Chandipura viral glycoprotein (CNV-G) promotes Gectosome generation and enables delivery of intracellular therapeutics.

Overexpression of vesicular stomatitis virus G protein (VSV-G) elevates the secretion of EVs known as gectosomes, which contain VSV-G. Such vesicles can be engineered to deliver therapeutic macromolecules. We investigated viral glycoproteins from several viruses for their potential in gectosome production and intracellular cargo delivery. Expression of the viral glycoprotein (viral glycoprotein from the Chandipura virus [CNV-G]) from the human neurotropic pathogen Chandipura virus in 293T cells significantly augments the production of CNV-G-containing gectosomes. In comparison with VSV-G gectosomes, CNV-G gectosomes exhibit heightened selectivity toward specific cell types, including primary cells and tumor cell lines. Consistent with the differential tropism between CNV-G and VSV-G gectosomes, cellular entry of CNV-G gectosome is independent of the Low-density lipoprotein receptor, which is essential for VSV-G entry, and shows varying sensitivity to pharmacological modulators. CNV-G gectosomes efficiently deliver diverse intracellular cargos for genomic modification or responses to stimuli in vitro and in the brain of mice in vivo utilizing a split GFP and chemical-induced dimerization system. Pharmacokinetics and biodistribution analyses support CNV-G gectosomes as a versatile platform for delivering macromolecular therapeutics intracellularly.

Liebig's law of the minimum in the TGF-ß/SMAD pathway.

Cells use signaling pathways to sense and respond to their environments. The transforming growth factor-ß (TGF-ß) pathway produces context-specific responses. Here, we combined modeling and experimental analysis to study the dependence of the output of the TGF-ß pathway on the abundance of signaling molecules in the pathway. We showed that the TGF-ß pathway processes the variation of TGF-ß receptor abundance using Liebig's law of the minimum, meaning that the output-modifying factor is the signaling protein that is most limited, to determine signaling responses across cell types and in single cells. We found that the abundance of either the type I (TGFBR1) or type II (TGFBR2) TGF-ß receptor determined the responses of cancer cell lines, such that the receptor with relatively low abundance dictates the response. Furthermore, nuclear SMAD2 signaling correlated with the abundance of TGF-ß receptor in single cells depending on the relative expression levels of TGFBR1 and TGFBR2. A similar control principle could govern the heterogeneity of signaling responses in other signaling pathways.

Cloflucarban Illuminates Specificity and Context-Dependent Activation of the PINK1-Parkin Pathway by Mitochondrial Complex Inhibition.

The PTEN-induced kinase 1 (PINK1)-Parkin pathway plays a vital role in maintaining a healthy pool of mitochondria in higher eukaryotic cells. While the downstream components of this pathway are well understood, the upstream triggers remain less explored. In this study, we conducted an extensive analysis of inhibitors targeting various mitochondrial electron transport chain (ETC) complexes to investigate their potential as activators of the PINK1-Parkin pathway. We identified cloflucarban, an antibacterial compound, as a novel pathway activator that simultaneously inhibits mitochondrial complexes III and V, and V. RNA interference (RNAi) confirmed that the dual inhibition of these complexes activates the PINK1-Parkin pathway. Intriguingly, we discovered that albumin, specifically bovine serum albumin (BSA) and human serum albumin (HSA) commonly present in culture media, can hinder carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced pathway activation. However, cloflucarban's efficacy remains unaffected by albumin, highlighting its reliability for studying the PINK1-Parkin pathway. This study provides insights into the activation of the upstream PINK1-Parkin pathway and underscores the influence of culture conditions on research outcomes. Cloflucarban emerges as a promising tool for investigating mitochondrial quality control and neurodegenerative diseases.

Characterization of a Novel Polysaccharide Derived from Rhizospheric Paecilomyces vaniformisi and Its Mechanism for Enhancing Salinity Resistance in Rice Seedlings.

