FKBP12 Research Articles

FKBP51 modulates hippocampal size and function in post-translational regulation of Parkin.

FK506-binding protein 51 (encoded by Fkpb51, also known as Fkbp5) has been associated with stress-related mental illness. To investigate its function, we studied the morphological consequences of Fkbp51 deletion. Artificial Intelligence-assisted morphological analysis revealed that male Fkbp51 knock-out (KO) mice possess more elongated dentate gyrus (DG) but shorter hippocampal height in coronal sections when compared to WT. Primary cultured Fkbp51 KO hippocampal neurons were shown to exhibit larger dendritic outgrowth than wild-type (WT) controls and pharmacological manipulation experiments suggest that this may occur through the regulation of microtubule-associated protein. Both in vitro primary culture and in vivo labeling support a role for FKBP51 in the regulation of microtubule-associated protein expression. Furthermore, Fkbp51 KO hippocampi exhibited decreases in βIII-tubulin, MAP2, and Tau protein levels, but a greater than 2.5-fold increase in Parkin protein. Overexpression and knock-down FKBP51 demonstrated that FKBP51 negatively regulates Parkin in a dose-dependent and ubiquitin-mediated manner. These results indicate a potential novel post-translational regulatory mechanism of Parkin by FKBP51 and the significance of their interaction on disease onset. KO has more flattened hippocampus using AI-assisted measurement Both pyramidal cell layer (PCL) of CA and granular cell layer (GCL) of DG distinguishable as two layers: deep cell layer and superficial layer.Distinct MAP2 expression between deep and superficial layer between KO and WT, Higher Parkin expression in KO brain Mechanism of FKBP51 inhibition resulting in Parkin, MAP2, Tau, and Tubulin expression differences between KO and WT mice, and resulting neurite outgrowth differences.

Relation of adipose tissue and skeletal muscle FKBP5 expression with insulin sensitivity and the regulation of FKBP5 by insulin and free fatty acids.

Recent studies suggest that FK506 binding protein 51 (FKBP51), a negative regulator of glucocorticoid response, encoded by FKBP5, may influence insulin action. The aim of the present study was to assess the relationship between subcutaneous adipose tissue (AT) and skeletal muscle FKBP5 expression in relation to insulin sensitivity in healthy individuals and to study its regulation by insulin and circulating free fatty acid (FFA) elevation. The study group comprised 96 male subjects, 49 normal-weight and 47 overweight/obese. Hyperinsulinemic clamp, subcutaneous AT and skeletal muscle biopsies were performed. In a subgroup of 20 subjects, two 6 h clamps were performed, with and without Intralipid/heparin infusion, and tissue biopsies were obtained before and after each clamp. AT FKBP5 expression was lower in overweight/obese individuals in comparison with normal-weight individuals (p = 0.004). Muscle FKBP5 expression did not differ between the groups, however, it was inversely related to insulin sensitivity (r = -0.32, p = 0.002). FKBP5 expression decreased in AT (p = 0.003) and increased in muscle (p < 0.0001) after insulin infusion. Intralipid/heparin diminished insulin-induced increase in muscle FKBP5. Our data show that lower AT FKBP5 expression is related to obesity, whereas muscle FKBP5 expression is associated with insulin resistance. AT and muscle FKBP5 expression is differentially regulated by insulin.

Circular noncoding RNA circ_0007865, serves as a competing endogenous RNA, targeting the miR-214-3p/FKBP5 axis to regulate oxygen-glucose deprivation-induced injury in brain microvascular endothelial cells.

