Performance evaluation of different albumin assays for the detection of analbuminemia.

Analbuminemia is characterized by the near absence of albumin in the plasma. Different methods are available for measuring albumin levels, but they do not necessarily agree with one another. It is a concern that analbuminemic samples could be falsely characterized due to the incorrect estimation of albumin. The objective of the work was to evaluate the performance of different assays in detecting analbuminemia. Albumin knockout (Alb-/-) mouse plasma was used to test the suitability of different albumin assays for their ability to properly characterize extreme albumin deficiency. Bromocresol green (BCG), bromocresol purple (BCP), enzyme-linked immunosorbent assay (ELISA), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and gel electrophoresis were tested. The LC-MS/MS assay exhibited broad coverage of the amino acid sequence of albumin and indicated 8,400-fold lower (P<0.0001) albumin expression in Alb-/- than wildtype (WT), demonstrating its suitability for identifying extreme albumin deficiency. ELISA estimated albumin at 1.5±0.1 g/dL in WT and was below the detection limit in all Alb-/- samples. Gel electrophoresis yielded consistent results with LC-MS/MS and ELISA. The BCG assay overestimated albumin with apparently appreciable albumin concentrations in Alb-/- mice, yet the assay still indicated a significant difference between genotypes (Alb-/-, 1.2±0.05 g/dL, WT, 3.7±0.1 g/dL, P<0.0001). BCP drastically overestimated albumin and could not successfully identify the known analbuminemic phenotype of Alb-/- mice. By using Alb-/- plasma as a reference material and LC-MS/MS as a reference method, ELISA and gel electrophoresis appear appropriate for identifying analbuminemia, while BCG and BCP are not suitable. It is concluded that dye-binding assays should be avoided when extreme hypoalbuminemia or analbuminemia is suspected.

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.

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.

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.

Anticancer peptides from induced tumor-suppressing cells for inhibiting osteosarcoma cells.

Osteosarcoma (OS) is the most frequent primary bone cancer, which is mainly suffered by children and young adults. While the current surgical treatment combined with chemotherapy is effective for the early stage of OS, advanced OS preferentially metastasizes to the lung and is difficult to treat. Here, we examined the efficacy of ten anti-OS peptide candidates from a trypsin-digested conditioned medium that was derived from the secretome of induced tumor-suppressing cells (iTSCs). Using OS cell lines, the antitumor capabilities of the peptide candidates were evaluated by assaying the alterations in metabolic activities, proliferation, motility, and invasion of OS cells. Among ten candidates, peptide P05 (ADDGRPFPQVIK), a fragment of aldolase A (ALDOA), presented the most potent OS-suppressing capabilities. Its efficacy was additive with standard-of-care chemotherapeutic agents such as cisplatin and doxorubicin, and it downregulated oncoproteins such as epidermal growth factor receptor (EGFR), Snail, and Src in OS cells. Interestingly, P05 did not present inhibitory effects on non-OS skeletal cells such as mesenchymal stem cells and osteoblast cells. Collectively, this study demonstrated that iTSC-derived secretomes may provide a source for identifying anticancer peptides, and P05 may warrant further evaluations for the treatment of OS.

Adverse developmental impacts in progeny of zebrafish exposed to the agricultural herbicide atrazine during embryogenesis.

