Producing highly effective extracellular vesicles using IBAR and talin F3 domain fusion.

Extracellular vesicles (EVs), transporting diverse cellular components, play a crucial role in intercellular communication in numerous physiological and pathological processes. EVs have also been recognized as a drug delivery platform for therapeutic purposes and cell-free regenerative medicine. While various approaches have focused on increasing EV production for efficient use therapeutic use of EVs, enhancing the quality of EVs, such as ensuring efficient uptake by their target cells, has not been widely explored. In this study, we linked a negative membrane curvature-forming inverse BAR (IBAR) domain with an integrin ß tail-binding talin F3 domain to create the IBAR-F3 fusion protein. We observed that IBAR-F3 can trigger filopodia-like membrane protrusions and attract integrins to those protrusion-rich regions, when expressed in Chinese hamster ovary cells expressing integrin αIIbß3. Surprisingly, the expression of IBAR-F3 also induced a robust production of EVs, which were then efficiently taken up by nearby cells in an integrin-dependent manner. Moreover, IBAR triggered integrin activation, presumably by inducing negative membrane curvature that likely disrupts the interaction between the integrin α and ß transmembrane domain. Therefore, we suggest that IBAR-F3 should be utilized to promote both EV production and efficient uptake mediated by integrins. Furthermore, the negative curvature-inducing integrin activation suggests that integrins on EVs can be activated by the nanoscale change in the curvature of the EV without the need for conventional machinery to activate integrin inside the EVs.

Human Probiotic Lactobacillus paracasei-Derived Extracellular Vesicles Improve Tumor Necrosis Factor-α-Induced Inflammatory Phenotypes in Human Skin.

Lactic acid bacteria (LAB), a probiotic, provide various health benefits. We recently isolated a new Lactobacillus paracasei strain with strong anti-inflammatory effects under lipopolysaccharide-induced conditions and proposed a new mode of action-augmenting the endoplasmic reticulum stress pathway for anti-inflammatory functions in host cells. The beneficial effects of the L. paracasei strains on the skin have been described; however, the effects of L. paracasei-derived extracellular vesicles (LpEVs) on the skin are poorly understood. Herein, we investigated whether LpEVs can improve inflammation-mediated skin phenotypes by determining their effects on primary human skin cells and a three-dimensional (3D) full-thickness human skin equivalent under tumor necrosis factor (TNF)-α-challenged inflammatory conditions. LpEVs were efficiently taken up by the human skin cells and were much less cytotoxic to host cells than bacterial lysates. Furthermore, low LpEV concentrations efficiently restored TNF-α-induced cellular phenotypes, resulting in increased cell proliferation and collagen synthesis, but decreased inflammatory factor levels (matrix metalloproteinase 1, interleukin 6, and interleukin 8) in the human dermal fibroblasts, which was comparable to that of retinoic acid, a representative antiaging compound. The beneficial effects of LpEVs were validated in a 3D full-thickness human skin equivalent model. LpEV treatment remarkably restored the TNF-α-induced epidermal malformation, abnormal proliferation of keratinocytes in the basal layer, and reduction in dermal collagen synthesis. Additionally, LpEVs penetrated and reached the deepest dermal layer within 24 h when overlaid on top of a 3D full-thickness human skin equivalent. Furthermore, they possessed superior antioxidant capacity compared with the human cell-derived EVs. Taken together, the anti-inflammatory probiotic LpEVs can be attractive antiaging and antioxidant substances for improving inflammation-induced skin phenotypes and disorders.

α-Hemolysin promotes uropathogenic E. coli persistence in bladder epithelial cells via abrogating bacteria-harboring lysosome acidification.

There is a growing consensus that a significant proportion of recurrent urinary tract infections are linked to the persistence of uropathogens within the urinary tract and their re-emergence upon the conclusion of antibiotic treatment. Studies in mice and human have revealed that uropathogenic Escherichia coli (UPEC) can persist in bladder epithelial cells (BECs) even after the apparent resolution of the infection. Here, we found that, following the entry of UPEC into RAB27b+ fusiform vesicles in BECs, some bacteria escaped into the cytoplasmic compartment via a mechanism involving hemolysin A (HlyA). However, these UPEC were immediately recaptured within LC3A/B+ autophagosomes that matured into LAMP1+ autolysosomes. Thereafter, HlyA+ UPEC-containing lysosomes failed to acidify, which is an essential step for bacterial elimination. This lack of acidification was related to the inability of bacteria-harboring compartments to recruit V-ATPase proton pumps, which was attributed to the defragmentation of cytosolic microtubules by HlyA. The persistence of UPEC within LAMP1+ compartments in BECs appears to be directly linked to HlyA. Thus, through intravesicular instillation of microtubule stabilizer, this host defense response can be co-opted to reduce intracellular bacterial burden following UTIs in the bladder potentially preventing recurrence.

