BPGAP1 is a multidomain Rho GTPase-activating protein (RhoGAP) that promotes Erk activation and cell motility. However, the molecular mechanism of how these two processes are linked and regulated remains unclear. Here, we show that the RhoGAP domain of BPGAP1 interacts with the peptidyl-prolyl cis/trans isomerase (PPI) Pin1, leading to enhanced GAP activity towards RhoA. BPGAP1 also interacted with wild-type and constitutively active Mek2, but not with its kinase-dead mutant. However, only active Mek2 could bind Pin1, acting as a scaffold to bridge Pin1 and BPGAP1 in a manner that involves the release of an autoinhibited proline-rich motif, 186-PPLP-189, proximal to the RhoGAP domain. This allows the non-canonical 186-PPLP-189 and 256-DDYGD-260 motifs of the proline-rich region and RhoGAP domain of BPGAP1 to become accessible to concerted binding by the WW and PPI domains of Pin1, respectively. Interestingly, Pin1 knockdown led to 'super-induction' of BPGAP1-induced acute, but not chronic, Erk activation upon epidermal growth factor stimulation, in a process independent of GAP modulation. Reintroducing Pin1, but not its catalytic or non-binding mutants, reversed the effect and inhibited cell migration induced by coexpression of BPGAP1 and active Mek2. Thus, Pin1 regulates BPGAP1 function in Rho and Erk signalling, with active Mek2 serving as a novel regulatory scaffold that promotes crosstalk between RhoGAP, Pin1 and Erk in the regulation of cell migration.

Oxidative signaling and oxidative stress contribute to aging, cancer, and diseases resulting from neurodegeneration. Pin1 is a proline isomerase that recognizes phosphorylated substrates and regulates the localization and conformation of its targets. Pin1(-/-) mice show phenotypes associated with premature aging, yet mouse embryonic fibroblasts (MEFs) from these mice are resistant to hydrogen peroxide (H(2)O(2))-induced cell death. We found that the abundance of phosphatidylinositol-5-phosphate (PtdIns5P) was increased in response to H(2)O(2), an effect that was enhanced in Pin1(-/-) MEFs. Reduction of H(2)O(2)-induced PtdIns5P compromised cell viability in response to oxidative stress, suggesting that PtdIns5P contributed to the enhanced cell viability of Pin1(-/-) MEFs exposed to oxidative stress. The increased PtdIns5P in the Pin1(-/-) MEFs stimulated the expression of genes involved in defense against oxidative stress and reduced the accumulation of reactive oxygen species. Pin1 and PtdIns5P 4-kinases (PIP4Ks), enzymes that phosphorylate and thereby reduce the amount of PtdIns5P, interacted in a manner dependent on the phosphorylation of PIP4K. Although reintroduction of Pin1 into the Pin1(-/-) MEFs reduced the amount of PtdIns5P produced in response to H(2)O(2), in vitro assays indicated that the isomerase activity of Pin1 inhibited PIP4K activity. Whether this isomerise-mediated inhibition of PIP4K occurs in cells remains an open question, but the data suggest that the regulation of PIP4K by Pin1 may be complex.

A functionally Pin1-like peptidyl-prolyl cis/trans isomerase (PPIase(1)) was isolated from proembryogenic masses (PEMs) of Digitalis lanata according to its enzymatic activity. Partial sequence analysis of the purified enzyme (DlPar13) revealed sequence homology to members of the parvulin family of PPIases. Similar to human Pin1 and yeast Ess1, it exhibits catalytic activity toward substrates containing (Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics with juglone. Unlike Pin1-type enzymes it lacks the phosphoserine or phosphothreonine binding WW domain. Western blotting with anti-DlPar13 serum recognized the endogenous form in nucleic and cytosolic fractions of the plant cells. Since the PIN1 homologue ESS1 is an essential gene, complementation experiments in yeast were performed. When overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1. In contrast, the human parvulin hPar14 is not able to rescue the lethal phenotype of this yeast strain at nonpermissive temperatures. These results suggest a function for DlPar13 rather similar to parvulins of the Pin1-type.