Soil salinity is an important limiting factor in agricultural production. Rhizospheric fungi can potentially enhance crop salinity tolerance, but the precise role of signaling substances is still to be systematically elucidated. A rhizospheric fungus identified as Paecilomyces vaniformisi was found to enhance the salinity tolerance of rice seedlings. In this study, a novel polysaccharide (PPL2b) was isolated from P. vaniformisi and identified as consisting of Manp, Glcp, GalpA, and Galp. In a further study, PPL2b showed significant activity in alleviating salinity stress-induced growth inhibition in rice seedlings. The results indicated that under salinity stress, PPL2b enhances seed germination, plant growth (height and biomass), and biochemical parameters (soluble sugar and protein contents). Additionally, PPL2b regulates genes such as SOS1 and SKOR to decrease K+ efflux and increase Na+ efflux. PPL2b increased the expression and activity of genes related to antioxidant enzymes and nonenzyme substances in salinity-induced oxidative stress. Further study indicated that PPL2b plays a crucial role in regulating osmotic substances, such as proline and betaine, in maintaining the osmotic balance. It also modulates plant hormones to promote rice seedling growth and enhance their tolerance to soil salinity. The variables interacted and were divided into two groups (PC1 77.39% and PC2 18.77%) based on their relative values. Therefore, these findings indicate that PPL2b from P. vaniformisi can alleviate the inhibitory effects of salinity stress on root development, osmotic adjustment, ion balance, oxidative stress balance, and growth of rice seedlings. Furthermore, it suggests that polysaccharides produced by rhizospheric fungi could be utilized to enhance crop tolerance to salinity.

Evaluation of negative effect of Naphthenic acids (NAs) on physiological metabolism and polycyclic aromatic hydrocarbons adsorption of Phragmites australis.

Toxic substances in the environment disturb the adsorption of pollutants in plants but little is known about the underlying mechanisms of these processes. This study evaluated the PAH adsorption by Phragmites australis under NAs stress. Results showed that Naphthenic acids (NAs) significantly decreased the adsorption of PAHs and had higher selectivity for type and structure. P. australis root cell growth and mitosis were significantly affected by NAs, which was accompanied by serious disturbances in mitochondrial function. The physiological evaluation showed the NAs could increase Reactive Oxygen Species (ROS) accumulation by around 16-fold and cause damage to the root cell normal redox equilibrium. The levels of three key related antioxidants, PLA, CAT and POD, decreased significantly to 35-50% under NAs stress and were dependent upon NAs concentration. Furthermore, NAs could significantly change the concentration and species of root exudates of P. ausralis. Autotoxic substances, including alcohol and amines, increased by 28.63% and 23.96, respectively. Sixteen compounds were identified and assumed as potential biomarkers. Galactonic, glyceric, and octadecanoic acid had the general effect of activating PAH in soil. The global view of the metabolic pathway suggests that NAs influenced the citric acid cycle, fatty acid synthesis, amino acid metabolism and the phenylpropanoid pathway. Detection data results indicated that the energy products cause hypoxia and oxidative stress, which are the main processes under the NAs. Furthermore, verification of these processes was fulfilled through gene expression and biomarkers quantification. Our results provide novel metabolic insights into the mechanisms of PAHs adsorption by P. australis under NAs disturbance, suggesting that monitoring NAs in phytoremediation applications is necessary.

Programmable Extracellular Vesicles for Macromolecule Delivery and Genome Modifications.

Getting large macromolecules through the plasma membrane and endosomal barriers remains a major challenge. Here, we report a generalizable method of delivering proteins and ribonucleoproteins (RNPs) to cells in vitro and mouse liver tissue in vivo with engineered ectosomes. These ectosomes, referred to as "Gectosomes," are designed to co-encapsulate vesicular stomatitis virus G protein (VSV-G) with bioactive macromolecules via split GFP complementation. We found that this method enables active cargo loading, improves the specific activity of cargo delivery, and facilitates Gectosome purification. Experimental and mathematical modeling analyses suggest that active cargo loading reduces non-specific encapsulation of cellular proteins, particularly nucleic-acid-binding proteins. Using Gectosomes that encapsulate Cre, Ago2, and SaCas9, we demonstrate their ability to execute designed modifications of endogenous genes in cell lines in vitro and mouse liver tissue in vivo, paving the way toward applications of this technology for the treatment of a wide range of human diseases.

Human Papillomavirus 16 oncoprotein E7 retards mitotic progression by blocking Mps1-MAP4 signaling cascade.

Human epithelial cells can be infected by more than 200 types of human papilloma viruses (HPVs), and persistent HPV infections lead to cervical cancer or other deadly cancers. It has been established that mitotic progression is critical for HPV16 infection, but the underlying mechanism remains unknown. Here, we report that oncoprotein E7 of HPV16 but not HPV18 retards mitotic progression in host cell by direct binding to the C terminus of Microtubule-Associated Protein 4 (MAP4). MAP4 is a novel bona fide target of HPV16E7 protein which binds and recruits the latter to spindle microtubule in mitosis. HPV16E7 protein promotes MAP4 stability by inhibiting MAP4 phosphorylation- mediated degradation to increase the stability of microtubule polymerization and cause an extend mitotic progression. We further uncovered that Mps1 is a kinase of MAP4, and E7-MAP4 binding blocks Mps1 phosphorylation of MAP4, thereby interrupting phosphorylation-dependent MAP4 degradation. Mutations of MAP4 at T927ES928E partially abolished E7-binding capacity and rescued mitotic progression in host cells. In conclusion, our study reveals a molecular mechanism by which HPV16E7 perturbs host mitotic progression by interfering Mps1-MAP4 signaling cascade, which results in an extended infection window and may facilitate the persistent HPV16 infection.

Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers.

Histone deacetylase inhibitors (HDACIs) are known to alter gene expression by both up- and down-regulation of protein-coding genes in normal and cancer cells. However, the exact regulatory mechanisms of action remain uncharacterized. Here we investigated genome wide dose-dependent epigenetic and transcriptome changes in response to HDACI largazole in a transformed and a non-transformed cell line. Exposure to low nanomolar largazole concentrations (

Oxidative stress induces mitotic arrest by inhibiting Aurora A-involved mitotic spindle formation.

Oxidative stress contributes to the oxidative modification of cellular components, including lipids, proteins and DNA, and results in DNA damage, cell cycle arrest, cellular dysfunction and apoptosis. However, the mechanism underlying oxidative stress-induced mitotic abnormalities is not fully understood. In this study, we demonstrated that exogenous and endogenous reactive oxygen species (ROS) promoted mitotic arrest. Delayed formation and abnormal function of the mitotic spindle, which directly impeded mitosis and promoted abnormal chromosome separation, was responsible for ROS-induced mitotic arrest. As a key regulator of mitotic spindle assembly, Aurora A kinase was hyperphosphorylated in early mitosis under oxidative stress, which may disturb the function of Aurora A in mitotic spindle formation. Our findings identified a mechanism by which ROS regulate mitotic progression and indicated a potential molecular target for the treatment of oxidative stress-related diseases.

The critical role of the central nervous system (pro)renin receptor in regulating systemic blood pressure.

The systemic renin-angiotensin system (RAS) has long been recognized as a critically important system in blood pressure (BP) regulation. However, extensive evidence has shown that a majority of RAS components are also present in many tissues and play indispensable roles in BP regulation. Here, we review evidence that RAS components, notably including the newly identified (pro)renin receptor (PRR), are present in the brain and are essential for the central regulation of BP. Binding of the PRR to its ligand, prorenin or renin, increases BP and promotes progression of cardiovascular diseases in an angiotensin II-dependent and -independent manner, establishing the PRR a promising antihypertensive drug target. We also review the existing PRR blockers, including handle region peptide and PRO20, and propose a rationale for blocking prorenin/PRR activation as a therapeutic approach that does not affect the actions of the PRR in vacuolar H(+)-ATPase and development. Finally, we summarize categories of currently available antihypertensive drugs and consider future perspectives.

A putative N-terminal nuclear export sequence is sufficient for Mps1 nuclear exclusion during interphase.

BACKGROUND: Mps1, an essential component of the mitotic checkpoint, is also an important interphase regulator and has roles in DNA damage response, cytokinesis and centrosome duplication. Mps1 predominantly resides in the cytoplasm and relocates into the nucleus at the late G2 phase. So far, the mechanism underlying the Mps1 translocation between the cytoplasm and nucleus has been unclear. RESULTS: In this work, a dynamic export process of Mps1 from the nucleus to cytoplasm in interphase was revealed- a process blocked by the Crm1 inhibitor, Leptomycin B, suggesting that export of Mps1 is Crm1 dependent. Consistent with this speculation, a direct association between Mps1 and Crm1 was found. Furthermore, a putative nuclear export sequence (pNES) motif at the N-terminal of Mps1 was identified by analyzing the motif of Mps1. This motif shows a high sequence similarity to the classic NES, a fusion of this motif with EGFP results in dramatic exclusion of the fusion protein from the nucleus. Additionally, Mps1 mutant loss of pNES integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm. CONCLUSION: Taken these findings together, it was concluded that the pNES sequence is sufficient for the Mps1 export from nucleus during interphase.

MAVS Promotes Inflammasome Activation by Targeting ASC for K63-Linked Ubiquitination via the E3 Ligase TRAF3.

