Functional regulation of the protein phosphatase PPM1M by phosphorylation at multiple sites with Ser/Thr-Pro motifs.

The imbalance in the phosphorylation and the dephosphorylation of proteins leads to various diseases. Therefore, in vivo, the functions of protein kinases and protein phosphatases are strictly regulated. Mg2+/Mn2+-dependent protein phosphatase PPM1M has been implicated in immunity and cancer; however, the regulation mechanism remains unknown. In this study, we show that PPM1M is regulated in different ways by multiple phosphorylation. PPM1M has four Ser/Thr-Pro motifs (Ser27, Ser43, Ser60, and Thr254) that are recognized by proline-directed kinases, and Ser60 was found to be phosphorylated by cyclin-dependent kinase 5 (CDK5) in the cell. The phospho-mimetic mutation of Ser27 and Ser43 in the N-terminal domain suppresses the nuclear localization of PPM1M and promotes its accumulation in the cytoplasm. The phospho-mimetic mutation of Ser60 decreases PPM1M activity; conversely, the phospho-mimetic mutation of Thr254 increases PPM1M activity. These results suggest that the subcellular localization and phosphatase activity of PPM1M are regulated by protein kinases, including CDK5, via phosphorylation at multiple sites. Thus, PPM1M is differentially regulated by proline-directed kinases, including CDK5.

Phase-Separated Structure of NBR/PVC Blends with Different Acrylonitrile Contents Investigated Using STEM-EDS Mapping Analysis.

We investigated the phase-separated structure of nitrile butadiene rubber (NBR)/polyvinyl chloride (PVC) blends with different acrylonitrile (AN) contents in the NBR, using dynamic mechanical analysis measurements and scanning-transmission-electron-microscopy (STEM)-energy-dispersive-X-ray-spectroscopy (EDS) elemental analysis. Two separate sharp tan δ peaks were observed in the blend at the lower AN content of 18.0%, whereas a broad peak was observed in the blends with the higher AN contents of 29.0 and 33.5%, due to the increase in miscibility, as expected from the decrease in the solubility parameter difference with the increasing AN content. The STEM-EDS elemental analysis for the concentration distribution showed that the NBR was mixed in the large PVC domains with a diameter of several micrometers, and the excluded PVC existed around the interface of the domain-matrix phases in the blend with the lower AN content, whereas small domains with a diameter of several tens of nanometers were dispersed in the blend with the higher AN content. The concentration difference in PVC between the PVC domain and the NBR matrix became smaller with increasing miscibility as the AN content increased although the blends contained the same PVC content of 40 wt%.

Partial Miscibility and Concentration Distribution of Two-Phase Blends of Crosslinked NBR and PVC.

We found that the blends of nitrile butadiene rubber (NBR) and polyvinyl chloride (PVC) exhibited lower critical solution temperature (LCST)-type phase behavior in which a single-phase blend tends to phase separate at elevated temperatures when the acrylonitrile content of NBR was 29.0%. The tan δ peaks, which originated from the glass transitions of the component polymers measured by dynamic mechanical analysis (DMA), were largely shifted and broader in the blends when the blends were melted in the two-phase region of the LCST-type phase diagram, suggesting that NBR and PVC are partially miscible in the two-phase structure. The TEM-EDS elemental mapping analysis using a dual silicon drift detector revealed that each component polymer existed in the partner polymer-rich phase, and the PVC-rich domains consisted of aggregated small PVC particles the size of several ten nanometers. The partial miscibility of the blends was explained by the lever rule for the concentration distribution in the two-phase region of the LCST-type phase diagram.

Development of Antimicrobial Peptide-Antibiotic Conjugates to Improve the Outer Membrane Permeability of Antibiotics Against Gram-Negative Bacteria.

Antibiotics have been widely used in the medical field as a treatment for infectious diseases, but they are not effective against all Gram-negative bacteria because of their low permeability to the outer membrane. One of the strategies to improve the antibacterial activity of antibiotics is the coadministration of antibiotics and membrane-perturbing antimicrobial peptides for their synergistic effects. However, because of their different pharmacokinetics, their coadministration may not exert expected effects in the clinical stage. Here, we designed various antimicrobial peptide-antibiotic conjugates as a novel approach to improve the antimicrobial activity of antibiotics. Ampicillin was chosen as a model antibiotic with poor outer membrane permeability, and the effects of the chemistry and position of conjugation and the choice of antimicrobial peptides were examined. One of the ampicillin conjugates exhibited significantly improved antimicrobial activity against ampicillin-resistant Gram-negative bacteria without exerting cytotoxicity against human cultured cells, demonstrating that our novel approach is an effective strategy to improve the antimicrobial activity of antibiotics with low outer membrane permeability.

BROMI/TBC1D32 together with CCRK/CDK20 and FAM149B1/JBTS36 contributes to intraflagellar transport turnaround involving ICK/CILK1.

Primary cilia are antenna-like organelles that contain specific proteins, and are crucial for tissue morphogenesis. Anterograde and retrograde trafficking of ciliary proteins are mediated by the intraflagellar transport (IFT) machinery. BROMI/TBC1D32 interacts with CCRK/CDK20, which phosphorylates and activates the intestinal cell kinase (ICK)/CILK1 kinase, to regulate the change in direction of the IFT machinery at the ciliary tip. Mutations in BROMI, CCRK, and ICK in humans cause ciliopathies, and mice defective in these genes are also known to demonstrate ciliopathy phenotypes. We show here that BROMI interacts not only with CCRK but also with CFAP20, an evolutionarily conserved ciliary protein, and with FAM149B1/ Joubert syndrome (JBTS)36, a protein in which mutations cause JBTS. In addition, we show that FAM149B1 interacts directly with CCRK as well as with BROMI. Ciliary defects observed in CCRK-knockout (KO), BROMI-KO, and FAM149B1-KO cells, including abnormally long cilia and accumulation of the IFT machinery and ICK at the ciliary tip, resembled one another, and BROMI mutants that are defective in binding to CCRK and CFAP20 were unable to rescue the ciliary defects of BROMI-KO cells. These data indicate that CCRK, BROMI, FAM149B1, and probably CFAP20 altogether regulate the IFT turnaround process under the control of ICK.

Effect of Molecular Architecture on Associating Behavior of Star-Like Amphiphilic Polymers Consisting of Plural Poly(ethylene oxide) and One Alkyl Chain.

Associating behavior of star-like amphiphilic polymers consisting of two or three poly(ethylene oxide) (PEO) chains and one stearyl chain (C18) was investigated. Although the aggregation number (Nagg) of linear analogue of amphiphilic polymers monotonically decreased with increasing number-average molecular weight of PEO (Mn,PEO), the Nagg of micelles of star-like amphiphilic polymers with Mn,PEO = 550 g/mol was smaller than that with Mn,PEO = 750 g/mol, whereas that with Mn,PEO ≥ 750 g/mol showed general Mn,PEO dependence. Small-angle X-ray scattering analyses revealed that the occupied area of one PEO chain on the interface between hydrophobic core and corona layer in the micelles of star-like polymers was much narrower than that in the linear amphiphilic polymers. This result indica ted the PEO chains of star-like polymers partially took unfavorable conformation near the core-corona interface in polymer micelles. The effect of local conformation of PEO chains near the interface on the associating behavior became significant as Mn,PEO decreased. Therefore, in polymer micelles of star-like amphiphilic polymers containing PEO with Mn,PEO = 550 g/mol, the enlargement of occupied area of PEO on the core-corona interface should be caused to avoid the formation of unfavorable conformations of partial PEO chains, resulting in a decrease in Naggs.