Peptidyl-prolyl Isomerase Pin1 Research Articles

A novel bivalent interaction mode underlies a non-catalytic mechanism for Pin1-mediated protein kinase C regulation.

Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and βII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of full-length Pin1 complexed to the C-terminal tail of PKCβII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a conformation in which it uses the WW and PPIase domains to engage two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, respectively. Hydrophobic motif is a non-canonical Pin1-interacting element. The structural information combined with the results of extensive binding studies and experiments in cultured cells suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.

Abstract 390: Direct interaction of Pin1 with HIF-prolyl hydroxylase 2: Implications for breast cancer cell growth and survival

Abstract Prolyl hydroxylase domain 2 (PHD2) is the main hypoxia-inducible factor (HIF)-prolyl hydroxylase. PHD2 in normoxia hydroxylates specific proline residues in HIF-1α and HIF-2α, which facilitates their ubiquitination and proteasomal degradation. Although the activity of PHD2 is reduced in hypoxia, significant levels of residual activity of this monooxygenase are still detected under hypoxic conditions. However, its role under hypoxia is poorly understood. Peptidyl-prolyl isomerase (Pin1) binds to target proteins containing phosphorylated serine or threonine residues followed by proline (pS/T-P). As PHD2 harbors several pS/T-P motifs, it may be a potential substrate of Pin1. We found for the first time interaction between Pin1 and PHD2 in human breast cancer MDA-MB-231 cells in normoxic and hypoxic conditions. Additionally, the breast cancer tissue array showed elevated expression of PHD2 as well as Pin1 in tumors compared to adjacent normal tissues. LC-MS/MS spectrometry identified three amino acid residues (S125, T168, and S174) of PHD2 undergoing phosphorylation. Among these, serine 125 was found to be the principal site required for Pin1 binding. As a novel binding partner of Pin1, oncogenic PHD2 can be explored as a therapeutic target for the treatment of breast cancer. Citation Format: Yanymee Nimesia Guillen Quispe, Su-Jung Kim, Soma Saeidi, Gyo-Jin Choi, Chaithanya Chelakkot, Tianchi Zhou, Sang-Beom Bang, Tae-Won Kim, Young-Kee Shin, Young-Joon Surh. Direct interaction of Pin1 with HIF-prolyl hydroxylase 2: Implications for breast cancer cell growth and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 390.

Stabilization of Pin1 by USP34 promotes Ubc9 isomerization and protein sumoylation in glioma stem cells

The peptidyl-prolyl cis-trans isomerase Pin1 is a pivotal therapeutic target in cancers, but the regulation of Pin1 protein stability is largely unknown. High Pin1 expression is associated with SUMO1-modified protein hypersumoylation in glioma stem cells (GSCs), but the underlying mechanisms remain elusive. Here we demonstrate that Pin1 is deubiquitinated and stabilized by USP34, which promotes isomerization of the sole SUMO E2 enzyme Ubc9, leading to SUMO1-modified hypersumoylation to support GSC maintenance. Pin1 interacts with USP34, a deubiquitinase with preferential expression and oncogenic function in GSCs. Such interaction is facilitated by Plk1-mediated phosphorylation of Pin1. Disruption of USP34 or inhibition of Plk1 promotes poly-ubiquitination and degradation of Pin1. Furthermore, Pin1 isomerizes Ubc9 to upregulate Ubc9 thioester formation with SUMO1, which requires CDK1-mediated phosphorylation of Ubc9. Combined inhibition of Pin1 and CDK1 with sulfopin and RO3306 most effectively suppresses orthotopic tumor growth. Our findings provide multiple molecular targets to induce Pin1 degradation and suppress hypersumoylation for cancer treatment.

