Protein Phosphatase 1δ with Nucleophosmin

Protein phosphorylation and dephosphorylation has been recognized as an essential mechanism in the regulation of cellular metabolism and function in various tissues. Serine and threonine protein phosphatases (PP) are divided into four categories: PP1, PP2A, PP2B, and PP2C. At least four isoforms of PP1 catalytic subunit in rat, PP1α, PP1γ1, PP1γ2, and PP1δ, were isolated. In the present study, we examined the localization and expression of PP1δ in human osteoblastic Saos-2 cells. Anti-PP1δ antibody recognized a protein present in the nucleolar regions in Saos-2 cells. Cellular fractionation revealed that PP1δ is a 37 kDa protein localized in the nucleolus. Nucleophosmin is a nucleolar phosphoprotein and located mainly in the nucleolus. Staining pattern of nucleophosmin in Saos-2 cells was similar to that of PP1δ. PP1δ and nucleophosmin were specifically stained as dots in the nucleus. Dual fluorescence images revealed that PP1δ and nucleophosmin were localized in the same regions in the nucleolus. Similar distribution patterns of PP1δ and nucleophosmin were observed in osteoblastic MG63 cells. The interaction of PP1δ and nucleophosmin was also shown by immunoprecipitation and Western analysis. These results indicated that PP1δ associate with nucleophosmin directly in the nucleolus and suggested that nucleophosmin is one of the candidate substrate for PP1δ

Role of Protein Phosphatase 2A in Osteoblast Differentiation and Function

The reversible phosphorylation of proteins plays hugely important roles in a variety of cellular processes, such as differentiation, proliferation, and apoptosis. These processes are strictly controlled by protein kinases (phosphorylation) and phosphatases (de-phosphorylation). Here we provide a brief history of the study of protein phosphorylation, including a summary of different types of protein kinases and phosphatases. One of the most physiologically important serine/threonine phosphatases is PP2A. This review provides a description of the phenotypes of various PP2A transgenic mice and further focuses on the known functions of PP2A in bone formation, including its role in osteoblast differentiation and function. A reduction in PP2A promotes bone formation and osteoblast differentiation through the regulation of bone-related transcription factors such as Osterix. Interestingly, downregulation of PP2A also stimulates adipocyte differentiation from undifferentiated mesenchymal cells under the appropriate adipogenic differentiation conditions. In osteoblasts, PP2A is also involved in the ability to control osteoclastogenesis as well as in the proliferation and metastasis of osteosarcoma cells. Thus, PP2A is considered to be a comprehensive factor in controlling the differentiation and function of cells derived from mesenchymal cells such as osteoblasts and adipocytes

Interaction of Protein Phosphatase 1δ with Nucleophosmin in Human Osteoblastic Cells

Protein phosphorylation and dephosphorylation has been recognized as an essential mechanism in the regulation of cellular metabolism and function in various tissues. Serine and threonine protein phosphatases (PP) are divided into four categories: PP1, PP2A, PP2B, and PP2C. At least four isoforms of PP1 catalytic subunit in rat, PP1α, PP1γ1, PP1γ2, and PP1δ, were isolated. In the present study, we examined the localization and expression of PP1δ in human osteoblastic Saos-2 cells. Anti-PP1δ antibody recognized a protein present in the nucleolar regions in Saos-2 cells. Cellular fractionation revealed that PP1δ is a 37 kDa protein localized in the nucleolus. Nucleophosmin is a nucleolar phosphoprotein and located mainly in the nucleolus. Staining pattern of nucleophosmin in Saos-2 cells was similar to that of PP1δ. PP1δ and nucleophosmin were specifically stained as dots in the nucleus. Dual fluorescence images revealed that PP1δ and nucleophosmin were localized in the same regions in the nucleolus. Similar distribution patterns of PP1δ and nucleophosmin were observed in osteoblastic MG63 cells. The interaction of PP1δ and nucleophosmin was also shown by immunoprecipitation and Western analysis. These results indicated that PP1δ associate with nucleophosmin directly in the nucleolus and suggested that nucleophosmin is one of the candidate substrate for PP1δ

GRam stain-guided Antibiotics ChoicE for Ventilator-Associated Pneumonia (GRACE-VAP) trial: rationale and study protocol for a randomised controlled trial

