The chaperone-like protein Cdc48 regulates ubiquitin-proteasome system in plants.

The degradation of misfolded proteins is mainly mediated by the ubiquitin-proteasome system (UPS). UPS can be assisted by the protein Cdc48 but the relationship between UPS and Cdc48 in plants has been poorly investigated. Here, we analysed the regulation of UPS by Cdc48 in tobacco thanks to two independent cell lines overexpressing Cdc48 constitutively and plant leaves overexpressing Cdc48 transiently. In the cell lines, the accumulation of ubiquitinated proteins was affected both quantitatively and qualitatively and the number of proteasomal subunits was modified, while proteolytic activities were unchanged. Similarly, the over-expression of Cdc48 in planta impacted the accumulation of ubiquitinated proteins. A similar process occurred in leaves overexpressing transiently Rpn3, a proteasome subunit. Cdc48 being involved in plant immunity, its regulation of UPS was also investigated in response to cryptogein, an elicitor of immune responses. In the cell lines stably overexpressing Cdc48 and in leaves transiently overexpressing Cdc48 and/or Rpn3, cryptogein triggered a premature cell death while no increase of the proteasomal activity occurred. Overall, this study highlights a role for Cdc48 in ubiquitin homeostasis and confirms its involvement, as well as that of Rpn3, in the processes underlying the hypersensitive response.

Nitric oxide production and signalling in algae.

Nitric oxide (NO) was the first identified gaseous messenger and is now well established as a major ubiquitous signalling molecule. The rapid development of our understanding of NO biology in embryophytes came with the partial characterization of the pathways underlying its production and with the decrypting of signalling networks mediating its effects. Notably, the identification of proteins regulated by NO through nitrosation greatly enhanced our perception of NO functions. In comparison, the role of NO in algae has been less investigated. Yet, studies in Chlamydomonas reinhardtii have produced key insights into NO production through the identification of NO-forming nitrite reductase and of S-nitrosated proteins. More intriguingly, in contrast to embryophytes, a few algal species possess a conserved nitric oxide synthase, the main enzyme catalysing NO synthesis in metazoans. This latter finding paves the way for a deeper characterization of novel members of the NO synthase family. Nevertheless, the typical NO-cyclic GMP signalling module transducing NO effects in metazoans is not conserved in algae, nor in embryophytes, highlighting a divergent acquisition of NO signalling between the green and the animal lineages.

The chaperone-like protein CDC48 regulates ascorbate peroxidase in tobacco.

There is increasing evidence that the chaperone-like protein CDC48 (cell division cycle 48) plays a role in plant immunity. Cytosolic ascorbate peroxidase (cAPX), which is a major regulator of the redox status of plant cells, has previously been shown to interact with CDC48. In this study, we examined the regulation of cAPX by the ATPase NtCDC48 during the cryptogein-induced immune response in tobacco cells. Our results not only confirmed the interaction between the proteins but also showed that it occurs in the cytosol. cAPX accumulation was modified in cells overexpressing NtCDC48, a process that was shown to involve post-translational modification of cAPX. In addition, cryptogein-induced increases in cAPX activity were suppressed in cells overexpressing NtCDC48 and the abundance of the cAPX dimer was below the level of detection. Furthermore, the levels of both reduced (GSH) and oxidized glutathione (GSSG) and the GSH/GSSG ratio decreased more rapidly in response to the elicitor in these cells than in controls. A decrease in cAPX activity was also observed in response to heat shock in the cells overexpressing NtCDC48, indicating that the regulation of cAPX by NtCDC48 is not specific to the immune response.

Functional characterization of the chaperon-like protein Cdc48 in cryptogein-induced immune response in tobacco.

