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Mammalian cell membrane proteins are attractive targets for engineering, because they are located at the interface between intracellular and extracellular milieus and play a pivotal role in controlling cell fates. Recent progresses in understanding the molecular mechanism of membrane proteins have enabled us to engineer these proteins for use in biomedical applications. The applications include cell fate control with artificial ligands, screening of membrane proteins, mapping of protein–protein interactions, cancer therapy, viral display methods for vaccination/screening, and smart protein labeling technologies using a chemical tool. This paper reviews current topics in the engineering of mammalian cell membrane proteins. [ABSTRACT FROM AUTHOR]

Summary: Homeobox domain-containing transcription factors are important regulators of hematopoiesis. Here, we report that increased levels of nonclustered H2.0-like homeobox (HLX) lead to loss of functional hematopoietic stem cells and formation of aberrant progenitors with unlimited serial clonogenicity and blocked differentiation. Inhibition of HLX reduces proliferation and clonogenicity of leukemia cells, overcomes the differentiation block, and leads to prolonged survival. HLX regulates a transcriptional program, including PAK1 and BTG1, that controls cellular differentiation and proliferation. HLX is overexpressed in 87% of patients with acute myeloid leukemia (AML) and independently correlates with inferior overall survival (n = 601, p = 2.3 × 10−6). Our study identifies HLX as a key regulator in immature hematopoietic and leukemia cells and as a prognostic marker and therapeutic target in AML. [ABSTRACT FROM AUTHOR]

Objective: In this study, we attempted a comprehensive analysis of MHC class I–bound peptides in adult T cell leukemia (ATL) cells in order to identify as many tumor-associated antigens (TAAs) as possible that could be used for CTL-based immunotherapy. Methods and Results: Using mass spectrometry combined with reversed-phase liquid chromatography, we could sequence 188 HLA class I–restricted candidate peptides from three ATL-derived cell lines. In accordance with the restrained expression of HTLV-I viral RNA in these cell lines, there were no HTLV-I-encoded peptides among these candidates. Based on the differential expression between ATL cells and normal CD4+ T cells, we selected 10 novel peptides as T cell epitopes of overexpressed source proteins. RT-PCR analysis revealed that 5 source proteins including PRAME, a known tumor-testis antigen, were highly expressed in the majority of 16 ATL cases. Furthermore we could induce PRAME-specific CTLs in vitro from an HLA-B62+ healthy donor that showed specific cytotoxicity against HLA-B62+ PRAME+ ATL cells. Conclusion: These results demonstrate that comprehensive analysis of HLA class I–bound peptides by mass spectrometry is useful for identification of TAA-derived peptides in ATL. Considering that expression patterns of leukemia/lymphoma-associated antigens vary from case to case, this approach appears to be suitable for the tailor-made immunotherapy of hematological malignancies. [Copyright &y& Elsevier]

Understanding the receptor activation mechanism is essential for the rational design of pharmacologically active ligand molecules. However, the activation mechanism of most cytokine receptors remains still unclear, and while agonism and antagonism have been described for ligand-mimetic peptides, there has been no report of inverse agonism that has been characterized for G protein-coupled receptors (GPCRs). To explore the activation mechanism of cytokine receptors, here we tried to investigate how agonism and antagonism could be altered by randomizing antibody variable region of an antibody/cytokine receptor chimera recognizing hen egg lysozyme (HEL) as an agonist. Based on our previous finding that the co-expression of VH-gp130 and VL-erythropoietin receptor (EpoR) chimeras transduced strict and efficient HEL-dependent cell growth signal, a VH-gp130 library encoding four randomized CDR2 residues was retrovirally infected to IL-3-dependent Ba/F3 cells already transfected with VL-EpoR. The selection without IL-3 resulted in a clonal expansion of the transduced cells, and interestingly some of which showed HEL dose-dependent growth suppression. Our results clearly indicate that agonism and antagonism of the antibody/cytokine receptor chimera can be readily switched by a subtle modification of the ligand binding domain as well as that of GPCRs, also implying the existence of inverse agonism in cytokine receptor superfamily. [Copyright &y& Elsevier]