Abstract Background Optimising the use of antibiotic agents is a pressing challenge to overcoming the rapid emergence and spread of multidrug-resistant pathogens in intensive care units (ICUs). Although Gram staining may possibly provide immediate information for predicting pathogenic bacteria, Gram stain-guided initial antibiotic treatment is not well established in the ICU setting. We planned the GRam stain-guided Antibiotics ChoicE for Ventilator-Associated Pneumonia (GRACE-VAP) trial to investigate whether Gram staining can safely restrict the use of broad-spectrum antibiotics in patients with ventilator-associated pneumonia (VAP), which is one of the most common hospital-acquired infections in ICUs. Methods/design The GRACE-VAP trial is a multicentre, randomised, open-label parallel-group trial to assess the non-inferiority of Gram stain-guided initial antibiotic treatment to guidelines-based initial antibiotic treatment for the primary endpoint of clinical response rate in patients with VAP. Secondary endpoints include the coverage rates of initial antibiotic therapies, the selected rates of anti-pseudomonal agents and anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) agents as initial antibiotic therapies, 28-day all-cause mortality, ICU-free days, ventilator-free days and adverse events. Patients are randomly assigned to receive Gram stain-guided treatment or guidelines-based treatment at a ratio of 1:1. In the Gram stain group, results of Gram staining of endotracheal aspirate are used to guide the selection of antibiotics. In the guidelines group, the combination of an anti-pseudomonal agent and an anti-MRSA agent is administered. A total sample size of 200 was estimated to provide a power of 80% with a one-sided alpha level of 2.5% and a non-inferiority margin of 20%, considering 10% non-evaluable patients. Discussion The GRACE-VAP trial is expected to reveal whether Gram staining can reduce the use of broad-spectrum antibiotics without impairing patient outcomes and thereby provide evidence for an antibiotic selection strategy in patients with VAP. Trial registration Clinicaltrials.gov, NCT03506113. Registered on 29 March 2018. University Hospital Medical Information Network, UMIN000031933. Registered on 26 March 2018

<i>In Vitro</i> Selection of Anti-Akt2 Thioether-Macrocyclic Peptides Leading to Isoform-Selective Inhibitors

The Akt kinase family, consisting of three isoforms in humans, is a well-validated class of drug target. Through various screening campaigns in academics and pharmaceutical industries, several promising inhibitors have been developed to date. However, due to the mechanistic and structural similarities of Akt kinases, it is yet a challenging task to discover selective inhibitors against a specific Akt isoform. We here report Akt-selective and also Akt2 isoform-selective inhibitors based on a thioether-macrocyclic peptide scaffold. Several anti-Akt2 peptides have been selected from a library by means of an <i>in vitro</i> display system, referred to as the RaPID (Random nonstandard Peptide Integrated Discovery) system. Remarkably, the majority of these “binding-active” anti-Akt2 peptides turned out to be “inhibitory active”, exhibiting IC₅₀ values of approximately 100 nM. Moreover, these peptides are not only selective to the Akt kinase family but also isoform-selective to Akt2. Particularly, one referred to as Pakti-L1 is able to discriminate Akt2 250- and 40-fold over Akt1 and Akt3, respectively. This proof-of-concept case study suggests that the RaPID system has a tremendous potential for the discovery of unique inhibitors with high family- and isoform-selectivity

Solid-Phase Synthesis of β‑Peptoids with Chiral Backbone Substituents Using Reductive Amination

A new submonomeric synthetic method of β-peptoids that allows introduction of chiral backbone substituents is established. The synthesis of β-peptoids with various backbone substituents on β-carbons and spectroscopic studies of synthesized oligomers are described

Peptoid-Based Reprogrammable Template for Cell-Permeable Inhibitors of Protein–Protein Interactions

The development of inhibitors of intracellular protein–protein interactions (PPIs) is of great significance for drug discovery, but the generation of a cell-permeable molecule with high affinity to protein is challenging. Oligo(N-substituted glycines) (oligo-NSGs), referred to as peptoids, are attractive as potential intracellular PPI inhibitors owing to their high membrane permeability. However, their intrinsically flexible backbones make the rational design of inhibitors difficult. Here, we propose a peptoid-based rational approach to develop cell-permeable PPI inhibitors using oligo(N-substituted alanines) (oligo-NSAs). The rigid structures of oligo-NSAs enable independent optimization of each N-substituent to improve binding affinity and membrane permeability, while preserving the backbone shape. A molecule with optimized N-substituents inhibited a target PPI in cells, which demonstrated the utility of oligo-NSA as a reprogrammable template to develop intracellular PPI inhibitors. </div

A Peptoid with Extended Shape in Water

“Peptoids” was proposed, over decades ago, as a term describing analogs of peptides that exhibit better physicochemical and pharmacokinetic properties than peptides. Oligo-(N-substituted glycines) (oligo-NSG) was previously proposed as a peptoid due to its high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible and is difficult to achieve a defined shape in water. This conformational flexibility is severely limiting biological application of oligo-NSG. Here, we propose oligo-(N-substituted alanines) (oligo-NSA) as a new peptoid that forms a defined shape in water. A synthetic method established in this study enabled the first isolation and conformational study of optically pure oligo-NSA. Computational simulations, crystallographic studies and spectroscopic analysis demonstrated the well-defined extended shape of oligo-NSA realized by backbone steric effects. The new class of peptoid achieves the constrained conformation without any assistance of N-substituents and serves as an ideal scaffold for displaying functional groups in well-defined three-dimensional space, which leads to effective biomolecular recognition. </div