The root stem cell niche defines the area that specifies and maintains the stem cells and is essential for the maintenance of root growth. Here, we characterize and examine the functional role of a quiescent center (QC)-expressed RAC/ROP GTPase activator, RopGEF7, in Arabidopsis thaliana. We show that RopGEF7 interacts with At RAC1 and overexpression of a C-terminally truncated constitutively active RopGEF7 (RopGEF7ΔC) activates RAC/ROP GTPases. Knockdown of RopGEF7 by RNA interference causes defects in embryo patterning and maintenance of the QC and leads to postembryonic loss of root stem cell population. Gene expression studies indicate that RopGEF7 is required for root meristem maintenance as it regulates the expression of PLETHORA1 (PLT1) and PLT2, which are key transcription factors that mediate the patterning of the root stem cell niche. Genetic analyses show that RopGEF7 interacts with PLT genes to regulate QC maintenance. Moreover, RopGEF7 is induced transcriptionally by auxin while its function is required for the expression of the auxin efflux protein PIN1 and maintenance of normal auxin maxima in embryos and seedling roots. These results suggest that RopGEF7 may integrate auxin-derived positional information in a feed-forward mechanism, regulating PLT transcription factors and thereby controlling the maintenance of root stem cell niches.

Peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) plays an important role in cell transformation and oncogenesis. Association between PIN1 promoter polymorphisms and cancer risk was reported in several cancers. This study aimed to evaluate the association between two single nucleotide polymorphisms (SNPs, -667T>C, rs2233679 and -842G>C, rs2233678) on PIN1 promoter and risk of nasopharyngeal carcinoma (NPC). The two SNPs were genotyped using polymerase chain reaction-restriction fragment length polymorphism in a total of 334 native Chinese subjects consisting of 178 cases and 156 controls. The results indicated that the -667CT heterozygote and -667CC homozygote exhibited a significantly decreased risk of nasopharyngeal carcinoma when compared with -667TT homozygote (OR = 0.639, 95% CI = 0.452-0.903, p = 0.011 for -667CT; and OR = 0.441, 95% CI = 0.213-0.915, p = 0.038 for -667CC, respectively). In the -842G>C polymorphism, compared with -842GG homozygote, only -842CG heterozygote but not -842CC homozygote had a significantly decreased risk of nasopharyngeal carcinoma (OR = 0.465, 95% CI = 0.249-0.871, p = 0.010). Genotype in the two SNPs in patients showed no significant associations with the clinicopathologic features examined. Our study showed that the minor genotypes of PIN1 promoter (-667CT, -667CC and -842CG) were associated with decreased risk of NPC in a Chinese population, suggested that PIN1 promoter polymorphisms might play an important role in NPC carcinogenesis.

The HER-2 oncogene, a member of the erythroblastosis oncogene B (ERBB)-like oncogene family, has been shown to be amplified in many types of cancer, including breast cancer. However, the molecular mechanism of HER-2 overexpression is not completely understood. The phosphorylation of proteins on the serine or threonine residues that immediately precede proline (pSer/Thr-Pro) is specifically catalyzed by the prolyl isomerase Pin1 and is a key signaling mechanism in cell proliferation and transformation. Here, we found that Pin1 interacts with mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) protein kinase 1, resulting in the induction of HER-2 expression. Pin1(-/-) mouse embryonic fibroblasts exhibited a decrease in epidermal growth factor (EGF)-induced MEK1/2 phosphorylation compared with Pin1(+/+) mouse embryonic fibroblast. In addition, a knockdown of Pin1 resulted in the inhibition of MEK1/2 phosphorylation induced by EGF in MCF-7 cells. Furthermore, PD98059, a specific inhibitor of MEK1/2, and Juglone, a potent Pin1 inhibitor, markedly suppressed the expression of activator protein-2alpha and the HER-2 promoter activity induced by EGF or 12-O-tetradecanoylphorbol-13-acetate in MCF-7 cells. Importantly, these inhibitors inhibited the neoplastic cell transformation induced by EGF in Pin1-overexpressing JB6 Cl41 cells, which showed enhanced cellular formation compared with the control cells. Therefore, Juglone and PD98059 inhibited the colony formation of MCF-7 breast cancer cells in soft agar. These results indicate that Pin1 amplifies EGF signaling in breast cancer cells through its interaction with MEK1 and then enhances HER-2 expression, suggesting that Pin1 plays an important role in the overexpression of HER-2 through Pin1-MEK1-activator protein-2alpha signaling in breast cancer.