Aluminum is environmentally abundant but not an essential trace element. Although there is increasing evidence suggesting the implication of aluminum in the pathogenesis of Alzheimer’s disease, it is still controversial. We found and report here that aluminum maltolate, a stable and hydrophilic aluminum complex, causes death of primary cultured rat hippocampal neurons in a time- and dose-dependent manner. Degenerated neurons were TUNEL-positive. Immunohistochemical detection of synapsin I and microtubule associated protein 2 revealed the synapse loss between neurons intoxicated by aluminum maltolate. To explore the mechanism underlying its neurotoxicity, we administered various pharmacological compounds prior to the application of aluminum maltolate, and found that brain-derived neurotrophic factor (BDNF) markedly attenuated the neurotoxicity. Furthermore, aluminum maltolate inhibited the elevation of intracellular calcium levels caused by BDNF. Our results suggest the involvement of BDNF in the molecular mechanism underlying neurotoxicity induced by aluminum maltolate. [Copyright &y& Elsevier]

The number of deaths from air pollution worldwide is estimated at 8.8 million per year, more than the number of deaths from smoking. Air pollutants, such as PM 2.5 , are known to induce respiratory and cardiovascular diseases by inducing oxidative stress. Thioredoxin (Trx) is a 12-kDa endogenous protein that exerts antioxidant activity by promoting dithiol disulfide exchange reactions. We previously synthesized human serum albumin-fused thioredoxin (HSA-Trx), which has a longer half-life in plasma compared with Trx, and demonstrated its efficacy against various diseases including respiratory diseases. Here, we examined the effect of HSA-Trx on urban aerosol-induced lung injury in mice. Urban aerosols induced lung injury and inflammatory responses in ICR mice, but intravenous administration of HSA-Trx markedly inhibited these responses. We next analyzed reactive oxygen species (ROS) production in murine lungs using an in vivo imaging system. The results show that intratracheal administration of urban aerosols induced ROS production that was inhibited by intravenously administered HSA-Trx. Finally, we found that HSA-Trx inhibited the urban aerosol-induced increase in levels of neutrophilic extracellular trap (NET) indicators (i.e., double-stranded DNA, citrullinated histone H3, and neutrophil elastase) in bronchoalveolar lavage fluid (BALF). Together, these findings suggest that HSA-Trx prevents urban aerosol-induced acute lung injury by suppressing ROS production and neutrophilic inflammation. Thus, HSA-Trx may be a potential candidate drug for preventing the onset or exacerbation of lung injury caused by air pollutants. Image 1 • HSA-Trx prevents urban aerosol-dependent lung injury. • HSA-Trx suppresses urban aerosol-induced reactive oxygen species (ROS) production. • HSA-Trx suppresses urban aerosol-induced neutrophilic extracellular traps (NETs) formation. [ABSTRACT FROM AUTHOR]

Since protein-protein interactions (PPIs) regulate a variety of cellular processes, the detection of PPIs is crucial for elucidating the underlying molecular mechanisms as well as developing therapeutics. In this study, we propose a novel system to detect PPIs using the distinct domains of focal adhesion kinase (FAK). In this system named "split FAK", the linker and kinase domains in native FAK are tethered separately to two target proteins of interest. The interaction between the target proteins brings the linker and kinase domains into proximity, which leads to phosphorylation at Y397 of the linker domain, recruitment of another tyrosine kinase Src, and phosphorylation at Y576 of the kinase domain. PPIs are readily detected by probing phosphorylation at Y397 and Y576 of these domains. To demonstrate this system, we designed a series of split FAK chimeras with different domain structures. Consequently, dimerizer-induced interaction between FK506-binding protein 12 (FKBP) and the T2098L mutant of FKBP12-rapamycin binding domain (FRB) was clearly detected by probing phosphorylation at the specific tyrosine residues of most of the split FAK chimeras. This is a novel PPI detection system based on a mechanism-inspired design of a trans-activated split kinase. [ABSTRACT FROM AUTHOR]

