Differential tissue-specific expression of NtAQP1 in Arabidopsis thaliana reveals a role for this protein in stomatal and mesophyll conductance of CO2 under standard and salt-stress conditions

The regulation of plant hydraulic conductance and gas conductance involves a number of different morphological, physiological and molecular mechanisms working in harmony. At the molecular level, aquaporins play a key role in the transport of water, as well as CO₂, through cell membranes. Yet, their tissue-related function, which controls whole-plant gas exchange and water relations, is less understood. In this study, we examined the tissue-specific effects of the stress-induced tobacco Aquaporin1 (NtAQP1), which functions as both a water and CO₂ channel, on whole-plant behavior. In tobacco and tomato plants, constitutive overexpression of NtAQP1 increased net photosynthesis (A _N), mesophyll CO₂ conductance (g _m) and stomatal conductance (g _s) and, under stress, increased root hydraulic conductivity (L _pr) as well. Our results revealed that NtAQP1 that is specifically expressed in the mesophyll tissue plays an important role in increasing both A _N and g _m. Moreover, targeting NtAQP1 expression to the cells of the vascular envelope significantly improved the plants’ stress response. Surprisingly, NtAQP1 expression in the guard cells did not have a significant effect under any of the tested conditions. The tissue-specific involvement of NtAQP1 in hydraulic and gas conductance via the interaction between the vasculature and the stomata is discussed.     

DAP-kinase and autophagy

DAP-kinase (DAPK) is a Ca²⁺-calmodulin regulated kinase with various, diverse cellular activities, including regulation of apoptosis and caspase-independent death programs, cytoskeletal dynamics, and immune functions. Recently, DAPK has also been shown to be a critical regulator of autophagy, a catabolic process whereby the cell consumes cytoplasmic contents and organelles within specialized vesicles, called autophagosomes. Here we present the latest findings demonstrating how DAPK modulates autophagy. DAPK positively contributes to the induction stage of autophagosome nucleation by modulating the Vps34 class III phosphatidyl inositol 3-kinase complex by two independent mechanisms. The first involves a kinase cascade in which DAPK phosphorylates protein kinase D, which then phosphorylates and activates Vps34. In the second mechanism, DAPK directly phosphorylates Beclin 1, a necessary component of the Vps34 complex, thereby releasing it from its inhibitor, Bcl-2. In addition to these established pathways, we will discuss additional connections between DAPK and autophagy and potential mechanisms that still remain to be fully validated. These include myosin-dependent trafficking of Atg9-containing vesicles to the sites of autophagosome formation, membrane fusion events that contribute to expansion of the autophagosome membrane and maturation through the endocytic pathway, and trafficking to the lysosome on microtubules. Finally, we discuss how DAPK's participation in the autophagic process may be related to its function as a tumor suppressor protein, and its role in neurodegenerative diseases.     

DAP‐kinase‐mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl‐XL and induction of autophagy

Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3‐domain‐only protein that binds to Bcl‐2 anti‐apoptotic family members. The dissociation of beclin 1 from its Bcl‐2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death‐associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin‐1‐dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl‐X_L and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation‐based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.     

Inhibition of effector function but not T cell activation and increase in FoxP3 expression in T cells differentiated in the presence of PP14

Background

T-helper polarization of naïve T cells is determined by a complex mechanism that involves many factors, eventually leading to activation of Th1, Th2, or Th17 responses or alternatively the generation of regulatory T cells. Placental Protein 14 (PP14) is a 28 kDa glycoprotein highly secreted in early pregnancy that is able to desensitize T cell receptor (TCR) signaling and modulate T cell activation.

Methodology/Principal Findings

Prolonged antigen-specific stimulation of T cells in the presence of PP14 resulted in an impaired secretion of IFN-γ, IL-5 and IL-17 upon restimulation, although the cells proliferated and expressed activation markers. Furthermore, the generation of regulatory CD4⁺CD25^highFoxp3⁺ T cells was induced in the presence of PP14, in both antigen-specific as well as polyclonal stimulation. In accordance with previous reports, we found that the induction of FoxP3 expression by PP14 is accompanied by down regulation of the PI3K-mTOR signaling pathway.

Conclusions/Significance

These data suggest that PP14 arrests T cells in a unique activated state that is not accompanied with the acquisition of effector function, together with promoting the generation of regulatory T cells. Taken together, our results may elucidate the role of PP14 in supporting immune tolerance in pregnancy by reducing T cell effector functions along with augmenting Treg differentiation.     