KPT6566 induces apoptotic cell death and suppresses the tumorigenicity of testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) frequently affect adolescent and young adult males. Although TGCT is more responsive to cisplatin-based chemotherapy than other solid tumors, some patients are nonresponders, and following treatment, many patients continue to experience acute and long-term cytotoxic effects from cisplatin-based chemotherapy. Consequently, it is imperative to develop new therapeutic modalities for treatment-resistant TGCTs. Peptidyl-prolyl isomerase (Pin1) regulates the activity and stability of many cancer-associated target proteins. Prior findings suggest that Pin1 contributes to the pathogenesis of multiple human cancers. However, the specific function of Pin1 in TGCTs has not yet been elucidated. TGCT cell proliferation and viability were examined using cell cycle analysis and apoptosis assays following treatment with KPT6566, a potent, selective Pin1 inhibitor that covalently binds to the catalytic domain of Pin1. A xenograft mouse model was used to assess the effect of KPT6566 on tumor growth in vivo. KPT6566 effectively suppressed cell proliferation, colony formation, and ATP production in P19 and NCCIT cells. Further, KPT6566 induced apoptotic cell death by generating cellular reactive oxygen species and downregulating the embryonic transcription factors Oct-4 and Sox2. Finally, KPT6566 treatment significantly reduced tumor volume and mass in P19 cell xenografts. The Pin1 inhibitor KPT6566 has significant antiproliferative and antitumor effects in TGCT cells. These findings suggest that Pin1 inhibitors could be considered as a potential therapeutic approach for TGCTs.

Regulation of eukaryotic protein kinases by Pin1, a peptidyl-prolyl isomerase.

The peptidyl-prolyl isomerase Pin1 cooperates with proline-directed kinases and phosphatases to regulate multiple oncogenic pathways. Pin1 specifically recognizes phosphorylated Ser/Thr-Pro motifs in proteins and catalyzes their cis-trans isomerization. The Pin1-catalyzed conformational changes determine the stability, activity, and subcellular localization of numerous protein substrates. We conducted a survey of eukaryotic protein kinases that are regulated by Pin1 and whose Pin1 binding sites have been identified. Our analyses reveal that Pin1 target sites in kinases do not fall exclusively within the intrinsically disordered regions of these enzymes. Rather, they fall into three groups based on their location: (i) within the catalytic kinase domain, (ii) in the C-terminal kinase region, and (iii) in regulatory domains. Some of the kinases downregulated by Pin1 activity are tumor-suppressing, and all kinases upregulated by Pin1 activity are functionally pro-oncogenic. These findings further reinforce the rationale for developing Pin1-specific inhibitors as attractive pharmaceuticals for cancer therapy.

The role of peptidyl-prolyl isomerase Pin1 in neuronal signaling in epilepsy.

Epilepsy is a common symptom of many neurological disorders and can lead to neuronal damage that plays a major role in seizure-related disability. The peptidyl-prolyl isomerase Pin1 has wide-ranging influences on the occurrence and development of neurological diseases. It has also been suggested that Pin1 acts on epileptic inhibition, and the molecular mechanism has recently been reported. In this review, we primarily focus on research concerning the mechanisms and functions of Pin1 in neurons. In addition, we highlight the significance and potential applications of Pin1 in neuronal diseases, especially epilepsy. We also discuss the molecular mechanisms by which Pin1 controls synapses, ion channels and neuronal signaling pathways to modulate epileptic susceptibility. Since neurotransmitters and some neuronal signaling pathways, such as Notch1 and PI3K/Akt, are vital to the nervous system, the role of Pin1 in epilepsy is discussed in the context of the CaMKII-AMPA receptor axis, PSD-95-NMDA receptor axis, NL2/gephyrin-GABA receptor signaling, and Notch1 and PI3K/Akt pathways. The effect of Pin1 on the progression of epilepsy in animal models is discussed as well. This information will lead to a better understanding of Pin1 signaling pathways in epilepsy and may facilitate development of new therapeutic strategies.

Molecular Insights into the Intrinsic Dynamics and Their Roles During Catalysis in Pin1 Peptidyl-prolyl Isomerase.

Proteins are intrinsically dynamic and change conformations over a wide range of time scales. While the conformational dynamics have been realized to be important for protein functions, e.g., in activity-stability trade-offs, how they play a role during enzyme catalysis has been of debate over decades. By studying Pin1 peptidyl-prolyl isomerase using extensive molecular dynamics simulations, here we discuss how the slow intrinsic dynamics of Pin1 observed in the NMR relaxation dispersion experiment occur and couple to isomerization reactions in molecular detail. In particular, we analyze the angular correlation functions of the backbone N-H bonds and find that slow conformational transitions occur at about the 310 helix in the apo state. These events at the helical region further affect the residues at about the ligand binding site. Unfolding of this helix leads to a tight hydrogen bond between the helical region and the ligand binding loop, thus forming a stable coiled structure. The helical and coiled structures are found to be characteristic of the Pin1-ligand complex with the ligand in the trans and cis states, respectively. These results indicate that the changes in the slow dynamics of Pin1 by the isomerization reaction occur via the shift in populations of the helical and coiled states, where the balance is dependent on the ligand isomerization states.

