Nuclear Phosphoproteome Reveals Prolyl Isomerase PIN1 as a Modulator of Oncogene-Induced Senescence.

Mammalian cells possess intrinsic mechanisms to prevent tumorigenesis upon deleterious mutations, including oncogene-induced senescence (OIS). The molecular mechanisms underlying OIS are, however, complex and remain to be fully characterized. In this study, we analyzed the changes in the nuclear proteome and phosphoproteome of human lung fibroblast IMR90 cells during the progression of OIS induced by oncogenic RASG12V activation. We found that most of the differentially regulated phosphosites during OIS contained prolyl isomerase PIN1 target motifs, suggesting PIN1 is a key regulator of several promyelocytic leukemia nuclear body proteins, specifically regulating several proteins upon oncogenic Ras activation. We showed that PIN1 knockdown promotes cell proliferation, while diminishing the senescence phenotype and hallmarks of senescence, including p21, p16, and p53 with concomitant accumulation of the protein PML and the dysregulation of promyelocytic leukemia nuclear body formation. Collectively, our data demonstrate that PIN1 plays an important role as a tumor suppressor in response to oncogenic ER:RasG12V activation.

Molecular Signatures of Neurodegenerative Diseases Identified by Proteomic and Phosphoproteomic Analyses in Aging Mouse Brain.

A central hallmark of neurodegenerative diseases is the irreversible accumulation of misfolded proteins in the brain by aberrant phosphorylation. Understanding the mechanisms underlying protein phosphorylation and its role in pathological protein aggregation within the context of aging is crucial for developing therapeutic strategies aimed at preventing or reversing such diseases. Here, we applied multi-protease digestion and quantitative mass spectrometry to compare and characterize dysregulated proteins and phosphosites in the mouse brain proteome using three different age groups: young-adult (3-4 months), middle-age (10 months), and old mice (19-21 months). Proteins associated with senescence, neurodegeneration, inflammation, cell cycle regulation, the p53 hallmark pathway, and cytokine signaling showed significant age-dependent changes in abundances and level of phosphorylation. Several proteins implicated in Alzheimer's disease (AD) and Parkinson's disease (PD) including tau (Mapt), Nefh, and Dpysl2 (also known as Crmp2) were hyperphosphorylated in old mice brain suggesting their susceptibility to the diseases. Cdk5 and Gsk3b, which are known to phosphorylate Dpysl2 at multiple specific sites, had also increased phosphorylation levels in old mice suggesting a potential crosstalk between them to contribute to AD. Hapln2, which promotes α-synuclein aggregation in patients with PD, was one of the proteins with highest abundance in old mice. CD9, which regulates senescence through the PI3K-AKT-mTOR-p53 signaling was upregulated in old mice and its regulation was correlated with the activation of phosphorylated AKT1. Overall, the findings identify a significant association between aging and the dysregulation of proteins involved in various pathways linked to neurodegenerative diseases with potential therapeutic implications.

Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP's enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

Proteomics analysis of round and wrinkled pea (Pisum sativum L.) seeds during different development periods.

Seed development is complex, influenced by genetic and environmental factors. Understanding proteome profiles at different seed developmental stages is key to improving seed composition and quality. We used label-free quantitative proteomics to analyze round and wrinkled pea seeds at five growth stages: 4, 7, 12, 15, and days after anthesis (DAA), and at maturity. Wrinkled peas had lower starch content (30%) compared to round peas (47%-55%). Proteomic analysis identified 3659 protein groups, with 21%-24% shared across growth stages. More proteins were identified during early seed development than at maturity. Statistical analysis found 735 significantly different proteins between wrinkled and round seeds, regardless of the growth stage. The detected proteins were categorized into 31 functional classes, including metabolic enzymes, proteins involved in protein biosynthesis and homeostasis, carbohydrate metabolism, and cell division. Cell division-related proteins were more abundant in early stages, while storage proteins were more abundant later in seed development. Wrinkled seeds had lower levels of the starch-branching enzyme (SBEI), which is essential for amylopectin biosynthesis. Seed storage proteins like legumin and albumin (PA2) were more abundant in round peas, whereas vicilin was more prevalent in wrinkled peas. This study enhances our understanding of seed development in round and wrinkled peas. The study highlighted the seed growth patterns and protein profiles in round and wrinkled peas during seed development. It showed how protein accumulation changed, particularly focusing on proteins implicated in cell division, seed reserve metabolism, as well as storage proteins and protease inhibitors. These findings underscore the crucial role of these proteins in seed development. By linking the proteins identified to Cameor-based pea reference genome, our research can open avenues for deeper investigations into individual proteins, facilitate their practical application in crop improvement, and advance our knowledge of seed development.

