Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling.

Analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. While there have been advances in techniques for proteome-wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatography (SEC) with label-free quantitative MS profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme T98G cells. We identified 1794 proteins that overlapped between two biological replicates of which 1244 proteins were characterized as existing within stably associated putative complexes. co-IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins and HDAC1 (histone deacetylase complex 1) and CHD4. HDAC1/2 also co-migrated with various SIN3A and nucleosome remodeling and deacetylase components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co-elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC-MS analysis for system-wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes.

Analysis of Protein Complexes in the Unicellular Cyanobacterium Cyanothece ATCC 51142.

The unicellular cyanobacterium Cyanothece ATCC 51142 is capable of oxygenic photosynthesis and biological N2 fixation (BNF), a process highly sensitive to oxygen. Previous work has focused on determining protein expression levels under different growth conditions. A major gap of our knowledge is an understanding on how these expressed proteins are assembled into complexes and organized into metabolic pathways, an area that has not been thoroughly investigated. Here, we combined size-exclusion chromatography (SEC) with label-free quantitative mass spectrometry (MS) and bioinformatics to characterize many protein complexes from Cyanothece 51142 cells grown under a 12 h light-dark cycle. We identified 1386 proteins in duplicate biological replicates, and 64% of those proteins were identified as putative complexes. Pairwise computational prediction of protein-protein interaction (PPI) identified 74 822 putative interactions, of which 2337 interactions were highly correlated with published protein coexpressions. Many sequential glycolytic and TCA cycle enzymes were identified as putative complexes. We also identified many membrane complexes that contain cytoplasmic domains. Subunits of NDH-1 complex eluted in a fraction with an approximate mass of ∼669 kDa, and subunits composition revealed coexistence of distinct forms of NDH-1 complex subunits responsible for respiration, electron flow, and CO2 uptake. The complex form of the phycocyanin beta subunit was nonphosphorylated, and the monomer form was phosphorylated at Ser20, suggesting phosphorylation-dependent deoligomerization of the phycocyanin beta subunit. This study provides an analytical platform for future studies to reveal how these complexes assemble and disassemble as a function of diurnal and circadian rhythms.

Diet Impacts Pre-implantation Histotroph Proteomes in Beef Cattle.

In ruminants, the period from fertilization to implantation is relatively prolonged, and the survival of embryos depends on uterine secretions known as histotroph. Our objective was to determine if the pre-breeding diet affected histotroph proteomes in beef cattle. Cows were assigned to one of four diets: a control diet (CON), a high-protein diet (PROT), a high-fat diet (OIL), or a high-protein and high-fat diet (PROT + OIL). After 185 days on these diets, an intravaginal progesterone implant (CIDR) was inserted for 7 days. At 9 days after CIDR removal, animals with a corpus luteum were selected ( n = 16; 4 per treatment). Proteins were isolated from the histotroph collected by uterine lavage and analyzed with liquid chromatography-tandem mass spectrometry. Over 2000 proteins were expressed ( n ≥ 3 cows per treatment), with 1239 proteins being common among all of the groups. There were 20, 37, 85, and 123 proteins unique to CON, PROT + OIL, PROT, and OIL, respectively. Relative to CON, 23, 14, and 51 proteins were differentially expressed in PROT + OIL, PROT, and OIL, respectively. Functional analysis found that 53% of histotroph proteins were categorized as extracellular exosome, 3.28% as cell-cell adhesion, and 17.4% in KEGG metabolic pathways. Differences in proteomes among treatments support the idea that pre-breeding diet affects histotroph. Understanding the impact of diet on histotroph proteins may help improve conception rates.

Proteomic Analysis Reveals That an Extract of the Plant Lippia origanoides Suppresses Mitochondrial Metabolism in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer is an aggressive subtype of breast cancer with low 5-year survival rates, high 3-year recurrence rates, and no known therapeutic targets. Recent studies have indicated that triple-negative breast cancers possess an altered metabolic state with higher rates of glycolysis, mitochondrial oxidative phosphorylation, and increased generation and utilization of tricarboxylic acid cycle intermediates. Here, we utilized label-free quantitative proteomics to gain insight into the anticancer mechanisms of a methanolic extract from the Central American plant Lippia origanoides on MDA-MB-231 triple-negative breast cancer cells. The L. origanoides extract dysregulated mitochondrial oxidative phosphorylation by suppressing the expression of several subunits of Complex I of the electron transport chain, and inhibited cellular metabolism by down-regulating key tricarboxylic acid cycle enzymes and mitochondrial lipid and amino-acid metabolic pathways. Our study also revealed that treatment with the extract activated the stress response and pathways related to cell-cycle progression and DNA repair. Overall, our results reveal compelling new evidence that the extract from L. origanodes triggers rapid irreversible apoptosis in MDA-MB-231 cells by effectively 'starving' the cells of metabolites and ATP. We continue to study the specific bioactive components of the extract in the search for novel, highly effective mitochondrial inhibitors to selectively target triple-negative breast cancer.

