MK-6, a novel not-α IL-2, elicits a potent antitumor activity by improving the effector to regulatory T cell balance.

IL-2 is a pleiotropic cytokine that regulates immune cell homeostasis. Its immunomodulatory function has been used clinically as an active immunotherapy agent for metastatic cancers. However, severe adverse effects, including the vascular leak syndrome and the preferential stimulation of anti-immunogenic Treg rather than effector T cells, have been obstacles.

A low-frequency IL4R locus variant in Japanese patients with intravenous immunoglobulin therapy-unresponsive Kawasaki disease.

Background: Kawasaki disease (KD) is a systemic vasculitis which may be associated with coronary artery aneurysms. A notable risk factor for the development of coronary artery aneurysms is resistance to intravenous immunoglobulin (IVIG) therapy, which comprises standard treatment for the acute phase of KD. The cause of IVIG resistance in KD is largely unknown; however, the contribution of genetic factors, especially variants in immune-related genes, has been suspected.

Physical interaction of junctophilin and the Ca1.1 C terminus is crucial for skeletal muscle contraction.

Close physical association of Ca1.1 L-type calcium channels (LTCCs) at the sarcolemmal junctional membrane (JM) with ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) is crucial for excitation-contraction coupling (ECC) in skeletal muscle. However, the molecular mechanism underlying the JM targeting of LTCCs is unexplored.

Angiotensin II activates Ca 1.2 Ca channels through β-arrestin2 and casein kinase 2 in mouse immature cardiomyocytes.

Key Points: Angiotensin II (AngII) is crucial in cardiovascular regulation in perinatal mammalians. Here we show that AngII increases twitch Ca transients of mouse immature but not mature cardiomyocytes by robustly activating Ca 1.2 L-type Ca channels through a novel signalling pathway involving angiotensin type 1 (AT ) receptors, β-arrestin2 and casein kinase 2.

Morphogenetic and molecular analyses of cyst wall components in the ciliated protozoan Colpoda cucullus Nag-1.

The cyst wall of the resting cyst of the ciliated protozoan Colpoda cucullus (Nag-1 strain) is composed of several layers of endocyst, a single layer of ectocyst associated with a mucous layer and lepidosomes composed of a fibrous or crystal-like structure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the ectocyst associated with lepidosomes and mucous materials contained proteins corresponding to 27, 31, 45 kDa and smear bands ranging from 50 to 60 kDa. Liquid chromatography-tandem mass spectrometry of these proteins revealed that the 45-kDa protein (p45) was elongation factor Tu (EF-Tu).

ESCRT-0 protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is targeted to endosomes independently of signal-transducing adaptor molecule (STAM) and the complex formation with STAM promotes its endosomal dissociation.

The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes.

A hydrophobic amino acid cluster inserted into the C-terminus of a recycling cell surface receptor functions as an endosomal sorting signal.

Cell surface receptors ubiquitylated after ligand stimulation are internalized and delivered to the lysosomal pathway for degradation. Ubiquitylated receptors are captured by ESCRT protein complexes that sort them to the lysosomal pathway. Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of endosomal sorting complexes required for transport (ESCRT)-0 that recognizes ubiquitin attached to receptors, indicating that it functions as a key molecule for ubiquitin-dependent endosomal sorting.

Identification of differentially expressed water-insoluble proteins in the encystment process of Colpoda cucullus by two-dimensional electrophoresis and LC-MS/MS analysis.

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process.

Excystment-dependent alteration of protein expression in terrestrial ciliate Colpoda cucullus.

Protein expression during the excystment of Colpoda cucullus was studied by SDS-PAGE. The expression levels of 60-, 50- and 49-kDa proteins were markedly changed from the early to later stage of excystment. The 60-kDa protein (p60) was temporarily expressed first, and its expression was inhibited by actinomycin D.

EF-1α and mitochondrial ATP synthase β chain: alteration of their expression in encystment-induced Colpoda cucullus.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total proteins contained in encystment-induced Colpoda cucullus showed that a 50-kDa protein (p50) disappeared, whereas the expression of a 49-kDa protein (p49) was enhanced in early phase of morphogenetic transformation into the resting cyst (i.e. 2-5 h after the onset of encystment induction).

