Identify Candidate Proteins Research Articles

Comparative proteomic analysis reveals novel insights into the continuous cropping induced response in Scrophularia ningpoensis.

Scrophularia ningpoensis is a well-known medicinal crop. Continuous cropping seriously affects the yield and quality, but little is known about the influence of continuous cropping on metabolic pathways. In this study, the difference in protein abundance between continuous cropping and non-continuous cropping of S. ningpoensis roots was studied by proteomics, and the molecular mechanism that protects S. ningpoensis against continuous cropping was explored. The results suggested that continuous cropping in S, ningpoensis altered the expression of proteins related to starch and sucrose metabolism, glycolysis/gluconeogenesis, pentose phosphate pathway, citric acid cycle, phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid biosynthesis, terpenoid backbone biosynthesis, monoterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, and steroid biosynthesis. Among these processes, the most affected were phenylpropanoid biosynthesis and starch and sucrose metabolism, which may be important for continuous cropping resistance. The effect of continuous cropping on S. ningpoensis was demonstrated at the proteome level in this work, and identified candidate proteins that may cause continuous cropping reactions. The paper provides the theoretical foundation and scientific reference for enhancing the continuous cropping resistance of S. ningpoensis. © 2022 Society of Chemical Industry.

Proteomics profiling identifies extracellular vesicles’ cargo associated with tumour cell induced platelet aggregation

BackgroundCancer patients have an increased risk of developing venous thromboembolism, with up to 30% dying within a month of their development. Some cancer cells are known to induce platelet aggregation, and this interaction is understood to contribute to thrombosis and haematogenous metastasis. Many researchers have reported on extracellular vesicles (EVs) released from platelets. However, less is known about how cancer cells’ EVs may affect platelet function. Here EVs released by triple-negative breast cancer (TNBC) cell line variants were extensively investigated in this regard.MethodsEVs were separated from conditioned media of TNBC Hs578T and Hs578Ts(i)8 cells using filtration and ultracentrifugation and were characterised by nanoparticle tracking analysis, immunoblots, and transmission electron microscopy. Blood samples from consenting donors were procured, and their platelets collected by differential centrifugation. Light transmission aggregometry and optical microscopy evaluated the potential interaction of TNBC cells and their EVs with platelets. Global proteomic analysis was performed on the EVs, by in-solution digestion and mass spectrometry. Data analysis included the use of Perseus, FunRich, and Vesiclepedia. Immunoblotting was used as a secondary method to investigate some key EV cargo proteins identified by the global proteomics approach.ResultsBoth TNBC cell variants induced platelet aggregation. Increasing cell numbers significantly reduced the time taken for platelet aggregation to occur. EVs released by the cells also resulted in platelet aggregation. The time to induce platelet aggregation was EV dose-dependent. Proteomics profiling and immunoblotting of the EVs’ cargo identified candidate proteins (including uPAR and PDGFRβ) that may be involved during this process.ConclusionsTNBC cells induce platelet aggregation. Furthermore, the cell-free EVs induced this undesirable effect. A number of EV cargo proteins were identified that may be relevant as therapeutic targets.

Identification of novel proteins associated with movement-related adverse antipsychotic effects by integrating GWAS data and human brain proteomes

Genome-wide association studies (GWAS) have identified some variants for movement-related adverse antipsychotic effects (MAAE), while how these variants confer MAAE remains unclear. We used the probabilistic Mendelian randomization (PMR) method to identify candidate proteins for MAAE by integrating MAAE GWASs and protein quantitative trait loci (pQTL) data. An independent pQTL data from the Banner project and brain-derived eQTL data were used to perform confirmatory PMR. A total of 56 proteins were identified as candidate targets for MAAE after false discovery rates (FDR) correction, such as GRIN2B, ADRA1A, and PED4B. 12 genes were replicated in the confirmatory PMR, and 18 genes had consistent evidence at the transcript level. Furthermore, we investigated the associations between candidate proteins and the motor symptoms of Parkinson's disease (PD). There were 24, 38, and 10 candidate proteins that were significantly associated with PD, PD motor subtypes, and PD motor progression, respectively. Enrichment analysis identified 34 GO terms and 17 pathways that may be involved in MAAE, such as glutamatergic synapse, glutamate receptor complex, and GABAergic synapse. Our study identified multiple candidate genes and pathways that were associated with MAAE, providing new insights into the biological mechanism of MAAE and targets for further mechanistic and therapeutic studies.

