Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4(+) T cells. We previously reported that HIV latency could be established in resting CD4(+) T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored.

In resting CD4(+) T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-κB, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4(+) T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4(+) T cells with mutant strains of HIV, lacking NF-κB binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1-115.4, p = 0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7-11, p>> 0.05) in fully activated CD4(+) T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4(+) T cells.

HIV integration in CCL19-treated resting CD4(+) T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection.

Hormone-dependent tumors are characterized by deregulated activity of specific steroid receptors, allowing aberrant expression of many genes involved in cancer initiation, progression and metastasis. In prostate cancer, the androgen receptor (AR) protein has pivotal functions, and over the years it has been the target of different drugs. AR is a nuclear receptor whose activity is regulated by a phosphorylation mechanism controlled by hormone and growth factors. Following phosphorylation, AR interacts with many cofactors that closely control its function. Among such cofactors, Pin1 is a peptidyl-prolyl isomerase that is involved in the control of protein phosphorylation and has a prognostic value in prostate cancer. In the present study, we demonstrate that ARSer81 is involved in the interaction with Pin1, and that this interaction is important for the transcriptional activity of AR. Since Pin1 expression positively correlates with tumor grade, our results suggest that Pin1 can participate in this process by modulating AR function.

The investigation of Rho-family GTPases has uncovered mechanisms for spatiotemporal control of cellular processes such as cell polarization, movement, morphogenesis and cell division. Now Rho GTPase plays another leading role in the discovery of a new signaling mechanism for auxin, a multifunctional hormone that regulates pattern formation in plants. Arabidopsis leaf epidermal pavement cells (PCs) develop the puzzle-piece cell shape with interlocking lobes and indentations via interdigitated cellular growth.1 Through the ABP1 (Auxin Binding Protein 1) cell surface receptor, auxin coordinately activates 2 mutually exclusive Rho GTPase signaling pathways that are activated in the complementary lobing and indenting sides of adjacent cells: the ROP2 pathway for lobe formation and the ROP6 pathway for promoting indentation. This new signaling mechanism also involves ROP2-dependent polar accumulation of PIN1 in the plasma membrane, a member of the PIN auxin efflux carrier family that is critical for the formation of various developmental patterns including the PC interdigitation pattern. This Rho-dependent auxin signaling mechanism explains how interdigitated cellular growth is coordinated. In this extra view, we propose that the same mechanism can also explain how a uniform auxin signal initiates the formation of the interdigitated pattern.

Plant growth and development is dependent on the specification and maintenance of pools of stem cells found in the meristems. Mutations in the Arabidopsis MERISTEM-DEFECTIVE (MDF) gene lead to a loss of stem cell and meristematic activity in the root and vegetative shoot. MDF encodes a putative RS domain protein with a predicted role in transcription or RNA processing control. mdf mutants exhibit decreased levels of PINFORMED2 (PIN2) and PIN4 mRNAs, which is associated with a reduction in PIN:GFP levels, and with a defective auxin maximum in the basal region of the developing mdf embryo and seedling root meristem. Seedling roots also exhibit reduced PLETHORA (PLT), SCARECROW and SHORTROOT gene expression, a loss of stem cell activity, terminal differentiation of the root meristem and defective cell patterning. MDF expression is not defective in the bodenlos, pin1 or eir1/pin2 auxin mutants, and is not modulated by exogenous auxin. plt1 plt2 double mutants have unaffected levels of MDF RNA, indicating that MDF acts upstream of PIN and PLT gene expression. Differentiation of the shoot stem cell pool also occurs in mdf mutants, associated with a reduced WUSCHEL (WUS) expression domain and expanded CLAVATA3 (CLV3) domain. Overexpression of MDF leads to the activation of markers of embryonic identity and ectopic meristem activity in vegetative tissues. These results demonstrate a requirement for the MDF-dependent pathway in regulating PIN/PLT- and WUS/CLV-mediated meristem activity.