Atrazine (ATZ) is an herbicide commonly used on crops in the Midwestern US and other select global regions. The US Environmental Protection Agency ATZ regulatory limit is 3 parts per billion (ppb; µg/L), but this limit is often exceeded. ATZ has a long half-life, is a common contaminant of drinking water sources, and is indicated as an endocrine disrupting chemical in multiple species. The zebrafish was used to test the hypothesis that an embryonic parental ATZ exposure alters protein levels leading to modifications in morphology and behavior in developing progeny. Zebrafish embryos (F1) were collected from adults (F0) exposed to 0, 0.3, 3, or 30 ppb ATZ during embryogenesis. Differential proteomics, morphology, and behavior assays were completed with offspring aged 120 or 144 h with no additional chemical treatment. Proteomic analysis identified differential expression of proteins associated with neurological development and disease; and organ and organismal morphology, development, and injury, specifically the skeletomuscular system. Head length and ratio of head length to total length was significantly increased in the F1 of 0.3 and 30 ppb ATZ groups (p < 0.05). Based on molecular pathway alterations, further craniofacial morphology assessment found decreased distance for cartilaginous structures, decreased surface area and distance between saccular otoliths, and a more posteriorly positioned notochord (p < 0.05), indicating delayed ossification and skeletal growth. The visual motor response assay showed hyperactivity in progeny of the 30 ppb treatment group for distance moved and of the 0.3 and 30 ppb treatment groups for time spent moving (p < 0.05). Due to the changes in saccular otoliths, an acoustic startle assay was completed and showed decreased response in the 0.3 and 30 ppb treatments (p < 0.05). These findings suggest that a single embryonic parental exposure alters cellular pathways in their progeny that lead to perturbations in craniofacial development and behavior.

Proteomic analysis of murine kidney proximal tubule sub-segment derived cell lines reveals preferences in mitochondrial pathway activity.

The proximal tubule (PT) is a nephron segment that is responsible for the majority of solute and water reabsorption in the kidney. Each of its sub-segments have specialized functions; however, little is known about the genes and proteins that determine the oxidative phosphorylation capacity of the PT sub-segments. This information is critical to understanding kidney function and will provide a comprehensive landscape of renal cell adaptations to injury, physiologic stressors, and development. This study analyzed three immortalized murine renal cell lines (PT S1, S2, and S3 segments) for protein content and compared them to a murine fibroblast cell line. All three proximal tubule cell lines generate ATP predominantly by oxidative phosphorylation while the fibroblast cell line is glycolytic. The proteomic data demonstrates that the most significant difference in proteomic signatures between the cell lines are proteins known to be localized in the nucleus followed by mitochondrial proteins. Mitochondrial metabolic substrate utilization assays were performed using the proximal tubule cell lines to determine substrate utilization kinetics thereby providing a physiologic context to the proteomic dataset. This data will allow researchers to study differences in nephron-specific cell lines, between epithelial and fibroblast cells, and between actively respiring cells and glycolytic cells. SIGNIFICANCE: Proteomic analysis of proteins expressed in immortalized murine renal proximal tubule cells was compared to a murine fibroblast cell line proteome. The proximal tubule segment specific cell lines: S1, S2 and S3 are all grown under conditions whereby the cells generate ATP by oxidative phosphorylation while the fibroblast cell line utilizes anaerobic glycolysis for ATP generation. The proteomic studies allow for the following queries: 1) comparisons between the proximal tubule segment specific cell lines, 2) comparisons between polarized epithelia and fibroblasts, 3) comparison between cells employing oxidative phosphorylation versus anaerobic glycolysis and 4) comparisons between cells grown on clear versus opaque membrane supports. The data finds major differences in nuclear protein expression and mitochondrial proteins. This proteomic data set will be an important baseline dataset for investigators who need immortalized renal proximal tubule epithelial cells for their research.

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

Mammalian polynucleotide kinase 3'-phosphatase (PNKP) is a dual-function DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, which generate 3'-OH and 5'-phosphate termini respectively, as substrates for DNA polymerase and DNA ligase to complete DNA repair. PNKP is thus 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, which involve distinct sets of proteins. In this study, we report that PNKP is acetylated at two lysine (K142 and K226) residues. While K142 (AcK142) is constitutively acetylated by p300, CBP acetylates K226 (AcK226) only after DSB induction. Co-immunoprecipitation analysis using antibodies specific for PNKP peptides containing AcK142 or AcK226 of PNKP showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP only with DSBR proteins. Although acetylation at these residues did not significantly affect the enzymatic activity of PNKP in vitro, cells expressing nonacetylable PNKP (K142R or K226R) accumulated DNA damage, specifically in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This observation is consistent with the reported degradation of CBP but not p300 in HD cells. Moreover, genomes of HD cells progressively accumulated DSBs specifically in the transcribed genes. Chromatin-immunoprecipitation analysis using anti-AcK142 or anti-AcK226 antibodies demonstrated an association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings collectively demonstrate that acetylation at two lysine residues located in different domains of PNKP regulates its functionally distinct role in BER/SSBR vs. DSBR.