Protein phosphorylation in encystment-induced Colpoda cucullus: localization and identification of phosphoproteins.

In Colpoda cucullus, the morphogenetic transformation was preceded by an enhancement of the in vivo protein phosphorylation level. Immunofluorescence microscopy using antiphosphoserine antibody showed that these phosphorylated proteins were localized in the macronucleus and other cytoplasmic regions. Biotinylated Phos-tag/ECL assays of isolated macronuclei showed that a 33-kDa protein (p33) was localized within them.

Hrs recognizes a hydrophobic amino acid cluster in cytokine receptors during ubiquitin-independent endosomal sorting.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of the ESCRT-0 protein complex that captures ubiquitylated cargo proteins and sorts them to the lysosomal pathway. Although Hrs acts as a key transporter for ubiquitin-dependent endosomal sorting, we previously reported that Hrs is also involved in ubiquitin-independent endosomal sorting of interleukin-2 receptor β (IL-2Rβ). Here, we show direct interactions between bacterially expressed Hrs and interleukin-4 receptor α (IL-4Rα), indicating that their binding is not required for ubiquitylation of the receptors, similar to the case for IL-2Rβ.

Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2 locus.

Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line.

Ubiquitin-independent binding of Hrs mediates endosomal sorting of the interleukin-2 receptor beta-chain.

Several lines of evidence have revealed that ubiquitylation of membrane proteins serves as a signal for endosomal sorting into lysosomes or lytic vacuoles. The hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) interacts with ubiquitylated cargoes through its ubiquitin-interacting-motif domain (UIM domain), and plays an essential early role in endosomal sorting. Here, we show that the C-terminal region of Hrs, which does not contain the UIM domain, can bind to interleukin-2 receptor beta (IL-2Rbeta).

Spatial orientation of mitochondrial processing peptidase and a preprotein revealed by fluorescence resonance energy transfer.

Mitochondrial processing peptidase (MPP), which is composed of heterodimeric alpha-MPP and beta-MPP subunits. It specifically recognizes mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs.

AMSH, an ESCRT-III associated enzyme, deubiquitinates cargo on MVB/late endosomes.

The appropriate sorting of vesicular cargo, including cell-surface proteins, is critical for many cellular functions. Ubiquitinated cargo is targeted to endosomes and digested by lysosomal enzymes. We previously identified AMSH, a deubiquitination enzyme (DUB), to be involved in vesicular transport.

Murine leukemia virus vector integration favors promoter regions and regional hot spots in a human T-cell line.

Genomic analysis of integration will be important in evaluating the safety of human gene therapy with retroviral vectors. Here, we investigated MLV vector integration sites in human T-cells, since they are amenable to gene transfer studies, and have been used therapeutically in clinical trials. We mapped 340 MLV vector integration sites in the infected human T-cell clones we established.

Synthesis and structure-activity relationships of novel parenteral carbapenems, CS-023 (R-115685) and related compounds containing an amidine moiety.

In order to design a new parenteral 1beta-methylcarbapenem antibiotic which has a broad antibacterial spectrum and improved plasma half-life, a series of 1beta-methylcarbapenems with 5-substituted pyrrolidine-3-ylthio groups including an amidine moiety at the C-2 position have been synthesized and structure-activity relationships were investigated. Among those carbapenem derivatives, CS-023 (R-115685) showed a broad spectrum and excellent antibacterial activity against Gram-positive and Gram-negative bacteria. This compound also showed sufficient dehydropeptidase-I (DHP-I) stability and high urinary recovery in animals after subcutaneous administration without cilastatin, a DHP-I inhibitor.

Determination of the cleavage site of the presequence by mitochondrial processing peptidase on the substrate binding scaffold and the multiple subsites inside a molecular cavity.

Mitochondrial processing peptidase (MPP) recognizes a large variety of basic presequences of mitochondrial preproteins and cleaves the single site, often including arginine, at the -2 position (P(2)). To elucidate the recognition and specific processing of the preproteins by MPP, we mutated to alanines at acidic residues conserved in a large internal cavity formed by the MPP subunits, alpha-MPP and beta-MPP, and analyzed the processing efficiencies for various preproteins. We report here that alanine mutations at a subsite in rat beta-MPP interacting with the P(2) arginine cause a shift in the processing site to the C-terminal side of the preprotein.