Pin1/Ess1p is a highly conserved WW domain-containing peptidyl-prolyl isomerase (PPIase); its WW domain binds specifically to phospho-Ser/Thr-Pro sequences and its catalytic domain isomerizes phospho-Ser/Thr-Pro bonds. Pin1 PPIase activity can alter protein conformation in a phosphorylation-dependent manner and/or promote protein dephosphorylation. Human Pin1 interacts with mitotic phosphoproteins, such as NIMA, Cdc25 and Wee1, and inhibits G(2)/M progression in Xenopus extracts. Depletion of Pin1 in HeLa cells and deletion of ESS1 in S. cerevisiae result in mitotic arrest. In addition, Pin1/Ess1p play roles in transcription in S. cerevisiae and in mammalian somatic cells. The S. pombe genome sequence has an open reading frame (ORF) that has 47% identity with Pin1. Expression of this ORF rescued the growth defect caused by ess1 deletion in S. cerevisiae, indicating that S. pombe Pin1p is a functional Pin1 homologue. Overexpression of pin1(+) in S. pombe caused slow growth and a G(1) delay. Deletion of pin1(+) (pin1 Delta) did not affect cell cycle progression or cell growth, but increased sensitivity to the cyclophilin inhibitor, cyclosporin A, suggesting that cyclophilin family PPIases have overlapping functions with the Pin1p PPIase. Deletion of pin1(+) did not affect the DNA replication checkpoint, but conferred a modest increase in UV sensitivity. Furthermore, the pin1 Delta allele caused a synthetic growth defect when combined with either cdc25-22 or wee1-50 but not the cdc24-1 temperature-sensitive mutant. The pin1 Delta strain showed increased sensitivity to the PP1/PP2A family phosphatase inhibitor, okadaic acid, suggesting that Pin1p plays a role in protein dephosphorylation as a result of its ability to increase the population of phospho-Ser/Thr-Pro peptide bonds in the trans conformation that is required for PP2A-mediated dephosphorylation. Our genetic data also suggest that Pin1p might function as a positive regulator of Cdc25p and Wee1p.

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H(2)O(2)) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H(2)O(2) and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.

The unique proline isomerase Pin1 is pivotal for protecting against age-dependent neurodegeneration in Alzheimer's disease (AD), with its inhibition providing a molecular link between tangle and plaque pathologies. Pin1 is oxidatively modified in human AD brains, but little is known about its regulatory mechanisms and pathological significance of such Pin1 modification. In this paper, our determination of crystal structures of oxidized Pin1 reveals a series of Pin1 oxidative modifications on Cys113 in a sequential fashion. Cys113 oxidization is further confirmed by generating antibodies specifically recognizing oxidized Cys113 of Pin1. Furthermore, Pin1 oxidation on Cys113 inactivates its catalytic activity in vitro, and Ala point substitution of Cys113 inactivates the ability of Pin1 to isomerize tau as well as to promote protein turnover of tau and APP. Moreover, redox regulation affects Pin1 subcellular localization and Pin1-mediated neuronal survival in response to hypoxia treatment. Importantly, Cys113-oxidized Pin1 is significantly increased in human AD brain comparing to age-matched controls. These results not only identify a novel Pin1 oxidation site to be the critical catalytic residue Cys113, but also provide a novel oxidative regulation mechanism for inhibiting Pin1 activity in AD. These results suggest that preventing Pin1 oxidization might help to reduce the risk of AD.

