This paper examines the folding mechanism of an individual beta-hairpin in the presence of other hairpins by using an off-lattice model of a small triple-stranded antiparallel beta-sheet protein, Pin1 WW domain. The turn zipper model and the hydrophobic collapse model originally developed for a single beta-hairpin in literature is confirmed to be useful in describing beta-hairpins in model Pin1 WW domain. We find that the mechanism for folding a specific hairpin is independent of whether it folds first or second, but the formation process are significantly dependent on temperature. More specifically, beta1-beta2 hairpin folds via the turn zipper model at a low temperature and the hydrophobic collapse model at a high temperature, while the folding of beta2-beta3 hairpin follows the turn zipper model at both temperatures. The change in folding mechanisms is interpreted by the interplay between contact stability (enthalpy) and loop lengths (entropy), the effect of which is temperature dependent.

Topoisomerase (Topo) IIalpha and condensin are essential for formation of mitotic chromosomes. However, the mechanism by which these two major components assemble during the chromosome condensation process had been unclear. Recent studies have revealed a coordinated and cooperative process, by which TopoIIalpha functions early to form an axis scaffold, whereas condensin complexes assemble at a later stage critical for chromosome integrity and subsequent segregation. Extending these observations, we recently found that the phosphorylation-dependent prolyl isomerase Pin1 is directly linked to the process. This conclusion is based on the observation of strong and extensive interactions of Pin1 with chromatin specifically at G2/M phase. Pin1 modulates the mitotic phosphorylation of TopoIIalpha by cdc2/cyclinB and promotes the association of phosphorylated TopoIIalpha with DNA elements to form an axis scaffold complex. The evidence highlights a critical role of Pin1 via its regulation of mitotic phosphorylation of key components in the chromosome condensation process.

Organosulfur compounds from garlic effectively inhibit growth of transplanted as well as spontaneous cancers in preclinical animal models without any adverse side effects. However, the mechanisms underlying anticancer effect of this class of compounds are not fully understood. This study reports, for the first time, that garlic organosulfide diallyl trisulfide (DATS) inhibits estrogen receptor-α (ER-α) activity in human breast cancer cells. Exposure of MCF-7 and T47D cells to DATS resulted in downregulation of ER-α protein, which peaked between 12- and 24-h post-treatment. DATS was relatively more effective in suppressing ER-α protein expression compared with its mono and disulfide analogs. The 17β-estradiol (E2)-induced expression of pS2 and cyclin D1, ER-α target gene products, was also decreased in the presence of DATS. Downregulation of ER-α protein expression resulting from DATS treatment was accompanied by a decrease in nuclear levels of ER-α protein, ER-α mRNA suppression, and inhibition of ERE2e1b-luciferase reporter activity. DATS-mediated inhibition of cell viability and apoptosis induction were not affected in the presence of E2. In agreement with these results, ectopic expression of ER-α in MDA-MB-231 cell line failed to confer any protection against cell proliferation inhibition or apoptosis induction resulting from DATS exposure. DATS treatment caused a decrease in protein levels of peptidyl-prolyl cis-trans isomerase (Pin1), and overexpression of Pin1 partially attenuated ER-α downregulation by DATS. DATS-induced apoptosis was modestly but significantly augmented by overexpression of Pin1. In conclusion, this study identifies ER-α as a novel target of DATS in mammary cancer cells.

BACKGROUND

The modest efficacy of available therapies for Hepatocellular carcinoma (HCC) indicates the need to develop novel therapeutic approaches. For the proteasome inhibitor Bortezomib (BZB), potentially attractive for HCC treatment, the mechanism of action is largely unknown. The BZB effect on E2Fs and the E2Fs control on the peptidylproline cis-trans isomerase (Pin1), prompted us to explore the BZB effect on the Pin1-E2F1 axis.

METHODS

The tumorigenic cell line HuH7 together with the non-tumorigenic cells IHH and the human pluripotent stem cell derived hepatocytes (hPSC-H), were used as cellular models of HCC and normal liver cells, respectively.

