Growth arrest specific protein 7 inhibits tau fibrillogenesis.

Here we show that Gas7 inhibits phosphorylated tau fibrillogenesis by binding to phosphorylated tau at its non-WW domain, presumably F-BAR domain. We revealed that Gas7 binds to the third repeat domain of tau, the core element of tau oligomerization and the C-terminal domain of tau and alters the conformation not to form fibrils. These results suggest that Gas7 may serve to protect against Alzheimer's disease and other tauopathies by preventing tau fibrillogenesis.

Prolyl isomerase Pin1 is required sperm production by promoting mitosis progression of spermatogonial stem cells.

A prolyl isomerase Pin1 deficient (Pin1-/-) male mice had severe testicular atrophy. We investigated the function of Pin1 in spermatogenesis by analyzing the Pin1-/- mice at reproductive age. Pin1-/- mice had lessαPLZF positive spermatogonia (undifferentiated spermatogonia) than wild type (WT). Nevertheless, the Pin1-/- testis contained approximately the same number of GFRα1 positive spermatogonia (SSCs in steady state) as the WT testis. Furthermore, degeneration of the spermatogenia appeared in seminiferous tubules of 10 months old Pin1-/- mouse testis, and abnormal shape GFRα1 positive spermatogonia were observed. In Pin1-/- spermatogonia, the ratio of the phospho-histone H3 positive cells (mitotic cells) in GFRα1-positive spermatogonia was higher than that of WT. These results suggest that Pin1 promotes the progression of the mitotic cell cycle of SSC in steady-state, which is required for the sperm production from SSCs.

Fluorescent resonance energy transfer -based biosensor for detecting conformational changes of Pin1.

Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), regulates the activity and stability of various phosphorylated proteins. Pin1 consists of a PPIase domain and WW domain, both of which are required for the function of Pin1. However, how the behavior of these domains changes upon binding to phosphorylated proteins has not been analyzed. We created a Fluorescent Resonance Energy Transfer (FRET)-based biosensor "CPinY", which is composed of Pin1 flanked by CFP and YFP, and analyzed the interaction between Pin1 and c-Myc. Our results indicated that the dual phosphorylation of c-Myc at Thr58 and Ser62 is essential for tight interaction with Pin1. Additionally, this interaction caused a significant conformational change in Pin1. Our CPinY biosensor also detected a novel type of inhibitor of Pin1 function. We believe that his biosensor will be a novel drug screening technology targeting Pin1.

Food polyphenols targeting peptidyl prolyl cis/trans isomerase Pin1.

We searched for inhibitors against prolyl isomerase Pin1 in order to develop functional foods to prevent and cure various Pin1 related diseases such as cancer, diabetes, cardiovascular disease, Alzheimers's disease, and so on. We created a polyphenol library consisting of ingredients in healthy foods and beverages, since polyphenols like epigallocatechin gallate (EGCG) in green tea and 974B in brown algae had been identified as its Pin1 inhibitors. Several polyphenols such as EGCG derivatives, caffeic acid derivatives and tannic acid inhibited Pin1 activity. These results provide a first step in development of the functional foods and beverage targeting Pin1 and its related diseases.

Visualization of NO3⁻/NO2⁻ Dynamics in Living Cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-component Regulatory System.

Nitrate (NO3(-)) and nitrite (NO2(-)) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO3(-)/NO2(-) exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO3(-)/NO2(-) for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO3(-)/NO2(-) in physiology), by employing NO3(-)/NO2(-)-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO3(-) and NO2(-), and its FRET signal is changed in response to NO3(-)/NO2(-) in the micromolar range. Furthermore, both an increase and decrease in cellular NO3(-) concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO3(-)/NO2(-) dynamics in various organisms, including plants and animals.

Prolyl isomerase Pin1 promotes proplatelet formation of megakaryocytes via tau.

Here we show that Pin1, a peptidyl-prolyl cis/trans isomerase which catalyzes the isomerization of phosphorylated Ser/Thr-Pro, is a regulatory molecule of thrombopoiesis. We found that mice lacking the Pin1 gene (Pin1-/- mice) formed more megakaryocytes (MKs) than wild type mice (WT mice), and that the proplatelet formation of MKs was poorer in Pin1-/- mice than WT mice. Treatment of Meg-01 cells, a megakaryoblastic floating cell line, with shRNA against Pin1 suppressed the proplatelet formation. Expression of tau, a microtubule associated protein was induced in MKs during proplatelet formation. Pin1 bound tau and promoted microtubule polymerization. Our results show that Pin1 serves as a positive regulatory molecule of proplatelet formation of MKs by enhancing the function of phosphorylated tau.

Prolyl isomerase Pin1 negatively regulates AMP-activated protein kinase (AMPK) by associating with the CBS domain in the γ subunit.