BACKGROUND

Cardiac hypertrophy results from the complex interplay of differentially regulated cascades based on the phosphorylation status of involved signaling molecules. Although numerous critical regulatory kinases and phosphatases have been identified in the myocardium, the intracellular mechanism for temporal regulation of signaling duration and intensity remains obscure. In the nonmyocyte context, control of folding, activity, and stability of proteins is mediated by the prolyl isomerase Pin1, but the role of Pin1 in the heart is unknown.

OBJECTIVE

To establish the role of Pin1 in the heart.

RESULTS

Here, we show that either genetic deletion or cardiac overexpression of Pin1 blunts hypertrophic responses induced by transaortic constriction and consequent cardiac failure in vivo. Mechanistically, we find that Pin1 directly binds to Akt, mitogen activated protein kinase (MEK), and Raf-1 in cultured cardiomyocytes after hypertrophic stimulation. Furthermore, loss of Pin1 leads to diminished hypertrophic signaling of Akt and MEK, whereas overexpression of Pin1 increases Raf-1 phosphorylation on the autoinhibitory site Ser259, leading to reduced MEK activation.

CONCLUSIONS

Collectively, these data support a role for Pin1 as a central modulator of the intensity and duration of 2 major hypertrophic signaling pathways, thereby providing a novel target for regulation and control of cardiac hypertrophy.

Pin1 is involved in eukaryotic cell proliferation by changing the structure and function of phosphorylated proteins. PiB, the Pin1 specific inhibitor, blocks cancer cell proliferation. However, low solubility of PiB in DMSO has limited studies of its effectiveness. We screened for additional Pin1 inhibitors and identified the DMSO-soluble compound dipentamethylene thiuram monosulfide (DTM) that inhibits Pin1 activity with an EC50 value of 4.1 microM. Molecular modeling and enzyme kinetic analysis indicated that DTM competitively inhibits Pin1 activity, with a K(i) value of 0.05 microM. The K(D) value of DTM with Pin1 was determined to be 0.06 microM by SPR technology. Moreover, DTM specifically inhibited peptidyl-prolyl cis/trans isomerase activity in HeLa cells. FACS analysis showed that DTM induced G0 arrest of the HCT116 cells. Our results suggest that DTM has the potential to guide the development of novel antifungal and/or anticancer drugs.

Microtubule-associated protein tau has long been known for its ability to promote microtubule assembly. A less known feature of tau is its existence as a non-microtubule associated protein. Here we review the interactions of tau with other proteins, some of which interact with the microtubule binding repeat region of tau. The tau interactions with Fyn and with Pin1 have attracted the most attention and both interactions have been thought to have a role in Alzheimer's disease. The fact that tau has unknown cellular functions is further evidenced by its involvement in cell cycle activated neurodegeneration. One possible route for additional investigations stems from the presence of tau in non-neuronal cells where its characteristics have been largely unknown, although there has been a correlation between tau levels and the response of some cancer cells to microtubule-targeting chemotherapy drugs. Our studies of prostate cancer cells indicate that these cells can provide a system with phosphorylated adult tau for functional studies. In fact, structural similarities exist between Alzheimer's disease tau and prostate cancer cell tau, raising the possibility that new tau functions uncovered in prostate cancer cells will have relevance to Alzheimer's disease.

The development of outgrowths from plant shoots depends on formation of epidermal sites of cell polarity convergence with high intracellular auxin at their centre. A parsimonious model for generation of convergence sites is that cell polarity for the auxin transporter PIN1 orients up auxin gradients, as this spontaneously generates convergent alignments. Here we test predictions of this and other models for the patterns of auxin biosynthesis and import. Live imaging of outgrowths from kanadi1 kanadi2 Arabidopsis mutant leaves shows that they arise by formation of PIN1 convergence sites within a proximodistal polarity field. PIN1 polarities are oriented away from regions of high auxin biosynthesis enzyme expression, and towards regions of high auxin importer expression. Both expression patterns are required for normal outgrowth emergence, and may form part of a common module underlying shoot outgrowths. These findings are more consistent with models that spontaneously generate tandem rather than convergent alignments.

