Ubiquitin is a peptide modifier able to form polymers of varying length and linkage as part of a powerful signaling system. Perhaps the best-known aspect of this protein's function is as the driver of targeted protein degradation through the Ubiquitin Proteasome System (UPS). Through the formation of lysine 48-linked polyubiquitin chains, it is able to direct the degradation of tagged proteins by the 26S proteasome, indirectly controlling many processes within the cell. However, recent research has indicated that ubiquitin performs a multitude of other roles within the cell beyond protein degradation. It is able to form 6 other "atypical" linkages though lysine residues at positions 6, 11, 27, 29, 33, and 63. These atypical chains perform a range of diverse functions, including the regulation of iron uptake in response to perceived deficiency, repair of double stranded breaks in the DNA, and regulation of the auxin response through the non-proteasomal degradation of auxin efflux carrier protein PIN1. This review explores the role ubiquitin chain topology plays in plant cellular function. We aim to highlight the importance of these varying functions and the future challenges to be encountered within this field.

The neuronal cytoskeleton is tightly regulated by phosphorylation and dephosphorylation reactions mediated by numerous associated kinases, phosphatases and their regulators. Defects in the relative kinase and phosphatase activities and/or deregulation of compartment-specific phosphorylation result in neurodegenerative disorders. The largest family of cytoskeletal proteins in mammalian cells is the superfamily of intermediate filaments (IFs). The neurofilament (NF) proteins are the major IFs. Aggregated forms of hyperphosphorylated tau and phosphorylated NFs are found in pathological cell body accumulations in the central nervous system of patients suffering from Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. The precise mechanisms for this compartment-specific phosphorylation of cytoskeletal proteins are not completely understood. In this review, we focus on the mechanisms of neurofilament phosphorylation in normal physiology and neurodegenerative diseases. We also address the recent breakthroughs in our understanding the role of different kinases and phosphatases involved in regulating the phosphorylation status of the NFs. In addition, special emphasis has been given to describe the role of phosphatases and Pin1 in phosphorylation of NFs.

Sterols are essential molecules for multiple biological processes, including embryogenesis, cell elongation, and endocytosis. The plant sterol biosynthetic pathway is unique in the involvement of two distinct sterol 4α-methyl oxidase (SMO) families, SMO1 and SMO2, which contain three and two isoforms, respectively, and are involved in sequential removal of the two methyl groups at C-4. In this study, we characterized the biological functions of members of the SMO2 gene family. SMO2-1 was strongly expressed in most tissues during Arabidopsis (Arabidopsis thaliana) development, whereas SMO2-2 showed a more specific expression pattern. Although single smo2 mutants displayed no obvious phenotype, the smo2-1 smo2-2 double mutant was embryonic lethal, and the smo2-1 smo2-2/+ mutant was dwarf, whereas the smo2-1/+ smo2-2 mutant exhibited a moderate phenotype. The phenotypes of the smo2 mutants resembled those of auxin-defective mutants. Indeed, the expression of DR5rev:GFP, an auxin-responsive reporter, was reduced and abnormal in smo2-1 smo2-2 embryos. Furthermore, the expression and subcellular localization of the PIN1 auxin efflux facilitator also were altered. Consistent with these observations, either the exogenous application of auxin or endogenous auxin overproduction (YUCCA9 overexpression) partially rescued the smo2-1 smo2-2 embryonic lethality. Surprisingly, the dwarf phenotype of smo2-1 smo2-2/+ was completely rescued by YUCCA9 overexpression. Gas chromatography-mass spectrometry analysis revealed a substantial accumulation of 4α-methylsterols, substrates of SMO2, in smo2 heterozygous double mutants. Together, our data suggest that SMO2s are important for correct sterol composition and function partially through effects on auxin accumulation, auxin response, and PIN1 expression to regulate Arabidopsis embryogenesis and postembryonic development.