Sulfotransferase 1C2 Increases Mitochondrial Respiration by Converting Mitochondrial Membrane Cholesterol to Cholesterol Sulfate.

HYPOTHESIS: In this communication, we test the hypothesis that sulfotransferase 1C2 (SULT1C2, UniProt accession no. Q9WUW8) can modulate mitochondrial respiration by increasing state-III respiration. METHODS AND RESULTS: Using freshly isolated mitochondria, the addition of SULT1C2 and 3-phosphoadenosine 5 phosphosulfate (PAPS) results in an increased maximal respiratory capacity in response to the addition of succinate, ADP, and rotenone. Lipidomics and thin-layer chromatography of mitochondria treated with SULT1C2 and PAPS showed an increase in the level of cholesterol sulfate. Notably, adding cholesterol sulfate at nanomolar concentration to freshly isolated mitochondria also increases maximal respiratory capacity. In vivo studies utilizing gene delivery of SULT1C2 expression plasmids to kidneys result in increased mitochondrial membrane potential and confer resistance to ischemia/reperfusion injury. Mitochondria isolated from gene-transduced kidneys have elevated state-III respiration as compared with controls, thereby recapitulating results obtained with mitochondrial fractions treated with SULT1C2 and PAPS. CONCLUSION: SULT1C2 increases mitochondrial respiratory capacity by modifying cholesterol, resulting in increased membrane potential and maximal respiratory capacity. This finding uncovers a unique role of SULT1C2 in cellular physiology and extends the role of sulfotransferases in modulating cellular metabolism.

Essential amino acids in the Plant-Conserved and Class-Specific Regions of cellulose synthases.

The Plant-Conserved Region (P-CR) and the Class-Specific Region (CSR) are two plant-unique sequences in the catalytic core of cellulose synthases (CESAs) for which specific functions have not been established. Here, we used site-directed mutagenesis to replace amino acids and motifs within these sequences predicted to be essential for assembly and function of CESAs. We developed an in vivo method to determine the ability of mutated CesA1 transgenes to complement an Arabidopsis (Arabidopsis thaliana) temperature-sensitive root-swelling1 (rsw1) mutant. Replacement of a Cys residue in the CSR, which blocks dimerization in vitro, rendered the AtCesA1 transgene unable to complement the rsw1 mutation. Examination of the CSR sequences from 33 diverse angiosperm species showed domains of high-sequence conservation in a class-specific manner but with variation in the degrees of disorder, indicating a nonredundant role of the CSR structures in different CESA isoform classes. The Cys residue essential for dimerization was not always located in domains of intrinsic disorder. Expression of AtCesA1 transgene constructs, in which Pro417 and Arg453 were substituted for Ala or Lys in the coiled-coil of the P-CR, were also unable to complement the rsw1 mutation. Despite an expected role for Arg457 in trimerization of CESA proteins, AtCesA1 transgenes with Arg457Ala mutations were able to fully restore the wild-type phenotype in rsw1. Our data support that Cys662 within the CSR and Pro417 and Arg453 within the P-CR of Arabidopsis CESA1 are essential residues for functional synthase complex formation, but our data do not support a specific role for Arg457 in trimerization in native CESA complexes.

Effects of Neighboring Phosphorylation Events on the Affinities of pT181-Tau Antibodies.

A tau variant phosphorylated on threonine 181 (pT181-tau) has been widely investigated as a potential Alzheimer's disease (AD) biomarker in cerebrospinal fluid (CSF) and blood. pT181-tau is present in neurofibrillary tangles (NFTs) of AD brains, and CSF levels of pT181-tau correlate with the overall NFT burden. Various immunobased analytical methods, including Western blotting and ELISA, have been used to quantify pT181-tau in human biofluids. The reliability of these methods is dependent on the affinity and binding specificity of the antibodies used to measure pT181-tau levels. Although both of these properties could, in principle, be affected by phosphorylation within or near the antibody's cognate antigen, such effects have not been extensively studied. Here, we developed a biolayer interferometry assay to determine the degree to which the affinity of pT181-tau antibodies is altered by the phosphorylation of serine or threonine residues near the target epitope. Our results revealed that phosphorylation near T181 negatively affected the binding of pT181-tau antibodies to their cognate antigen to varying degrees. In particular, two of three antibodies tested showed a complete loss of affinity for the pT181 target when S184 or S185 was phosphorylated. These findings highlight the importance of selecting antibodies that have been thoroughly characterized in terms of affinity and binding specificity, addressing the potential disruptive effects of post-translational modifications in the epitope region to ensure accurate biomarker quantitation.

Proteomic analysis reveals that aging rabbit vocal folds are more vulnerable to changes caused by systemic dehydration.

