Abnormal folding of the microtubule-associated protein tau leads to aggregation of tau into paired helical filaments (PHFs) and neurofibrillary tangles, the major hallmark of Alzheimer's disease (AD). We have recently shown that grape seed polyphenol extract (GSPE) reduces tau pathology in the TMHT mouse model of tauopathy (Wang et al., 2010). In the present studies we assessed the impact of GSPE exposure on the ultrastructure of PHFs isolated from Alzheimer's disease brain. Transmission electron microscopy revealed that GSPE induced profound dose- and time-dependent alterations in the morphology of PHFs with partial disintegration of filaments. Filaments showed ∼2-fold enlargement in width and displayed numerous protrusions and splayed ends consistent with unfolding of tau and diminished structural stability. In addition, GSPE induced a reduction in immunogold labeling with antibodies against the C-terminal half (12E8, PHF-1) and the middle region of tau (AT8, Tau5, pSer214 tau, and AT180) but not the C-terminal end (Tau46). In comparison, labeling of N-terminus (Alz50) was enhanced. It is unlikely that alterations in immunogold labeling were due to biochemical alterations, e.g., protein phosphatase or proteolytic activities potentially stimulated by GSPE, because western blotting studies have shown the preservation of full length polypeptides of tau and their phospho-epitopes in GSPE-treated samples. The GSPE mechanism may include a noncovalent interaction of polyphenols with proline residues in the proline-rich domain of tau, with Pin1 sites at P213 and P232 most seriously affected as judged by suppression of labeling. Collectively, our results suggest that GSPE has a significant potential for therapeutic development by neutralizing phospho-epitopes and disrupting fibrillary conformation leading to disintegration of PHFs.

The hemolytic activity of six cationic amphipathic peptides (Oxki1, Oxki2, Pin1, Pin2, IsCT1 and IsCT2) from arachnids strongly depends on the source of red blood cells. The hemolytic activity of the amphipathic peptides was correlated to the phosphocholine-to-sphingomyelin ratio (PC/SM) content, the potency order of which on mammal erythrocytes ranked as follows Guinea pig>pig>sheep. The spider peptides, Oxki1 and Oxki2, prefer small unilamellar vesicles (SUV) composed of PC, but they could not disrupt SUVs made of SM only. Moreover, the membrane-disrupting activity of the scorpion peptide Pin1 was affected by increasing concentrations of SM. Only the scorpion hemolytic peptide Pin2 was able to disrupt SUVs composed merely of SM at high concentrations. Finally, the short scorpion peptides IsCT1 and IsCT2 seem to tolerate high concentrations of SM in the presence of PC for disruption of SUVs; however, the disrupting activities of IsCT1 and IsCT2 are much lower than that of the other four hemolytic peptides. The hemolytic activity caused by all six cationic peptides in mammalian erythrocytes was positively correlated to increases in temperature and increases in the concentration of benzyl alcohol, a membrane fluidizing agent. It was concluded that the hemolytic activity of the cationic peptides strongly depends on the PC/SM content of mammalian erythrocytes, in which cell membranes with a low PC/SM ratio (i.e., of low fluidity) were less disturbed than membranes with a high PC/SM ratio (i.e., of high fluidity).

Sphingolipids play an essential role in the functioning of the secretory pathway in eukaryotic organisms. Their importance in the functional organization of plant cells has not been studied in any detail before. The sphingolipid synthesis inhibitor fumonisin B1 (FB1), a mycotoxin acting as a specific inhibitor of ceramide synthase, was tested for its effects on cell growth, cell polarity, cell shape, cell cycle and on the ultrastructure of BY2 cells. We used cell lines expressing different GFP-tagged markers for plant cell compartments, as well as a Golgi marker fused to the photoconvertible protein Kaede. Light and electron microscopy, combined with flow cytometry, were applied to analyse the morphodynamics and architecture of compartments of the secretory pathway. The results indicate that FB1 treatment had severe effects on cell growth and cell shape, and induced a delay in cell division processes. The cell changes were accompanied by the formation of the endoplasmic reticulum (ER)-derived tubular aggregates (FB1-induced compartments), together with an inhibition of cargo transport from the ER to the Golgi apparatus. A change in polar localization of the auxin transporter PIN1 was also observed, but endocytic processes were little affected. Electron microscopy studies confirmed that molecular FB1 targets were distinct from brefeldin A (BFA) targets. We propose that the reported effects of inhibition of ceramide biosynthesis reflect the importance of sphingolipids during cell growth and establishment of cell polarity in higher plant cells, notably through their contribution to the functional organization of the ER or its differentiation into distinct compartments.

