Oncogenes Neu/HER2/ErbB2 and Ras can induce mammary tumorigenesis via upregulation of cyclin D1. One major regulatory mechanism in these oncogenic signaling pathways is phosphorylation of serines or threonines preceding proline (pSer/Thr-Pro). Interestingly, the pSer/Thr-Pro motifs in proteins exist in two completely distinct cis and trans conformations, whose conversion is catalyzed specifically by the essential prolyl isomerase Pin1. By isomerizing pSer/Thr-Pro bonds, Pin1 can regulate the conformation and function of certain phosphorylated proteins. We have previously shown that Pin1 is overexpressed in breast tumors and positively regulates cyclin D1 by transcriptional activation and posttranslational stabilization. Moreover, in Pin1 knockout mice, mammary epithelial cells fail to undergo massive proliferation during pregnancy, as is the case in cyclin D1 null mice. These results indicate that Pin1 is upregulated in breast cancer and may be involved in mammary tumors. However, the mechanism of Pin1 overexpression in cancer and its significance in cell transformation remain largely unknown. Here we demonstrate that PIN1 expression is mediated by the transcription factor E2F and enhanced by c-Neu and Ha-Ras via E2F. Furthermore, overexpression of Pin1 not only confers transforming properties on mammary epithelial cells but also enhances the transformed phenotypes of Neu/Ras-transformed mammary epithelial cells. In contrast, inhibition of Pin1 suppresses Neu- and Ras-induced transformed phenotypes, which can be fully rescued by overexpression of a constitutively active cyclin D1 mutant that is refractory to the Pin1 inhibition. Thus, Pin1 is an E2F target gene that is essential for the Neu/Ras-induced transformation of mammary epithelial cells through activation of cyclin D1.

Plants have many polarized cell types, but relatively little is known about the mechanisms that establish polarity. The orc mutant was identified originally by defects in root patterning, and positional cloning revealed that the affected gene encodes STEROL METHYLTRANSFERASE1, which is required for the appropriate synthesis and composition of major membrane sterols. smt1(orc) mutants displayed several conspicuous cell polarity defects. Columella root cap cells revealed perturbed polar positioning of different organelles, and in the smt1(orc) root epidermis, polar initiation of root hairs was more randomized. Polar auxin transport and expression of the auxin reporter DR5-beta-glucuronidase were aberrant in smt1(orc). Patterning defects in smt1(orc) resembled those observed in mutants of the PIN gene family of putative auxin efflux transporters. Consistently, the membrane localization of the PIN1 and PIN3 proteins was disturbed in smt1(orc), whereas polar positioning of the influx carrier AUX1 appeared normal. Our results suggest that balanced sterol composition is a major requirement for cell polarity and auxin efflux in Arabidopsis.

Auxin transport is mediated at the cellular level by three independent mechanisms that are characterised by the PIN-formed (PIN), P-glycoprotein (ABCB/PGP) and AUX/LAX transport proteins. The PIN and ABCB transport proteins, best represented by PIN1 and ABCB19 (PGP19), have been shown to coordinately regulate auxin efflux. When PIN1 and ABCB19 coincide on the plasma membrane, their interaction enhances the rate and specificity of auxin efflux and the dynamic cycling of PIN1 is reduced. However, ABCB19 function is not regulated by the dynamic cellular trafficking mechanisms that regulate PIN1 in apical tissues, as localisation of ABCB19 on the plasma membrane was not inhibited by short-term treatments with latrunculin B, oryzalin, brefeldin A (BFA) or wortmannin--all of which have been shown to alter PIN1 and/or PIN2 plasma membrane localisation. When taken up by endocytosis, the styryl dye FM4-64 labels diffuse rather than punctuate intracellular bodies in abcb19 (pgp19), and some aggregations of PIN1 induced by short-term BFA treatment did not disperse after BFA washout in abcb19. Although the subcellular localisations of ABCB19 and PIN1 in the reciprocal mutant backgrounds were like those in wild type, PIN1 plasma membrane localisation in abcb19 roots was more easily perturbed by the detergent Triton X-100, but not other non-ionic detergents. ABCB19 is stably associated with sterol/sphingolipid-enriched membrane fractions containing BIG/TIR3 and partitions into Triton X-100 detergent-resistant membrane (DRM) fractions. In the wild type, PIN1 was also present in DRMs, but was less abundant in abcb19 DRMs. These observations suggested a rationale for the observed lack of auxin transport activity when PIN1 is expressed in a non-plant heterologous system. PIN1 was therefore expressed in Schizosaccharomyces pombe, which has plant-like sterol-enriched microdomains, and catalysed auxin transport in these cells. These data suggest that ABCB19 stabilises PIN1 localisation at the plasma membrane in discrete cellular subdomains where PIN1 and ABCB19 expression overlaps.

