Novel Protein Interactions with Endoglin and Activin Receptor-like Kinase 1: Potential Role in Vascular Networks

Endoglin and activin receptor-like kinase 1 are specialized transforming growth factor-beta (TGF-β) superfamily receptors, primarily expressed in endothelial cells. Mutations in the corresponding ENG or ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT1 and HHT2 respectively). To discover proteins interacting with endoglin, ACVRL1 and TGF-β receptor type 2 and involved in TGF-β signaling, we applied LUMIER, a high-throughput mammalian interactome mapping technology. Using stringent criteria, we identified 181 novel unique and shared interactions with ACVRL1, TGF-β receptor type 2, and endoglin, defining potential novel important vascular networks. In particular, the regulatory subunit B-beta of the protein phosphatase PP2A (PPP2R2B) interacted with all three receptors. Interestingly, the PPP2R2B gene lies in an interval in linkage disequilibrium with HHT3, for which the gene remains unidentified. We show that PPP2R2B protein interacts with the ACVRL1/TGFBR2/endoglin complex and recruits PP2A to nitric oxide synthase 3 (NOS3). Endoglin overexpression in endothelial cells inhibits the association of PPP2R2B with NOS3, whereas endoglin-deficient cells show enhanced PP2A-NOS3 interaction and lower levels of endogenous NOS3 Serine 1177 phosphorylation. Our data suggest that endoglin regulates NOS3 activation status by regulating PPP2R2B access to NOS3, and that PPP2R2B might be the HHT3 gene. Furthermore, endoglin and ACVRL1 contribute to several novel networks, including TGF-β dependent and independent ones, critical for vascular function and potentially defective in HHT.Transforming growth factor-β (TGF-β)¹ superfamily ligands, including TGF-βs, activins and bone morphogenic proteins (BMPs), regulate several pathways essential for vascular development and function (1). Responses to these ligands are controlled by type I and II serine kinase receptors, coreceptors and signaling SMAD intermediates. Endothelial cells express the coreceptor, endoglin, and the specialized type I receptor, ACVRL1 (activin receptor-like kinase 1 or ALK1); both molecules are critical for regulation of angiogenesis and vasomotor function by TGF-β superfamily ligands (2, 3).Mutations in ENG and ACVRL1 genes lead to hereditary hemorrhagic telangiectasia (HHT), types 1 and 2, respectively (4). HHT affects 1 in 5000–8000 people worldwide and is characterized by arteriovenous malformations (AVMs) in multiple organs, potentially leading to severe hemorrhages and strokes (4). Haploinsufficiency is the underlying cause of HHT, indicating that reduced levels of functional endoglin or ACVRL1 (ALK1) proteins predispose to endothelial dysfunction and AVMs (5). Although the mechanisms responsible for AVMs remain unclear, the elucidation of how members of the TGF-β superfamily and their molecular networks regulate vascular integrity is vital for future treatments of HHT.We have demonstrated that endoglin interacts with endothelial nitric oxide synthase (NOS3 or eNOS) and regulates its activation (2). NOS3 is a Ca⁺² and calmodulin-regulated enzyme that produces NO● in response to humoral and mechanical stimuli via dynamic interactions with various allosteric regulators such as heat shock protein 90 (HSP90). NOS3 is also regulated by dynamic changes in its phosphorylation status. For example, effects of the vascular endothelial growth factor (VEGF) on angiogenesis, vascular permeability and vasomotor tone are mediated in part through Akt-dependent phosphorylation of NOS3 Ser1177 and by increased NOS3-HSP90 association (6). Although phosphorylation of NOS3 Ser1177 is indicative of agonist-induced activation, it is preceded by dephosphorylation at Thr495. TGF-β1 and -β3 but not -β2 responses can sensitize NOS3 for activation by inducing dephosphorylation at Thr495, and therefore contribute to NOS3 activation and NO-dependent vasorelaxation (7). Endoglin regulates TGF-β1 and -β3 but not -β2 responses, and is required for their induction of NOS3 Thr495 dephosphorylation (7, 8).In the vascular endothelium of HHT patients and in Eng and Alk1 heterozygous mice, impaired association of NOS3 with HSP90 renders the enzyme uncoupled, causing production of superoxide (●O₂⁻) instead of NO● (2, 3, 9) and leading to endothelial damage. Interestingly, TGF-β1 and -β3 do not induce phosphorylation at NOS3 Ser1177, yet NOS3 activation in response to TGF-β1 is abolished in endoglin-deficient cells, impairing vasomotor function (3). ACVRL1 (or ALK1) also interacts with NOS3, and its reduced levels in endothelial cells similarly cause NOS3-derived oxidative stress (3, 9).In view of the crucial roles of endoglin and ACVRL1 in the development and maintenance of the normal vasculature and the definite contribution of their mutated state to HHT, we used the LUMIER high-throughput technology (10) to identify novel protein interactions and molecular networks for these predominantly endothelial receptors. We included TGFBR2 to further define TGF-β protein networks potentially important for vascular function, and attempt to distinguish the TGF-β networks from those associated with BMP9/BMP10 and mediated by ACVRL1 in a complex with BMPR2 and endoglin (11, 12).One of identified proteins interacting with all three receptors was protein phosphatase 2A (PP2A), implicated in multiple pathways. PP2A is a holoenzyme with one structural subunit (PPP2R1A or PPP2R1B) associated with one catalytic subunit (PPP2CA or PPP2CB) and one of 19 regulatory B subunits, the latter conferring specificity to the enzyme by recruiting interacting proteins (13, 14). Of interest, PP2A interacts with NOS3 to regulate Ser1177 phosphorylation and NO● production (15). However, the mechanisms governing recruitment of PP2A to NOS3 and the contribution of TGF-β/BMP receptor complexes are unknown. Recently, the human PPP2R2B gene coding for PPP2R2B protein (also known as PP2A-Bβ regulatory subunit) was mapped to chromosome 5q31-q32, in an interval in linkage disequilibrium with the HHT3 locus (16, 17). We now report that PPP2R2B interacts with the ACVRL1/TGFBR2/endoglin complex and that endoglin governs NOS3 phosphorylation and activation status by hindering PP2A access to NOS3 via the PPP2R2B subunit. Loss of endoglin leads to constitutive reduction in NOS3 phosphorylation and likely to changes in several networks with consequent endothelial dysfunction.     

Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium

Conidiation of the entomopathogenic fungus Metarhizium acridum on agar media was investigated. M. acridum CQMa102 exhibits two different conidiation patterns on agar media: normal conidiation in which conidia are formed on extended hyphae and microcycle conidiation in which conidiation occurs directly after conidia germination. Microcycle conidiation resulted in a mass of conidia produced via budding by accelerated development at the inoculation site. The mean total conidial yield (conidiation at day 10) was 4–5-fold greater after microcycle conidiation than during normal conidiation. Insect pathology assays indicated that microcycle conidia produced on SYA agar were as effective as normal aerial conidia against the locust. Ultraviolet (UV)-resistance tests showed no significant differences between the two types of cell propagules. However, microcycle conidia were more heat resistant than normal aerial conidia, and accumulated higher levels of trehalose in response to heat induction compared to normal aerial conidia.     

Transformation of Metarhizium anisopliae with benomyl resistance and green fluorescent protein genes provides a tag for genetically engineered strains

A plasmid, pBGFP, carrying green fluorescent protein (gfp) and benomyl-resistance genes was constructed and transformed into Metarhizium anisopliae. The transformants grew normally and GFP fluorescence was detected. No change was found in virulence for the transformants. Fluorescence was detected in hyphae from the haemolymph of the infected locust, and the benomyl-resistance was maintained. Results suggested that the two markers provided a useful tool for screening and monitoring the engineered strains even after infection.     