NMR spectroscopy was used to explore the different aspects of the normal and pathological functions of tau, but proved challenging because the protein contains 441 amino acids and has poor signal dispersion. We have set out to dissect the phosphorylation patterns of tau in order to understand better its role in the aggregation process and microtubule-binding regulation. Our current knowledge on the functional consequences of specific phosphorylations is still limited, mainly because producing and assessing quantitatively phosphorylated tau samples is far from straightforward, even in vitro. We use NMR spectroscopy as a proteomics tool to characterize the phosphorylation patterns of tau, after in vitro phosphorylation by recombinant kinases. The phosphorylated tau can next be use for functional assays or interaction assays with phospho-dependent protein partners, such as the prolyl cis-trans isomerase Pin1.

Ectrodactyly, or Split-Hand/Foot Malformation (SHFM), is a congenital condition characterized by the loss of central rays of hands and feet. The p63 and the DLX5;DLX6 transcription factors, expressed in the embryonic limb buds and ectoderm, are disease genes for these conditions. Mutations of p63 also cause the ectodermal dysplasia-ectrodactyly-cleft lip/palate (EEC) syndrome, comprising SHFM. Ectrodactyly is linked to defects of the apical ectodermal ridge (AER) of the developing limb buds. FGF8 is the key signaling molecule in this process, able to direct proximo-distal growth and patterning of the skeletal primordial of the limbs. In the limb buds of both p63 and Dlx5;Dlx6 murine models of SHFM, the AER is poorly stratified and FGF8 expression is severely reduced. We show here that the FGF8 locus is a downstream target of DLX5 and that FGF8 counteracts Pin1-ΔNp63α interaction. In vivo, lack of Pin1 leads to accumulation of the p63 protein in the embryonic limbs and ectoderm. We show also that ΔNp63α protein stability is negatively regulated by the interaction with the prolyl-isomerase Pin1, via proteasome-mediated degradation; p63 mutant proteins associated with SHFM or EEC syndromes are resistant to Pin1 action. Thus, DLX5, p63, Pin1 and FGF8 participate to the same time- and location-restricted regulatory loop essential for AER stratification, hence for normal patterning and skeletal morphogenesis of the limb buds. These results shed new light on the molecular mechanisms at the basis of the SHFM and EEC limb malformations.

Mutations of isocitrate dehydrogenase 1 (IDH1) and IDH2 in acute myeloid leukemia (AML) cells produce the oncometabolite R-2-hydroxyglutarate (R-2HG) to induce epigenetic alteration and block hematopoietic differentiation. However, the effect of R-2HG released by IDH-mutated AML cells on the bone marrow microenvironment is unclear. Here, we report that R-2HG induces IκB kinase-independent activation of NF-κB in bone marrow stromal cells. R-2HG acts via a reactive oxygen species/extracellular signal-regulated kinase (ERK)-dependent pathway to phosphorylate NF-κB on the Thr254 residue. This phosphorylation enhances the interaction of NF-κB and the peptidyl-prolyl cis-trans isomerase PIN1 and increases the protein stability and transcriptional activity of NF-κB. As a consequence, R-2HG enhances NF-κB-dependent expression of cytokines including IL-6, IL-8 and complement 5a to stimulate proliferation of AML cells. In addition, R-2HG also upregulates vascular endothelial adhesion molecule 1 and CXCR4 in stromal cells to enhance the contact between AML and stromal cells and attenuates chemotherapy-induced apoptosis. More importantly, we validated the R-2HG-activated gene signature in the primary bone marrow stromal cells isolated from IDH-mutated AML patients. Collectively, our results suggest that AML cell-derived R-2HG may be helpful for the establishment of a supportive bone marrow stromal niche to promote AML progression via paracrine stimulation.

