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Ferroptosis, characterized by iron-dependent cell death, has recently emerged as a critical defense mechanism against microbial infections. The present study aims to investigate the involvement of exosomes in the induction of ferroptosis and the inhibition of bacterial infection in crustaceans. Our findings provide compelling evidence for the pivotal role of exosomes in the immune response of crustaceans, wherein they facilitate intracellular iron accumulation and activate the ferroptotic pathways. Using RNA-seq and bioinformatic analysis, we demonstrate that cytochrome P450 (CYP) can effectively trigger ferroptosis. Moreover, by conducting an analysis of exosome cargo proteins, we have identified the participation of six-transmembrane epithelial antigen of prostate 4 in the regulation of hemocyte ferroptotic sensitivity. Subsequent functional investigations unveil that six-transmembrane epithelial antigen of prostate 4 enhances cellular Fe2+ levels, thereby triggering Fenton reactions and accelerating CYPmediated lipid peroxidation, ultimately culminating in ferroptotic cell death. Additionally, the Fe2+-dependent CYP catalyzes the conversion of arachidonic acid into 20-hydroxyeicosatetraenoic acid, which activates the peroxisome proliferator-activated receptor. Consequently, the downstream target of peroxisome proliferator-activated receptor, cluster of differentiation 36, promotes intracellular fatty acid accumulation, lipid peroxidation, and ferroptosis. These significant findings shed light on the immune defense mechanisms employed by crustaceans and provide potential strategies for combating bacterial infections in this species. [ABSTRACT FROM AUTHOR]

Mitotic phosphorylation of the conserved GRASP domain of GRASP65 disrupts its self-association, leading to a loss of Golgi membrane tethering, cisternal unlinking, and Golgi breakdown. Recently, the structural basis of the GRASP self-interaction was determined, yet the mechanism by which phosphorylation disrupts this activity is unknown. Here, we present the crystal structure of a GRASP phosphomimic containing an aspartic acid substitution for a serine residue (Ser-189) that in GRASP65 is phosphorylated by PLK1, causing a block in membrane tethering and Golgi ribbon formation. The structure revealed a conformational change in the GRASP internal ligand that prevented its insertion into the PDZ binding pocket, and gel filtration assays showed that this phosphomimic mutant exhibited a significant reduction in dimer formation. Interestingly, the structure also revealed an apparent propagation of conformational change from the site of phosphorylation to the shifted ligand, and alanine substitution of two residues (Glu-145 and Ser-146) at penultimate positions in this chain rescued dimer formation by the phosphomimic. These data reveal the structural basis of the phosphoinhibition of GRASP-mediated membrane tethering and provide a mechanism for its allosteric regulation. [ABSTRACT FROM AUTHOR]

Our previous study demonstrated that transforming growth factor (TGF)-β activates β-catenin signaling through Smad3 interaction with β-catenin in chondrocytes. In the present studies, we further investigated the detailed molecular mechanism of the cross-talk between TGF-β/Smad3 and Wnt/β-catenin signaling pathways. We found that C-terminal Smad3 interacted with both the N-terminal region and the middle region of β-catenin protein in a TGF-IJ-dependent manner. Both Smad3 and Smad4 were required for the interaction with β-catenin and protected β-catenin from an ubiquitin-proteasome-dependent degradation. In addition, the formation of the Smad3-Smad4-βcatenin protein complex also mediated β-catenin nuclear translocation. This Smad3-mediated regulatory mechanism of β-catenin protein stability enhanced the activity of β-catenin to activate downstream target genes during chondrogenesis. Our findings demonstrate a novel mechanism between TGF-β and Wnt/β-catenin signaling pathways during chondrocyte development. [ABSTRACT FROM AUTHOR]