Environment-Sensitive Ectodomain Shedding of Epithin/PRSS14 Increases Metastatic Potential of Breast Cancer Cells by Producing CCL2.

Epithin/PRSS14 is a membrane serine protease that plays a key role in tumor progression. The protease exists on the cell surface until its ectodomain shedding, which releases most of the extracellular domain. Previously, we showed that the remaining portion on the membrane undergoes intramembrane proteolysis, which results in the liberation of the intracellular domain and the intracellular domainmediated gene expression. In this study, we investigated how the intramembrane proteolysis for the nuclear function is initiated. We observed that ectodomain shedding of epithin/PRSS14 in mouse breast cancer 4T1 cells increased depending on environmental conditions and was positively correlated with invasiveness of the cells and their proinvasive cytokine production. We identified selenite as an environmental factor that can induce ectodomain shedding of the protease and increase C-C motif chemokine ligand 2 (CCL2) secretion in an epithin/PRSS14-dependent manner. Additionally, by demonstrating that the expression of the intracellular domain of epithin/PRSS14 is sufficient to induce CCL2 secretion, we established that epithin/PRSS14- dependent shedding and its subsequent intramembrane proteolysis are responsible for the metastatic conversion of 4T1 cells under these conditions. Consequently, we propose that epithin/PRSS14 can act as an environment-sensing receptor that promotes cancer metastasis by liberating the intracellular domain bearing transcriptional activity under conditions promoting ectodomain shedding.

Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain.

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

TRIM28 functions as a negative regulator of aggresome formation.

Selective recognition and elimination of misfolded polypeptides are crucial for protein homeostasis. When the ubiquitin-proteasome system is impaired, misfolded polypeptides tend to form small cytosolic aggregates and are transported to the aggresome and eventually eliminated by the autophagy pathway. Despite the importance of this process, the regulation of aggresome formation remains poorly understood. Here, we identify TRIM28/TIF1ß/KAP1 (tripartite motif containing 28) as a negative regulator of aggresome formation. Direct interaction between TRIM28 and CTIF (cap binding complex dependent translation initiation factor) leads to inefficient aggresomal targeting of misfolded polypeptides. We also find that either treatment of cells with poly I:C or infection of the cells by influenza A viruses triggers the phosphorylation of TRIM28 at S473 in a way that depends on double-stranded RNA-activated protein kinase. The phosphorylation promotes association of TRIM28 with CTIF, inhibits aggresome formation, and consequently suppresses viral proliferation. Collectively, our data provide compelling evidence that TRIM28 is a negative regulator of aggresome formation.Abbreviations: BAG3: BCL2-associated athanogene 3; CTIF: CBC-dependent translation initiation factor; CED: CTIF-EEF1A1-DCTN1; DCTN1: dynactin subunit 1; EEF1A1: eukaryotic translation elongation factor 1 alpha 1; EIF2AK2: eukaryotic translation initiation factor 2 alpha kinase 2; HDAC6: histone deacetylase 6; IAV: influenza A virus; IP: immunoprecipitation; PLA: proximity ligation assay; polypeptidyl-puro: polypeptidyl-puromycin; qRT-PCR: quantitative reverse-transcription PCR; siRNA: small interfering RNA.

UXT chaperone prevents proteotoxicity by acting as an autophagy adaptor for p62-dependent aggrephagy.

p62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.

Insight Into Pathological Integrin αIIbß3 Activation From Safeguarding The Inactive State.