The purpose of this study is to evaluate the efficacy and safety of neoadjuvant treatment with carboplatin and eribulin in patients with early-stage triple negative breast cancer (TNBC), and to explore biomarkers based on DNA and protein expression profiles as predictors of response. Patients with histologically confirmed early-stage TNBC received carboplatin AUC 6 iv every 21 days, and eribulin 1.4 mg/m(2) day 1 and day 8 every 21 days for four cycles. The primary endpoint of the study was pathologic complete response (pCR), with secondary endpoints including clinical response and safety of the combination. Exploratory studies assessed DNA-based biomarkers [homologous recombination deficiency (HRD) score, and HR deficiency status (HRD score + BRCA1/BRCA2 mutation status)], protein-based biomarkers (Ki67, TP53, androgen receptor, Cyclin E, CDK2, Cyclin D, CDK4, Pin1 and Smad3), and clinical pretreatment factors as predictors of pCR. 13/30 (43.3 %) patients enrolled in the study achieved pCR. 24 (80.0 %) had a clinical complete or partial response. The combination was safe with mostly grade 1 and 2 toxicities. HRD score (P = 0.0024) and HR deficiency status (P = 0.0012) significantly predicted pCR. Pretreatment cytoplasmic CDK2 was also associated with pCR (P = 0.021). Significant differences in pre- versus post-treatment expression levels of nuclear Cyclin D (P = 0.020), nuclear CDK4 (P = 0.0030), and nuclear Smad3 (P = 0.015) were detected. The combination of carboplatin and eribulin is safe and efficacious in the treatment of early-stage TNBC. HRD score, HR deficiency status, and cytoplasmic CDK2 predicted pCR in this patient population.

A complete characterization of the proteome of seminal plasma (SP) is an essential step to understand how SP influences sperm function and fertility after artificial insemination (AI). The purpose of this study was to identify which among characterized proteins in boar SP were differently expressed among AI boars with significantly different fertility outcomes. A total of 872 SP proteins, 390 of them belonging specifically to Sus Scrofa taxonomy, were identified (Experiment 1) by using a novel proteomic approach that combined size exclusion chromatography and solid-phase extraction as prefractionation steps prior to Nano LC-ESI-MS/MS analysis. The SP proteomes of 26 boars showing significant differences in farrowing rate (n = 13) and litter size (n = 13) after the AI of 10 526 sows were further analyzed (Experiment 2). A total of 679 SP proteins were then quantified by the SWATH approach, where the penalized linear regression LASSO revealed differentially expressed SP proteins for farrowing rate (FURIN, AKR1B1, UBA1, PIN1, SPAM1, BLMH, SMPDL3A, KRT17, KRT10, TTC23, and AGT) and litter size (PN-1, THBS1, DSC1, and CAT). This study extended our knowledge of the SP proteome and revealed some SP proteins as potential biomarkers of fertility in AI boars.

Root system architecture is a major determinant of water and nutrient acquisition as well as stress tolerance in plants. The Mediator complex is a conserved multiprotein complex that acts as a universal adaptor between transcription factors and the RNA polymerase II. In this article, we characterize possible roles of the MEDIATOR8 (MED8) and MED25 subunits of the plant Mediator complex in the regulation of root system architecture in Arabidopsis (Arabidopsis thaliana). We found that loss-of-function mutations in PHYTOCHROME AND FLOWERING TIME1 (PFT1)/MED25 increase primary and lateral root growth as well as lateral and adventitious root formation. In contrast, PFT1/MED25 overexpression reduces these responses, suggesting that PFT1/MED25 is an important element of meristematic cell proliferation and cell size control in both lateral and primary roots. PFT1/MED25 negatively regulates auxin transport and response gene expression in most parts of the plant, as evidenced by increased and decreased expression of the auxin-related reporters PIN-FORMED1 (PIN1)::PIN1::GFP (for green fluorescent protein), DR5:GFP, DR5:uidA, and BA3:uidA in pft1-2 mutants and in 35S:PFT1 seedlings, respectively. No alterations in endogenous auxin levels could be found in pft1-2 mutants or in 35S:PFT1-overexpressing seedlings. However, detailed analyses of DR5:GFP and DR5:uidA activity in wild-type, pft1-2, and 35S:PFT1 seedlings in response to indole-3-acetic acid, naphthaleneacetic acid, and the polar auxin transport inhibitor 1-N-naphthylphthalamic acid indicated that PFT1/MED25 principally regulates auxin transport and response. These results provide compelling evidence for a new role for PFT1/MED25 as an important transcriptional regulator of root system architecture through auxin-related mechanisms in Arabidopsis.

