Immunophilin FKB20-2 participates in oligomerization of Photosystem I in Chlamydomonas.

PSI is a sophisticated photosynthesis protein complex that fuels the light reaction of photosynthesis in algae and vascular plants. While the structure and function of PSI have been studied extensively, the dynamic regulation on PSI oligomerization and high light response is less understood. In this work, we characterized a high light-responsive immunophilin gene FKB20-2 (FK506-binding protein 20-2) required for PSI oligomerization and high light tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Biochemical assays and 77-K fluorescence measurement showed that loss of FKB20-2 led to the reduced accumulation of PSI core subunits and abnormal oligomerization of PSI complexes and, particularly, reduced PSI intermediate complexes in fkb20-2. It is noteworthy that the abnormal PSI oligomerization was observed in fkb20-2 even under dark and dim light growth conditions. Coimmunoprecipitation, MS, and yeast 2-hybrid assay revealed that FKB20-2 directly interacted with the low molecular weight PSI subunit PsaG, which might be involved in the dynamic regulation of PSI-light-harvesting complex I supercomplexes. Moreover, abnormal PSI oligomerization caused accelerated photodamage to PSII in fkb20-2 under high light stress. Together, we demonstrated that immunophilin FKB20-2 affects PSI oligomerization probably by interacting with PsaG and plays pivotal roles during Chlamydomonas tolerance to high light.

Chemical remodeling of a cellular chaperone to target the active state of mutant KRAS.

The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product-inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRASG12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRASG12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRASG12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).

Conserved requirement of autophagy-related effectors during coronavirus replication.

The recurrence of zoonotic transmission events highlights the need for novel treatment strategies against emerging coronaviruses (CoVs), namely SARS-CoV, MERS-CoV and most notably SARS-CoV-2. Our recently performed genome-wide CRISPR knockout screen revealed a list of conserved pan-coronavirus as well as MERS-CoV or HCoV-229E-specific host dependency factors (HDF) essential during the viral life cycle. Intriguingly, we identified the macroautophagy/autophagy pathway-regulating immunophilins FKBP8, TMEM41B, and MINAR1 as conserved MERS-CoV, HCoV-229E, SARS-CoV, and SARS-CoV-2 host factors, which further constitute potential targets for therapeutic intervention by clinically approved drugs.

Functions of the Hsp90-Binding FKBP Immunophilins.

The Hsp90 chaperone is known to interact with a diverse array of client proteins. However, in every case examined, Hsp90 is also accompanied by a single or several co-chaperone proteins. One class of co-chaperone contains a tetratricopeptide repeat (TPR) domain that targets the co-chaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family. Despite the common association of FKBP co-chaperones with Hsp90, it is abundantly clear that the client protein influences, and is often influenced by, the particular FKBP bound to Hsp90. Examples include Xap2 in aryl hydrocarbon receptor complexes and FKBP52 in steroid receptor complexes. In this chapter, we discuss the known functional roles played by FKBP co-chaperones and, where possible, relate distinctive functions to structural differences between FKBP members.

Immunophilin CYN28 is required for accumulation of photosystem II and thylakoid FtsH protease in Chlamydomonas.

Excess light causes severe photodamage to photosystem II (PSII) where the primary charge separation for electron transfer takes place. Dissection of mechanisms underlying the PSII maintenance and repair cycle in green algae promotes the usage of genetic engineering and synthetic biology to improve photosynthesis and biomass production. In this study, we systematically analyzed the high light (HL) responsive immunophilin genes in Chlamydomonas (Chlamydomonas reinhardtii) and identified one chloroplast lumen-localized immunophilin, CYN28, as an essential player in HL tolerance. Lack of CYN28 caused HL hypersensitivity, severely reduced accumulation of PSII supercomplexes and compromised PSII repair in cyn28. The thylakoid FtsH (filamentation temperature-sensitive H) is an essential AAA family metalloprotease involved in the degradation of photodamaged D1 during the PSII repair cycle and was identified as one potential target of CYN28. In the cyn28 mutant, the thylakoid FtsH undergoes inefficient turnover under HL conditions. The CYN28-FtsH1/2 interaction relies on the FtsH N-terminal proline residues and is strengthened particularly under HL. Further analyses demonstrated CYN28 displays peptidyl-prolyl isomerase (PPIase) activity, which is necessary for its physiological function. Taken together, we propose that immunophilin CYN28 participates in PSII maintenance and regulates the homeostasis of FtsH under HL stress via its PPIase activity.

