Prohibitin 2 Is an Inner Mitochondrial Membrane Mitophagy Receptor.

The removal of unwanted or damaged mitochondria by autophagy, a process called mitophagy, is essential for key events in development, cellular homeostasis, tumor suppression, and prevention of neurodegeneration and aging. However, the precise mechanisms of mitophagy remain uncertain. Here, we identify the inner mitochondrial membrane protein, prohibitin 2 (PHB2), as a crucial mitophagy receptor involved in targeting mitochondria for autophagic degradation. PHB2 binds the autophagosomal membrane-associated protein LC3 through an LC3-interaction region (LIR) domain upon mitochondrial depolarization and proteasome-dependent outer membrane rupture. PHB2 is required for Parkin-induced mitophagy in mammalian cells and for the clearance of paternal mitochondria after embryonic fertilization in C. elegans. Our findings pinpoint a conserved mechanism of eukaryotic mitophagy and demonstrate a function of prohibitin 2 that may underlie its roles in physiology, aging, and disease.

N-acetylgalactosaminyltransferase GALNT6 is a potential therapeutic target of clear cell renal cell carcinoma progression.

High expression of truncated O-glycans Tn antigen predicts adverse clinical outcome in patients with clear cell renal cell carcinoma (ccRCC). To understand the biosynthetic underpinnings of Tn antigen changes in ccRCC, we focused on N-acetylgalactosaminyltransferases (GALNTs, also known as GalNAcTs) known to be involved in Tn antigen synthesis. Data from GSE15641 profile and local cohort showed that GALNT6 was significantly upregulated in ccRCC tissues. The current study aimed to determine the role of GALNT6 in ccRCC, and whether GALNT6-mediated O-glycosylation aggravates malignant behaviors. Gain- and loss-of-function experiments showed that overexpression of GALNT6 accelerated ccRCC cell proliferation, migration, and invasion, as well as promoted ccRCC-derived xenograft tumor growth and lung metastasis. In line with this, silencing of GALNT6 yielded the opposite results. Mechanically, high expression of GALNT6 led to the accumulation of Tn antigen in ccRCC cells. By undertaking immunoprecipitation coupled with liquid chromatography/mass spectrometry, vicia villosa agglutinin blot, and site-directed mutagenesis assays, we found that O-glycosylation of prohibitin 2 (PHB2) at Ser161 was required for the GALNT6-induced ccRCC cell proliferation, migration, and invasion. Additionally, we identified lens epithelium-derived growth factor (LEDGF) as a key regulator of GALNT6 transcriptional induction in ccRCC growth and an upstream contributor to ccRCC aggressive behavior. Collectively, our findings indicate that GALNT6-mediated abnormal O-glycosylation promotes ccRCC progression, which provides a potential therapeutic target in ccRCC development.

Progesterone-induced progesterone receptor membrane component 1 rise-to-decline changes are essential for decidualization.

BACKGROUND: Decidualization of endometrial cells is the prerequisite for embryo implantation and subsequent placenta formation and is induced by rising progesterone levels following ovulation. One of the hormone receptors contributing to endometrial homeostasis is Progesterone Receptor Membrane Component 1 (PGRMC1), a non-classical membrane-bound progesterone receptor with yet unclear function. In this study, we aimed to investigate how PGRMC1 contributes to human decidualization. METHODS: We first analyzed PGRMC1 expression profile during a regular menstrual cycle in RNA-sequencing datasets. To further explore the function of PGRMC1 in human decidualization, we implemented an inducible decidualization system, which is achieved by culturing two human endometrial stromal cell lines in decidualization-inducing medium containing medroxyprogesterone acetate and 8-Br-cAMP. In our system, we measured PGRMC1 expression during hormone induction as well as decidualization status upon PGRMC1 knockdown at different time points. We further conferred proximity ligation assay to identify PGRMC1 interaction partners. RESULTS: In a regular menstrual cycle, PGRMC1 mRNA expression is gradually decreased from the proliferative phase to the secretory phase. In in vitro experiments, we observed that PGRMC1 expression follows a rise-to-decline pattern, in which its expression level initially increased during the first 6 days after induction (PGRMC1 increasing phase) and decreased in the following days (PGRMC1 decreasing phase). Knockdown of PGRMC1 expression before the induction led to a failed decidualization, while its knockdown after induction did not inhibit decidualization, suggesting that the progestin-induced 'PGRMC1 increasing phase' is essential for normal decidualization. Furthermore, we found that the interactions of prohibitin 1 and prohibitin 2 with PGRMC1 were induced upon progestin treatment. Knocking down each of the prohibitins slowed down the decidualization process compared to the control, suggesting that PGRMC1 cooperates with prohibitins to regulate decidualization. CONCLUSIONS: According to our findings, PGRMC1 expression followed a progestin-induced rise-to-decline expression pattern during human endometrial decidualization process; and the correct execution of this expression program was crucial for successful decidualization. Thereby, the results of our in vitro model explained how PGRMC1 dysregulation during decidualization may present a new perspective on infertility-related diseases.