V(D)J recombination is a process integral to lymphocyte development. However, this process is not always benign, since certain lymphoid malignancies exhibit recurrent chromosomal abnormalities, such as translocations and deletions, that harbor molecular signatures suggesting an origin from aberrant V(D)J recombination. Translocations involving LMO2, TAL1, Ttg-1, and Hox11, as well as a recurrent interstitial deletion at 1p32 involving SIL/SCL, are cited examples of illegitimate V(D)J recombination. Previous studies using extrachromosomal substrates reveal that cryptic recombination signal sequences (cRSSs) identified near the translocation breakpoint in these examples support V(D)J recombination with efficiencies ranging from about 30- to 20,000-fold less than bona fide V(D)J recombination signals. To understand the molecular basis for these large differences, we investigated the binding and cleavage of these cRSSs by the RAG1/2 proteins that initiate V(D)J recombination. We find that the RAG proteins comparably bind all cRSSs tested, albeit more poorly than a consensus RSS. We show that four cRSSs that support levels of V(D)J recombination above background levels in cell culture (LMO2, TAL1, Ttg-1, and SIL) are also cleaved by the RAG proteins in vitro with efficiencies ranging from 18 to 70% of a consensus RSS. Cleavage of LMO2 and Ttg-1 by the RAG proteins can also be detected in cell culture using ligation-mediated PCR. In contrast, Hox11 and SCL are nicked but not cleaved efficiently in vitro, and cleavage at other adventitious sites in plasmid substrates may also limit the ability to detect recombination activity at these cRSSs in cell culture. [ABSTRACT FROM AUTHOR]

Rab/Ypt GTPases play key roles in the regulation of vesicular trafficking. They perform most of their functions in a GTP-bound form by interacting with specific downstream effectors. The exocyst is a complex of eight polypeptides involved in constitutive secretion and functions as an effector for multiple Ras-related small GTPases, including the Rab protein Sec4p in yeast. In this study, we have examined the localization and function of the Sec15 exocyst subunit in mammalian cells. Overexpressed Sec15 associated with clusters of tubular/vesicular elements that were concentrated in the perinuclear region. The tubular/vesicular clusters were dispersed throughout the cytoplasm upon treatment with the microtubule-depolymerizing agent nocodazole and were accessible to endocytosed transferrin, but not exocytic cargo (vesicular stomatitis virus glycoprotein). Consistent with these observations, Sec15 colocalized selectively with the recycling endosome marker Rabl1 and exhibited a GTP-dependent interaction with the Rab11 GTPase, but not with Rab4, Rab6, or Rab7. These findings provide the first evidence that the exocyst functions as a Rab effector complex in mammalian cells. [ABSTRACT FROM AUTHOR]

Reports that alpha-11-beta-1 integrin recognizes the GFOGER in interstitial collagens. Cell adhesion to collagen; Stereo view of the interactions between the arginine from chain C of the collagen mimetic tripeptide and integrin alpha-2 and alpha-1 domains; Mechanism of integrin/collagen binding.

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H+-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca2+ influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R.Wefurther show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion. [ABSTRACT FROM AUTHOR]

PAX6 is a master regulatory gene involved in neuronal cell fate specification. It also plays a critical role in early eye field and subsequent limbal stem cell (LSC) determination during eye development. Defects in Pax6 cause aniridia and LSC deficiency in humans and the Sey (Small eye) phenotype in mice (Massé, K., Bhamra, S., Eason, R., Dale, N., and Jones, E. A. (2007) Nature 449, 1058-1062). However, how PAX6 specifies LSC and corneal fates during eye development is not well understood. Here, we show that PAX6 is expressed in the primitive eye cup and later in corneal tissue progenitors in early embryonic development. In contrast, p63 expression commences after that of PAX6 in ocular adnexal and skin tissue progenitors and later in LSCs. Using an in vitro feeder-free culture system, we show that PAX6 knockdown in LSCs led to up-regulation of skin epidermis-specific keratins concomitant with differentiation to a skin fate. Using gene expression analysis, we identified the involvement of Notch, Wnt, and TGF-α signaling pathways in LSC fate determination. Thus, loss of PAX6 converts LSCs to epidermal stem cells, as demonstrated by a switch in the keratin gene expression profile and by the appearance of congenital dermoid tissue. [ABSTRACT FROM AUTHOR]