The inhibition of physiological activation pathways of the platelet adhesion receptor integrin αIIbß3 may fail to prevent fatal thrombosis, suggesting that the receptor is at risk of activation by yet an unidentified pathway. Here, we report the discovery and characterization of a structural motif that safeguards the receptor by selectively destabilizing its inactive state. At the extracellular membrane border, an overpacked αIIb(W968)-ß3(I693) contact prevents αIIb(Gly972) from optimally assembling the αIIbß3 transmembrane complex, which maintains the inactive state. This destabilization of approximately 1.0 kcal/mol could be mitigated by hydrodynamic forces but not physiological agonists, thereby identifying hydrodynamic forces as pathological activation stimulus. As reproductive life spans are not generally limited by cardiovascular disease, it appears that the evolution of the safeguard was driven by fatal, hydrodynamic force-mediated integrin αIIbß3 activation in the healthy cardiovascular system. The triggering of the safeguard solely by pathological stimuli achieves an effective increase of the free energy barrier between inactive and active receptor states without incurring an increased risk of bleeding. Thus, integrin αIIbß3 has evolved an effective way to protect receptor functional states that indicates the availability of a mechanical activation pathway when hydrodynamic forces exceed physiological margins.

Selection and identification of a novel bone-targeting peptide for biomedical imaging of bone.

The global burden of bone-related diseases is increasing in the aging society; thus, improved bone targeted imaging for their early identification and treatment are needed. In this study, we screened novel peptide ligands for hydroxyapatite, a major inorganic component of teeth and bones, and identified a peptide enabling in vivo bone targeting and real-time fluorescence bone detection. To isolate peptides highly specific for hydroxyapatite, we used negative and positive selection from a randomized 8-mer peptide phage library and identified hydroxyapatite-specific peptides (HA-pep2, HA-pep3, and HA-pep7). Among these three peptides, HA-pep3 showed the highest binding capacity and superior dissociation constant towards hydroxyapatite surfaces over time (~ 88.3% retained on hydroxyapatite after two weeks). Furthermore, HA-pep3 was highly specific for hydroxyapatite compared to other calcium salt-based materials. Using this superior specificity, HA-pep3 showed higher accumulation in skull, spine, and joints in comparison with scrambled control peptide during real-time whole-body imaging. Ex vivo analysis of the major organs and bone from mice demonstrated that the fluorescence intensity in bone was about 3.32 folds higher in the case of HA-pep3 than the one exhibited by the scrambled control peptide. Our study identified a novel approach for targeting ligands for bone specific imaging and can be useful for drug delivery applications.

Inhibition of Smooth Muscle Cell Proliferation and Migration by a Talin Modulator Attenuates Neointimal Formation after Femoral Arterial Injury.

BACKGROUND AND OBJECTIVES: Vascular smooth muscle cell (SMC) proliferation and migration play a critical role in neointimal formation. Focal adhesion is involved in cell proliferation and migration, and talin is known to be a key regulator of these processes. We synthesized a new talin modulator that binds to the talin protein, and investigated its effects on SMCs and neointimal formation after vascular injury. METHODS: Human aortic SMCs (HAoSMCs) were treated with a newly synthesized talin modulator. Apolipoprotein E knockout (ApoE KO) mice were subjected to left femoral arterial injury and orally administered with the talin modulator daily. Laser Doppler imager was used to compare the blood flow, and injured femoral arteries and blood serum were analyzed after 28 days. RESULTS: The talin modulator significantly inhibited cell proliferation in a concentration-dependent manner and suppressed the migration of HAoSMCs. Treatment with a talin modulator resulted in a significant reduction in the phosphorylation of focal adhesion molecules and downstream signaling molecules related to cell proliferation and migration. The effects of the talin modulator in HAoSMCs were found to be reversible, as evidenced by the reactivation of signaling pathways upon its removal. After 28 days of administration of the talin modulator, an improvement in the blood flow and reduction in neointimal formation in the injured femoral arteries were observed. CONCLUSIONS: We demonstrated the inhibitory effects of a talin modulator on SMC proliferation and migration, and that were associated with downregulation of signaling pathways, resulting in the attenuation of neointimal formation in ApoE KO mice.

Intramembrane proteolysis of an extracellular serine protease, epithin/PRSS14, enables its intracellular nuclear function.