Plants continuously extend their root and shoot systems through the action of meristems at their growing tips. By regulating which meristems are active, plants adjust their body plans to suit local environmental conditions. The transport network of the phytohormone auxin has been proposed to mediate this systemic growth coordination, due to its self-organising, environmentally sensitive properties. In particular, a positive feedback mechanism termed auxin transport canalization, which establishes auxin flow from active shoot meristems (auxin sources) to the roots (auxin sinks), has been proposed to mediate competition between shoot meristems and to balance shoot and root growth. Here we provide strong support for this hypothesis by demonstrating that a second hormone, strigolactone, regulates growth redistribution in the shoot by rapidly modulating auxin transport. A computational model in which strigolactone action is represented as an increase in the rate of removal of the auxin export protein, PIN1, from the plasma membrane can reproduce both the auxin transport and shoot branching phenotypes observed in various mutant combinations and strigolactone treatments, including the counterintuitive ability of strigolactones either to promote or inhibit shoot branching, depending on the auxin transport status of the plant. Consistent with this predicted mode of action, strigolactone signalling was found to trigger PIN1 depletion from the plasma membrane of xylem parenchyma cells in the stem. This effect could be detected within 10 minutes of strigolactone treatment and was independent of protein synthesis but dependent on clathrin-mediated membrane trafficking. Together these results support the hypothesis that growth across the plant shoot system is balanced by competition between shoot apices for a common auxin transport path to the root and that strigolactones regulate shoot branching by modulating this competition.

In contrast to FK506 binding proteins and cyclophilins, the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases; E.C. 5.2.1.8) cannot be inhibited by either FK506 or cyclosporin A. We have found that juglone, 5-hydroxy-1,4-naphthoquinone, irreversibly inhibits the enzymatic activity of several parvulins, like the E. coli parvulin, the yeast Ess1/Ptf1, and human Pin1, in a specific manner, thus allowing selective inactivation of these enzymes in the presence of other PPIases. The mode of action was studied by analyzing the inactivation kinetics and the nature of products of the reaction of E. coli parvulin and its Cys69Ala variant with juglone. For all parvulins investigated, complete inactivation was obtained by a slow process that is characterized by pseudo-first-order rate constants in the range of 5.3 x 10(-)4 to 4. 5 x 10(-)3 s-1. The inactivated parvulin contains two juglone molecules that are covalently bound to the side chains of Cys41 and Cys69 because of a Michael addition of the thiol groups to juglone. Redox reactions did not contribute to the inactivation process. Because thiol group modification was shown to proceed 5-fold faster than the rate of enzyme inactivation, it was considered as a necessary but not sufficient condition for inactivation. When measured by far-UV circular dichroism (CD), the rate of structural alterations following thiol group modification parallels exactly the rate of inactivation. Thus, partial unfolding of the active site of the parvulins was thought to be the cause of the deterioration of PPIase activity.

Bim, a "BH3-only" protein, is expressed de novo following withdrawal of serum survival factors and promotes cell death. We have shown previously that activation of the ERK1/2 pathway promotes phosphorylation of Bim(EL), targeting it for degradation via the proteasome. However, the nature of the kinase responsible for Bim(EL) phosphorylation remained unclear. We now show that Bim(EL) is phosphorylated on at least three sites in response to activation of the ERK1/2 pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK1/2-dependent phosphorylation of Bim(EL) occurs at (S/T)P motifs. ERK1/2 phosphorylates Bim(EL), but not Bim(S) or Bim(L), in vitro, and mutation of Ser(65) to alanine blocks the phosphorylation of Bim(EL) by ERK1/2 in vitro and in vivo and prevents the degradation of the protein following activation of the ERK1/2 pathway. We also find that ERK1/2, but not JNK, can physically associate with GST-Bim(EL), but not GST-Bim(L) or GST-Bim(S), in vitro. ERK1/2 also binds to full-length Bim(EL) in vivo, and we have localized a potential ERK1/2 "docking domain" lying within a 27-amino acid stretch of the Bim(EL) protein. Our findings provide new insights into the post-translational regulation of Bim(EL) and the role of the ERK1/2 pathway in cell survival signaling.