BACKGROUND: Older adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol. RESULTS: Young and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of - 5.4% and - 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration. CONCLUSIONS: Water restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

Global landscape of protein complexes in postprandial-state livers from diet-induced obese and lean mice.

Obesity is associated with a spectrum of nonalcoholic fatty liver disease (NAFLD) which is characterized by steatosis. Prolonged fat deposition aggravates liver dysfunctions leading to an advanced form of NAFLD such as steatohepatitis and cirrhosis. As liver function in the postprandial state is critical for macronutrient metabolism and energy homeostasis, we sought to determine the differences in protein complex profiles in lean and fatty livers in the postprandial state. Protein complex profiling is of interest as proteins often do not function alone and the information on the interactions may reveal novel etiology of NAFLD, which is currently limited compared with proteome profiles or RNA-sequencing profiles. To this end, we fractionated liver lysates using size-exclusion chromatography (SEC) and analyzed each fraction using untargeted LC-MS/MS. We identified 1172 proteins that were discovered in lean and fatty livers, and their elution profiles were compared. We found that the majority of liver proteins were present as putative complexes. Also, the fatty liver protein elution profile showed great conservations as lean liver despite the metabolic disease state. Yet, we discovered a few proteins that showed different elution patterns in the fatty liver, including Acadm, Aldh1a7, Aldh1a1, Akr1a1, Eif3l, Fkbp2, G6pdx, Gm20441, Hao1, Pcna, Pkm, Ppif, Prdx4, Stmn1, Tagln, Tubb4b, Ubqln2, and Usp14, which may be involved in high fat diet-induced alterations of protein oligomerization and hepatic functions. Overall, our protein complex profiling could expand our understanding of hepatic abnormalities that cannot be uncovered by simple quantitation of protein expression.

Glycome and Proteome Components of Golgi Membranes Are Common between Two Angiosperms with Distinct Cell-Wall Structures.

The plant endoplasmic reticulum-Golgi apparatus is the site of synthesis, assembly, and trafficking of all noncellulosic polysaccharides, proteoglycans, and proteins destined for the cell wall. As grass species make cell walls distinct from those of dicots and noncommelinid monocots, it has been assumed that the differences in cell-wall composition stem from differences in biosynthetic capacities of their respective Golgi. However, immunosorbence-based screens and carbohydrate linkage analysis of polysaccharides in Golgi membranes, enriched by flotation centrifugation from etiolated coleoptiles of maize (Zea mays) and leaves of Arabidopsis (Arabidopsis thaliana), showed that arabinogalactan-proteins and arabinans represent substantial portions of the Golgi-resident polysaccharides not typically found in high abundance in cell walls of either species. Further, hemicelluloses accumulated in Golgi at levels that contrasted with those found in their respective cell walls, with xyloglucans enriched in maize Golgi, and xylans enriched in Arabidopsis. Consistent with this finding, maize Golgi membranes isolated by flotation centrifugation and enriched further by free-flow electrophoresis, yielded >200 proteins known to function in the biosynthesis and metabolism of cell-wall polysaccharides common to all angiosperms, and not just those specific to cell-wall type. We propose that the distinctive compositions of grass primary cell walls compared with other angiosperms result from differential gating or metabolism of secreted polysaccharides post-Golgi by an as-yet unknown mechanism, and not necessarily by differential expression of genes encoding specific synthase complexes.

Expression profiles of an inactive aspartic protease (Bla g 2 allergen) in different tissues and developmental stages of the German cockroach (Blattella germanica).

Tergal glands are found in many insect species and contain constituents such as pheromones, sugars, proteins, and so forth. Preliminary studies have revealed that tergal gland secretions in the German cockroach (Blattella germanica L.) contain the human allergen Bla g 2 (B. germanica allergen 2), an inactive aspartic protease. Although Bla g 2 protein expression has been detected previously in various German cockroach body parts, including male tergal glands, studies that link protein expression in various life stages and tissues with mRNA and protein abundance have not been conducted. Therefore, the goal of this study was to measure the relative abundances of Bla g 2 protein and mRNA in different tissues and life stages of B. germanica using immunoblotting, quantitative PCR, and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantitative profiling. We found that Bla g 2 protein was detected in every sampled tissue, including the male tergal glands. Protein abundance was relatively high in adult males and their tergal glands in comparison to nymphs and virgin females. Similarly, Bla g 2 mRNA transcript levels were also comparatively higher in male tergal glands and adult males. In conclusion, this study provides new information on the relative abundance and distribution of Bla g 2 allergen, a medically significant protein, in different tissues and developmental stages of the German cockroach and lays the foundation for future studies that aim to determine the function of this protein in B. germanica development.