Circ-U2AF1 promotes human glioma via derepressing neuro-oncological ventral antigen 2 by sponging hsa-miR-7-5p

The prognosis for human glioma, a malignant tumor of the central nervous system, is poor due to its rapid growth, genetic heterogeneity, and inadequate understanding of its underlying molecular mechanisms. Circular RNAs composed of exonic sequences, represent an understudied form of noncoding RNAs (ncRNAs) that was discovered more than a decade ago, function as microRNA sponges. We aimed to assess the relationship between circ-U2AF1 (CircRNA ID: hsa_circ_0061868) and hsa-mir-7-5p and examine their effects on proliferation, apoptosis, and the metastatic phenotype of glioma cells regulated by neuro-oncological ventral antigen 2 (NOVA2). We found that the expression levels of circ-U2AF1 and NOVA2 were upregulated, while hsa-miR-7-5p was downregulated in human glioma tissues and glioma cell lines. Our data and bioinformatic analysis indicated the association of these molecules with glioma grade, a positive correlation between circ-U2AF1 and NOVA2 expression levels and a negative correlation of hsa-miR-7-5p with both circ-U2AF1 and NOVA2, respectively. In addition, silencing of circ-U2AF1 expression resulted in increased hsa-miR-7-5p expression and decreased NOVA2 expression both in vitro and in vivo. Luciferase assay confirmed hsa-miR-7-5p as a direct target of circ-U2AF1 and NOVA2 as a direct target of hsa-miR-7-5p. Functionally, silencing of circ-U2AF1 inhibits glioma development by repressing NOVA2 via upregulating hsa-miR-7-5p both in vitro and in vivo. Thus, we assumed that circ-U2AF1 promotes glioma malignancy via derepressing NOVA2 by sponging hsa-miR-7-5p. Taken together, we suggest that circ-U2AF1 can be a prognostic biomarker and the circ-U2AF1/hsa-miR-7-5p/NOVA2 regulatory pathway may be a novel therapeutic target for treating gliomas.     

Expression of scorpion toxin LqhIT2 increases the virulence of Metarhizium acridum towards Locusta migratoria manilensis

LqhIT2 is an insect-specific neurotoxin from the venom of scorpion. In this study, the LqhIT2 gene was introduced into the entomopathogenic fungus, Metarhizium acridum. The virulence of the genetically modified strain MaLqhIT2 was then evaluated against locusts (Locusta migratoria manilensis). Compared with the wild-type strain, the median lethal cell density (LC₅₀) for MaLqhIT2 was a 22.6-fold lower, and the median times to death (LT₅₀) for MaLqhIT2 were reduced by 30.3 and 29.6 %, respectively, after topical inoculation and injection. MaLqhIT2 also grew significantly faster in the hemolymph than wild-type strain. There were no significant differences in germination, appressorium formation and sporulation in locust carcasses between the MaLqhIT2 and wild-type strain. These results indicate that LqhIT2 increased the virulence of M. acridum towards locusts by shortening the in vivo infection period, without affecting cuticle penetration or conidia formation in the carcasses. LqhIT2 thus shows considerable potential for increasing fungal virulence against locusts.     

Establishment and characterization of a novel ‘double‐hit’ follicular lymphoma cell line,FL‐SJC

About 5 per cent of follicular lymphoma (FL) cases are double‐hit (DH) lymphomas. Double‐hit follicular lymphoma (DHFL) cell lines can improve our understanding and drug development on FL. But there are only few DHFL cell lines. Here, we established a new MYC/BCL2 DHFL cell line, FL‐SJC. The cells were obtained from the hydrothorax of a patient with MYC/BCL2 DHFL and cultured for 140 passages in vitro. FL‐SJC cells demonstrated CD19⁺⁺, CD20⁺, CD22⁺⁺, HLA‐DR⁺, CD10⁺, CD38⁺, Lambda⁺ CD23^‐, CD5^‐ and Kappa^‐. The chromosome karyotypic analysis confirmed the co‐existence of t(8;22)(q24;q11) and t(14;18)(q32;q21), as well as additional abnormalities involving chromosomes 2 and 3. Fluorescence in situ hybridization analysis (FISH) showed IGH/BCL2 fusion gene and the MYC rearrangement. In addition, the FL‐SJC cells displayed KMT2D/MLL2 and CREBBP gene mutations. After subcutaneous inoculation of FL‐SJC cells, the SCID mice developed solid tumour masses within 6‐8 weeks. FL‐SJC cells were proven to be free of Epstein‐Barr (EB) virus infection and be multidrug‐resistant. In a conclusion, the FL‐SJC cell line has been identified as a novel MYC/BCL2 double‐hit follicular lymphoma that can be used as a potentially available tool for the clinical and basic research, together with the drug development for MYC/BCL2 DHFL.     

