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.

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.

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 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.

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.

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.

Toward the understanding of the role of CDC48, a major component of the protein quality control, in plant immunity.

The evolutionally conserved chaperone-like protein CDC48 (cell division cycle 48) is a major component of ubiquitin-dependent protein degradation pathways in animal and yeast and, more generally, of the protein quality control machinery. In plants, CDC48 plays essential regulatory functions in development and the possibly that it contributes to protein degradation through the ubiquitin-proteasome system (UPS) and the endoplasmic reticulum-associated protein degradation (ERAD) system has been reported. In this review we described recent findings highlighting a role for CDC48 in plant immunity. First data indicated that CDC48 is S-nitrosylated in plant cells undergoing an immune response, regulates the turnover of immune receptors and mediates the degradation of viral proteins. Furthermore its overexpression was associated to an exacerbated hypersensitive-like cell death. We also designed and reported here the first CDC48 interactome. The corresponding data confirm the closed interaction of CDC48 with components of the UPS and shed light on its putative regulatory function of S-adenosyl-methionine synthesis and metabolism. More generally, these investigations further support the concept that plant cells facing pathogen attack finely regulate the protein quality control machinery.

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.

NO signaling in plant immunity: a tale of messengers.

Nitric oxide (NO) is a free radical gas involved in a myriad of plant physiological processes including immune responses. How NO mediates its biological effects in plant facing microbial pathogen attack is an unresolved question. Insights into the molecular mechanisms by which it propagates signals reveal the contribution of this simple gas in complex signaling pathways shared with reactive oxygen species (ROS) and the second messenger Ca(2+). Understanding of the subtle cross-talks operating between these signals was greatly improved by the recent identification and the functional analysis of proteins regulated through S-nitrosylation, a major NO-dependent post-translational protein modification. Overall, these findings suggest that NO is probably an important component of the mechanism coordinating and regulating Ca(2+) and ROS signaling in plant immunity.

Protein S-nitrosylation: specificity and identification strategies in plants.

The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the biotin switch technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identified. Functional studies focused on specific proteins provided preliminary comprehensive views of how this PTM impacts the structure and function of proteins and, more generally, of how NO might regulate biological plant processes. The aim of this review is to detail the basic principle of protein S-nitrosylation, to provide information on the biochemical and structural features of the S-nitrosylation sites and to describe the proteomic strategies adopted to investigate this PTM in plants. Limits of the current approaches and tomorrow's challenges are also discussed.

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.

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.

COG5-CDG: expanding the clinical spectrum.

BACKGROUND: The Conserved Oligomeric Golgi (COG) complex is involved in the retrograde trafficking of Golgi components, thereby affecting the localization of Golgi glycosyltransferases. Deficiency of a COG-subunit leads to defective protein glycosylation, and thus Congenital Disorders of Glycosylation (CDG). Mutations in subunits 1, 4, 5, 6, 7 and 8 have been associated with CDG-II. The first patient with COG5-CDG was recently described (Paesold-Burda et al. Hum Mol Genet 2009; 18:4350-6). Contrary to most other COG-CDG cases, the patient presented a mild/moderate phenotype, i.e. moderate psychomotor retardation with language delay, truncal ataxia and slight hypotonia. METHODS: CDG-IIx patients from our database were screened for mutations in COG5. Clinical data were compared. Brefeldin A treatment of fibroblasts and immunoblotting experiments were performed to support the diagnosis. RESULTS AND CONCLUSION: We identified five new patients with proven COG5 deficiency. We conclude that the clinical picture is not always as mild as previously described. It rather comprises a broad spectrum with phenotypes ranging from mild to very severe. Interestingly, on a clinical basis some of the patients present a significant overlap with COG7-CDG, a finding which can probably be explained by subunit interactions at the protein level.

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.

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.

PUGNAc treatment leads to an unusual accumulation of free oligosaccharides in CHO cells.

Free oligosaccharides (fOS) are generated as the result of N-glycoproteins catabolism that occurs in two distinct principal pathways: the endoplasmic reticulum-associated degradation (ERAD) of misfolded newly synthesized N-glycoproteins and the mature N-glycoproteins turnover pathway. The O-(2-acetamidO-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc) is a potent inhibitor of the O-GlcNAcase (OGA) catalysing the cleavage of ß-O-linked 2-acetamido-2-deoxy-ß-D-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationaly O-GlcNAc modified proteins. In order to estimate the impact of O-GlcNAc modification on N-glycoproteins catabolism, fOS were analysed by mass spectrometry (MS). MS analysis revealed the appearance of an unusual population of fOS after PUGNAc treatment. The structures representing this population have been identified as containing non-reducing end GlcNAc residues resulting from incomplete lysosomal fOS degradation. Only observed after PUGNAc treatment, the NButGt, another OGA inhibitor, did not lead to the appearance of this population. These abnormal fOS structures have clearly been shown to accumulate in membrane fractions as the consequence of lysosomal ß-hexosaminidases inhibition by PUGNAc. As lysosomal storage disorders (LSD) are characterized by the accumulation of storage material as fOS in lysosomes, our study evokes that the use of PUGNAc could mimic a LSD. This study clearly points out another off target effects of PUGNAc that need to be taken into account in the use of this drug.

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.