A glycomic workflow for LC-MS/MS analysis of urine glycosaminoglycan biomarkers in mucopolysaccharidoses.

In recent years, several rational designed therapies have been developed for treatment of mucopolysaccharidoses (MPS), a group of inherited metabolic disorders in which glycosaminoglycans (GAGs) are accumulated in various tissues and organs. Thus, improved disease-specific biomarkers for diagnosis and monitoring treatment efficacy are of paramount importance. Specific non-reducing end GAG structures (GAG-NREs) have become promising biomarkers for MPS, as the compositions of the GAG-NREs depend on the nature of the lysosomal enzyme deficiency, thereby creating a specific pattern for each subgroup. However, there is yet no straightforward clinical laboratory platform which can assay all MPS-related GAG-NREs in one single analysis. Here, we developed and applied a GAG domain mapping approach for analyses of urine samples of ten MPS patients with various MPS diagnoses and corresponding aged-matched controls. We describe a nano-LC-MS/MS method of GAG-NRE profiling, utilizing 2-aminobenzamide reductive amination labeling to improve the sensitivity and the chromatographic resolution. Diagnostic urinary GAG-NREs were identified for MPS types IH/IS, II, IIIc, IVa and VI, corroborating GAG-NRE as biomarkers for these known enzyme deficiencies. Furthermore, a significant reduction of diagnostic urinary GAG-NREs in MPS IH (n = 2) and MPS VI (n = 1) patients under treatment was demonstrated. We argue that this straightforward glycomic workflow, designed for the clinical analysis of MPS-related GAG-NREs in one single analysis, will be of value for expanding the use of GAG-NREs as biomarkers for MPS diagnosis and treatment monitoring.

Subtyping of cardiac amyloidosis by mass spectrometry-based proteomics of endomyocardial biopsies.

BACKGROUND: Cardiac amyloidosis is a severe condition leading to restrictive cardiomyopathy and heart failure. Mass spectrometry-based methods for cardiac amyloid subtyping have become important diagnostic tools but are currently used only in a few reference laboratories. Such methods include laser-capture microdissection to ensure the specific analysis of amyloid deposits. Here we introduce a direct proteomics-based method for subtyping of cardiac amyloidosis. METHODS: Endomyocardial biopsies were retrospectively analysed from fresh frozen material of 78 patients with cardiac amyloidosis and from 12 biopsies of unused donor heart explants. Cryostat sections were digested with trypsin and analysed with liquid chromatography - mass spectrometry, and data were evaluated by proteomic software. RESULTS: With a diagnostic threshold set to 70% for each of the four most common amyloid proteins affecting the heart (LC κ, LC λ, TTR and SAA), 65 of the cases (87%) could be diagnosed, and of these, 61 cases (94%) were in concordance with the original diagnoses. The specimens were also analysed for the summed intensities of the amyloid signature proteins (ApoE, ApoA-IV and SAP). The intensities were significantly higher (p < 0.001) for all assigned cases compared with controls. CONCLUSION: Cardiac amyloidosis can be successfully subtyped without the prior enrichment of amyloid deposits with laser microdissection.

Expansion of nickel binding- and histidine-rich proteins during gastric adaptation of Helicobacter species.