TP53 missense mutations significantly influence the development and progression of various human cancers via their gain of new functions (GOF) through different mechanisms. Here we report a unique mechanism underlying the GOF of p53-R249S (p53-RS), a p53 mutant frequently detected in human hepatocellular carcinoma (HCC) that is highly related to hepatitis B infection and aflatoxin B1. A CDK inhibitor blocks p53-RS's nuclear translocation in HCC, whereas CDK4 interacts with p53-RS in the G1/S phase of the cells, phosphorylates it, and enhances its nuclear localization. This is coupled with binding of a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) to p53-RS, but not the p53 form with mutations of four serines/threonines previously shown to be crucial for PIN1 binding. As a result, p53-RS interacts with c-Myc and enhances c-Myc-dependent rDNA transcription key for ribosomal biogenesis. These results unveil a CDK4-PIN1-p53-RS-c-Myc pathway as a novel mechanism for the GOF of p53-RS in HCC.

Development and organogenesis in both dicot and monocot plants are highly dependent on polar auxin transport (PAT), which requires the proper asymmetric localization of both auxin influx and efflux carriers. In the model dicot plant Arabidopsis thaliana, the trafficking and localization of auxin efflux facilitators such as PIN-FORMED1 (PIN1) are mediated by GNOM, a guanine-nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, but molecular regulators of the auxin influx facilitators remain unknown. Here, we show that over-expression of OsAGAP, an ARF-GTPase-activating protein (ARF-GAP) in rice, impaired PAT and interfered with both primary and lateral root development. The lateral root phenotype could be rescued by the membrane-permeable auxin 1-naphthyl acetic acid, but not by indole 3-acetic acid (IAA) or by 2,4-dichloro-phenoxyacetic acid, which require influx facilitators to enter the cells. OsAGAP-over-expressing plants had alterations in vesicle trafficking and localization of the presumptive A. thaliana auxin-influx carrier AUX1, but not in the localization of the auxin efflux facilitators. Together, our data suggest that OsAGAP has a specific role in regulating vesicle trafficking pathways such as the auxin influx pathway, which in turn controls auxin-dependent root growth in plants.

Kras is a decisive oncogene in pancreatic ductal adenocarcinoma (PDAC). PIN1 is a key effector involved in the Kras/ERK axis, synergistically mediating various cellular events. However, the underlying mechanism by which PIN1 promotes the development of PDAC remains unclear. Here we sought to elucidate the effect of PIN1 on redox homeostasis in Kras-driven PDAC. PIN1 was prevalently upregulated in PDAC and predicted the prognosis of the disease, especially Kras-mutant PDAC. Downregulation of PIN1 inhibited PDAC cell growth and promoted apoptosis, partially due to mitochondrial dysfunction. Silencing of PIN1 damaged basal mitochondrial function by significantly increasing intracellular ROS. Furthermore, PIN1 maintained redox balance via synergistic activation of c-Myc and NRF2 to upregulate expression of antioxidant response element (ARE)-driven genes in PDAC cells. This study elucidates a new mechanism by which Kras/ERK/NRF2 promotes tumor growth and identifies PIN1 as a decisive target in therapeutic strategies aimed at disturbing the redox balance in pancreatic cancer.

Fruit length is a prominent agricultural trait during cucumber (Cucumis sativus) domestication and diversifying selection; however, the regulatory mechanisms of fruit elongation remain elusive. We identified two alleles of the FRUITFULL (FUL)-like MADS-box gene CsFUL1 with 3393^C/A Single Nucleotide Polymorphism variation among 150 cucumber lines. Whereas CsFUL1^A was specifically enriched in the long-fruited East Asian type cucumbers (China and Japan), the CsFUL1^C allele was randomly distributed in cucumber populations, including wild and semiwild cucumbers. CsFUL1^A knockdown led to further fruit elongation in cucumber, whereas elevated expression of CsFUL1^A resulted in significantly shorter fruits. No effect on fruit elongation was detected when CsFUL1^C expression was modulated, suggesting that CsFUL1^A is a gain-of-function allele in long-fruited cucumber that acts as a repressor during diversifying selection of East Asian cucumbers. Furthermore, CsFUL1^A binds to the CArG-box in the promoter region of SUPERMAN, a regulator of cell division and expansion, to repress its expression. Additionally, CsFUL1^A inhibits the expression of auxin transporters PIN-FORMED1 (PIN1) and PIN7, resulting in decreases in auxin accumulation in fruits. Together, our work identifies an agriculturally important allele and suggests a strategy for manipulating fruit length in cucumber breeding that involves modulation of CsFUL1^A expression.