BACKGROUND: Epithin/PRSS14, a type II transmembrane serine protease, is an emerging target of cancer therapy because of its critical roles in tumor progression and metastasis. In many circumstances, the protease, through its ectodomain shedding, exists as a soluble form and performs its proteolytic functions in extracellular environments increasing cellular invasiveness. The seemingly functional integrity of the soluble form raises the question of why the protease is initially made as a membrane-associated protein. RESULTS: In this report, we show that the epithin/PRSS14 intracellular domain (EICD) can be released from the membrane by the action of signal peptide peptidase-like 2b (SPPL2b) after ectodomain shedding. The EICD preferentially localizes in the nucleus and can enhance migration, invasion, and metastasis of epithelial cancer when heterologously expressed. Unbiased RNA-seq analysis and subsequent antibody arrays showed that EICD could control the gene expression of chemokines involved in cell motility, by increasing their promoter activities. Finally, bioinformatics analysis provided evidence for the clinical significance of the intramembrane proteolysis of epithin/PRSS14 by revealing that the poor survival of estrogen receptor (ER)-negative breast cancer patients with high epithin/PRSS14 expression is further worsened by high levels of SPPL2b. CONCLUSIONS: These results show that ectodomain shedding of epithin/PRSS14 can initiate a unique and synchronized bidirectional signal for cancer metastasis: extracellularly broadening proteolytic modification of the surrounding environment and intracellularly reprogramming the transcriptome for metastatic conversion. Clinically, this study also suggests that the intracellular function of epithin/PRSS14 should be considered for targeting this protease for anti-cancer treatment.

A JUN N-terminal kinase inhibitor induces ectodomain shedding of the cancer-associated membrane protease Prss14/epithin via protein kinase CßII.

Serine protease 14 (Prss14)/epithin is a transmembrane serine protease that plays essential roles in tumor progression and metastasis and therefore is a promising target for managing cancer. Prss14/epithin shedding may underlie its activity in cancer and worsen outcomes; accordingly, a detailed understanding of the molecular mechanisms in Prss14/epithin shedding may inform the design of future cancer therapies. On the basis of our previous observation that an activator of PKC, phorbol 12-myristate 13-acetate (PMA), induces Prss14/epithin shedding, here we further investigated the intracellular signaling pathway involved in this process. While using mitogen-activated protein kinase inhibitors to investigate possible effectors of downstream PKC signaling, we unexpectedly found that an inhibitor of c-Jun N-terminal kinase (JNK), SP600125, induces Prss14/epithin shedding even in the absence of PMA. SP600125-induced shedding, like that stimulated by PMA, was mediated by tumor necrosis factor-α-converting enzyme. In contrast, a JNK activator, anisomycin, partially abolished the effects of SP600125 on Prss14/epithin shedding. Moreover, the results from loss-of-function experiments with specific inhibitors, short hairpin RNA-mediated knockdown, and overexpression of dominant-negative PKCßII variants indicated that PKCßII is a major player in JNK inhibition- and PMA-mediated Prss14/epithin shedding. SP600125 increased phosphorylation of PKCßII and tumor necrosis factor-α-converting enzyme and induced their translocation into the plasma membrane. Finally, in vitro cell invasion experiments and bioinformatics analysis of data in The Cancer Genome Atlas breast cancer database revealed that JNK and PKCßII are important for Prss14/epithin-mediated cancer progression. These results provide important information regarding strategies against tumor metastasis.

Topological Adaptation of Transmembrane Domains to the Force-Modulated Lipid Bilayer Is a Basis of Sensing Mechanical Force.

Cells can sense and respond to various mechanical stimuli from their surrounding environment. One of the explanations for mechanosensitivity, a lipid-bilayer model, suggests that a stretch of the membrane induced by mechanical force alters the physical state of the lipid bilayer, driving mechanosensors to assume conformations better matched to the altered membrane. However, mechanosensors of this class are restricted to ion channels. Here, we reveal that integrin αIIbß3, a prototypic adhesion receptor, can be activated by various mechanical stimuli including stretch, shear stress, and osmotic pressure. The force-induced integrin activation was not dependent on its known intracellular activation signaling events and was even observed in reconstituted cell-free liposomes. Instead, these mechanical stimuli were found to alter the lipid embedding of the integrin ß3 transmembrane domain (TMD) and subsequently weaken the αIIb-ß3 TMD interaction, which results in activation of the receptor. Moreover, artificial modulation of the membrane curvature near integrin αIIbß3 can induce its activation in cells as well as in lipid nanodiscs, suggesting that physical deformation of the lipid bilayer, either by mechanical force or curvature, can induce integrin activation. Thus, our results establish the adhesion receptor as a bona fide mechanosensor that directly senses and responds to the force-modulated lipid environment. Furthermore, this study expands the lipid-bilayer model by suggesting that the force-induced topological change of TMDs and subsequent alteration in the TMD interactome is a molecular basis of sensing mechanical force transmitted via the lipid bilayer.

