scholarly journals Structural and mechanistic insights into secretagogin-mediated exocytosis

2020 ◽  
Vol 117 (12) ◽  
pp. 6559-6570 ◽  
Author(s):  
Jiao Qin ◽  
Qi Liu ◽  
Zhe Liu ◽  
Yun-Zu Pan ◽  
Luis Sifuentes-Dominguez ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Helical Structure ◽  
Snare Complex ◽  
Dependent Manner ◽  
Ef Hand ◽  
Multiple Cell ◽  
Ef Hands ◽  
Underlying Mechanisms ◽  
Close Homolog

Secretagogin (SCGN) is a hexa–EF-hand protein that is highly expressed in the pancreas, brain, and gastrointestinal tract. SCGN is known to modulate regulated exocytosis in multiple cell lines and tissues; however, its exact functions and underlying mechanisms remain unclear. Here, we report that SCGN interacts with the plasma membrane SNARE SNAP-25, but not the assembled SNARE complex, in a Ca2+-dependent manner. The crystal structure of SCGN in complex with a SNAP-25 fragment reveals that SNAP-25 adopts a helical structure and binds to EF-hands 5 and 6 of SCGN. SCGN strongly inhibits SNARE-mediated vesicle fusion in vitro by binding to SNAP-25. SCGN promotes the plasma membrane localization of SNAP-25, but not Syntaxin-1a, in SCGN-expressing cells. Finally, SCGN controls neuronal growth and brain development in zebrafish, likely via interacting with SNAP-25 or its close homolog, SNAP-23. Our results thus provide insights into the regulation of SNAREs and suggest that aberrant synapse functions underlie multiple neurological disorders caused by SCGN deficiency.

scholarly journals Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

2004 ◽  
Vol 167 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Brenton L. Scott ◽  
Jeffrey S. Van Komen ◽  
Hassan Irshad ◽  
Song Liu ◽  
Kirilee A. Wilson ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Snare Complex ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Bud Neck ◽  
Precise Role

Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

scholarly journals HDAC6 inhibitor WT161 performs antitumor effect on osteosarcoma and synergistically interacts with 5-FU

2021 ◽  
Author(s):  
Jun Sun ◽  
Wei Wu ◽  
Xiaofeng Tang ◽  
Feifei Zhang ◽  
Cheng Ju ◽  
...  
Keyword(s):  
Dependent Manner ◽  
Osteosarcoma Cells ◽  
Synergistic Inhibition ◽  
Proliferative Effect ◽  
Xenograft Models ◽  
Hdac6 Inhibitor ◽  
Underlying Mechanisms ◽  
Induced Apoptosis

Background: WT161, as a selective HDAC6 inhibitor, has been shown to play anti-tumor effects on several kinds of cancers. The aim of this study is to explore the roles of WT161 in osteosarcoma and its underlying mechanisms. Methods: The anti-proliferative effect of WT161 on osteosarcoma cells was examined using MTT assay and colony formation assay. Cell apoptosis was analyzed using flow cytometer. The synergistic effect was evaluated by isobologram analysis using CompuSyn software. The osteosarcoma xenograft models were established to evaluate the anti-proliferative effect of WT161 in vivo. Results: WT161 suppressed the cell growth and induced apoptosis of osteosarcoma cells in a dose- and time-dependent manner. Mechanistically, we found that WT161 treatment obviously increased the protein level of PTEN and decreased the phosphorylation level of AKT. More importantly, WT161 show synergistic inhibition with 5-FU on osteosarcoma cells in vitro and in vivo. Conclusions: These results indicate that WT161 inhibits the growth of osteosarcoma through PTEN and has a synergistic efficiency with 5-FU.

scholarly journals TB-4 Antitumor effects of a novel curcumin derivative curcumin monoglucuronide on glioblastoma cells in vitro and in vivo

2021 ◽  
Vol 3 (Supplement_6) ◽  
pp. vi6-vi6
Author(s):  
Takashi Fujii ◽  
Shun Yamamuro ◽  
Masamichi Takahashi ◽  
Akihide Kondo ◽  
Yoshitaka Narita ◽  
...  
Keyword(s):  
Cell Death ◽  
Water Soluble ◽  
Dependent Manner ◽  
Antitumor Effects ◽  
Multiple Cell ◽  
Human Gbm ◽  
Dose Dependent ◽  
Novel Therapeutic