Pin1 regulates a subset of phosphoproteins by isomerizing phospho-Ser/Thr-Pro motifs via a 'post-phosphorylation' mechanism. Here, we characterize TR3 as a novel Pin1 substrate, and the mitogenic function of TR3 depends on Pin1-induced isomerization. There are at least three phospho-Ser-Pro motifs on TR3 that bind to Pin1. The Ser95-Pro motif of TR3 is the key site through which Pin1 enhances TR3 stability by retarding its degradation. Pin1 can also catalyze TR3 through phospho-Ser431-Pro motif, which is phosphorylated by extracellular signal-regulated kinase 2 (ERK2), resulting in enhanced TR3 transactivation. Furthermore, Pin1 not only facilitates TR3 targeting to the promoter of cyclin D2, a novel downstream target of TR3, but also promotes TR3 to recruit p300, thereby inducing cell proliferation. Importantly, we found that Pin1 is indispensable for TR3 to promote tumor growth both in vitro and in vivo. Our study thus suggests that Pin1 has an important role in cell proliferation by isomerizing TR3.

The WW module of the peptidyl-prolyl cis/trans isomerase Pin1 targets specifically phosphorylated proteins involved in the cell cycle through the recognition of phospho-Thr(Ser)-Pro motifs. When the microtubule-associated Tau protein becomes hyperphosphorylated, it equally becomes a substrate for Pin1, with two recognition sites described around the phosphorylated Thr212 and Thr231. The Pin1 WW domain binds both sites with moderate affinity, but only the Thr212-Pro213 bond is isomerized by the catalytic domain of Pin1. We show here that, in a peptide carrying a single recognition site, the WW module increases significantly the enzymatic isomerase activity of Pin1. However, with addition of a second recognition motif, the affinity of both the WW and catalytic domain for the substrate increases, but the isomerization efficacy decreases. We therefore conclude that the WW domain can act as a negative regulator of enzymatic activity when multiple phosphorylation is present, thereby suggesting a subtle mechanism of its functional regulation.

The human parvulin Pin1 is a member of the peptidyl-prolyl cis-trans isomerase group of proteins, which modulate the assembly, folding, activity, and transport of essential cellular proteins. Pin1 is a mitotic regulator interacting with a range of proteins that are phosphorylated before cell division. In addition, an involvement of Pin1 in the tau-related neurodegenerative brain disorders has recently been shown. In this context, Pin1 becomes depleted from the nucleus in Alzheimer's disease (AD) neurons when it is redirected to the large amounts of hyperphosphorylated tau associated with the neurofibrillary tangles. This depletion from the nucleus may ultimately contribute to neuron cell death. Recently we have devised a novel methodology in which exogenous Pin1 is used as a TEM probe for its target proteins. Here we extend this methodology to provide further evidence that Pin1 binds at enhanced levels to mitotic nuclear proteins and to hyperphosphorylated tau in AD brain. We suggest that exogenous Pin1 labeling can be used to elucidate the phosphorylation status of its target proteins in general and could specifically provide important insights into the development of tau-related neurodegenerative brain disorders.

An N-terminally truncated and cooperatively folded version (residues 6-39) of the human Pin1 WW domain (hPin1 WW hereafter) has served as an excellent model system for understanding triple-stranded beta-sheet folding energetics. Here we report that the negatively charged N-terminal sequence (Met1-Ala-Asp-Glu-Glu5) previously deleted, and which is not conserved in highly homologous WW domain family members from yeast or certain fungi, significantly increases the stability of hPin1 WW (approximately 4 kJ mol(-1) at 65 degrees C), in the context of the 1-39 sequence based on equilibrium measurements. N-terminal truncations and mutations in conjunction with a double mutant cycle analysis and a recently published high-resolution X-ray structure of the hPin1 cis/trans-isomerase suggest that the increase in stability is due to an energetically favorable ionic interaction between the negatively charged side chains in the N terminus of full-length hPin1 WW and the positively charged epsilon-ammonium group of residue Lys13 in beta-strand 1. Our data therefore suggest that the ionic interaction between Lys13 and the charged N terminus is the optimal solution for enhanced stability without compromising function, as ascertained by ligand binding studies. Kinetic laser temperature-jump relaxation studies reveal that this stabilizing interaction has not formed to a significant extent in the folding transition state at near physiological temperature, suggesting a differential contribution of the negatively charged N-terminal sequence to protein stability and folding rate. As neither the N-terminal sequence nor Lys13 are highly conserved among WW domains, our data further suggest that caution must be exercised when selecting domain boundaries for WW domains for structural, functional, or thermodynamic studies.

