Discovery of a septin-4 covalent binder with antimetastatic activity in a mouse model of melanoma.

Cancer spreading through metastatic processes is one of the major causes of tumour-related mortality. Metastasis is a complex phenomenon which involves multiple pathways ranging from cell metabolic alterations to changes in the biophysical phenotype of cells and tissues. In the search for new effective anti-metastatic agents, we modulated the chemical structure of the lead compound AA6, in order to find the structural determinants of activity, and to identify the cellular target responsible of the downstream anti-metastatic effects observed. New compounds synthesized were able to inhibit in vitro B16-F10 melanoma cell invasiveness, and one selected compound, CM365, showed in vivo anti-metastatic effects in a lung metastasis mouse model of melanoma. Septin-4 was identified as the most likely molecular target responsible for these effects. This study showed that CM365 is a promising molecule for metastasis prevention, remarkably effective alone or co-administered with drugs normally used in cancer therapy, such as paclitaxel.

Biallelic loss-of-function mutations in SEPTIN4 (C17ORF47), encoding a conserved annulus protein, cause thin midpiece spermatozoa and male infertility in humans.

Asthenoteratozoospermia is the primary cause of infertility in humans. However, the genetic etiology remains largely unknown for those suffering from severe asthenoteratozoospermia caused by thin midpiece defects. In this study, we identified two biallelic loss-of-function variants of SEPTIN4 (previously SEPT4) (Patient 1: c.A721T, p.R241* and Patient 2: c.C205T, p.R69*) in two unrelated individuals from two consanguineous Chinese families. SEPT4 is a conserved annulus protein that is critical for male fertility and the structural integrity of the sperm midpiece in mice. SEPT4 mutations disrupted the formation of SEPT-based annulus and localization of SEPTIN subunits in sperms from patients. The ultrastructural analysis demonstrated striking thin midpiece spermatozoa defects owing to annulus loss and disorganized mitochondrial sheath. Immunofluorescence and immunoblotting analyses of the mitochondrial sheath proteins TOMM20 and HSP60 further indicated that the distribution and abundance of mitochondria were impaired in men harboring biallelic SEPT4 variants. Additionally, we found that the precise localization of SLC26A8, a testis-specific anion transporter that colocalizes with SEPT4 at the sperm annulus, was missing without SEPT4. Moreover, the patient achieved a good pregnancy outcome following intracytoplasmic sperm injection. Overall, our study demonstrated for the first time that SEPT4 variants that induced thin midpiece spermatozoa defects were directly associated with human asthenoteratozoospermia.

Orai3 mediates Orai channel remodelling to activate fibroblast in pulmonary fibrosis.

Orai family are a calcium channel of cell membrane extracellular Ca2+ influx which participates in tissue fibrosis. But the roles of Orai3 have less attention on the mechanism of regulating lung fibrosis. In this study, we found that Orai3 expression was increased significantly in BLM-induced lung fibrosis. The knockdown of Orai3 decreased TGF-ß1-induced fibroblast proliferation, ECM production, activation of NFAT1 and Calpain/ERK signal pathway and glycolysis levels. Orai3 interacting with Orai1 was increased in BLM-induced lung fibrosis and TGF-ß1-induced fibroblast, while the Stim1 interacting with Orai1 and SOCE activity was suppressed, leading in a high and stable extracellular Ca2+ influx. Furthermore, the over-expression of Orai3 did not enhance Orai3 interacting with Orai1 under TGF-ß1 free fibroblast. And then, the deeper mechanism of TGF-ß1-induced increased SEPTIN4 promoted Orai3 interacting with Orai1. Our results indicated that Orai3 could be one of the therapy targets for PF in which remodels Orai channel, suppresses SOCE activity and activated fibroblast to alleviate fibrosis progress.

Septin 4 controls CCNB1 stabilization via APC/CCDC20 during meiotic G2/M transition in mouse oocytes.