RESULTS

BZB reduces HuH7 growth as shown by cell counting, cell vitality test and cell cycle analysis; this is paralleled by the decrease of Pin1, E2F1, cyclin A2 and of the hyper-phosphorylated pRB. Pin1-E2F1 axis impairment justifies the anti-proliferative effect since Pin-E2F1 depletion decreases HuH7 growth while the over-expression rescues BZB-induced inhibition of proliferation. Moreover, Pin1-E2F1 promote HuH7 growth via the up-regulation of cyclin D1, cyclin E, cyclin A2, E2F2 and in part E2F3. Finally, in the control cells IHH and hPSC-H, BZB effect on cell vitality is not irrelevant, a fact correlated to the cellular proliferation rate. Thus, BZB effect on healthy liver tissue may not be entirely negligible hence caution should be exercised in its use in liver regeneration processes.

CONCLUSIONS

For the first time we prove the functional involvement of the Pin1-E2F1 axis in the anti-proliferative effect of BZB indicating Pin1-E2F as an attractive target to control HCC cell growth.

Pin1 specifically recognizes and catalyzes the cis-trans isomerization of phosphorylated-Ser/Thr-Pro bonds, which modulate the stability, localization, and function of numerous Pin1 targets involved in tumor progression. However, the role of Pin1 in cancer remains enigmatic as the gene is located on chromosome 19p13.2, which is a region subject to loss of heterozygosity in several tumors. Since Pin1 protein is frequently under-expressed in kidney cancer, we have explored its role in human clear cell renal cell carcinoma (ccRCC). Here we show evidence for PIN1 gene deletion and mRNA under-expression as a mechanism of Pin1 reduction in ccRCC tumors. We demonstrate that restoration of Pin1 in cell lines found to be deficient in Pin1 protein expression can attenuate the growth of ccRCC cells in soft agar and a xenograft tumor model. Moreover, this ability of Pin1 to negatively influence tumor growth in ccRCC cells may be dependent on the presence of functional p53, which is infrequently mutated in ccRCC. These observations suggest Pin1 may have a mild tumor suppressive role in ccRCC.

The WW domain of the human Pin1 protein for its simple topology and large amount of experimental data is an ideal candidate to assess theoretical approaches to protein folding. The purpose of this work is to compare the reliability of the chemically based Sorenson/Head-Gordon (SHG) model and a standard native centric model in reproducing, through molecular dynamics simulations, some of the well known features of the folding transition of this small domain. Our results show that the Gō model correctly reproduces the cooperative, two-state, folding mechanism of the WW-domain, while the SHG model predicts a transition occurring in two stages: a collapse, followed by a structural rearrangement. The lack of a cooperative folding in the SHG simulations appears to be related to the nonfunnel shape of the energy landscape featuring a partitioning of the native valley in subbasins corresponding to different chain chiralities. However, the SHG approach remains more reliable in estimating the phi-values with respect to Gō-like description. This may suggest that the WW-domain folding process is stirred by energetic and topological factors as well, and it highlights the better suitability of chemically based models in simulating mutations.

Circular versions of a large number of proteins have been designed by connecting the N and C termini via peptide linkers. A motivation for these designs is the assumed enhancement in folding stability, because backbone cyclization reduces the chain entropy of the unfolded state. Here, it is recognized that backbone cyclization also reduces the chain entropy of a flexible peptide linker in the folded state. Specifically, the end-to-end distance of the linker is restricted to fluctuations around the average displacement between the N and C termini of the folded protein. The balance of the chain-entropy reductions in the folded and unfolded states is used to predict the change in the unfolding free energy, deltadeltaG(cycl), by backbone cyclization. Predicted values of deltadeltaG(cycl) are in quantitative agreement with results of a careful study on cyclizing the 34 residue PIN1 WW domain by linkers with two to seen residues. The experimental results of an optimal linker length l=4 and a maximum stabilization of 1.7 kcal/mol are reproduced. Calculations of deltadeltaG(cycl) for a broad selection of circular proteins suggest that the stabilizing effect of backbone cyclization is modest, reflecting entropy reductions in both the unfolded and the folded states.