PHB2 Maintains the Contractile Phenotype of VSMCs by Counteracting PKM2 Splicing.

BACKGROUND: Phenotypic transition of vascular smooth muscle cells (VSMCs) accounts for the pathogenesis of a variety of vascular diseases during the early stage. Recent studies indicate the metabolic reprogramming may be involved in VSMC phenotypic transition. However, the definite molecules that link energy metabolism to distinct VSMC phenotype remain elusive. METHODS: A carotid artery injury model was used to study postinjury neointima formation as well as VSMC phenotypic transition in vivo. RNA-seq analysis, cell migration assay, collagen gel contraction assay, wire myography assay, immunoblotting, protein interactome analysis, co-immunoprecipitation, and mammalian 2-hybrid assay were performed to clarify the phenotype and elucidate the molecular mechanisms. RESULTS: We collected cell energy-regulating genes by using Gene Ontology annotation and applied RNA-Seq analysis of transforming growth factor-ß or platelet-derived growth factor BB stimulated VSMCs. Six candidate genes were overlapped from energy metabolism-related genes and genes reciprocally upregulated by transforming growth factor-ß and downregulated by platelet-derived growth factor BB. Among them, prohibitin 2 has been reported to regulate mitochondrial oxidative phosphorylation. Indeed, prohibitin 2-deficient VSMCs lost the contractile phenotype as evidenced by reduced contractile proteins. Consistently, Phb2SMCKO mice were more susceptible to postinjury VSMC proliferation and neointima formation compared with Phb2flox/flox mice. Further protein interactome analysis, co-immunoprecipitation, and mammalian 2-hybrid assay revealed that prohibitin 2, through its C-terminus, directly interacts with hnRNPA1, a key modulator of pyruvate kinase M1/2 (PKM) mRNA splicing that promotes PKM2 expression and glycolysis. Prohibitin 2 deficiency facilitated PKM1/2 mRNA splicing and reversion from PKM1 to PKM2, and enhanced glycolysis in VSMCs. Blocking prohibitin 2-hnRNPA1 interaction resulted in increased PKM2 expression, enhanced glycolysis, repressed contractile marker genes expression in VSMCs, as well as aggravated postinjury neointima formation in vivo. CONCLUSIONS: Prohibitin 2 maintains VSMC contractile phenotype by interacting with hnRNPA1 to counteract hnRNPA1-mediated PKM alternative splicing and glucose metabolic reprogramming.

Targeting PHB2 mediated mitophagy alleviates non-esterified fatty acid-induced mitochondrial dysfunction in bovine mammary epithelial cells.