Acquisition and homeostasis of essential metals during host colonization by bacterial pathogens rely on metal uptake, trafficking, and storage proteins. How these factors have evolved within bacterial pathogens is poorly defined. Urease, a nickel enzyme, is essential for Helicobacter pylori to colonize the acidic stomach. Our previous data suggest that acquisition of nickel transporters and a histidine-rich protein (HRP) involved in nickel storage in H. pylori and gastric Helicobacter spp. have been essential evolutionary events for gastric colonization. Using bioinformatics, proteomics, and phylogenetics, we extended this analysis to determine how evolution has framed the repertoire of HRPs among 39 Epsilonproteobacteria; 18 gastric and 11 non-gastric enterohepatic (EH) Helicobacter spp., as well as 10 other Epsilonproteobacteria. We identified a total of 213 HRPs distributed in 22 protein families named orthologous groups (OGs) with His-rich domains, including 15 newly described OGs. Gastric Helicobacter spp. are enriched in HRPs (7.7 ± 1.9 HRPs/strain) as compared to EH Helicobacter spp. (1.9 ± 1.0 HRPs/strain) with a particular prevalence of HRPs with C-terminal histidine-rich domains in gastric species. The expression and nickel-binding capacity of several HRPs was validated in five gastric Helicobacter spp. We established the evolutionary history of new HRP families, such as the periplasmic HP0721-like proteins and the HugZ-type heme oxygenases. The expansion of histidine-rich extensions in gastric Helicobacter spp. proteins is intriguing but can tentatively be associated with the presence of the urease nickel enzyme. We conclude that this HRP expansion is associated with unique properties of organisms that rely on large intracellular nickel amounts for their survival.

Dataset for suppressors of amyloid-ß toxicity and their functions in recombinant protein production in yeast.

The production of recombinant proteins at high levels often induces stress-related phenotypes by protein misfolding or aggregation. These are similar to those of the yeast Alzheimer's disease (AD) model in which amyloid-ß peptides (Aß42) were accumulated [1], [2]. We have previously identified suppressors of Aß42 cytotoxicity via the genome-wide synthetic genetic array (SGA) [3] and here we use them as metabolic engineering targets to evaluate their potentiality on recombinant protein production in yeast Saccharomyces cerevisiae. In order to investigate the mechanisms linking the genetic modifications to the improved recombinant protein production, we perform systems biology approaches (transcriptomics and proteomics) on the resulting strain and intermediate strains. The RNAseq data are preprocessed by the nf-core/RNAseq pipeline and analyzed using the Platform for Integrative Analysis of Omics (PIANO) package [4]. The quantitative proteome is analyzed on an Orbitrap Fusion Lumos mass spectrometer interfaced with an Easy-nLC1200 liquid chromatography (LC) system. LC-MS data files are processed by Proteome Discoverer version 2.4 with Mascot 2.5.1 as a database search engine. The original data presented in this work can be found in the research paper titled "Suppressors of Amyloid-ß Toxicity Improve Recombinant Protein Production in yeast by Reducing Oxidative Stress and Tuning Cellular Metabolism", by Chen et al. [5].

Optimized sample preparation and data analysis for TMT proteomic analysis of cerebrospinal fluid applied to the identification of Alzheimer's disease biomarkers.

BACKGROUND: Cerebrospinal fluid (CSF) is an important biofluid for biomarkers of neurodegenerative diseases such as Alzheimer's disease (AD). By employing tandem mass tag (TMT) proteomics, thousands of proteins can be quantified simultaneously in large cohorts, making it a powerful tool for biomarker discovery. However, TMT proteomics in CSF is associated with analytical challenges regarding sample preparation and data processing. In this study we address those challenges ranging from data normalization over sample preparation to sample analysis. METHOD: Using liquid chromatography coupled to mass-spectrometry (LC-MS), we analyzed TMT multiplex samples consisting of either identical or individual CSF samples, evaluated quantification accuracy and tested the performance of different data normalization approaches. We examined MS2 and MS3 acquisition strategies regarding accuracy of quantification and performed a comparative evaluation of filter-assisted sample preparation (FASP) and an in-solution protocol. Finally, four normalization approaches (median, quantile, Total Peptide Amount, TAMPOR) were applied to the previously published European Medical Information Framework Alzheimer's Disease Multimodal Biomarker Discovery (EMIF-AD MBD) dataset. RESULTS: The correlation of measured TMT reporter ratios with spiked-in standard peptide amounts was significantly lower for TMT multiplexes composed of individual CSF samples compared with those composed of aliquots of a single CSF pool, demonstrating that the heterogeneous CSF sample composition influences TMT quantitation. Comparison of TMT reporter normalization methods showed that the correlation could be improved by applying median- and quantile-based normalization. The slope was improved by acquiring data in MS3 mode, albeit at the expense of a 29% decrease in the number of identified proteins. FASP and in-solution sample preparation of CSF samples showed a 73% overlap in identified proteins. Finally, using optimized data normalization, we present a list of 64 biomarker candidates (clinical AD vs. controls, p < 0.01) identified in the EMIF-AD cohort. CONCLUSION: We have evaluated several analytical aspects of TMT proteomics in CSF. The results of our study provide practical guidelines to improve the accuracy of quantification and aid in the design of sample preparation and analytical protocol. The AD biomarker list extracted from the EMIF-AD cohort can provide a valuable basis for future biomarker studies and help elucidate pathogenic mechanisms in AD.

