Phosphorylation detection of protein DGCR8 between between control and coilin knockdown samples
Ontology highlight
ABSTRACT: The project is for identification and quantification of phosphorylated peptides of protein DGCR8 between control and coilin knockdown samples.
Project description:Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6,000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.
Project description:The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C-terminal fragments of the protein TDP-43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS-PRM) can generate a high-resolution map of pathological TDP-43 peptide ratios to form the basis for quantitation of abnormal C-terminal TDP-43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of phosphoTDP-43, p-tau, alpha-synuclein and beta-amyloid pathology, were biochemically fractionated and analysed by immunoblot and MS for detection of full-length and truncated (disease-specific) TDP-43 peptides. This informed synthesis of heavy isotope-labelled peptides for the absolute quantification of TDP-43 by MS-PRM across 16 ALS, 8 Parkinson’s and 8 Alzheimer’s disease and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C-terminal fragments in ALS-TDP urea fractions. Subsequent MS analysis resolved specific TDP-43 N- and C-terminal peptides, including a novel N-terminal truncation site-specific peptide. Absolute quantification of peptides by MS-PRM showed an increased C:N-terminal TDP-43 peptide ratio in ALS-TDP brain compared to normal and disease controls. A C:N-terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6-100) and specificity of 100% (CI 68-100), and from Parkinson’s and Alzheimer’s disease with a sensitivity of 93% (CI 70-100) and specificity of 100% (CI 68-100). N-terminal Truncation site-specific peptides were increased in ALS in line with C-terminal fragment enrichment, but also found in a proportion of Alzheimer cases with normal C:N-terminal ratio but coexistent TDP-43 pathology. In conclusion this is a novel, sensitive and specific method to quantify the enrichment of pathological TDP-43 fragments in human brain, which could form the basis for an antibody-free assay. Our methodology has the potential to help clarify if specific pathological TDP-43 peptide signatures are associated with primary or secondary TDP-43 proteinopathies.
Project description:While aging is well established as an important risk factor for the development and progression of atherosclerosis, the underlying molecular mechanisms of this relationship remain poorly defined, and its role in atherosclerosis regression is unknown. We investigated the response of bone marrow-derived macrophages isolated from old and young mice to atherogenic aggregated LDL.
Project description:Leucyl and cystinyl aminopeptidase (LNPEP/oxytocinase) is a key regulator of oxytocin. Our objectives were to measure serum concentrations of LNPEP and determine the presence of LNPEP and oxytocin (OXT) in equine tissue. LNPEP was measured by ELISA in serum obtained: every other day from ovulation (D0) until D21-22 in nonpregnant and pregnant mares; and at around 320 days of gestation, at 24 hours before foaling, and 20 min and 2 hrs post-foaling. Placenta from the pregnant horn (PH), nonpregnant horn (NPH) and body (B) and from tissues of 6 nonpregnant mares were homogenized and extracts obtained for protein determination and LNPEP ELISA assay. Identification of LNPEP and OXT protein in tissues was also performed via western blot (WB), immunohistochemistry (IHC) and liquid chromatography-mass spectrometry (LC-MS/MS). Furthermore, in situ hybridization was performed for LNPEP and OXT on endometrium, myometrium, pituitary and corpus luteum (CL). No difference in serum LNPEP concentration was identified between any of the groups. The NPH had the lowest quantities of LNPEP (P<0.05). The highest to lowest LNPEP U/mg protein by tissue were: myometrium> follicle wall> endometrium> kidney>CL> liver. Oxytocin was identified in the equine pituitary, CL and placenta. LNPEP may regulate the availability of luteal OXT locally.
Project description:Candida albicans is the most common fungal pathogen in humans. C.albicans tolerates aneuploidy of all of its chromosomes. Genome plasticity is a hallmark of C.albicans. It is an adaptation strategy of this species. But questions like the extent of such genomic variability, which genes contribute to the divergence, and what mechanisms drive the adaptive genetic change, are not well answered yet. We used array-based comparative genomic hybridization (array CGH) to investigate the diversity of gene contents of 10 clinical C.albicans strains, of various anatomical origins and genotypes. One self to self hybridization was included as a control.