The tumor-suppressor protein promyelocytic leukemia (PML) is aberrantly degraded in multiple types of human cancers through mechanisms that are incompletely understood. Here, we show that the phosphatase SCP1 and its isoforms SCP2/3 dephosphorylate PML at S518, thereby blocking PML ubiquitination and degradation mediated by the prolyl isomerase Pin1 and the ubiquitin ligase KLHL20. Clinically, SCP1 and SCP3 are downregulated in clear cell renal cell carcinoma (ccRCC) and these events correlated with PMLS518 phosphorylation, PML turnover, and high-grade tumors. Restoring SCP1-mediated PML stabilization not only inhibited malignant features of ccRCC, including proliferation, migration, invasion, tumor growth, and tumor angiogenesis, but also suppressed the mTOR-HIF pathway. Furthermore, blocking PML degradation in ccRCC by SCP1 overexpression or Pin1 inhibition enhanced the tumor-suppressive effects of the mTOR inhibitor temsirolimus. Taken together, our results define a novel pathway of PML degradation in ccRCC that involves SCP downregulation, revealing contributions of this pathway to ccRCC progression and offering a mechanistic rationale for combination therapies that jointly target PML degradation and mTOR inhibition for ccRCC treatment.

SMRT (silencing mediator for retinoic acid and thyroid hormone receptors) is a transcriptional co-repressor that mediates the repressive function of nuclear hormone receptors such as the estrogen receptor (ER). Decreased SMRT levels correlate with acquired tamoxifen resistance in breast cancer, and SMRT restoration might resensitize breast cancer cells to tamoxifen. A new study demonstrates that SMRT protein stability is regulated by phosphorylation-dependent Pin1-catalyzed prolyl-isomerization. Pin1 functions downstream of HER2, positioning it as an important modulator of the crosstalk between ER and growth factor signaling.

Bone is constantly formed and resorbed throughout life by coordinated actions of osteoblasts and osteoclasts. However, the molecular mechanisms involved in osteoblast function remain incompletely understood. Here we show, for the first time, that the peptidyl-prolyl isomerase PIN1 controls the osteogenic activity of osteoblasts. Pin1 null mice exhibited an age-dependent decrease in bone mineral density and trabecular bone formation without alteration in cortical bone. Further analysis identified a defect in BMP signaling in Pin1 null osteoblasts but normal osteoclast function. PIN1 interacted with SMAD5 and was required for the expression by primary osteoblasts of osteoblast specific transcription factors (CBFA1 and OSX), ECM (collagen I and OCN) and the formation of bone nodules. Our results thus uncover a novel aspect of the molecular underpinning of osteoblast function and identify a new therapeutic target for bone diseases.

Mitotic clonal expansion (MCE) is one of the important events taking place at the early stage during 3T3-L1 adipocyte differentiation. To investigate the mechanism underlying this process, we carried out a temporal proteomic analysis to profile the dynamic changes in MCE. Using 8-plex-iTRAQ-2DLC-MS/MS analysis, 3152 proteins were quantified during the initial 28 h of 3T3-L1 adipogenesis. Functional analysis was performed on 595 proteins with maximum or minimum quantities at 20 h of adipogenic induction that were potentially involved in MCE, which identified PI3K/AKT/mTOR as the most relevant pathway. Among the 595 proteins, PKM2 (Pyruvate kinase M2), a patterned protein identified as a potential target gene of C/EBPβ in our previous work, was selected for further investigation. Network analysis suggested positive correlations among C/EBPβ, PIN1, and PKM2, which may be related with the PI3K-AKT pathway. Knockdown of PKM2 with siRNA inhibited both MCE and adipocyte differentiation of 3T3-L1 cells. Moreover, PKM2 was down-regulated at both the mRNA level and the protein level upon the knockdown of C/EBPβ. And overexpressed PKM2 can partially restore MCE, although it did not restore terminal adipocyte differentiation, which was inhibited by siC/EBPβ. Thus, PKM2, potentially regulated by C/EBPβ, is involved in MCE during adipocyte differentiation. The dynamic proteome changes quantified here provide a promising basis for revealing molecular mechanism regulating adipogenesis.

The cell adhesion molecule Neuroligin2 (NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis-trans isomerase Pin1. This signalling cascade negatively regulates NL2's ability to interact with gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of NL2, gephyrin and GABAA receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1-/-) associated with an increase in amplitude of spontaneous GABAA-mediated post-synaptic currents. Our results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/gephyrin interaction.