Targeting metastatic breast cancer with peptide epitopes derived from autocatalytic loop of Prss14/ST14 membrane serine protease and with monoclonal antibodies.

BACKGROUND: In order to develop a new immunotherapeutic agent targeting metastatic breast cancers, we chose to utilize autocatalytic feature of the membrane serine protease Prss14/ST14, a specific prognosis marker for ER negative breast cancer as a target molecule. METHODS: The study was conducted using three mouse breast cancer models, 4 T1 and E0771 mouse breast cancer cells into their syngeneic hosts, and an MMTV-PyMT transgenic mouse strain was used. Prss14/ST14 knockdown cells were used to test function in tumor growth and metastasis, peptides derived from the autocatalytic loop for activation were tested as preventive metastasis vaccine, and monoclonal and humanized antibodies to the same epitope were tested as new therapeutic candidates. ELISA, immunoprecipitation, Immunofluorescent staining, and flow cytometry were used to examine antigen binding. The functions of antibodies were tested in vitro for cell migration and in vivo for tumor growth and metastasis. RESULTS: Prss14/ST14 is critically involved in the metastasis of breast cancer and poor survival rather than primary tumor growth in two mouse models. The epitopes derived from the specific autocatalytic loop region of Prss14/ST14, based on structural modeling acted as efficient preventive metastasis vaccines in mice. A new specific monoclonal antibody mAb3F3 generated against the engineered loop structure could reduce cell migration, eliminate metastasis in PyMT mice, and can detect the Prss14/ST14 protein expressed in various human cancer cells. Humanized antibody huAb3F3 maintained the specificity and reduced the migration of human breast cancer cells in vitro. CONCLUSION: Our study demonstrates that Prss14/ST14 is an important target for modulating metastasis. Our newly developed hybridoma mAbs and humanized antibody can be further developed as new promising candidates for the use in diagnosis and in immunotherapy of human metastatic breast cancer.

Cellular machinery for sensing mechanical force.

For mechanical force to induce changes in cellular behaviors, two main processes are inevitable; perception of the force and response to it. Perception of mechanical force by cells, or mechanosensing, requires mechanical force-induced conformational changes in mechanosensors. For this, at least one end of the mechanosensors should be anchored to relatively fixed structures, such as extracellular matrices or the cytoskeletons, while the other end should be pulled along the direction of the mechanical force. Alternatively, mechanosensors may be positioned in lipid bilayers, so that conformational changes in the embedded sensors can be induced by mechanical force-driven tension in the lipid bilayer. Responses to mechanical force by cells, or mechanotransduction, require translation of such mechanical force-induced conformational changes into biochemical signaling. For this, protein-protein interactions or enzymatic activities of mechanosensors should be modulated in response to force-induced structural changes. In the last decade, several molecules that met the required criteria of mechanosensors have been identified and proven to directly sense mechanical force. The present review introduces examples of such mechanosensors and summarizes their mechanisms of action. [BMB Reports 2018; 51(12): 623-629].

Membrane Anchoring of α-Helical Proteins: Role of Tryptophan.

The function of membrane proteins relies on a defined orientation of protein relative to lipid. In apparent correlation to protein anchoring, tryptophan residues are enriched in the lipid headgroup region. To characterize the thermodynamic and structural basis of this relationship in α-helical membrane proteins, we examined the role of three conserved tryptophans in the folding of the heterodimeric integrin αIIbß3 transmembrane (TM) complex in phospholipid bicelles and mammalian membranes. In the homogenous lipid environment of bicelles, tryptophan was replaceable by residues of distinct polarities. The appropriate polarity was guided by the electrostatic potential of the tryptophan surrounding, suggesting that tryptophan can complement diverse environments by adjusting the orientation of its anisotropic side chain to achieve site-specific anchoring. As a sole membrane anchor, tryptophan made a contribution of 0.4 kcal/mol to TM complex stability in bicelles. In membranes, it proved more difficult to replace tryptophan even by tyrosine, indicating a superior capacity to interact with heterogeneous lipids of biological membranes. Interestingly, at intracellular TM helix ends, where integrin activation is initiated, sequence motifs that interact with lipids via opposing polarity patterns were found to restrict TM helix orientations beyond tryptophan anchoring. In contrast to bicelles, phenylalanine became the least accepted substitute in membranes, demonstrating an increased role of the hydrophobic effect. Altogether, our study implicates a wide amphiphilic range of tryptophan, membrane complexity, and the hydrophobic effect to be important factors in tryptophan membrane anchoring.