A Comparative In Vivo Study of Albumin-Coated Paclitaxel Nanocrystals and Abraxane.

Nanoparticulate drug carriers exploit the enhanced permeability of tumor vasculature to achieve selective delivery of chemotherapeutic drugs. For this purpose, nanoparticles (NPs) need to circulate with a long half-life, enter tumors via the permeable vasculature and stay in tumors via favorable interactions with tumor cells. To fulfill these requirements, albumin-coated nanocrystal formulation of paclitaxel (PTX), Cim-F-alb, featuring high drug loading content, physical stability in serum, and surface-bound albumin in its native conformation is prepared. The pharmacokinetic and biodistribution (PK/BD) profiles of Cim-F-alb in a mouse model of B16F10 melanoma show that Cim-F-alb exhibits a longer plasma half-life and a greater PTX deposition in tumors than Abraxane by ≈1.5 and ≈4.6 fold, respectively. Biolayer interferometry analysis indicates that Cim-F-alb has less interaction with serum proteins than nanocrystals lacking albumin coating, indicating the protective effect of the surface-bound albumin against opsonization in the initial deposition phase. With the advantageous PK/BD profiles, Cim-F-alb shows greater and longer-lasting anticancer efficacy than Abraxane at the equivalent dose. This study demonstrates the significance of controlling circulation stability and surface property of NPs in efficient drug delivery to tumors and enhanced anticancer efficacy.

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma.

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Wolbachia Endosymbionts Modify Drosophila Ovary Protein Levels in a Context-Dependent Manner.

UNLABELLED: Endosymbiosis is a unique form of interaction between organisms, with one organism dwelling inside the other. One of the most widespread endosymbionts is Wolbachia pipientis, a maternally transmitted bacterium carried by insects, crustaceans, mites, and filarial nematodes. Although candidate proteins that contribute to maternal transmission have been identified, the molecular basis for maternal Wolbachia transmission remains largely unknown. To investigate transmission-related processes in response to Wolbachia infection, ovarian proteomes were analyzed from Wolbachia-infected Drosophila melanogaster and D. simulans. Endogenous and variant host-strain combinations were investigated. Significant and differentially abundant ovarian proteins were detected, indicating substantial regulatory changes in response to Wolbachia Variant Wolbachia strains were associated with a broader impact on the ovary proteome than endogenous Wolbachia strains. The D. melanogaster ovarian environment also exhibited a higher level of diversity of proteomic responses to Wolbachia than D. simulans. Overall, many Wolbachia-responsive ovarian proteins detected in this study were consistent with expectations from the experimental literature. This suggests that context-specific changes in protein abundance contribute to Wolbachia manipulation of transmission-related mechanisms in oogenesis. IMPORTANCE: Millions of insect species naturally carry bacterial endosymbionts called Wolbachia. Wolbachia bacteria are transmitted by females to their offspring through a robust egg-loading mechanism. The molecular basis for Wolbachia transmission remains poorly understood at this time, however. This proteomic study identified specific fruit fly ovarian proteins as being upregulated or downregulated in response to Wolbachia infection. The majority of these protein responses correlated specifically with the type of host and Wolbachia strain involved. This work corroborates previously identified factors and mechanisms while also framing the broader context of ovarian manipulation by Wolbachia.

Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS).

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

Harnessing nerve-muscle cell interactions for biomaterials-based skeletal muscle regeneration.