A large-scale polygenic risk score analysis identified candidate proteins associated with anxiety, depression and neuroticism

Psychiatric disorders and neuroticism are closely associated with central nervous system, whose proper functioning depends on efficient protein renewal. This study aims to systematically analyze the association between anxiety / depression / neuroticism and each of the 439 proteins. 47,536 pQTLs of 439 proteins in brain, plasma and cerebrospinal fluid (CSF) were collected from recent genome-wide association study. Polygenic risk scores (PRS) of the 439 proteins were then calculated using the UK Biobank cohort, including 120,729 subjects of neuroticism, 255,354 subjects of anxiety and 316,513 subjects of depression. Pearson correlation analyses were performed to evaluate the correlation between each protein and each of the mental traits by using calculated PRSs as the instrumental variables of protein. In general population, six correlations were identified in plasma and CSF such as plasma protease C1 inhibitor (C1-INH) with neuroticism score (r = − 0.011, P = 2.56 × 10− 9) in plasma, C1-INH with neuroticism score (r = -0.010, P = 3.09 × 10− 8) in CSF, and ERBB1 with self-reported depression (r = − 0.012, P = 4.65 × 10− 5) in CSF. C1-INH and ERBB1 may induce neuroticism and depression by affecting brain function and synaptic development. Gender subgroup analyses found that BST1 was correlated with neuroticism score in male CSF (r = − 0.011, P = 1.80 × 10− 5), while CNTN2 was correlated with depression score in female brain (r = − 0.013, P = 6.43 × 10− 4). BST1 and CNTN2 may be involved in nervous system metabolism and brain health. Six common candidate proteins were associated with all three traits (P < 0.05) and were confirmed in relevant proteomic studies, such as C1-INH in plasma, CNTN2 and MSP in the brain. Our results provide novel clues for revealing the roles of proteins in the development of anxiety, depression and neuroticism.

Network‐based quantitative proteomics identified significant proteins associated with growth heterosis in triploid fish

Triploid fish show significant growth heterosis compared to their parents, but its underlying molecular mechanisms are still largely unknown. In this study, Sequential Window Acquisition of All Theoretical Mass Spectra-based quantitative proteomic profiling was performed on the liver tissues from triploid crucian carp (TCC) and its parents red crucian carp (RCC) and allotetraploid (4nAT). Comparative proteomics analysis revealed that several causal proteins were differentially regulated between TCC and its parents. Particularly, proteins eno3 (p = 1.18E-3, ratio = 2.86 between TCC and RCC; p = 4.76E-4, ratio = 4.92 between TCC and 4nAT) and glud1a (p = 4.4E-7, ratio = 9.21 between TCC and RCC; p = 7.59E-7, ratio = 4.57 between TCC and 4nAT) were significantly upregulated in triploid fish. Multiple bioinformatics analysis including functional enrichment, pathway analysis and protein–protein interaction network construction further suggested that these candidate proteins play crucial roles in regulating growth heterosis by mediating important metabolic pathways including ‘carbohydrate metabolic process’, ‘glycolytic process’ and ‘glycolysis/gluconeogenesis’ and ‘carbon metabolism’. They may accelerate the processes of substrate and energy metabolism to meet the requirement of body growth. The identified candidate proteins such as eno3, glud1a in this study may serve as significant target indicators for further research involved in hybrid breeding and variety improvement.

RNF213-associated urticarial lesions with hypercytokinemia

Urticarial lesions are observed in both cutaneous and systemic disorders. Familial forms of urticarial syndromes are rare and can be encountered in systemic autoinflammatory diseases. We sought to investigate a large family with dominantly inherited chronic urticarial lesions associated with hypercytokinemia. We performed a genetic linkage analysis in 14 patients from a 5-generation family, as well as whole-exome sequencing, cytokine profiling, and transcriptomic analyses on samples from 2 patients. The identified candidate protein was studied after invitro expression of the corresponding normal and mutated recombinant proteins. An unsupervised proteomic approach was used to unveil the associated protein network. The disease phenotype of the most affected family members is characterized by chronic urticarial flares associated with extremely high plasma levels of proinflammatory (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-10 and IL-1 receptor antagonist [IL-1RA]) cytokines, with no secondary organ dysfunction, no susceptibility to infections, no fever, and normal C-reactive protein levels. Monocyte transcriptomic analyses identified an immunotolerant profile in the most affected patient. The affected family members carried a loss-of-function mutation in RNF213 that encodes mysterin, a protein with a poorly known physiologic role. We identified the deubiquitinase CYLD, a major regulator of inflammation, as an RNF213 partner and showed that CYLD expression is inhibited by wild-type but not mutant RNF213. We identified a new entity characterized by chronic urticarial lesions associated with a clinically blunted hypercytokinemia. This disease, which is due to loss of function of RNF213, reveals mysterin's key role in the complex molecular network of innate immunity.