The cis/trans isomerization of the peptide bond preceding proline is an intrinsically slow process, although important in many biological processes in both prokaryotes and eukaryotes. In vivo, this isomerization is catalyzed by peptidyl-prolyl cis/trans-isomerases (PPIases). Here, we present the molecular and biochemical characterization of parvulin-type PPIase family members of the model legume Lotus japonicus, annotated as LjPar1, LjPar2, and LjPar3. Although LjPar1 and LjPar2 were found to be homologous to PIN1 (Protein Interacting with NIMA)-type parvulins and hPar14 from human, respectively, LjPar3 represents a novel multidomain parvulin, apparently present only in plants, that contains an active carboxyl-terminal sulfurtransferase domain. All Lotus parvulins were heterologously expressed and purified from Escherichia coli, and purified protein verification measurements used a liquid chromatography-mass spectrometry-based proteomic method. The biochemical characterization of the recombinant Lotus parvulins revealed that they possess PPIase activity toward synthetic tetrapeptides, although they exhibited different substrate specificities depending on the amino acid amino terminal to proline. These differences were also studied in a structural context using molecular modeling of the encoded polypeptides. Real-time reverse transcription-polymerase chain reaction revealed that the three parvulin genes of Lotus are ubiquitously expressed in all plant organs. LjPar1 was found to be up-regulated during the later stages of nodule development. Subcellular localization of LjPar-enhanced Yellow Fluorescence Protein (eYFP) fusions expressed in Arabidopsis (Arabidopsis thaliana) leaf epidermal cells revealed that LjPar1- and LjPar2-eYFP fusions were localized in the cytoplasm and in the nucleus, in contrast to LjPar3-eYFP, which was clearly localized in plastids. Divergent substrate specificities, expression profiles, and subcellular localization indicate that plant parvulin-type PPIases are probably involved in a wide range of biochemical and physiological processes.

In contrast with the wealth of recent reports about the function of μ-adaptins and clathrin adaptor protein (AP) complexes, there is very little information about the motifs that determine the sorting of membrane proteins within clathrin-coated vesicles in plants. Here, we investigated putative sorting signals in the large cytosolic loop of the Arabidopsis (Arabidopsis thaliana) PIN-FORMED1 (PIN1) auxin transporter, which are involved in binding μ-adaptins and thus in PIN1 trafficking and localization. We found that Phe-165 and Tyr-280, Tyr-328, and Tyr-394 are involved in the binding of different μ-adaptins in vitro. However, only Phe-165, which binds μA(μ2)- and μD(μ3)-adaptin, was found to be essential for PIN1 trafficking and localization in vivo. The PIN1:GFP-F165A mutant showed reduced endocytosis but also localized to intracellular structures containing several layers of membranes and endoplasmic reticulum (ER) markers, suggesting that they correspond to ER or ER-derived membranes. While PIN1:GFP localized normally in a μA (μ2)-adaptin mutant, it accumulated in big intracellular structures containing LysoTracker in a μD (μ3)-adaptin mutant, consistent with previous results obtained with mutants of other subunits of the AP-3 complex. Our data suggest that Phe-165, through the binding of μA (μ2)- and μD (μ3)-adaptin, is important for PIN1 endocytosis and for PIN1 trafficking along the secretory pathway, respectively.

BACKGROUND

Obstructive jaundice (OJ) is an important clinical consideration associated with a high risk of bacteremia. Hepatocyte nuclear factor-kappa B (NF-kappaB) activation confers an antiapoptotic function. Although the occurrence of hepatocyte apoptosis has been shown in OJ, the activation and role of NF-kappaB over the time course of OJ in conjunction with endotoxemia have not yet been well defined. We hypothesized that NF-kappaB activation may be decreased over the time course of OJ and endotoxemia, which leads to severe liver injury. The aim of the current study was to examine whether NF-kappaB activation can decrease hepatocyte apoptosis and liver injury over the time course of OJ in response to lipopolysaccharide (LPS) administration.