The peptidyl-proline isomerase Protein Never in Mitosis Gene A Interacting-1 (PIN1) increases the level or activity of several transcription factors that can induce the inducible nitric oxide (NO) synthase (iNOS). PIN1 can also regulate mRNA and protein turnover. Here, the effect of depletion of PIN1 on induction of iNOS by Escherichia coli endotoxin (LPS) and interferon-gamma (IFNgamma) in murine aortic endothelial cells (MAEC) was determined. Suppression of PIN1 by 85% with small hairpin RNA enhanced the induction of NO and iNOS protein by LPS-IFNgamma. There was no effect on induction of iNOS mRNA, suggesting a posttranscriptional effect. The enhanced levels of iNOS protein were functionally significant since LPS-IFNgamma was cytotoxic to MAEC lacking PIN1 but not MAEC harboring an inactive control construct, and because cytotoxicity was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Consistent with posttranscriptional action, knockdown of PIN1 increased the stability of iNOS protein in cycloheximide-treated cells. Furthermore, loss of iNOS was blocked by the calpain inhibitor carbobenzoxy-valinyl-phenylalaninal but not by the selective proteasome inhibitor epoxomicin. Immunoprecipitation indicated that PIN1 can interact with iNOS. Pull down of iNOS with a wild-type glutathione-S-transferase-PIN1 fusion protein, but not with a mutant of the amino terminal phospho-(serine/threonine)-proline binding WW domain of PIN1, indicated that this domain mediates interaction. The results suggest that PIN1 associates with iNOS and can limit its induction by facilitating calpain-mediated degradation in MAEC.

We isolated three recessive mutants of Arabidopsis (Arabidopsis thaliana) showing ectopic expression of the xylem-specific marker, pAtxyn3::YFP. Genetic analysis indicated that the phenotypes were caused by mutations in three different genes, designated Abnormal Tracheary Element formation-related gene expression (ate1-3). The ate1 mutants showed a normal DR5::GUS gene expression pattern, and the ate1 mutation did not affect the abnormal vascular pattern formation in the van3 and pin1 mutants, indicating that the ate1 mutation does not affect the vascular pattern organization governed by auxin. The ate mutants showed ectopic lignin deposition, patterned secondary wall thickenings, and cell death, which are characteristic of mature tracheary elements (TEs) in cells ectopically expressing the pAtxyn3::YFP gene. Ectopic TE formation was rapidly induced in parenchymal tissue of the ate mutants in a TE-inducible system with excised hypocotyl. Furthermore, reverse transcription-polymerase chain reaction experiments showed that the expression of TE formation-related genes is up-regulated in the ate mutants. The ate1 mutation also caused ectopic expression of another xylem-specific marker gene, pAt3g62160::YFP. Overall, our results suggest that the ATE genes are responsible for the in situ repression of transdifferentiation into TEs in Arabidopsis and could be participants in the transdifferentiation-masking system.

The impact of transient carbon depletion on reproductive growth in Arabidopsis was investigated by transferring long-photoperiod-grown plants to continuous darkness and returning them to a light-dark cycle. After 2 days of darkness, carbon reserves were depleted in reproductive sinks, and RNA in situ hybridization of marker transcripts showed that carbon starvation responses had been initiated in the meristem, anthers and ovules. Dark treatments of 2 or more days resulted in a bare-segment phenotype on the floral stem, with 23-27 aborted siliques. These resulted from impaired growth of immature siliques and abortion of mature and immature flowers. Depolarization of PIN1 protein and increased DII-VENUS expression pointed to rapid collapse of auxin gradients in the meristem and inhibition of primordia initiation. After transfer back to a light-dark cycle, flowers appeared and formed viable siliques and seeds. A similar phenotype was seen after transfer to sub-compensation point irradiance or CO2 . It also appeared in a milder form after a moderate decrease in irradiance and developed spontaneously in short photoperiods. We conclude that Arabidopsis inhibits primordia initiation and aborts flowers and very young siliques in C-limited conditions. This curtails demand, safeguarding meristem function and allowing renewal of reproductive growth when carbon becomes available again.