Effect of Immunophilin Inhibitors on Cochlear Fibroblasts and Spiral Ganglion Cells.

INTRODUCTION: Loss of hair cells and degeneration of spiral ganglion neurons (SGN) lead to severe hearing loss or deafness. The successful use of a cochlear implant (CI) depends among other factors on the number of surviving SGN. Postoperative formation of fibrous tissue around the electrode array causes an increase in electrical impedances at the stimulating contacts. The use of immunophilin inhibitors may reduce the inflammatory processes without suppressing the immune response. Here, we report on in vitro experiments with different concentrations of immunophilin inhibitors MM284 and compound V20 regarding a possible application of these substances in the inner ear. METHODS: Standard cell lines (NIH/3T3 fibroblasts), freshly isolated SGN, and fibroblasts from neonatal rat cochleae (p3-5) were incubated with different concentrations of immunophilin inhibitors for 48 h. Metabolic activity of fibroblasts was investigated by MTT assay and cell survival by counting of immunochemically stained neurons and compared to controls. RESULTS: MM284 did not affect SGN numbers and neurite growth at concentrations of 4 × 10-5 mol/L and below, whereas V20 had no effect at 8 × 10-6 mol/L and below. Metabolic activity of fibroblasts was unchanged at these concentrations. CONCLUSION: Especially MM284 might be considered as a possible candidate for application within the cochlea.

Apoptosis and glucocorticoid-related genes mRNA expression is modulated by coenzyme Q10 supplementation during in vitro maturation and vitrification of bovine oocytes and cumulus cells.

Oocyte in vitro maturation (IVM) and vitrification procedures lead to detrimental effects on the overall oocyte quality. The addition of antioxidants during IVM, such as the coenzyme Q10 (Q10), has been demonstrated to positively impact on the cumulus-oocyte complexes due to its role in protection from oxidative damage and modulating gene transcription. Furthermore, glucocorticoids (GC) regulate gene transcription, energy metabolism and apoptosis during the early steps of reproduction. In this sense, most GC actions are mediated by the glucocorticoid receptor (NR3C1), a transcription factor. However, the specific roles of GC in ovarian physiology and oocyte maturation are still unknown. In this regard, a better knowledge on the expression of GC-related and apoptosis-related genes during IVM and cryopreservation procedures could potentially benefit the refinement of assisted reproductive techniques in the bovine species. The present study aims to explore the expression of NR3C1 mRNA in fresh and vitrified bovine oocytes and cumulus cells in response to Q10 (50 µM), and the effect of cortisol addition (0.25 µM, 0.5 µM) on the expression of NR3C1. We also studied the mRNA expression of NR3C1-related genes belonging to the GC regulation pathway, such as hydroxysteroid dehydrogenases (HSD11B1; HSD11B2), immunophilins (FKBP4; FKBP5), signal transducers and activators of transcription (STAT3; STAT5A), the mineralocorticoid receptor (NR3C2), and to the apoptosis pathway, such as the anti- (BCL2) and pro-apoptotic (BAX) mRNA transcripts in oocytes and cumulus cells 1) after IVM, and 2) after vitrification, both in presence or absence of Q10 supplementation during IVM. Our results show that there is an increase in the NR3C1 receptor expression after vitrification of oocytes, but not after exogenous cortisol supplementation during IVM. In addition, Q10 reduces the mRNA expression of HSD11B1 and FKBP5 in oocytes at levels of immature oocytes (HSD11B1 mRNA expression also in cumulus cells), and the BAX:BCL2 ratio mRNA expression. After vitrification in the presence of Q10, HSD11B2 mRNA expression increases in cumulus cells, while HSD11B1 and BAX:BCL2 mRNA expression decreases significantly both in oocytes and cumulus cells. In conclusion, our results show for the first time the effect of IVM, vitrification and Q10 supplementation on the mRNA relative expression of GC-related and apoptosis genes, and the effect of vitrification in the protein expression of NR3C1.