Potential Proteins Interactions with Bombyx mori Nucleopolyhedrovirus Revealed by Co-Immunoprecipitation.

Simple SummaryThe Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical model baculovirus, representing one of the major pathogens of the silkworm. We reconstituted the virus in vitro and used it as a bait for immunoprecipitation experiments on cells and silkworm bodies, obtaining a database of proteins potentially interacting with BmNPV. This study provides a protein data reference for the screening of BmNPV receptors and the study of proteins that play a key role in the replication of BmNPV. It is also important to deal with the prevention of BmNPV in silkworms at the molecular level.Virus–host interactions are critical for virus replication, virulence, and pathogenicity. The Bombyx mori nucleopolyhedrovirus (BmNPV) is a typical model baculovirus, representing one of the most common and harmful pathogens in sericulture. Herein, we used co-immunoprecipitation to identify candidate proteins with potential interactions with BmNPV. First, a recombinant BV virus particle rBmBV-egfp-p64-3×flag-gp64sp was constructed using a MultiBac baculovirus multigene expression system. Co-immunoprecipitation experiments were then performed with the recombinant BV virus infected with BmN cells and Dazao silkworms. LC-MS/MS analysis revealed a total of 845 and 1368 candidate proteins were obtained from BmN cells and silkworm samples, respectively. Bioinformatics analysis (Gene Ontology, KEGG Pathway) was conducted for selection of proteins with significant enrichment for further confirmation of the effects on BmNPV replication. Overall, the results showed that SEC61 and PIC promoted the replication of BmNPV, while FABP1 inhibited the replication of BmNPV. In summary, this study reveals the potential proteins involved in BmNPV invasion and proliferation in the host and provides a platform for identifying the potential receptor proteins of BmNPV.

Proteomic Profiling of Cryoglobulinemia.

ObjectiveWe aimed to explore and identify candidate protein biomarkers of cryoglobulinemia (CGE) in disease control patients with negative cryoglobulin (DC) or healthy controls (HCs).MethodsThe tandem mass tag (TMT)-labeled serum quantitative proteomics approach was used to identify differentially expressed proteins between the CGE and DC groups. Ingenuity pathway analysis was used for functional annotation of differentially expressed proteins. Biomarker candidates were validated in another cohort using the parallel reaction monitoring (PRM) method. Apolipoprotein A1 (APOA1), apolipoprotein CIII (APOC3), adiponectin, and proprotein convertase subtilisin/kexin type-9 (PCSK9), which represent key proteins involved in the cholesterol metabolism pathway, were further verified in an increased number of samples by enzyme-linked immunosorbent assay (ELISA).ResultsA total of 1004 proteins were identified, of which 109 proteins were differentially expressed between the CGE and DC groups. These differentially expressed proteins were primarily involved in hepatic fibrosis/hepatic stellate cell activation and immune/inflammation-related pathways. In the disease and biofunction analysis, these proteins were mainly associated with the adhesion of blood cells, leukocyte migration, cholesterol transport, and transport of lipids. Twelve candidate biomarkers were validated by PRM-based proteomics, and proteins involved in the cholesterol metabolism pathway were further verified. APOA1, APOC3, adiponectin and PCSK9 concentrations were increased in CGE patients compared with healthy controls (P=0.0123, 0.1136, 0.5760, and 0.0019, respectively).ConclusionThis report describes the first application of a TMT-PRM-ELISA workflow to identify and validate CGE-specific biomarkers in serum. APOA1 and PCSK9 have been confirmed to be increased in CGE patients, demonstrating that proteins involved in cholesterol metabolism are also implicated in the development of CGE. These findings contribute to pathogenesis research and biomarker discovery in CGE.

Molecular Characterization of Irreversible Pulpitis: A Protocol Proposal and Preliminary Data