Abstract The therapeutic outcome of glioblastomas (GBMs) is still very poor. Therefore, invention of novel therapeutic methods against GBM cases is considered urgent. The antitumor effects of naturally-derived compounds are attracting attention recently, and therapeutic efficacy of curcumin, a plant-derived compound previously used for multiple purpose, has been indicated in many cancer systems; however, clinical application of curcumin is considered difficult because of its poor bioavailability (under 1 %). Curcumin monoglucuronide (CMG), a water-soluble prodrug of curcumin recently developed for overcoming this weakness, has been demonstrated excellent antitumor effects for several malignancies in vitro and in vivo; therefore, we investigated the effects of CMG against GBM cells. CMG induced cell death of human GBM cells lines (T98G, U251MG, and U87MG) by dose dependent manner by triggering multiple forms of cell death such as apoptosis and perthanatos. Immunoblotting of CMG-treated GBM cell lysates demonstrated activation of multiple cell death signaling. Furthermore, immunodeficiency mice harboring intracerebral U87MG cell xenografts systemically treated by CMG showed significantly prolonged survival compared with control mice. These results suggest CMG would be a novel therapeutic agent against GBM cases.

scholarly journals Membrane Estrogen Receptor-Dependent Extracellular Signal-Regulated Kinase Pathway Mediates Acute Activation of Endothelial Nitric Oxide Synthase by Estrogen in Uterine Artery Endothelial Cells

Endocrinology ◽  
2004 ◽  
Vol 145 (1) ◽  
pp. 113-125 ◽  
Author(s):  
Dong-bao Chen ◽  
Ian M. Bird ◽  
Jing Zheng ◽  
Ronald R. Magness
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Estrogen Receptor ◽  
Plasma Membrane ◽  
Uterine Artery ◽  
Fluorescein Isothiocyanate ◽  
Estrogen Receptor Α ◽  
Dependent Manner ◽  
Enos Phosphorylation

Abstract Rapid uterine vasodilatation after estrogen administration is believed to be mediated by endothelial production of nitric oxide (NO) via endothelial NO synthase (eNOS). However, the mechanism(s) by which estrogen activates eNOS in uterine artery endothelial cells (UAEC) is unknown. In this study, we observed that estradiol-17β (E2) and E2-BSA rapidly (<2 min) increased total NOx production in UAEC in vitro. This was associated with rapid eNOS phosphorylation and activation but was unaltered by pretreatment with actinomycin-D. estrogen receptor-α protein was detectable in isolated plasma membrane proteins by immunoblotting, and E2-BSA-fluorescein isothiocyanate binding was evident on the plasma membrane of UAEC. E2 did not mobilize intracellular Ca2+, but E2 and ionomycin in combination induced greater eNOS phosphorylation than either E2 or ionomycin alone. E2 did not stimulate rapid Akt phosphorylation. E2 stimulated rapid ERK2/1 activation in a time- and dose-dependent manner, with maximal responses observed at 5–10 min with E2 (10 nm to 1 μm) treatment. Acute activation of eNOS and NOx production by E2 could be inhibited by PD98059 but not by LY294002. When E2-BSA was applied, similar responses in NOx production, eNOS, and ERK2/1 activation to those of E2 were achieved. In addition, E2 and E2-BSA-induced ERK2/1 activation and ICI 182,780 could inhibit NOx production by E2. Thus, acute activation of eNOS to produce NO in UAEC by estrogen is at least partially through an ERK pathway, possibly via estrogen receptor localized on the plasma membrane. This pathway may provide a novel mechanism for NO-mediated rapid uterine vasodilatation by estrogen.

scholarly journals p-Coumaric Acid Protects Human Lens Epithelial Cells against Oxidative Stress-Induced Apoptosis by MAPK Signaling