In mammals, oocytes are arrested at G2/prophase for a long time, which is called germinal vesicle (GV) arrest. After puberty, fully-grown oocytes are stimulated by a gonadotropin surge to resume meiosis as indicated by GV breakdown (GVBD). CCNB1 is accumulated to a threshold level to trigger the activation of maturation promoting factor (MPF), inducing the G2/M transition. It is generally recognized that the anaphase-promoting complex/cyclosome (APC/C) and its cofactor CDH1 (also known as FZR1) regulates the accumulation/degradation of CCNB1. Here, by using small interfering RNA (siRNA) and messenger RNA (mRNA) microinjection, immunofluorescence and confocal microscopy, immunoprecipitation, time-lapse live imaging, and immunoblotting analysis, we showed that Septin 4 regulates the G2/M transition by regulating the accumulation of CCNB1 via APC/CCDC20 . Depletion of Septin 4 caused GV arrest by reducing CCNB1 accumulation. Unexpectedly, the expression level of CDC20 was higher in Septin 4 siRNA-injected oocytes than in control oocytes, but there was no significant change in the expression level of CDH1. Importantly, the reduced GVBD after Septin 4 depletion could be rescued not only by over-expressing CCNB1 but also could be partially rescued by depleting CDC20. Taken together, our results demonstrate that Septin 4 may play a critical role in meiotic G2/M transition by indirect regulation of CCNB1 stabilization in mouse oocytes.

Septin4 as an autophagy modulator regulates Angiotensin-II mediated VSMCs proliferation and migration.

Vascular smooth muscle cells (VSMCs) proliferation and migration play a fundamental role during the process of hypertensive angiopathy. Angiotensin-II (Ang-II) is one of the robust phenotype-modulating agents, which changes VSMCs to efficiently proliferate and migrate. The mechanism of the proliferation and migration is not well understood yet. Septin4, as a member of GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be an essential component of the cytoskeleton which is involved in many important physiological processes. We approved that Septin4 expression was upregulated in mouse aorta by continuous infusion of Ang-II and in cultured VSMCs treated with Ang-II. Overexpression of Septin4 led to lower level of autophagy and decreased capacity of proliferation and migration. In order to identify the mechanism by which Septin4 interacts with these processes, we blocked autophagy by chloroquine (CQ). After inhibiting the autophagy, the ability of proliferation and migration was further restrained in the Septin4 overexpression VSMCs. In conclusion, our results indicated that during the process of VSMCs proliferation and migration induced by Ang-II, Septin4 modulated autophagy and thus regulated the activity of proliferation and migration.

Septin4 as a novel binding partner of PARP1 contributes to oxidative stress induced human umbilical vein endothelial cells injure.

Oxidative stress induced vascular endothelial cell injure is one of the key and initial event in the development of atherosclerosis. Septin4, as a member of GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be an essential component of the cytoskeleton which is involved in many important physiological processes. However, whether Septin4 is involved in cardiovascular diseases, such as oxidative stress inducted endothelial cell injury still unclear. PARP1 as a DNA repair enzyme can be activated by identifying DNA damaged fragments, which consumes high levels of energy and leads to vascular endothelial cell apoptosis. Here, our results first found that Septin4 is involved in oxidative stress induced endothelial cell ROS production and apoptosis through knock-down and over-expression Septin4 approaches. Furthermore, to explore how Septin4 is involved in oxidative stress induced endothelial cells injure, we first identified that Septin4 is a novel PARP1 interacting protein and the interaction is enhanced under oxidative stress. In conclusions, our founding indicates that Septin4 is a novel essential factor involved in oxidative stress induced vascular endothelial cell injury by interacting with apoptosis-related protein PARP1.

The correlation of Septin4 gene expression with sperm quality, DNA damage, and oxidative stress level in infertile patients.