Biostratigraphic correlation,paleoenvironment stress,and subrosion pipe collapse: Dutch Rhaetian shales uncover their secrets

A subrosion pipe or sinkhole is a geologic phenomenon that occurs due to dissolution of strata in the subsurface causing the overlying sediments to collapse. The subrosion pipe in the Winterswijk quarry complex in the eastern Netherlands yielded rare, dark-colored shales. Bivalves and palynomorphs indicate that the shales were deposited during the Rhaetian (uppermost Triassic). In addition, detailed correlation with other NW European localities in Great Britain, Austria, and Germany further constrained the age of the shales to the middle of the Rhaetian. The shales were deposited in a near-coastal environment and contained a low diverse macroinvertebrate fauna with bivalves and some brittle stars that lived in a hostile environment, probably caused by low salinity and oxygen levels. These middle Rhaetian shales were mixed with dark-colored middle to late Hettangian sediments, both overlying Middle Triassic (Anisian) strata, which is present in the pipe as well. The presence of Rhaetian sediments in the subrosion pipe reopened the discussion on its age of formation. We suggest that a collapse in the Middle Eocene is most likely. This research expands the knowledge of the marine realms in the uppermost Triassic in Europe, just prior to the Permian–Triassic extinction event.     

GLTSCR2/PICT-1, a Putative Tumor Suppressor Gene Product,Induces the Nucleolar Targeting of the Kaposi's Sarcoma-Associated Herpesvirus KS-Bcl-2 Protein