IntroductionAn attempt to determine the association of a large array of inflammatory proteins in pulpitis with precise measurement of clinical signs and symptoms, and to correlate these findings with levels in peripheral blood has not been reported. Such an analysis could serve to identify key clinical findings and potential biomarkers to predict the prognosis of vital pulp therapy. The aim of this study was to undertake a preliminary, proof-of-concept study to correlate the levels of key inflammatory mediators in cariously exposed dental pulp of adults with reversible or irreversible pulpitis, and no apical periodontitis, with a panel of subjective and objective diagnostic clinical findings as well as the status of the pulp upon exposure. Pulpal and peripheral blood inflammatory mediators were also compared.MethodsDental pulp and peripheral blood were sampled. The Luminex technology was used to assess the expression of a panel of 45 inflammatory proteins to determine their association with clinical signs and symptoms of reversible or irreversible pulpitis.ResultsData from three pulpal and three peripheral blood samples were used for the analysis. The correlation of levels of the 45 proteins in the inflamed dental pulp and peripheral blood was 0.87. The pulp had significantly higher levels of these proteins collectively than peripheral blood (t-test, p = 0.047). The following proteins had correlated at a level of ≥0.8 with the duration of pain with cold: MMP-12, MMP-9, RANTES, MIP-2, MCP-1, MMP-2, MMP-1, and P-Selectin. Relatively high correlations (0.5-0.75) were also present between these proteins and presenting pain level.ConclusionsSeveral pulpal proteins correlated well with spontaneous and evoked pain parameters. Peripheral blood may not be necessary in future similar studies. Finally, additional data is needed to identify candidate proteins to be investigated as potential markers of truly irreversible pulp inflammation.

Cathepsin D as biomarker in cerebrospinal fluid of nusinersen-treated patients with spinal muscular atrophy.

The therapeutic landscape of spinal muscular atrophy (SMA) has changed dramatically during the past 4years, but treatment responses differ remarkably between individuals, and therapeutic decision-making remains challenging, underlining the persistent need for validated biomarkers. We applied untargeted proteomic analyses to determine biomarkers in cerebrospinal fluid (CSF) samples of SMA patients under treatment with nusinersen. Identified candidate proteins were validated in CSF samples of SMA patients by Western blot and enzyme-linked immunosorbent assay. Furthermore, levels of peripheral neurofilament heavy and light chain were determined. Untargeted proteomic analysis of CSF samples of three SMA type 1 patients revealed the lysosomal protease cathepsin D as a candidate biomarker. Subsequent validation analysis in a larger cohort of 31 pediatric SMA patients (type 1, n=12; type 2, n=9; type 3, n=6; presymptomatically treated, n=4; age = 0-16years) revealed a significant decline of cathepsin D levels in SMA patients aged ≥2months at the start of treatment. Although evident in all older age categories, this decline was only significant in the group of patients who showed a positive motor response. Moreover, downregulation of cathepsin D was evident in muscle biopsies of SMA patients. We identified a decline of cathepsin D levels in CSF samples of SMA patients under nusinersen treatment that was more pronounced in the group of "treatment responders" than in "nonresponders." We believe that our results indicate a suitability of cathepsin D levels as a possible biomarker in SMA also in older patients, in combination with analysis of peripheral neurofilament light chain in adolescents or alone in adult patients.

Proteomic Analysis of Control and Disrupted Endothelial Monolayers

The microvascular wall regulates normal blood‐tissue exchange, but under inflammatory conditions, elevated endothelial permeability leads to edema, poor tissue oxygenation, and disrupted tissue homeostasis. Much remains unknown about the mechanisms involved. Using an unbiased proteomic analysis of confluent human umbilical vein endothelial cell (HUVEC) monolayers that were stimulated with 0.1 U/mL thrombin, which produces a time‐dependent increase in permeability that peaks 30 min and recovers to baseline levels at 120 min, novel candidate proteins that may participate in this mechanism were identified. Cell lysates were collected, proteins digested, and the peptides were characterized using a Thermo Q‐exactive‐HF mass spectrometer coupled to a Thermo Easy nLC 1200 to get label‐free quantitation (LFQ) of relative protein expression. High‐resolution mass spectrometric data were analyzed on the MaxQuant processing suite, and the protein groups file subsequently analyzed with the Perseus processing suite. The analysis revealed identified 4627 unique proteins present in HUVEC. Ratios of the LFQ intensities of 30 min. thrombin vs. control and 120 min. thrombin vs. control were generated. Statistical significance (p &lt; 0.05) for 30 min. thrombin vs. control or 120 min. thrombin vs. control was determined using Welch’s t‐test of log(2)‐transformed LFQ intensities. In addition, a z‐score cutoff calculated as [(t test difference of individual protein) − (median t test difference of dataset)] / (standard deviation of t test difference of dataset) was used. The criteria of |z‐score| &gt; 1 and p &lt; 0.05 with Welch’s t test were utilized to filter the dataset, identifying 214 proteins to have altered expression after 30 min thrombin treatment, and 172 proteins having altered expression after 120 min thrombin. When entering the filtered datasets into String‐db, tight junction signaling was identified as enriched with thrombin treatment. Employing a more stringent filtering approach to identify candidate proteins for further study (|z‐score| &gt; 2), we found that the tight junction protein claudin‐11 (CLDN11) had significantly decreased expression after 30 min thrombin (73% decrease from control) but was not significant changed at 120 min. Expression of CLDN11 was confirmed to be decreased by thrombin at 30 min using SimpleWestern (WES) analysis. To determine whether experimental depletion of CLDN11 expression decreases endothelial barrier function, selective CLDN11 siRNA was used. However, evaluation of barrier function using electric cell‐substrate impedance sensing showed no significant changes for up to 48 h after transfection with the siRNA, when CLDN11 protein levels were significantly decreased. Immunofluorescence labeling of CLDN11 in confluent HUVEC revealed that while some CLDN11 could be found in junctions, most labeling was punctate in the cytoplasm, possibly residing in vesicles. Collectively, the results suggest that thrombin significantly alters endothelial tight junctions and reduces CLDN11 expression. However, selective reduction of CLDN11 levels alone does not appear to be sufficient to alter HUVEC monolayer barrier function, and this may be because most CLDN11 fails to localize in junctions in this cultured endothelial cell model. Future studies will be directed at evaluating CLDN11 localization in postcapillary venules and determining its potential role in microvascular barrier function using intact microvessels and in vivo.