Septin4 belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. Since, Septin4 is expressed specifically in the testis, we aimed to determine the association between Septin4 gene expression with sperm quality, DNA damage, and stress oxidative level in infertile patients. The present study included 60 semen samples that grouped into three groups: normozoospermia (n=20), asthenozoospermia (n=20), astheno-teratozoospermia (n=20). Initially, semen parameters were analyzed by using the World Health Organization protocol. The mRNA expression of Septin4 in sperm was examined using reverse transcription-polymerase chain reaction. Oxidative stress markers, i.e., total antioxidant capacity, superoxide dismutase, catalase, glutathione peroxidase, and malondialdehyde, were determined by ELISA kit. The current study showed a statistically significant highly positive correlation in Septin4 gene expression with sperm motility, normal morphology, viability, capacity, and sperm mitochondrial membrane potential (MMP). However, it showed significant negative correlation with sperm DNA fragmentation. Septin4 had a significant correlation with stress oxidative factor and antioxidant enzyme levels. In conclusion, Septin4 gene expression provides clinical useful information for the diagnosis of male infertility. It might be a marker for discrimination between fertile and infertile patients. The current study showed a statistically significant highly positive correlation in Septin4 gene expression with sperm motility, normal morphology, viability, capacity, and sperm MMP. However, it shows significant negative correlation with sperm DNA fragmentation. Septin4 had a significant correlation with stress oxidative factor and antioxidant enzyme levels.

Identification and Structure Prediction of Human Septin-4 as a Biomarker for Diagnosis of Asthenozoospermic Infertile Patients-Critical Finding Toward Personalized Medicine.

Semen parameters are been found as a key factor to evaluate the count and morphology in the given semen sample. The deep knowledge of male infertility will unravel with semen parameters correlated with molecular and biochemical parameters. The current research study is to identify the motility associated protein and its structure through the in-silico approach. Semen samples were collected and initial analysis including semen parameters was analyzed by using the World Health Organization protocol. Semen biochemical parameters, namely, seminal plasma protein concentration, fructose content, and glucosidase content were calculated and evaluated for correlation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) were carried out for identification of Septin-4 presence in the semen sample. Mascot search was done for protein conformation and in-silico characterization of Septin-4 by structural modeling in Iterative Threading Assembly Refinement (I-TASSER). Twenty-five nanoseconds molecular dynamics (MD) simulations results showed the stable nature of Septin-4 in the dynamic system. Overall, our results showed the presence of motility-associated protein in normospermia and control samples and not in the case of asthenospermia and oligoasthenospermia. Molecular techniques characterized the presence of Septin-4 and as a novel biomarker for infertility diagnosis.

Septin 4 activates PPARγ/LXRα signaling by upregulating ABCA1 and ABCG1 expression to inhibit the formation of THP-1 macrophage-derived foam cells.

Septin 4 is a member of a family of GTP-binding proteins that has been previously reported regulate cytoskeletal organization. In addition, it has been suggested to serve a role in atherosclerosis. Therefore, the present study aimed to investigate the effects of Septin 4 on foam cell formation. THP-1 cells were first exposed to phorbol-12-myristate-13-acetate for differentiation into macrophages before being transformed into foam cells by treatment with oxidized low-density lipoprotein (ox-LDL). Septin 4 expression was then knocked down or overexpressed in THP-1 cells using transfection, whilst peroxisome proliferator activated receptor γ (PPARγ) was also inhibited using its selective antagonist (T0070907) in the presence of Septin 4 overexpression. Oil red staining was used to detect lipid uptake, and total cholesterol (TC), free cholesterol (FC) and ATP binding cassette subfamily A/G member 1 (ABCA1/G1) protein expression were also measured. The results demonstrated that upon ox-LDL stimulation, macrophages that were derived from THP-1 cells transformed into foam cells, where Septin 4 was highly expressed in ox-LDL-induced foam cells. Septin 4 knockdown promoted TC and FC levels, but reduced ABCA1/G1 protein expression. The protein expression levels of PPARγ and liver X receptor α (LXRα) were also decreased after Septin 4 knockdown. However, Septin 4 overexpression resulted in the opposite results being observed. Additionally, blocking PPARγ activity using its inhibitor T0070907 or knocking down LXRα expression using short hairpin RNA reversed the effects of Septin 4 overexpression on foam cell formation and cholesterol handling. In conclusion, Septin 4 may serve an important role in preventing foam cell formation by activating PPARγ/LXRα signaling and subsequently enhancing ABCA1/G1 expression.

Septin4 Prevents PDGF-BB-induced HAVSMC Phenotypic Transformation, Proliferation and Migration by Promoting SIRT1-STAT3 Deacetylation and Dephosphorylation.

