Transcription factors BZR1 and PAP1 cooperate to promote anthocyanin biosynthesis in Arabidopsis shoots.

Anthocyanins play critical roles in protecting plant tissues against diverse stresses. The complicated regulatory networks induced by various environmental factors modulate the homeostatic level of anthocyanins. Here, we show that anthocyanin accumulation is induced by brassinosteroids (BRs) in Arabidopsis (Arabidopsis thaliana) shoots and shed light on the underlying regulatory mechanism. We observed that anthocyanin levels are altered considerably in BR-related mutants, and BRs induce anthocyanin accumulation by up-regulating the expression of anthocyanin biosynthetic genes. Our genetic analysis indicated that BRASSINAZOLE RESISTANT 1 (BZR1) and PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) are essential for BR-induced anthocyanin accumulation. The BR-responsive transcription factor BZR1 directly binds to the PAP1 promoter, regulating its expression. In addition, we found that intense anthocyanin accumulation caused by the pap1-D dominant mutation is significantly reduced in BR mutants, implying that BR activity is required for PAP1 function after PAP1 transcription. Moreover, we demonstrated that BZR1 physically interacts with PAP1 to cooperatively regulate the expression of PAP1 target genes, such as TRANSPARENT TESTA 8 (TT8), DIHYDROFLAVONOL 4-REDUCTASE (DFR), and LEUKOANTHOCYANIDIN DIOXYGENASE (LDOX). Our findings indicate that BZR1 functions as an integral component of the PAP1-containing transcription factor complex, contributing to increased anthocyanin biosynthesis. Notably, we also show that functional interaction of BZR1 with PAP1 is required for anthocyanin accumulation induced by low nitrogen stress. Taken together, our results demonstrate that BR-regulated BZR1 promotes anthocyanin biosynthesis through cooperative interaction with PAP1 of the MBW complex.

Regulation of Phosphorylation of Glycogen Synthase Kinase 3α and the Correlation with Sperm Motility in Human.

PURPOSE: To unravel the mechanism regulating the phosphorylation of glycogen synthase kinase 3 (GSK3) and the correlation between the inhibitory phosphorylation of GSK3α and sperm motility in human. MATERIALS AND METHODS: The phosphorylation and priming phosphorylated substrate-specific kinase activity of GSK3 were examined in human spermatozoa with various motility conditions. RESULTS: In human spermatozoa, GSK3α/ß was localized in the head, midpiece, and principal piece of tail and p-GSK3α(Ser21) was enriched in the midpiece. The ratio of p-GSK3α(Ser21)/GSK3α was positively coupled with normal sperm motility criteria of World Health Organization. In high-motility spermatozoa, p-GSK3α(Ser21) phosphotyrosine (p-Tyr) proteins but p-GSK3α(Tyr279) markedly increased together with decreased kinase activity of GSK3 after incubation in Ca2+ containing medium. In high-motility spermatozoa, p-GSK3α(Ser21) levels were negatively coupled with kinase activity of GSK3, and which was deregulated in low-motility spermatozoa. In high-motility spermatozoa, 6-bromo-indirubin-3'-oxime, an inhibitor of kinase activity of GSK3 increased p-GSK3α(Ser21) and p-Tyr proteins. p-GSK3α(Ser21) and p-Tyr protein levels were decreased by inhibition of PKA and Akt. Calyculin A, a protein phosphatase-1/2A inhibitor, markedly increased the p-GSK3α(Ser21) and p-Tyr proteins, and significantly increased the motility of low-motility human spermatozoa. CONCLUSIONS: Down regulation of kinase activity of GSK3α by inhibitory phosphorylation was positively coupled with human sperm motility, and which was regulated by Ca2+, PKA, Akt, and PP1. Small-molecule inhibitors of GSK3 and PP1 can be considered to potentiate human sperm motility.

Cyclized proteins with tags as permeable and stable cargos for delivery into cells and liposomes.