Stringent control of inflammasome signaling pathway is important for maintaining immunological balance, yet the molecular mechanisms responsible for its tight regulation are still poorly understood. In this study, we found that the signaling pathway dependent on mitochondrial antiviral signaling protein (MAVS) was required for the optimal activation of apoptosis-associated specklike protein (ASC)-dependent inflammasome. In particular, TNFR-associated factor 3 was found to be a direct E3 ligase for ASC. Ubiquitination of ASC at Lys(174) was critical for speck formation and inflammasome activation. Deficiency in MAVS or TNFR-associated factor 3 impaired ASC ubiquitination and cytosolic aggregates formation, resulting in reduced inflammasome response upon RNA virus infection. This study has identified a previously unrecognized role of MAVS in the regulation of inflammasome signaling and provided molecular insight into the mechanisms by which ubiquitination of ASC controls inflammasome activity through the formation of ASC specks.

Downregulation of microRNA miR-526a by enterovirus inhibits RIG-I-dependent innate immune response.

UNLABELLED: Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that induces type I interferon-mediated host-protective innate immunity against viral infection. Although cylindromatosis (CYLD) has been shown to negatively regulate innate antiviral response by removing K-63-linked polyubiquitin from RIG-I, the regulation of its expression and the underlying regulatory mechanisms are still incompletely understood. Here we show that RIG-I activity is regulated by inhibition of CYLD expression mediated by the microRNA miR-526a. We found that viral infection specifically upregulates miR-526a expression in macrophages via interferon regulatory factor (IRF)-dependent mechanisms. In turn, miR-526a positively regulates virus-triggered type I interferon (IFN-I) production, thus suppressing viral replication, the underlying mechanism of which is the enhancement of RIG-I K63-linked ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic miR-526a expression inhibits the replication of EV71 virus. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and suggest a novel mechanism for the evasion of the innate immune response controlled by EV71. IMPORTANCE: RNA virus infection upregulates the expression of miR-526a in macrophages through IRF-dependent pathways. In turn, miR-526a positively regulates virus-triggered type I IFN production and inhibits viral replication, the underlying mechanism of which is the enhancement of RIG-I K-63 ubiquitination by miR-526a via suppression of the expression of CYLD. Remarkably, virus-induced miR-526a upregulation and CYLD downregulation are blocked by enterovirus 71 (EV71) 3C protein; cells with overexpressed miR-526a were highly resistant to EV71 infection. The collective results of this study suggest a novel mechanism of the regulation of RIG-I activity during RNA virus infection by miR-526a and propose a novel mechanism for the evasion of the innate immune response controlled by EV71.

Overexpression of Mps1 in colon cancer cells attenuates the spindle assembly checkpoint and increases aneuploidy.

The spindle assembly checkpoint kinase Mps1 is highly expressed in several types of cancers, but its cellular involvement in tumorigenesis is less defined. Herein, we confirm that Mps1 is overexpressed in colon cancer tissues. Further, we find that forced expression of Mps1 in the colon cancer cell line SW480 enables cells to become resistant to both Mps1 inhibition-induced checkpoint depletion and cell death. Overexpression of Mps1 also increases genome instability in tumor cells owing to a weakened spindle assembly checkpoint. Collectively, our findings suggest that high levels of Mps1 contribute to tumorigenesis by attenuating the spindle assembly checkpoint.

Elevated expression of TANK-binding kinase 1 enhances tamoxifen resistance in breast cancer.

Resistance to antiestrogens is one of the major challenges in breast cancer treatment. Although phosphorylation of estrogen receptor α (ERα) is an important factor in endocrine resistance, the contributions of specific kinases in endocrine resistance are still not fully understood. Here, we report that an important innate immune response kinase, the IκB kinase-related TANK-binding kinase 1 (TBK1), is a crucial determinant of resistance to tamoxifen therapies. We show that TBK1 increases ERα transcriptional activity through phosphorylation modification of ERα at the Ser-305 site. Ectopic TBK1 expression impairs the responsiveness of breast cancer cells to tamoxifen. By studying the specimens from patients with breast cancer, we find a strong positive correlation of TBK1 with ERα, ERα Ser-305, and cyclin D1. Notably, patients with tumors highly expressing TBK1 respond poorly to tamoxifen treatment and show high potential for relapse. Therefore, our findings suggest that TBK1 contributes to tamoxifen resistance in breast cancer via phosphorylation modification of ERα.

MAVS regulates apoptotic cell death by decreasing K48-linked ubiquitination of voltage-dependent anion channel 1.