KS-Bcl-2, encoded by Kaposi''s sarcoma-associated herpesvirus (KSHV), is a structural and functional homologue of the Bcl-2 family of apoptosis regulators. Like several other Bcl-2 family members, KS-Bcl-2 protects cells from apoptosis and autophagy. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we identified a novel KS-Bcl-2-interacting protein, referred to as protein interacting with carboxyl terminus 1 (PICT-1), encoded by a candidate tumor suppressor gene, GLTSCR2. Confocal laser scanning microscopy revealed nucleolar localization of PICT-1, whereas KS-Bcl-2 was located mostly at the mitochondrial membranes with a small fraction in the nucleoli. Ectopic expression of PICT-1 resulted in a large increase in the nucleolar fraction of KS-Bcl-2, and only a minor fraction remained in the cytoplasm. Furthermore, knockdown of endogenous PICT-1 abolished the nucleolar localization of KS-Bcl-2. However, ectopically expressed PICT-1 did not alter the cellular distribution of human Bcl-2. Subsequent analysis mapped the crucial amino acid sequences of both KS-Bcl-2 and PICT-1 required for their interaction and for KS-Bcl-2 targeting to the nucleolus. Functional studies suggest a correlation between nucleolar targeting of KS-Bcl-2 by PICT-1 and reduction of the antiapoptotic activity of KS-Bcl-2. Thus, these studies demonstrate a cellular mechanism to sequester KS-Bcl-2 from the mitochondria and to downregulate its virally encoded antiapoptotic activity. Additional characterization of the interaction of KS-Bcl-2 and PICT-1 is likely to shed light on the functions of both proteins.Kaposi''s sarcoma (KS)-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), is a gamma 2 herpesvirus implicated in several cancers, including KS, primary effusion lymphoma (PEL), and a subset of multicentric Castleman''s disease. Among human viruses, KSHV is most closely related to the Epstein-Barr virus (EBV), a tumorigenic gamma 1 herpesvirus known to be associated with lymphomas and nasopharyngeal carcinoma (10, 12).KSHV open reading frame 16 (orf16) encodes the KS-Bcl-2 protein, which shares sequence and functional homology with the Bcl-2 family (9, 31). Members of the Bcl-2 family are defined by the presence of up to four conserved domains known as the Bcl-2 homology (BH) domains. Several members also possess a carboxy-terminal transmembrane domain that mediates their association with intracellular membranes, such as the endoplasmic reticulum or mitochondria. Bcl-2 proteins are thought to serve primarily as cell death agonists or antagonists that integrate diverse survival and death signals, which are generated outside and within the cell (15, 37), yet Bcl-2 proteins also modulate cell cycle checkpoints, DNA repair/recombination pathways, calcium homeostasis, and cellular bioenergetics.All gammaherpesviruses encode Bcl-2 proteins that generally share 20 to 30% homology with one another and with their cellular counterparts (8, 11). The conservation of Bcl-2 homologues in these viruses indicates their importance for viral infection, with an evolutionarily conserved function of unknown nature. KS-Bcl-2, like most herpesvirus homologues of Bcl-2, contains a transmembrane domain and demonstrates conservation of sequences in both BH1 and BH2 but has only a low degree of homology with other regions of cellular Bcl-2 (18, 22). Still, KS-Bcl-2 shares 3-dimensional structural conservation with Bcl-2 family members and includes the conserved BH3 binding groove and a hydrophobic membrane anchor domain that also contains a mitochondrial outer membrane targeting signal (18). The BH3 binding cleft of KS-Bcl-2 binds with high affinity to peptides encoding BH3 domains present on the proapoptotic proteins Noxa, Bik, PUMA, Bak, Bax, Bid, Bim, and, to a much lesser extent, Bad (13, 18, 22). Based on these characteristics, KS-Bcl-2 has been suggested to have the closest resemblance to the cellular Bcl-2 family member Mcl-1 (13).Previous studies have demonstrated that KS-Bcl-2 protects various cell types from apoptosis mediated by the expression of BAX, tBid, or Bim through Sindbis virus infection or by ectopic expression of KSHV-cyclin-CDK6 (9, 13, 25, 31). However, unlike the cellular Bcl-2, KS-Bcl-2 is not a substrate for KSHV-cyclin-CDK6 phosphorylation (25) and cannot be converted into a proapoptotic protein via caspase cleavage (3). KS-Bcl-2 is able to form a stable complex with the cellular protein Aven, which binds Apaf-1 and is known as a regulator of caspase 9 and ataxia-telangiectasia (ATM) activation (7, 16). Like the cellular and other virus-encoded Bcl-2 proteins, KS-Bcl-2 binds Beclin and disrupts its lysosomal degradation pathway of autophagy (21, 29). However, since KS-Bcl-2 lacks the nonstructured loop located between the BH4 and BH3 domains, its binding to BH3-containing proapoptotic proteins and to the BH3-containing proautophagy protein Beclin is not modulated by phosphorylation (38).KS-Bcl-2 is transcribed during lytic virus infection (30, 31). Thus, inhibition of apoptosis and autophagy by KS-Bcl-2 may provide an attractive mechanism for prolonging the life span of KSHV-infected cells, which in turn enables increased virus production or establishment of latency. Whether the function of KS-Bcl-2 is necessary for KSHV-mediated oncogenesis is still unknown. Nevertheless, the KS-Bcl-2 protein is expressed in late-stage KS lesions but has not been detected in latent or in lytic KSHV-infected PEL cells (39).To explore the role of KS-Bcl-2 in cell signaling, we searched for its potential cellular-protein partners. In the present study, we describe a novel interaction between KS-Bcl-2 and the protein interacting with carboxyl terminus 1 (PICT-1) cellular protein, encoded by a candidate tumor suppressor gene, GLTSCR2. We show that this interaction specifically targets KS-Bcl-2 to the nucleolus and decreases its antiapoptotic activity.(Portions of this work were submitted to Bar Ilan Univeristy, Ramat Gan, Israel, by I. Kalt and T. Borodianskiy-Shteinberg in partial fulfillment of the requirements for the degree of Doctor of Philosophy.)     

Abnormal vasculature interferes with optic fissure closure in lmo2 mutant zebrafish embryos

Ocular coloboma is a potentially blinding congenital eye malformation caused by failure of optic fissure closure during early embryogenesis. The optic fissure is a ventral groove that forms during optic cup morphogenesis, and through which hyaloid artery and vein enter and leave the developing eye, respectively. After hyaloid artery and vein formation, the optic fissure closes around them. The mechanisms underlying optic fissure closure are poorly understood, and whether and how this process is influenced by hyaloid vessel development is unknown. Here we show that a loss-of-function mutation in lmo2, a gene specifically required for hematopoiesis and vascular development, results in failure of optic fissure closure in zebrafish. Analysis of ocular blood vessels in lmo2 mutants reveals that some vessels are severely dilated, including the hyaloid vein. Remarkably, reducing vessel size leads to rescue of optic fissure phenotype. Our results reveal a new mechanism leading to coloboma, whereby malformed blood vessels interfere with eye morphogenesis.     