Development of an Anti-canine PD-L1 Antibody and Caninized PD-L1 Mouse Model as Translational Research Tools for the Study of Immunotherapy in Humans.

Immune checkpoint blockade therapy, one of the most promising cancer immunotherapies, has shown remarkable clinical impact in multiple cancer types. Despite the recent success of immune checkpoint blockade therapy, however, the response rates in patients with cancer are limited (∼20%-40%). To improve the success of immune checkpoint blockade therapy, relevant preclinical animal models are essential for the development and testing of multiple combination approaches and strategies. Companion dogs naturally develop several types of cancer that in many respects resemble clinical cancer in human patients. Therefore, the canine studies of immuno-oncology drugs can generate knowledge that informs and prioritizes new immuno-oncology therapy in humans. The challenge has been, however, that immunotherapeutic antibodies targeting canine immune checkpoint molecules such as canine PD-L1 (cPD-L1) have not been commercially available. Here, we developed a new cPD-L1 antibody as an immuno-oncology drug and characterized its functional and biological properties in multiple assays. We also evaluated the therapeutic efficacy of cPD-L1 antibodies in our unique caninized PD-L1 mice. Together, these in vitro and in vivo data, which include an initial safety profile in laboratory dogs, support development of this cPD-L1 antibody as an immune checkpoint inhibitor for studies in dogs with naturally occurring cancer for translational research. Our new therapeutic antibody and caninized PD-L1 mouse model will be essential translational research tools in raising the success rate of immunotherapy in both dogs and humans. Significance: Our cPD-L1 antibody and unique caninized mouse model will be critical research tools to improve the efficacy of immune checkpoint blockade therapy in both dogs and humans. Furthermore, these tools will open new perspectives for immunotherapy applications in cancer as well as other autoimmune diseases that could benefit a diverse and broader patient population.

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis.

Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42 nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.

Proteomics and metabolomics profiling of meat exudate to determine the impact of postmortem aging on oxidative stability of beef muscles.

The objective of this study was to characterize the major proteomes and metabolites in beef exudate and determine their relationship to color and oxidative quality of beef muscles. Beef loin (LD) and tenderloin (PM) muscles were cut into sections, individually vacuum-packaged, and aged for 9, 16 and 23 days at 2 °C. Following aging, beef exudates were collected and analyzed for both proteomics and metabolomics profiles. Proteome analysis indicated clustering by muscle types, while metabolomics profiling further clustered the samples based on the aging periods. The PM exudate had a greater concentration of oxidative enzymes, while the LD exudate contained more glycolytic enzymes. Greater lipid, nucleotide, carnitine and glucoside metabolites were observed in LD and 23d exudates. HSP70 and laminin proteins, together with glucosides metabolites, were correlated to muscle oxidative stability. The results indicated that meat exudate could be a viable analytical matrix to determine changes in quality attributes of meat with aging.

Proteomic and phosphoproteomic analyses of Jurkat T-cell treated with 2'3' cGAMP reveals various signaling axes impacted by cyclic dinucleotides.