Cdc48, a molecular chaperone conserved in different kingdoms, is a member of the AAA+ family contributing to numerous processes in mammals including proteins quality control and degradation, vesicular trafficking, autophagy and immunity. The functions of Cdc48 plant orthologues are less understood. We previously reported that Cdc48 is regulated by S-nitrosylation in tobacco cells undergoing an immune response triggered by cryptogein, an elicitin produced by the oomycete Phytophthora cryptogea. Here, we inv estigated the function of NtCdc48 in cryptogein signalling and induced hypersensitive-like cell death. NtCdc48 was found to accumulate in elicited cells at both the protein and transcript levels. Interestingly, only a small proportion of the overall NtCdc48 population appeared to be S-nitrosylated. Using gel filtration in native conditions, we confirmed that NtCdc48 was present in its hexameric active form. An immunoprecipitation-based strategy following my mass spectrometry analysis led to the identification of about a hundred NtCdc48 partners and underlined its contribution in cellular processes including targeting of ubiquitylated proteins for proteasome-dependent degradation, subcellular trafficking and redox regulation. Finally, the analysis of cryptogein-induced events in NtCdc48-overexpressing cells highlighted a correlation between NtCdc48 expression and hypersensitive cell death. Altogether, this study identified NtCdc48 as a component of cryptogein signalling and plant immunity.

Structure and functions of the chaperone-like p97/CDC48 in plants.

BACKGROUND: The chaperone-like p97 is a member of the AAA+ ATPase enzyme family that contributes to numerous cellular activities. P97 has been broadly studied in mammals (VCP/p97) and yeasts (CDC48: Cell Division Cycle 48/p97) and numerous investigations highlighted that this protein is post-translationally regulated, is structured in homohexamer and interacts with partners and cofactors that direct it to distinct cellular signalization pathway including protein quality control and degradation, cell cycle regulation, genome stability, vesicular trafficking, autophagy and immunity. SCOPE OF REVIEW: p97 is also conserved in plants (CDC48) but its functions are less understood. In the present review we intended to present the state of the art of the structure, regulation and functions of CDC48 in plants. MAJOR CONCLUSIONS: Evidence accumulated underline that CDC48 plays a crucial role in development, cell cycle regulation and protein turnover in plants. Furthermore, its involvement in plant immunity has recently emerged and first interacting partners have been identified, shedding light on its putative cellular activities. GENERAL SIGNIFICANCE: Identification of emerging functions of CDC48 in plants opens new roads of research in immunity and provides new insights into the mechanisms of protein quality control.

Impact of disease-causing mutations on TMEM165 subcellular localization, a recently identified protein involved in CDG-II.

TMEM165 has recently been identified as a novel protein involved in CDG-II. TMEM165 has no biological function described so far. Different mutations were recently found in patients with Golgi glycosylation defects and harboring a peculiar skeletal phenotype. In this study, we examined the effect of naturally occurring mutations on the intracellular localization of TMEM165 and their abilities to complement the TMEM165-deficient yeast, gdt1▵. Wild-type TMEM165 was present within Golgi compartment, plasma membrane and late endosomes/lysosomes, whereas mutated TMEM165 were found differentially localized according to the mutations. We demonstrated that, in the yeast functional assay with TMEM165 ortholog Gdt1, the homozygous point mutation correlating with a mild phenotype restores the yeast functional assay, whereas the truncated mutation, associated with severe disease, failed to restore Gdt1 function. These studies highly suggest that these clinically relevant point mutations do not affect the protein function but critically changes the subcellular protein localization. Moreover, the data point to a critical role of the YNRL motif in TMEM165 subcellular localization.

TMEM165 deficiency causes a congenital disorder of glycosylation.

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.

CDC48 in plants and its emerging function in plant immunity.

Protein homeostasis, namely the balance between protein synthesis and degradation, must be finely controlled to ensure cell survival, notably through the ubiquitin-proteasome system (UPS). In all species, including plants, homeostasis is disrupted by biotic and abiotic stresses. A key player in the maintenance of protein balance, the protein CDC48, shows emerging functions in plants, particularly in response to biotic stress. In this review on CDC48 in plants, we detail its highly conserved structure, describe a gene expansion that is only present in Viridiplantae, discuss its various functions and regulations, and finally highlight its recruitment, still not clear, during the plant immune response.

CDC48 regulates immunity pathway in tobacco plants.