Ischemic stroke (IS) is a major cause of permanent morbidity and lifelong disability worldwide. Circular RNA (circRNA) circ_0007865 has been reported to be upregulated in acute ischemic stroke (AIS) patients. Also, AIS patients exhibited increased death of human brain microvascular endothelial cells (HBMECs). This study is designed to explore the role and mechanism of circ_0007865 in the oxygen-glucose deprivation (OGD)-induced cell damage in AIS. Circ_0007865, microRNA-214-3p (miR-214-3p), and FK506-binding protein 5 (FKBP5) levels were detected by real-time quantitative PCR. Cell proliferative angiogenesis, migration, and apoptosis were assessed by Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, colony formation, tube formation, wound healing, transwell, and flow cytometry assays. B-cell lymphoma-2 (Bcl-2), Bcl-2-related X protein (Bax), cleaved caspase-3, and FKBP5 protein levels were determined by western blot assay. The binding relationship between miR-214-3p and circ_0007865 or FKBP5 was predicted by StarBase, and verified by a dual-luciferase reporter, RNA pull-down assay. Circ_0007865 and FKBP5 were increased, and miR-214-3p was decreased in OGD-treated HBMECs. Furthermore, the silencing of circ_0007865 could promote cell proliferative angiogenesis, migration, and inhibit apoptosis in OGD-triggered HBMECs in vitro. Mechanically, circ_0007865 acted as a sponge of miR-214-3p to regulate FKBP5. According to these results, circ_0007865 deficiency could attenuate OGD-induced HBMEC damage by modulating the miR-214-3p/FKBP5 axis, hinting at a promising therapeutic target for future acute IS therapy.

Physiological Genomics Plays a Crucial Role in Response to Stressful Life Events, the Development of Aggressive Behaviours, and Post-Traumatic Stress Disorder (PTSD).

Physiological genomics plays a crucial role in responding to stressful life events, such as violence and traumatic stress. This exposure to traumatic stress can trigger several physiological pathways, which are associated with genetic variability. Exposure to traumatic stress can result in the development of behavioural and psychiatric disorders, such as aggressive behaviour and anxiety disorders. Several genes play a crucial role in the neurophysiological response to chronic stress and trauma. These essential genes include monoamine oxidase A (MAOA), solute carrier family 6 member 4 (SLC6A4), brain-derived neurotrophic factor (BDNF), catechol-O-methyltransferase (COMT), dopamine receptor 2 and 4 (DRD2 and DRD4), and FK506 binding protein 5 (FKBP5). Genetic variations in several genes have been found to have altered physiological response, which associates with the development of several behavioural traits. Interestingly, previous studies show that there is an interplay between aggressive behaviour and anxiety disorders, which may be associated with physiological genomics structure. The physiological responses are based on genetic architecture and its molecular reaction. Understanding physiological genomics may show underpinnings related to the development of aggressive behaviours and their interaction with anxiety disorders. This review aims to discuss the association between different physiological genes and the development of psychiatric disorders related to aggressive behaviours and anxiety disorders, such as post-traumatic stress disorder.

FKBP52 in Neuronal Signaling and Neurodegenerative Diseases: A Microtubule Story.

The FK506-binding protein 52 (FKBP52) belongs to a large family of ubiquitously expressed and highly conserved proteins (FKBPs) that share an FKBP domain and possess Peptidyl-Prolyl Isomerase (PPIase) activity. PPIase activity catalyzes the isomerization of Peptidyl-Prolyl bonds and therefore influences target protein folding and function. FKBP52 is particularly abundant in the nervous system and is partially associated with the microtubule network in different cell types suggesting its implication in microtubule function. Various studies have focused on FKBP52, highlighting its importance in several neuronal microtubule-dependent signaling pathways and its possible implication in neurodegenerative diseases such as tauopathies (i.e., Alzheimer disease) and alpha-synucleinopathies (i.e., Parkinson disease). This review summarizes our current understanding of FKBP52 actions in the microtubule environment, its implication in neuronal signaling and function, its interactions with other members of the FKBPs family and its involvement in neurodegenerative disease.

FRET-based trilateration resolves distinct structural states and transitions of calmodulin bound to ryanodine receptors

We used fluorescence lifetime FRET (FLT-FRET) and trilateration to map the location of calmodulin (CaM) bound to skeletal and cardiac ryanodine receptor (RyR) Ca2+ channels at resting vs. peak contracting [Ca2+] (nM vs. μM). Eight single-Cys variants of the 12.6-kDa FK506-binding protein (FKBP) were labeled with FRET donor (AlexaFluor 488), and targeted to RyR1 and RyR2. Four single-Cys variants of WT-CaM or the Ca2+-insensitive CaM1234, two N-lobe and two C-lobe sites, were labelled with acceptor (AlexaFluor 568).