The immunophilin CYCLOPHILIN28 affects PSII-LHCII supercomplex assembly and accumulation in Arabidopsis thaliana.

In plant chloroplasts, photosystem II (PSII) complexes, together with light-harvesting complex II (LHCII), form various PSII-LHCII supercomplexes (SCs). This process likely involves immunophilins, but the underlying regulatory mechanisms are unclear. Here, by comparing Arabidopsis thaliana mutants lacking the chloroplast lumen-localized immunophilin CYCLOPHILIN28 (CYP28) to wild-type and transgenic complemented lines, we determined that CYP28 regulates the assembly and accumulation of PSII-LHCII SCs. Compared to the wild type, cyp28 plants showed accelerated leaf growth, earlier flowering time, and enhanced accumulation of high molecular weight PSII-LHCII SCs under normal light conditions. The lack of CYP28 also significantly affected the electron transport rate. Blue native-polyacrylamide gel electrophoresis analysis revealed more Lhcb6 and less Lhcb4 in M-LHCII-Lhcb4-Lhcb6 complexes in cyp28 versus wild-type plants. Peptidyl-prolyl cis/trans isomerase (PPIase) activity assays revealed that CYP28 exhibits weak PPIase activity and that its K113 and E187 residues are critical for this activity. Mutant analysis suggested that CYP28 may regulate PSII-LHCII SC accumulation by altering the configuration of Lhcb6 via its PPIase activity. Furthermore, the Lhcb6-P139 residue is critical for PSII-LHCII SC assembly and accumulation. Therefore, our findings suggest that CYP28 likely regulates PSII-LHCII SC assembly and accumulation by altering the configuration of P139 of Lhcb6 via its PPIase activity.

In Silico Characterization of Calcineurin from Pathogenic Obligate Intracellular Trypanosomatids: Potential New Biological Roles.

Calcineurin (CaN) is present in all eukaryotic cells, including intracellular trypanosomatid parasites such as Trypanosoma cruzi (Tc) and Leishmania spp. (Lspp). In this study, we performed an in silico analysis of the CaN subunits, comparing them with the human (Hs) and looking their structure, post-translational mechanisms, subcellular distribution, interactors, and secretion potential. The differences in the structure of the domains suggest the existence of regulatory mechanisms and differential activity between these protozoa. Regulatory subunits are partially conserved, showing differences in their Ca2+-binding domains and myristoylation potential compared with human CaN. The subcellular distribution reveals that the catalytic subunits TcCaNA1, TcCaNA2, LsppCaNA1, LsppCaNA1_var, and LsppCaNA2 associate preferentially with the plasma membrane compared with the cytoplasmic location of HsCaNAα. For regulatory subunits, HsCaNB-1 and LsppCaNB associate preferentially with the nucleus and cytoplasm, and TcCaNB with chloroplast and cytoplasm. Calpain cleavage sites on CaNA suggest differential processing. CaNA and CaNB of these trypanosomatids have the potential to be secreted and could play a role in remote communication. Therefore, this background can be used to develop new drugs for protozoan pathogens that cause neglected disease.

Corticosteroid receptors as a model for the Hsp90•immunophilin-based transport machinery.