The chromosome number, karyotype and genome size of the desert plant diploid Reaumuria soongorica (Pall.) Maxim

Extreme drought and salt resistant plant Reaumuria soongorica is of great potential for revealing genetic bases unique to naturally stress-tolerant plants. A preliminary genome survey, including chromosome number, karyotype, chromosomal localization of 45S rDNA loci and genome size was conducted with R. soongorica collected from Lanzhou, China. Chromosome counting showed that R. soongorica is diploid with chromosome number of 22. Karyotypical analysis illustrated that the chromosomes size ranges from 3.38 to 5.51 μm, and the chromosomal formula is 2n = 2x = 22 = 4 m + 14sm + 4st. Fluorescence in situ hybridization revealed that four pairs of 45SrDNA signals were detected at the end of R. soongorica chromosomes. The flow cytometry analysis indicated that the mean C value of R. soongorica is 0.806 pg with predicted genome size of about 778 Mb. The results indicate that the extreme drought and salt resistance of R. soongorica was not attributed to a big and complicate genome and also offer some clues in resolving the problems of taxonomy and evolution in Tamaricaceae.     

Purification and characterization of a novel thermostable extracellular protein tyrosine phosphatase from Metarhizium anisopliae strain CQMa102

An extracellular phosphatase was purified to homogeneity from the entomopathogenic fungus Metarhizium anisopliae with a 41.0% yield. The molecular mass and isoelectric point of the purified enzyme were about 82.5 kDa and 9.5 respectively. The optimum pH and temperature were about 5.5 and 75 degrees C when using O-phospho-L-tyrosine as substrate. The protein displayed high stability in a pH range 3.0-9.5 at 30 degrees C and was remarkably thermostable at 70 degrees C. The purified enzyme showed high activity on O-phospho-L-tyrosine and protein tyrosine phosphatase substrate monophosphate (a specific substrate of protein tyrosine phosphatase). Although one peptide of the phosphatase shared identity with one alkaline phosphatase of Neurospora crassa, its substrate specificity and inhibitor sensitivity indicate that the enzyme is a protein tyrosine phosphatase.     

Diabetes influences cardiac extracellular matrix remodelling after myocardial infarction and subsequent development of cardiac dysfunction

This study was conducted to examine the influence of acute streptozotocin‐induced diabetes on cardiac remodelling and function in mice subjected to myocardial infarction (MI) by coronary artery ligation. Echocardiography analysis indicated that diabetes induced deleterious cardiac functional changes as demonstrated by the negative differences of ejection fraction, fractional shortening, stroke volume, cardiac output and left ventricular volume 24 hrs after MI. Temporal analysis for up to 2 weeks after MI showed higher mortality in diabetic animals because of cardiac wall rupture. To examine extracellular matrix remodelling, we used fluorescent molecular tomography to conduct temporal studies and observed that total matrix metalloproteinase (MMP) activity in hearts was higher in diabetic animals at 7 and 14 days after MI, which correlated well with the degree of collagen deposition in the infarct area visualized by scanning electron microscopy. Gene arrays indicated temporal changes in expression of distinct MMP isoforms after 1 or 2 weeks after MI, particularly in diabetic mice. Temporal changes in cardiac performance were observed, with a trend of exaggerated dysfunction in diabetic mice up to 14 days after MI. Decreased radial and longitudinal systolic and diastolic strain rates were observed over 14 days after MI, and there was a trend towards altered strain rates in diabetic mouse hearts with dyssynchronous wall motion clearly evident. This correlated with increased collagen deposition in remote areas of these infarcted hearts indicated by Masson's trichrome staining. In summary, temporal changes in extracellular matrix remodelling correlated with exaggerated cardiac dysfunction in diabetic mice after MI.