Pin1 is a unique regulator that catalyzes the conversion of a specific phospho-Ser/Thr-Pro-containing motif in target proteins. Herein, we identified AMP-activated protein kinase (AMPK) as a Pin1-binding protein. Pin1 wild-type, but not Pin1 mutant at serine 16 (S16A), associated with AMPK. Reciprocally, the constitutively active form of AMPK (AMPK-CA), but not the dominant negative form of AMPK (AMPK-DN), interacted with Pin1 wild type. In addition, mutation of Ser176 site in AMPK led to a significant loss of binding between AMPK and Pin1. Ablation of the Pin1 gene in MEFs enhanced AMPK phosphorylation induced by AICAR. Pin1 overexpression in Pin1(-/-) MEFs and SK-HEP-1 cells attenuated AMPK phosphorylation induced by EGF, whereas gene knockdown of Pin1 by siRNA enhanced it. The association between Pin1 and AMPK was increased by EGF, leading to their interaction with protein phosphatase-2A (PP2A). Furthermore, Pin1 increased the PP2A activity induced by EGF. In addition, AMPK-WT and AMPK-CA, but not AMPK-DN, inhibited EGF-induced neoplastic cell transformation of JB6 Cl41 cells and tumorigenicity of SK-HEP-1 cells. The overexpression of Pin1 in JB6 Cl41 cells and SK-HEP-1 cells attenuated the inhibitory effect of AMPK in EGF-induced neoplastic cell transformation of JB6 Cl41 and tumorigenicity of SK-HEP-1 cells, respectively. Taken together, these results indicate that Pin1 plays a pivotal role in EGF-induced carcinogenesis through downregulation of AMPK activity in hepatocarcinoma cells.

Pin1 is an essential mitotic regulator consisting of a peptidyl-prolyl isomerase (PPIase) domain flexibly tethered to a smaller Trp-Trp (WW) binding domain. Communication between these domains is important for Pin1 in vivo activity; however, the atomic basis for this communication has remained elusive. Our previous nuclear magnetic resonance (NMR) studies of Pin1 functional dynamics suggested that weak interdomain contacts within Pin1 enable allosteric communication between the domain interface and the distal active site of the PPIase domain.1,2 A necessary condition for this hypothesis is that the intrinsic properties of the PPIase domain should be sensitive to interdomain contact. Here, we test this sensitivity by generating a Pin1 mutant, I28A, which weakens the wild-type interdomain contact while maintaining the overall folds of the two domains. Using NMR, we show that I28A leads to altered substrate binding affinity and isomerase activity. Moreover, I28A causes long-range perturbations to conformational flexibility in both domains, for both the apo and substrate-complexed states of the protein. These results show that the distribution of conformations sampled by the PPIase domain is sensitive to interdomain contact and strengthen the hypothesis that such contact supports interdomain allosteric communication in Pin1. Other modular systems may exploit interdomain interactions in a similar manner.

Histone H1 plays a crucial role in stabilizing higher order chromatin structure. Transcriptional activation, DNA replication, and chromosome condensation all require changes in chromatin structure and are correlated with the phosphorylation of histone H1. In this study, we describe a novel interaction between Pin1, a phosphorylation-specific prolyl isomerase, and phosphorylated histone H1. A sub-stoichiometric amount of Pin1 stimulated the dephosphorylation of H1 in vitro and modulated the structure of the C-terminal domain of H1 in a phosphorylation-dependent manner. Depletion of Pin1 destabilized H1 binding to chromatin only when Pin1 binding sites on H1 were present. Pin1 recruitment and localized histone H1 phosphorylation were associated with transcriptional activation independent of RNA polymerase II. We thus identify a novel form of histone H1 regulation through phosphorylation-dependent proline isomerization, which has consequences on overall H1 phosphorylation levels and the stability of H1 binding to chromatin.