Mitochondrial dysfunction has been reported to occur in the mammary gland of dairy cows suffering from ketosis. Prohibitin 2 (PHB2) plays a crucial role in regulating mitophagy, which clears impaired mitochondria to maintain normal mitochondrial function. Therefore, the current study aimed to investigate how PHB2 mediates mitophagy, thereby influencing mitochondrial function in the bovine mammary epithelial cell MAC-T. First, mammary gland tissue and blood samples were collected from healthy cows (control; n = 15, BHB <0.6 mM) and cows with clinical ketosis (CK; n = 15, BHB >3.0 mM). Compared with the control group, the CK group exhibited lower dry matter intake (DMI), milk production, milk protein, milk lactose, and serum glucose. In contrast, milk fat, serum nonesterified fatty acids (NEFA) and BHB were greater in CK group. The protein abundance of PHB2, peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α), mitofusin 2 (MFN2) in whole cell lysates (WCL), as well as PHB2, sequestosome-1 (SQSTM1, also called p62), microtubule-associated protein 1 light chain 3-II (LC3-II), and ubiquitinated proteins in mitochondrial fraction were significantly lower in the CK group. ATP content of mammary gland tissue in CK group was lower than that of healthy cows. Second, MAC-T were cultured and treated with NEFA (0, 0.3, 0.6, 1.2 mM). MAC-T treated with 1.2 mM NEFA displayed decreased protein abundance of PHB2, PGC-1α, MFN2 in WCL, as well as protein abundance of PHB2, p62, LC3-II, and ubiquitinated proteins in mitochondrial fraction. The content of ATP and JC-1 aggregates in 1.2 mM NEFA group were lower than in the 0 mM NEFA group. Additionally, 1.2 mM NEFA disrupted the fusion between mitochondria and lysosomes. MAC-T were then pretreated with 100 nM rapamycin, followed by treatment with or without NEFA. Rapamycin alleviated impaired mitophagy and mitochondria dysfunction induced by 1.2 mM NEFA. Third, MAC-T were transfected with small interfering RNA to silence PHB2 or a plasmid for overexpression of PHB2, followed by treatment with or without NEFA. The silencing of PHB2 aggravated 1.2 mM NEFA induced impaired mitophagy and mitochondrial dysfunction, whereas the overexpression of PHB2 alleviated these effects. Overall, this study provides evidence that PHB2, in regulation of mitophagy, is a mechanism for bovine mammary epithelial cells to counteract NEFA-induced mitochondrial dysfunction.

The expression of PHB2 in the cochlea: Possible relation to age-related hearing loss.

Age-related hearing loss (ARHL) is the most prevalent sensory deficit in the elderly, but its mechanism remains unclear. Scaffold protein prohibitin 2 (PHB2) has been widely involved in aging and neurodegeneration. However, the role of PHB2 in ARHL is undeciphered to date. To investigate the expression pattern and the role of PHB2 in ARHL, we used C57BL/6 mice and HEI-OC1 cell line as models. In our study, we have found PHB2 exists in the cochlea and is expressed in hair cells, spiral ganglion neurons, and HEI-OC1 cells. In mice with ARHL, mitophagy is reduced and correspondingly the expression level of PHB2 is decreased. Moreover, after H2 O2 treatment the mitophagy is activated and the PHB2 expression is increased. These findings indicate that PHB2 may exert an important role in ARHL through mitophagy. Findings from this study will be helpful for elucidating the mechanism underlying the ARHL and for providing a new target for ARHL treatment.

Small-molecule screening yields a compound that inhibits the cancer-associated transcription factor Hes1 via the PHB2 chaperone.

The transcription factor Hes family basic helix-loop-helix transcription factor 1 (Hes1) is a downstream effector of Notch signaling and plays a crucial role in orchestrating developmental processes during the embryonic stage. However, its aberrant signaling in adulthood is linked to the pathogenesis of cancer. In the present study, we report the discovery of small organic molecules (JI051 and JI130) that impair the ability of Hes1 to repress transcription. Hes1 interacts with the transcriptional corepressor transducing-like enhancer of split 1 (TLE1) via an interaction domain comprising two tryptophan residues, prompting us to search a chemical library of 1,800 small molecules enriched for indole-like π-electron-rich pharmacophores for a compound that blocks Hes1-mediated transcriptional repression. This screening identified a lead compound whose extensive chemical modification to improve potency yielded JI051, which inhibited HEK293 cell proliferation with an EC50 of 0.3 µm Unexpectedly, using immunomagnetic isolation and nanoscale LC-MS/MS, we found that JI051 does not bind TLE1 but instead interacts with prohibitin 2 (PHB2), a cancer-associated protein chaperone. We also found that JI051 stabilizes PHB2's interaction with Hes1 outside the nucleus, inducing G2/M cell-cycle arrest. Of note, JI051 dose-dependently reduced cell growth of the human pancreatic cancer cell line MIA PaCa-2, and JI130 treatment significantly reduced tumor volume in a murine pancreatic tumor xenograft model. These results suggest a previously unrecognized role for PHB2 in the regulation of Hes1 and may inform potential strategies for managing pancreatic cancer.