Regulation of XPO5 phosphorylation by PP2A in hepatocellular carcinoma.

Exportin 5 (XPO5) is a shuttle protein that mediates precursor miRNA (pre‐miRNA) export from the nucleus to the cytoplasm, an important step in miRNA maturation. We previously demonstrated that XPO5 was phosphorylated by ERK kinase and subsequently underwent conformation change by the peptidyl‐prolyl isomerase Pin1, leading to the reduced miRNA expression in hepatocellular carcinoma (HCC). Protein phosphorylation modification serves as a reversible regulatory mechanism precisely governed by protein kinases and phosphatases. Here we identified that the phosphatase PP2A catalyzed XPO5 dephosphorylation. PP2A holoenzyme is a ternary complex composed of a catalytic subunit, a scaffold subunit, and a regulatory subunit that determines substrate specificity. In this study, we characterized the involvement of B55β subunit in XPO5 dephosphorylation that favored the distribution of XPO5 into the cytoplasm and promoted miRNA expression, leading to HCC inhibition in vitro and in vivo. Our study demonstrates the regulatory role of B55β‐containing PP2A in miRNA expression and may shed light on HCC pathogenesis.

Pin1 as Molecular Switch in Vascular Endothelium: Notes on Its Putative Role in Age-Associated Vascular Diseases.

By controlling the change of the backbones of several cellular substrates, the peptidyl-prolyl cis-trans isomerase Pin1 acts as key fine-tuner and amplifier of multiple signaling pathways, thereby inducing several biological consequences, both in physiological and pathological conditions. Data from the literature indicate a prominent role of Pin1 in the regulating of vascular homeostasis. In this review, we will critically dissect Pin1’s role as conformational switch regulating the homeostasis of vascular endothelium, by specifically modulating nitric oxide (NO) bioavailability. In this regard, Pin1 has been reported to directly control NO production by interacting with bovine endothelial nitric oxide synthase (eNOS) at Ser116-Pro117 (human equivalent is Ser114-Pro115) in a phosphorylation-dependent manner, regulating its catalytic activity, as well as by regulating other intracellular players, such as VEGF and TGF-β, thereby impinging upon NO release. Furthermore, since Pin1 has been found to act as a critical driver of vascular cell proliferation, apoptosis, and inflammation, with implication in many vascular diseases (e.g., diabetes, atherosclerosis, hypertension, and cardiac hypertrophy), evidence indicating that Pin1 may serve a pivotal role in vascular endothelium will be discussed. Understanding the role of Pin1 in vascular homeostasis is crucial in terms of finding a new possible therapeutic player and target in vascular pathologies, including those affecting the elderly (such as small and large vessel diseases and vascular dementia) or those promoting the full expression of neurodegenerative dementing diseases.

Abstract 14033: Inhibition of the Prolyl Isomerase Pin1 Improves Endothelial Function and Attenuates Vascular Remodelling in Pulmonary Hypertension by Inhibiting TGF-β Signalling

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by obstructive pulmonary vascular remodelling ultimately leading to right ventricular (RV) failure and death. Disturbed transforming growth factor-β (TGF-β) / bone morphogenetic protein (BMP) signalling, endothelial cell dysfunction, increased proliferation of smooth muscle cells and fibroblasts, and inflammation contribute to this abnormal remodelling. Peptidyl prolyl isomerase Pin1 has been identified as a critical driver of proliferation and inflammation in vascular cells, but its role in the disturbed TGF-β/BMP signalling, endothelial cell dysfunction and vascular remodelling in PAH is unknown. Methods and Results: Pin1 expression is increased in cultured pulmonary microvascular endothelial cells (MVECs) and lung tissue of PAH patients. Pin1 inhibitor, juglone significantly decreased TGF-β signalling, increased BMP signalling, normalized their hyper-proliferative, and inflammatory phenotype. Juglone treatment reversed vascular remodelling through reducing TGF-β signalling in monocrotaline + shunt-PAH rat model. Juglone treatment decreased Fulton index, but did not affect or harm cardiac function and remodelling in rats with RV pressure load induced by pulmonary artery banding. Conclusions: Our study demonstrates that inhibition of Pin1 reversed the PAH phenotype in PAH-MVECs in vitro and in PAH rats in vivo, potentially through modulation of TGF-β/BMP signalling pathways. Selective inhibition of Pin1 could be a novel therapeutic option for the treatment of PAH.

