Search

Background Cancer stem cells (CSCs) have been proposed to be responsible for tumor recurrence and chemo-resistance. Previous studies suggested that miR-153 played essential roles in lung cancer. However, the molecular mechanism of miR-153 in regulating the stemness of non-small cell lung cancer (NSCLC) remains poorly understood. In this study, we investigated the role of miR-153 in regulation of the stemness of NSCLC. Methods The stemness property of lung stem cancer cells was detected by sphere formation assay, immunofluorescence, and Western blot. Luciferase reporter assay was performed to investigate the direct binding of miR-153 to the 3′-UTR of JAG1 mRNA. Animal study was conducted to evaluate the effect of miR-153 on tumor growth in vivo. The clinical relevance of miR-153 in NSCLC was evaluated by Rt-PCR and Kaplan-Meier analysis. Results MiR-153 expression was decreased in lung cancer tissues. Reduced miR-153 expression was associated with lung metastasis and poor overall survival of lung cancer patients. Jagged1, one of the ligands of Notch1, is targeted by miR-153 and inversely correlates with miR-153 in human lung samples. More importantly, we found that miR-153 inhibited stem cell-like phenotype and tumor growth of lung adenocarcinoma through inactivating the Jagged1/Notch1 axis. Conclusion MiR-153 suppresses the stem cell-like phenotypes and tumor growth of lung adenocarcinoma by targeting Jagged1 and provides a potential therapeutic target in lung cancer therapy.

Calcium-dependent proteolytic calpains are implicated in a variety of physiological processes, as well as pathologies associated with calcium overload. However, the mechanism by which calpain is activated remains elusive since intracellular calcium levels under physiological conditions do not reach the high concentration range required to trigger calpain activation. From a candidate screening using the abundance of the calpain target glutamate receptor GluRIIA at the Drosophila neuromuscular junction as a readout, we uncovered that calpain activity was inhibited upon knockdown of Ttm50, a subunit of the Tim23 complex known to be involved in the import of proteins across the mitochondrial inner membrane. Unexpectedly, Ttm50 and calpain are co-localized at calcium stores Golgi and endoplasmic reticulum (ER), and Ttm50 interacts with calpain via its C-terminal domain. This interaction is required for calpain localization at Golgi/ER, and increases calcium sensitivity of calpain by roughly an order of magnitude. Our findings reveal the regulation of calpain activation by Ttm50, and shed new light on calpain-associated pathologies.

Calpains are calcium-dependent, cytosolic proteinases active at neutral pH. They do not degrade but cleave substrates at limited sites. Calpains are implicated in various pathologies, such as ischemia, injuries, muscular dystrophy, and neurodegeneration. Despite so, the physiological function of calpains remains to be clearly defined. Using the neuromuscular junction of Drosophila of both sexes as a model, we performed RNAi screening and uncovered that calpains negatively regulated protein levels of the glutamate receptor GluRIIA but not GluRIIB. We then showed that calpains enrich at the postsynaptic area, and the calcium-dependent activation of calpains induced cleavage of GluRIIA at Q788 of its C terminus. Further genetic and biochemical experiments revealed that different calpains genetically and physically interact to form a protein complex. The protein complex was required for the proteinase activation to downregulate GluRIIA. Our data provide a novel insight into the mechanisms by which different calpains act together as a complex to specifically control GluRIIA levels and consequently synaptic function. SIGNIFICANCE STATEMENT Calpain has been implicated in neural insults and neurodegeneration. However, the physiological function of calpains in the nervous system remains to be defined. Here, we show that calpain enriches at the postsynaptic area and negatively and specifically regulates GluRIIA, but not IIB, level during development. Calcium-dependent activation of calpain cleaves GluRIIA at Q788 of its C terminus. Different calpains constitute an active protease complex to cleave its target. This study reveals a critical role of calpains during development to specifically cleave GluRIIA at synapses and consequently regulate synaptic function.

Altered expression of the E3 ubiquitin ligase UBE3A, which is involved in protein degradation through the proteasome-mediated pathway, is associated with neurodevelopmental and behavioral defects observed in Angelman syndrome (AS) and autism. However, little is known about the neuronal function of UBE3A and the pathogenesis of UBE3A-associated disorders. To understand the in vivo function of UBE3A in the nervous system, we generated multiple mutations of ube3a, the Drosophila ortholog of UBE3A. We found a significantly increased number of total boutons and satellite boutons in conjunction with compromised endocytosis in the neuromuscular junctions (NMJs) of ube3a mutants compared to the wild type. Genetic and biochemical analysis showed upregulation of bone morphogenetic protein (BMP) signaling in the nervous system of ube3a mutants. An immunochemical study revealed a specific increase in the protein level of Thickveins (Tkv), a type I BMP receptor, but not other BMP receptors Wishful thinking (Wit) and Saxophone (Sax), in ube3a mutants. Ube3a was associated with and specifically ubiquitinated lysine 227 within the cytoplasmic tail of Tkv, and promoted its proteasomal degradation in Schneider 2 cells. Negative regulation of Tkv by Ube3a was conserved in mammalian cells. These results reveal a critical role for Ube3a in regulating NMJ synapse development by repressing BMP signaling. This study sheds new light onto the neuronal functions of UBE3A and provides novel perspectives for understanding the pathogenesis of UBE3A-associated disorders., Author Summary Angelman syndrome (AS), characterized by severe mental retardation, developmental delay, ataxia, seizures, speech impairment, and happy disposition, is caused by mutation of E3 ubiquitin ligase UBE3A; a critical enzyme involved in proteasome-mediated protein degradation. Increasing evidence demonstrates that overexpression or hyperactivation of UBE3A is associated with autism. Thus, both loss and gain of UBE3A functions result in neurodevelopmental and cognitive defects. However, the neuronal functions of UBE3A and the mechanism by which altered expression of UBE3A leads to developmental and cognitive defects are poorly understood. Using Drosophila melanogaster as a model system in conjunction with an array of biochemical and physiological assays, we showed that mutants of ube3a had excess synaptic boutons and endocytic defects at the neuromuscular junction terminals due to an elevated level of bone morphogenetic protein (BMP) signaling. Specifically, Ube3a directly binds and ubiquitinates the BMP receptor Thickveins for proteasomal degradation; a function that is conserved in mammalian cells. Negative regulation of BMP signaling by UBE3A suggests a previously unknown molecular mechanism that underlies the pathogenesis of UBE3A-associated AS and autism.