Nerve cells secrete neurotrophic factors that play a critical role in neuronal survival, proliferation, and regeneration. However, their role in regulating myoblast behavior and skeletal muscle repair remains largely unexplored. In the present study, we investigated the effects of PC12 secreted signaling factors in modulating C2C12 myoblast behavior under physiologically relevant conditions. We showed that PC12 conditioned media modulated myoblast proliferation and differentiation in both 2D culture and 3D aligned electrospun fiber scaffold system in a dose-dependent manner. We further developed a biomimetic, tunable hydrogel consisting of hyaluronic acid, chondroitin sulfate, and polyethylene glycol as a 3D matrix encapsulating PC12 cells. The hydrogel-encapsulated PC12 cells promoted survival and proliferation of myoblasts in co-culture. Further proteomics analysis identified a total of 2,088 proteins from the secretome of the encapsulated PC12 cells and revealed the biological role and overlapping functions of nerve-secreted proteins for skeletal muscle regeneration, potentially through regulating myoblast behavior, nerve function, and angiogenesis. These experiments provide insights into the nerve-muscle interactions and pave the way for developing advanced biomaterials strategies incorporating nerve cell secretome for accelerated skeletal muscle regeneration.

Proteomic identification of aerobic glycolysis as a potential metabolic target for methylglyoxal in adipocytes.

Obesity is often accompanied by metabolic changes in adipocytes that are closely associated with metabolic disease. Although high sugar consumption contributes to obesity, it may also directly affect adipocytes by increasing the rate of glycolysis and formation of the glycolytic by-product methylglyoxal (MG). MG is a reactive dicarbonyl that irreversibly damages proteins and other cellular components. Although the accumulation of MG is clinically associated with hyperglycemia and diabetic complications, a better understanding of how proteins are regulated by MG is needed to evaluate its role in the pathogenesis of metabolic disease. Because adipocytes rely heavily on glycolysis for glucose disposal, we hypothesized that prolonged MG treatment at nontoxic concentrations would impact the landscape of proteins involved in glucose metabolism. To test this hypothesis, we treated 3T3-L1 adipocytes with MG (100 µmol/L) and used comparative proteomics to assess the effects. We identified 25 differentially expressed proteins in adipocytes treated with MG compared to the control. Our results suggested that MG induced metabolic changes typically associated with aerobic glycolysis, including a lowered expression of proteins involved in oxidative metabolism and increased expression of the glycolytic enzymes L-lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The detection of increased lactate secreted into the culture media of adipocytes treated with MG further supported these findings, as did gene expression analysis. In summary, these results indicate MG as a metabolic contributor to aerobic glycolysis in adipocytes, a potential adaptive response to increased glucose flux which over time could lead to permanent metabolic changes.

Proteomic and metabolomic profiling reveals the involvement of apoptosis in meat quality characteristics of ovine M. longissimus from different callipyge genotypes.

Proteome and metabolome changes in muscles from callipyge mutation (+/C) and non-callipyge phenotype (+/+, C/+, and C/C) lambs were profiled to provide insight into the biochemical changes affecting meat quality attributes. M. longissimus thoracis from lambs with all four possible callipyge genotype (n = 4, C/+, C/C, +/C, and +/+) were collected after 3d aging and analyzed using mass-spectrometry based platforms. Among identified proteomes, cytochrome c (pro-apoptotic protein) was detected with significantly lower abundances in +/C. Anti-apoptotic HSP70, BAG3, and PARK7 were over-abundant in +/C, which could result in delayed apoptosis and possibly attributed to tougher meat in callipyge lambs. Eight glycolysis enzymes were overabundant in +/C lambs, whereas 3 enzymes involved in TCA cycle were overabundant in non-callipyge ones (C/C and/or C/+). Twenty-five metabolites were affected by genotypes (P < .05), including metabolic co-factors, polyphenols, and AA/short peptides. Our omics results provided insightful information for revealing the differences in biochemical attributes caused by callipyge mutation.

Global Proteomic Analyses of STING-Positive and -Negative Macrophages Reveal STING and Non-STING Differentially Regulated Cellular and Molecular Pathways.

PURPOSE: Cyclic guanosine monophosphate-adenosine monophosphate and other bacterial-derived cyclic di-guanosine monophosphate or cyclic di-adenosine monophosphate trigger innate immune responses through binding to stimulator of interferon genes (STING). Thus in chronic infection, such as in periodontitis, immune cells can be exposed to bacterial DNA and/or cyclic dinucleotides, potentially activating STING to cause inflammation. Thus far the cyclic GMP-AMP synthase-STING- TANK-binding kinase 1 pathway has been well characterized but a global perspective of how the presence or lack of STING affect the proteome is lacking. The aim of this study is to identify macrophage proteins that are affected by STING. EXPERIMENTAL DESIGN: Proteins are extracted from a macrophage cell line harboring STING (RAW-Blue ISG) as well as a STING knockout (STING KO) cell line (RAW-Lucia ISG-KO-STING) and global proteomics analyses are performed. RESULTS: Proteins related to kinase and phosphatase signaling, spliceosome, terpenoid backbone biosynthesis, glycosylation, ubiquitination, and phagocytosis are affected by STING knock out. CONCLUSIONS AND CLINICAL RELEVANCE: STING pathway in macrophages is related to the regulation of several proteins that are known as potent biomarkers of various cancers and autoimmune diseases. Moreover, the relation between STING and phagocytosis is demonstrated for the first time. Further validation studies will help identify molecules and pathways that may function as diagnostic or therapeutic targets.

Rab11b-mediated integrin recycling promotes brain metastatic adaptation and outgrowth.

Breast cancer brain metastases (BCBM) have a 5-20 year latency and account for 30% of mortality; however, mechanisms governing adaptation to the brain microenvironment remain poorly defined. We combine time-course RNA-sequencing of BCBM development with a Drosophila melanogaster genetic screen, and identify Rab11b as a functional mediator of metastatic adaptation. Proteomic analysis reveals that Rab11b controls the cell surface proteome, recycling proteins required for successful interaction with the microenvironment, including integrin ß1. Rab11b-mediated control of integrin ß1 surface expression allows efficient engagement with the brain ECM, activating mechanotransduction signaling to promote survival. Lipophilic statins prevent membrane association and activity of Rab11b, and we provide proof-of principle that these drugs prevent breast cancer adaptation to the brain microenvironment. Our results identify Rab11b-mediated recycling of integrin ß1 as regulating BCBM, and suggest that the recycleome, recycling-based control of the cell surface proteome, is a previously unknown driver of metastatic adaptation and outgrowth.

Temporal analysis of vaginal proteome reveals developmental changes in lower reproductive tract of gilts across the first two weeks postnatal.

In swine the upper reproductive tract undergoes early postnatal development, however little is known about the lower reproductive tract. Our objective was to measure cytology and proteome of vaginal swab samples taken on postnatal day (PND) 2 and 16 in gilts to determine if temporal changes occurred in cell and protein profiles during the first two weeks after birth. The posterior vagina was swabbed using a cytology brush on PND 0, 2 and 16 and slides were prepared. The proportion of anuclear and superficial cells increased and parabasal decreased (P < 0.05) from PND 0 to 16. Proteins isolated from vaginal swabs taken on PND 2 and 16 from six gilts across three litters were measured using LC-MS/MS. Over 1500 proteins were identified, with 881 differentially expressed (P-adj < 0.05) between PND 2 and 16. One-third of proteins upregulated between days were categorized as secreted, including lipocalins. Categories enriched by downregulated proteins included cell-cell adherens junction, translation and ER to Golgi vesicle-mediated transport, and reflected increased cornification of stratified epithelium and thus mirrored changes in cytology. Changes in cytology and proteome over the first two weeks after birth support that the porcine vagina continues to develop postnatal.

An Integrative Proteomic/Lipidomic Analysis of the Gold Nanoparticle Biocorona in Healthy and Obese Conditions.

Introduction: When nanoparticles (NPs) enter a physiological environment, a coating of biomolecules or biocorona (BC) forms on the surface. Formation of the NP-BC is dependent on NP properties, the physiological environment, and time. The BC influences NP properties and biological interactions such as cellular internalization, immune responses, biodistribution, and others, leading to pharmacological and toxicological consequences. To date, examination of the NP-BC has focused primarily on protein components and healthy conditions. Therefore, we evaluated the protein and lipid content of BCs that formed on physicochemically distinct gold nanoparticles (AuNPs) under healthy and obese conditions. A comprehensive understanding of the NP-BC is necessary for the translation of in vitro toxicity assessments to clinical applications. Materials and Methods: AuNPs with two coatings (poly-N-vinylpyrrolidone [PVP] or citrate) and diameters (20 or 100 nm) were incubated in pooled human serum, and an integrated proteomic/lipidomic approach was used to evaluate BC composition. Macrophages were utilized to evaluate differential immune responses due to variations in the AuNP-BC. Results: AuNPs form distinct BCs based on physicochemical properties and the surrounding environment, with the obese BC containing more proteins and fewer lipids than the healthy BC. Differential macrophage inflammatory responses were observed based on AuNP properties and BC composition. Discussion and Conclusion: Overall, these findings demonstrate that AuNP size and coating, as well as physiological environment, influence the protein and lipid composition of the BC, which impacts cellular responses following exposure. These findings demonstrate that incorporation of BCs representing distinct physiological conditions may enhance the translatability of nanosafety in vitro studies.

Complementary use of mass spectrometry and cryo-electron microscopy to assess the maturity of live attenuated dengue vaccine viruses.

Dengue virus (DENV) infection is a global health threat with the potential to affect at least 3.6 billion people living in areas of risk. No specific curative treatments against dengue disease are available and vaccines are currently the only way to prevent the disease. The tetravalent dengue vaccine developed by Sanofi Pasteur has demonstrated significant efficacy in phase III studies and is now licensed in several countries for the prevention of disease in dengue-seropositives over 9 years of age. The vaccine is composed of four recombinant, live, attenuated vaccines (CYD 1-4) based on a yellow fever vaccine 17D (YFV 17D) backbone, each expressing the pre-membrane (prM) and envelope (E) genes of one of the four DENV serotypes. Virus maturity could impact the biological activity of the vaccine viruses. To address this question, the maturity of the four vaccine viruses used in phase III clinical studies was assessed by two complementary techniques: mass spectrometry (MS) and cryo-electron microscopy (cryoEM). MS assessed viral maturity at the molecular level by quantifying specifically the prM, and M proteins. CryoEM provided information at the particle level, allowing visualizing the different phenotypes of viral particles: spiky (immature), smooth/bumpy (mature), and mixed (partially mature). Results of the two assays used in this study show that all four CYD dengue vaccine viruses present in lots used in phase III efficacy trials, display in the majority a mature phenotype.

Maternal high-fat diet exposure during gestation, lactation, or gestation and lactation differentially affects intestinal morphology and proteome of neonatal mice.

Offspring nutrition depends on the mother during gestation and lactation; thus, maternal nutrition and metabolism can affect their development. We hypothesized that maternal exposure to high-fat (HF) diet affects neonate's gastrointestinal tract development. Our objective was to determine the effect of maternal HF diet during gestation and lactation on neonate's duodenum histomorphology and proteome. Female mice were fed either a control (C, 10% kcal fat) or an HF (60% kcal fat) diet for 4 weeks and bred. On postnatal day 2, half the pups were cross-fostered to dams fed on different diet, creating 4 treatments: C-C, C-HF, HF-C, and HF-HF, indicating maternal diet during gestation-lactation, respectively. On postnatal day 12, pups' duodenum was excised and prepared for histology and liquid chromatography-tandem mass spectrometry analysis of proteome. Villi were significantly longer in HF-HF pups, and crypt cell proliferation rate was not different among treatments. Between C-C and HF-HF, HF-C, or C-HF, 812, 601, or 894 proteins were differentially expressed (Tukey adjusted P < .05), respectively. Functional analysis clustered proteins upregulated in HF-HF vs C-C in fat digestion and absorption, extracellular matrix, cell adhesion, immune response, oxidation-reduction processes, phagocytosis, and transport categories. Proteins downregulated were classified as RNA splicing, translation, protein folding, endocytosis, and transport. There was evidence for a carryover effect of exposure to HF diet during gestation to the postnatal period. Alterations in proteome relative to HF exposure potentially reflect long-term changes in the functioning of the duodenum.

Proteomic Analysis of 3T3-L1 Adipocytes Treated with Insulin and TNF-α.