Mitophagy Reduces Oxidative Stress Via Keap1 (Kelch-Like Epichlorohydrin-Associated Protein 1)/Nrf2 (Nuclear Factor-E2-Related Factor 2)/PHB2 (Prohibitin 2) Pathway After Subarachnoid Hemorrhage in Rats.

Background and Purpose- Mitoquinone has been reported as a mitochondria-targeting antioxidant to promote mitophagy in various chronic diseases. Here, our aim was to study the role of mitoquinone in mitophagy activation and oxidative stress-induced neuronal death reduction after subarachnoid hemorrhage (SAH) in rats. Methods- Endovascular perforation was used for SAH model of male Sprague-Dawley rats. Exogenous mitoquinone was injected intraperitoneally 1 hour after SAH. ML385, an inhibitor of Nrf2 (nuclear factor-E2-related factor 2), was given intracerebroventricularly 24 hours before SAH. Small interfering RNA for PHB2 (prohibitin 2) was injected intracerebroventricularly 48 hours before SAH. Nuclear, mitochondrial, and cytoplasmic fractions were gathered using nucleus and mitochondria isolation kits. SAH grade evaluation, short- and long- term neurological function tests, oxidative stress, and apoptosis measurements were performed. Pathway related proteins were investigated with Western blot and immunofluorescence staining. Results- Expression of Keap1 (Kelch-like epichlorohydrin-associated protein 1, 2.84× at 24 hours), Nrf2 (2.78× at 3 hours), and LC3II (light chain 3-II; 1.94× at 24 hours) increased, whereas PHB2 (0.46× at 24 hours) decreased after SAH compared with sham group. Mitoquinone treatment attenuated oxidative stress and neuronal death, both short-term and long-term. Administration of mitoquinone resulted in a decrease in expression of Keap1 (0.33×), Romo1 (reactive oxygen species modulator 1; 0.24×), Bax (B-cell lymphoma-2 associated X protein; 0.31×), Cleaved Caspase-3 (0.29×) and an increase in Nrf2 (2.13×), Bcl-xl (B-cell lymphoma-extra large; 1.67×), PINK1 (phosphatase and tensin-induced kinase 1; 1.67×), Parkin (1.49×), PHB2 (1.60×), and LC3II (1.67×) proteins compared with SAH+vehicle group. ML385 abolished the treatment effects of mitoquinone on behavior and protein levels. PHB2 small interfering RNA reversed the outcomes of mitoquinone administration through reduction in protein expressions downstream of PHB2. Conclusions- Mitoquinone inhibited oxidative stress-related neuronal death by activating mitophagy via Keap1/Nrf2/PHB2 pathway after SAH. Mitoquinone may serve as a potential treatment to relieve brain injury after SAH.

Prohibitin 2-mediated mitophagy attenuates renal tubular epithelial cells injury by regulating mitochondrial dysfunction and NLRP3 inflammasome activation.