The tyrosine kinase c-Abl (or Abl) and the prolyl-isomerase Pin1 cooperatively activate the transcription factor p73 by enhancing recruitment of the acetyltransferase p300. As the transcription factor c-Myc (or Myc) is a known target of Pin1 and p300, we hypothesized that it might be regulated in a similar manner. Consistent with this hypothesis, overexpression of Pin1 augmented the interaction of Myc with p300 and transcriptional activity. The action of Abl, however, was more complex than predicted. On one hand, Abl indirectly enhanced phosphorylation of Myc on Ser 62 and Thr 58, its association with Pin1 and p300 and its acetylation by p300. These effects of Abl were exerted through phosphorylation of substrate(s) other than Myc itself. On the other hand, Abl interacted with the C-terminal domain of Myc and phosphorylated up to five tyrosine residues in its N-terminus, the principal of which was Y74. Indirect immunofluorescence or immunohistochemical staining suggested that the Y74-phosphorylated form of Myc (Myc-pY74) localized to the cytoplasm and coexisted either with active Abl in a subset of mammary carcinomas or with Bcr-Abl in chronic myeloid leukemia. In all instances, Myc-pY74 constituted a minor fraction of the cellular Myc protein. Thus, our data unravel two potential effects of Abl on Myc: first, Abl signaling can indirectly augment acetylation of Myc by p300, and most likely also its transcriptional activity in the nucleus; second, Abl can directly phosphorylate Myc on tyrosine: the resulting form of Myc appears to be cytoplasmic, and its presence correlates with Abl activation in cancer.

BACKGROUND

Atherosclerosis is a major cause of mortality in patients with diabetes. However, novel breakthrough therapies have yet to be approved in this setting. Prolyl-isomerase-1 (Pin1) is emerging as a key molecule implicated in vascular oxidative stress and inflammation. In the present study, we investigate whether pharmacological inhibition of Pin1 may protect against diabetes-induced oxidative stress, endothelial dysfunction and vascular inflammation.

RESULTS

Experiments were performed in human aortic endothelial cells (HAECs) exposed to normal (5 mmol/L) or high glucose (25 mmol/L) concentrations, in the presence of Pin1 inhibitor Juglone (10 μM) or vehicle (<1% ethanol). In parallel, streptozotocin-induced diabetic mice were treated with Juglone i.p. every other day for 30 days (1mg/Kg). Organ chamber experiments were performed in aortic rings to assess endothelium-dependent relaxations to acetylcholine (Ach 10(-9) to 10(-6)mol/L). Mitochondrial oxidative stress, organelle integrity as well as NF-kB-dependent inflammatory signatures were determined both in HAECs and aortas from diabetic mice. In HAECs, ambient hyperglycemia increased mitochondrial superoxide anion generation while treatment with Juglone prevented this phenomenon. Pharmacological inhibition of Pin1 also preserved mitochondrial integrity, nitric oxide availability and endothelial expression of adhesion molecules. Interestingly enough, endothelial dysfunction, oxidative stress and NF-kB-driven inflammation were significantly attenuated in diabetic mice chronically treated with Juglone as compared to vehicle-treated animals.

CONCLUSIONS

Pharmacological inhibition of Pin1 by Juglone prevents hyperglycemia-induced vascular dysfunction. Taken together, our findings may set the stage for novel therapeutic approaches to prevent vascular complications in patients with diabetes.

This study tested the hypothesis that priming the neutrophil respiratory burst requires both granule exocytosis and activation of the prolyl isomerase Pin1. Fusion proteins containing the TAT cell permeability sequence and either the SNARE domain of syntaxin-4 or the N-terminal SNARE domain of SNAP-23 were used to examine the role of granule subsets in TNF-mediated respiratory burst priming using human neutrophils. Concentration-inhibition curves for exocytosis of individual granule subsets and for priming of fMLF-stimulated superoxide release and phagocytosis-stimulated H2O2 production were generated. Maximal inhibition of priming ranged from 72 to 88%. Linear regression lines for inhibition of priming versus inhibition of exocytosis did not differ from the line of identity for secretory vesicles and gelatinase granules, while the slopes or the y-intercepts were different from the line of identity for specific and azurophilic granules. Inhibition of Pin1 reduced priming by 56%, while exocytosis of secretory vesicles and specific granules was not affected. These findings indicate that exocytosis of secretory vesicles and gelatinase granules and activation of Pin1 are independent events required for TNF-mediated priming of neutrophil respiratory burst.