Steroid receptors form soluble heterocomplexes with the 90-kDa heat-shock protein (Hsp90) and other chaperones and co-chaperones. The assembly and composition of the oligomer is influenced by the presence and nature of the bound steroid. Although these receptors shuttle dynamically in and out of the nucleus, their primary localization in the absence of steroid can be mainly cytoplasmic, mainly nuclear, or partitioned into both cellular compartments. Upon steroid binding, receptors become localized to the nucleus via the transportosome, a retrotransport molecular machinery that comprises Hsp90, a high-molecular-weight immunophilin, and dynein motors. This molecular machinery, first evidenced in steroid receptors, can also be used by other soluble proteins. In this review, we dissect the complete model of this transport machinery system.

Role of Mitochondrial Heat-shock Proteins and Immunophilins in Neuro Degenerative Diseases.

Pathophysiologic conditions of neurodegenerative diseases are unquestionably related to protein misfolding. The accumulation of misfolded proteins into relatively ordered structures such as fibrillar intracellular and extracellular amyloids results in tissue lesions that lead to neuronal loss and brain damage. In these pathologies, the occurrence of protein aggregates suggests certain inefficient or insufficient cellular responses of those molecular chaperones that should properly assist the folding of the client proteins. In this regard, most experimental models for neurodegenerative diseases have demonstrated that the overexpression of molecular chaperones provides effective neuroprotection. A subset of these molecular chaperones corresponds to a group of proteins that exhibit peptidylprolyl isomerase enzymatic activity, the immunophilins. Most of the family members of the latter group were first described as being responsible for the immunosuppressive response or they were reported as members of the chaperone complex associated with HSP90 in steroid receptor oligomers. In this article, we review some aspects of the liaison between molecular chaperones and neurodegenerative diseases, in particular heat-shock proteins and immunophilins with demonstrated influence on the proper function of mitochondria. This article is intended to address a field that represents a yet critical unmet clinical need for the development of neuroprotective molecules focused on potentially novel molecular targets.

The immunophilin Zonda controls regulated exocytosis in endocrine and exocrine tissues.

Exocytosis is a fundamental process in physiology, that ensures communication between cells, organs and even organisms. Hormones, neuropeptides and antibodies, among other cargoes are packed in exocytic vesicles that need to reach and fuse with the plasma membrane to release their content to the extracellular milieu. Hundreds of proteins participate in this process and several others in its regulation. We report here a novel component of the exocytic machinery, the Drosophila transmembrane immunophilin Zonda (Zda), previously found to participate in autophagy. Zda is highly expressed in secretory tissues, and regulates exocytosis in at least three of them: the ring gland, insulin-producing cells and the salivary gland. Using the salivary gland as a model system, we found that Zda is required at final steps of the exocytic process for fusion of secretory granules to the plasma membrane. In a genetic screen we identified the small GTPase RalA as a crucial regulator of secretory granule exocytosis that is required, similarly to Zda, for fusion between the secretory granule and the plasma membrane.

A twist in the ABC: regulation of ABC transporter trafficking and transport by FK506-binding proteins.

Post-transcriptional regulation of ATP-binding cassette (ABC) proteins has been so far shown to encompass protein phosphorylation, maturation, and ubiquitination. Yet, recent accumulating evidence implicates FK506-binding proteins (FKBPs), a type of peptidylprolyl cis-trans isomerase (PPIase) proteins, in ABC transporter regulation. In this perspective article, we summarize current knowledge on ABC transporter regulation by FKBPs, which seems to be conserved over kingdoms and ABC subfamilies. We uncover striking functional similarities but also differences between regulatory FKBP-ABC modules in plants and mammals. We dissect a PPIase- and HSP90-dependent and independent impact of FKBPs on ABC biogenesis and transport activity. We propose and discuss a putative new mode of transient ABC transporter regulation by cis-trans isomerization of X-prolyl bonds.

Nuclear immunophilin FKBP39 from Drosophila melanogaster drives spontaneous liquid-liquid phase separation.

