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.

LINC01588 regulates WWP2-mediated cardiomyocyte injury by interacting with HNRNPL.

Cardiomyocyte dysfunction and apoptosis induced by ischemia-hypoxia are common features of many acute and chronic heart diseases. WW domain-containing E3 ubiquitin ligase (WWP2) has been identified as an important regulator in pathogenesis of some health-threatening diseases. Although a couple of recent reports prompted the potential role of WWP2 in heart dysfunction, however, its exact role and how its expression was regulated in ischemic-hypoxic cardiomyocytes are still elusive. Here, we found that WWP2 protein level was induced in anoxia/reoxygenation (A/R) treated cardiomyocytes in a time-dependent manner, accompanied by synchronous expression of LINC01588 and HNRNPL. Knockdown of LINC01588 increased cardiomyocyte apoptosis, the level of oxidative stress, and expression of pro-inflammatory cytokine genes, down-regulated the expression of WWP2 and promoted expression of SEPT4 gene that contributed to cardiomyocyte dysfunction and was a target gene of WWP2. LINC01588 overexpression improved the functions of A/R treated cardiomyocytes, up-regulated WWP2 and reduced SEPT4 expression. In the mechanism exploration, we found that LINC01588 could directly bind with HNRNPL protein that could interact with WWP2, suggesting that WWP2 was involved in the regulation of LINC01588 in A/R treated cardiomyocytes. Moreover, WWP2 inhibition declined the protective role of LINC01588 in cardiomyocyte dysfunction induced by A/R. Finally, we demonstrated that LINC01588 overexpression improved acute myocardial infarction in mice in vivo. In conclusion, LINC01588 improved A/R-induced cardiomyocyte dysfunction by interacting with HNRNPL and promoting WWP2-mediated degradation of SEPT4.

Electro-hyperthermia up-regulates tumour suppressor Septin 4 to induce apoptotic cell death in hepatocellular carcinoma.

PURPOSE: Modulated electro-hyperthermia (mEHT) has been shown to be effective against various types of human tumours, including hepatocellular carcinoma (HCC). Here we aimed to investigate the molecular mechanism underlying the cytotoxic effects of mEHT to HCC cells. MATERIALS AND METHODS: Human liver cancer cell lines, Huh7 and HepG2, were treated with mEHT (42 °C/60 min) three times at 2-day intervals. Growth inhibition and apoptotic induction were evaluated using MTS, microscopic analysis, a clonogenic assay, annexin V/PI staining and a ccK18 ELISA. Global changes in gene expression were examined using RNA sequencing to obtain insights into molecular changes in response to mEHT. For in vivo evaluation of mEHT we used HepG2 HCC xenografts grown in nude mice. RESULTS: mEHT suppressed HCC cell proliferation and long-term colony formation through induction of apoptosis. The growth inhibitory effects are induced through a subset of molecular changes. Notably the expression level of septin 4 (SEPT4) (involved in pro-apoptotic activity and growth suppression) was up-regulated, whereas a key regulator of invasiveness G-Protein coupled receptor 64 (GPR64) was repressed. Subsequent Western blotting confirmed that the common increase in tumour suppressor SEPT4 in both Huh7 and HepG2 cells is accompanied by the restoration of cyclin-dependent kinase (CDK) inhibitor p21 and decrease in pro-caspase 7 and pro-caspase 3, thereby accelerating apoptotic signalling in HCC cells. Additionally, mEHT significantly inhibited the growth of human HCC xenografts in nude mice. CONCLUSIONS: These findings suggest that apoptotic cell death induced by mEHT is mediated by the up-regulation of tumour suppressor SEPT4 in human HCC cells.

The ARTS of p53-dependent mitochondrial apoptosis.