The apical-basal axis of the Arabidopsis gynoecium is established early during development and is divided into four elements from the bottom to the top: the gynophore, the ovary, the style, and the stigma. Currently, it is proposed that the hormone auxin plays a critical role in the correct apical-basal patterning through a concentration gradient from the apical to the basal part of the gynoecium, as chemical inhibition of polar auxin transport through 1-N-naphtylphtalamic acid (NPA) application, severely affects the apical-basal patterning of the gynoecium. In this work, we show that the apical-basal patterning of gynoecia is also sensitive to exogenous cytokinin (benzyl amino purine, BAP) application in a similar way as to NPA. BAP and NPA treatments were performed in different mutant backgrounds where either cytokinin perception or auxin transport and perception were affected. We observed that cytokinin and auxin signaling mutants are hypersensitive to NPA treatment, and auxin transport and signaling mutants are hypersensitive to BAP treatment. BAP effects in apical-basal gynoecium patterning are very similar to the effects of NPA, therefore, it is possible that BAP affects auxin transport in the gynoecium. Indeed, not only the cytokinin-response TCS::GFP marker, but also the auxin efflux carrier PIN1 (PIN1::PIN1:GFP) were both affected in BAP-induced valveless gynoecia, suggesting that the BAP treatment producing the morphological changes has an impact on both in the response pattern to cytokinin and on auxin transport. In summary, we show that cytokinin affects proper apical-basal gynoecium patterning in Arabidopsis in a similar way to the inhibition of polar auxin transport, and that auxin and cytokinin mutants and markers suggest a relation between both hormones in this process.

FK506-binding proteins (FKBPs) are members of the immunophilins, enzymes that assist protein folding with their peptidyl-prolyl isomerase (PPIase) activity. Some non-immunosuppressive inhibitors of these enzymes have neuroregenerative and neuroprotective properties with an unknown mechanism of action. We have previously shown that FKBPs accelerate the aggregation of α-synuclein (α-SYN) in vitro and in a neuronal cell culture model for synucleinopathy. In this study we investigated whether acceleration of α-SYN aggregation is specific for the FKBP or even the PPIase family. Therefore, we studied the effect of several physiologically relevant PPIases, namely FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, on α-SYN aggregation in vitro and in neuronal cell culture. Among all PPIases tested in vitro, FKBP12 accelerated α-SYN aggregation the most. Furthermore, only FKBP12 accelerated α-SYN fibril formation at subnanomolar concentrations, pointing toward an enzymatic effect. Although stable overexpression of various FKBPs enhanced the aggregation of α-SYN and cell death in cell culture, they were less potent than FKBP12. When FKBP38, FKBP52, and FKBP65 were overexpressed in a stable FKBP12 knockdown cell line, they could not fully restore the number of α-SYN inclusion-positive cells. Both in vitro and cell culture data provide strong evidence that FKBP12 is the most important PPIase modulating α-SYN aggregation and validate the protein as an interesting drug target for Parkinson disease.