Accumulating evidence demonstrates that mitochondrial dysfunction and inflammasome activation play a critical role in the pathogenesis of renal tubular injury through the production of reactive oxygen species and cytokines. Prohibitin 2 (PHB2) is a newly identified intracellular receptor of mitophagy (a type of autophagy) that mediates selective removal of damaged mitochondria, and it could possibly play a renoprotective role in kidney disease. In this study, we confirmed that autophagy is activated in tubular epithelial cells treated with angiotensin II and that inhibition of autophagy results in tubular cell injury. Strikingly, PHB2 knockdown reduced the level of mitophagy and augmented cell death, while overexpression of PHB2 provided protection against pyrin domain-containing protein 3 (NLRP3)-induced inflammatory pathways through amelioration of mitochondrial dysfunction. Our research is the first to experimentally demonstrate the role of PHB2 in renal proximal tubular cells and thereby to provide a better understanding of how autophagy modulates inflammation in renal tubules. These data highlight PHB2 as a therapeutic target in the future treatment of CKD.

Biophysical characterization of the breast cancer-related BIG3-PHB2 interaction: Effect of non-conserved loop region of BIG3 on the structure and the interaction.

Brefeldin A-inhibited guanine nucleotide-exchange protein 3 (BIG3) interacts with and inhibits the tumor suppressor function of prohibitin-2 (PHB2), and recent in vivo studies have demonstrated that the BIG3-PHB2 interaction is a promising target for breast cancer therapy. However, little biophysical characterization on BIG3 and its interaction with PHB2 has been reported. Here we compared the calculated 8-class secondary structure of the N-terminal domains of BIG family proteins and identified a loop region unique to BIG3. Our biophysical characterization demonstrated that this loop region significantly affects the colloidal and thermodynamic stability of BIG3 and the thermodynamic and kinetic profile of its interaction with PHB2. These results establish a model for the BIG3-PHB2 interaction and an entry for drug discovery for breast cancer.

Prohibitin-2 is a novel regulator of p21WAF1/CIP1 induced by depletion of γ-glutamylcyclotransferase.

Previous studies show that gamma-glutamylcyclotransferase (GGCT) is expressed at high levels in various cancer tissues and that its knockdown inhibits MCF7 cancer cell growth via upregulation of p21WAF1/CIP1 (p21). However, the detailed underlying mechanism is unclear. Here, we used yeast two-hybrid screening and co-immunoprecipitation to identify Prohibitin-2 (PHB2) as a novel protein that interacts with GGCT. We also show that nuclear expression of PHB2 in MCF7 cells falls upon GGCT knockdown, and that overexpression of PHB2 inhibits p21 upregulation. A chromatin immunoprecipitation assay revealed that nuclear PHB2 proteins bind to the p21 promoter, and that this interaction is abrogated by GGCT knockdown. Moreover, knockdown of PHB2 alone led to significant upregulation of p21 and mimicked the cellular events induced by GGCT depletion, including G0/G1 arrest, cellular senescence, and growth inhibition, in a p21 induction-dependent manner. Taken together, the results indicate that PHB2 plays a central role in p21 upregulation following GGCT knockdown and as such may promote deregulated proliferation of cancer cells by suppressing p21.

Differential binding of prohibitin-2 to estrogen receptor α and to drug-resistant ERα mutants.

Endocrine resistance is one of the most challenging problems in estrogen receptor alpha (ERα)-positive breast cancer. The transcriptional activity of ERα is controlled by several coregulators, including prohibitin-2 (PHB2). Because of its ability to repress the transcriptional activity of activated ERα, PHB2 is a promising antiproliferative agent. In this study, were analyzed the interaction of PHB2 with ERα and three mutants (Y537S, D538G, and E380Q) that are frequently associated with a lack of sensitivity to hormonal treatments, to help advance novel drug discovery. PHB2 bound to ERα wild-type (WT), Y537S, and D538G, but did not bind to E380Q. The binding thermodynamics of Y537S and D538G to PHB2 were favorably altered entropically compared with those of WT to PHB2. Our results show that PHB2 binds to the ligand binding domain of ERα with a conformational change in the helix 12 of ERα.

Prohibitin-2 binding modulates insulin-like growth factor-binding protein-6 (IGFBP-6)-induced rhabdomyosarcoma cell migration.