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Jiao Peng ◽  
Ting-ting Zheng ◽  
Yue Liang ◽  
Li-fang Duan ◽  
Yao-dong Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Mapk Signaling ◽  
Human Lens ◽  
Lens Epithelial Cells ◽  
Dependent Manner ◽  
Coumaric Acid ◽  
Underlying Mechanisms ◽  
Induced Apoptosis

To protect against oxidative stress-induced apoptosis in lens epithelial cells is a potential strategy in preventing cataract formation. The present study aimed at studying the protective effect and underlying mechanisms of p-coumaric acid (p-CA) on hydrogen peroxide- (H2O2-) induced apoptosis in human lens epithelial (HLE) cells (SRA 01–04). Cells were pretreated with p-CA at a concentration of 3, 10, and 30 μM before the treatment of H2O2 (275 μM). Results showed that pretreatment with p-CA significantly protected against H2O2-induced cell death in a dose-dependent manner, as well as downregulating the expressions of both cleaved caspase-3 and cleaved caspase-9 in HLE cells. Moreover, p-CA also greatly suppressed H2O2-induced intracellular ROS production and mitochondrial membrane potential loss and elevated the activities of T-SOD, CAT, and GSH-Px of H2O2-treated cells. As well, in vitro study showed that p-CA also suppressed H2O2-induced phosphorylation of p-38, ERK, and JNK in HLE cells. These findings demonstrate that p-CA suppresses H2O2-induced HLE cell apoptosis through modulating MAPK signaling pathways and suggest that p-CA has a potential therapeutic role in the prevention of cataract.

scholarly journals A novel Netrin-1–sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

2014 ◽  
Vol 205 (2) ◽  
pp. 217-232 ◽  
Author(s):  
Cortney C. Winkle ◽  
Leslie M. McClain ◽  
Juli G. Valtschanoff ◽  
Charles S. Park ◽  
Christopher Maglione ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cortical Neurons ◽  
Direct Interaction ◽  
Vesicle Fusion ◽  
Dependent Manner ◽  
Axon Branching ◽  
Spatial Regulation ◽  
Novel Model

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

Reticulon 3-mediated Chk2/p53 activation suppresses hepatocellular carcinogenesis and is blocked by hepatitis B virus

Gut ◽  
2020 ◽  
pp. gutjnl-2020-321386
Author(s):  
Shushu Song ◽  
Yinghong Shi ◽  
Weicheng Wu ◽  
Hao Wu ◽  
Lei Chang ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Dependent Manner ◽  
Mice Model ◽  
Calcium Dependent ◽  
B Virus ◽  
P53 Activation ◽  
Underlying Mechanisms ◽  
Induced Apoptosis

ObjectiveDysfunction of endoplasmic reticulum (ER) proteins is closely related to homeostasis disturbance and malignant transformation of hepatocellular carcinoma (HCC). Reticulons (RTN) are a family of ER-resident proteins critical for maintaining ER function. Nevertheless, the precise roles of RTN in HCC remain largely unclear. The aim of the study is to examine the effect of reticulon family member RTN3 on HCC development and explore the underlying mechanisms.DesignClinical HCC samples were collected to assess the relationship between RTN3 expression and patients’ outcome. HCC cell lines were employed to examine the effects of RTN3 on cellular proliferation, apoptosis and signal transduction in vitro. Nude mice model was used to detect the role of RTN3 in modulating tumour growth in vivo.ResultsWe found that RTN3 was highly expressed in normal hepatocytes but frequently downregulated in HCC. Low RTN3 expression predicted poor outcome in patients with HCC in TP53 gene mutation and HBV infection status-dependent manner. RTN3 restrained HCC growth and induced apoptosis by activating p53. Mechanism studies indicated that RTN3 facilitated p53 Ser392 phosphorylation via Chk2 and enhanced subsequent p53 nuclear localisation. RTN3 interacted with Chk2, recruited it to ER and promoted its activation in an ER calcium-dependent manner. Nevertheless, the tumour suppressive effects of RTN3 were abrogated in HBV-positive cells. HBV surface antigen competed with Chk2 for RTN3 binding and blocked RTN3-mediated Chk2/p53 activation.ConclusionThe findings suggest that RTN3 functions as a novel suppressor of HCC by activating Chk2/p53 pathway and provide more clues to better understand the oncogenic effects of HBV.