Zinc plays an essential role in the regulation of pancreatic β cell function, affecting important processes including insulin biosynthesis, glucose-stimulated insulin secretion, and cell viability. Mutations in the zinc efflux transport protein ZnT8 have been linked with both type 1 and type 2 diabetes, further supporting an important role for zinc in glucose homeostasis. However, very little is known about how cytosolic zinc is controlled by zinc influx transporters (ZIPs). In this study, we examined the β cell and islet ZIP transcriptome and show consistent high expression of ZIP6 (Slc39a6) and ZIP7 (Slc39a7) genes across human and mouse islets and MIN6 β cells. Modulation of ZIP6 and ZIP7 expression significantly altered cytosolic zinc influx in pancreatic β cells, indicating an important role for ZIP6 and ZIP7 in regulating cellular zinc homeostasis. Functionally, this dysregulated cytosolic zinc homeostasis led to impaired insulin secretion. In parallel studies, we identified both ZIP6 and ZIP7 as potential interacting proteins with GLP-1R by a membrane yeast two-hybrid assay. Knock-down of ZIP6 but not ZIP7 in MIN6 β cells impaired the protective effects of GLP-1 on fatty acid-induced cell apoptosis, possibly via reduced activation of the p-ERK pathway. Therefore, our data suggest that ZIP6 and ZIP7 function as two important zinc influx transporters to regulate cytosolic zinc concentrations and insulin secretion in β cells. In particular, ZIP6 is also capable of directly interacting with GLP-1R to facilitate the protective effect of GLP-1 on β cell survival. [ABSTRACT FROM AUTHOR]

Previous studies had implicated the IFN-γ transcription factor signal transducer and activator of transcription 1 (STAT1) as a tumor suppressor. However, accumulating evidence has correlated increased STAT1 activation with increased tumor progression in multiple types of cancer, including breast cancer. Indeed, we present evidence that tumor up-regulation of STAT1 activity in human and mouse mammary tumors correlates with increasing disease progression to invasive carcinoma. A microarray analysis comparing low aggressive TM40D and highly aggressive TM40D-MB mouse mammary carcinoma cells revealed significantly higher STAT1 activity in the TM40D-MB cells. Ectopic overexpression of constitutively active STAT1 in TM40D cells promoted mobilization of myeloid-derived suppressor cells (MDSCs) and inhibition of antitumor T cells, resulting in aggressive tumor growth in tumor-transplanted, immunocompetent mice. Conversely, gene knockdown of STAT1 in the metastatic TM40D-MB cells reversed these events and attenuated tumor progression. Importantly, we demonstrate that in human breast cancer, the presence of tumor STAT1 activity and tumor-recruited CD33+ myeloid cells correlates with increasing disease progression from ductal carcinoma in situ to invasive carcinoma. We conclude that STAT1 activity in breast cancer cells is responsible for shaping an immunosuppressive tumor microenvironment, and inhibiting STAT1 activity is a promising immune therapeutic approach. [ABSTRACT FROM AUTHOR]

Maspin is a member of the serine protease inhibitor (serpin) superfamily and displays tumor-suppressing activity by controlling cell migration, proliferation, apoptosis, and adhesion. Here, we provide evidence that maspin acts as a reactive oxygen species (ROS) scavenger through oxidation of three structurally exposed cysteine thiols to sulfenic acid. Ablation of these cysteine residues in maspin resulted in a significant increase in total ROS production in mouse mammary TM40D cells. Also, cells containing a triple-cysteine mutant of maspin showed elevated ERK1/2 activity, a downstream target of ROS, and enhanced proliferation and colony formation. These findings establish a novel mechanism by which maspin utilizes its cysteine thiols to inhibit oxidative stress and cell growth. [ABSTRACT FROM AUTHOR]

Very long chain polyunsaturated fatty acid (VLC-PUFA)-containing glycerophospholipids are highly enriched in the retina; however, details regarding the specific synthesis and function of these highly unusual retinal glycerophospholipids are lacking. Elongation of very long chain fatty acids-4 (ELOVL4) has been identified as a fatty acid elongase protein involved in the synthesis of VLC-PUFAs. Mutations in ELOVL4 have also been implicated in an autosomal dominant form of Stargardt disease (STGD3), a type of juvenile macular degeneration. We have generated photoreceptor-specific conditional knock-out mice and used high performance liquid chromatography-mass spectrometry (HPLC-MS) to examine and analyze the fatty acid composition of retinal membrane glycerophosphatidylcholine and glycerophosphatidylethanolamine species. We also used immunofluorescent staining and histology coupled with electrophysiological data to assess retinal morphology and visual response. The conditional knock-out mice showed a significant decrease in retinal glycerophospholipids containing VLC-PUFAs, specifically contained in the sn-1 position of glycerophosphatidylcholine, implicating the role of Elovl4 in their synthesis. Conditional knock-out mice were also found to have abnormal accumulation of lipid droplets and lipofuscin-like granules while demonstrating photoreceptor-specific abnormalities in visual response, indicating the critical role of Elovl4 for proper rod or cone photoreceptor function. Altogether, this study demonstrates the essential role of ELOVL4 in VLCPUFA synthesis and retinal function. [ABSTRACT FROM AUTHOR]