Despite the therapeutic potential of recombinant proteins, their cell permeabilities and stabilities remain significant challenges. Here we demonstrate that cyclized recombinant proteins can be used as universal cargos for permeable and stable delivery into cells and polydiacetylene liposomes. Utilizing a split intein-mediated process, cyclized model fluorescent proteins containing short tetraarginine (R4) and hexahistidine (H6) tags were generated without compromising their native protein functions. Strikingly, as compared to linear R4/H6-tagged proteins, the cyclized counterparts have substantially increased permeabilities in both cancer cells and synthetic liposomes, as well as higher resistances to enzymatic degradation in cancer cells. These properties are likely a consequence of structural constraints imposed on the proteins in the presence of short functional peptides. Additionally, photodynamic therapy by cyclized photoprotein-loaded liposomes in cancer cells was significantly improved in comparison to that by their non-cyclized counterparts. These findings suggest that our strategy will be universally applicable to intercellular delivery of proteins and therapeutics.

Photosensitizing deep-seated cancer cells with photoprotein-conjugated upconversion nanoparticles.

To resolve the problem of target specificity and light transmission to deep-seated tissues in photodynamic therapy (PDT), we report a cancer cell-targeted photosensitizer using photoprotein-conjugated upconversion nanoparticles (UCNPs) with high target specificity and efficient light transmission to deep tissues. Core-shell UCNPs with low internal energy back transfer were conjugated with recombinant proteins that consists of a photosensitizer (KillerRed; KR) and a cancer cell-targeted lead peptide (LP). Under near infrared (NIR)-irradiating condition, the UCNP-KR-LP generated superoxide anion radicals as reactive oxygen species via NIR-to-green light conversion and exhibited excellent specificity to target cancer cells through receptor-mediated cell adhesion. Consequently, this photosensitizing process facilitated rapid cell death in cancer cell lines (MCF-7, MDA-MB-231, and U-87MG) overexpressing integrin beta 1 (ITGB1) receptors but not in a cell line (SK-BR-3) with reduced ITGB1 expression and a non-invasive normal breast cell line (MCF-10A). In contrast to green light irradiation, NIR light irradiation exhibited significant PDT efficacy in cancer cells located beneath porcine skin tissues up to a depth of 10 mm, as well as in vivo tumor xenograft mouse models. This finding suggests that the designed nanocomposite is useful for sensing and targeting various deep-seated tumors.

A local water molecular-heating strategy for near-infrared long-lifetime imaging-guided photothermal therapy of glioblastoma.

Owing to the strong absorption of water in the near-infrared (NIR) region near 1.0 µm, this wavelength is considered unsuitable as an imaging and analytical signal in biological environments. However, 1.0 µm NIR can be converted into heat and used as a local water-molecular heating strategy for the photothermal therapy of biological tissues. Herein, we describe a Nd-Yb co-doped nanomaterial (water-heating nanoparticles (NPs)) as strong 1.0 µm emissive NPs to target the absorption band of water. Furthermore, introducing Tm ions into the water-heating NPs improve the NIR lifetime, enabling the development of a NIR imaging-guided water-heating probe (water-heating NIR NPs). In the glioblastoma multiforme male mouse model, tumor-targeted water-heating NIR NPs reduce the tumor volume by 78.9% in the presence of high-resolution intracranial NIR long-lifetime imaging. Hence, water-heating NIR NPs can be used as a promising nanomaterial for imaging and photothermal ablation in deep-tissue-bearing tumor therapy.

Activatable Peptides for Rapid and Simple Visualization of Protease Activity Secreted in Living Cells.

Activity-based monitoring of cell-secreted proteases has gained significant interest due to the implication of these substances in diverse cellular functions. Here, we demonstrated a cell-based method of monitoring protease activity using fluorescent cell-permeable peptides. The activatable peptide consists of anionic (EEEE), cleavable, and cationic sequences (RRRR) that enable intracellular delivery by matrix metalloproteinase-2 (MMP2), which is secreted by living cancer cells. Compared to HT-29 cells (MMP2-negative), HT-1080 cells (MMP2-positive) showed a strong fluorescence response to the short fluorescent peptide via cell-secreted protease activation. Our approach is expected to find applications for the rapid visualization of protease activity in living cells.

Gold nanoparticle-assisted SELEX as a visual monitoring platform for the development of small molecule-binding DNA aptasensors.

