Proteomics data of SNF1-related protein kinase 2.4 interacting proteins revealed by immunoprecipitation-mass spectrometry.

Identification of kinase substrates is a prerequisite for elucidating the mechanism by which a kinase transduces internal or external stimuli to cellular responses. Conventional methods to profile this type of protein-protein interaction typically deal with one kinase-substrate pair at a time. Mass spectrometry-based proteomics, on the other hand, can determine putative kinase-substrate pairs at a large-scale in an unbiased manner. In this study, we identified the interacting partners of SNF1-related protein kinase 2.4 (SnRK2.4) via immunoprecipitation coupled with mass spectrometry. Proteins from stable transgenic Arabidopsis plants overexpressing a FLAG-tagged SnRK2.4 (cloned from Brassica napus) were pulled down using an anti-FLAG antibody. The protein components were then identified by mass spectrometry. In parallel, proteins from wild type plants were also analyzed, providing a list of nonspecific binding proteins that were further removed from the candidate SnRK2.4-interacting protein list. Our data identified over 30 putative SnRK2.4 interacting partners, which included many key players in stress responses, transport, and cellular metabolic processes.

Proteomic characterization of MPK4 signaling network and putative substrates.

KEY MESSAGE: Combining genetic engineering of MPK4 activity and quantitative proteomics, we established an in planta system that enables rapid study of MPK4 signaling networks and potential substrate proteins. Mitogen activated protein kinase 4 (MPK4) is a multifunctional kinase that regulates various signaling events in plant defense, growth, light response and cytokinesis. The question of how a single protein modulates many distinct processes has spurred extensive research into the physiological outcomes resulting from genetic perturbation of MPK4. However, the mechanism by which MPK4 functions is still poorly understood due to limited data on the MPK4 networks including substrate proteins and downstream pathways. Here we introduce an experimental system that combines genetic engineering of kinase activity and quantitative proteomics to rapidly study the signaling networks of MPK4. First, we transiently expressed a constitutively active (MPK4CA) and an inactive (MPK4IN) version of a Brassica napus MPK4 (BnMPK4) in Nicotiana benthamiana leaves. Proteomics analysis revealed that BnMPK4 activation affects multiple pathways (e.g., metabolism, redox regulation, jasmonic acid biosynthesis and stress responses). Furthermore, BnMPK4 activation also increased protein phosphorylation in the phosphoproteome, from which putative MPK4 substrates were identified. Using protein kinase assay, we validated that a transcription factor TCP8-like (TCP8) and a PP2A regulatory subunit TAP46-like (TAP46) were indeed phosphorylated by BnMPK4. Taken together, we demonstrated the utility of proteomics and phosphoproteomics in elucidating kinase signaling networks and in identification of downstream substrates.

MPK4 Phosphorylation Dynamics and Interacting Proteins in Plant Immunity.

Arabidopsis MAP kinase 4 (MPK4) has been proposed to be a negative player in plant immunity, and it is also activated by pathogen-associated molecular patterns (PAMPs), such as flg22. The molecular mechanisms by which MPK4 is activated and regulates plant defense remain elusive. In this study, we investigated Arabidopsis defense against a bacterial pathogen Pseudomonas syringae pv tomato ( Pst) DC3000 when Brassica napus MPK4 ( BnMPK4) is overexpressed. We showed an increase in pathogen resistance and suppression of jasmonic acid (JA) signaling in the BnMPK4 overexpressing (OE) plants. We also showed that the OE plants have increased sensitivity to flg22-triggered reactive oxygen species (ROS) burst in guard cells, which resulted in enhanced stomatal closure compared to wild-type (WT). During flg22 activation, dynamic phosphorylation events within and outside of the conserved TEY activation loop were observed. To elucidate how BnMPK4 functions during the defense response, we used immunoprecipitation coupled with mass spectrometry (IP-MS) to identify BnMPK4 interacting proteins in the absence and presence of flg22. Quantitative proteomic analysis revealed a shift in the MPK4-associated protein network, providing insight into the molecular functions of MPK4 at the systems level.

Quantitative proteomics reveals a role of JAZ7 in plant defense response to Pseudomonas syringae DC3000.