Inhibition of the prolyl isomerase Pin1 improves endothelial function and attenuates vascular remodelling in pulmonary hypertension by inhibiting TGF-\u03b2 signalling

Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by obstructive pulmonary vascular remodelling ultimately leading to right ventricular (RV) failure and death. Disturbed transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signalling, endothelial cell dysfunction, increased proliferation of smooth muscle cells and fibroblasts, and inflammation contribute to this abnormal remodelling. Peptidyl-prolyl isomerase Pin1 has been identified as a critical driver of proliferation and inflammation in vascular cells, but its role in the disturbed TGF-β/BMP signalling, endothelial cell dysfunction, and vascular remodelling in PAH is unknown. Here, we report that Pin1 expression is increased in cultured pulmonary microvascular endothelial cells (MVECs) and lung tissue of PAH patients. Pin1 inhibitor, juglone significantly decreased TGF-β signalling, increased BMP signalling, normalized their hyper-proliferative, and inflammatory phenotype. Juglone treatment reversed vascular remodelling through reducing TGF-β signalling in monocrotaline + shunt-PAH rat model. Juglone treatment decreased Fulton index, but did not affect or harm cardiac function and remodelling in rats with RV pressure load induced by pulmonary artery banding. Our study demonstrates that inhibition of Pin1 reversed the PAH phenotype in PAH MVECs in vitro and in PAH rats in vivo, potentially through modulation of TGF-β/BMP signalling pathways. Selective inhibition of Pin1 could be a novel therapeutic option for the treatment of PAH.

Mixed Lineage Kinase 3 phosphorylates prolyl-isomerase PIN1 and potentiates GLI1 signaling in pancreatic cancer development.

The transcription factor Glioma-Associated Oncogene Homolog 1 (GLI1) is activated by sonic hedgehog (SHH) cascade and is an established driver of pancreatic ductal adenocarcinoma (PDAC). However, therapies targeting upstream hedgehog signaling have shown little to no efficacy in clinical trials. Here, we identify Mixed Lineage Kinase 3 (MLK3) as a druggable regulator of oncogenic GLI1. Earlier, we reported that MLK3 phosphorylated a peptidyl-prolyl isomerase PIN1 on the S138 site, and the PIN1-pS138 translocated to the nucleus. In this report, we identify GLI1 as one of the targets of PIN1-pS138 and demonstrate that PIN1-pS138 is upregulated in human PDAC and strongly associates with the upregulation of GLI1 and MLK3 expression. Moreover, we also identified two new phosphorylation sites on GLI1, T394, and S1089, which are directly phosphorylated by MLK3 to promote GLI1 nuclear translocation, transcriptional activity, and cell proliferation. Additionally, pharmacological inhibition of MLK3 by CEP-1347 promoted apoptosis in PDAC cell lines, reduced tumor burden, extended survival, and reduced GLI1 expression in the Pdx1-Cre x LSL-KRASG12D x LSL-TP53R172H (KPC) mouse model of PDAC. These findings collectively suggest that MLK3 is an important regulator of oncogenic GLI1 and that therapies targeting MLK3 warrant consideration in the management of PDAC patients.

Pin1 inhibition improves the efficacy of ralaniten compounds that bind to the N-terminal domain of androgen receptor

Therapies for lethal castration-resistant prostate cancer (CRPC) are an unmet medical need. One mechanism underlying CRPC and resistance to hormonal therapies is the expression of constitutively active splice variant(s) of androgen receptor (AR-Vs) that lack its C-terminus ligand-binding domain. Transcriptional activities of AR-Vs and full-length AR reside in its N-terminal domain (NTD). Ralaniten is the only drug proven to bind AR NTD, and it showed promise of efficacy in Phase 1 trials. The peptidyl-prolyl isomerase Pin1 is frequently overexpressed in prostate cancer. Here we show that Pin1 interacted with AR NTD. The inhibition of Pin1 expression or its activity selectively reduced the transcriptional activities of full-length AR and AR-V7. Combination of Pin1 inhibitor with ralaniten promoted cell cycle arrest and had improved antitumor activity against CRPC xenografts in vivo compared to individual monotherapies. These findings support the rationale for therapy that combines a Pin1 inhibitor with ralaniten for treating CRPC.

The fission yeast Pin1 peptidyl-prolyl isomerase promotes dissociation of Sty1 MAPK from RNA polymerase II and recruits Ssu72 phosphatase to facilitate oxidative stress induced transcription.