To resolve time-consuming and imperceptible monitoring problems in the traditional systematic evolution of ligands by exponential enrichment (SELEX), we report gold nanoparticle-assisted SELEX (GNP-SELEX) as a visual, proofreading, and self-monitoring platform and its application to small molecule-binding single-stranded DNA (ssDNA) aptasensors. Through the colorimetric changes between rounds, GNP-SELEX enabled the rapid determination of target-specific aptamer library enrichment with neither target modification nor extra monitoring process. We identified ssDNA aptamers with high selectivity and binding affinity by targeting two small molecules (brassinolide; BL and bisphenol A; BPA) as a model. The rational design of selected aptamers by 3D molecular simulation increased their ability to detect BL or BPA in real samples as bioreceptors. These results suggest that GNP-SELEX is useful as a self-monitoring platform to discover ssDNA aptamers as well as to develop aptasensors for diverse targets in a rapid and simple way.

Tailoring photosensitive ROS for advanced photodynamic therapy.

Photodynamic therapy (PDT) has been considered a noninvasive and cost-effective modality for tumor treatment. However, the complexity of tumor microenvironments poses challenges to the implementation of traditional PDT. Here, we review recent advances in PDT to resolve the current problems. Major breakthroughs in PDTs are enabling significant progress in molecular medicine and are interconnected with innovative strategies based on smart bio/nanomaterials or therapeutic insights. We focus on newly developed PDT strategies designed by tailoring photosensitive reactive oxygen species generation, which include the use of proteinaceous photosensitizers, self-illumination, or oxygen-independent approaches. While these updated PDT platforms are expected to enable major advances in cancer treatment, addressing future challenges related to biosafety and target specificity is discussed throughout as a necessary goal to expand the usefulness of PDT.

Self-luminescent photodynamic therapy using breast cancer targeted proteins.

Despite the potential of photodynamic therapy (PDT), its comprehensive use in cancer treatment has not been achieved because of the nondegradable risks of photosensitizing drugs and limits of light penetration and instrumentation. Here, we present bioluminescence (BL)-induced proteinaceous PDT (BLiP-PDT), through the combination of luciferase and a reactive oxygen species (ROS)-generating protein (Luc-RGP), which is self-luminescent and degradable. After exposure to coelenterazine-h as a substrate for luciferase without external light irradiation, Luc-RGP fused with a small lead peptide-induced breast cancer cell death through the generation of BL-sensitive ROS in the plasma membrane. Even with extremely low light energy, BLiP-PDT exhibited targeted effects in primary breast cancer cells from patients and in in vivo tumor xenograft mouse models. These findings suggest that BLiP-PDT is immediately useful as a promising theranostic approach against various cancers.

Collagen-Immobilized Extracellular FRET Reporter for Visualizing Protease Activity Secreted by Living Cells.

Despite the diverse roles of cell-secreted proteases in the extracellular matrix (ECM), classical methods to analyze protease activity have not been explored at the cell culture site. Here, we report a stable, matrix-sticky, and protease-sensitive extracellular reporter that comprises a collagen-binding protein and a Förster resonance energy transfer (FRET) coupler of an enhanced green fluorescent protein and a small dye molecule. The extracellular FRET reporter via split intein-mediated protein trans-splicing is able to adhere to collagen matrices, leading to fluorescence changes by matrix metalloproteinase-2 (MMP2) activity during living cell culture without impeding cell viability. When a proMMP2 mutant (Y581A) with altered protease secretion and activity was transfected into cancer cells, the reporter revealed a dramatic reduction in MMP2 activity in both two- and three-dimensional culture systems, compared with cells transfected with wild-type proMMP2. Our reporter is immediately amenable to monitor protease activity in diverse ECM-resident cells as well as to study protease-related extracellular signaling and tissue remodeling.

Microbial Redox Regulator-Enabled Pulldown for Rapid Analysis of Plasma Low-Molecular-Weight Biothiols.