Jasmonate ZIM-domain (JAZ) proteins are key transcriptional repressors regulating various biological processes. Although many studies have studied JAZ proteins by genetic and biochemical analyses, little is known about JAZ7-associated global protein networks and how JAZ7 contributes to bacterial pathogen defense. In this study, we aim to fill this knowledge gap by conducting unbiased large-scale quantitative proteomics using tandem mass tags (TMT). We compared the proteomes of a JAZ7 knock-out line, a JAZ7 overexpression line, as well as the wild type Arabidopsis plants in the presence and absence of Pseudomonas syringae DC3000 infection. Both pairwise comparison and multi-factor analysis of variance reveal that differential proteins are enriched in biological processes such as primary and secondary metabolism, redox regulation, and response to stress. The differential regulation in these pathways may account for the alterations in plant size, redox homeostasis and accumulation of glucosinolates. In addition, possible interplay between genotype and environment is suggested as the abundance of seven proteins is influenced by the interaction of the two factors. Collectively, we demonstrate a role of JAZ7 in pathogen defense and provide a list of proteins that are uniquely responsive to genetic disruption, pathogen infection, or the interaction between genotypes and environmental factors. SIGNIFICANCE: We report proteomic changes as a result of genetic perturbation of JAZ7, and the contribution of JAZ7 in plant immunity. Specifically, the similarity between the proteomes of a JAZ7 knockout mutant and the wild type plants confirmed the functional redundancy of JAZs. In contrast, JAZ7 overexpression plants were much different, and proteomic analysis of the JAZ7 overexpression plants under Pst DC3000 infection revealed that JAZ7 may regulate plant immunity via ROS modulation, energy balance and glucosinolate biosynthesis. Multiple variate analysis for this two-factor proteomics experiment suggests that protein abundance is determined by genotype, environment and the interaction between them.

Identification of MAPK Substrates Using Quantitative Phosphoproteomics.

Activation of mitogen-activated protein kinases (MAPKs) under diverse biotic and abiotic factors and identification of an array of downstream MAPK target proteins are hot topics in plant signal transduction. Through interactions with a plethora of substrate proteins, MAPK cascades regulate many physiological processes in the course of plant growth, development, and response to environmental factors. Identification and quantification of potential MAPK substrates are essential, but have been technically challenging. With the recent advancement in phosphoproteomics, here we describe a method that couples metal dioxide for phosphopeptide enrichment with tandem mass tags (TMT) mass spectrometry (MS) for large-scale MAPK substrate identification and quantification. We have applied this method to a transient expression system carrying a wild type (WT) and a constitutive active (CA) version of a MAPK. This combination of genetically engineered MAPKs and phosphoproteomics provides a high-throughput, unbiased analysis of MAPK-triggered phosphorylation changes on the proteome scale. Therefore, it is a robust method for identifying potential MAPK substrates and should be applicable in the study of other kinase cascades in plants as well as in other organisms.

Distinct immunological activation profiles of dSLIM® and ProMune® depend on their different structural context.

INTRODUCTION: DNA-based TLR9 agonists are potent activators of the immune system. ProMune® and dSLIM® belong to different families of TLR9 agonists and both have been established as cancer immunotherapeutics in clinical proof-of-concept studies. Unfortunately, ProMune® failed in pivotal oncological trials. dSLIM®, the active ingredient of Lefitolimod (MGN1703), successfully finished a double-blinded, placebo-controlled phase II study in patients with advanced colorectal cancer, exhibiting improved progression-free survival and durable disease control. METHODS: To explain the different systemic efficacies of dSLIM® and ProMune®, both TLR9 agonists and chimeric molecules thereof are analyzed side-by-side in a panel of in vitro assays for immune activation. RESULTS AND CONCLUSIONS: Indeed, dSLIM® exposure results in an IFN-α dependent broad activation of immune cells whereas ProMune® strongly stimulates B cells. Moreover, all functional effects of dSLIM® strictly depend on the presence of CG-motifs within its dumbbell-shaped, covalently closed structural context. Conversely, several immunological effects of ProMune® like IL-8 secretion are independent of CG-motifs and could be ascribed to the phosphorothioate-modifications of its DNA backbone, which may have caused the side effects of ProMune® in clinical trials. Finally, we showed that the implementation of ProMune® (ODN2006) base sequence into the characteristic dSLIM® dumbbell form resulted in dSLIM2006 with all beneficial effects for immunostimulation combined from both TLR9 classes without any CG-independent effects.

Identification of thioredoxin targets in guard cell enriched epidermal peels using cysTMT proteomics.