The mitochondrial antiviral signaling protein MAVS (IPS-1, VISA, or Cardif) plays an important role in the host defense against viral infection by inducing type I interferon. Recent reports have shown that MAVS is also critical for virus-induced apoptosis. However, the mechanism of MAVS-mediated apoptosis induction remains unclear. Here, we show that MAVS binds to voltage-dependent anion channel 1 (VDAC1) and induces apoptosis by caspase-3 activation, which is independent of its role in innate immunity. MAVS modulates VDAC1 protein stability by decreasing its degradative K48-linked ubiquitination. In addition, MAVS knockout mouse embryonic fibroblasts (MEFs) display reduced VDAC1 expression with a consequent reduction of the vesicular stomatitis virus (VSV)-induced apoptosis response. Notably, the upregulation of VDAC1 triggered by VSV infection is completely abolished in MAVS knockout MEFs. We thus identify VDAC1 as a target of MAVS and describe a novel mechanism of MAVS control of virus-induced apoptotic cell death.

UV-C irradiation delays mitotic progression by recruiting Mps1 to kinetochores.

The effect of UV irradiation on replicating cells during interphase has been studied extensively. However, how the mitotic cell responds to UV irradiation is less well defined. Herein, we found that UV-C irradiation (254 nm) increases recruitment of the spindle checkpoint proteins Mps1 and Mad2 to the kinetochore during metaphase, suggesting that the spindle assembly checkpoint (SAC) is reactivated. In accordance with this, cells exposed to UV-C showed delayed mitotic progression, characterized by a prolonged chromosomal alignment during metaphase. UV-C irradiation also induced the DNA damage response and caused a significant accumulation of γ-H2AX on mitotic chromosomes. Unexpectedly, the mitotic delay upon UV-C irradiation is not due to the DNA damage response but to the relocation of Mps1 to the kinetochore. Further, we found that UV-C irradiation activates Aurora B kinase. Importantly, the kinase activity of Aurora B is indispensable for full recruitment of Mps1 to the kinetochore during both prometaphase and metaphase. Taking these findings together, we propose that UV irradiation delays mitotic progression by evoking the Aurora B-Mps1 signaling cascade, which exerts its role through promoting the association of Mps1 with the kinetochore in metaphase.

Identification of tyrosine-9 of MAVS as critical target for inducible phosphorylation that determines activation.

BACKGROUND: Innate immunity to viruses involves receptors such as RIG-I, which senses viral RNA and triggers an IFN-ß signaling pathway involving the outer mitochondrial membrane protein MAVS. However, the functional status of MAVS phosphorylation remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate for the first time that MAVS undergoes extensive tyrosine phosphorylation upon viral infection, indicating that MAVS phosphorylation might play an important role in MAVS function. A tyrosine-scanning mutational analysis revealed that MAVS tyrosine-9 (Y9) is a phosphorylation site that is required for IFN-ß signaling. Indeed, MAVS Y9F mutation severely impaired TRAF3/TRAF6 recruitment and displayed decreased tyrosine phosphorylation in response to VSV infection compared to wild type MAVS. Functionally, MAVS Y9 phosphorylation contributed to MAVS antiviral function without interfering with its apoptosis property. CONCLUSIONS/SIGNIFICANCE: These experiments identify a novel residue of MAVS that is crucially involved in the recruitment of TRAF3/TRAF6 and in downstream propagation of MAVS signaling.

Two LXXLL motifs in the N terminus of Mps1 are required for Mps1 nuclear import during G(2)/M transition and sustained spindle checkpoint responses.

Spindle assembly checkpoint kinase Mps1 is spatially and temporally regulated during cell cycle progression. Mps1 is predominately localized to the cytosol in interphase cells, whereas it is concentrated on kinetochores in prophase and prometaphase cells. The timing and mechanism of Mps1 redistribution during cell cycle transition is currently poorly understood. Here, we show that Mps1 relocates from the cytosol to the nucleus at the G 2/M boundary prior to nuclear envelope breakdown (NEB). This timely translocation depends on two tandem LXXLL motifs in the N terminus of Mps1, and mutations in either motif abolish Mps1 nuclear accumulation. Furthermore, we found that phosphorylation of Mps1 Ser80 (which is located between the two LXXLL motifs) also plays a role in regulating timely nuclear entry of Mps1. Mps1 that is defective in LXXLL motifs has near wild-type kinase activity. Moreover, the kinase activity of Mps1 appears to be dispensable for nuclear translocation, as inhibition of Mps1 by a highly specific small-molecule inhibitor did not perturb its nuclear entry. Remarkably, translocation-deficient Mps1 can mediate activation of spindle assembly checkpoint response; however, it fails to support a sustained mitotic arrest upon prolonged treatment with nocodazole. The mitotic slippage can be attributed to precocious degradation of Mps1 in the arrested cells. Our studies reveal a novel cell cycle-dependent nuclear translocation signal in the N terminus of Mps1 and suggest that timely nuclear entry could be important for sustaining spindle assembly checkpoint responses.