FK506-binding protein-like and FK506-binding protein 8 regulate dual leucine zipper kinase degradation and neuronal responses to axon injury

The dual leucine zipper kinase (DLK) is a key regulator of axon regeneration and degeneration in response to neuronal injury; however, regulatory mechanisms of the DLK function via its interacting proteins are largely unknown. To better understand the molecular mechanism of DLK function, we performed yeast two-hybrid screening analysis and identified FK506-binding protein-like (FKBPL, also known as WAF-1/CIP1 stabilizing protein 39) as a DLK-binding protein. FKBPL binds to the kinase domain of DLK and inhibits its kinase activity. In addition, FKBPL induces DLK protein degradation through ubiquitin-dependent pathways. We further assessed other members in the FKBP protein family and found that FK506-binding protein 8 (FKBP8) also induced DLK degradation. We identified the lysine 271 residue in the kinase domain as a major site of DLK ubiquitination and SUMO3 conjugation and was thus responsible for regulating FKBP8-mediated proteasomal degradation that was inhibited by the substitution of the lysine 271 to arginine. FKBP8-mediated degradation of DLK is mediated by autophagy pathway because knockdown of Atg5 inhibited DLK destabilization. We show that in vivo overexpression of FKBP8 delayed the progression of axon degeneration and suppressed neuronal death after axotomy in sciatic and optic nerves. Taken together, this study identified FKBPL and FKBP8 as novel DLK-interacting proteins that regulate DLK stability via the ubiquitin-proteasome and lysosomal protein degradation pathways.

FKBP8 is a novel molecule that participates in the regulation of the autophagic pathway

Autophagy is a homeostatic process by which misfolded proteins, organelles and cytoplasmic material are engulfed in autophagosomal vesicles and degraded through a lisosomal pathway. FKBP8 is a member of the FK506-binding proteins family (FKBP) usually found in mitochondria and the endoplasmic reticulum. This protein plays a critical role in cell functions such as protein trafficking and folding. In the present report we demonstrate that the depletion of FKBP8 abrogated autophagy activation induced by starvation, whereas the overexpression of this protein triggered the autophagy cascade. We found that FKBP8 co-localizes with ATG14L and BECN1, both members of the VPS34 lipid kinase complex, which regulates the initial steps in the autophagosome formation process. We have also demonstrated that FKBP8 is necessary for VPS34 activity. Our findings indicate that the regulatory function of FKBP8 in the autophagy process depends of its transmembrane domain. Surprisingly, this protein was not found in autophagosomal vesicles, which reinforces the notion that the FKBP8 only participates in the initial steps of the autophagosome formation process. Taken together, our data provide evidence that FKBP8 modulates the early steps of the autophagosome formation event by interacting with the VPS34 lipid kinase complex. SummaryIn this article, the protein FKBP38 is reported to be a novel modulator of the initial steps of the autophagic pathway, specifically in starvation-induced autophagy. FKBP38 interacts with the VPS34 lipid kinase complex, with the transmembrane domain of FKBP38 being critical for its biological function.

Effect of FKBP12-Derived Intracellular Peptides on Rapamycin-Induced FKBP-FRB Interaction and Autophagy.

Intracellular peptides (InPeps) generated by proteasomes were previously suggested as putative natural regulators of protein–protein interactions (PPI). Here, the main aim was to investigate the intracellular effects of intracellular peptide VFDVELL (VFD7) and related peptides on PPI. The internalization of the peptides was achieved using a C-terminus covalently bound cell-penetrating peptide (cpp; YGRKKRRQRRR). The possible inhibition of PPI was investigated using a NanoBiT® luciferase structural complementation reporter system, with a pair of plasmids vectors each encoding, simultaneously, either FK506-binding protein (FKBP) or FKBP-binding domain (FRB) of mechanistic target of rapamycin complex 1 (mTORC1). The interaction of FKBP–FRB within cells occurs under rapamycin induction. Results shown that rapamycin-induced interaction between FKBP–FRB within human embryonic kidney 293 (HEK293) cells was inhibited by VFD7-cpp (10–500 nM) and FDVELLYGRKKRRQRRR (VFD6-cpp; 1–500 nM); additional VFD7-cpp derivatives were either less or not effective in inhibiting FKBP–FRB interaction induced by rapamycin. Molecular dynamics simulations suggested that selected peptides, such as VFD7-cpp, VFD6-cpp, VFAVELLYGRKKKRRQRRR (VFA7-cpp), and VFEVELLYGRKKKRRQRRR (VFA7-cpp), bind to FKBP and to FRB protein surfaces. However, only VFD7-cpp and VFD6-cpp induced changes on FKBP structure, which could help with understanding their mechanism of PPI inhibition. InPeps extracted from HEK293 cells were found mainly associated with macromolecular components (i.e., proteins and/or nucleic acids), contributing to understanding InPeps’ intracellular proteolytic stability and mechanism of action-inhibiting PPI within cells. In a model of cell death induced by hypoxia-reoxygenation, VFD6-cpp (1 µM) increased the viability of mouse embryonic fibroblasts cells (MEF) expressing mTORC1-regulated autophagy-related gene 5 (Atg5), but not in autophagy-deficient MEF cells lacking the expression of Atg5. These data suggest that VFD6-cpp could have therapeutic applications reducing undesired side effects of rapamycin long-term treatments. In summary, the present report provides further evidence that InPeps have biological significance and could be valuable tools for the rational design of therapeutic molecules targeting intracellular PPI.