Inhibitor-2 (I-2) is the most ancient protein that selectively recognizes type-1 protein phosphatase and is phosphorylated by CDK1-cyclinB during mitosis at Thr72 in a conserved PXTP site. Pin1 is a peptide prolyl cis/trans isomerase conserved among eukaryotes that specifically reacts with proteins phosphorylated at Ser/Thr-Pro sites. We tested phospho-T72-I-2 as a substrate for Pin1 and discovered that unphosphorylated I-2 bound Pin1 with micromolar affinity and phosphorylation of the PXTP site or truncation of I-2 reduced binding 10-fold. Ectopic Pin1 coprecipitated endogenous I-2 and ectopic I-2 coprecipitated endogenous Pin1, but only in the absence of detergents, which may account for the interaction not being detected previously. Endogenous I-2 and Pin1 colocalized in HeLa cells and showed nuclear-cytoplasmic redistribution in response to cell density, suggestive of their association in living cells. Recombinant Pin1 binding to different phosphoproteins in mitotic cell extracts was modulated by I-2, and binding to individual mitotic phosphoproteins was increased, decreased or unaffected by I-2, showing that I-2 allosterically modifies Pin1 specificity. This was confirmed by mutation of Ser16 to Ala in the Pin1 WW domain that eliminated I-2 binding and abrogated I-2 effects on Pin1 binding to different phosphoproteins. A S16E mutation to mimic Pin1 phosphorylation restored binding to both I-2 and phospho-T72-I-2, indicating that phosphorylation of both proteins governs their interaction. The results reveal a novel function for I-2, and suggest phosphorylation-dependent regulation of Pin1 specificity during entry and exit of mitosis, in other phases of the cell cycle, and in multiple cell signaling processes.

Haploinsufficiency for the p53 family member p73 causes behavioral and neuroanatomical correlates of neurodegeneration in aging mice, including the appearance of aberrant phospho-tau-positive aggregates. Here, we show that these aggregates and tau hyperphosphorylation, as well as a generalized dysregulation of the tau kinases GSK3β, c-Abl, and Cdk5, occur in the brains of aged p73+/- mice. To investigate whether p73 haploinsufficiency therefore represents a general risk factor for tau hyperphosphorylation during neurodegeneration, we crossed the p73+/- mice with 2 mouse models of neurodegeneration, TgCRND8+/Ø mice that express human mutant amyloid precursor protein, and Pin1-/- mice. We show that haploinsufficiency for p73 leads to the early appearance of phospho-tau-positive aggregates, tau hyperphosphorylation, and activation of GSK3β, c-Abl, and Cdk5 in the brains of both of these mouse models. Moreover, p73+/-;TgCRND8+/Ø mice display a shortened lifespan relative to TgCRND8+/Ø mice that are wild type for p73. Thus, p73 is required to protect the murine brain from tau hyperphosphorylation during aging and degeneration.

Interactions involving phosphorylated Ser/Thr-Pro motifs in proteins play a key role in numerous regulatory processes in the cell. Here, we investigate potential ligands of the WW binding domain of Pin1 in order to inhibit protein-protein interactions between Pin1 and phosphopeptides. Our structure-based strategy implies the synthesis of analogues of the Ac-Thr(PO(3)H(2))-Pro-NH(2) dipeptide and relies on high resolution NMR spectroscopy to accurately measure the affinity constants even in the high micromolar range.

Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G(1)-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G(1) and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCF(Fbw7) (also known as SCF(Cdc4)), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7α is nucleoplasmic but excluded from the nucleolus, Fbw7β is cytoplasmic, and Fbw7γ is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7α and Fbw7γ, respectively. In vitro reconstitution showed that the role of SCF(Fbw7α) in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCF(Fbw7γ). Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7γ drive nucleolar localization of cyclin E, where it is ubiquitylated by SCF(Fbw7γ) prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation.