Thioredoxins (Trx) play central roles in cellular redox regulation. Although hundreds of Trx targets have been identified using different approaches, the capture of targets in a quantitative and efficient manner is challenging. Here we report a high-throughput method using cysteine reactive tandem mass tag (cysTMT) labeling followed by liquid chromatography (LC)-mass spectrometry (MS) to screen for Trx targets. Compared to existing methods, this approach allows for i) three replicates of pairwise comparison in a single LC-MS run to reduce run-to-run variation; ii) efficient enrichment of cysteine-containing peptides that requires low protein input; and iii) accurate quantification of the cysteine redox status and localization of the Trx targeted cysteine residues. Application of this method in guard cell-enriched epidermal peels from Brassica napus revealed 80 Trx h targets involved in a broad range of processes, including photosynthesis, stress response, metabolism and cell signaling. The adaption of this protocol in other systems will greatly improve our understanding of the Trx function in regulating cellular redox homeostasis. BIOLOGICAL SIGNIFICANCE: Redox homeostasis is tightly regulated for proper cellular activities. Specific protein-protein interactions between redox active molecules such as thioredoxin (Trx) and target proteins constitute the basis for redox-regulated biological processes. The use of cysTMT quantitative proteomics for studying Trx reactions enabled identification of potential Trx targets that provide important insights into the redox regulation in guard cells, a specialized plant cell type responsible for sensing of environmental signals, gas exchange and plant productivity.

Effects of 2 polishing techniques and reglazing on the surface roughness of dental porcelain.

The aim of this study was to compare the effect of 2 polishing systems and reglazing of dental porcelain through a quantitative and qualitative analysis of surface roughness using a stylus profilometer and scanning electron microscope. Fifteen porcelain specimens (10 x 3 x 3 mm) were used. On 1 surface of each block, a layer of glaze was applied, and surface roughness (Ra) was analyzed. All specimens were ground with aluminum oxide sandpaper until the shine was removed and the resulting Ra values were obtained. Afterwards, they were randomly divided into 3 treatment groups (n = 5): Group I (GI), polished with diamond-impregnated rubber wheels; Group II (GII), polished with silicon carbide-impregnated rubber wheels; and Group III (GIII), reglazed firing procedure alone. After the treatments, new Ra measurements were done. Data were submitted to analysis of variance (ANOVA), and Tukey tests at 5%. Comparisons between ground surface and treated surface were made by paired t-test. The ground and treated Ra values (µm) were determined as follows: GI: 0.66 ± 0.14, 0.35 ± 0.06; GII: 0.60 ± 0.04, 0.09 ± 0.03; and GIII: 0.67 ± 0.05, 0.75 ± 0.24. Significant differences were found between the ground and treated values for all groups. After the treatments, all groups differed statistically (P < 0.05). The silicon carbide system re-established the initial surface smoothness, while polishing with diamond-impregnated rubber or reglazing alone were not able to achieve a satisfactory smoothness.

FMRP regulates actin filament organization via the armadillo protein p0071.

Loss of fragile X mental retardation protein (FMRP) causes synaptic dysfunction and intellectual disability. FMRP is an RNA-binding protein that controls the translation or turnover of a subset of mRNAs. Identifying these target transcripts is an important step toward understanding the pathology of the disease. Here, we show that FMRP regulates actin organization and neurite outgrowth via the armadillo protein p0071. In mouse embryonic fibroblasts (MEFs) lacking FMRP (Fmr1-), the actin cytoskeleton was markedly reorganized with reduced stress fibers and F-actin/G-actin ratios compared to fibroblasts re-expressing the protein. FMRP interfered with the translation of the p0071 mRNA in a 3'-UTR-dependent manner. Accordingly, FMRP-depleted cells revealed elevated levels of p0071 protein. The knockdown of p0071 in Fmr1- fibroblasts restored stress fibers and an elongated cell shape, thus rescuing the Fmr1- phenotype, whereas overexpression of p0071 in Fmr1+ cells mimicked the Fmr1- phenotype. Moreover, p0071 and FMRP regulated neurite outgrowth and branching in a diametrically opposed way in agreement with the negative regulation of p0071 by FMRP. These results identify p0071 as an important and novel FMRP target and strongly suggest that impaired actin cytoskeletal functions mediated by an excess of p0071 are key aspects underlying the fragile X syndrome.

Alpha-synuclein aggregation involves a bafilomycin A 1-sensitive autophagy pathway.

Synucleinopathies like Parkinson disease and dementia with Lewy bodies (DLB) are characterized by α-synuclein aggregates within neurons (Lewy bodies) and their processes (Lewy neurites). Whereas α-synuclein has been genetically linked to the disease process, the pathological relevance of α-synuclein aggregates is still debated. Impaired degradation is considered to result in aggregation of α-synuclein. In addition to the ubiquitin-proteasome degradation, the autophagy-lysosomal pathway (ALP) is involved in intracellular degradation processes for α-synuclein. Here, we asked if modulation of ALP affects α-synuclein aggregation and toxicity. We have identified an induction of the ALP markers LAMP-2A and LC3-II in human brain tissue from DLB patients, in a transgenic mouse model of synucleinopathy, and in a cell culture model for α-synuclein aggregation. ALP inhibition using bafilomycin A 1 (BafA1) significantly potentiates toxicity of aggregated α-synuclein species in transgenic mice and in cell culture. Surprisingly, increased toxicity is paralleled by reduced aggregation in both in vivo and in vitro models. The dichotomy of effects on aggregating and nonaggregating species of α-synuclein was specifically sensitive to BafA1 and could not be reproduced by other ALP inhibitors. The present study expands on the accumulating evidence regarding the function of ALP for α-synuclein degradation by isolating an aggregation specific, BafA1-sensitive, ALP-related pathway. Our data also suggest that protein aggregation may represent a detoxifying event rather than being causal for cellular toxicity.