Molecular insights into the maturation of phosphodiesterase 6 by the specialized chaperone complex of HSP90 with AIPL1

Phosphodiesterase 6 (PDE6) is a key effector enzyme in vertebrate phototransduction, and its maturation and function are known to critically depend on a specialized chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). Defects in PDE6 and AIPL1 underlie several severe retinal diseases, including retinitis pigmentosa and Leber congenital amaurosis. Here, we characterize the complex of AIPL1 with HSP90 and demonstrate its essential role in promoting the functional conformation of nascent PDE6. Our analysis suggests that AIPL1 preferentially binds to HSP90 in the closed state with a stoichiometry of 1:2, with the tetratricopeptide repeat domain and the tetratricopeptide repeat helix 7 extension of AIPL1 being the main contributors to the AIPL1/HSP90 interface. We demonstrate that mutations of these determinants markedly diminished both the affinity of AIPL1 for HSP90 and the ability of AIPL1 to cochaperone the maturation of PDE6 in a heterologous expression system. In addition, the FK506-binding protein (FKBP) domain of AIPL1 encloses a unique prenyl-binding site that anchors AIPL1 to posttranslational lipid modifications of PDE6. A mouse model with rod PDE6 lacking farnesylation of its PDE6A subunit revealed normal expression, trafficking, and signaling of the enzyme. Furthermore, AIPL1 was unexpectedly capable of inducing the maturation of unprenylated cone PDE6C, whereas mutant AIPL1 deficient in prenyl binding competently cochaperoned prenylated PDE6C. Thus, we conclude neither sequestration of the prenyl modifications is required for PDE6 maturation to proceed, nor is the FKBP-lipid interaction involved in the conformational switch of the enzyme into the functional state.

Engineering a Rapid Insulin Release System Controlled By Oral Drug Administration.

Rapid insulin release plays an essential role in maintaining blood‐glucose homeostasis in mammalians. Patients diagnosed with type‐I diabetes mellitus experience chronic and remarkably high blood‐sugar levels, and require lifelong insulin injection therapy, so there is a need for more convenient and less invasive insulin delivery systems to increase patients’ compliance and also to enhance their quality of life. Here, an endoplasmic‐reticulum‐localized split sec‐tobacco etch virus protease (TEVp)‐based rapamycin‐actuated protein‐induction device (RAPID) is engineered, which is composed of the rapamycin‐inducible dimerization domains FK506 binding protein (FKBP) and FKBP‐rapamycin binding protein fused with modified split sec‐TEVp components. Insulin accumulation inside the endoplasmic reticulum (ER) is achieved through tagging its C‐terminus with KDEL, an ER‐retention signal, spaced by a TEVp cleavage site. In the presence of rapamycin, the split sec‐TEVp‐based RAPID components dimerize, regain their proteolytic activity, and remove the KDEL retention signal from insulin. This leads to rapid secretion of accumulated insulin from cells within few minutes. Using liver hydrodynamic transfection methodology, it is shown that RAPID quickly restores glucose homeostasis in type‐1‐diabetic (T1DM) mice treated with an oral dose of clinically licensed rapamycin. This rapid‐release technology may become the foundation for other cell‐based therapies requiring instantaneous biopharmaceutical availability.