The FKBP39 from Drosophila melanogaster is a multifunctional regulatory immunophilin. It contains two globular domains linked by a highly charged disordered region. The N-terminal domain shows homology to the nucleoplasmin core domain, and the C-terminal domain is characteristic for the family of the FKBP immunophilin ligand binding domain. The specific partially disordered structure of the protein inspired us to investigate whether FKBP39 can drive spontaneous liquid-liquid phase separation (LLPS). Preliminary analyses using CatGranule and Pi-Pi contact predictors suggested a propensity for LLPS. Microscopy observations revealed that FKBP39 can self-concentrate to form liquid condensates. We also found that FKBP39 can lead to LLPS in the presence of RNA and peptides containing Arg-rich linear motifs derived from selected nuclear and nucleolar proteins. These heterotypic interactions have a stronger propensity for driving LLPS when compared to the interactions mediated by self-associating FKBP39 molecules. To investigate whether FKBP39 can drive LLPS in the cellular environment, we analysed it in fusion with YFP in COS-7 cells. The specific distribution and diffusion kinetics of FKBP39 examined by FRAP experiments provided evidence that immunophilin is an important driver of phase separation. The ability of FKBP39 to go into heterotypic interaction may be fundamental for ribosome subunits assembly.

VEGF-A-Cleavage by FSAP and Inhibition of Neo-Vascularization.

Alternative splicing leads to the secretion of multiple forms of vascular endothelial growth factor-A (VEGF-A) that differ in their activity profiles with respect to neovascularization. FSAP (factor VII activating protease) is the zymogen form of a plasma protease that is activated (FSAPa) upon tissue injury via the release of histones. The purpose of the study was to determine if FSAPa regulates VEGF-A activity in vitro and in vivo. FSAP bound to VEGF165, but not VEGF121, and VEGF165 was cleaved in its neuropilin/proteoglycan binding domain. VEGF165 cleavage did not alter its binding to VEGF receptors but diminished its binding to neuropilin. The stimulatory effects of VEGF165 on endothelial cell proliferation, migration, and signal transduction were not altered by FSAP. Similarly, proliferation of VEGF receptor-expressing BAF3 cells, in response to VEGF165, was not modulated by FSAP. In the mouse matrigel model of angiogenesis, FSAP decreased the ability of VEGF165, basic fibroblast growth factor (bFGF), and their combination, to induce neovascularization. Lack of endogenous FSAP in mice did not influence neovascularization. Thus, FSAP inhibited VEGF165-mediated angiogenesis in the matrigel model in vivo, where VEGF's interaction with the matrix and its diffusion are important.

Histone Chaperone Paralogs Have Redundant, Cooperative, and Divergent Functions in Yeast.

Gene duplications increase organismal robustness by providing freedom for gene divergence or by increasing gene dosage. The yeast histone chaperones Fpr3 and Fpr4 are paralogs that can assemble nucleosomes in vitro; however, the genomic locations they target and their functional relationship is poorly understood. We refined the yeast synthetic genetic array approach to enable the functional dissection of gene paralogs. Applying this method to Fpr3 and Fpr4 uncovered redundant, cooperative, and divergent functions. While Fpr3 is uniquely involved in chromosome segregation, Fpr3 and Fpr4 cooperate to regulate genes involved in polyphosphate metabolism and ribosome biogenesis. We find that the TRAMP5 RNA exosome is critical for fitness in Δfpr3Δfpr4 yeast and leverage this information to identify an important role for Fpr4 at the 5' ends of protein coding genes. Additionally, Fpr4 and TRAMP5 negatively regulate RNAs from the nontranscribed spacers of ribosomal DNA. Yeast lacking Fpr3 and Fpr4 exhibit a genome instability phenotype at the ribosomal DNA, which implies that these histone chaperones regulate chromatin structure and DNA access at this location. Taken together. we provide genetic and transcriptomic evidence that Fpr3 and Fpr4 operate separately, cooperatively, and redundantly to regulate a variety of chromatin environments.