Insulin-like growth factor (IGF)-binding protein (IGFBP)-6 decreases cancer cell proliferation and survival by inhibiting the effects of IGF-II. More recently, IGFBP-6 was found to promote the migration of rhabdomyosarcoma (RMS) cells in an IGF-independent manner, and MAPK pathways were involved in this process. However, the precise molecular mechanisms of these IGF-independent migratory actions of IGFBP-6 are largely unknown. Here, we report that prohibitin-2 (PHB2), a single-span membrane protein, is a key regulator of IGFBP-6-induced RMS cell migration. PHB2 and IGFBP-6 co-localize on the RMS cell surface, and they specifically interact, as demonstrated by affinity chromatography, co-immunoprecipitation, biosensor analysis, and confocal microscopy. Binding affinities for PHB2 are 9.0 ± 1.0 nM for IGFBP-6 and 10.2 ± 0.5 nM for mIGFBP-6, a non-IGF-binding mutant of IGFBP-6. The C-domain but not the N-domain of IGFBP-6 is involved in PHB2 binding. In addition, IGFBP-6 indirectly increases PHB2 tyrosine phosphorylation on RMS membranes. Importantly, PHB2 knockdown completely abolished IGFBP-6-mediated RMS cell migration. In contrast, IGFBP-6-induced MAPK pathway activation was not affected, suggesting that PHB2 may act as a downstream effector of these pathways. These results indicate that PHB2 plays a key role in this IGF-independent action of IGFBP-6 and suggest a possible therapeutic target for RMS.

Prohibitin-2 promotes hepatocellular carcinoma malignancy progression in hypoxia based on a label-free quantitative proteomics strategy.

The rapid growth of hepatocellular carcinoma (HCC) leading to tumor hypoxia is a common pathological phenomenon. Meanwhile, tumor hypoxia can promote a change in the biological properties of tumor cells. It may enhance the survival of tumor cells under stress conditions, resulting in resistance to apoptosis and angiogenesis. The moleculars that could modulate the malignant phenotypes of HCC cells remain largely unknown. Based on label-free quantitative proteomic data, we found a significant upregulation of prohibitin-2 (PHB2) in HCC tissues. Treatment of hepatoma cells with small interfering RNAs against PHB2 suppressed cell growth and colony formation, led to G1 phase arrest and sensitized HCC cells to apoptosis. Moreover, inhibition of PHB2 expression dramatically repressed the ability of HCC cells to adapt to hypoxic microenvironments and resist chemotherapy-induced apoptosis. Thus, PHB2 in HCC supports the development and progression of hepatocellular malignancy to hypoxia, and implicates the potential antagonist function of PHB2 in transarterial chemoembolization treatment.

Oxidative stress suppresses PHB2-mediated mitophagy in ß-cells via the Nrf2/PHB2 pathway.

AIMS/INTRODUCTION: Mitochondrial damage caused by oxidative stress is a main driver of pancreatic ß-cell dysfunction in the pathogenesis of type 2 diabetes mellitus. Prohibitin2 (PHB2) is a vital inner mitochondrial membrane protein that participates in mitophagy to remove the damaged mitochondria. This study aimed to investigate the role and mechanisms of PHB2-mediated mitophagy in oxidative stress-induced pancreatic ß-cell dysfunction. MATERIALS AND METHODS: PHB2 and mitophagy-related protein expression were analyzed by real-time polymerase chain reaction and western blotting in RINm5F cells treated with H2O2 and islets of diabetic rats. Mitophagy was observed by mitochondrial and lysosome colocalization. RINm5F cells were transfected by phb2 siRNA or overexpression plasmid to explore the role of PHB2 in mitophagy of RINm5F cells. The mechanism of Nrf2 regulating PHB2 was explored by Nrf2 inhibitor and agonist. RESULTS: The expression of PHB2, mitophagy related protein PINK1, and Parkin were decreased in RINm5F cells incubated with H2O2 and in islets of diabetic rats. Overexpression of PHB2 protected ß-cells from oxidative stress by promoting mitophagy and inhibiting cell apoptosis, whereas transfection with PHB2 siRNA suppressed mitophagy. Furthermore, PHB2-mediated mitophagy induced by oxidative stress was through the Nrf2/PHB2 pathway in ß-cells. Antioxidant NAC alleviated oxidative stress injury by promoting PHB2-mediated mitophagy. CONCLUSION: Our study suggested that PHB2-mediated mitophagy can protect ß-cells from apoptosis via the Nrf2/PHB2 pathway under oxidative stress. Antioxidants may protect ß-cell from oxidative stress by prompting PHB2-mediated mitophagy. PHB2-mediated mitophagy as a potential mechanism takes part in the oxidative stress induced ß-cell injury.