Human placental proteomics and exon variant studies link AAT/SERPINA1 with spontaneous preterm birth

BackgroundPreterm birth is defined as live birth before 37 completed weeks of pregnancy, and it is a major problem worldwide. The molecular mechanisms that lead to onset of spontaneous preterm birth are incompletely understood. Prediction and evaluation of the risk of preterm birth is challenging as there is a lack of accurate biomarkers. In this study, our aim was to identify placental proteins that associate with spontaneous preterm birth.MethodsWe analyzed the proteomes from placentas to identify proteins that associate with both gestational age and spontaneous labor. Next, rare and potentially damaging gene variants of the identified protein candidates were sought for from our whole exome sequencing data. Further experiments we performed on placental samples and placenta-associated cells to explore the location and function of the spontaneous preterm labor-associated proteins in placentas.ResultsExome sequencing data revealed rare damaging variants in SERPINA1 in families with recurrent spontaneous preterm deliveries. Protein and mRNA levels of alpha-1 antitrypsin/SERPINA1 from the maternal side of the placenta were downregulated in spontaneous preterm births. Alpha-1 antitrypsin was expressed by villous trophoblasts in the placenta, and immunoelectron microscopy showed localization in decidual fibrinoid deposits in association with specific extracellular proteins. siRNA knockdown in trophoblast-derived HTR8/SVneo cells revealed that SERPINA1 had a marked effect on regulation of the actin cytoskeleton pathway, Slit–Robo signaling, and extracellular matrix organization.ConclusionsAlpha-1 antitrypsin is a protease inhibitor. We propose that loss of the protease inhibition effects of alpha-1 antitrypsin renders structures critical to maintaining pregnancy susceptible to proteases and inflammatory activation. This may lead to spontaneous premature birth.

Proteomic Analysis of Autoimmune Retinopathy Implicates Neuronal Cell Adhesion Molecule as a Potential Biomarker

PurposeTo identify vitreous molecular biomarkers associated with autoimmune retinopathy (AIR).DesignCase-control study.ParticipantsWe analyzed 6 eyes from 4 patients diagnosed with AIR and 8 comparative controls diagnosed with idiopathic macular holes (IMHs) and epiretinal membranes (ERMs).MethodsVitreous biopsies were collected from the participants and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) or multiplex enzyme-linked immunoassay (ELISA).Main Outcome MeasuresProtein expression changes were evaluated by 1-way analysis of variance (significant P value < 0.05), hierarchical clustering, and pathway analysis to identify candidate protein biomarkers.ResultsThere were 16 significantly upregulated and 17 significantly downregulated proteins in the vitreous of 3 patients with AIR compared with controls. The most significantly upregulated proteins included lysozyme C, zinc-alpha-2-glycoprotein, complement factor D, transforming growth factor-ß (TGF-ß)–induced protein, beta-crystallin B2, and alpha-crystallin A chain. The most significantly downregulated proteins included DIP2C, retbindin, and amyloid beta precursor-like protein 2. Pathway analysis revealed that vascular endothelial growth factor (VEGF) signaling was a top represented pathway in the vitreous of patients with AIR compared with controls. In comparison with a different cohort of 3 patients with AIR analyzed by multiplex ELISA, a commonly differentially expressed protein was neuronal cell adhesion molecule (NrCAM) with P values of 0.027 in the LC-MS/MS dataset and 0.035 in the ELISA dataset.ConclusionsProtein biomarkers in the vitreous, such as NrCAM, may eventually help diagnose AIR.

Microautophagy Mediates Vacuolar Delivery of Storage Proteins in Maize Aleurone Cells.