SIRT1 and STAT3 are key to human aortic vascular smooth muscle cells (HAVSMCs) proliferation, migration and phenotypic transformation, but the regulatory mechanism of SIRT1-STAT3 in this process is still unclear. Septin4 is a cytoskeleton-related protein that regulates oxidative stress-vascular endothelial injury. However, the role and underlying mechanism of Septin4 in atherosclerosis remains unknown. Here, we revealed the role and mechanism of Septin4 in regulating SIRT1-STAT3 in atherosclerosis. We determined that the expression of Septin4 were markedly increased in Apoe-/- atherosclerosis mice and PDGF-BB-induced HAVSMCs. Knockdown of Septin4 significantly increased PDGF-BB-induced HAVSMCs proliferation, migration and phenotypic transformation, while overexpression of Septin4 had the opposite effects. Mechanically, co-immunoprecipitation results demonstrated that Septin4 was a novel interacting protein of STAT3 and SIRT1. Septin4 formed a complex with SIRT1-STAT3, enhancing the interaction between SIRT1 and STAT3, ensuing promoting SIRT1-regulated STAT3-K685 deacetylation and STAT3-Y705 dephosphorylation, which inhibited PDGF-BB-induced HAVSMCs proliferation, migration and phenotype transformation. Therefore, our findings provide novel insights into the prevention and treatment of atherosclerosis.

Septin4 promotes cell death in human colon cancer cells by interacting with BAX.

Septin4 is a tumor suppressor protein that promotes cell programmed death in various cell types through specifically antagonizing XIAP (X linked inhibitor of apoptosis), little is known its other novel binding partner and role in colorectal cancer. In this study, we found that Septin4 significantly expressed lower in human colon cancer when compared to peri-tumor benign cells, and its low expression was significantly associated with worse prognostic outcomes. Furthermore, Septin4 participated in DOX-induced colon cancer cell death in vitro. Septin4-overexpressing colon cancer cells displayed augmented apoptotic cell death and ROS production. Additionally, Septin4-knockdown cells revealed a resistance of DOX-induced cell death and reduced ROS production. Importantly, we first identified that BAX is a novel Septin4 binding partner and the interaction is enhanced under DOX treatment. Finally, Septin4-knockdown promoted colon cells growth in vivo. These observations suggest that Septin4 as an essential molecule contribute to the occurrence and development of human colon cancer and provide new technical approaches for targeted treatment of this disease.

Role of WW domain E3 ubiquitin protein ligase 2 in modulating ubiquitination and Degradation of Septin4 in oxidative stress endothelial injury.

Oxidative stress-associated endothelial injury is the initial event and major cause of multiple cardiovascular diseases such as atherosclerosis and hypertensive angiopathy. A protein homeostasis imbalance is a critical cause of endothelial injury, and homologous to E6AP C-terminus (HECT)-type E3 ubiquitin ligases are the core factors controlling protein homeostasis. Although HECT-type E3 ubiquitin ligases are involved in the regulation of cardiac development and diseases, their roles in endothelial injury remain largely unknown. This study aimed to identify which HECT-type E3 ubiquitin ligase is involved in endothelial injury and clarify the mechanisms at molecular, cellular, and organism levels. We revealed a novel role of the HECT-type E3 ubiquitin ligase WWP2 in regulating endothelial injury and vascular remodeling after endothelial injury. Endothelial/myeloid-specific WWP2 knockout in mice significantly aggravated angiotensin II/oxidative stress-induced endothelial injury and vascular remodeling after endothelial injury. The same results were obtained from in vitro experiments. Mechanistically, the endothelial injury factor Septin4 was identified as a novel physiological substrate of WWP2. In addition, WWP2 interacted with the GTPase domain of Septin4, ubiquitinating Septin4-K174 to degrade Septin4 through the ubiquitin-proteasome system, which inhibited the Septin4-PARP1 endothelial damage complex. These results identified the first endothelial injury-associated physiological pathway regulated by HECT-type E3 ubiquitin ligases in vivo as well as a unique proteolytic mechanism through which WWP2 controls endothelial injury and vascular remodeling after endothelial injury. These findings might provide a novel treatment strategy for oxidative stress-associated atherosclerosis and hypertensive vascular diseases.

Septin4 regulates endoplasmic reticulum stress and apoptosis in melatonin­induced osteoblasts.