Cyclic dinucleotides (CDNs), such as 2'3'-cGAMP, bind to STING to trigger the production of cytokines and interferons, mainly via activation of TBK1. STING activation by CDN also leads to the release and activation of Nuclear Factor Kappa-light-chain-enhancer of activated B cells (NF-κB) via the phosphorylation of Inhibitor of NF-κB (IκB)-alpha (IκBα) by IκB Kinase (IKK). Beyond the canonical TBK1 or IKK phosphorylations, little is known about how CDNs broadly affect the phosphoproteome and/or other signaling axes. To fill this gap, we performed an unbiased proteome and phosphoproteome analysis of Jurkat T-cell treated with 2'3'-cGAMP or vehicle control to identify proteins and phosphorylation sites that are differentially modulated by 2'3'-cGAMP. We uncovered different classes of kinase signatures associated with cell response to 2'3'-cGAMP. 2'3'-cGAMP upregulated Arginase 2 (Arg2) and the antiviral innate immune response receptor RIG-I as well as proteins involved in ISGylation, E3 ISG15-protein ligase HERC5 and ubiquitin-like protein ISG15, while downregulating ubiquitin-conjugating enzyme UBE2C. Kinases that play a role in DNA double strand break repair, apoptosis, and cell cycle regulation were differentially phosphorylated. Overall, this work demonstrates that 2'3'-cGAMP has a much broader effects on global phosphorylation events than currently appreciated, beyond the canonical TBK1/IKK signaling. SIGNIFICANCE: The host cyclic dinucleotide, 2'3'-cGAMP is known to bind to Stimulator of Interferon Genes (STING) to trigger the production of cytokines and interferons in immune cells via STING-TBK1-IRF3 pathway. Beyond the canonical phosphorelay via the STING-TBK1-IRF3 pathway, little is known about how this second messenger broadly affects the global proteome. Using an unbiased phosphoproteomics, this study identifies several kinases and phosphosites that are modulated by cGAMP. The study expands our knowledge about how cGAMP modulates global proteome and also global phosphorylations.

Proteomic analysis of canine vaccines.

OBJECTIVE: To use proteomic analysis to identify qualitatively and quantitatively mammalian protein components of commercial veterinary vaccines against canine distemper, leptospirosis, borreliosis, and rabies. SAMPLE: 25 licensed veterinary vaccines (from 4 different manufacturers) against canine distemper and leptospirosis, borreliosis, and rabies (3-year and 1-year durations of immunity). PROCEDURES: Duplicate samples from a single-lot vial of each vaccine were prepared by acetone precipitation and proteolysis with trypsin and Lys-C protease mix. Peptides mixtures (1 µg) were analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap Fusion Lumos mass spectrometer. Liquid chromatography-tandem mass spectroscopy data were searched against a Bos taurus protein database using MaxQuant to identify and quantify mammalian proteins in the vaccines. Identified proteins were classified by function and network analysis to visualize interactions. RESULTS: The largest number of mammalian proteins was identified in 3-year rabies vaccines (median, 243 proteins; range, 184 to 339 proteins) and 1-year rabies vaccines (median, 193 proteins; range, 169 to 350 proteins). Borrelia and leptospirosis-distemper (L&D) vaccines had the lowest number of proteins. Rabies vaccines had the highest number of identified proteins in common (n = 316); 33 were unique to 1-year products and 44 were found in 3-year products. Borrelia and L&D vaccines had 16 and 22 uniquely identified proteins, respectively. The protein classifications were primarily modulators of protein-binding activity, enzymes, transfer-carrier proteins, cytoskeletal proteins, defense-immunity proteins, calcium-binding proteins, and extracellular matrix proteins. CLINICAL RELEVANCE: This study demonstrates proteomics application to evaluate quality differences among different vaccines, identifying potential stimulants of desirable and undesirable immune responses.

Characterization of sow milk N-linked glycoproteome over the course of lactation.

Milk proteins serve as nutrition and affect neonate development and immunity through their bioactivity. Post-translational modifications of proteins affect their bioactivity. Glycosylation is the attachment of sugar moieties to proteins, with attachment of glycans to asparagine indicated as N-linked glycosylation. Our objective was to characterize N-linked glycosylated proteins in homogenate swine milk samples collected from sows (n = 5/6) during farrowing to represent colostrum and on days 3 and 14 post-farrowing to represent transitional and mature milk, respectively. Glycopeptides were isolated with lectin-based extraction and treated with Peptide N-glycosidase F (PNGase F) to identify N-linked glycosylation sites. Purified glycopeptides were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant software was used to align spectra to Sus scrofa Uniport database to identify proteins and measure their relative abundances. Analysis of variance and Welch's t-test analysis identified glycoproteins differentially abundant between colostrum, transitional, and mature milk (false discovery rate <0.05). Shotgun proteome analysis identified 545 N-linked and glutamine, Q, -linked, glycosylation (P > 0.75 for deamidation) sites on 220 glycoproteins in sow milk. Glycoproteins were found across all three phases of swine milk production and varied by number of glycosylation sites (1-14) and in abundance and distribution between colostrum, transitional, and mature milk. Polymeric immunoglobulin receptor was the most glycosylated protein with 14 sites identified. Also highly glycosylated were casein and mucin proteins. These data are described and the relevance of glycosylated milk proteins in neonate development, such as protection against pathogens, is discussed.