Proficiency of PCR in hospital settings for nonculture diagnosis of invasive meningococcal infections.

BACKGROUND: Meningococcal meningitis requires rapid diagnosis and immediate management which is enhanced by the use of PCR for the ascertainment of these infections. However, its use is still restricted to reference laboratories. METHODS: We conducted an inter-laboratory study to assess the implementation and the performance of PCR in ten French hospital settings in 2010. RESULTS: Our data are in favour of this implementation. Although good performance was obtained in identifying Neisseria meningitidis positive samples, the main issue was reported in identifying other species (Streptococcus pneumoniae and Haemophilus influenzae) which are also involved in bacterial meningitis cases. CONCLUSIONS: Several recommendations are required and, mainly, PCR should target the major etiological agents (N. meningitidis, S. pneumonia, and H. influenzae) of acute bacterial meningitis. Moreover, PCR should predict the most frequent serogroups of Neisseria meningitidis according to local epidemiology.

Olfactory neuron-specific expression of A30P α-synuclein exacerbates dopamine deficiency and hyperactivity in a novel conditional model of early Parkinson's disease stages.

Mutations in the N-terminus of the gene encoding α-synuclein (α-syn) are linked to autosomal dominantly inherited Parkinson's disease (PD). The vast majority of PD patients develop neuropsychiatric symptoms preceding motor impairments. During this premotor stage, synucleinopathy is first detectable in the olfactory bulb (OB) and brain stem nuclei; however its impact on interconnected brain regions and related symptoms is still less far understood. Using a novel conditional transgenic mouse model, displaying region-specific expression of human mutant α-syn, we evaluated effect and reversibility of olfactory synucleinopathy. Our data showed that induction of mutant A30P α-syn expression increased transgenic deposition into somatodendritic compartment of dopaminergic neurons, without generating fibrillar inclusions. We found reversibly reduced levels of dopamine and metabolites in the OB, suggesting an impact of A30P α-syn on olfactory neurotransmitter content. We further showed that mutant A30P expression led to neurodegenerative changes on an ultrastructural level and a behaviorally hyperactive response correlated with novelty, odor processing and stress associated with an increased dopaminergic tone in midbrain regions. Our present data indicate that mutant (A30P) α-syn is directly implicated in reduction of dopamine signaling in OB interneurons, which mediates further alterations in brain regions without transgenic expression leading functionally to a hyperactive response. These modulations of neurotransmission may underlie in part some of the early neuropsychiatric symptoms in PD preceding dysfunction of the nigrostriatal dopaminergic system.

Use of recombinant human erythropoietin and risk of severe retinopathy in extremely low-birth-weight infants.

STUDY OBJECTIVES: To evaluate the frequency and severity of retinopathy of prematurity in extremely low-birth-weight (ELBW) infants who received recombinant human erythropoietin (rHuEPO), and to compare the frequency of blood cell transfusions these infants required with a matched control group who did not receive rHuEPO. DESIGN: Retrospective cohort analysis. SETTING: Level III neonatal intensive care unit in a large academic medical center. PATIENTS: One hundred thirty-eight ELBW infants who received rHuEPO and 138 ELBW infants who did not (control group) but who were matched by birth weight, gestational age, sex, and year of birth and who survived to the first ophthalmologic examination. MEASUREMENTS AND MAIN RESULTS: The rHuEPO was started before the 8th day of life in 115 (83%) of the 138 infants. Stages III-V retinopathy of prematurity occurred with similar frequency in both groups of infants (rHuEPO group 19% [26 infants] vs control group 20% [27 infants], p>0.05). Infants in the rHuEPO group received fewer transfusions on average during their hospitalization compared with those in the control group (4.2 vs 6.1 transfusions, p<0.01). CONCLUSION: Use of rHuEPO for prevention or treatment of anemia of prematurity in ELBW infants does not increase the frequency of severe retinopathy of prematurity and reduces the number of transfusions.