The co-chaperone FKBP51 modulates HPA axis activity and age-related maladaptation of the stress system in pituitary proopiomelanocortin cells

Glucocorticoid (GC)-mediated negative feedback of the hypothalamic-pituitary-adrenal (HPA) axis, the body’s physiological stress response system, is tightly regulated and essential for appropriate termination of this hormonal cascade. Disturbed regulation and maladaptive response of this axis are fundamental components of multiple stress-induced psychiatric and metabolic diseases and aging. The co-chaperone FK506 binding protein 51 (FKBP51) is a negative regulator of the GC receptor (GR), is highly stress responsive, and its polymorphisms have been repeatedly associated with stress-related disorders and dysfunctions in humans and rodents. Proopiomelanocortin (Pomc)-expressing corticotropes in the anterior pituitary gland are one of the key cell populations of this closed-loop GC-dependent negative feedback regulation of the HPA axis in the periphery. However, the cell type-specific role of FKBP51 in anterior pituitary corticotrope POMC cells and its impact on age-related HPA axis disturbances are yet to be elucidated. Here, using a combination of endogenous knockout and viral rescue, we show that male mice lacking FKBP51 in Pomc-expressing cells exhibit enhanced GR-mediated negative feedback and are protected from age-related disruption of their diurnal corticosterone (CORT) rhythm. Our study highlights the complexity of tissue- and cell type-specific, but also cross-tissue effects of FKBP51 in the rodent stress response at different ages and extends our understanding of potential targets for pharmacological intervention in stress- and age-related disorders.

Engineering a Fluorescent Protein Color Switch Using Entropy-Driven β-Strand Exchange.

Protein conformational switches are widely used in biosensing. They are often composed of an input domain (which binds a target ligand) fused to an output domain (which generates an optical readout). A central challenge in designing such switches is to develop mechanisms for coupling the input and output signals via conformational changes. Here, we create a biosensor in which binding-induced folding of the input domain drives a conformational shift in the output domain that results in a sixfold green-to-yellow ratiometric fluorescence change in vitro and a 35-fold intensiometric fluorescence increase in cultured cells. The input domain consists of circularly permuted FK506 binding protein (cpFKBP) that folds upon binding its target ligand (FK506 or rapamycin). cpFKBP folding induces the output domain, an engineered green fluorescent protein (GFP) variant, to replace one of its β-strands (containing T203 and specifying green fluorescence) with a duplicate β-strand (containing Y203 and specifying yellow fluorescence) in an intramolecular exchange reaction. This mechanism employs the loop-closure entropy principle, embodied by the folding of the partially disordered cpFKBP domain, to couple ligand binding to the GFP color shift. This study highlights the high-energy barriers present in GFP folding which cause β-strand exchange to be slow and are also likely responsible for the shift from the β-strand exchange mechanism in vitro to ligand-induced chromophore maturation in cells. The proof-of-concept design has the advantages of full genetic encodability and potential for modularity. The latter attribute is enabled by the natural coupling of binding and folding and circular permutation of the input domain, which theoretically allows different binding domains to be compatible for insertion into the GFP surface loop.

FKBP-type peptidyl-prolyl cis-trans isomerase interacts with the movement protein of tomato leaf curl New Delhi virus and impacts viral replication in Nicotiana benthamiana.

Begomoviruses belonging to the family Geminiviridae are plant‐infecting DNA viruses. Begomoviral movement protein (MP) has been reported to be required for virus movement, host range determination, and symptom development. In the present study, the FK506‐binding protein (FKBP)‐type peptidyl‐prolyl cis‐trans isomerase (NbFKPPIase) of Nicotiana benthamiana was identified by a yeast two‐hybrid screening system using the MP of tomato leaf curl New Delhi virus (ToLCNDV) oriental melon (OM) isolate (MPOM) as bait. Transient silencing of the gene encoding NbFKPPIase increased replication of three test begomoviruses, and transient overexpression decreased viral replication, indicating that NbFKPPIase plays a role in defence against begomoviruses. However, infection of N. benthamiana by ToLCNDV‐OM or overexpression of the gene encoding MPOM drastically reduced the expression of the gene encoding NbFKPPIase. Fluorescence resonance energy transfer analysis revealed that MPOM interacted with NbFKPPIase in the periphery of cells. Expression of the gene encoding NbFKPPIase was induced by salicylic acid but not by methyl jasmonate or ethylene. Moreover, the expression of the gene encoding NbFKPPIase was down‐regulated in response to 6‐benzylaminopurine and up‐regulated in response to gibberellin or indole‐3‐acetic acid, suggesting a role of NbFKPPIase in plant development. Transcriptome analysis and comparison of N. benthamiana transient silencing and overexpression of the gene encoding MPOM led to the identification of several differentially expressed genes whose functions are probably associated with cell cycle regulation. Our results indicate that begomoviruses could suppress NbFKPPIase‐mediated defence and biological functions by transcriptional inhibition and physical interaction between MP and NbFKPPIase to facilitate infection.