Facile Method to Prepare for the Ni2P Nanostructures with Controlled Crystallinity and Morphology as Anode Materials of Lithium-Ion Batteries.

Conversion reaction materials (transition metal oxides, sulfides, phosphides, etc.) are attractive in the field of lithium-ion batteries because of their high theoretical capacity and low cost. However, the realization of these materials in lithium-ion batteries is impeded by large voltage hysteresis, high polarization, inferior cycle stability, rate capability, irreversible capacity loss in first cycling, and dramatic volume change during redox reactions. One method to overcome these problems is the introduction of amorphous materials. This work introduces a facile method to synthesize amorphous and crystalline dinickel phosphide (Ni2P) nanoparticle clusters with identical morphology and presents a direct comparison of the two materials as anode materials for rechargeable lithium-ion batteries. To assess the effect of crystallinity and hierarchical structure of nanomaterials, it is crucial to conserve other factors including size, morphology, and ligand of nanoparticles. Although it is rarely studied about synthetic methods of well-controlled Ni2P nanomaterials to meet the above criteria, we synthesized amorphous, crystalline Ni2P, and self-assembled Ni2P nanoparticle clusters via thermal decomposition of nickel-surfactant complex. Interestingly, simple modulation of the quantity of nickel acetylacetonate produced amorphous, crystalline, and self-assembled Ni2P nanoparticles. A 0.357 M nickel-trioctylphosphine (TOP) solution leads to a reaction temperature limitation (∼315 °C) by the nickel precursor, and crystalline Ni2P (c-Ni2P) nanoparticles clusters are generated. On the contrary, a lower concentration (0.1 M) does not accompany a temperature limitation and hence high reaction temperature (330 °C) can be exploited for the self-assembly of Ni2P (s-Ni2P) nanoparticle clusters. Amorphous Ni2P (a-Ni2P) nanoparticle clusters are generated with a high concentration (0.714 M) of nickel-TOP solution and a temperature limitation (∼290 °C). The a-Ni2P nanoparticle cluster electrode exhibits higher capacities and Coulombic efficiency than the electrode based on c-Ni2P nanoparticle clusters. In addition, the amorphous structure of Ni2P can reduce irreversible capacity and voltage hysteresis upon cycling. The amorphous morphology of Ni2P also improves the rate capability, resulting in superior performance to those of c-Ni2P nanoparticle clusters in terms of electrode performance.

Disruption of TACE-filamin interaction can inhibit TACE-mediated ectodomain shedding.

Ectodomain shedding regulates functions of many membrane proteins through the cleavage of their juxtamembrane region mainly by a disintegrin and metalloproteinase family proteinases. Tumor necrosis factor-alpha converting enzyme (TACE) is known to be responsible for phorbol myristate acetate (PMA)-induced shedding of various membrane proteins. How PMA regulates TACE-dependent shedding and how TACE exhibits substrate specificity without proteolysis of other membrane proteins are questionable. Here, we show that TACE can interact with an actin-binding protein, filamin, through 20th filamin repeat. We found that the interaction between TACE and filamin was increased by PMA treatment. In addition, loss of filamin or specific disruption of TACE-filamin interaction inhibited ectodomain shedding of representative TACE substrates, CD44 and amyloid protein precursor. From these data, we suggest that filamin may work as a scaffold that can recruit TACE and its substrates in a PMA-dependent manner to achieve substrate specificity for TACE.

Epigallocatechin gallate has pleiotropic effects on transmembrane signaling by altering the embedding of transmembrane domains.

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbß3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbß3 activation, but it activates αIIbß3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbß3 is tightly regulated by the topology of ß3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin ß3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-ß TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.