Pin1 is a peptidyl-prolyl cis/trans isomerase that was initially discovered as an important mitotic regulator. In keeping with this, it targets specifically phosphorylated ser- or thr-pro dipeptides, which are found in numerous mitotic proteins as a result of cyclin-dependent kinase activity. But Pin1 also modulates the activity of numerous other proteins, and a strong candidate for such regulation has been RNA polymerase II (RNAP II). The unique C-terminal domain of the RNAP II largest subunit contains as many as 100 ser-pro dipeptides laid out in an essentially linear array, and which are subject to different patterns of phosphorylation. Here we summarize findings indicating that Pin1 can indeed directly modulate the structure and function of RNAP II, and propose that this activity plays a role both in mitotic gene silencing and during the transcription cycle.

Aetiology of neurodegenerative mechanisms underlying Alzheimer's disease (AD) are still under elucidation. The contribution of cerebrovascular deficiencies (such as cerebral ischemia/stroke) has been strongly endorsed in recent years. Reduction of blood supply leading to hypoxic condition is known to activate cellular responses mainly controlled by hypoxia-inducible transcription factor-1 (HIF-1). Thus alterations of oxygen responsive HIF-1α subunit in the central nervous system may contribute to the cognitive decline, especially influencing mechanisms associated to amyloid precursor protein (APP) amyloidogenic metabolism. Although HIF-1α protein level is known to be regulated by von Hippel-Lindau (VHL) ubiquitin-proteasome system, it has been recently suggested that glycogen synthase kinase-3β (Gsk-3β) promotes a VHL-independent HIF-1α degradation. Here we provide evidences that in rat primary hippocampal cell cultures, HIF-1α degradation might be mediated by a synergic action of Gsk-3β and peptidyl-prolyl cis/trans isomerase (Pin1). In post-ischemic conditions, such as those mimicked with oxygen glucose deprivation (OGD), HIF-1α protein level increases remaining unexpectedly high for long time after normal condition restoration jointly with the increase of lactate dehydrogenase (LDH) and β-secretase 1 (BACE1) protein expression (70 and 140% respectively). Interestingly the Pin1 activity decreases about 40-60% and Pin1(S16) inhibitory phosphorylation significantly increases, indicating that Pin1 binding to its substrate and enzymatic activity are reduced by treatment. Co-immunoprecipitation experiments demonstrate that HIF-1α/Pin1 in normoxia are associated, and that in presence of specific Pin1 and Gsk-3β inhibitors their interaction is reduced in parallel to an increase of HIF-1α protein level. Thus we suggest that in post-OGD neurons the high level of HIF-1α might be due to Pin1 binding ability and activity reduction which affects HIF-1α degradation: an event that may highlight the relevance of ischemia/HIF-1α as a risk factor in AD pathogenesis.

Phosphorylation on serine or threonine residue preceding proline (Ser/Thr-Pro) is a key regulatory mechanism. The conformation of certain phosphorylated Ser/Thr-Pro bonds is regulated specifically by the prolyl isomerase Pin1. It has been reported that Pin1 is strikingly overexpressed in a subset of human tumors. A differential-display screen reveals that Pin1 increases the transcription of several beta-catenin target genes, including those encoding cyclin D1 and c-Myc. Pin1 cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1. We have previously reported that Pin1 is overexpressed in oral squamous cell carcinoma (OSCC) and its level correlates with Cyclin D1 expression. However, the analysis of the relationship between Pin1 and other cyclin genes has not been demonstrated in human OSCC. We examined Pin1 mRNA and protein expressions in OSCC cell lines, and analyzed Pin1/cyclins expression by RT-PCR. We report that Pin1 mRNA correlates with Cyclin D1 mRNA expression and the expression of many cyclin genes is associated with lymph node metastasis in OSCC. These results indicate that Pin1 is a regulator of Cyclin D1 expression in OSCC and might have a role in oncogenesis; and the expression of many cyclin genes will be an indicator of lymph node metastasis in OSCC.