Anchor Away: A System for Fast Inhibition of Proteins in Drosophila.

Anchor away is a sequestering method designed to acutely and timely abrogate the function of a protein of interest by anchoring to a cell compartment different from its target. This method induces the binding of the target protein to the anchor by either the addition of rapamycin to Drosophila food or cell media. Rapamycin mediates the formation of a ternary complex between the anchor, which is tagged with the FK506-binding protein (FKBP12), and the target protein fused with the FKB12 rapamycin-binding (FRB) domain of mammalian target of rapamycin (mTOR). The rapamycin-bound target protein stays sequestered away from its compartment, where it cannot perform its biological function.

Molecular Tension Probe for In Vitro Bioassays.

Cell-free bioassays (CFBs) provide their own distinctive merits over cell-based bioassays (CBBs) including (i) rapid and on-site applicability, (ii) long-term utility, and (iii) bioanalytical versatility. The authors previously introduced a unique bioluminescent imaging probe for illuminating molecular tension appended by protein-protein interactions (PPIs) of interest. In this chapter, we exemplify that a full-length artificial luciferase is sandwiched between FRB (FKBP-rapamycin-binding domain of FKBP12-rapamycin-associated protein) and FKBP (FK506-binding protein) via minimal flexible linkers, named FRB-A23-FKBP. The rapamycin-activated PPIs between FRB and FKBP append molecular tension to the sandwiched luciferase, enhancing the enzymatic activity in a quantitative manner. The fusion protein, FRB-A23-FKBP, is three-step column-purified and the bioanalytical utility is characterized in various CFB conditions. This chapter guides the detailed protocols from the purification to the practical bioassays of FRB-A23-FKBP.

Reversion of glucocorticoid-induced senescence and collagen synthesis decrease by LY294002 is mediated through p38 in skin

Glucocorticoids (GCs) are the most common treatment for inflammatory skin disorders; however, they show several adverse side effects, including atrophy and collagen decrease following chronic treatment. In particular, transcription factors and p38 signaling for collagen synthesis have been shown to be suppressed by the active glucocorticoid receptor (GR). LY294002 (LY), a phosphoinositide 3-kinase (PI3K) inhibitor, has been reported to protect keratinocytes in epidermis against GC-induced hypoplasia; however, its protective effect in dermis remains unclear. Furthermore, clobetasol propionate (CP) is the most used commercial synthetic GC, yet studies on how CP causes side effects in dermal fibroblasts are limited. In this study, dermal atrophy was modeled using CP in human dermal fibroblasts (HDFs) and C57BL/6 mice. CP treatment significantly upregulated FK506 binding protein 5 (FKBP51), an atrophy marker (2.4 ± 0.25 and 3.3 ± 0.3 fold in in vitro and in vivo, respectively), phosphorylated GR (1.96 ± 0.08 and 2.29 ± 0.25 fold in in vitro and in vivo, respectively), decreased fibroblast proliferation (82.71 ± 1.95% in in vitro), reduced collagen synthesis (0.36 ± 0.05 and 0.3 ± 0.1 fold in in vitro and in vivo, respectively), and induced aging, all of which were reversed by LY treatment (from 1.43 ± 0.08 to 2.8 ± 0.12 fold) without showing growth inhibition and exerting the anti-inflammation of CP. Interestingly, the protective effect of LY was dose-dependently reversed by treatment with a p38 inhibitor and reached 2.9 ± 0.15 fold at dose 20 µM. Taken together, our results demonstrate that LY reduced CP-induced upregulation of the atrophy marker FKBP51, GR phosphorylation, and GR nuclear translocation via the activation of p38, whilst maintaining the anti-inflammatory effect of glucocorticoids.