Nuclear factor of activated T cells (NFAT) plays a key role in T cell activation. The activation of NFAT involves calcium- and calcineurin-dependent dephosphorylation and nuclear translocation from the cytoplasm, a process that is opposed by protein kinases. We show here that the peptidyl prolyl cis-trans isomerase Pin1 interacts specifically with the phosphorylated form of NFAT. The NFAT-Pin1 interaction is mediated through the WW domain of Pin1 and the serine-proline-rich domains of NFAT. Furthermore, binding of Pin1 to NFAT inhibits the calcineurin-mediated dephosphorylation of NFAT in vitro, and overexpression of Pin1 in T cells inhibits calcium-dependent activation of NFAT in vivo. These results suggest a possible role for Pin1 in the regulation of NFAT in T cells.

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser(98) and Ser(110), which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.

Pin1 is an emerging oncology target strongly implicated in Ras and ErbB2-mediated tumourigenesis. Pin1 isomerizes bonds linking phospho-serine/threonine moieties to proline enabling it to play a key role in proline-directed kinase signalling. Here we report a novel series of Pin1 inhibitors based on a phenyl imidazole acid core that contains sub-μM inhibitors. Compounds have been identified that block prostate cancer cell growth under conditions where Pin1 is essential.

Cep55 is a mitotic phosphoprotein that plays an important role in cytokinesis, the final stage of cell division during which physical separation of the two daughter cells is accomplished. We recently demonstrated that the peptidyl-prolyl isomerase Pin1 regulates this cell cycle event by enhancing the Plk1-dependent phosphorylation of Cep55. We show here that Cep55 is stabilized post-translationally during mitosis and that siRNA-mediated knockdown of Pin1 prevents this stabilization. Consistent with this, Cep55 is unstable in Pin1 knockout mouse embryonic fibroblasts. Moreover, mutation of the Pin1 binding sites in Cep55 reduces its stability during mitosis. Mutation of the Plk1 phosphorylation site also lowers Cep55 stability, whereas overexpression of Plk1 increases Cep55 levels, in keeping with Pin1 regulating Plk1-mediated phosphorylation of Cep55. Importantly, expression of wild-type Cep55 at levels similar to that of the phosphorylation mutants only partially reverts the cytokinesis defect induced by depletion of Cep55, indicating that inadequate levels of Cep55 prevent proper execution of cytokinesis. Taken together, these data provide more insight into the regulation of the final stages of cell division. As cytokinesis defects can cause chromosomal instability, knowledge about the processes that regulate normal cytokinesis adds to our understanding of events that lead to tumorigenesis.

Fmoc-pSer-Psi[(Z)CHC]-Pro-(2)-N-(3)-ethylaminoindole 1, showed moderate inhibition towards the mitotic regulator, Pin1 (IC(50)=28.3microM). To improve the cell permeability, the charged phosphate was masked as the bis-pivaloyloxymethyl (POM) phosphate in Fmoc-(bisPOM)-pSer-Psi[(Z)CHC]-Pro-(2)-N-(3)-ethylaminoindole 2. Antiproliferative activity towards A2780 ovarian cancer cells of 1 (IC(50)=46.2microM) was improved significantly in 2 (IC(50)=26.9microM), comparable to the IC(50) of 1 towards Pin1 enzymatic activity.

c-Jun N-terminal kinase (JNK) is activated by dual phosphorylation of both threonine and tyrosine residues in the phosphorylation loop of the protein in response to several stress factors. However, the precise molecular mechanisms for activation after phosphorylation remain elusive. Here we show that Pin1, a peptidyl-prolyl isomerase, has a key role in the JNK1 activation process by modulating a phospho-Thr-Pro motif in the phosphorylation loop. Pin1 overexpression in human breast cancer cell lines correlates with increased JNK activity. In addition, small interfering RNA (siRNA) analyses showed that knockdown of Pin1 in a human breast cancer cell line decreased JNK1 activity. Pin1 associates with JNK1, and then catalyzes prolyl isomerization of the phospho-Thr-Pro motif in JNK1 from trans- to cis-conformation. Furthermore, Pin1 enhances the association of JNK1 with its substrates. As a result, Pin1(-/-) cells are defective in JNK activation and resistant to oxidative stress. These results provide novel insights that, following stress-induced phosphorylation of Thr in the Thr-Pro motif of JNK1, JNK1 associates with Pin1 and undergoes conformational changes to promote the binding of JNK1 to its substrates, resulting in cellular responses from extracellular signals.