scholarly journals ROS-Induced GATA4 and GATA6 Downregulation Inhibits StAR Expression in LPS-Treated Porcine Granulosa-Lutein Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Xiaolu Qu ◽  
Leyan Yan ◽  
Rihong Guo ◽  
Hui Li ◽  
Zhendan Shi
Keyword(s):  
Molecular Mechanisms ◽  
Cell Model ◽  
Animal Husbandry ◽  
Dependent Manner ◽  
Progesterone Production ◽  
Progesterone Synthesis ◽  
Porcine Granulosa Cell ◽  
Underlying Mechanisms ◽  
Dose Dependent

LPS is a major endotoxin produced by gram-negative bacteria, and exposure to it commonly occurs in animal husbandry. Previous studies have shown that LPS infection disturbs steroidogenesis, including progesterone production, and subsequently decreases animal reproductive performance. However, little information about the underlying mechanisms is available thus far. In the present study, an in vitro-luteinized porcine granulosa cell model was used to study the underlying molecular mechanisms of LPS treatment. We found that LPS significantly inhibits progesterone production and downregulates the expressions of progesterone synthesis-associated genes (StAR, CYP11A1, and 3β-HSD). Furthermore, the levels of ROS were significantly increased in an LPS dose-dependent manner. Moreover, transcriptional factors GATA4 and GATA6, but not NR5A1, were significantly downregulated. Elimination of LPS-stimulated ROS by melatonin or vitamin C could restore the expressions of GATA4, GATA6, and StAR. In parallel, StAR expression was also inhibited by the knockdown of GATA4 and GATA6. Based on these data, we conclude that LPS impairs StAR expression via the ROS-induced downregulation of GATA4 and GATA6. Collectively, these findings provide new insights into the understanding of reproductive losses in animals suffering from bacterial infection and LPS exposure.

scholarly journals Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells

1999 ◽  
Vol 10 (11) ◽  
pp. 3979-3990 ◽  
Author(s):  
Anastasiya D. Blagoveshchenskaya ◽  
Eric W. Hewitt ◽  
Daniel F. Cutler
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Brefeldin A ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Protein Traffic ◽  
C Terminus ◽  
Targeting Signal

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRP-tyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents.

scholarly journals AN INTEGRATIVE APPROACH DISCOVERS A NOVEL ANTI-LEUKEMIC PEPTIDE FROM HUMAN MILK

2021 ◽  
Author(s):  
Wararat Chiangjong ◽  
Jirawan Panachan ◽  
Thitinee Vanichapol ◽  
Nutkridta Pongsakul ◽  
Tassanee Lerksuthirat ◽  
...  
Keyword(s):  
Childhood Leukemia ◽  
Leukemic Cell ◽  
Helical Structure ◽  
Leukemic Cells ◽  
Integrative Approach ◽  
Human Breast Milk ◽  
Dependent Manner ◽  
Leukemic Cell Lines ◽  
Late Morbidity

AbstractChemotherapy in childhood leukemia is associated with late morbidity in leukemic survivors, while certain patient subsets relatively resistant to standard chemotherapy. Identifying new agents with the sensitivity and selectivity toward leukemic cells with less systemic toxicity is a warrant. Peptide-based therapeutics is gaining attention during the last few years. Here, we used an integrative workflow combining mass spectrometric peptide library construction, in silico anticancer peptide screening, and in vitro leukemic cell studies to discover a novel anti-leukemic peptide owning 3+charges and alpha-helical structure, namely HMP-S7, from human breast milk. HMP-S7 showed cytotoxic activity against four distinct leukemic cell lines in a dose-dependent manner but had no affected on solid malignancies or representative normal cells. HMP-S7 induced leukemic cell death by penetrating the plasma membrane into the cytoplasm, causing lactate dehydrogenase leakage, thereby defining membranolytic action. In conclusion, HMP-S7 is the selective anti-leukemic peptide promising for further validation in preclinical and clinical studies.

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