CgPHB2 involved in the haemocyte mitophagy in response to Vibrio splendidus stimulation in Pacific oyster Crassostrea gigas.

Prohibitin2 (PHB2) is recently identified as a novel inner membrane mitophagy receptor to mediate mitophagy. In the present study, the function of CgPHB2 in mediating mitophagy in response to Vibrio splendidus stimulation was investigated in Crassostrea gigas. CgPHB2 protein was mainly distributed in the cytoplasm of three subpopulations of haemocytes. After V. splendidus stimulation, the expressions of CgPHB2 mRNA in haemocytes were up-regulated significantly at 6, 12 and 24 h, and the abundance of CgPHB2 protein was also enhanced at 12-24 h compared to control group. Furthermore, the green signals of CgPHB2 were colocalized respectively with the red signals of mitochondria and CgLC3 in the haemocytes at 12 h after V. splendidus stimulation, and the co-localization value of CgPHB2 and mtphagy Dye was significantly increased. The direct interaction between CgPHB2 and CgLC3 was simulated by molecular docking. In PHB2-inhibitor Fluorizoline-treated oysters, the mRNA expressions of mitophagy-related genes and the ratio of mitophagy were significantly decreased in haemocytes of oysters after V. splendidus stimulation. All the results collectively suggested that CgPHB2 participated in mediating the haemocyte mitophagy in the antibacterial immune response of oysters.

Selection and identification of a prohibitin 2-binding DNA aptamer for tumor tissue imaging and targeted chemotherapy.

Tumor cell-targeting molecules play a vital role in cancer diagnosis, targeted therapy, and biomarker discovery. Aptamers are emerging as novel targeting molecules with unique advantages in cancer research. In this work, we have developed several DNA aptamers through cell-based systematic evolution of ligands by exponential enrichment (Cell-SELEX). The selected SYL-6 aptamer can bind to a variety of cancer cells with high signal. Tumor tissue imaging demonstrated that SYL-6-Cy5 fluorescent probe was able to recognize multiple clinical tumor tissues but not the normal tissues, which indicates great potential of SYL-6 for clinical tumor diagnosis. Meanwhile, we identified prohibitin 2 (PHB2) as the molecular target of SYL-6 using mass spectrometry, pull-down and RNA interference assays. Moreover, SYL-6 can be used as a delivery vehicle to carry with doxorubicin (Dox) chemotherapeutic agents for antitumor targeted chemotherapy. The constructed SYL-6-Dox can not only selectively kill tumor cells in vitro, but also inhibit tumor growth with reduced side effects in vivo. This work may provide a general tumor cell-targeting molecule and a potential biomarker for cancer diagnosis and targeted therapy.

Prx1/PHB2 axis mediates mitophagy in oral leukoplakia cellular senescence.