Project description:Cold-sensitive Cavendish Banana and relatively cold-tolerant Dajiao (Musa spp.) comprise an important part of diets for millions of people around the globe. Low temperature is one of the key environment stresses which greatly affect the global banana production. However, little is known about the changes of global protein phosphorylation in Musa spp. and their regulatory roles in response to cold stress. In this study, we employed a TMT6-plex quantitative analysis to conduct a global phosphoproteome profiling between Cavendish Banana and Dajiao subject to the cold stress for 0 hour and 3 hour. A total of 679 phosphopeptides containing 772 distinct phosphorylated sites from 529 phosphoproteins were identified in Cavendish Banana, 180 phosphorylation sites (belonging to 147 phosphoproteins) were differentially changed after 3 h cold stress. While in Dajiao 241 phosphopeptides with 271 individual phosphosites from 207 phosphoproteins were confidently identified, and 83 phosphorylation sites from 63 phosphoproteins were differentially changed under 3 h cold stress. Bioinformatic analysis of protein interaction network indicated that Mitogen-activated protein kinase kinase 2 (MKK2) was located in the center of the MAPK signaling network along with 7 other members whose phosphorylated site abundances were remarkably differentiated between Cavendish Banana and Dajiao in response to cold stress. Western blotting of MKK2 protein and its T31 phosphorylated site showed the increased expression of MKK2 in the time course of cold stress, with no detectable T31 phosphorylation in Cavendish Banana. On the contrary, the decreased MKK2 expression with increased T31 phosphorylation was consistently observed in Dajiao. These results suggest that the MKK2 interaction network in Dajiao, along with other cold-specific phosphoproteins found in this study, appears to play an important role in the molecular mechanisms of Dajiao being high tolerance to cold stress. The results also provide new evidence that cellular MKK2 phosphorylation as a signaling pathway plays an important role in abiotic stress tolerance that serves as a universal plant cold tolerance mechanism. To the best of our knowledge, this is the first report of MKK2 network involved in the regulatory of the Musa spp. response to cold stress.
Project description:Posttranslational modifications with ubiquitin alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the periphery of the ischemic territory, wherein cells remain viable. Revealing the identity of ubiquitinated proteins, their cellular location, and the functional consequences of ubiquitin modification may shed light on the role of ubiquitination in ischemic injury. Here, we employed a proteomics approach to identify proteins ubiquitinated following ischemic stroke, induced by transient middle cerebral artery occlusion (tMCAO) in mice. We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD93, PSD95 and Shank3, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with high post-ischemic ubiquitination levels were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, the aberrant activity of which contributes to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered phosphorylation patterns associated with the PSD, indicative of modified PSD kinase activity following stroke. CaMKII, PKC, and Cdk5 activity was decreased while Pyk2 activity was increased at the PSD after stroke, accompanied by the hypo- and hyper-phosphorylation of downstream targets, respectively. Removal of ubiquitin restored kinases’ activity to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of post-ischemic ubiquitination in the regulation of essential kinases involved in ischemic injury, and presents targeting ubiquitination as a potential new avenue for the treatment of ischemic stroke.
Project description:Primary human AML cells (newly diagnosed, prior to treatment initation) were obtained from donor after consent and AML blasts were isolated by standard Ficoll centrifugation. AML blasts were treated with FLT3 inhibitor gilteritinib (or DMSO vehicle control) at 10 nM concentration for 6 h, followed by harvest for LC/MS.
Project description:Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25% of acute myeloid leukemia (AML) patients and lead to constitutive activation of FLT3, driving leukemia cell survival and proliferation. Quizartinib, crenolanib and gilteritinib are second-generation FLT3 inhibitors (FLT3i) in phase III trials or clinical use for the targeted treatment of FLT3-ITD+ AML. However, they demonstrated only limited benefit and were not curative. A full understanding of cellular resistance factors contributing to this poor response is lacking. Here, we examined cell-autonomous pathways modulated by FLT3i using global translatome and phosphoproteome proteomics to identify non-genetic resistance mechanisms.
Project description:Cognitive decline in aging is a major issue, causing both personal and economic hardship in an increasingly aging society. There are several known individual misfolded proteins that cause issues with age, such as amyloid beta and alpha synuclein. However, many studies have found that the proteostasis network, which works to keep proteins properly folded, is impaired with age, suggesting that there may be more global protein structural changes. We used limited-proteolysis mass spectrometry (LiP-MS) to investigate protein structural changes proteome-wide in a rodent model of aging. We compared hippocampi from aged rodents with normal cognition (aged unimpaired, AU) to hippocampi from aged rodents with impaired cognition (aged impaired, AI). We identified several hundred proteins as Cognition-Associated Structural Changes (CASCs), which are structurally different between the AU and AI populations. We found correlations between these trends and those of protein refoldability, a separate measure of how well a protein can independently refold to its native state after complete denaturation. CASCs were enriched with nonrefoldable proteins. Potential confounding factors of our study such as LiP reproducibility and post-translational modifications were assessed. Searches for oxidation and phosphorylation did not yield significant differences between AU and AI samples within each hippocampal region. Our study overall suggests that neuronal protein structural changes are global in nature and are more often intrinsically nonrefoldable, which may partially explain their susceptibility to structural changes due to proteostasis network breakdown in age.