Alzheimer's disease (AD) is a neurodegenerative disorder contributing to rapid decline in cognitive function and ultimately dementia. Most cases of AD occur in elderly and later years. There is a growing need for understanding the relationship between aging and AD to identify shared and unique hallmarks associated with the disease in a region and cell-type specific manner. Although genomic studies on AD have been performed extensively, the molecular mechanism of disease progression is still not clear. The major objective of our study is to obtain a higher-order network-level understanding of aging and AD, and their relationship using the hippocampal gene expression profiles of young (20-50 years), aging (70-99 years), and AD (70-99 years). The hippocampus is vulnerable to damage at early stages of AD and altered neurogenesis in the hippocampus is linked to the onset of AD. We combined the weighted gene co-expression network and weighted protein-protein interaction network-level approaches to study the transition from young to aging to AD. The network analysis revealed the organization of co-expression network into functional modules that are cell-type specific in aging and AD. We found that modules associated with astrocytes, endothelial cells and microglial cells are upregulated and significantly correlate with both aging and AD. The modules associated with neurons, mitochondria and endoplasmic reticulum are downregulated and significantly correlate with AD than aging. The oligodendrocytes module does not show significant correlation with neither aging nor disease. Further, we identified aging- and AD-specific interactions/subnetworks by integrating the gene expression with a human protein-protein interaction network. We found dysregulation of genes encoding protein kinases (FYN, SYK, SRC, PKC, MAPK1, ephrin receptors) and transcription factors (FOS, STAT3, CEBPB, MYC, NFKβ, and EGR1) in AD. Further, we found genes that encode proteins with neuroprotective function (14-3-3 proteins, PIN1, ATXN1, BDNF, VEGFA) to be part of the downregulated AD subnetwork. Our study highlights that simultaneously analyzing aging and AD will help to understand the pre-clinical and clinical phase of AD and aid in developing the treatment strategies.

Melatonin (N-acetyl-5-methoxytryptamine) plays important roles in regulating both biotic and abiotic stress tolerance, biological rhythms, plant growth and development. Sharing the same substrate (tryptophan) for the biosynthesis, melatonin and auxin also have similar effects in plant development. However, the specific function of melatonin in modulating plant root growth and the relationship between melatonin and auxin as well as underlying mechanisms are still unclear. In this study, we found high concentration of melatonin remarkably inhibited root growth in Arabidopsis by reducing root meristem size. Further studies showed that melatonin negatively regulated auxin biosynthesis, the expression of PINFORMED (PIN) proteins as well as auxin response in Arabidopsis. Moreover, the root growth of the triple mutant pin1pin3pin7 was more tolerant than that of wild-type in response to melatonin treatment, suggesting the essential role of PIN1/3/7 in melatonin-mediated root growth. Combination treatment of melatonin and 5-Triiodobenzoic acid (TIBA) did not enhance melatonin-mediated reduction of root meristem size, indicating that polar auxin transport (PAT) may be necessary for the regulation of root meristem size by melatonin treatment. Taken together, this study indicates that melatonin regulates root growth in Arabidopsis, through auxin synthesis and polar auxin transport, at least partially.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma and primary effusion lymphoma. KSHV-infected cells are predominantly latent, with a subset undergoing lytic reactivation. Rta is the essential lytic switch protein that reactivates virus by forming transactivation-competent complexes with the Notch effector protein RBP-Jk and promoter DNA. Strikingly, Rta homolog analysis reveals that prolines constitute 17% of conserved residues. Rta is also highly phosphorylated in vivo. We previously demonstrated that proline content determines Rta homotetramerization and function. We hypothesize that proline-directed modifications regulate Rta function by controlling binding to peptidyl-prolyl cis/trans isomerases (PPIases). Cellular PPIase Pin1 binds specifically to phosphoserine- or phosphothreonine-proline (pS/T-P) motifs in target proteins. Pin1 dysregulation is implicated in myriad human cancers and can be subverted by viruses. Our data show that KSHV Rta protein contains potential pS/T-P motifs and binds directly to Pin1. Rta transactivation is enhanced by Pin1 at two delayed early viral promoters in uninfected cells. Pin1's effect, however, suggests a rheostat-like influence on Rta function. We show that in infected cells, endogenous Pin1 is active during reactivation and enhances Rta-dependent early protein expression induced by multiple signals, as well as DNA replication. Surprisingly, ablation of Pin1 activity by the chemical juglone or dominant-negative Pin1 enhanced late gene expression and production of infectious virus, while ectopic Pin1 showed inhibitory effects. Our data thus suggest that Pin1 is a unique, dose-dependent molecular timer that enhances Rta protein function, but inhibits late gene synthesis and virion production, during KSHV lytic reactivation.