Cadmium regulates FKBP5 through miR-9-5p and induces carp lymphocyte apoptosis

Cadmium (Cd) is an environmental pollutant produced by industrial activities, which has no known physiological benefits to organisms. In our previous study, the transcriptomic profiles of carp head kidney exposed to Cd was analyzed by genomics technique, and confirmed that miRNAs are important in the head kidney injury of carp induced by Cd, but the specific biological mechanism was unclear. In order to further explore the effect of Cd on carp head kidney lymphocyte damage, we established a model of Cd exposure in vitro. The results showed that Cd could increase the expression of Bax (Bcl‐2 associated X protein), Caspase9 (Cysteinyl aspartate specific proteinase 9) and Caspase3 (Cysteinyl aspartate specific proteinase 3), inhibit the expression of Bcl-2 (B cell lymphoma/leukemia 2), and induce apoptosis of carp head kidney lymphocytes. In our previous study, we screened the differentially expressed miRNA in Cd-treated lymphocytes by high-throughput sequencing, and found that there was a significant difference in the expression of miR-9-5p. The expression trend of miR-9-5p in the vitro model was the same as that of high-throughput sequencing. We screened the differentially expressed gene FKBP5 (FK506-binding protein 51) in lymphocytes treated with Cd. It was confirmed by double luciferase reporter gene analysis that FKBP5 was the target gene of miR-9-5p. We established the overexpression/knockdown model of miR-9-5p in carp head kidney lymphocyte in vitro. The results showed that miR-9-5p could inhibit the expression of FKBP5, increase the phosphorylation level of Akt, inhibit apoptosis and improve the cell survival rate in carp head kidney lymphocytes. Together, Cd could down-regulate the expression of miR-9-5p, target up-regulate the expression of FKBP5, inhibit the phosphorylation of Akt, and promote the apoptosis of carp head kidney lymphocytes through mitochondrial pathway.

Thermococcus sp. KS-1 PPIase as a fusion partner improving soluble production of aromatic amino acid decarboxylase

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS−1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS−1 (PPIase KS−1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS−1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS−1. The recombinant E. coli cells expressing the PPIase KS−1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS−1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS−1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.

Esterase D stabilizes FKBP25 to suppress mTORC1

BackgroundEsterase D (ESD) is a nonspecific esterase that detoxifies formaldehyde. Many reports have stated that ESD activity is associated with a variety of physiological and pathological processes. However, the detailed signaling pathway of ESD remains poorly understood.MethodsConsidering the advantages of the small chemical molecule, our recent work demonstrated that 4-chloro-2-(5-phenyl-1-(pyridin-2-yl)-4,5-dihydro-1H-pyrazol-3-yl) phenol (FPD5) activates ESD, and will be a good tool for studying ESD further. Firstly, we determined the interaction between ESD and FK506 binding protein 25 (FKBP25) by yeast two-hybrid assay and co-immunoprecipitation (CO-IP) and analyzed the phosphorylation levels of mTORC1, P70S6K and 4EBP1 by western blot. Furthermore, we used the sulforhodamine B (SRB) and chick chorioallantoic membrane (CAM) assay to analyze cell viability in vitro and in vivo after treatment with ESD activator FPD5.ResultsWe screened FKBP25 as a candidate protein to interact with ESD by yeast two-hybrid assay. Then we verified the interaction between ESD and endogenous FKBP25 or ectopically expressed GFP-FKBP25 by CO-IP. Moreover, the N-terminus (1–90 aa) domain of FKBP25 served as the crucial element for their interaction. More importantly, ESD reduced the K48-linked poly-ubiquitin chains of FKBP25 and thus stabilized cytoplasmic FKBP25. ESD also promoted FKBP25 to bind more mTORC1, suppressing the activity of mTORC1. In addition, ESD suppressed tumor cell growth in vitro and in vivo through autophagy.ConclusionsThese findings provide novel evidence for elucidating the molecular mechanism of ESD and ubiquitination of FKBP25 to regulate autophagy and cancer cell growth. The ESD/FKBP25/mTORC1 signaling pathway is involved in inhibiting tumor cell growth via regulating autophagy.