METHODS

Male C57BL/6 mice were subjected to bile duct ligation and were administered LPS intravenously at 3 days (OJ3) or 14 days (OJ14) after bile duct ligation. NF-kappaB activation; protein expressions of NF-kappaB p65, IkappaB-alpha, Ikappabeta-b, and Pin1; immunohistochemistry of poly adenosine diphosphate (ADP)-ribose polymerase p85 fragment (PARP); and serum alanine transaminase (ALT) levels were examined.

RESULTS

Hepatocyte NF-kappaB activation was observed during OJ. After LPS administration, the hepatic NF-kappaB activation defined by electrophoretic mobility shift assay was decreased in the OJ14 group compared with the OJ3 group, which is consistent with a decrease in NF-kappaB p65 protein expression. Changes in phosphorylated Ikappa-B-beta but not phosphorylated IkappaB-alpha mirrored these results. Significant hepatocyte apoptosis defined by PARP immunohistochemistry was observed in the LPS-treated OJ14 relative to the LPS-treated OJ3. Hepatic expressions of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the LPS OJ14 mice were upregulated relative to those in the LPS OJ3. Serum ALT levels increased significantly in the LPS OJ14 relative to other mice. The survival rate was significantly less in the LPS OJ14 relative to other mice.

CONCLUSIONS

After prolonged OJ, exposure to endotoxemia was associated with a decrease in hepatocyte NF-kappaB activation and an increase in hepatocyte apoptosis and secondary necrosis, thus resulting in liver dysfunction.

Inflammatory responses and osteoclast differentiation play pivotal roles in the pathogenesis of osteolytic bone diseases such as periodontitis. Although overexpression or inhibition of peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (PIN1) offers a possible therapeutic strategy for chronic inflammatory diseases, the role of PIN1 in periodontal disease is unclear. The aim of the present study was to evaluate PIN1 expression in periodontitis patients as well as the effects of PIN1 inhibition by juglone or PIN1 small-interfering RNA (siRNA) and of PIN1 overexpression using a recombinant adenovirus encoding PIN1 (Ad-PIN1) on the inflammatory response and osteoclastic differentiation in lipopolysaccharide (LPS)- and nicotine-stimulated human periodontal ligament cells (PDLCs). PIN1 was up-regulated in chronically inflamed PDLCs from periodontitis patients and in LPS- and nicotine-exposed PDLCs. Inhibition of PIN1 by juglone or knockdown of PIN1 gene expression by siRNA markedly attenuated LPS- and nicotine-stimulated prostaglandin E2 (PGE2) and nitric oxide (NO) production, as well as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, whereas PIN1 overexpression by Ad-PIN1 increased it. LPS- and nicotine-induced nuclear factor (NF)-κB activation was blocked by juglone and PIN1 siRNA but increased by Ad-PIN1. Conditioned medium prepared from LPS- and nicotine-treated PDLCs increased the number of tartrate-resistant acid phosphatase-stained osteoclasts and osteoclast-specific gene expression. These responses were blocked by PIN1 inhibition and silencing but stimulated by Ad-PIN1. Furthermore, juglone and PIN1 siRNA inhibited LPS- and nicotine-induced osteoclastogenic cytokine expression in PDLCs. This study is the first to demonstrate that PIN1 inhibition exhibits anti-inflammatory effects and blocks osteoclastic differentiation in LPS- and nicotine-treated PDLCs. PIN1 inhibition may be a therapeutic strategy for inflammatory osteolysis in periodontal disease.