A Label-free Mass Spectrometry Method to Predict Endogenous Protein Complex Composition.

Information on the composition of protein complexes can accelerate mechanistic analyses of cellular systems. Protein complex composition identifies genes that function together and provides clues about regulation within and between cellular pathways. Cytosolic protein complexes control metabolic flux, signal transduction, protein abundance, and the activities of cytoskeletal and endomembrane systems. It has been estimated that one third of all cytosolic proteins in leaves exist in an oligomeric state, yet the composition of nearly all remain unknown. Subunits of stable protein complexes copurify, and combinations of mass-spectrometry-based protein correlation profiling and bioinformatic analyses have been used to predict protein complex subunits. Because of uncertainty regarding the power or availability of bioinformatic data to inform protein complex predictions across diverse species, it would be highly advantageous to predict composition based on elution profile data alone. Here we describe a mass spectrometry-based protein correlation profiling approach to predict the composition of hundreds of protein complexes based on biochemical data. Extracts were obtained from an intact organ and separated in parallel by size and charge under nondenaturing conditions. More than 1000 proteins with reproducible elution profiles across all replicates were subjected to clustering analyses. The resulting dendrograms were used to predict the composition of known and novel protein complexes, including many that are likely to assemble through self-interaction. An array of validation experiments demonstrated that this new method can drive protein complex discovery, guide hypothesis testing, and enable systems-level analyses of protein complex dynamics in any organism with a sequenced genome.

Quantitative Proteomics and Phosphoproteomics Reveal TNF-α-Mediated Protein Functions in Hepatocytes.

Increased secretion of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), is often associated with adipose tissue dysregulation, which often accompanies obesity. High levels of TNFα have been linked to the development of insulin resistance in several tissues and organs, including skeletal muscle and the liver. In this study, we examined the complex regulatory roles of TNFα in murine hepatocytes utilizing a combination of global proteomic and phosphoproteomic analyses. Our results show that TNFα promotes extensive changes not only of protein levels, but also the dynamics of their downstream phosphorylation signaling. We provide evidence that TNFα induces DNA replication and promotes G1/S transition through activation of the MAPK pathway. Our data also highlight several other novel proteins, many of which are regulated by phosphorylation and play a role in the progression and development of insulin resistance in hepatocytes.

Ubiquitin C-Terminal Hydrolase L1: Biochemical and Cellular Characterization of a Covalent Cyanopyrrolidine-Based Inhibitor.

The deubiquitinase (DUB) ubiquitin C-terminal hydrolase L1 (UCHL1) is expressed primarily in the central nervous system under normal physiological conditions. However, UCHL1 is overexpressed in various aggressive forms of cancer with strong evidence supporting UCHL1 as an oncogene in lung, glioma, and blood cancers. In particular, the level of UCHL1 expression in these cancers correlates with increased invasiveness and metastatic behavior, as well as poor patient prognosis. Although UCHL1 is considered an oncogene with potential as a therapeutic target, there remains a significant lack of useful small-molecule probes to pharmacologically validate in vivo targeting of the enzyme. Herein, we describe the characterization of a new covalent cyanopyrrolidine-based UCHL1 inhibitory scaffold in biochemical and cellular studies to better understand the utility of this inhibitor in elucidating the role of UCHL1 in cancer biology.

Gas-Phase Sequencing of Cyclotides: Introduction of Selective Ring Opening at Dehydroalanine via Ion/Ion Reaction.

The gas-phase linearization of cyclotides via site-selective ring opening at dehydroalanine residues and its application to cyclotide sequencing is presented. This strategy relies on the ability to incorporate dehydroalanine into macrocyclic peptide ions, which is easily accomplished through an ion/ion reaction. Triply protonated cyclotide cations are transformed into radical cations via ion/ion reaction with the sulfate radical anion. Subsequent activation of the cyclotide radical cation generates dehydroalanine at a single cysteine residue, which is easily identified by the odd-electron loss of ·SCH2CONH2. The presence of dehydroalanine in cyclotides provides a site-selective ring-opening pathway that, in turn, generates linear cyclotide analogues in the gas phase. Unlike cyclic variants, product ions derived from the linear peptides provide rich sequence information. The sequencing capability of this strategy is demonstrated with four known cyclotides found in Viola inconspicua, where, in each case, greater than 93% sequence coverage was observed. Furthermore, the utility of this method is highlighted by the partial de novo sequencing of an unknown cyclotide with much greater sequence coverage than that obtained with a conventional Glu-C digestion approach. This method is particularly well-suited for cyclotide species that are not abundant enough to characterize with traditional methods.

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 proteomic strategy for global analysis of plant protein complexes.

Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.