Although low-molecular-weight (LMW) biothiols function as a disease indicator in plasma, rapidly and effectively analyzing them remains challenging in the extracellular oxidative environment due to technical difficulties. Here, we report a newly designed, affinity pulldown platform using a Bacillus subtilis-derived organic hydroperoxide resistance regulatory (OhrRBS) protein and its operator dsDNA for rapid and cost-effective analyses of plasma LMW biothiols. In the presence of organic hydroperoxide, LMW biothiols triggered the rapid dissociation of FAM-labeled dsDNA from FLAG-tagged OhrRBS via S-thiolation of OhrRBS on anti-FLAG antibody-coated beads, which led to a strong increase of fluorescence intensity in the supernatant after pulldown. This method was easily extended by using a reducing agent to detect free and total LMW biothiols simultaneously in mouse plasma. Unlike free plasma LMW biothiols, total plasma LMW biothiols were more elevated in ΔLDLR mice than those in normal mice. Owing to the rapid dissociation of OhrR/dsDNA complexes in response to LMW biothiols, this pulldown platform is immediately suitable for monitoring rapid redox changes in plasma LMW biothiols as well as studying oxidative stress and diseases in blood.

Rapid and sensitive determination of bisphenol A using aptamer and split DNAzyme.

Despite the increasing concern regarding bisphenol A (BPA) as an endocrine disrupting chemical (EDC) upon environmental or human exposure, development of simple method for BPA detection has been hampered, due to the lack of a stable bioreceptor and signal generator. Here, we report a nucleic acid-based rapid and sensitive method for BPA detection, which constitutes a ssDNA aptamer and ssDNAzyme. When the peroxidase-like DNAzyme sequence was split into two parts (one incorporated into the anti-BPA aptamer as a target recognition element and the other into the complementary sequence as a bait), the presence of BPA hindered the association of the split DNA sequence, leading to a reduced signal in the DNAzyme-triggered chemiluminescence (CL). Thus, this NA-based CL measurement permitted the detection of BPA at as low as 5 nM with a broad dynamic range of five orders and with high selectivity towards BPA over other EDCs with structural similarity. With the development of aptamers, our detection method is expected to facilitate studies to monitor EDCs with high simplicity and sensitivity in the field of environmental science.

Correction to: ECM1 regulates cell proliferation and trastuzumab resistance through activation of EGF-signaling.

After the publication of this work [1] errors were found in Figs. 1a and 5d.

Identification of the Thioredoxin-Like 2 Autoantibody as a Specific Biomarker for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has a higher risk of death within 5 years of being diagnosed than the other forms of breast cancer. It is the second leading cause of death due to cancer among women. Currently, however, no diagnostic blood-based biomarker exists to identify the early stages of TNBC. To address this point, we utilized a human protein microarray system to identify serum autoantibodies that showed different expression patterns between TNBC and normal serum samples, and identified five autoantibodies showing TNBC-specific expression. Among them, we selected the thioredoxin-like 2 (TXNL2) autoantibody and evaluated its diagnostic relevance by dot blot analysis with the recombinant TXNL2 protein. We demonstrated that the TXNL2 autoantibody showed 2- to 6-fold higher expression in TNBC samples than in normal samples suggesting that serum TXNL2 autoantibodies are potential biomarkers for TNBC.

Detection of Matrix Metalloproteinase Activity by Bioluminescence via Intein-Mediated Biotinylation of Luciferase.

We report bioluminescence analysis of matrix metalloproteinase (MMP) activity in biological substances using a surface-bound luciferase probe. Intein-fused luciferase protein enables site-specific biotinylation of luciferase in the presence of N-terminus cysteine-biotin via intein-mediated splicing process, resulting in a strong association with high bioluminescence signal onto a NeutrAvidin-coated surface. When the peptide substrate for MMP-7 was inserted into a region between luciferase and intein, the biotinylated probe detected MMP-7 activity by cleaving the peptide, and surface-induced bioluminescence signal was strongly reduced in the MMP-secreted media or mouse tissue extracts, compared with that in MMP-deficient control set. Our approach is anticipated to be useful for generating biotinylated proteins and for their applications in diagnosing MMP activity in human diseases.

Facile Determination of Sodium Ion and Osmolarity in Artificial Tears by Sequential DNAzymes.