The membrane disruption mechanism of pandinin 1 (pin1), an antimicrobial peptide isolated from the venom of the African scorpion, was studied using 31P, 13C, 1H solid-state and multidimensional solution-state NMR spectroscopy. A high-resolution NMR solution structure of pin1 showed that the two distinct alpha-helical regions move around the central hinge region, which contains Pro19. 31P NMR spectra of lipid membrane in the presence of pin1, at various temperatures, showed that pin1 induces various lipid phase behaviors depending on the acyl chain length and charge of phospholipids. Notably, it was found that pin1 induced formation of the cubic phase in shorter lipid membranes above Tm. Further, the 13C NMR spectra of pin1 labeled at Leu28 under magic angle spinning (MAS) indicated that the motion of pin1 bound to the lipid bilayer was very slow, with a correlation time of the order of 10(-3) s. 31P NMR spectra of dispersions of four saturated phosphatidyl-cholines in the presence of three types of pin1 derivatives, [W4A, W6A, W15A]-pin1, pin1(1-18), and pin1(20-44), at various temperatures demonstrated that all three pin1 derivatives have a reduced ability to trigger the cubic phase. 13C chemical shift values for pin1(1-18) labeled at Val3, Ala10, or Ala11 under static or slow MAS conditions indicate that pin1(1-18) rapidly rotates around the average helical axis, and the helical rods are inclined at approximately 30 degrees to the lipid long axis. 13C chemical shift values for pin1(20-44) labeled at Gly25, Leu28, or Ala31 under static conditions indicate that pin1(20-44) may be isotropically tumbling. 1H MAS chemical shift measurements suggest that pin1 is located at the membrane-water interface approximately parallel to the bilayer surface. Solid-state NMR results correlated well with the observed biological activity of pin1 in red blood cells and bacteria.

In Agave tequilana, reproductive failure or inadequate flower development stimulates the formation of vegetative bulbils at the bracteoles, ensuring survival in a hostile environment. Little is known about the signals that trigger this probably unique phenomenon in agave species. Here we report that auxin plays a central role in bulbil development and show that the localization of PIN1-related proteins is consistent with altered auxin transport during this process. Analysis of agave transcriptome data led to the identification of the A. tequilana orthologue of PIN1 (denoted AtqPIN1) and a second closely related gene from a distinct clade reported as 'Sister of PIN1' (denoted AtqSoPIN1). Quantitative real-time reverse transcription-PCR (RT-qPCR) analysis showed different patterns of expression for each gene during bulbil formation, and heterologous expression of the A. tequilana PIN1 and SoPIN1 genes in Arabidopsis thaliana confirmed functional differences between these genes. Although no free auxin was detected in induced pedicel samples, changes in the levels of auxin precursors were observed. Taken as a whole, the data support the model that AtqPIN1 and AtqSoPIN1 have co-ordinated but distinct functions in relation to auxin transport during the initial stages of bulbil formation.

In seed plants, the body organization is established during embryogenesis and is uniform across gymnosperms and angiosperms, despite differences during early embryogeny. Evidence from angiosperms implicates the plant hormone auxin and its polar transport, mainly established by the PIN family of auxin efflux transporters, in the patterning of embryos. Here, PaPIN1 from Norway spruce (Picea abies [L.] Karst.), a gene widely expressed in conifer tissues and organs, was characterized and its expression and localization patterns were determined with reverse transcription polymerase chain reaction and in situ hybridization during somatic embryo development and in seedlings. PaPIN1 shares the predicted structure of other PIN proteins, but its central hydrophilic loop is longer than most PINs. In phylogenetic analyses, PaPIN1 clusters with Arabidopsis thaliana (L.) Heynh. PIN3, PIN4 and PIN7, but its expression pattern also suggests similarity to PIN1. The PaPIN1 expression signal was high in the protoderm of pre-cotyledonary embryos, but not if embryos were pre-treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This, together with a high auxin immunolocalization signal in this cell layer, suggests a role of PaPIN1 during cotyledon formation. At later stages, high PaPIN1 expression was observed in differentiating procambium, running from the tip of incipient cotyledons down through the embryo axis and to the root apical meristem (RAM), although the mode of RAM specification in conifer embryos differs from that of most angiosperms. Also, the PaPIN1 in situ signal was high in seedling root tips including root cap columella cells. The results thus suggest that PaPIN1 provides an ancient function associated with auxin transport and embryo pattern formation prior to the separation of angiosperms and gymnosperms, in spite of some morphological differences.