Genome-wide association study (GWAS) has identified genetic variants in the promoter region of the high temperature requirement factor A1 (HTRA1) gene associated with age-related macular degeneration (AMD). As a secreted serine protease, HTRA1 has been reported to interact with members of the transforming growth factor-β (TGF-β) family and regulate their signaling pathways. Growth differentiation factor 6 (GDF6), a member of the TGF-β family, is involved in ectoderm patterning and eye development. Mutations in GDF6 have been associated with abnormal eye development that may result in microphthalmia and anophthalmia. In this report, we identified a single nucleotide polymorphism (SNP) rs6982567 A/G near the GDF6 gene that is significantly associated withAMD(p value=3.54 × 10-8). We demonstrated that the GDF6 AMD risk allele (rs6982567 A) is associated with decreased expression of the GDF6 and increased expression of HTRA1. Similarly, the HTRA1 AMD risk allele (rs10490924 T) is associated with decreased GDF6 and increased HTRA1 expression. We observed decreased vascular development in the retina and significant up-regulation of GDF6 gene in the RPE layer, retinal and brain tissues in HTRA1 knock-out (htra1-/-) mice as compared with the wild-type counterparts. Furthermore, we showed enhanced SMAD signaling in htra1-/- mice. Our data suggests a critical role of HTRA1 in the regulation of angiogenesis via TGF-β signaling and identified GDF6 as a novel disease gene for AMD. [ABSTRACT FROM AUTHOR]

Innate immune response after transient ischemia is the most common cause of myocardial inflammation and may contribute to injury, yet the detailed signaling mechanisms leading to such a response are not well understood. Herein we tested the hypothesis that myocardial ischemia activates interleukin receptor-associated kinase-1 (IRAK-1), a kinase critical for the innate immune signaling such as that of Toll-like receptors (TLRs), via a mechanism that involves heat shock proteins (HSPs) and TLRs. Coronary artery occlusion induced a rapid myocardial IRAK-1 activation within 30 min in wild-type (WT), TLR2-/-, or Trif-/- mice, but not in TLR4def or MyD88-/- mice. HSP60 protein was markedly increased in serum or in perfusate of isolated heart following ischemia/reperfusion (I/R). In vitro, recombinant HSP60 induced IRAK-1 activation in cells derived from WT, TLR2-/-, or Trif-/- mice, but not from TLR4def or MyD88-/- mice. Both myocardial ischemia- and HSP60-induced IRAK-1 activation was abolished by anti-HSP60 antibody. Moreover, HSP60 treatment of cardiomyocytes (CMs) led to marked activation of caspase-8 and -3, but not -9. Expression of dominant-negative mutant of Fas-associated death domain protein or a caspase-8 inhibitor completely blocked HSP60-induced caspase-8 activation, suggesting that HSP60 likely activates an apoptotic program via the death-receptor pathway. In vivo, I/R-induced myocardial apoptosis and cytokine expression were significantly attenuated in TLR4def mice or in WT mice treated with anti-HSP60 antibody compared with WT controls. Taken together, the current study demonstrates that myocardial ischemia activates an innate immune signaling via HSP60 and TLR4, which plays an important role in mediating apoptosis and inflammation during I/R. [ABSTRACT FROM AUTHOR]

Maspin is a non-inhibitory serine protease inhibitor (serpin) that influences many cellular functions including adhesion, migration, and invasion. The underlying molecular mechanisms that facilitate these actions are still being elucidated. In this study we determined the mechanism by which maspin mediates increased MCF10A cell adhesion. Utilizing competition peptides and mutation analyses, we discovered two unique regions (amino acid residues 190-202 and 260-275) involved in facilitating the increased adhesion function of maspin. In addition, we demonstrate that the urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) complex is required for the localization and adhesion function of maspin. Finally, we showed that maspin, uPAR, and β1 integrin co-immunoprecipitate, suggesting a novel maspin-uPA-uPAR-β1 integrin mega-complex that regulates mammary epithelial cell adhesion. [ABSTRACT FROM AUTHOR]