Pin1 and Par14 are parvulin-type peptidyl-prolyl cis/trans isomerases. Although numerous proteins have been identified as Pin1 substrates, the target proteins of Par14 remain largely unknown. Par14 expression levels are increased in the livers and embryonic fibroblasts of Pin1 KO mice, suggesting a compensatory relationship between the functions of Pin1 and Par14. In this study, the association of Par14 with insulin receptor substrate 1 (IRS-1) was demonstrated in HepG2 cells overexpressing both as well as endogenously in the mouse liver. The analysis using deletion-mutated Par14 and IRS-1 constructs revealed the N-terminal portion containing the basic domain of Par14 and the two relatively C-terminal portions of IRS-1 to be involved in these associations, in contrast to the WW domain of Pin1 and the SAIN domain of IRS-1. Par14 overexpression in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events, PI3K binding with IRS-1 and Akt phosphorylation. In contrast, treating HepG2 cells with Par14 siRNA suppressed these events. In addition, overexpression of Par14 in the insulin-resistant ob/ob mouse liver by adenoviral transfer significantly improved hyperglycemia with normalization of hepatic PEPCK and G6Pase mRNA levels, and gene suppression of Par14 using shRNA adenovirus significantly exacerbated the glucose intolerance in Pin1 KO mice. Therefore, although Pin1 and Par14 associate with different portions of IRS-1, the prolyl cis/trans isomerization in multiple sites of IRS-1 by these isomerases appears to be critical for efficient insulin receptor-induced IRS-1 phosphorylation. This process is likely to be one of the major mechanisms regulating insulin sensitivity and also constitutes a potential therapeutic target for novel insulin-sensitizing agents.

The cdk5/p35 complex has been implicated in a variety of functions related to brain development, including axonal outgrown and neuronal migration. In this study, by co-immunoprecipitation and pull-down experiments, we have shown that the cdk5/p35 complex associates with and phosphorylates the neuronal delta-catenin. Immunocytochemical studies of delta-catenin and the cdk5-activator p35 in primary cortical neurons indicated that these proteins co-localize in the cell body of neuronal cells. In addition, cdk5 co-localized with beta-catenin in the cell-cell contacts and plasma membrane of undifferentiated and differentiated N2A cells. In this context, we identified Ser(191) and Ser(246) on beta-catenin structure as specific phosphorylation sites for cdk5/p35 complex. Moreover, Pin1, a peptidyl-prolyl isomerase (PPIase) directly bound to both, beta- and delta-catenin, once they have been phosphorylated by the cdk5/p35 complex. Studies indicate that the cdk5/p35 protein kinase system is directly involved in the regulatory mechanisms of neuronal beta- and delta-catenin.

Phosphorylation on certain Ser/Thr-Pro motifs is a major oncogenic mechanism. The conformation and function of phosphorylated Ser/Thr-Pro motifs are further regulated by the prolyl isomerase Pin1. Pin1 has been shown to be prevalently overexpressed in human breast cancer cell lines and cancer tissues and to play a critical role in the transformation of mammary epithelial cells by activating multiple oncogenic pathways. Pin1 expression was found to be an excellent independent prognostic marker in prostate cancer. However, little is known about Pin1 and its downstream targets beta-catenin and cyclin D1 expressions in human oral cancers. In the present study, we quantified Pin1 expression in 74 paired normal/tumor human oral cancer samples as well as oral cancer cell lines. Pin1 was overexpressed in oral squamous cell carcinoma (OSCC) and its level correlated with beta-catenin accumulation and cyclin D1 expression. Moreover, we examined Pin1 mRNA expression in OSCC and cancer cell lines by RT-PCR analysis. The results showed that there is concordance in the relationship between the Pin1 mRNA level and Pin1 protein expression. The up-regulation of Pin1 mRNA expression in tumor part when comparing with that in non-tumor part was in agreement with that of the Pin1 protein overexpressed in OSCC. In addition, we showed that the molecular and immunohistochemical profiles of Pin1 overexpression were associated with progression of OSCC. Taken together, these results indicate that Pin1 is a regulator of cyclin D1 expression in OSCC and might play a role in oral oncogenesis. The overexpression of Pin1 can be used as an indicator for pathological diagnosis of OSCC.

