mouse embryo fibroblasts exhibit enhanced cell proliferation with a significant alteration in phospho-FADD(Ser191). These results suggest that AK2 is an associated activator of DUSP26 and suppresses cell proliferation by FADD dephosphorylation, postulating AK2 as a negative regulator of tumour growth.

All gammaherpesviruses express homologues of antiapoptotic B-cell lymphoma-2 (BCL-2) to counter the clearance of infected cells by host antiviral defense machineries. To gain insights into the action mechanisms of these viral BCL-2 proteins, we carried out structural and biochemical analyses on the interactions of M11, a viral BCL-2 of murine gamma-herpesvirus 68, with a fragment of proautophagic Beclin1 and BCL-2 homology 3 (BH3) domain-containing peptides derived from an array of proapoptotic BCL-2 family proteins. Mainly through hydrophobic interactions, M11 bound the BH3-like domain of Beclin1 with a dissociation constant of 40 nanomole, a markedly tighter affinity compared to the 1.7 micromolar binding affinity between cellular BCL-2 and Beclin1. Consistently, M11 inhibited autophagy more efficiently than BCL-2 in NIH3T3 cells. M11 also interacted tightly with a BH3 domain peptide of BAK and those of the upstream BH3-only proteins BIM, BID, BMF, PUMA, and Noxa, but weakly with that of BAX. These results collectively suggest that M11 potently inhibits Beclin1 in addition to broadly neutralizing the proapoptotic BCL-2 family in a similar but distinctive way from cellular BCL-2, and that the Beclin1-mediated autophagy may be a main target of the virus.

Fibrillar aggregates of amyloid-β (Aβ) are the main component of plaques lining the cerebrovasculature in cerebral amyloid angiopathy. As the predominant Aβ isoform in vascular deposits, Aβ40 is a valuable target in cerebral amyloid angiopathy research. However, the slow process of Aβ40 aggregation in vitro is a bottleneck in the search for Aβ-targeting molecules. In this study, we sought a method to accelerate the aggregation of Aβ40 in vitro, to improve experimental screening procedures. We evaluated the aggregating ability of bicine, a biological buffer, using various in vitro methods. Our data suggest that bicine promotes the aggregation of Aβ40 with high speed and reproducibility, yielding a mixture of aggregates with significant β-sheet-rich fibril formation and toxicity.