The Ets family of transcription factors control a myriad of cellular processes and contribute to the underlying genetic loss of cellular homeostasis resulting in cancer. PDEF (prostate-derived Ets factor) has been under investigation for its role in tumor development and progression. However, the role of PDEF in cancer development has been controversial. Some reports link PDEF to tumor promoter, and others show tumor-suppressing functions in various systems under different conditions. So far, there has been no conclusive evidence from in vivo experiments to prove the role of PDEF. We have used both in vitro and in vivo systems to provide a conclusive role of PDEF in the progression process. PDEF-expressing cells block the cell growth rate, and this retardation was reversible when PDEF expression was silenced with PDEF-specific small interfering RNA. When these PDEF-expressing cells were orthotopically implanted into the mouse mammary gland, tumor incidence and growth rate were significantly retarded. Cell cycle analysis revealed that PDEF expression partially blocked cell cycle progression at G1/S without an effect on apoptosis. PDEF overexpression resulted in an increase in p21/CIP1 at both the mRNA and protein levels, resulting in decreased Cdk2 activity. Promoter deletion analysis, electrophoresis mobility shift assays, and chromatin immunoprecipitation studies identified the functional Ets DNA binding site at -2118 bp of the p21/CIPJ gene promoter. This site is capable of binding and responding to PDEF. Furthermore, we silenced p21/CIP1 expression in PDEFoverexpressing cells by small interfering RNA. p21-silenced PDEF cells exhibited significantly increased cell growth in vitro and in vivo, demonstrating the p21 regulation by PDEF as a key player. These experiments identified PDEF as a new transcription factor that directly regulates p21/CIP1 expression under non-stressed conditions. This study conclusively proves that PDEF is a breast tumor suppressor for the first time using both in vitro and in vivo systems. PDEF can be further developed as a target for designing therapeutic intervention of breast cancer. [ABSTRACT FROM AUTHOR]

MEKK3 serves as a critical intermediate signaling molecule in lysophosphatidic acid-mediated nuclear factor-κB (NF-κB) activation. However, the precise regulation for MEKK3 activation at the molecular level is still not fully understood. Here we report the identification of two regulatory phosphorylation sites at Thr-516 and Ser-520 within the kinase activation loop that is essential for MEKK3-mediated IκB kinase β (IKKβ)/NF-κB activation. Substitution of these two residues with alanine abolished the ability of MEKK3 to activate IKKβ/NF-κB, whereas replacement with acidic residues rendered MEKK3 constitutively active. Furthermore, substitution of these two residues with alanine abolished the ability of MEKK3 to mediate lysophosphatidic acid-induced optimal IKKβ/NF-κB activation. [ABSTRACT FROM AUTHOR]

BMP-2 plays an essential role in osteoblast and chondrocyte differentiation, but its signaling mechanism has not been fully defined. In the present studies, we investigated the mechanism through which BMP-2 activates the Smad6 gene. A -2006/+45 Smad6 promoter-luciferase construct was generated along with deletions and Runx2 binding site mutations to examine the role of Smad1 and Runx2 signaling following BMP-2 stimulation in osteoblasts. Transfection of Runx2 or treatment with BMP-2-stimulated promoter activity of the -2006/+45 and -11911+45 reporters but not the -829/+45 and -374/+45 reporters. No Smad1/5 binding site is present in the -1191/ -829 region of the Smad6 promoter. Mutation of the OSE2-a site (-1036/-1031) completely abolished the stimulatory effect of Runx2 as well as BMP-2 on the -20061+45 and -11911+45 Smad6 reporters. Gel shift and chromatin immunoprecipitation (ChIP) assays showed that Runx2 binds the OSE2-a element. ChIP assays demonstrated that Smadi also interacts with the OSE2-a site at the Smad6 promoter through Runx2. The protein degradation of Runx2 is mediated by the E3 ubiquitin ligase Smurf1. In the present studies, we found that Smurf1 binds the OSE2-a site through Runx2 and inhibits Smad6 gene transcription. Treatment with BMP-2 and transfection of Smadi abolished Smurf1 binding to the OSE2 site. These results show that Smadi binding excludes Smurf1 interaction with the OSE2 site and promotes Smad6 gene transcription. [ABSTRACT FROM AUTHOR]

It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-ΚB ligand (RANKL) is a critical osteoclastegenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/ xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells. [ABSTRACT FROM AUTHOR]