BACKGROUND: Oral leukoplakia (OLK) is the most common oral potentially malignant disorder (OPMD), which can be malignantly transformed into oral squamous cell carcinoma (OSCC). Peroxiredoxin1(Prx1) has been predicted to bind to Prohibitin2 (PHB2), which confers to affect OLK progression; however, the mechanism of Prx1/PHB2 mediated mitophagy involved in OLK remains unclear. METHODS: This study aimed to explore the mechanism of the Prx1/PHB2 axis on senescence in OLK through mediating mitophagy. The positive rate of Ki67 and the expression of p21, p16, PHB2, and LC3 in human normal, OLK, and OSCC tissues were detected by immunohistochemical staining. The mitophagy and mitochondrial function changes were then analyzed in Prx1 knockdown and Prx1C52S mutations in dysplastic oral keratinocyte (DOK) cells treated with H2O2. In situ Proximity Ligation Assay combined with co-immunoprecipitation was used to detect the interaction between Prx1 and PHB2. RESULTS: Clinically, the positive rate of Ki67 progressively increased from normal to OLK, OLK with dysplasia, and OSCC. Higher p21, p16, PHB2, and LC3 expression levels were observed in OLK with dysplasia than in normal and OSCC tissues. In vitro, PHB2 and LC3II expression gradually increased with the degree of DOK cell senescence. Prx1/PHB2 regulated mitophagy and affected senescence in H2O2-induced DOK cells. Furthermore, Prx1C52S mutation specifically reduced interaction between Prx1 and PHB2. Prx1Cys52 is associated with mitochondrial reactive oxygen species (ROS) accumulated and cell cycle arrest. CONCLUSION: Prx1Cys52 functions as a redox sensor that binds to PHB2 and regulates mitophagy in the senescence of OLK, suggesting its potential as a clinical target.

CRISPR-editing of the virus vector Aedes albopictus cell line C6/36, illustrated by prohibitin 2 gene knockout.

Aedes mosquitoes are important virus vectors. We provide a toolkit for CRISPR-Cas9-editing of difficult-to-knockdown gene previously shown to be refractory to siRNA silencing in mosquito cells, which is pivotal in understanding vector biology, vector competence, host-pathogen interactions and in gene annotations. Starting from database searches of Ae. albopictus and the C6/36 cell line whole genome shotgun sequences for the prohibitin 2 (PHB2) gene, primers were designed to confirm the gene sequence in our laboratory-passaged C6/36 cell line for the correct design and cloning of CRISPR RNA into an insect plasmid vector to create a single guide RNA for the PHB2 gene target. After transfection of this plasmid vector into the C6/36 cells, cell clones selected by puromycin and/or limiting dilution were analyzed for insertions and deletions (INDELs) using PCR, sequencing and computational sequence decomposition. From this, we have identified mono-allelic and bi-allelic knockout cell clones. Using a mono-allelic knockout cell clone as an example, we characterized its INDELs by molecular cloning and computational analysis. Importantly, mono-allelic knockout was sufficient to reduce >80 % of PHB2 expression, which led to phenotypic switching and the propensity to form foci but was insufficient to affect growth rate or to inhibit Zika virus infection.•We provide a toolkit for CRISPR-Cas9-editing of the virus vector, Aedes albopictus C6/36 cell line•We validate this using a difficult-to-knockdown gene prohibitin 2•This toolkit is pivotal in understanding vector biology, vector competence, host-pathogen interactions and in gene annotations.

PHB2 binds to ERß to induce the autophagy of porcine ovarian granulosa cells through mTOR phosphorylation.

Autophagy of ovarian granulosa cells is one of the reasons which results in follicular atresia. PHB2 regulates many fundamental biological processes and is pivotal in the mitophagy of cells; nevertheless, the autophagy in the porcine ovary and how PHB2 regulates the follicular cells are unknown. Here we report a protein complex that induces autophagy in porcine granulosa cells (PGCs) through the direct interaction of ERß and PHB2. In this study, we aimed to elucidate the autophagy and the role of PHB2 in porcine ovaries using porcine primary ovarian granulosa cells (PGCs). The results showed that PHB2 induces PGCs autophagy because of the change in related genes and protein expression levels. In addition, the results of Co-IP and the distribution of the combination of PHB2 and ERß showed that this complex is also indicated as an essential role of PHB2 in PGCs autophagy. Based on our results, it can be concluded that PHB2 combined with ERß induces PGCs autophagy by targeting the mTOR pathway. This study pinpoints a novel regulatory mechanism of autophagy and demonstrates the existence of a protein complex that may underlie its roles in autophagy in PGCs.