The CDC48 protein, highly conserved in the living kingdom, is a player of the ubiquitin proteasome system and contributes to various cellular processes. In plants, CDC48 is involved in cell division, plant growth and, as recently highlighted in several reports, in plant immunity. In the present study, to further extend our knowledge about CDC48 functions in plants, we analysed the incidence of its overexpression on tobacco development and immune responses. CDC48 overexpression disrupted plant development and morphology, induced changes in plastoglobule appearance and exacerbated ROS production. In addition, levels of salicylic acid (SA) and glycosylated SA were higher in transgenic plants, both in the basal state and in response to cryptogein, a protein produced by the oomycete Phytophthora cryptogea triggering defence responses. The expression of defence genes, notably those coding for some pathogenesis-related (PR) proteins, was also exacerbated in the basal state in transgenic plant lines. Finally, tobacco plants overexpressing CDC48 did not develop necrosis in response to tobacco mosaic virus (TMV) infection, suggesting a role for CDC48 in virus resistance.

Hepatitis C virus NS5B and host cyclophilin A share a common binding site on NS5A.

Nonstructural protein 5B (NS5B) is essential for hepatitis C virus (HCV) replication as it carries the viral RNA-dependent RNA polymerase enzymatic activity. HCV replication occurs in a membrane-associated multiprotein complex in which HCV NS5A and host cyclophilin A (CypA) have been shown to be present together with the viral polymerase. We used NMR spectroscopy to perform a per residue level characterization of the molecular interactions between the unfolded domains 2 and 3 of NS5A (NS5A-D2 and NS5A-D3), CypA, and NS5B(Δ21). We show that three regions of NS5A-D2 (residues 250-262 (region A), 274-287 (region B), and 306-333 (region C)) interact with NS5B(Δ21), whereas NS5A-D3 does not. We show that both NS5B(Δ21) and CypA share a common binding site on NS5A that contains residues Pro-306 to Glu-323. No direct molecular interaction has been detected by NMR spectroscopy between HCV NS5B(Δ21) and host CypA. We show that cyclosporine A added to a sample containing NS5B(Δ21), NS5A-D2, and CypA specifically inhibits the interaction between CypA and NS5A-D2 without altering the one between NS5A-D2 and NS5B(Δ21). A high quality heteronuclear NMR spectrum of HCV NS5B(Δ21) has been obtained and was used to characterize the binding site on the polymerase of NS5A-D2. Moreover these data highlight the potential of using NMR of NS5B(Δ21) as a powerful tool to characterize in solution the interactions of the HCV polymerase with all kinds of molecules (proteins, inhibitors, RNA). This work brings new insights into the comprehension of the molecular interplay between NS5B, NS5A, and CypA, three essentials proteins for HCV replication.

Glycosylation disorders of membrane trafficking.

During evolution from prokaryotic to eukaryotic cells, compartmentalization of cellular functions has been achieved with a high degree of complexity. Notably, all secreted and transmembrane proteins travel through endoplasmic reticulum (ER) and Golgi apparatus, where they are synthesized, folded and subjected to covalent modifications, most particularly glycosylation. N-glycosylation begins in the ER with synthesis and transfer of glycan onto nascent protein and proceeds in Golgi apparatus where maturation occurs. This process not only requires the precise localization of glycosyltransferases, glycosidases and substrates but also an efficient, finely regulated and bidirectional vesicular trafficking among membrane-enclosed organelles. Basically, it is no surprise that alterations in membrane transport or related pathways can lead to glycosylation abnormalities. During the last few years, this has particularly been highlighted in genetic diseases called CDG (Congenital Disorders of Glycosylation). Alterations in mechanisms of vesicle formation due to COPII coat component SEC23B deficiency, or in vesicles tethering, caused by defects of the COG complex, but also impaired Golgi pH homeostasis due to ATP6V0A2 defects have been discovered in CDG patients. This mini review will summarize these fascinating discoveries.

Interaction proteomics analysis of polycomb proteins defines distinct PRC1 complexes in mammalian cells.