The molecular machinery orchestrating microautophagy, whereby eukaryotic cells sequester autophagic cargo by direct invagination of the vacuolar/lysosomal membrane, is still largely unknown, especially in plants. Here, we demonstrate microautophagy of storage proteins in the maize aleurone cells of the endosperm and analyzed proteins with potential regulatory roles in this process. Within the cereal endosperm, starchy endosperm cells accumulate storage proteins (mostly prolamins) and starch whereas the peripheral aleurone cells store oils, storage proteins, and specialized metabolites. Although both cell types synthesize prolamins, they employ different pathways for their subcellular trafficking. Starchy endosperm cells accumulate prolamins in protein bodies within the endoplasmic reticulum (ER), whereas aleurone cells deliver prolamins to vacuoles via an autophagic mechanism, which we show is by direct association of ER prolamin bodies with the tonoplast followed by engulfment via microautophagy. To identify candidate proteins regulating this process, we performed RNA-seq transcriptomic comparisons of aleurone and starchy endosperm tissues during seed development and proteomic analysis on tonoplast-enriched fractions of aleurone cells. From these datasets, we identified 10 candidate proteins with potential roles in membrane modification and/or microautophagy, including phospholipase-Dα5 and a possible EUL-like lectin. We found that both proteins increased the frequency of tonoplast invaginations when overexpressed in Arabidopsis leaf protoplasts and are highly enriched at the tonoplast surface surrounding ER protein bodies in maize aleurone cells, thus supporting their potential connections to microautophagy. Collectively, this candidate list now provides useful tools to study microautophagy in plants.

The antioxidant protein ZmPrx5 contributes resistance to maize stalk rot

Maize (Zea mays L.) stalk rot is a devastating disease worldwide, causing severe yield losses. Although previous studies have focused on the genetic dissection of maize resistance to stalk rot, the mechanisms of resistance remain largely unknown. We used a comparative proteomics approach to identify candidate proteins associated with stalk rot resistance. Statistical analyses revealed 763 proteins differentially accumulated between Fusarium graminearum and mock-inoculated plants. Among them, the antioxidant protein ZmPrx5, which was up-accumulated in diseased plants, was selected for further study. ZmPrx5 transcripts were present in root, stalk, leaf, ear, and reproductive tissues. The expression of ZmPrx5 in three inbred lines increased significantly upon F. graminearum infection. ZmPrx5 was localized in the cytoplasm. Compared to control plants, maize plants overexpressing ZmPrx5 showed increased resistance to F. graminearum infection, and ZmPrx5 mutant plants were more susceptible than wild-type plants. Defense-associated pathways including plant–pathogen interactions, phenylalanine metabolism, and benzoxazinoid and flavonoid biosynthesis were suppressed in ZmPrx5 homozygous mutant plants compared with wild-type plants. We suggest that ZmPrx5 positively regulates resistance against stalk rot in maize, likely through defense-oriented transcriptome reprogramming. These results lay a foundation for further research on the roles of Prx5 subfamily proteins in resistance to plant fungal diseases, and provide a potential genetic resource for breeding disease-resistance maize lines.