Insulin resistance is an indication of early stage Type 2 diabetes (T2D). Insulin resistant adipose tissues contain higher levels of insulin than the physiological level, as well as higher amounts of intracellular tumor necrosis factor-α (TNF-α) and other cytokines. However, the mechanism of insulin resistance remains poorly understood. To better understand the roles played by insulin and TNF-α in insulin resistance, we performed proteomic analysis of differentiated 3T3-L1 adipocytes treated with insulin (Ins), TNF-α (TNF), and both (Ins + TNF). Out of the 693 proteins identified, the abundances of 78 proteins were significantly different (p < 0.05). Carnitine parmitoyltransferase-2 (CPT2), acetyl CoA carboxylase 1 (ACCAC-1), ethylmalonyl CoA decarboxylase (ECHD1), and methylmalonyl CoA isomerase (MCEE), enzymes required for fatty acid ß-oxidation and respiratory electron transport, and ß-glucuronidase, an enzyme responsible for the breakdown of complex carbohydrates, were down-regulated in all the treatment groups, compared to the control group. In contrast, superoxide dismutase 2 (SOD2), protein disulfide isomerase (PDI), and glutathione reductase, which are the proteins responsible for cytoskeletal structure, protein folding, degradation, and oxidative stress responses, were up-regulated. This suggests higher oxidative stress in cells treated with Ins, TNF, or both. We proposed a conceptual metabolic pathway impacted by the treatments and their possible link to insulin resistance or T2D.

YopT domain of the PfhB2 toxin from Pasteurella multocida: protein expression, characterization, crystallization and crystallographic analysis.

Pasteurella multocida causes respiratory-tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multidomain toxin called PfhB2, which contains a YopT-like cysteine protease domain at its C-terminus. The YopT domain of PfhB2 contains a well conserved Cys-His-Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. To date, only one crystal structure of a YopT family member has been reported; however, additional structural information is needed to help characterize the varied substrate specificity and enzymatic action of this large protease family. Here, a catalytically inactive C3733S mutant of PfhB2 YopT that provides enhanced protein stability was used with the aim of gaining structural insight into the diversity within the YopT protein family. To this end, the C3733S mutant of PfhB2 YopT has been successfully cloned, overexpressed, purified and crystallized. Diffraction data sets were collected from native crystals to 3.5 Šresolution and a single-wavelength anomalous data set was collected from an iodide-derivative crystal to 3.2 Šresolution. Data pertaining to crystals belonging to space group P31, with unit-cell parameters a = 136.9, b = 136.9, c = 74.7 Šfor the native crystals and a = 139.2, b = 139.2, c = 74.7 Šfor the iodide-derivative crystals, are discussed.

Embryonic atrazine exposure elicits proteomic, behavioral, and brain abnormalities with developmental time specific gene expression signatures.

Atrazine (ATZ), the second most commonly used herbicide in the United States, is an endocrine disrupting chemical linked to cancer and a common drinking water contaminant. This study further investigates ATZ-related developmental toxicity by testing the following hypotheses in zebrafish: the effects of embryonic ATZ exposure are dependent on timing of exposure; embryonic ATZ exposure alters brain development and function; and embryonic ATZ exposure changes protein abundance in carcinogenesis-related pathways. After exposing embryos to 0, 0.3, 3, or 30 parts per billion (ppb) ATZ, we monitored the expression of cytochrome P450 family 17 subfamily A member 1 (cyp17a1), glyoxalase I (glo1), ring finger protein 14 (rnf14), salt inducible kinase 2 (sik2), tetratricopeptide domain 3 (ttc3), and tumor protein D52 like 1 (tpd52l1) at multiple embryonic time points to determine normal expression and if ATZ exposure altered expression. Only cyp17a1 had normal dynamic expression, but ttc3 and tpd52l1 had ATZ-related expression changes before 72 h. Larvae exposed to 0.3 ppb ATZ had increased brain length, while larvae exposed to 30 ppb ATZ were hypoactive. Proteomic analysis identified altered protein abundance in pathways related to cellular function, neurodevelopment, and genital-tract cancer. The results indicate embryonic ATZ toxicity involves interactions of multiple pathways. SIGNIFICANCE: This is the first report of proteomic alterations following embryonic exposure to atrazine, an environmentally persistent pesticide and common water contaminant. Although the transcriptomic alterations in larval zebrafish with embryonic atrazine exposure have been reported, neither the time at which gene expression changes occur nor the resulting proteomic changes have been investigated. This study seeks to address these knowledge gaps by evaluating atrazine's effect on gene expression through multiple time points during embryogenesis, and correlating changes in gene expression to pathological alterations in brain length and functional changes in behavior. Finally, pathway analysis of the proteomic alterations identifies connections between the molecular changes and functional outcomes associated with embryonic atrazine exposure.