Accumulating evidence suggests that endoplasmic reticulum (ER) stress plays a major role in the development of many diseases. A previous study indicated that the apoptotic regulator p53 is significantly increased in response to ER stress and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood. Here, we investigated whether p53 contributes to the impairment of Pin1 signaling under ER stress. We found that treatment with thapsigargin, a stimulator of p53 expression and an inducer of ER stress, decreased Pin1 expression in HCT116 cells. Also, we identified functional p53 response elements (p53REs) in the Pin1 promoter. Overexpression of p53 significantly decreased Pin1 expression in HCT116 cells while abolition of p53 gene expression induced Pin1 expression. Pin1 expression was significantly increased by treatment with the p53 inhibitor pifithrin-α or down-regulation of p53 expression. Taken together, ER stress decreased Pin1 expression through p53 activation, and this mechanism may be associated with ER stress-induced cell death. These data reported here support the importance of Pin1 as a potential target molecule mediating tumor development.

The trans-Golgi network (TGN) in plant cells is an independent organelle, displaying rapid association and dissociation with Golgi bodies. In plant cells, the TGN is the site where secretory and endocytic membrane trafficking meet. Cell wall components, signaling molecules and auxin transporters have been found to undergo intracellular trafficking around the TGN. However, how different trafficking pathways are regulated and how different cargoes are sorted in the TGN is poorly defined in plant cells. Using a combined approach of genetic and in vivo imaging, we recently demonstrated that Arabidopsis TRAPPII acts in the TGN and is required for polar targeting of PIN2, but not PIN1, auxin efflux carrier in root tip cells. Here, we report that, TRAPPII in Arabidopsis is required for polar distribution of AUX1, an auxin influx carrier in protophloem cells and epidermal cells of Arabidopsis root tips. In yeast cells, TRAPPII serves as a guanine-nucleotide exchange factor (GEF) for Ypt1 and Ypt31/32 in late Golgi trafficking, while in mammalian cells, TRAPPII acts as a GEF for Rab1 (homolog of yeast Ypt1) in early Golgi trafficking. We show here that TRAPPII in Arabidopsis is functionally linked to Rab-A proteins, homologs of yeast Ypt31/32, but not Rab-D proteins, homologs of yeast Ypt1 and animal Rab1 proteins.

Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue-residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link.

The phytohormone auxin participates in lateral root formation and primary root growth in plants. The auxin gradient formation is mainly regulated by the direction of polar auxin transport (PAT). PAT requires PIN family proteins, which are auxin transport facilitators and contribute to the establishment and maintenance of auxin gradients and mediate multiple developmental processes. Here, we report the effect of the 3', 5'-cyclic guanosine monophosphate (cGMP), an important second messenger, on postembryonic developmental of Arabidopsis lateral root. We find that enhanced cGMP level through the application of the membrane permeable cGMP analog 8-Br-cGMP, promotes the initiation of lateral root primordia and formation of lateral root. 6-Anilino-5,8-quinolinedione (Ly83583, the guanylate cyclase inhibitor) negatively regulates the process. cGMP also mediates acropetal auxin transport and basipetal auxin transport in the root. We further find that 8-Br-cGMP and Ly83583 change the expression of auxin transport genes and alter the polar localization and expression of PIN1 and PIN2 proteins. Moreover, Ly83583 affects actin organization and localization. Taken together, we propose that cGMP affects auxin transport and auxin gradient through modulation PINs proteins localization and expression. cGMP regulates postembryonic formation of Arabidopsis lateral root through the crosstalk with PAT.