Targeting Bclxl Mitigates Mcl1 Chemoresistance in Multiple Myeloma

Aim:Despite the adoption of novel therapeutic modalities, Multiple Myeloma (MM) remains incurable. The Bcl2 inhibitor Venetoclax is active in several haematologic malignancies, but the benefits in MM patients are limited to those with the t(11;14) and/or high Bcl2 expression. These results underscore the significance of Bcl2 alternative anti-apoptotic proteins (Mcl1 and BclxL) for the survival of myeloma cells.Method:We validated the anti MM effect of the Mcl1 inhibitor S63845 both in vitro utilising 11 human myeloma cell lines (HMCL) and ex vivo against n=30 primary MM tumours. Comparative analysis of RNAseq between S63845 sensitive and resistant HMCL was undertaken to identify candidate proteins that potentially modulate resistance to S63845. Treatment with S63845 and rationally selected combination partners was further evaluated in vitro, ex vivo and in vivo with flow cytometry, immunoblotting and live imaging mitochondria fitness monitoring.Results:RNAseq identified BclxL as potential mediator of resistance to S63845 in HMCL. Immunoblotting confirmed high BclxL expression and high BclxL/BclS in S63845 resistant HMCL. Five S63845 resistant HMCL (U266, ANBL6, KMS28PE, EJM, MM1R) and primary tumours were treated with S63845 combined with the BclxL inhibitor A1331852 . Combined treatment of the HMCL demonstrated a high Bliss synergy score for all the HMCL tested (54, 42, 24, 47, 45 for U266, EJM, KMS28PE, MM1R and ANBL6 respectively) and induced synergistic killing of 80% of the primary tumours treated. Dual inhibition in U266 induced an 80% drop in intracellular ATP at 4h with an increase in active Caspases 9 and 8 (4.5 and 5 fold, respectively). Similarly, the combination induced a 78% drop in mitochondrial transmembrane potential (TMRE intensity) by 4h with live imaging revealing striking mitochondrial damage as early as 40 minutes after exposure (figure). These changes were associated with a reduction of both Mcl1 and, BclxL proteins and Bim and Bid protein levels. No changes were seen in the level of Bcl2, Bak or Bax protein expression. The combination of S63845 and A1331852 in healthy NSG mice at 12.5mg/kg proved lethal due to hepatotoxicity, arguing against the clinical utility of such an approach. However, this observed anti-MM synergistic activity was recapitulated when S63845 was combined with the already approved anti-MM therapeutic panobinostat, with the induction of a significant reduction in both BclxL and Myc protein levels at 24h, and synergistic killing of 56% of primary tumours.Conclusion:High BclxL expression and BclxL/BclxS ratio correlates with resistance to the Mcl1 inhibitor S63845. A combinatorial approach targeting Mcl1 and BclxL induced immediate and significant anti-MM effect both in vitro and ex vivo but proved to be toxic in vivo. Combination of the anti-MM therapeutic panobinostat in combination with S635845 recapitulated the anti-MM activity seen with A1331852 and warrants further evaluation. [Display omitted] DisclosuresSpencer: Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria.

Establishing the Role of EPS15, DVL2 and Cortactin in CALM-AF10 Leukemogenesis

Background:Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoblastic leukemia (T-ALL)as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXA genes. To identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS).Methods:We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with BioID2-CALM-AF10 and grown in the presence or absence of biotin. MS was performed to identify candidate interacting proteins. We validated direct interactions of candidate proteins with CALM-AF10 using co-immunoprecipitation experiments in HEK293 cells transfected with a CALM-AF10 plasmid. We confirmed that candidate proteins are present in murine CALM-AF10 leukemia cells via Western blotting. In order to efficiently knockout (KO) candidate proteins, we have generated a human U937 cell line (which harbors a t(10;11) CALM-AF10 translocation) with a stable incorporated Cas9. To assess whether KO of EPS15, DVL2 or CTTN affects HOXA5 expression, we performed RT-qPCR in U937-Cas9 cells lines with confirmed KO.Results:We carried out three independent transfections/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 12 candidates were common to all three experiments . Importantly, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that interacts with CRM1 and that is involved in leukemogenic translocations. We chose EPS15, DVL2 and CTTN for further study, as each of these proteins plays a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that all three proteins co-precipitate with CALM-AF10. Western blotting showed that all three proteins are expressed in a murine CALM-AF10 leukemia cell line. We effectively knocked out EPS15 protein expression in U937 cells, and showed that HOXA5 expression is reduced in the setting of EPS15 knockout.Conclusion:We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate three candidate proteins - EPS15, DVL2 and CTTN - all of which are involved in leukemogenic transformation. We have shown that EPS15, DVL2 and CTTN are expressed in murine CALM-AF10 leukemia cells and directly interact with the CALM-AF10 fusion protein. Knockout of EPS15 in U937 cells results in decreased HOXA5 expression, suggesting the importance of EPS15 in CALM-AF10 leukemogenesis. Evaluation of the roles of these proteins in leukemogenesis may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

The MttB superfamily member MtyB from the human gut symbiont Eubacterium limosum is a cobalamin-dependent γ-butyrobetaine methyltransferase