Pin1 is a peptidyl-prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II (Pol II) through interaction with the C-terminal domain (CTD) of Rpb1, the largest subunit of Pol II. We demonstrated that this function is important for cellular response to oxidative stress in the fission yeast Schizosaccharomyces pombe. In response to oxidative stress, the Atf1 transcription factor targets Sty1, the mitogen-activated protein kinase (MAPK), to specific stress-responsive promoters. Anchored Sty1 recruits Pol II through direct association with Rpb1-CTD and phosphorylates the reiterated heptad sequence at Serine 5. Pin1 binds phosphorylated CTD to promote dissociation of Sty1 from it, and directly recruits Ssu72 phosphatase to facilitate dephosphorylation of CTD for transcription elongation. In the absence of Pin1, the association of Sty1-Atf1 with Rpb1 persists on stress-responsive promoters failed to generate transcripts of the corresponding genes effectively. The identified characteristic features of the fission yeast Pin1 are conserved in humans. We demonstrated that elevated Pin1 level in cancer cells might help to sustain survival under oxidative stress generated from their altered metabolic pathways. Together, these results suggest a conserved function of Pin1 in cellular response to oxidative stress among eukaryotic cells that might have clinical implication.

Roles of peptidyl-prolyl isomerase Pin1 in disease pathogenesis.

Pin1 belongs to the peptidyl-prolyl cis-trans isomerases (PPIases) superfamily and catalyzes the cis-trans conversion of proline in target substrates to modulate diverse cellular functions including cell cycle progression, cell motility, and apoptosis. Dysregulation of Pin1 has wide-ranging influences on the fate of cells; therefore, it is closely related to the occurrence and development of various diseases. This review summarizes the current knowledge of Pin1 in disease pathogenesis.

Genetic interactions and transcriptomics implicate fission yeast CTD prolyl isomerase Pin1 as an agent of RNA 3' processing and transcription termination that functions via its effects on CTD phosphatase Ssu72.

The phosphorylation pattern of Pol2 CTD Y1S2P3T4S5P6S7 repeats comprises an informational code coordinating transcription and RNA processing. cis–trans isomerization of CTD prolines expands the scope of the code in ways that are not well understood. Here we address this issue via analysis of fission yeast peptidyl-prolyl isomerase Pin1. A pin1Δ allele that does not affect growth per se is lethal in the absence of cleavage-polyadenylation factor (CPF) subunits Ppn1 and Swd22 and elicits growth defects absent CPF subunits Ctf1 and Dis2 and termination factor Rhn1. Whereas CTD S2A, T4A, and S7A mutants thrive in combination with pin1Δ, a Y1F mutant does not, nor do CTD mutants in which half the Pro3 or Pro6 residues are replaced by alanine. Phosphate-acquisition genes pho1, pho84 and tgp1 are repressed by upstream lncRNAs and are sensitive to changes in lncRNA 3′ processing/termination. pin1Δ hyper-represses PHO gene expression and erases the de-repressive effect of CTD-S7A. Transcriptional profiling delineated sets of 56 and 22 protein-coding genes that are down-regulated and up-regulated in pin1Δ cells, respectively, 77% and 100% of which are downregulated/upregulated when the cis-proline-dependent Ssu72 CTD phosphatase is inactivated. Our results implicate Pin1 as a positive effector of 3′ processing/termination that acts via Ssu72.

Dissecting the Dynamics during Enzyme Catalysis: A Case Study of Pin1 Peptidyl-Prolyl Isomerase.

Free energy surfaces have played a central role in studying protein conformational changes and enzymatic reactions over decades. Yet, free energy barriers and kinetics are highly dependent on the coordinates chosen to define the surface, and furthermore, the dynamics during the reactions are often overlooked. Our recent study on the Pin1-catalyzed isomerization reaction has indicated that the isomerization transition events remarkably deviate from the free energy path, highlighting the need to understand the reaction dynamics in more detail. To this end, here we investigate the reaction coordinates that describe the transition states of the free energy and transition pathways by minimizing the cross-entropy function. We show that the isomerization transition events can be expressed by the concerted changes in the improper torsion angle ζ and nearby backbone torsional angles of the ligand, whereas the transition state of the free energy surface involves changes in a broad range of coordinates including multiple protein-ligand interactions. The current result supports the previous finding that the isomerization transitions occur quickly from the conformational excited states, which is in sharp contrast to the slow and collective changes suggested from the free energy path. Our results further indicate that the coordinates derived from the transition trajectories are not sufficient for finding the transition states on the free energy surfaces due to the lack of information from conformational excited states.