Polycomb group (PcG) proteins maintain transcriptional repression of hundreds of genes involved in development, signaling or cancer using chromatin-based epigenetic mechanisms. Biochemical studies in Drosophila have revealed that PcG proteins associate in at least two classes of protein complexes known as Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Drosophila core PRC1 is composed of four subunits, Polycomb (Pc), Sex combs extra (Sce), Polyhomeotic (Ph), and Posterior sex combs (Psc). Each of these proteins has multiple orthologs in vertebrates classified respectively as the CBX, RING1/RNF2, PHC, and BMI1/PCGF families. Mammalian genomes encode five CBX family members (CBX2, CBX4, CBX6, CBX7, and CBX8) that are believed to have distinct biological functions. Here, we applied a tandem affinity purification (TAP) approach coupled with tandem mass spectrometry (MS/MS) methodologies in order to identify interacting partners of CBX family proteins under the same experimental conditions. Our analysis identified with high confidence about 20 proteins co-eluted with CBX2 and CBX7 tagged proteins, about 40 with CBX4, and around 60 with CBX6 and CBX8. We provide evidences that the CBX family proteins are mutually exclusive and define distinct PRC1-like protein complexes. CBX proteins also interact with different efficiencies with the other PRC1 components. Among the novel CBX interacting partners, protein kinase 2 associates with all CBX-PRC1 protein complexes, whereas 14-3-3 proteins specifically bind to CBX4. 14-3-3 protein binding to CBX4 appears to modulate the interaction between CBX4 and the BMI1/PCGF components of PRC1, but has no effect on CBX4-RING1/RNF2 interaction. Finally, we suggest that differences in CBX protein interactions would account, at least in part, for distinct subnuclear localization of the CBX family members.

Functional characterization of human Polycomb-like 3 isoforms identifies them as components of distinct EZH2 protein complexes.

PcG (Polycomb group) proteins are conserved transcriptional repressors essential to regulate cell fate and to maintain epigenetic cellular memory. They work in concert through two main families of chromatin-modifying complexes, PRC1 (Polycomb repressive complex 1) and PRC2-4. In Drosophila, PRC2 contains the H3K27 histone methyltransferase E(Z) whose trimethylation activity towards PcG target genes is stimulated by PCL (Polycomb-like). In the present study, we have examined hPCL3, one of its three human paralogues. Through alternative splicing, hPCL3 encodes a long isoform, hPCL3L, containing an N-terminal TUDOR domain and two PHDs (plant homeodomains) and a smaller isoform, hPCL3S, lacking the second PHD finger (PHD2). By quantitative reverse transcription-PCR analyses, we showed that both isoforms are widely co-expressed at high levels in medulloblastoma. By co-immunoprecipitation analyses, we demonstrated that both isoforms interact with EZH2 through their common TUDOR domain. However, the hPCL3L-specific PHD2 domain, which is better conserved than PHD1 in the PCL family, is also involved in this interaction and implicated in the self-association of hPCL3L. Finally, we have demonstrated that both hPCL3 isoforms are physically associated with EZH2, but in different complexes. Our results provide the first evidence that the two hPCL3 isoforms belong to different complexes and raise important questions about their relative functions, particularly in tumorigenesis.

Reliable reference genes and abiotic stress marker genes in Klebsormidium nitens.

Microalgae have recently emerged as a key research topic, especially as biological models. Among them, the green alga Klebsormidium nitens, thanks to its particular adaptation to environmental stresses, represents an interesting photosynthetic eukaryote for studying the transition stages leading to the colonization of terrestrial life. The tolerance to different stresses is manifested by changes in gene expression, which can be monitored by quantifying the amounts of transcripts by RT-qPCR. The identification of optimal reference genes for experiment normalization was therefore necessary. In this study, using four statistical algorithms followed by the RankAggreg package, we determined the best reference gene pairs suitable for normalizing RT-qPCR data in K. nitens in response to three abiotic stresses: high salinity, PEG-induced dehydration and heat shock. Based on these reference genes, we were able to identify marker genes in response to the three abiotic stresses in K. nitens.

S-Nitrosation of Arabidopsis thaliana Protein Tyrosine Phosphatase 1 Prevents Its Irreversible Oxidation by Hydrogen Peroxide.