A key feature of plants (as opposed to animals) is their ability to establish new organs not only during embryogenesis, but also throughout their development. A master regulator of organ initiation in plants is the phytohormone auxin. Auxin acts locally as a morphogen and is directionally transported from cell to cell by polarized auxin efflux carriers, termed PIN-FORMED (PIN) proteins. Here we report that the Arabidopsis ortholog of the yeast and mammalian vacuolar protein sorting 29 (VPS29), a member of the retromer complex, mediates the formation of new axes of development. Furthermore, we show that VPS29 is required for endosome homeostasis, PIN protein cycling, and dynamic PIN1 repolarization during development. We propose a model that links VPS29 function, PIN1 polarity, and organ initiation in plants.

Embryo patterning in Arabidopsis thaliana is highly affected when KANADI or Class III HD-Zip genes are compromised. Triple loss-of-function kan1 kan2 kan4 embryos exhibit striking defects in the peripheral-central axis, developing lateral leaf-like organs from the hypocotyls, whereas loss of Class III HD-Zip gene activity results in a loss of bilateral symmetry. Loss of KANADI activity in a Class III HD-Zip mutant background mitigates the defects in bilateral symmetry, implying that the two gene families act antagonistically during embryonic pattern formation. Dynamic patterns of auxin concentration and flux contribute to embryo patterning. Polar cellular distribution of PIN-FORMED1 (PIN1) mediates auxin flow throughout embryogenesis and is required for establishment of the apical-basal axis and bilateral symmetry. Defects in the pattern of PIN1 expression are evident when members of either the KANADI or Class III HD-Zip gene families are compromised. Abnormal expression patterns of PIN1 in KANADI or Class III HD-Zip multiple mutants and the phenotype of plants in which members of both gene families are mutated suggest that pattern formation along the central-peripheral axis results from interplay between auxin and the KANADI and Class III HD-Zip transcription factors, whose defined spatial and temporal expression patterns may also be influenced by auxin.

PIN1 is a peptidyl-prolyl isomerase that can alter the conformation of phosphoproteins and so affect protein function and/or stability. PIN1 regulates a number of proteins important for cell-cycle progression and, based on gain- and loss-of-function studies, is presumed to operate as a molecular timer of this important process. Therefore, it seems logical that alterations in the level of PIN1 can influence hyperproliferative diseases such as cancer. However, the precise role of PIN1 in cancer remains controversial.

The B-Raf kinase is a Ras pathway effector activated by mutation in numerous human cancers and certain developmental disorders. Here we report that normal and oncogenic B-Raf proteins are subject to a regulatory cycle of extracellular signal-regulated kinase (ERK)-dependent feedback phosphorylation, followed by PP2A- and Pin1-dependent dephosphorylation/recycling. We identify four S/TP sites of B-Raf phosphorylated by activated ERK and find that feedback phosphorylation of B-Raf inhibits binding to activated Ras and disrupts heterodimerization with C-Raf, which is dependent on the B-Raf pS729/14-3-3 binding site. Moreover, we find that events influencing Raf heterodimerization can alter the transforming potential of oncogenic B-Raf proteins possessing intermediate or impaired kinase activity but have no significant effect on proteins with high kinase activity, such as V600E B-Raf. Mutation of the feedback sites or overexpression of the Pin1 prolyl-isomerase, which facilitates B-Raf dephosphorylation/recycling, resulted in increased transformation, whereas mutation of the S729/14-3-3 binding site or expression of dominant negative Pin1 reduced transformation. Mutation of each feedback site caused increased transformation and correlated with enhanced heterodimerization and activation of C-Raf. Finally, we find that B-Raf and C-Raf proteins containing mutations identified in certain developmental disorders constitutively heterodimerize and that their signaling activity can also be modulated by feedback phosphorylation.

The 2.7 A structure of Candida albicans RNA guanylyltransferase Cgt1 cocrystallized with a carboxy-terminal domain (CTD) peptide composed of four Ser5-PO4 YSPTSPS heptad repeats illuminates distinct CTD-docking sites localized to the Cgt1 N-terminal nucleotidyl transferase domain. Tyr1, Pro3, Pro6, and Ser5-PO4 side chains from each of two YSPTSPS repeats contribute to the interface. Comparison to the Pin1-CTD structure shows that the CTD can assume markedly different conformations that are templated by particular binding partners. Structural plasticity combined with remodeling of CTD primary structure by kinases and phosphatases provides a versatile mechanism by which the CTD can recruit structurally dissimilar proteins during transcription. A binding site for the RNA triphosphatase component of the capping apparatus was also uncovered within the Cgt1 OB domain.