The peptidyl prolyl isomerase (Pin1) that induces cis-trans isomerization of the peptide bond involving serine/threonine-proline has recently been shown to regulate the activity of many phosphoproteins including the ones involved in damage response pathways. We investigated Pin1 as a potential target for enhancing the efficacy of anticancer therapy by studying the effects of juglone, a Pin1 inhibitor on the cytotoxicity of etoposide (a widely used anticancer drug that targets topoisomerase IIα) in human tumor cell lines. Treatment of cells with juglone synergistically enhanced the cytotoxicity of etoposide (loss of clonogenicity) with a tenfold increase when etoposide treatment preceded juglone exposure. On the other hand, the toxicity was than additive when the treatment protocol was reversed (i.e exposure to juglone followed by etoposide treatment). This suggests that Pin1 inhibition possibly reduces the induction of initial DNA damage by etoposide, which was supported by a decrease in the levels of chromatin bound topoIIα. Increase in the etoposide induced toxicity by juglone appeared to be mainly due to enhanced mitotic cell death linked to cytogenetic damage, although a moderate increase in interphase (apoptotic) death was also evident as revealed by DNA degradation (hypodiploid population and TUNEL assay). Since the level of Pin1 is found to be higher in cancer cells, this enzyme could be a potential target for developing an adjuvant to enhance the efficacy of anticancer therapies.

The aim of the present study was to examine the expression and significance of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), β‑catenin and cyclin D1 in hepatocellular carcinoma (HCC). A total of 24 samples of HCC and adjacent normal tissues were analyzed. The expression of Pin1, β‑catenin and cyclin D1 in HCC were detected using immunohistochemistry, western blot analysis, polymerase chain reaction and immunofluorescence. The expression of Pin1, β‑catenin and cyclin D1 in HCC tissues were significantly higher than that in adjacent tissues. Pin1 was not markedly expressed in the adjacent normal tissues, while expression in the cytoplasm and nucleus of HCC cells was high. However, β‑catenin and cyclin D1 only revealed a weak expression in the cytoplasm and nucleus of HCC cells. Immunoprecipitation analyses demonstrated two clear bands at 19 and 34 kDa, and a brown band at 55 kDa as expected. Immunofluorescence analysis of HCC cells indicated that Pin1 was present in the cytoplasm and nucleus, and β‑catenin and cyclin D1 were present in the nucleus. In conclusion, the present study indicated that Pin1, β‑catenin and cyclin D1 were highly expressed in HCC. Therefore, detection of the expression of Pin1, β‑catenin and cyclin D1 may be useful for the development of novel diagnostic and treatment strategies for HCC.

The developmental programme of the pistil is under the control of both auxin and cytokinin. Crosstalk between these factors converges on regulation of the auxin carrier PIN-FORMED 1 (PIN1). Here, we show that in the triple transcription factor mutant cytokinin response factor 2 (crf2) crf3 crf6 both pistil length and ovule number were reduced. PIN1 expression was also lower in the triple mutant and the phenotypes could not be rescued by exogenous cytokinin application. pin1 complementation studies using genomic PIN1 constructs showed that the pistil phenotypes were only rescued when the PCRE1 domain, to which CRFs bind, was present. Without this domain, pin mutants resemble the crf2 crf3 crf6 triple mutant, indicating the pivotal role of CRFs in auxin-cytokinin crosstalk.

After protein phosphorylation on certain serine or threonine residues preceding a proline (pSer/Thr-Pro), the function of certain phosphorylated protein is further regulated by cis-trans conformational change. Due to the lack of any tool to detect such two conformations in cells, however, it is not even known whether any cis or trans conformation exists in vivo, not to mention their conformation-specific functions or regulation. We developed a novel peptide chemistry technology to generate the first pair of antibodies that can distinguish cis from trans pThr231-Pro tau. Cis, but not trans, pThr231-tau appears early in mild cognitive impairment (MCI) neurons and further accumulates in only degenerating neurons as Alzheimer disease (AD) progresses, localizing to dystrophic neurites, which are known to correlate well with memory loss. Unlike trans p-tau, the cis cannot promote microtubule assembly, and is more resistant to dephosphorylation and degradation and more prone to aggregation. Pin1 accelerates cis to trans isomerization to prevent tau pathology in AD. Thus, during MCI and AD development, cis pThr231-Pro tau is the earliest detectable pathogenic tau conformation and antibodies and vaccines against the pathogenic cis p-tau may be used for the early diagnosis and treatment of AD. These findings offer in vivo approach to study conformational regulation of Pro-directed phosphorylation signaling.