Effects of Destruxin A on Silkworm's Immunophilins.

Destruxin A (DA), a major secondary metabolite of Metarhizium anisopliae, has anti-immunity to insects. However, the detailed mechanism and its interactions with target proteins are elusive. Previously, three immunophilins, peptidyl-prolyl cis-trans isomerase (BmPPI), FK506 binding-protein 45 (BmFKBP45) and BmFKBP59 homologue, were isolated from the silkworm, Bombyx mori Bm12 cell line following treatment with DA, which suggested that these proteins were possible DA-binding proteins. To validate the interaction between DA and the three immunophilins, we performed bio-layer interferometry (BLI) assay, and the results showed that DA has interaction with BmPPI, whose affinity constant value is 1.98 × 10-3 M and which has no affinity with FKBP45 and FKBP59 homologue in vitro. Furthermore, we investigated the affinity between DA and human PPI protein (HsPPIA) and the affinity constant (KD) value is 2.22 × 10-3 M. Additionally, we compared the effects of silkworm and human PPI proteins produced by DA and immunosuppressants, cyclosporine A (CsA), and tacrolimus (FK506), by employing I2H (insect two-hybrid) in the SF-9 cell line. The results indicated that in silkworm, the effects created by DA and CsA were stronger than FK506. Furthermore, the effects created by DA in silkworm were stronger than those in humans. This study will offer new thinking to elucidate the molecular mechanism of DA in the immunity system of insects.

FKBPL and its peptide derivatives inhibit endocrine therapy resistant cancer stem cells and breast cancer metastasis by downregulating DLL4 and Notch4.

BACKGROUND: Optimising breast cancer treatment remains a challenge. Resistance to therapy is a major problem in both ER- and ER+ breast cancer. Tumour recurrence after chemotherapy and/or targeted therapy leads to more aggressive tumours with enhanced metastatic ability. Self-renewing cancer stem cells (CSCs) have been implicated in treatment resistance, recurrence and the development of metastatic disease. METHODS: In this study, we utilised in vitro, in vivo and ex vivo breast cancer models using ER+ MCF-7 and ER- MDA-MB-231 cells, as well as solid and metastatic breast cancer patient samples, to interrogate the effects of FKBPL and its peptide therapeutics on metastasis, endocrine therapy resistant CSCs and DLL4 and Notch4 expression. The effects of FKBPL overexpression or peptide treatment were assessed using a t-test or one-way ANOVA with Dunnett's multiple comparison test. RESULTS: We demonstrated that FKBPL overexpression or treatment with FKBPL-based therapeutics (AD-01, pre-clinical peptide /ALM201, clinical peptide) inhibit i) CSCs in both ER+ and ER- breast cancer, ii) cancer metastasis in a triple negative breast cancer metastasis model and iii) endocrine therapy resistant CSCs in ER+ breast cancer, via modulation of the DLL4 and Notch4 protein and/or mRNA expression. AD-01 was effective at reducing triple negative MDA-MB-231 breast cancer cell migration (n ≥ 3, p < 0.05) and invasion (n ≥ 3, p < 0.001) and this was translated in vivo where AD-01 inhibited breast cancer metastasis in MDA-MB-231-lucD3H1 in vivo model (p < 0.05). In ER+ MCF-7 cells and primary breast tumour samples, we demonstrated that ALM201 inhibits endocrine therapy resistant mammospheres, representative of CSC content (n ≥ 3, p < 0.05). Whilst an in vivo limiting dilution assay, using SCID mice, demonstrated that ALM201 alone or in combination with tamoxifen was very effective at delaying tumour recurrence by 12 (p < 0.05) or 21 days (p < 0.001), respectively, by reducing the number of CSCs. The potential mechanism of action, in addition to CD44, involves downregulation of DLL4 and Notch4. CONCLUSION: This study demonstrates, for the first time, the pre-clinical activity of novel systemic anti-cancer therapeutic peptides, ALM201 and AD-01, in the metastatic setting, and highlights their impact on endocrine therapy resistant CSCs; both areas of unmet clinical need.