When directed to the nucleus by TGF-β or BMP signals, Smad proteins undergo cyclin-dependent kinase 8/9 (CDK8/9) and glycogen synthase kinase-3 (GSK3) phosphorylations that mediate the binding of YAP and Pin1 for transcriptional action, and of ubiquitin ligases Smurf1 and Nedd4L for Smad destruction. Here we demonstrate that there is an order of events-Smad activation first and destruction later-and that it is controlled by a switch in the recognition of Smad phosphoserines by WW domains in their binding partners. In the BMP pathway, Smad1 phosphorylation by CDK8/9 creates binding sites for the WW domains of YAP, and subsequent phosphorylation by GSK3 switches off YAP binding and adds binding sites for Smurf1 WW domains. Similarly, in the TGF-β pathway, Smad3 phosphorylation by CDK8/9 creates binding sites for Pin1 and GSK3, then adds sites to enhance Nedd4L binding. Thus, a Smad phosphoserine code and a set of WW domain code readers provide an efficient solution to the problem of coupling TGF-β signal delivery to turnover of the Smad signal transducers.

The Arabidopsis GNOM gene encodes an ARF GDP/GTP exchange factor involved in embryonic axis formation and polar localisation of the auxin efflux regulator PIN1. To examine whether GNOM also plays a role in post-embryonic development and to clarify its involvement in auxin transport, we have characterised newly isolated weak gnom alleles as well as trans-heterozygotes of complementing strong alleles. These genotypes form a phenotypic series of GNOM activity in post-embryonic development, with auxin-related defects, especially in the maintenance of primary root meristem activity and in the initiation and organisation of lateral root primordia. Our results suggest a model for GNOM action mediating auxin transport in both embryogenesis and post-embryonic organ development.

Protein folding barriers result from a combination of factors including unavoidable energetic frustration from nonnative interactions, natural variation and selection of the amino acid sequence for function, and/or selection pressure against aggregation. The rate-limiting step for human Pin1 WW domain folding is the formation of the loop 1 substructure. The native conformation of this six-residue loop positions side chains that are important for mediating protein-protein interactions through the binding of Pro-rich sequences. Replacement of the wild-type loop 1 primary structure by shorter sequences with a high propensity to fold into a type-I' beta-turn conformation or the statistically preferred type-I G1 bulge conformation accelerates WW domain folding by almost an order of magnitude and increases thermodynamic stability. However, loop engineering to optimize folding energetics has a significant downside: it effectively eliminates WW domain function according to ligand-binding studies. The energetic contribution of loop 1 to ligand binding appears to have evolved at the expense of fast folding and additional protein stability. Thus, the two-state barrier exhibited by the wild-type human Pin1 WW domain principally results from functional requirements, rather than from physical constraints inherent to even the most efficient loop formation process.

Although it is known that proteins are delivered to and recycled from the plasma membrane (PM) via endosomes, the nature of the compartments and pathways responsible for cargo and vesicle sorting and cellular signaling is poorly understood. To define and dissect specific recycling pathways, chemical effectors of proteins involved in vesicle trafficking, especially through endosomes, would be invaluable. Thus, we identified chemicals affecting essential steps in PM/endosome trafficking, using the intensely localized PM transport at the tips of germinating pollen tubes. The basic mechanisms of this localized growth are likely similar to those of non-tip growing cells in seedlings. The compound endosidin 1 (ES1) interfered selectively with endocytosis in seedlings, providing a unique tool to dissect recycling pathways. ES1 treatment induced the rapid agglomeration of the auxin translocators PIN2 and AUX1 and the brassinosteroid receptor BRI1 into distinct endomembrane compartments termed "endosidin bodies"; however, the markers PIN1, PIN7, and other PM proteins were unaffected. Endosidin bodies were defined by the syntaxin SYP61 and the V-ATPase subunit VHA-a1, two trans-Golgi network (TGN)/endosomal proteins. Interestingly, brassinosteroid (BR)-induced gene expression was inhibited by ES1 and treated seedlings displayed a brassinolide (BL)-insensitive phenotype similar to a bri1 loss-of-function mutant. No effect was detected in auxin signaling. Thus, PIN2, AUX1, and BRI1 use interactive pathways involving an early SYP61/VHA-a1 endosomal compartment.

To understand how auxin regulates root growth, we quantified cell division and elemental elongation, and examined actin organization in the primary root of Arabidopsis thaliana. In treatments for 48 h that inhibited root elongation rate by 50%, we find that auxins and auxin-transport inhibitors can be divided into two classes based on their effects on cell division, elongation and actin organization. Indole acetic acid (IAA), 1-naphthalene acetic acid (NAA) and tri-iodobenzoic acid (TIBA) inhibit root growth primarily through reducing the length of the growth zone rather than the maximal rate of elemental elongation and they do not reduce cell production rate. These three compounds have little effect on the extent of filamentous actin, as imaged in living cells or by chemical fixation and immuno-cytochemistry, but tend to increase actin bundling. In contrast, 2,4-dichlorophenoxy-acetic acid (2,4-D) and naphthylphthalamic acid (NPA) inhibit root growth primarily by reducing cell production rate. These compounds remove actin and slow down cytoplasmic streaming, but do not lead to mislocalization of the auxin-efflux proteins, PIN1 or PIN2. The effects of 2,4-D and NPA were mimicked by the actin inhibitor, latrunculin B. The effects of these compounds on actin were also elicited by a 2 h treatment at higher concentration but were not seen in two mutants, eir1-1 and aux1-7, with deficient auxin transport. Our results show that IAA regulates the size of the root elongation zone whereas 2,4-D affects cell production and actin-dependent processes; and, further, that elemental elongation and localization of PINs are appreciably independent of actin.

Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized. The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion. Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings. Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport. We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow. Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.

Multiple small molecule hormones contribute to growth promotion or restriction in plants. Brassinosteroids (BRs), acting specifically in the epidermis, can both drive and restrict shoot growth. However, our knowledge of how BRs affect meristem size is scant. Here, we study the root meristem and show that BRs are required to maintain normal cell cycle activity and cell expansion. These two processes ensure the coherent gradient of cell progression, from the apical to the basal meristem. In addition, BR activity in the meristem is not accompanied by changes in the expression level of the auxin efflux carriers PIN1, PIN3 and PIN7, which are known to control the extent of mitotic activity and differentiation. We further demonstrate that BR signaling in the root epidermis and not in the inner endodermis, quiescent center (QC) cells or stele cell files is sufficient to control root meristem size. Interestingly, expression of the QC and the stele-enriched MADS-BOX gene AGL42 can be modulated by BRI1 activity solely in the epidermis. The signal from the epidermis is probably transmitted by a different component than BES1 and BZR1 transcription factors, as their direct targets, such as DWF4 and BRox2, are regulated in the same cells that express BRI1. Taken together, our study provides novel insights into the role of BRs in controlling meristem size.

Genetic evidence links the Arabidopsis MONOPTEROS (MP) and PIN-FORMED1 (PIN1) genes to the patterning of leaf veins. To elucidate their potential functions and interactions in this process, we have assessed the dynamics of MP and PIN1 expression during vascular patterning in Arabidopsis leaf primordia. Both genes undergo a dynamic process of gradual refinement of expression into files one to two cells wide before overt vascular differentiation. The subcellular distribution of PIN1 is also gradually refined from a non-polar distribution in isodiametric cells to strongly polarized in elongated procambial cells and provides an indication of overall directions of auxin flow. We found evidence that MP expression can be activated by auxin exposure and that PIN1 as well as DR5::GUS expression is defective in mp mutant leaves. Taken together the results suggest a feedback regulatory loop that involves auxin, MP and PIN1 and provide novel experimental support for the canalization-of-auxin-flow hypothesis.

Cytokinin is an important regulator of plant growth and development. In Arabidopsis thaliana, the two-component phosphorelay mediated through a family of histidine kinases and response regulators is recognized as the principal cytokinin signal transduction mechanism activating the complex transcriptional response to control various developmental processes. Here, we identified an alternative mode of cytokinin action that uses endocytic trafficking as a means to direct plant organogenesis. This activity occurs downstream of known cytokinin receptors but through a branch of the cytokinin signaling pathway that does not involve transcriptional regulation. We show that cytokinin regulates endocytic recycling of the auxin efflux carrier PINFORMED1 (PIN1) by redirecting it for lytic degradation in vacuoles. Stimulation of the lytic PIN1 degradation is not a default effect for general downregulation of proteins from plasma membranes, but a specific mechanism to rapidly modulate the auxin distribution in cytokinin-mediated developmental processes.