Interleukin 33 (IL-33) is among the earliest-released cytokines in response to allergens that orchestrate type 2 immunity. The prolyl cis-trans isomerase PIN1 is known to induce cytokines for eosinophil survival and activation by stabilizing cytokines mRNAs, but the function of PIN1 in upstream signaling pathways in asthma is unknown. Here we show that interleukin receptor associated kinase M (IRAK-M) is a PIN1 target critical for IL-33 signaling in allergic asthma. NMR analysis and docking simulations suggest that PIN1 might regulate IRAK-M conformation and function in IL-33 signaling. Upon IL-33-induced airway inflammation, PIN1 is activated for binding with and isomerization of IRAK-M, resulting in IRAK-M nuclear translocation and induction of selected proinflammatory genes in dendritic cells. Thus, the IL-33-PIN1-IRAK-M is an axis critical for dendritic cell activation, type 2 immunity and IL-33 induced airway inflammation.

PIN1 participates in the regulation of a number of signalling pathways in the cell involving protein phosphorylation/dephosphorylation. Its role seems to be an essential control level in addition to the protein phosphorylation by proline-directed kinases. Its cellular function includes regulation of the cell cycle by interaction with phosphorylated mitotic proteins such as Cdc25 and transcription factors such as p53. PIN1 was shown to be involved in the malignant transformation of cells in breast cancer, by up regulation of cyclinD1 and is thought to be involved in the development of the AD by regulating the function of phosphorylated Tau. We propose here to discuss the molecular function of PIN1 at the atomic level based on data from the recent literature and our own results obtained by the technique of Nuclear Magnetic Resonance. PIN1 specifically interacts with pThr/pSer-Pro motifs and is constituted by two domains: a WW N-terminal domain that binds pThr/pSer-Pro epitopes and a prolyl cis/trans isomerase C-terminal catalytic domain. An exception to this organisation is found in the plant PIN1 homologous enzymes, like PIN1At from Arabidopsis thaliana, that are constituted of the sole catalytic domain. The molecular function of PIN1, binding to and isomerization of pThr/pSer-Pro bonds, are thought to lead to several functional consequences. In a first mode of action, exemplified by its competition with the CKS protein, the interaction with PIN1 prevents interaction with other regulatory proteins, like ubiquitin-ligases that lead to degradation pathways. In a second mode of action, the idea is largely accepted that the local isomerization modifies the global conformation of the protein substrate and hence its intrinsic activity, although this has never been directly demonstrated. Finally, isomerization catalysis is thought to regulate the (de)phosphorylation of specific pThr/pSer-Pro motifs, exemplified by the stimulation of the dephosphorylation of pThr231 of Tau by the PP2A phosphatase.

The changes in external K+ concentration affect plant root growth. However, the molecular mechanism for perceiving a K+ signal to modulate root growth remains unknown. It is hypothesized that the K+ channel AKT1 is involved in low K+ sensing in the Arabidopsis root and subsequent regulation of root growth. Along with the decline of external K+ concentration, the primary root growth of wild-type plants was gradually inhibited. However, the primary root of the akt1 mutant could still grow under low K+ (LK) conditions. Application of NAA inhibited akt1 root growth, but promoted wild-type root growth under LK conditions. By using the ProDR5:GFP and ProPIN1:PIN1-GFP lines, we found that LK treatment reduced auxin accumulation in wild-type root tips by degrading PIN1 proteins, which did not occur in the akt1 mutant. The LK-induced PIN1 degradation may be due to the inhibition of vesicle trafficking of PIN1 proteins. In conclusion, our findings indicate that AKT1 is required for an Arabidopsis response to changes in external K+ , and subsequent regulation of K+ -dependent root growth by modulating PIN1 degradation and auxin redistribution in the root.