In hormone receptor-positive breast cancers, most tumors in the early stages of development depend on the activity of the estrogen receptor and its ligand, estradiol. Anti-estrogens, such as tamoxifen, have been used as the first line of therapy for over three decades due to the fact that they elicit cell cycle arrest. Unfortunately, after an initial period, most cells become resistant to hormonal therapy. Peptidylprolyl isomerase 1 (Pin1), a protein overexpressed in many tumor types including breast, has been demonstrated to modulate ERalpha activity and is involved in resistance to hormonal therapy. Here we show a new mechanism through which CDK2 drives an ERalpha-Pin1 interaction under hormone- and growth factor-free conditions. The PI3K/AKT pathway is necessary to activate CDK2, which phosphorylates ERalphaSer294, and mediates the binding between Pin1 and ERalpha. Site-directed mutagenesis demonstrated that ERalphaSer294 is essential for Pin1-ERalpha interaction and modulates ERalpha phosphorylation on Ser118 and Ser167, dimerization and activity. These results open up new drug treatment opportunities for breast cancer patients who are resistant to anti-estrogen therapy.

BACKGROUND

The purpose of this study was to investigate the association between the peptidyl-propyl-cis/trans isomerase 1 (PIN1) -842(G>> C) and -667(T>> C) polymorphic variants and breast cancer risk among Malaysian ethnic groups namely the Malays, Chinese and Indians, as well as clinico-pathological characteristics of the patients.

METHODS

The polymerase chain reaction-restriction fragment length polymorphism was used to genotype 387 breast cancer patients and 252 normal and healthy women who had no history of any malignancy.

RESULTS

The distribution of -842(G>> C) and -667(T>> C) genotypes and alleles frequencies between breast cancer cases and normal individuals showed lack of statistical significance among the Malays (p>> 0.05), Chinese (p>> 0.05) and Indians (p>> 0.05), respectively. Multivariate logistic regression analysis showed that the Malay, Chinese and Indian women who were -842CC homozygotes (p = 0.198, 0.089, 0.620), -842GC heterozygotes (p = 0.492, 0.176, 0.377) and -842C allele carriers (P = 0.226, 0.059, 0.669), respectively, were not associated with breast cancer risk. Furthermore Malay, Chinese and Indian women who were heterozygous (p = 0.777, 0.319, 0.710) and homozygous (p = 0.864, 0.986, 0.954) for -667C allele or carriers of -667C allele (p = 0.977, 0.915, 0.880), respectively, were not associated with an increased risk of breast cancer. None of the -842C and -667C allele genotypes were significantly associated with the clinico-pathological characteristics.

CONCLUSIONS

Our findings suggest that the polymorphic variants of -842(G>> C) and -667(T>> C) genes may not appear to have an influence on breast cancer risk among Malaysian Malay, Chinese and Indian women.

The generation of vascular pattern formation in plants is an interesting process of pattern formation in organisms. It is well known that the plant hormone auxin is involved in plant vascular differentiation and that the PIN1 protein, an auxin efflux carrier, localizes to one side of the cell membrane. Several hypotheses have been proposed to explain the formation of leaf venation. One is the canalization hypothesis that is based on the assumption that a positive feedback regulation exists between the flow of a signal molecule and the capacity of its flow. Here, we attempted to integrate the canalization hypothesis and experimental data. We investigated models of the positive feedback regulation between the auxin flow and PIN1 localization. Model 1, with conserved PIN1 amount in each cell, can generate a branching pattern similar to that of plant leaf venation. We introduced the diffusible enhancer "e" into the model as unknown factor. The obtained patterns show a quasi-periodic distribution of auxin flow paths, when the model dynamics includes domain growth. In order to understand the early initiation process that generates an inhomogeneity from an almost homogeneous distribution, we introduced model 2, a simplified version of model 1. Model 2 can generate inhomogeneity with a parameter dependency similar to that of model 1. To analyse parameter condition required for pattern development, approximated equations are obtained from model 2. The isocline analysis of the equations without spatial structure shows that the inhomogeneous distribution occurs from an almost homogeneous distribution. This parameter condition for generating inhomogeneity is consistent with the results of models 1 and 2.

Mammary gland development, various stages of mammary tumorigenesis and breast cancer progression have the peptidyl-prolyl cis/trans isomerase PIN1 at their centerpiece, in virtue of the ability of this unique enzyme to fine-tune the dynamic crosstalk between multiple molecular pathways. PIN1 exerts its action by inducing conformational and functional changes on key cellular proteins, following proline-directed phosphorylation. Through this post-phosphorylation signal transduction mechanism, PIN1 controls the extent and direction of the cellular response to a variety of inputs, in physiology and disease. This review discusses PIN1's roles in normal mammary development and cancerous progression, as well as the clinical impact of targeting this enzyme in breast cancer patients.

Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes.