Activation of Ca2+ transport in cardiac microsomes enriches functional sets of ER and SR proteins.

The importance of sarcoplasmic reticulum (SR) Ca2+-handling in heart has led to detailed understanding of Ca2+-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca2+-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Proteins from 20.0 µg of MV, MedSR, and HighSR protein were fractionated using SDS-PAGE, then trypsinized from 20 separate gel pieces, and analyzed by LC-MS/MS to determine protein content. From 62,000 individual peptide spectra obtained, we identified 1105 different proteins, of which 354 were enriched ≥ 2.0-fold in SR fractions compared to the crude membrane preparation. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca2+ leak co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins constituting other putative ER/SR subdomains also exhibited average Esub enrichment values (mean ± S.D.) that spanned the range of possible Esub values, suggesting that functional sets of proteins are localized to the same areas of the ER/SR membrane. We conclude that active Ca2+ loading of cardiac microsomes, reflecting the combined activities of Ca2+ uptake by SERCA, and Ca2+ leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.

Extracellular vesicles from retinal pigment epithelial cells expressing R345W-Fibulin-3 induce epithelial-mesenchymal transition in recipient cells.

We have shown previously that expression of R345W-Fibulin-3 induces epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W-Fibulin-3 mutation are sufficient to induce EMT in recipient cells. ARPE-19 cells were infected with luciferase-tagged wild-type (WT)- Fibulin-3 or luciferase-tagged R345W-Fibulin-3 (R345W) using lentiviruses. EVs were isolated from the media by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC-MS/MS based proteomics. EV-associated transforming growth factor beta 1 (TGFß1) protein was measured by enzyme-linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT- or R345W-EVs. The role of EV-bound TGFß was determined by pre-incubation of EVs with a pan-TGFß blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W- EVs, while EMT drivers were enriched. Additionally, R345W-EVs had higher concentrations of TGFß1 compared to control. Critically, treatment with R345W-EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV-increased cell migration was significantly inhibited by pre-incubation of EVs with pan-TGFß-neutralising antibody. In conclusion, the expression of R345W-Fibulin-3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFß dependent manner. These results suggest that EV-bound TGFß plays a critical role in the induction of EMT in RPE cells.

Activation of Ca transport in cardiac microsomes enriches functional sets of ER and SR proteins.

The importance of sarcoplasmic reticulum (SR) Ca-handling in heart has led to detailed understanding of Ca-release and re-uptake protein complexes, while less is known about other endoplasmic reticulum (ER) functions in the heart. To more fully understand cardiac SR and ER functions, we analyzed cardiac microsomes based on their increased density through the actions of the SR Ca-ATPase (SERCA) and the ryanodine receptor that are highly active in cardiomyocytes. Crude cardiac microsomal vesicles loaded with Ca oxalate produced two higher density subfractions, MedSR and HighSR. Analyses of protein enrichments from the 3 membrane preparations (crude microsomes, MedSR, and HighSR), showed that only a third of microsomal proteins in heart, or 354 proteins, were enriched ≥2.0-fold in SR. Previously studied SR proteins were all enriched, as were proteins associated with canonical ER functions. Contractile, mitochondrial, and sarcolemmal proteins were not enriched. Comparing the levels of SERCA-positive SR proteins in MedSR versus HighSR vesicles produced a range of SR subfraction enrichments signifying differing levels of Ca leak (ryanodine receptor) co-localized in the same membrane patch. All known junctional SR proteins were more enriched in MedSR, while canonical ER proteins were more enriched in HighSR membrane. Proteins from other putative ER/SR subdomains also showed characteristic distributions among SR subpopulations. We conclude that active Ca loading of cardiac microsomes, reflecting the combined activities of Ca uptake by SERCA, and Ca leak by RyR, permits evaluation of multiple functional ER/SR subdomains. Sets of proteins from these subdomains exhibited similar enrichment patterns across membrane subfractions, reflecting the relative levels of SERCA and RyR present within individual patches of cardiac ER and SR.

Enriching extracellular vesicles for mass spectrometry.

Extracellular vesicles from plasma, other body fluids and cell culture media hold great promise in the search for biomarkers. Exosomes in particular, the vesicle type that is secreted after being produced in the endocytic pathway and having a diameter of 30-150 nm, are considered to be a conveyance for signaling molecules and, therefore, to hold valuable information regarding the health and activity status of the cells from which they are released. The vesicular nature of exosomes is central to all methods used to separate them from the highly abundant proteins in plasma and other fluids. The enrichment of the vesicles is essential for mass spectrometry-based analysis as they represent only a very small component of all plasma proteins. The progression of isolation techniques for exosomes from ultracentrifugation through chromatographic separation using hydrophobic packing materials shows that effective enrichment is possible and that high throughput approaches to exosome enrichment are achievable.

Deletion of mdig enhances H3K36me3 and metastatic potential of the triple negative breast cancer cells.

In this report, we provide evidence showing diminished expression of the mineral dust-induced gene (mdig), a previously identified oncogenic gene, in human triple negative breast cancer (TNBC). Using a mouse model of orthotopic xenograft of the TNBC MDA-MB-231 cells, we demonstrate that mdig promotes the growth of primary tumors but inhibits metastasis of these cells in vivo. Knockout of mdig resulted in an enhancement of H3K36me3 in the genome and upregulation of some X chromosome-linked genes for cell motility, invasion, and metastasis. Silencing MAGED2, one of the most upregulated and H3K36me3-enriched genes resulted from mdig depletion, can partially reverse the invasive migration of the mdig knockout cells. The anti-metastatic and inhibitory role of mdig on H3K36me3 was cross-validated in another cell line, A549 lung cancer cells. Together, our data suggest that mdig is antagonist against H3K36me3 that enforces expression of genes, such as MAGED2, for cell invasion and metastasis.

Depletion of Mdig Changes Proteomic Profiling in Triple Negative Breast Cancer Cells.

Triple-negative breast cancers are highly aggressive with an overall poor prognosis and limited therapeutic options. We had previously investigated the role of mdig, an oncogenic gene induced by some environmental risk factors, on the pathogenesis of breast cancer. However, a comprehensive analysis of the proteomic profile affected by mdig in triple-negative breast cancer has not been determined yet. Using label-free bottom-up quantitative proteomics, we compared wildtype control and mdig knockout MDA-MB-231 cells and identified the proteins and pathways that are significantly altered with mdig deletion. A total of 904 differentially expressed (p < 0.005) proteins were identified in the KO cells. Approximately 30 pathways and networks linked to the pathogenicity of breast cancer were either up- or downregulated, such as EIF2 signaling, the unfolded protein response, and isoleucine degradation I. Ingenuity Pathway Analysis established that the differentially expressed proteins have relevant biological actions in cell growth, motility, and malignancy. These data provide the first insight into protein expression patterns in breast cancer associated with a complete disruption of the mdig gene and yielded substantial information on the key proteins, biological processes, and pathways modulated by mdig that contribute to breast cancer tumorigenicity and invasiveness.

Pattern Analysis of Organellar Maps for Interpretation of Proteomic Data.

Localization of organelle proteins by isotope tagging (LOPIT) maps are a coordinate-directed representation of proteome data that can aid in biological interpretation. Analysis of organellar association for proteins as displayed using LOPIT is evaluated and interpreted for two types of proteomic data sets. First, test and control group protein abundances and fold change data obtained in a proximity labeling experiment are plotted on a LOPIT map to evaluate the likelihood of true protein interactions. Selection of true positives based on co-localization of proteins in the organellar space is shown to be consistent with carboxylase enrichment which serves as a positive control for biotinylation in streptavidin affinity selected proteome data sets. The mapping in organellar space facilitates discrimination between the test and control groups and aids in identification of proteins of interest. The same representation of proteins in organellar space is used in the analysis of extracellular vesicle proteomes for which protein abundance and fold change data are evaluated. Vesicular protein organellar localization patterns provide information about the subcellular origin of the proteins in the samples which are isolates from the extracellular milieu. The organellar localization patterns are indicative of the provenance of the vesicular proteome origin and allow discrimination between proteomes prepared using different enrichment methods. The patterns in LOPIT displays are easy to understand and compare which aids in the biological interpretation of proteome data.

The human type 2 diabetes-specific visceral adipose tissue proteome and transcriptome in obesity.

Dysfunctional visceral adipose tissue (VAT) in obesity is associated with type 2 diabetes (DM) but underlying mechanisms remain unclear. Our objective in this discovery analysis was to identify genes and proteins regulated by DM to elucidate aberrant cellular metabolic and signaling mediators. We performed label-free proteomics and RNA-sequencing analysis of VAT from female bariatric surgery subjects with DM and without DM (NDM). We quantified 1965 protein groups, 23 proteins, and 372 genes that were differently abundant in DM vs. NDM VAT. Proteins downregulated in DM were related to fatty acid synthesis and mitochondrial function (fatty acid synthase, FASN; dihydrolipoyl dehydrogenase, mitochondrial, E3 component, DLD; succinate dehydrogenase-α, SDHA) while proteins upregulated in DM were associated with innate immunity and transcriptional regulation (vitronectin, VTN; endothelial protein C receptor, EPCR; signal transducer and activator of transcription 5B, STAT5B). Transcriptome indicated defects in innate inflammation, lipid metabolism, and extracellular matrix (ECM) function, and components of complement classical and alternative cascades. The VAT proteome and transcriptome shared 13 biological processes impacted by DM, related to complement activation, cell proliferation and migration, ECM organization, lipid metabolism, and gluconeogenesis. Our data revealed a marked effect of DM in downregulating FASN. We also demonstrate enrichment of complement factor B (CFB), coagulation factor XIII A chain (F13A1), thrombospondin 1 (THBS1), and integrins at mRNA and protein levels, albeit with lower q-values and lack of Western blot or PCR confirmation. Our findings suggest putative mechanisms of VAT dysfunction in DM.

C-reactive Protein Levels in Plasma and Chronic Venous Ulcer Exudate of Persons Who Inject Drugs: A Pilot Study.

BACKGROUND: Persons who inject drugs (PWID) in the groin, legs, and/or feet are at high risk for chronic venous ulcers (CVUs). The plasma C-reactive protein (CRP) level is a marker of systemic inflammation. OBJECTIVE: This pilot study examined CRP levels in plasma and CVU exudate of PWID. The aims were to (1) compare levels of CRP in plasma and exudate; (2) examine if the CRP level in exudate changed over 4 weeks with wound treatment; and (3) examine the relationship of the exudate CRP level with CVU area, CVU age, number of CVUs, and number of comorbidities. MATERIALS AND METHODS: Persons who inject drugs seeking wound care were enrolled in this Institutional Review Board approved prospective, longitudinal, descriptive study. A blood sample was collected on the first visit (week 1); the plasma was then separated. Wound exudate was collected on swabs during the first visit (week 1) and 4 weeks later (week 4). All samples were stored at -80° C. Samples were eluted from swabs using mass spectrometry grade water then aliquoted for CRP analysis. RESULTS: The participants of the study included 14 PWID (mean age, 62.14 ± 4.52 years; mean number of comorbidities, 5.71 ± 1.90; and mean number of ulcers 2.07 ± 1.07 that were present for a mean of 7.96 ± 11.91 years without healing). C-reactive protein level in plasma was a mean of 6.47 ± 8.56 mg/L, with lower levels found in wound exudate but highly correlated (rho = .925). Exudate CRP levels decreased from week 1 to week 4, and the 2 were highly correlated (rho = .895). Exudate CRP level week 1 was not significantly related to wound area, wound age, number of ulcers, or number of comorbidities. CONCLUSIONS: Plasma and exudate CRP levels were highly correlated. Exudate CRP levels decreased across time. Future large-scale wound healing studies should examine CRP levels over a longer duration and as they correlate to wound healing.

Effects of Glycyrrhizin on Multi-Drug Resistant Pseudomonas aeruginosa.

The effects of glycyrrhizin (GLY) on multi-drug resistant (MDR) systemic (MDR9) vs. ocular (B1045) Pseudomonas aeruginosa clinical isolates were determined. Proteomes of each isolate with/without GLY treatment were profiled using liquid chromatography mass spectrometry (LC-MS/MS). The effect of GLY on adherence of MDR isolates to immortalized human (HCET) and mouse (MCEC) corneal epithelial cells, and biofilm and dispersal was tested. Both isolates were treated with GLY (0.25 minimum inhibitory concentration (MIC), 10 mg/mL for MDR9 and 3.75 mg/mL for B1045) and subjected to proteomic analysis. MDR9 had a greater response to GLY (51% of identified proteins affected vs. <1% in B1045). In MDR9 vs. controls, GLY decreased the abundance of proteins for: antibiotic resistance, biofilm formation, and type III secretion. Further, antibiotic resistance and type III secretion proteins had higher control abundances in MDR9 vs. B1045. GLY (5 and 10 mg/mL) significantly reduced binding of both isolates to MCEC, and B1045 to HCET. MDR9 binding to HCET was only reduced at 10 mg/mL GLY. GLY (5 and 10 mg/mL) enhanced dispersal for both isolates, at early (6.5 h) but not later times (24-72 h). This study provides evidence that GLY has a greater effect on the proteome of MDR9 vs. B1045, yet it was equally effective at disrupting adherence and early biofilm dispersal.

4-Hydroxy-2-nonenal attenuates 8-oxoguanine DNA glycosylase 1 activity.

Elevated cellular oxidative stress and oxidative DNA damage are key contributors to impaired cardiac function in diabetes. During chronic inflammation, reactive oxygen species (ROS)-induced lipid peroxidation results in the formation of reactive aldehydes, foremost of which is 4-hydroxy-2-nonenal (4HNE). 4HNE forms covalent adducts with proteins, negatively impacting cellular protein function. During conditions of elevated oxidative stress, oxidative DNA damage such as modification by 8-hydroxydeoxyguanosine (8OHdG) is repaired by 8-oxoguanine glycosylase-1 (OGG-1). Based on these facts, we hypothesized that 4HNE forms adducts with OGG-1 inhibiting its activity, and thus, increases the levels of 8OHG in diabetic heart tissues. To test our hypothesis, we evaluated OGG-1 activity, 8OHG and 4HNE in the hearts of leptin receptor deficient db/db mice, a type-2 diabetic model. We also treated the recombinant OGG-1 with 4HNE to measure direct adduction. We found decreased OGG-1 activity (P > .05), increased 8OHG (P > .05) and increased 4HNE adducts (P > .05) along with low aldehyde dehydrogenase-2 activity (P > .05). The increased colocalization of OGG-1 and 4HNE in cardiomyocytes suggest 4HNE adduction on OGG-1. Furthermore, colocalization of 8OHG and OGG-1 with mitochondrial markers TOM 20 and aconitase, respectively, indicated significant levels of oxidatively-induced mtDNA damage and implicated a role for mitochondrial OGG-1 function. In vitro exposure of recombinant OGG-1 (rOGG-1) with increasing concentrations of 4HNE resulted in a concentration-dependent decrease in OGG-1 activity. Mass spectral analysis of trypsin digests of 4HNE-treated rOGG-1 identified 4HNE adducts on C28, C75, C163, H179, H237, C241, K249, H270, and H282. In silico molecular modeling of 4HNE-K249 OGG-1 and 4HNE-H270 OGG-1 mechanistically supported 4HNE-mediated enzymatic inhibition of OGG-1. In conclusion, these data support the hypothesis that inhibition of OGG-1 by direct modification by 4HNE contributes to decreased OGG-1 activity and increased 8OHG-modified DNA that are present in the diabetic heart.

The anti-MRSA compound 3-O-alpha-L-(2″,3″-di-p-coumaroyl)rhamnoside (KCR) inhibits protein synthesis in Staphylococcus aureus.

Methicillin-resistant S aureus (MRSA) contributes to patient mortality and extended hospital stays. 3-O-alpha-L-(2″,3″-di-p-coumaroyl)rhamnoside (KCR) is a natural product antibiotic that is effective against MRSA but has no known mechanism of action (MOA). We used proteomics to identify the MOA for KCR. Methicillin sensitive S aureus and a mixture of four KCR stereoisomers were tested. A time-kill assay was used to choose a 4 h treatment using KCR at 5× its MIC for proteomic analysis. S aureus was treated in triplicate with KCR, oxacillin or vehicle and quantitative proteomic analysis was carried out using isobaric tags and mass spectrometry. 1190 proteins were identified and 552 were affected by KCR (q < 0.01). Ontology analysis identified 6 distinct translation-related categories that were affected by KCR (PIANO, 10% false-discovery rate) including structural constituent of ribosome, translation, rRNA binding, tRNA binding, tRNA processing and aminoacyl-tRNA ligase activity. Median fold changes (KCR vs Control) for small and large ribosomal components were 1.46 and 1.43 respectively. KCR inhibited the production of luciferase protein in an in vitro assay (IC50 39.6 µg/ml). Upregulation of translation-related proteins in response to KCR indicates that KCR acts to disrupt S aureus protein synthesis. This was confirmed with an in vitro transcription/translation assay. SIGNIFICANCE: Methicillin-resistant S aureus (MRSA) contributes to patient mortality and extended hospital stays. 3-O-alpha-L-(2″,3″-di-p-coumaroyl)rhamnoside (KCR) is a natural product antibiotic that is effective against MRSA but has no known mechanism of action (MOA). Using proteomic analysis we determined that KCR acts by inhibiting protein synthesis. KCR is an exciting novel antibiotic and this work represents an important step in its development towards clinical use.

Chronic Low Dose Oral Exposure to Microcystin-LR Exacerbates Hepatic Injury in a Murine Model of Non-Alcoholic Fatty Liver Disease.

Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Leprdb/J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Leprdb/J mice were gavaged with 50 µg/kg, 100 µg/kg MC-LR or vehicle every 48 h for 4 weeks (n = 15-17 mice/group). Early mortality was observed in both the 50 µg/kg (1/17, 6%), and 100 µg/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice.

Exosome-enriched fractions from MS B cells induce oligodendrocyte death.

Objective: To identify whether factors toxic to oligodendrocytes (OLs), released by B cells from patients with MS, are found in extracellular microvesicles enriched in exosomes. Methods: Conditioned medium (Sup) was obtained from cultures of blood B cells of patients with MS and normal controls (NCs). Exosome-enriched (Ex-En) fractions were prepared by solvent precipitation from Sup containing bovine serum and from serum-free Sup by ultracentrifugation (UC) or immunoprecipitation (IP) with antibodies to CD9. Ex-En fractions were diluted 1:4 with OL culture medium and screened for toxic effects on cultured rat OLs as measured by trypan blue uptake. Proteomic analysis was performed on Sup fractions. Results: MS B cell-derived Ex-En fractions prepared from Sup by solvent extraction, UC, or IP induced OL death, whereas corresponding Ex-En fractions from NC showed little toxicity. Proteomic analysis of Sup demonstrated enrichment of proteins characteristic of exosomes from both NC and MS B-cell Sup. Ontology enrichment analysis suggested differences in the types and cargo of exosomes from MS Sup compared with NC, with proteins related to cell surface, extracellular plasma membrane, and gliogenesis enriched in MS. Conclusions: Much of the in vitro toxicity of Sup from B cells of patients with relapsing-remitting MS is found in Ex-En fractions, as confirmed by 3 methods. Proteomic analysis of B-cell Sup indicates multiple differences between MS and NC.

Single Amino Acid Variant Discovery in Small Numbers of Cells.

We have performed deep proteomic profiling down to as few as 9 Panc-1 cells using sample fractionation, TMT multiplexing, and a carrier/reference strategy. Off line fractionation of the TMT-labeled sample pooled with TMT-labeled carrier Panc-1 whole cell proteome was achieved using alkaline reversed phase spin columns. The fractionation in conjunction with the carrier/reference (C/R) proteome allowed us to detect 47 414 unique peptides derived from 6261 proteins, which provided a sufficient coverage to search for single amino acid variants (SAAVs) related to cancer. This high sample coverage is essential in order to detect a significant number of SAAVs. In order to verify genuine SAAVs versus false SAAVs, we used the SAVControl pipeline and found a total of 79 SAAVs from the 9-cell Panc-1 sample and 174 SAAVs from the 5000-cell Panc-1 C/R proteome. The SAAVs as sorted into high confidence and low confidence SAAVs were checked manually. All the high confidence SAAVs were found to be genuine SAAVs, while half of the low confidence SAAVs were found to be false SAAVs mainly related to PTMs. We identified several cancer-related SAAVs including KRAS, which is an important oncoprotein in pancreatic cancer. In addition, we were able to detect sites involved in loss or gain of glycosylation due to the enhanced coverage available in these experiments where we can detect both sites of loss and gain of glycosylation.

Urine proteomic profiling in patients with nephrolithiasis and cystinuria.

PURPOSE: The purpose of the study was to assess the differences in the concentration and function of urinary proteins between patients with cystine stones (CYS) and healthy controls (HC). We postulated that CYS and HC groups would demonstrate different proteomic profiles. METHODS: A pilot study was performed comparing urinary proteomes of 10 patients with CYS and 10 age- and gender-matched HC, using liquid chromatography-mass spectrometry. Proteins which met the selection criteria (i) ≥ 2 unique peptide identifications; (ii) ≥ twofold difference in protein abundance; and (iii) ≤ 0.05 p value for the Fisher's Exact Test were analyzed using Gene Ontology classifications. RESULTS: Of the 2097 proteins identified by proteomic analysis, 398 proteins were significantly different between CYS and HC. Of those, 191 were involved in transport processes and 61 in inflammatory responses. The majority were vesicle-mediated transport proteins (78.5%), and 1/3 of them were down-regulated; of those, 12 proteins were involved in endosomal transport (including 6 charged multivesicular body proteins (CHMP) and 3 vacuolar sorting-associated proteins) and 9 in transmembrane transport. Myosin-2 and two actin-related proteins were significantly up-regulated in the vesicle-mediated transport group. CONCLUSION: We provide proteomic evidence of impaired endocytosis, dysregulation of actin and myosin cytoskeleton, and inflammation in CYS. Endosomal transport proteins were down-regulated mainly through defective CHMP. These findings may contribute to further understanding of the pathogenesis of CYS, potentially affecting its management.

Human Skeletal Muscle Cells on Engineered 3D Platform Express Key Growth and Developmental Proteins.

Current approaches in regenerative medicine to develop human skeletal muscle replicating native tissue for engrafts and high-throughput drug screening and gene therapy are still in their infancy and have not proven to recapitulate the behavior and regulatory processes present in endogenous skeletal muscle tissue. This stems at least in part from the lack of a comprehensive understanding of the emergent properties of in vitro skeletal muscle growth and development. To address this gap in our current knowledge, we have developed a stretchable micropatterned 3D human skeletal muscle platform that recapitulates organized and parallel growth of muscle cells and fibers as opposed to the randomly oriented cells growth on a 2D glass surface. Mass spectrometry of the muscle cells growing on the 3D platform express key myogenic proteins such as myoferlin for myoblast fusion required in the formation of muscle tissue, and proteins involved in mitochondrial health and biogenesis, in contrast to cells growing on 2D glass surface. These results demonstrate that the engineered human muscle cells grown on the 3D platform holds great promise to further establish the emergent properties of in vitro skeletal muscle growth and development for a wide range of biomedical applications.

The predictive performance of short-linear motif features in the prediction of calmodulin-binding proteins.

BACKGROUND: The prediction of calmodulin-binding (CaM-binding) proteins plays a very important role in the fields of biology and biochemistry, because the calmodulin protein binds and regulates a multitude of protein targets affecting different cellular processes. Computational methods that can accurately identify CaM-binding proteins and CaM-binding domains would accelerate research in calcium signaling and calmodulin function. Short-linear motifs (SLiMs), on the other hand, have been effectively used as features for analyzing protein-protein interactions, though their properties have not been utilized in the prediction of CaM-binding proteins. RESULTS: We propose a new method for the prediction of CaM-binding proteins based on both the total and average scores of known and new SLiMs in protein sequences using a new scoring method called sliding window scoring (SWS) as features for the prediction module. A dataset of 194 manually curated human CaM-binding proteins and 193 mitochondrial proteins have been obtained and used for testing the proposed model. The motif generation tool, Multiple EM for Motif Elucidation (MEME), has been used to obtain new motifs from each of the positive and negative datasets individually (the SM approach) and from the combined negative and positive datasets (the CM approach). Moreover, the wrapper criterion with random forest for feature selection (FS) has been applied followed by classification using different algorithms such as k-nearest neighbors (k-NN), support vector machines (SVM), naive Bayes (NB) and random forest (RF). CONCLUSIONS: Our proposed method shows very good prediction results and demonstrates how information contained in SLiMs is highly relevant in predicting CaM-binding proteins. Further, three new CaM-binding motifs have been computationally selected and biologically validated in this study, and which can be used for predicting CaM-binding proteins.

Global Signaling Profiling in a Human Model of Tumorigenic Progression Indicates a Role for Alternative RNA Splicing in Cellular Reprogramming.

Intracellular signaling is controlled to a large extent by the phosphorylation status of proteins. To determine how human breast cells can be reprogrammed during tumorigenic progression, we profiled cell lines in the MCF10A lineage by phosphoproteomic analyses. A large cluster of proteins involved in RNA splicing were hypophosphorylated as cells progressed to a hyperplastic state, and then hyperphosphorylated after progression to a fully metastatic phenotype. A comprehensive transcriptomic approach was used to determine whether alterations in splicing factor phosphorylation status would be reflected in changes in mRNA splicing. Results indicated that the degree of mRNA splicing trended with the degree of tumorigenicity of the 4 cell lines tested. That is, highly metastatic cell cultures had the greatest number of genes with splice variants, and these genes had greater fluctuations in expression intensities. Genes with high splicing indices were mapped against gene ontology terms to determine whether they have known roles in cancer. This group showed highly significant associations for angiogenesis, cytokine-mediated signaling, cell migration, programmed cell death and epithelial cell differentiation. In summary, data from global profiling of a human model of breast cancer development suggest that therapeutics should be developed which target signaling pathways that regulate RNA splicing.

Low level Hg2+ exposure modulates the B-cell cytoskeletal phosphoproteome.

Exposure of Wehi-231 B-cells to Hg2+ for 5min resulted in concentration dependent changes in protein phosphorylations. Phosphorylation was quantified using mass spectrometry to analyze TiO2 and anti-pTyr antibody selected phosphopeptides from Wehi-231 digests. The most frequent and largest amplitude responses to Hg2+ exposure were increased phosphorylation although a decrease was observed for 1% of phosphoproteins detected in the untreated cells. A subset of proteins responded with an increase in phosphorylation to Hg2+ exposure at low micromolar concentrations. The majority of proteins required Hg2+ over 20µM in order to increase phosphorylation. Ser/Thr phosphorylations are prominent in the cytoskeletal organization and the GTPase signaling systems and these systems are notable as the primary ones responding to the lowest concentrations of Hg2+. Systems that required higher concentrations of Hg2+ to increase phosphorylation included immune receptor signaling. The proteins for which an increase in phosphorylation occurred at Hg2+ above 20µM have a higher proportion of pTyr sites. Anti Ig stimulation of Wehi-231 cells confirmed that cytoskeletal protein phosphorylation and GTPase signaling are modulated in physiologically relevant B-cell receptor activation. Candidate kinases that respond to Hg2+ exposure at the low µM concentrations include MAP Kinase 1, CaM Kinase II delta and PAK2. SIGNIFICANCE: Mercury (Hg) is a wide spread environmental toxicant. Epidemiological and laboratory studies suggest that exposure to environmental Hg at current levels, which have been perceived to be non-toxic, may contribute to immune system dysfunction and autoimmune disease in humans and animals respectively. While we have previously shown that exposure of B lymphocytes to low levels of mercury interferes with B-cell receptor signaling mediated by post transcriptional phosphorylation events, overall the mechanism that is responsible for increased autoimmunity in mercury exposed human or animal populations is not well understood. The current study evaluated the dose dependent actions of mercury to change phosphorylation in the Wehi-231 cell line, an immature B-cell model in which actions of mercury on development of cell function can be evaluated. The study identified the cytoskeletal proteins as the most sensitive to modulation by mercury with changes in Ser/Thr phosphorylation being observed at the lowest concentrations of mercury. These findings indicate that the actions of mercury on B-cell immune function and development are at least in part likely mediated through changes in cytoskeletal protein phosphorylation.