Lateral plant organs, particularly leaves, initiate at the flanks of the shoot apical meristem (SAM) following auxin maxima signals; however, little is known about the underlying mechanisms. Here, we show that tomato leafless (lfs) mutants fail to produce cotyledons and leaves and grow a naked pin while maintaining an active SAM. A similar phenotype was observed among pin-like shoots induced by polar auxin transport inhibitors such as 2,3,5-triiodobenzoic acid (TIBA). Both types of pin-like shoots showed reduced expression of primordia markers as well as abnormal auxin distribution, as evidenced by expression of the auxin reporters pPIN1:PIN1:GFP and DR5:YFP Upon auxin microapplication, both lfs meristems and TIBA-pin apices activated DR5:YFP expression with similar kinetics; however, only lfs plants failed to concurrently initiate leaf primordia. We found that LFS encodes the single tomato ortholog of Arabidopsis DORNRONSCHEN (DRN) and DRN-like (DRNL) genes and is transiently expressed at incipient and young primordia, overlapping with auxin response maxima. LFS is rapidly induced by auxin application, implying feed-forward activity between LFS and auxin signals. However, driving LFS at auxin response maxima sites using the DR5 promoter fails to fully rescue lfs plants, suggesting that additional, auxin-independent regulation is needed. Indeed, extended GCC-box elements upstream of LFS drove primordia-specific expression in a LFS-dependent but auxin-independent manner. We thus suggest that LFS transiently acts at the site of primordia initiation, where it provides a specific context to auxin response maxima culminating in leaf primordia initiation.

Leaves develop from the shoot apical meristem (SAM) via recruitment of leaf founder cells. Unlike eudicots, most monocot leaves display parallel venation and sheathing bases wherein the margins overlap the stem. Here we utilized computed tomography (CT) imaging, localization of PIN-FORMED1 (PIN1) auxin transport proteins, and in situ hybridization of leaf developmental transcripts to analyze the ontogeny of monocot leaf morphology in maize (Zea mays). CT imaging of whole-mounted shoot apices illustrates the plastochron-specific stages during initiation of the basal sheath margins from the tubular disc of insertion (DOI). PIN1 localizations identify basipetal auxin transport in the SAM L1 layer at the site of leaf initiation, a process that continues reiteratively during later recruitment of lateral leaf domains. Refinement of these auxin transport domains results in multiple, parallel provascular strands within the initiating primordium. By contrast, auxin is transported from the L2 toward the L1 at the developing margins of the leaf sheath. Transcripts involved in organ boundary formation and dorsiventral patterning accumulate within the DOI, preceding the outgrowth of the overlapping margins of the sheathing leaf base. We suggest a model wherein sheathing bases and parallel veins are both patterned via the extended recruitment of lateral maize leaf domains from the SAM.

Synchronized tissue polarization during regeneration or de novo vascular tissue formation is a plant-specific example of intercellular communication and coordinated development. According to the canalization hypothesis, the plant hormone auxin serves as polarizing signal that mediates directional channel formation underlying the spatio-temporal vasculature patterning. A necessary part of canalization is a positive feedback between auxin signaling and polarity of the intercellular auxin flow. The cellular and molecular mechanisms of this process are still poorly understood, not the least, because of a lack of a suitable model system. We show that the main genetic model plant, Arabidopsis (Arabidopsis thaliana) can be used to study the canalization during vascular cambium regeneration and new vasculature formation. We monitored localized auxin responses, directional auxin-transport channels formation, and establishment of new vascular cambium polarity during regenerative processes after stem wounding. The increased auxin response above and around the wound preceded the formation of PIN1 auxin transporter-marked channels from the primarily homogenous tissue and the transient, gradual changes in PIN1 localization preceded the polarity of newly formed vascular tissue. Thus, Arabidopsis is a useful model for studies of coordinated tissue polarization and vasculature formation after wounding allowing for genetic and mechanistic dissection of the canalization hypothesis.