AMP-activated protein kinase (AMPK) plays a critical role in metabolic regulation. In this study, first, it was revealed that Pin1 associates with any isoform of γ, but not with either the α or the ß subunit, of AMPK. The association between Pin1 and the AMPK γ1 subunit is mediated by the WW domain of Pin1 and the Thr(211)-Pro-containing motif located in the CBS domain of the γ1 subunit. Importantly, overexpression of Pin1 suppressed AMPK phosphorylation in response to either 2-deoxyglucose or biguanide stimulation, whereas Pin1 knockdown by siRNAs or treatment with Pin1 inhibitors enhanced it. The experiments using recombinant Pin1, AMPK, LKB1, and PP2C proteins revealed that the protective effect of AMP against PP2C-induced AMPKα subunit dephosphorylation was markedly suppressed by the addition of Pin1. In good agreement with the in vitro data, the level of AMPK phosphorylation as well as the expressions of mitochondria-related genes, such as PGC-1α, which are known to be positively regulated by AMPK, were markedly higher with reduced triglyceride accumulation in the muscles of Pin1 KO mice as compared with controls. These findings suggest that Pin1 plays an important role in the pathogenic mechanisms underlying impaired glucose and lipid metabolism, functioning as a negative regulator of AMPK.

Prolyl isomerase Pin1 regulates doxorubicin-inducible P-glycoprotein level by reducing Foxo3 stability.

It has been known that the phosphoSer/Thr-Pro-specific peptidyl prolyl cis/trans isomerase Pin1 regulates a variety of intracellular signaling pathways, including the response to the genotoxic drug doxorubicin. Pin1 binds phosphorylated p53 and stabilizes p53 to cause cell cycle arrest and apoptosis quickly in response to doxorubicin. Here we show another mechanism of Pin1 to maintain cell sensitivity to genotoxic stress, irrespective of whether p53 is present or not. In response to the genotoxic drug, Pin1 binds and decreases levels of the phosphorylated Foxo3, the positive transcription factor of P-glycoprotein (P-gp) gene. Through this mechanism of action, Pin1 decreases the level of P-gp and signals the cell to pump the genotoxic drugs out. This shows that Pin1 is implemented in maintaining the susceptibility to the genotoxic drugs by controlling P-gp level as well as p53-dependent apoptosis and cell cycle signaling pathways.

Pin1 promotes neuronal death in stroke by stabilizing Notch intracellular domain.

OBJECTIVE: Stroke is a leading cause of mortality and disability. The peptidyl-prolyl cis/trans isomerase Pin1 regulates factors involved in cell growth. Recent evidence has shown that Pin1 plays a major role in apoptosis. However, the role of Pin1 in ischemic stroke remains to be investigated. METHODS: We used Pin1 overexpression and knockdown to manipulate Pin1 expression and explore the effects of Pin1 in cell death on ischemic stress in vitro and in a mouse stroke model. We also used Pin 1 inhibitor, γ-secretase inhibitor, Notch1 intracellular domain (NICD1)-deleted mutant cells, and Pin1 mutant cells to investigate the underlying mechanisms of Pin1-NICD1-mediated cell death. RESULTS: Our findings indicate that Pin1 facilitates NICD1 stability and its proapoptotic function following ischemic stroke. Thus, overexpression of Pin1 increased NICD1 levels and enhanced its potentiation of neuronal death in simulated ischemia. By contrast, depletion or knockout of Pin1 reduced the NICD1 level, which in turn desensitized neurons to ischemic conditions. Pin1 interacted with NICD1 and increased its stability by inhibiting FBW7-induced polyubiquitination. We also demonstrate that Pin1 and NICD1 levels increase following stroke. Pin1 heterozygous (+/-) and knockout (-/-) mice, and also wild-type mice treated with an inhibitor of Pin1, each showed reduced brain damage and improved functional outcomes in a model of focal ischemic stroke. INTERPRETATION: These results suggest that Pin1 contributes to the pathogenesis of ischemic stroke by promoting Notch signaling, and that inhibition of Pin1 is a novel approach for treating ischemic stroke.

Differential regulation of cellular senescence and differentiation by prolyl isomerase Pin1 in cardiac progenitor cells.

Autologous c-kit(+) cardiac progenitor cells (CPCs) are currently used in the clinic to treat heart disease. CPC-based regeneration may be further augmented by better understanding molecular mechanisms of endogenous cardiac repair and enhancement of pro-survival signaling pathways that antagonize senescence while also increasing differentiation. The prolyl isomerase Pin1 regulates multiple signaling cascades by modulating protein folding and thereby activity and stability of phosphoproteins. In this study, we examine the heretofore unexplored role of Pin1 in CPCs. Pin1 is expressed in CPCs in vitro and in vivo and is associated with increased proliferation. Pin1 is required for cell cycle progression and loss of Pin1 causes cell cycle arrest in the G1 phase in CPCs, concomitantly associated with decreased expression of Cyclins D and B and increased expression of cell cycle inhibitors p53 and retinoblastoma (Rb). Pin1 deletion increases cellular senescence but not differentiation or cell death of CPCs. Pin1 is required for endogenous CPC response as Pin1 knock-out mice have a reduced number of proliferating CPCs after ischemic challenge. Pin1 overexpression also impairs proliferation and causes G2/M phase cell cycle arrest with concurrent down-regulation of Cyclin B, p53, and Rb. Additionally, Pin1 overexpression inhibits replicative senescence, increases differentiation, and inhibits cell death of CPCs, indicating that cell cycle arrest caused by Pin1 overexpression is a consequence of differentiation and not senescence or cell death. In conclusion, Pin1 has pleiotropic roles in CPCs and may be a molecular target to promote survival, enhance repair, improve differentiation, and antagonize senescence.