Biological Actions of the Hsp90-binding Immunophilins FKBP51 and FKBP52

Immunophilins are a family of proteins whose signature domain is the peptidylprolyl-isomerase domain. High molecular weight immunophilins are characterized by the additional presence of tetratricopeptide-repeats (TPR) through which they bind to the 90-kDa heat-shock protein (Hsp90), and via this chaperone, immunophilins contribute to the regulation of the biological functions of several client-proteins. Among these Hsp90-binding immunophilins, there are two highly homologous members named FKBP51 and FKBP52 (FK506-binding protein of 51-kDa and 52-kDa, respectively) that were first characterized as components of the Hsp90-based heterocomplex associated to steroid receptors. Afterwards, they emerged as likely contributors to a variety of other hormone-dependent diseases, stress-related pathologies, psychiatric disorders, cancer, and other syndromes characterized by misfolded proteins. The differential biological actions of these immunophilins have been assigned to the structurally similar, but functionally divergent enzymatic domain. Nonetheless, they also require the complementary input of the TPR domain, most likely due to their dependence with the association to Hsp90 as a functional unit. FKBP51 and FKBP52 regulate a variety of biological processes such as steroid receptor action, transcriptional activity, protein conformation, protein trafficking, cell differentiation, apoptosis, cancer progression, telomerase activity, cytoskeleton architecture, etc. In this article we discuss the biology of these events and some mechanistic aspects.

BDNF (Brain-Derived Neurotrophic Factor) Promotes Embryonic Stem Cells Differentiation to Endothelial Cells Via a Molecular Pathway, Including MicroRNA-214, EZH2 (Enhancer of Zeste Homolog 2), and eNOS (Endothelial Nitric Oxide Synthase).

Objective- The NTs (neurotrophins), BDNF (brain-derived neurotrophic factor) and NT-3 promote vascular development and angiogenesis. This study investigated the contribution of endogenous NTs in embryonic stem cell (ESC) vascular differentiation and the potential of exogenous BDNF to improve the process of ESC differentiation to endothelial cells (ECs). Approach and Results- Mouse ESCs were differentiated into vascular cells using a 2-dimensional embryoid body (EB) model. Supplementation of either BDNF or NT-3 increased EC progenitors' abundance at day 7 and enlarged the peripheral vascular plexus with ECs and SM22α+ (smooth muscle 22 alpha-positive) smooth muscle cells by day 13. Conversely, inhibition of either BDNF or NT-3 receptor signaling reduced ECs, without affecting smooth muscle cells spread. This suggests that during vascular development, endogenous NTs are especially relevant for endothelial differentiation. At mechanistic level, we have identified that BDNF-driven ESC-endothelial differentiation is mediated by a pathway encompassing the transcriptional repressor EZH2 (enhancer of zeste homolog 2), microRNA-214 (miR-214), and eNOS (endothelial nitric oxide synthase). It was known that eNOS, which is needed for endothelial differentiation, can be transcriptionally repressed by EZH2. In turn, miR-214 targets EZH2 for inhibition. We newly found that in ESC-ECs, BDNF increases miR-214 expression, reduces EZH2 occupancy of the eNOS promoter, and increases eNOS expression. Moreover, we found that NRP-1 (neuropilin 1), KDR (kinase insert domain receptor), and pCas130 (p130 Crk-associated substrate kinase), which reportedly induce definitive endothelial differentiation of pluripotent cells, were increased in BDNF-conditioned ESC-EC. Mechanistically, miR-214 mediated the BDNF-induced expressional changes, contributing to BDNF-driven endothelial differentiation. Finally, BDNF-conditioned ESC-ECs promoted angiogenesis in vitro and in vivo. Conclusions- BDNF promotes ESC-endothelial differentiation acting via miR-214.