The tumor-suppressive activity of p53 is largely attributed to its ability to induce cell death, including apoptosis, through transcription-dependent and transcription-independent mechanisms. On the one hand, nuclear p53 transcriptionally activates the expression of a myriad of pro-apoptotic BCL-2 family genes, such as NOXA, PUMA, BID, BAD, BIK, BAX, etc., whereas it inactivates the expression of anti-apoptotic BCL-2, BCL-XL, and MCL1, leading to mitochondrial apoptosis. On the other hand, cytoplasmic p53 also promotes mitochondrial apoptosis by directly associating with multiple BCL-2 family proteins in the mitochondria. Apoptosis-related protein in TGF-ß signaling pathway (ARTS), a mitochondria-localized pro-apoptotic protein encoded by an alternative spliced variant of the SEPT4 gene, triggers apoptosis by facilitating proteasomal degradation of BCL-2 and XIAP upon pro-apoptotic stimuli. We recently identified SEPT4/ARTS as a new p53 target gene in response to genotoxic stress. ARTS in turn binds to p53, drives its mitochondrial localization, and enhances the interaction between p53 and BCL-XL, thereby promoting mitochondrial apoptosis. This review will illustrate the mechanisms of p53-induced mitochondrial apoptosis, offer some recently discovered new insights into the functions of ARTS in regulating mitochondrial cell death, and discuss the clinical significance of ARTS in cancer and non-cancer diseases.

Evaluation of SEPT2 and SEPT4 transcript contents in spermatozoa from men with asthenozoospermia and teratozoospermia.

BACKGROUND AND AIMS: Motility and morphological defects of spermatozoa can cause male infertility. Sperm RNAs are related to sperm quality. They are considered to have clinical values as a biomarker for assessing sperm quality and fertility potential. The annulus, located in the mammalian sperm tail, is required for motility and terminal differentiation of the spermatozoa. SEPT2, 4, 6, 7, and 12 proteins are the main components of the annulus in the sperm tail. The study aimed to evaluate SEPT2 and SEPT4 mRNA contents in the spermatozoa of patients with asthenozoospermia and teratozoospermia. METHODS: We evaluated transcript levels of SEPT2 and SEPT4 in the sperm samples of 20 asthenozoospermic, 20 teratozoospermic, and 20 normozoospermic samples using quantitative PCR. RESULTS: The SEPT2 transcript level was significantly decreased in the asthenozoospermia samples compared with the normal group (P = .013). However, SEPT4 was not significantly different between these two groups. The transcript levels of SEPT2 and SEPT4 were not statistically different between teratozoospermic and normozoospermic groups. CONCLUSION: In conclusion, downregulation of SEPT2 in patients with asthenozoospermia appears to be associated with poor sperm motility.

PIP2 and septin control STIM1/Orai1 assembly by regulating cytoskeletal remodeling via a CDC42-WASP/WAVE-ARP2/3 protein complex.

Store-operated calcium entry (SOCE) is triggered by assembly of Orai1 with STIM proteins in ER-PM junctions. Plasma membrane PIP2 as well as PIP2-binding protein, SEPT4, significantly impact Orai1-STIM1 interaction. While septins and PIP2 can organize the actin cytoskeleton, it is unclear whether the status of actin within the junctions contributes to SOCE. We report herein that actin remodeling modulates STIM1 clustering. Our findings show that a PIP2- and SEPT4-dependent mechanism involving CDC42, WASP/WAVE, and ARP2 regulates actin remodeling into a ring-like structure around STIM1 puncta. CDC42 localization in the ER-plasma membrane region is enhanced following ER-Ca2+ store depletion. PIP2 depletion or knockdown of SEPT4 attenuate the recruitment of CDC42 to the ER-PM region. Importantly, knockdown of SEPT4, or CDC42+ARP2, disrupts the organization of actin as well as STIM1 clustering. Consequently, Orai1 recruitment to STIM1 puncta, SOCE, and NFAT translocation to the nucleus are all attenuated. Ca2+ influx induced by STIM1-C terminus is not affected by CDC42 knockdown. In aggregate, our findings reveal that PIP2 and SEPT4 affect Orai1/STIM1 clustering by coordinating actin remodeling within ER-PM junctions. This dynamic reorganization of actin has an important role in regulation of SOCE and downstream Ca2+-dependent effector functions.