DNA damage leads to stabilization and accumulation of p53, which plays a pivotal role in transcriptional activation of p21 and cell cycle arrest. The increase in p53 stability depends critically on its phosphorylation on serine/threonine residues, including those preceding a proline (Ser(P)/Thr-Pro). The Ser(P)/Thr-Pro moiety exists in the two distinct cis and trans conformations and their conversion is catalyzed specifically by the prolyl isomerase Pin1. Pin1 regulates the conformation and function of certain phosphorylated proteins and plays an important role in cell cycle regulation, oncogenesis, and Alzheimer's disease. However, nothing is known about the role of Pin1 in DNA damage. Here we found that DNA damage enhanced the interaction between Pin1 and p53, which depended on the WW domain in Pin1 and Ser(33/46)-Pro motifs in p53. Furthermore, Pin1 regulates the stability of p53 and its transcriptional activity toward the p21 promoter. As a result, p53 and p21 barely increased after DNA damage in Pin1 knock-out embryonic fibroblasts or in neoplastic cells depleted of Pin1. Moreover, Pin1 null cells displayed significant defects in cell cycle checkpoints induced by DNA damage. These results demonstrate a new role of Pin1 in regulating p53 function during DNA damage.

In seed plants, leaves are born on radial shoots, but unlike shoots, they are determinate dorsiventral organs made of flat lamina. YABBY genes are found only in seed plants and in all cases studied are expressed primarily in lateral organs and in a polar manner. Despite their simple expression, Arabidopsis thaliana plants lacking all YABBY gene activities have a wide range of morphological defects in all lateral organs as well as the shoot apical meristem (SAM). Here, we show that leaves lacking all YABBY activities are initiated as dorsiventral appendages but fail to properly activate lamina programs. In particular, the activation of most CINCINNATA-class TCP genes does not commence, SAM-specific programs are reactivated, and a marginal leaf domain is not established. Altered distribution of auxin signaling and the auxin efflux carrier PIN1, highly reduced venation, initiation of multiple cotyledons, and gradual loss of the SAM accompany these defects. We suggest that YABBY functions were recruited to mold modified shoot systems into flat plant appendages by translating organ polarity into lamina-specific programs that include marginal auxin flow and activation of a maturation schedule directing determinate growth.

Alzheimer disease (AD) is characterized neuropathologically by intracellular neurofibrillary tangles (NFT) and of extracellular senile plaques (SP), the central core of which is amyloid beta-peptide (Abeta) derived from amyloid precursor protein (APP), a transmembrane protein. AD brain has been reported to be under oxidative stress that may play an important role in the pathogenesis and progression of AD. The present proteomics study is focused on identification of a specific target of protein oxidation in AD hippocampus that has relevance to the role of oxidative stress in AD. Here, we report that the protein, Pin1, is significantly down-regulated and oxidized in AD hippocampus. The identity of Pin1 was confirmed immunochemically. Analysis of Pin1 activity in AD brain and separately as oxidized pure Pin1 demonstrated that oxidation of Pin1 led to loss of activity. Pin1 has been implicated in multiple aspects of cell cycle regulation and dephosphorylation of tau protein as well as in AD. The in vivo oxidative modification of Pin1 as found by proteomics in AD hippocampus in the present study suggests that oxidative modification may be related to the known loss of Pin1 isomerase activity that could be crucial in AD neurofibrillary pathology. Taken together, these results provide evidence supporting a direct link between oxidative damage to neuronal Pin1 and the pathobiology of AD.

cis-trans isomerization of proteins phosphorylated by proline-directed kinases is proposed to control numerous signaling molecules and is implicated in the pathogenesis of Alzheimer's and other diseases. However, there is no direct evidence for the existence of cis-trans protein isomers in vivo or for their conformation-specific function or regulation. Here we develop peptide chemistries that allow the generation of cis- and trans-specific antibodies and use them to raise antibodies specific for isomers of phosphorylated tau. cis, but not trans, p-tau appears early in the brains of humans with mild cognitive impairment, accumulates exclusively in degenerated neurons, and localizes to dystrophic neurites during Alzheimer's progression. Unlike trans p-tau, the cis isomer cannot promote microtubule assembly, is more resistant to dephosphorylation and degradation, and is more prone to aggregation. Pin1 converts cis to trans p-tau to prevent Alzheimer's tau pathology. Isomer-specific antibodies and vaccines may therefore have value for the early diagnosis and treatment of Alzheimer's disease.