Protein phosphorylation by proline-directed protein kinases plays an essential role in triggering a programmed set of cell cycle events. We have recently isolated an essential and conserved mitotic regulator, Pin1. Pin1 is a phosphorylation-dependent prolyl isomerase that specifically isomerizes the phosphorylated serine/threonine-proline bond. Pin1 also binds and regulates the function of a conserved set of mitosis-specific phosphoproteins. These results suggest phosphorylation-dependent prolyl isomerization to be a novel cell cycle regulatory mechanism. This new post-translational regulation may allow the general increase in protein phosphorylation to be converted into the organised and programmed set of structural modifications that occur during mitosis. In addition, since inhibition of Pin1 induces mitotic arrest and apoptosis, Pin1 may be a potential new drug target.

Pin1 [Protein Interacting with NIMA (never in mitosis A)] is a peptidyl prolyl cis-trans isomerase that isomerizes phospho-Serine/Threonine-Proline [p(S/T)-P] motifs of its target proteins. Pin1 functions in concert with proline directed kinases such as cyclin-dependent protein kinases, extracellular signal-regulated kinases, and c-Jun N- terminal kinase, and protein phosphatases such as protein phosphatase 2A (PP2A) and PP2B, in the regulation of a wide range of cellular processes including cell division, DNA damage response, and gene transcription, and in susceptibility to cancer and neurodegenerative diseases. This review focuses on the roles of Pin1 in neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Frontotemporal dementia associated with parkinsonism linked to chromosome 17. Pin1 interacts with neuronal cytoskeletal proteins such as tau, amyloid-beta protein precursor, alpha-synuclein, and neurofilaments, often in association with phosphorylation events that influence their functions in the neuronal cytoskeleton. Overexpression of Pin1 reduces WT tau stability but increases P301L mutant tau stability. Pin1 associates with neurofilament H (NF-H) and modulates excitotoxic and oxidative stress induced perikaryal phosphorylation of NF-H. Pin1 mediates the neural specific apoptosis machinery. The specific inhibitors of Pin1 may have potential therapeutic implications in neurodegeneration.

Numerous signal molecules move through the phloem to regulate development, including proteins, secondary metabolites, small RNAs and full-length transcripts. Several full-length mRNAs have been identified that move long distances in a shootward or rootward direction through the plant vasculature to modulate both floral and vegetative processes of growth. Here we discuss two recently discovered examples of long-distance transport of full-length mRNAs into roots and the potential target genes and pathways for these mobile signals. In both cases, the mobile RNAs regulate root growth. Previously, RNA movement assays demonstrated that transcripts of StBEL5, a transcription factor from the three-amino-loop-extension superclass, move through the phloem to stolon tips to enhance tuber formation in potato (Solanum tuberosum L.). StBEL5 mRNA originates in the leaf and its movement to stolons is induced by a short-day photoperiod. Movement of StBEL5 RNA to roots correlated with increased growth and the accumulation of several transcripts associated with hormone metabolism, including GA2-oxidase1, YUCCA1a and -c, several Aux/IAA types, and PIN1, -2, and -4 was observed. In another example, heterografting techniques were used to identify phloem-mobile Aux/IAA transcripts in Arabidopsis. Movement assays confirmed that these Aux/IAA transcripts are transported into the root system where they suppress lateral root formation. Phloem transport of both StBEL5 and Aux/IAA RNAs are linked to hormone metabolism and both target auxin synthesis genes or auxin signaling processes. The mechanisms of transport for these mobile RNAs, the impact they have on controlling root growth, and a potential transcriptional connection between the BEL1/KNOX complex and Aux/IAA genes are discussed.

Cucurbit seedlings potentially develop a peg on each side of the transition zone between the hypocotyl and root. Seedlings grown in a horizontal position suppress the development of the peg on the upper side of the transition zone in response to gravity. It is suggested that this suppression occurs due to a reduction in auxin levels to below the threshold value. We show in this study that the free indole-3-acetic acid (IAA) content is low, while IAA conjugates are significantly more abundant in the upper side of the transition zone of gravistimulated seedlings, compared to the lower side. A transient increase in mRNA of the auxin-inducible gene, CS-IAA1, was observed in the excised transition zone. The result suggests that the transition zone is a source of auxin. Cucumber seedlings treated with auxin-transport inhibitors exhibited agravitropic growth and developed a peg on each side of the transition zone. Auxin-transport inhibitors additionally caused an increase in CS-IAA1 mRNA accumulation at the transition zone, indicating a rise in intracellular auxin concentrations due to a block of auxin efflux. To study the involvement of the auxin transport system in peg formation, we isolated the cDNAs of a putative auxin influx carrier, CS-AUX1, and putative efflux carrier, CS-PIN1, from cucumber (Cucumis sativus L.) plants. Both genes (CS-AUX1 in particular) were auxin-inducible. Accumulation of CS-AUX1 and CS-PIN1 mRNAs was observed in vascular tissue, cortex and epidermis of the transition zone. A reduced level of CS-AUX1 mRNA was observed in the upper side of the gravistimulated transition zone, compared with the lower side. It is therefore possible that a balance in the activities of auxin influx and efflux carriers controls intracellular auxin concentration at the transition zone, which results in lateral placement of a peg in cucumber seedlings.

Local efflux-dependent auxin gradients and maxima mediate organ and tissue development in plants. Auxin efflux is regulated by dynamic expression and subcellular localization of the PIN auxin-efflux proteins, which appears to be established not only through a self-organizing auxin-mediated polarization mechanism, but also through other means, such as cell fate determination and auxin-independent mechanisms. Here, we show that the Arabidopsis thaliana NO VEIN (NOV) gene, encoding a novel, plant-specific nuclear factor, is required for leaf vascular development, cellular patterning and stem cell maintenance in the root meristem, as well as for cotyledon outgrowth and separation. nov mutations affect many aspects of auxin-dependent development without directly affecting auxin perception. NOV is required for provascular PIN1 expression and region-specific expression of PIN7 in leaf primordia, cell type-specific expression of PIN3, PIN4, and PIN7 in the root, and PIN2 polarity in the root cortex. NOV is specifically expressed in developing embryos, leaf primordia, and shoot and root apical meristems. Our data suggest that NOV function underlies cell fate decisions associated with auxin gradients and maxima, thus establishing cell type-specific PIN expression and polarity. We propose that NOV mediates the acquisition of competence to undergo auxin-dependent coordinated cell specification and patterning, thereby eliciting context-dependent auxin-mediated developmental responses.

We have found a novel cDNA encoding a 265 amino acid protein possessing a WW domain in our full-length cDNA bank. The WW domain was sandwiched between an acidic region and an acidic-basic amino acid repetitive region. In vitro transcription/translation of the cDNA produced a 38 kDa product that was also found in the cell lysate by western blot analysis. Thus this protein is named the nuclear protein containing a WW domain with a molecular mass of 38 kDa, Npw38. Immunofluorescence studies and expression of a fusion protein to a green fluorescent protein revealed that this protein is localized in the nucleus. Npw38 was shown to be capable of binding to a poly(rG) resin. Interestingly, the WW domain of Npw38 was found to function as a transcriptional activator in CHO cells using the GAL4 DNA-binding fusion system. Furthermore, the WW domains of human YAP and Pin1 were demonstrated to have a similar transcription-promoting activity. Combined mutation of the conserved first and second Trp residues and a hydrophobic triplet of TyrTyrTrp in the WW domain of Npw38 abolished the transcription-promoting activity, but single mutations of these sites did not. These results suggest that some WW domains potentially possess transcription-promoting activity in mammalian cells.

Fibroblast growth factor 2 (FGF2) signaling plays a pivotal role in bone growth/differentiation through the activation of osteogenic master transcription factor Runx2, which is mediated by the ERK/MAPK-dependent phosphorylation and the p300-dependent acetylation of Runx2. In this study, we found that Pin1-dependent isomerization of Runx2 is the critical step for FGF2-induced Runx2 transactivation function. We identified four serine or threonine residues in the C-terminal domain of Runx2 that are responsible for Pin1 binding and structural modification. Confocal imaging studies indicated that FGF2 treatment strongly stimulated the focal accumulation of Pin1 in the subnuclear area, which recruited Runx2. In addition, active forms of RNA polymerase-II also colocalized in the same subnuclear compartment. Dipentamethylene thiuram monosulfide, a Pin1 inhibitor, strongly attenuated their focal accumulation as well as Runx2 transactivation activity. The Pin1-mediated structural modification of Runx2 is an indispensable step connecting phosphorylation and acetylation and, consequently, transcriptional activation of Runx2 by FGF signaling. Thus, the modulation of Pin1 activity may be a target for the regulation of bone formation.