Tyrosine-specific protein tyrosine phosphatases (Tyr-specific PTPases) are key signaling enzymes catalyzing the removal of the phosphate group from phosphorylated tyrosine residues on target proteins. This post-translational modification notably allows the regulation of mitogen-activated protein kinase (MAPK) cascades during defense reactions. Arabidopsis thaliana protein tyrosine phosphatase 1 (AtPTP1), the only Tyr-specific PTPase present in this plant, acts as a repressor of H2O2 production and regulates the activity of MPK3/MPK6 MAPKs by direct dephosphorylation. Here, we report that recombinant histidine (His)-AtPTP1 protein activity is directly inhibited by H2O2 and nitric oxide (NO) exogenous treatments. The effects of NO are exerted by S-nitrosation, i.e., the formation of a covalent bond between NO and a reduced cysteine residue. This post-translational modification targets the catalytic cysteine C265 and could protect the AtPTP1 protein from its irreversible oxidation by H2O2. This mechanism of protection could be a conserved mechanism in plant PTPases.

The MtSNF4b subunit of the sucrose non-fermenting-related kinase complex connects after-ripening and constitutive defense responses in seeds of Medicago truncatula.

Dormant seeds are capable of remaining alive in the hydrated state for extended periods of time without losing vigor, until environmental cues or after-ripening result in the release of dormancy. Here, we investigated the possible role of the regulatory subunit of the sucrose non-fermenting-related kinase complex, MtSNF4b, in dormancy of Medicago truncatula seeds. Expression of MtSNF4b and its involvement in a high-molecular-weight complex are found in dormant seeds, whereas imbibition of fully after-ripened, non-dormant seeds leads to dissociation of the complex. MtSNF4b is capable of complementing the yeast Delta snf4 mutant and of interacting with the MtSnRK1 alpha-subunit in a double hybrid system. Transcriptome analyses on freshly harvested and after-ripened RNAi Mtsnf4b and wild-type embryos implicate MtSNF4b in the defense response in hydrated dormant embryonic tissues, affecting the expression of genes encoding enzymes of flavonoid and phenylpropanoid metabolism, WRKY transcription factors and pathogenesis-related proteins. Silencing MtSNF4b also increased the speed of after-ripening during dry storage, an effect that appears to be related to a change in base water potential. No significant difference in ABA content or sensitivity was detected between mutant and wild-type seeds. Pharmacological studies using hexoses and sugar analogs revealed that mannose restored germination behavior and expression of the genes PAL, CHR and IFR in RNAi Mtsnf4b seeds towards that of the wild-type, suggesting that MtSNF4b might act upstream of sugar-sensing pathways. Overall, the results suggest that MtSNF4b participates in regulation of a constitutively activated defense response in hydrated, dormant seeds.

Analysis of the human HP1 interactome reveals novel binding partners.

Heterochromatin protein 1 (HP1) has first been described in Drosophila as an essential component of constitutive heterochromatin required for stable epigenetic gene silencing. Less is known about the three mammalian HP1 isotypes CBX1, CBX3 and CBX5. Here, we applied a tandem affinity purification approach coupled with tandem mass spectrometry methodologies in order to identify interacting partners of the mammalian HP1 isotypes. Our analysis identified with high confidence about 30-40 proteins co-eluted with CBX1 and CBX3, and around 10 with CBX5 including a number of novel HP1-binding partners. Our data also suggest that HP1 family members are mainly associated with a single partner or within small protein complexes composed of limited numbers of components. Finally, we showed that slight binding preferences might exist between HP1 family members.

Identification of Partner Proteins of the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in Eukaryotic Photosynthetic Organisms.