Autophagy gets a brake: DAP1, a novel mTOR substrate,is activated to suppress the autophagic process

Autophagy, a highly regulated catabolic process, is controlled by the action of positive and negative regulators. While many of the positive mediators of autophagy have been identified, very little is known about negative regulators that might counterbalance the process. We recently identified death-associated protein 1 (DAP1) as a suppressor of autophagy and as a novel direct substrate of mammalian target of rapamycin (mTOR). We found that DAP1 is functionally silent in cells growing under rich nutrient supplies through mTOR-dependent inhibitory phosphorylation on two sites, which were mapped to Ser3 and Ser51. During amino acid starvation, mTOR activity is turned off resulting in a rapid reduction in the phosphorylation of DAP1. This caused the conversion of the protein into a suppressor of autophagy, thus providing a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under conditions of nutrient deprivation. Based on these studies we propose the “gas and brake” concept in which mTOR, the main sensor that regulates autophagy in response to amino acid deprivation, also controls the activity of a specific balancing brake to prevent the overactivation of autophagy.Key words: DAP1, mTOR, autophagy, amino acid starvation, phosphorylationIn recent years, many of the genes controlling and executing the autophagic process have been identified. Most of these genes act as positive mediators of the various steps of the process, including the ULK1 complex, which regulates the induction step, the Vps34-Beclin 1 complex that participates in the vesicle nucleation step and two ubiquitin-like pathways, the Atg12-Atg5 and the LC3-phosphatidylethanolamine (PE) conjugation steps, which play a central role in the vesicle elongation process. To date, only a few negative regulators of autophagy have been identified, including mTOR and the anti-apoptotic Bcl-2 family members. mTOR Ser/Thr kinase is a central suppressor of autophagy acting at the initiating regulatory steps of the process. Many signaling pathways act to inhibit mTOR activity, thus relieving its inhibitory effects on autophagy. The anti-apoptotic Bcl-2 and Bcl-X_L proteins, on the other hand, act at the nucleation step, by directly binding to Beclin 1''s BH3 domain, thus reducing the activation of Vps34 and subsequent autophagy. This inhibition can be relieved through dissociation of the complex, following either JNK-1 mediated phosphorylation of Bcl-2 or DAP kinase-mediated phosphorylation of the BH3 domain of Beclin 1.DAP1 is a small (∼15 kDa), ubiquitously expressed protein, rich in prolines and lacking known functional motifs. DAP1 was isolated more than a decade ago in our laboratory using a functional approach to gene cloning aimed at identifying novel mediators of IFNγ-induced cell death in mammalian cell cultures. Until recently, very little was known about the cellular and molecular functions of DAP1, mainly due to the lack of homology to other known proteins and the lack of functional motifs that could indicate a possible cellular function and studies in mammalian systems were missing.Recently, we discovered that DAP1 is another negative regulator of autophagy; yet, interestingly, its suppressive activity is selectively turned on during the autophagic process. Moreover, we found that DAP1 suppressive activity is tightly linked to the status of mTOR kinase activity. Under nutrient-rich culture conditions, DAP1 is phosphorylated by mTOR on two sites, Ser3 and Ser51, resulting in its inactivation. In response to nutrient deprivation, mTOR is inhibited and DAP1 undergoes rapid dephosphorylation. By knocking down the endogenous DAP1 and introducing either the phosphomimetic or the nonphosphorylatible DAP1 mutants, we found that the dephosphorylation leads to activation of the autophagic suppressive function of DAP1, whereas the phophorylated form is inactive. These results led to a “gas and brake” model, in which at the same time that autophagy is induced, some brakes such as DAP1 are also activated to provide a buffering mechanism that counterbalances the autophagic flux and prevents its overactivation under nutrient-deprivation conditions (Fig. 1). Notably, balancing autophagy is extremely important, since deregulated or excessive autophagy has been implicated in the pathogenesis of diverse diseases, such as certain types of neuronal degeneration and cancer and also in cellular aging.Open in a separate windowFigure 1“Gas and brake” model. During nutrient-rich conditions, active mTORC1 phosphorylates and inactivates the components of the ULK1 complex, ULK1 and Atg13, thus preventing the induction of autophagy. DAP1 is also inactivated simultaneously by mTORC1-mediated phosphorylation on Ser3 and Ser51. In addition, mTORC1 phosphorylates and activates p70S6K and 4E-BP1, which mediate the protein translation and cell growth activities of mTOR. Upon nutrient starvation, mTORC1 activity is attenuated, leading to dephosphorylation and activation of ULK1. ULK1, in turn, undergoes autophosphorylation and phosphorylates Atg13 and FIP200 resulting in ULK1 complex activation and induction of autophagy. On the other hand, activation of DAP1 by dephosphorylation, results in suppression of autophagy, thus inserting a brake into the process of autophagy. Note that the inactive proteins/complexes are faded out.The current challenge is to identify the molecular basis of the suppressive functions of DAP1 on autophagy. We have recently shown that DAP1 knockdown enhances LC3 lipidation and autophagosome accumulation both during amino acid starvation and rapamycin treatment. In addition, preliminary data indicate that the knockdown of DAP1 has no effect on mTOR complex 1 (mTORC1) activity in cells, at least during the first hours of starvation. Accordingly, DAP1 may function between the mTORC1 and the LC3 conjugation systems. The potential targets may fall into one of the multiprotein complexes functioning downstream of mTOR such as the ULK1 complex, the Vps34-Beclin 1 complex and more. Future studies will be performed to identify the molecular mechanism by which DAP1 suppresses autophagy. The lack of known functional motifs in the DAP1 protein sequence suggests that this small proline-rich protein may function as an adaptor blocking autophagy by binding to critical protein partners that still await identification.Although autophagy is primarily a protective process for the cell, it can also play a role in cell death. In response to prolonged starvation, autophagy can act either as a cell survival mechanism or be recruited as a cell death executer. In the future it would be interesting to examine whether the autophagy enhancement resulting from DAP1 knockdown contributes to increased cell death in our system or even may convert the survival properties of autophagy into death induction. This will fit the “gas and brake” model, in which autophagy, which is initially recruited as a cell survival mechanism, is converted into cell death machinery when a certain threshold is crossed due to the loss of the “brake” by the knockdown of DAP1.To date, very little is known about the putative mechanisms that restrict the intensity of the autophagic flux to maintain the continuous benefits of this process under stress. Therefore, the ability of DAP1 to counterbalance and buffer the process in a manner that is tightly linked to the status of a central player in autophagy (i.e., mTOR) is an important discovery in this field and provides a target for future drug design.     