Despite high relevance of tear osmolarity and eye abnormality, numerous methods for detecting tear osmolarity rely upon expensive osmometers. We report a reliable method for simply determining sodium ion-based osmolarity in artificial tears using sequential DNAzymes. When sodium ion-specific DNAzyme and peroxidase-like DNAzyme were used as a sensing and detecting probe, respectively, the concentration of Na⁺ in artificial tears could be measured by absorbance or fluorescence intensity, which was highly correlated with osmolarity over the diagnostic range (R² > 0.98). Our approach is useful for studying eye diseases in relation to osmolarity.

C-terminal dimerization of apo-cyclic AMP receptor protein validated in solution.

Although cyclic AMP receptor protein (CRP) has long served as a typical example of effector-mediated protein allostery, mechanistic details into its regulation have been controversial due to discrepancy between the known crystal structure and NMR structure of apo-CRP. Here, we report that the recombinant protein corresponding to its C-terminal DNA-binding domain (CDD) forms a dimer. This result, together with structural information obtained in the present NMR study, is consistent with the previous crystal structure and validates its relevance also in solution. Therefore, our findings suggest that dissociation of the CDD may be critically involved in cAMP-induced allosteric activation of CRP.

On-Chip Peptide Mass Spectrometry Imaging for Protein Kinase Inhibitor Screening.

Protein kinases are enzymes that are important targets for drug discovery because of their involvement in regulating the essential cellular processes. For this reason, the changes in protein kinase activity induced by each drug candidate (the inhibitor in this case) need to be accurately determined. Here, an on-chip secondary ion mass spectrometry (SIMS) imaging technique of the peptides was developed for determining protein kinase activity and inhibitor screening without a matrix. In our method, cysteine-tethered peptides adsorbed onto a gold surface produced changes in the relative peak intensities of the phosphorylated and unphosphorylated substrate peptides, which were quantitatively dependent on protein kinase activity. Using mass spectrometry imaging of multiple compartments on the gold surface in the presence of a peptide substrate, we screened 13,727 inhibitors, of which seven were initially found to have inhibitor efficiencies that surpassed 50%. Of these, we were able to identify a new breakpoint cluster region-abelson (BCR-ABL)T315I kinase inhibitor, henceforth referred to as KR135861. KR135861 showed no cytotoxicity and was subsequently confirmed to be superior to imatinib, a commercial drug marketed as Gleevec. Moreover, KR135861 exhibited a greater inhibitory effect on the BCR-ABLT315I tyrosine kinase, with an IC50 value as low as 1.3 µM. In in vitro experiments, KR135861 reduced the viability of both Ba/F3 cells expressing wild-type BCR-ABL and BCR-ABLT315I, in contrast to imatinib's inhibitory effects only on Ba/F3 cells expressing wild-type BCR-ABL. Due to the surface sensitivity and selectivity of SIMS imaging, it is anticipated that our approach will make it easier to validate the small modifications of a substrate in relation to enzyme activity as well as for drug discovery. This mass spectrometry imaging analysis enables efficient screening for protein kinase inhibitors, thus permitting high-throughput drug screening with high accuracy, sensitivity, and specificity.

Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

Rapid Detection of Glycogen Synthase Kinase-3 Activity in Mouse Sperm Using Fluorescent Gel Shift Electrophoresis.

Assaying the glycogen synthase kinase-3 (GSK3) activity in sperm is of great importance because it is closely implicated in sperm motility and male infertility. While a number of studies on GSK3 activity have relied on labor-intensive immunoblotting to identify phosphorylated GSK3, here we report the simple and rapid detection of GSK3 activity in mouse sperm using conventional agarose gel electrophoresis and a fluorescent peptide substrate. When a dye-tethered and prephosphorylated (primed) peptide substrate for GSK3 was employed, a distinct mobility shift in the fluorescent bands on the agarose was observed by GSK3-induced phosphorylation of the primed peptides. The GSK3 activity in mouse testes and sperm were quantifiable by gel shift assay with low sample consumption and were significantly correlated with the expression levels of GSK3 and p-GSK3. We suggest that our assay can be used for reliable and rapid detection of GSK3 activity in cells and tissue extracts.