The repressor element 1 (RE1) silencing transcription factor (REST) in stem cells represses hundreds of genes essential to neuronal function. During neurogenesis, REST is degraded in neural progenitors to promote subsequent elaboration of a mature neuronal phenotype. Prior studies indicate that part of the degradation mechanism involves phosphorylation of two sites in the C terminus of REST that require activity of beta-transducin repeat containing E3 ubiquitin protein ligase, βTrCP. We identify a proline-directed phosphorylation motif, at serines 861/864 upstream of these sites, which is a substrate for the peptidylprolyl cis/trans isomerase, Pin1, as well as the ERK1/2 kinases. Mutation at S861/864 stabilizes REST, as does inhibition of Pin1 activity. Interestingly, we find that C-terminal domain small phosphatase 1 (CTDSP1), which is recruited by REST to neuronal genes, is present in REST immunocomplexes, dephosphorylates S861/864, and stabilizes REST. Expression of a REST peptide containing S861/864 in neural progenitors inhibits terminal neuronal differentiation. Together with previous work indicating that both REST and CTDSP1 are expressed to high levels in stem cells and down-regulated during neurogenesis, our results suggest that CTDSP1 activity stabilizes REST in stem cells and that ERK-dependent phosphorylation combined with Pin1 activity promotes REST degradation in neural progenitors.

Ultraviolet photodissociation (UVPD) mass spectrometry (MS) was used to characterize the sequences of proteins in native protein-ligand and protein-protein complexes and to provide auxiliary information about the binding sites of the ligands and protein-protein interfaces. UVPD outperformed collisional induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron transfer dissociation (ETD) in terms of yielding the most comprehensive diagnostic primary sequence information about the proteins in the complexes. UVPD also generated noncovalent fragment ions containing a portion of the protein still bound to the ligand which revealed some insight into the nature of the binding sites of myoglobin/heme, eIF4E/m(7)GTP, and human peptidyl-prolyl cis-trans isomerase 1 (Pin1) in complex with the peptide derived from the C-terminal domain of RNA polymerase II (CTD). Noncovalently bound protein-protein fragment ions from oligomeric β-lactoglobulin dimers and hexameric insulin complexes were also produced upon UVPD, providing some illumination of tertiary and quaternary protein structural features.

Pin1 actively regulates diverse biological/pathological processes, but little is known about the regulatory mechanisms of its cellular localization. In this study, we report that the endogenous Pin1 is distributed in both nucleus and cytoplasm. We found that point mutations of several basic amino acids in the PPIase domain of Pin1 significantly compromise its nuclear localization. Such inhibition is independent of Pin1 enzymatic activity, and is mainly due to the defects in the nuclear import. A novel sequence harboring these residues was identified as a putative nuclear localization signal (NLS) of Pin1. Importin alpha5 of the nuclear import machinery was found to interact with Pin1.

Pin1 is a peptidyl prolyl cis-trans isomerase that only binds to and isomerizes phosphorylated serine/threonine-proline motifs, inducing conformational changes that alter target protein function and phosphorylation. We have shown previously that deficiency of another peptidyl prolyl isomerase, FK506 binding protein 12/12.6, alters endothelial NO synthase phosphorylation and causes endothelial dysfunction and hypertension. Endothelial NO synthase contains the Pin1 binding sequence at (p)serine 116-proline 117 and phosphorylation of endothelial NO synthase serine 116 inhibits NO production; however, whether Pin1 deficiency alters vascular function and blood pressure is unknown. We hypothesized that Pin1 isomerizes p-endothelial NO synthase serine 116, which enables dephosphorylation and stimulates NO production. Immunoprecipitation of endothelial NO synthase and probing for Pin1 in rat aortic endothelial cells confirmed the interaction between the two. Pin1 knockdown via small interfering RNA or inhibition by juglone increased endothelial NO synthase serine 116 phosphorylation and prevented vascular endothelial growth factor-induced serine 116 dephosphorylation in endothelial cells. Acute treatment of isolated mouse aortas with juglone increased endothelial NO synthase serine 116 phosphorylation and decreased NO production and relaxation responses. Mice treated with juglone for 2 weeks, as well as Pin1 knockout mice, exhibited increased aortic endothelial NO synthase serine 116 phosphorylation, endothelial dysfunction, and hypertension. These data demonstrate that Pin1 binds endothelial NO synthase and enables dephosphorylation of serine 116, which increases NO production and endothelium-dependent dilation, leading to blood pressure maintenance.