In animals, NO is synthesized from L-arginine by three isoforms of nitric oxide synthase (NOS) enzyme. NO production and effects have also been reported in plants but the identification of its sources, especially the enzymatic ones, remains one of the critical issues in the field. NOS-like activities have been reported, although there are no homologs of mammalian NOS in the land plant genomes sequenced so far. However, several NOS homologs have been found in algal genomes and transcriptomes. A first study has characterized a functional NOS in the chlorophyte Ostreococcus tauri and the presence of NOS homologs was later confirmed in a dozen algae. These results raise the questions of the significance of the presence of NOS and their molecular diversity in algae. We hypothesize that comparisons among protein structures of the two KnNOS, together with the identification of their interacting partner proteins, might allow a better understanding of the molecular diversification and functioning of NOS in different physiological contexts and, more generally, new insights into NO signaling in photosynthetic organisms. We recently identified two NOS homologs sequences in the genome of the streptophyte Klebsormidium nitens, a model alga in the study of plant adaptation to terrestrial life. The first sequence, named KnNOS1, contains canonical NOS signatures while the second, named KnNOS2, presents a large C-ter extension including a globin domain. In order to identify putative candidates for KnNOSs partner proteins, we draw the protein-protein interaction networks of the three human NOS using the BioGRID database and hypothesized on the biological role of K. nitens orthologs. Some of these conserved partners are known to be involved in mammalian NOSs regulation and functioning. In parallel, our methodological strategy for the identification of partner proteins of KnNOS1 and KnNOS2 by in vitro pull-down assay is presented.

Cell adhesion properties on chemically micropatterned boron-doped diamond surfaces.

The adhesion properties of living cells were investigated on a range of chemically modified boron-doped diamond (BDD) surfaces. We studied the influence of oxidized, H-, amine- (NH(2)-), methyl- (CH(3)-), trifluoromethyl- (CF(3)-) and vinyl- (CH(2)═CH-) terminated BDD surfaces on human osteosarcoma U2OS and mouse fibroblast L929 cells behavior. Cell-surface interactions were analyzed by fluorescence microscopy in terms of cell attachment, spreading and proliferation. U2OS cells poorly adhered on hydrophobic surfaces and their growth was blocked. In contrast, L929 cells were mainly influenced by the presence of perfluoroalkyl chains in regard to their morphology. The results were subsequently applied to selectively micropattern U2OS cells on dual hydrophobic/hydrophilic surfaces prepared by a UV/ozone lithographic approach. U2OS cells colonized preferentially hydrophilic (oxide-terminated) motifs, forming confluent arrays with distinguishable edges separating the alkyl regions.

The histone methyltransferase SUV420H2 and Heterochromatin Proteins HP1 interact but show different dynamic behaviours.

BACKGROUND: Histone lysine methylation plays a fundamental role in chromatin organization and marks distinct chromatin regions. In particular, trimethylation at lysine 9 of histone H3 (H3K9) and at lysine 20 of histone H4 (H4K20) governed by the histone methyltransferases SUV39H1/2 and SUV420H1/2 respectively, have emerged as a hallmark of pericentric heterochromatin. Controlled chromatin organization is crucial for gene expression regulation and genome stability. Therefore, it is essential to analyze mechanisms responsible for high order chromatin packing and in particular the interplay between enzymes involved in histone modifications, such as histone methyltransferases and proteins that recognize these epigenetic marks. RESULTS: To gain insights into the mechanisms of SUV420H2 recruitment at heterochromatin, we applied a tandem affinity purification approach coupled to mass spectrometry. We identified heterochromatin proteins HP1 as main interacting partners. The regions responsible for the binding were mapped to the heterochromatic targeting module of SUV420H2 and HP1 chromoshadow domain. We studied the dynamic properties of SUV420H2 and the HP1 in living cells using fluorescence recovery after photobleaching. Our results showed that HP1 proteins are highly mobile with different dynamics during the cell cycle, whereas SUV420H2 remains strongly bound to pericentric heterochromatin. An 88 amino-acids region of SUV420H2, the heterochromatic targeting module, recapitulates both, HP1 binding and strong association to heterochromatin. CONCLUSION: FRAP experiments reveal that in contrast to HP1, SUV420H2 is strongly associated to pericentric heterochromatin. Then, the fraction of SUV420H2 captured and characterized by TAP/MS is a soluble fraction which may be in a stable association with HP1. Consequently, SUV420H2 may be recruited to heterochromatin in association with HP1, and stably maintained at its heterochromatin sites in an HP1-independent fashion.