Synaptic connections must be precisely controlled to ensure proper neural circuit formation. In Drosophila melanogaster, bone morphogenetic protein (BMP) promotes growth of the neuromuscular junction (NMJ) by binding and activating the BMP ligand receptors wishful thinking (Wit) and thickveins (Tkv) expressed in motor neurons. We report here that an evolutionally conserved, previously uncharacterized member of the S6 kinase (S6K) family S6K like (S6KL) acts as a negative regulator of BMP signaling. S6KL null mutants were viable and fertile but exhibited more satellite boutons, fewer and larger synaptic vesicles, larger spontaneous miniature excitatory junctional potential (mEJP) amplitudes, and reduced synaptic endocytosis at the NMJ terminals. Reducing the gene dose by half of tkv in S6KL mutant background reversed the NMJ overgrowth phenotype. The NMJ phenotypes of S6KL mutants were accompanied by an elevated level of Tkv protein and phosphorylated Mad, an effector of the BMP signaling pathway, in the nervous system. In addition, Tkv physically interacted with S6KL in cultured S2 cells. Furthermore, knockdown of S6KL enhanced Tkv expression, while S6KL overexpression downregulated Tkv in cultured S2 cells. This latter effect was blocked by the proteasome inhibitor MG132. Our results together demonstrate for the first time that S6KL regulates synaptic development and function by facilitating proteasomal degradation of the BMP receptor Tkv., Author Summary Bi-directional signaling between neurons and their target cells is critical for synapse formation, growth, and plasticity, as well as for neuronal survival. Bone morphogenetic protein (BMP) acts as a retrograde signal promoting synaptic growth at the Drosophila neuromuscular junction (NMJ), but little is known about proteins that regulate BMP signaling by controlling BMP release, receptor expression, and signal transduction. We report here that a previously uncharacterized and evolutionally conserved member of the S6 kinase (S6K) family S6K like (S6KL) inhibits BMP signaling by interacting with and promoting proteasome-mediated degradation of the BMP receptor Thickveins (Tkv). In S6KL mutants, there was an elevated level of Tkv protein, together with overgrown NMJs characterized by excess satellite boutons. Reducing the gene dose of tkv by half in S6KL null background restored normal NMJ morphology, suggesting that S6KL normally serves to suppress Tkv-mediated BMP signaling. Biochemically, S6KL interacted with Tkv. Overexpression of S6KL down-regulated Tkv and this effect was inhibited by blocking the proteasomal degradation pathway. Collectively, our data demonstrate that S6KL regulates NMJ synapse development by promoting the proteasomal degradation of Tkv. Thus, we have identified a novel negative regulator of BMP signaling in the Drosophila nervous system.

The nanos gene encodes a zinc-finger protein which is required for the migration and differentiation of primordial germ cells as well as for their fate maintenance. In this study, a 1913 bp nanos gene was cloned and characterized in silkworm (Bombyx mori). RT-PCR and Western blot analysis showed that the nanos was expressed in developing embryos and various silkworm larval tissues. The expression patterns of Nanos and Vasa in silkworm larval gonads were analyzed using immunohistochemistry. It was found that, in silkworm larval ovaries, the Nanos and Vasa proteins were expressed in oocytes. While in testes, high expression of Nanos and Vasa was detected in spermatogonia and relatively weaker expression was found in spermatocytes at latter stages.

Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5'-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mori and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5. 38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee (Apis mellifera). This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.

The vacuolar ATPase (V-ATPase) is a multifunctional enzyme that consists of several subunits. Subunit B is a part of the catalytic domain of the enzyme. The result of the RTPCR suggested that the V-ATPase B subunit is a ubiquitous gene. 24 h after the larvae were infected with the Bombyx mori nucleopolyhedrovirus (BmNPV), the expression level of the V-ATPase B subunit in the midgut of the resistant strain NB was about 3 times higher than in the susceptible strain 306, and then the expression level of the V-ATPase B subunit decreased rapidly to a very low level. This indicated that the virus may cause a lot of changes of physiological conditions in the midgut. Localization of the V-ATPase B subunit was attempted in midgut cells of Bombyx mori by immunohistochemistry. The immunohistochemical localization with the antibody against the B subunit revealed a positive staining in goblet cell apical membranes of Bombyx mori midgut cells as well as in the midgut of Manduca sexta. This sequence has been registered in GenBank under the accession number EU727173.