BACKGROUND

A peptidyl prolyl cis/trans isomerase, Pin1, regulates insulin signal transduction. Pin1 reduces responses to insulin stimulation by binding CRTC2 (CREB-regulated transcriptional co-activator 2) and PPARγ (peroxisome prolifereator- activated receptor γ), but conversely enhances insulin signaling by binding IRS-1 (insulin receptor substrate-1), Akt kinase, and Smad3. Therefore, it is still unclear whether Pin1 inhibits or enhances adipose cell differentiation.

RESULTS

Pin1(-/-) and wild-type mice were fed with high fat diets and adipose tissue weight was measured. Compared to wild-type mice, Pin1(-/-) mice had lower adipose tissue weight, while the weight of other tissues was similar. Mouse embryo fibroblasts (MEFs), prepared from both groups of mice, were induced to differentiate into adipose cells by stimulation with insulin. However, the rate of differentiation of MEFs from Pin1(-/-) mice was less than that of MEFs from wild-type mice. The rate of insulin-induced MEF cell differentiation in Pin1(-/-) mice was restored by increasing expression of Pin1. We found that Pin1 binds to phosphoThr172- and phosphoSer271-Pro sites in CREB suppress the activity in COS-7 cells.

CONCLUSIONS

Pin1 enhanced the uptake of triglycerides and the differentiation of MEF cells into adipose cells in response to insulin stimulation. Results of this study suggest that Pin1 down-regulation could be a potential approach in obesity-related dysfunctions, such as high blood pressure, diabetes, non-alcoholic steatohepatitis.

In addition to oncogenic drivers, signaling nodes can critically modulate cancer-related cellular networks to strength tumor hallmarks. We identify G-protein-coupled receptor kinase 2 (GRK2) as a relevant player in breast cancer. GRK2 is up-regulated in breast cancer cell lines, in spontaneous tumors in mice, and in a proportion of invasive ductal carcinoma patients. Increased GRK2 functionality promotes the phosphorylation and activation of the Histone Deacetylase 6 (HDAC6) leading to de-acetylation of the Prolyl Isomerase Pin1, a central modulator of tumor progression, thereby enhancing its stability and functional interaction with key mitotic regulators. Interestingly, a correlation between GRK2 expression and Pin1 levels and de-acetylation status is detected in breast cancer patients. Activation of the HDAC6-Pin1 axis underlies the positive effects of GRK2 on promoting growth factor signaling, cellular proliferation and anchorage-independent growth in both luminal and basal breast cancer cells. Enhanced GRK2 levels promote tumor growth in mice, whereas GRK2 down-modulation sensitizes cells to therapeutic drugs and abrogates tumor formation. Our data suggest that GRK2 acts as an important onco-modulator by strengthening the functionality of key players in breast tumorigenesis such as HDAC6 and Pin1.

We reported previously that Pin1 facilitates human immunodeficiency virus type 1 (HIV-1) uncoating by interacting with the capsid core through the phosphorylated Ser(16)-Pro(17) motif. However, the specific kinase responsible for Ser(16) phosphorylation has remained unknown. Here, we showed that virion-associated extracellular signal-regulated kinase 2 (ERK2) phosphorylates Ser(16). The characterization of immature virions produced by exposing chronically HIV-1LAV-1-infected CEM/LAV-1 cells to 10 µM saquinavir indicated that Ser(16) is phosphorylated after the initiation of Pr55(Gag) processing. Furthermore, a mass spectrometry-based in vitro kinase assay demonstrated that ERK2 specifically phosphorylated the Ser(16) residue in the Ser(16)-Pro(17) motif-containing substrate. The treatment of CEM/LAV-1 cells with the ERK2 inhibitor sc-222229 decreased the Ser(16) phosphorylation level inside virions, and virus partially defective in Ser(16) phosphorylation showed impaired reverse transcription and attenuated replication owing to attenuated Pin1-dependent uncoating. Furthermore, the suppression of ERK2 expression by RNA interference in CEM/LAV-1 cells resulted in suppressed ERK2 packaging inside virions and decreased the Ser(16) phosphorylation level inside virions. Interestingly, the ERK2-packaging-defective virus showed impaired reverse transcription and attenuated HIV-1 replication. Taken together, these findings provide insights into the as-yet-obscure processes in Pin1-dependent HIV-1 uncoating.