Non-invasive monitoring of temperature elevations inside tumor tissue is imperative for the oncological thermotherapy known as hyperthermia. In the present study, two cancer patients, one with a developing right renal cell carcinoma and the other with pseudomyxoma peritonei, underwent hyperthermia. The two patients were irradiated with radiofrequency current for 40 min during hyperthermia. We report the results of our clinical trial study in which the temperature increases inside the tumor tissues of patients with right renal cell carcinoma and pseudomyxoma peritonei induced by radiofrequency current irradiation for 40 min could be detected by statistical analysis of ultrasonic scattered echoes. The Nakagami shape parameter m varies depending on the temperature of the medium. We calculated the Nakagami shape parameter m by statistical analysis of the ultrasonic echoes scattered from the tumor tissues. The temperature elevations inside the tumor tissues were expressed as increases in brightness on 2-D hot-scale maps of the specific parameter αmod, indicating the absolute values of the percentage changes in m values. In the αmod map for each tumor tissue, the brightness clearly increased with treatment time. In quantitative analysis, the mean values of αmod were calculated. The mean value of αmod for the right renal cell carcinoma increased to 1.35 dB with increasing treatment time, and the mean value of αmod for pseudomyxoma peritonei increased to 1.74 with treatment time. The increase in both αmod brightness and the mean value of αmod implied temperature elevations inside the tumor tissues induced by the radiofrequency current; thus, the acoustic method is promising for monitoring temperature elevations inside tumor tissues during hyperthermia. [ABSTRACT FROM AUTHOR]

Zinc (Zn) is a co-factor for a vast number of enzymes, and functions as a regulator for immune mechanism and protein synthesis. However, excessive Zn release induced in pathological situations such as stroke or transient global ischemia is toxic. Previously, we demonstrated that the interaction of Zn and copper (Cu) is involved in the pathogenesis of Alzheimer’s disease and vascular dementia. Furthermore, oxidative stress has been shown to play a significant role in the pathogenesis of various metal ions induced neuronal death. Thioredoxin-Albumin fusion (HSA-Trx) is a derivative of thioredoxin (Trx), an antioxidative protein, with improved plasma retention and stability of Trx. In this study, we examined the effect of HSA-Trx on Cu 2+ /Zn 2+ -induced neurotoxicity. Firstly, HSA-Trx was found to clearly suppress Cu 2+ /Zn 2+ -induced neuronal cell death in mouse hypothalamic neuronal cells (GT1-7 cells). Moreover, HSA-Trx markedly suppressed Cu 2+ /Zn 2+ -induced ROS production and the expression of oxidative stress related genes, such as heme oxygenase-1. In contrast, HSA-Trx did not affect the intracellular levels of both Cu 2+ and Zn 2+ after Cu 2+ /Zn 2+ treatment. Finally, HSA-Trx was found to significantly suppress endoplasmic reticulum (ER) stress response induced by Cu 2+ /Zn 2+ treatment in a dose dependent manner. These results suggest that HSA-Trx counteracted Cu 2+ /Zn 2+ -induced neurotoxicity by suppressing the production of ROS via interfering the related gene expressions, in addition to the highly possible radical scavenging activity of the fusion protein. Based on these findings, HSA-Trx has great potential as a promising therapeutic agent for the treatment of refractory neurological diseases. [ABSTRACT FROM AUTHOR]

Renal ischemia/reperfusion (I/R) injury is a major clinical problem because it can cause acute kidney injury (AKI) or lead to the transition from AKI to chronic kidney disease (CKD). Oxidative stress, which involves the production of reactive oxygen species (ROS), plays an important role in the development and exacerbation of I/R-induced kidney injury. However, we have previously reported that lecithinized superoxide dismutase (PC-SOD), a SOD derivative with high tissue affinity and high stability in plasma, has beneficial effects in various disease models because of its inhibitory effect on ROS production. Therefore, we aimed to determine the effects of intravenous PC-SOD administration in a mouse model of renal injury induced by I/R. PC-SOD markedly ameliorated the I/R-induced increases in markers of renal damage (urea nitrogen, creatinine, neutrophil gelatinase-associated lipocalin, and interleukin-6) and tubular necrosis 48 h after the intervention. We also found that PC-SOD significantly ameliorated the I/R-induced increase in ROS production, using an ex vivo imaging system. Furthermore, PC-SOD inhibited the increases in expression of markers of fibrosis (α-smooth muscle actin and collagen 1A1) 96 h after, and renal fibrosis 25 days after I/R was induced. Finally, we found that PC-SOD ameliorated the I/R-induced AKI in mice with high-fat diet-induced prediabetes. These results suggest that PC-SOD inhibits AKI and the transition from AKI to CKD through the inhibition of ROS production. Therefore, we believe that PC-SOD may represent an effective therapeutic agent for I/R-induced renal injury. [ABSTRACT FROM AUTHOR]