Milk is essential for healthy growth and development of neonates, with proteins in milk serving as key nutrients and regulators of these processes. Protein activity is affected by modifications made to their structure including the addition of sugar groups called glycans. Here we present the characterization of sow milk proteins modification with glycan groups on asparagine and glutamine amino acids in colostrum, transitional, and mature milk of pigs. We found 220 high confidences (found in at least two sows on one day) glycoproteins, and that the abundance of glycosylated proteins varied by stage of milk production and number of glycosylated sites.

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.

Prostate cancer-associated urinary proteomes differ before and after prostatectomy.

Background: A wide range of disorders can be detected in the urine. Tumor-modifying proteins in the urine may serve as a diagnostic tool for cancer patients and the alterations in their profiles may indicate efficacies of chemotherapy, radiotherapy, and surgery. Methods: We focused on urinary proteomes of patients with prostate cancer and identified tumor-modifying proteins in the samples before and after prostatectomy. Protein array analysis was conducted to evaluate a differential profile of tumor-promoting cytokines, while mass spectrometry-based global proteomics was conducted to identify tumor-suppressing proteins. Results: The result revealed striking differences by prostatectomy. Notably, the urine from the post-prostatectomy significantly decreased the tumorigenic behaviors of prostate tumor cells as well as breast cancer cells. We observed that angiogenin, a stimulator of blood vessel formation, was reduced in the post-prostatectomy urine. By contrast, the levels of three cell-membrane proteins such as prostasin (PRSS8), nectin 2 (PVRL2), and nidogen 1 (NID1) were elevated and they acted as extracellular tumor-suppressing proteins. These three proteins, given extracellularly, downregulated tumorigenic genes such as Runx2, Snail, and transforming growth factor beta and induced apoptosis of tumor cells. However, the role of NID1 differed depending on the location, and intracellular NID1 was tumorigenic and reduced the percent survival. Conclusions: This study demonstrated that prostatectomy remarkably altered the profile of urinary proteomes, and the post-prostatectomy urine provided tumor-suppressive proteomes. The result sheds novel light on the dynamic nature of the urinary proteomes and a unique strategy for predicting tumor suppressors.

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.

Erratum: Preventing tumor progression to the bone by induced tumor-suppressing MSCs: Erratum.

[This corrects the article DOI: 10.7150/thno.58779.].

PI3K-activated MSC proteomes inhibit mammary tumors via Hsp90ab1 and Myh9.

Despite the advance in medications in the past decade, aggressive breast cancer such as triple-negative breast cancer is difficult to treat. Here, we examined a counter-intuitive approach to converting human bone marrow-derived mesenchymal stem cells (MSCs) into induced tumor-suppressing cells by administering YS49, a PI3K/Akt activator. Notably, PI3K-activated MSCs generated tumor-suppressive proteomes, while PI3K-inactivated MSCs tumor-promotive proteomes. In a mouse model, the daily administration of YS49-treated MSC-derived CM decreased the progression of primary mammary tumors as well as the colonization of tumor cells in the lung. In the ex vivo assay, the size of freshly isolated human breast cancer tissues, including estrogen receptor positive and negative as well as human epidermal growth factor receptor 2 (HER2) positive and negative, was decreased by YS49-treated MSC-derived CM. Hsp90ab1 was enriched in CM as an atypical tumor-suppressing protein and immunoprecipitated a non-muscle myosin, Myh9. Extracellular Hsp90ab1 and Myh9 exerted the anti-tumor action and inhibited the maturation of bone-resorbing osteoclasts. Collectively, this study demonstrated that the activation of PI3K generated tumor-suppressive proteomes in MSCs and supported the possibility of using patient-derived MSCs for the treatment of breast cancer and bone metastasis.