An Alternative Pin1 Binding and Isomerization Site in the N-Terminus Domain of PSD-95.

Phosphorylation-dependent peptidyl-prolyl cis-trans isomerization plays key roles in cell cycle progression, the pathogenesis of cancer, and age-related neurodegeneration. Most of our knowledge about the role of phosphorylation-dependent peptidyl-prolyl cis-trans isomerization and the enzyme catalyzing this reaction, the peptidyl-prolyl isomerase (Pin1), is largely limited to proteins not present in neurons. Only a handful of examples have shown that phosphorylation-dependent peptidyl-prolyl cis-trans isomerization, Pin1 binding, or Pin1-mediated peptidyl-prolyl cis-trans isomerization regulate proteins present at excitatory synapses. In this work, I confirm previous findings showing that Pin1 binds postsynaptic density protein-95 (PSD-95) and identify an alternative binding site in the phosphorylated N-terminus of the PSD-95. Pin1 associates via its WW domain with phosphorylated threonine (T19) and serine (S25) in the N-terminus domain of PSD-95 and this association alters the local conformation of PSD-95. Most importantly, I show that proline-directed phosphorylation of the N-terminus domain of PSD-95 alters the local conformation of this region. Therefore, proline-directed phosphorylation of the N-terminus of PSD-95, Pin1 association, and peptidyl-prolyl cis-trans isomerization may all play a role in excitatory synaptic function and synapse development.

Pin1 Binding to Phosphorylated PSD-95 Regulates the Number of Functional Excitatory Synapses.

The post-synaptic density protein 95 (PSD-95) plays a central role in excitatory synapse development and synaptic plasticity. Phosphorylation of the N-terminus of PSD-95 at threonine 19 (T19) and serine 25 (S25) decreases PSD-95 stability at synapses; however, a molecular mechanism linking PSD-95 phosphorylation to altered synaptic stability is lacking. Here, we show that phosphorylation of T19/S25 recruits the phosphorylation-dependent peptidyl-prolyl cis–trans isomerase (Pin1) and reduces the palmitoylation of Cysteine 3 and Cysteine 5 in PSD-95. This reduction in PSD-95 palmitoylation accounts for the observed loss in the number of dendritic PSD-95 clusters, the increased AMPAR mobility, and the decreased number of functional excitatory synapses. We find the effects of Pin1 overexpression were all rescued by manipulations aimed at increasing the levels of PSD-95 palmitoylation. Therefore, Pin1 is a key signaling molecule that regulates the stability of excitatory synapses and may participate in the destabilization of PSD-95 following the induction of synaptic plasticity.

Carotid body type I cells engage flavoprotein and Pin1 for oxygen sensing.

Carotid body (CB) type I cells sense the blood Po2 and generate a nervous signal for stimulating ventilation and circulation when blood oxygen levels decline. Three oxygen-sensing enzyme complexes may be used for this purpose: 1) mitochondrial electron transport chain metabolism, 2) heme oxygenase 2 (HO-2)-generating CO, and/or 3) an NAD(P)H oxidase (NOX). We hypothesize that intracellular redox changes are the link between the sensor and nervous signals. To test this hypothesis type I cell autofluorescence of flavoproteins (Fp) and NAD(P)H within the mouse CB ex vivo was recorded as Fp/(Fp+NAD(P)H) redox ratio. CB type I cell redox ratio transiently declined with the onset of hypoxia. Upon reoxygenation, CB type I cells showed a significantly increased redox ratio. As a control organ, the non-oxygen-sensing sympathetic superior cervical ganglion (SCG) showed a continuously reduced redox ratio upon hypoxia. CN-, diphenyleneiodonium, or reactive oxygen species influenced chemoreceptor discharge (CND) with subsequent loss of O2 sensitivity and inhibited hypoxic Fp reduction only in the CB but not in SCG Fp, indicating a specific role of Fp in the oxygen-sensing process. Hypoxia-induced changes in CB type I cell redox ratio affected peptidyl prolyl isomerase Pin1, which is believed to colocalize with the NADPH oxidase subunit p47phox in the cell membrane to trigger the opening of potassium channels. We postulate that hypoxia-induced changes in the Fp-mediated redox ratio of the CB regulate the Pin1/p47phox tandem to alter type I cell potassium channels and therewith CND.