Nuclear and plastid genetic engineering of plants: Comparison of opportunities and challenges

Plant genetic engineering is one of the key technologies for crop improvement as well as an emerging approach for producing recombinant proteins in plants. Both plant nuclear and plastid genomes can be genetically modified, yet fundamental functional differences between the eukaryotic genome of the plant cell nucleus and the prokaryotic-like genome of the plastid will have an impact on key characteristics of the resulting transgenic organism. So, which genome, nuclear or plastid, to transform for the desired transgenic phenotype? In this review we compare the advantages and drawbacks of engineering plant nuclear and plastid genomes to generate transgenic plants with the traits of interest, and evaluate the pros and cons of their use for different biotechnology and basic research applications, ranging from generation of commercial crops with valuable new phenotypes to ‘bioreactor’ plants for large-scale production of recombinant proteins to research model plants expressing various reporter proteins.     

Large‐scale analysis of secondary structure changes in proteins suggests a role for disorder‐to‐order transitions in nucleotide binding proteins

Conformational changes in proteins often involve secondary structure transitions. Such transitions can be divided into two types: disorder‐to‐order changes, in which a disordered segment acquires an ordered secondary structure (e.g., disorder to α‐helix, disorder to β‐strand), and order‐to‐order changes, where a segment switches from one ordered secondary structure to another (e.g., α‐helix to β‐strand, α‐helix to turn). In this study, we explore the distribution of these transitions in the proteome. Using a comprehensive, yet highly conservative method, we compared solved three‐dimensional structures of identical protein sequences, looking for differences in the secondary structures with which they were assigned. Protein chains in which such secondary structure transitions were detected, were classified into two sets according to the type of transition that is involved (disorder‐to‐order or order‐to‐order), allowing us to characterize each set by examining enrichment of gene ontology terms. The results reveal that the disorder‐to‐order set is significantly enriched with nucleotide binding proteins, whereas the order‐to‐order set is more diverse. Remarkably, further examination reveals that >22% of the purine nucleotide binding proteins include segments which undergo disorder‐to‐order transitions, suggesting that such transitions play an important role in this process. Proteins 2010. © 2009 Wiley‐Liss, Inc.