The production of trimethylamine (TMA) from quaternary amines such as l-carnitine or γ-butyrobetaine (4-(trimethylammonio)butanoate) by gut microbial enzymes has been linked to heart disease. This has led to interest in enzymes of the gut microbiome that might ameliorate net TMA production, such as members of the MttB superfamily of proteins, which can demethylate TMA (e.g., MttB) or l-carnitine (e.g., MtcB). Here, we show that the human gut acetogen Eubacterium limosum demethylates γ-butyrobetaine and produces MtyB, a previously uncharacterized MttB superfamily member catalyzing the demethylation of γ-butyrobetaine. Proteomic analyses of E. limosum grown on either γ-butyrobetaine or dl-lactate were employed to identify candidate proteins underlying catabolic demethylation of the growth substrate. Three proteins were significantly elevated in abundance in γ-butyrobetaine-grown cells: MtyB, MtqC (a corrinoid-binding protein), and MtqA (a corrinoid:tetrahydrofolate methyltransferase). Together, these proteins act as a γ-butyrobetaine:tetrahydrofolate methyltransferase system, forming a key intermediate of acetogenesis. Recombinant MtyB acts as a γ-butyrobetaine:MtqC methyltransferase but cannot methylate free cobalamin cofactor. MtyB is very similar to MtcB, the carnitine methyltransferase, but neither was detectable in cells grown on carnitine nor was detectable in cells grown with γ-butyrobetaine. Both quaternary amines are substrates for either enzyme, but kinetic analysis revealed that, in comparison to MtcB, MtyB has a lower apparent Km for γ-butyrobetaine and higher apparent Vmax, providing a rationale for MtyB abundance in γ-butyrobetaine-grown cells. As TMA is readily produced from γ-butyrobetaine, organisms with MtyB-like proteins may provide a means to lower levels of TMA and proatherogenic TMA-N-oxide via precursor competition.

Multi-tiered analyses of honey bees that resist or succumb to parasitic mites and viruses

BackgroundVarroa destructor mites, and the numerous viruses they vector to their honey bee hosts, are among the most serious threats to honey bee populations, causing mortality and morbidity to both the individual honey bee and colony, the negative effects of which convey to the pollination services provided by honey bees worldwide. Here we use a combination of targeted assays and deep RNA sequencing to determine host and microbial changes in resistant and susceptible honey bee lineages. We focus on three study sets. The first involves field sampling of sympatric western bees, some derived from resistant stock and some from stock susceptible to mites. The second experiment contrasts three colonies more deeply, two from susceptible stock from the southeastern U.S. and one from mite-resistant bee stock from Eastern Texas. Finally, to decouple the effects of mites from those of the viruses they vector, we experimentally expose honey bees to DWV in the laboratory, measuring viral growth and host responses.ResultsWe find strong differences between resistant and susceptible bees in terms of both viral loads and bee gene expression. Interestingly, lineages of bees with naturally low levels of the mite-vectored Deformed wing virus, also carried lower levels of viruses not vectored by mites. By mapping gene expression results against current ontologies and other studies, we describe the impacts of mite parasitism, as well as viruses on bee health against two genetic backgrounds. We identify numerous genes and processes seen in other studies of stress and disease in honey bee colonies, alongside novel genes and new patterns of expression.ConclusionsWe provide evidence that honey bees surviving in the face of parasitic mites do so through their abilities to resist the presence of devastating viruses vectored by these mites. In all cases, the most divergence between stocks was seen when bees were exposed to live mites or viruses, suggesting that gene activation, rather than constitutive expression, is key for these interactions. By revealing responses to viral infection and mite parasitism in different lineages, our data identify candidate proteins for the evolution of mite tolerance and virus resistance.

PDGF as an Important Initiator for Neurite Outgrowth Associated with Fibrovascular Membranes in Proliferative Diabetic Retinopathy

ABSTRACT Purpose The formation of fibrovascular membranes (FVMs) is a serious sight-threatening complication of proliferative diabetic retinopathy (PDR) that may result in retinal detachment and eventual blindness. During the formation of these membranes, neurite/process outgrowth occurs in retinal neurons and glial cells, which may both serve as a scaffold and have guiding or regulatory roles. To further understand this process, we investigated whether previously identified candidate proteins, from vitreous of PDR patients with FVMs, could induce neurite outgrowth in an experimental setting. Materials and methods Retinal explants of C57BL6/N mouse pups on postnatal day 3 (P3) were cultured in poly-L-lysine- and laminin-coated dishes. Outgrowth stimulation experiments were performed with the addition of potential inducers of neurite outgrowth. Automated analysis of neurite outgrowth was performed by measuring β–tubulin-immunopositive neurites using Image J. Expression of PDGF receptors was quantified by RT-PCR in FVMs of PDR patients. Results Platelet-derived growth factor (PDGF) induced neurite outgrowth in a concentration-dependent manner, whilst neuregulin 1 (NRG1) and connective tissue growth factor (CTGF) did not. When comparing three different PDGF dimers, treatment with PDGF-AB resulted in the highest neurite induction, followed by PDGF-AA and -BB. In addition, incubation of retinal explants with vitreous from PDR patients resulted in a significant induction of neurite outgrowth as compared to non-diabetic control vitreous from patients with macular holes, which could be prevented by addition of CP673451, a potent PDGF receptor (PDGFR) inhibitor. Abundant expression of PDGF receptors was detected in FVMs. Conclusion Our findings suggest that PDGF may be involved in the retinal neurite outgrowth, which is associated with the formation of FVMs in PDR.