Idiopathic scoliosis (IS) is a spinal 3­dimensional deformity with an unknown cause. Melatonin is secreted by the pineal body and contributes to the occurrence and progression of IS. In our previous preliminary study, it was reported that high concentrations of melatonin can induce osteoblast apoptosis, thus acting as an IS treatment, but the mechanism of action is unknown. Therefore, the present study was performed to further investigate the possible mechanism underlying the efficacy of melatonin as a treatment for IS. The present results indicated that high concentrations of melatonin mediate endoplasmic reticulum stress (ERS)­induced apoptosis in hFOB 1.19 cells, and this resulted in a significant and dose­dependent increase in the expression of Septin4, as well as the expression levels of glucose­regulated protein (GRP)78, GRP94 and cleaved caspase­3. Furthermore, osteoblasts were overexpressed with Septin4 and the mechanism via which melatonin induces osteoblast ERS was demonstrated to be via the regulation of Septin4. In addition, it was indicated that cytoskeleton destruction, cell morphology changes and the decrease in the number of cells were aggravated after osteoblasts were overexpressed with Septin4, as indicated by phalloidin and DAPI staining. Collectively, the present results suggest that the Septin4 protein may be a target of ERS in melatonin­induced osteoblast apoptosis, which is involved in bone metabolism diseases, thus providing novel evidence for clinical melatonin treatment of IS.

Adenovirus-mediated over-expression of Septin4 ameliorates hepatic fibrosis in mouse livers infected with Schistosoma japonicum.

Septin4 (Sept4) belongs to Septin family and may be involved in apoptosis, vesicle trafficking and other cell processes. In this study, we attempted to investigate the effect of Sept4 in hepatic fibrosis induced by Schistosoma japonicum. ICR mice infected with S. japonicum for 12weeks were treated with PBS, Ad-ctr and Ad-Sept4, respectively. All mice were killed at 2weeks after injection, and the changes in the fibrotic livers were detected via H&E staining, Sirius red staining, qRT-PCR, western blot and TUNEL analysis. In addition, pcDNA3.1-Sept4 plasmid was transfected into LX-2 cells to observe the effect of Sept4 on apoptosis of HSCs in vitro. Ad-Sept4 could ameliorate liver fibrosis, as detected by H&E staining and Sirius red staining. The number of TUNEL-positive cells was increased in the Ad-Sept4 treated group. The expression of Sept4 and cleaved-caspase-3 were all augmented, while the expression of α-SMA, Col1α1 and IL-13 were reduced in the Ad-Sept4 treated group, compared with that expressed in the Ad-ctr group. Over-expression of Sept4 in LX-2 cells could promote apoptosis of LX-2 cells in vitro. In conclusion, Ad-Sept4 can attenuate the development of liver fibrosis induced by S. japonicum through apoptosis.

Septin4_i1 regulates apoptosis in hepatic stellate cells through peroxisome proliferator-activated receptor-γ/Akt/B-cell lymphoma 2 pathway.

Apoptosis of activated hepatic stellate cells (HSCs) has been verified as a potential mechanism to aid in hepatic fibrosis remission. Earlier research suggests that Septin4_i1 may sensitize hepatocellular carcinoma cells to serum starvation-induced apoptosis. Here, we aimed to investigate the effect of Septin4_i1 on HSC apoptosis and explore the associated signaling pathways. We found that Septin4_i1 can induce apoptosis in LX-2 cells and that this is accompanied by an up-regulation in cleaved-caspase-3 and peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and a down-regulation in α-SMA expression. Over-expression of Septin4_i1 reduced phosphorylated Akt and B-cell lymphoma 2 (Bcl-2) expression but had no effect on the expression of p53 and death receptor (DR)-5. The decreased expression of Bcl-2 and the increased expression of cleaved-caspase-3 induced by Sept4_i1 could be reversed by GW501516, a PPAR-ß/δ agonist that has been reported by others to enhance Akt signaling. In addition, GW9662, an antagonist of PPAR-γ, could also inhibit apoptosis in LX-2 cells induced by Sept4_i1. In conclusion, our data suggest that Sept4_i1 induces HSC apoptosis by inhibiting Akt and Bcl-2 expression and up-regulating PPAR-γ expression.