Suppressors of amyloid-ß toxicity improve recombinant protein production in yeast by reducing oxidative stress and tuning cellular metabolism.

High-level production of recombinant proteins in industrial microorganisms is often limited by the formation of misfolded proteins or protein aggregates, which consequently induce cellular stress responses. We hypothesized that in a yeast Alzheimer's disease (AD) model overexpression of amyloid-ß peptides (Aß42), one of the main peptides relevant for AD pathologies, induces similar phenotypes of cellular stress. Using this humanized AD model, we previously identified suppressors of Aß42 cytotoxicity. Here we hypothesize that these suppressors could be used as metabolic engineering targets to alleviate cellular stress and improve recombinant protein production in the yeast Saccharomyces cerevisiae. Forty-six candidate genes were individually deleted and twenty were individually overexpressed. The positive targets that increased recombinant α-amylase production were further combined leading to an 18.7-fold increased recombinant protein production. These target genes are involved in multiple cellular networks including RNA processing, transcription, ER-mitochondrial complex, and protein unfolding. By using transcriptomics and proteomics analyses, combined with reverse metabolic engineering, we showed that reduced oxidative stress, increased membrane lipid biosynthesis and repressed arginine and sulfur amino acid biosynthesis are significant pathways for increased recombinant protein production. Our findings provide new insights towards developing synthetic yeast cell factories for biosynthesis of valuable proteins.

Age and sex effects across the blood proteome after ionizing radiation exposure can bias biomarker screening and risk assessment.

Molecular biomarkers of ionizing radiation (IR) exposure are a promising new tool in various disciplines: they can give necessary information for adaptive treatment planning in cancer radiotherapy, enable risk projection for radiation-induced survivorship diseases, or facilitate triage and intervention in radiation hazard events. However, radiation biomarker discovery has not yet resolved the most basic features of personalized medicine: age and sex. To overcome this critical bias in biomarker identification, we quantitated age and sex effects and assessed their relevance in the radiation response across the blood proteome. We used high-throughput mass spectrometry on blood plasma collected 24 h after 0.5 Gy total body irradiation (15 MV nominal photon energy) from male and female C57BL/6 N mice at juvenile (7-weeks-old) or adult (18-weeks-old) age. We also assessed sex and strain effects using juvenile male and female BALB/c nude mice. We showed that age and sex created significant effects in the proteomic response regarding both extent and functional quality of IR-induced responses. Furthermore, we found that age and sex effects appeared non-linear and were often end-point specific. Overall, age contributed more to differences in the proteomic response than sex, most notably in immune responses, oxidative stress, and apoptotic cell death. Interestingly, sex effects were pronounced for DNA damage and repair pathways and associated cellular outcome (pro-survival vs. pro-apoptotic). Only one protein (AHSP) was identified as a potential general biomarker candidate across age and sex, while GMNN, REG3B, and SNCA indicated some response similarity across age. This low yield advocated that unisex or uniage biomarker screening approaches are not feasible. In conclusion, age- and sex-specific screening approaches should be implemented as standard protocol to ensure robustness and diagnostic power of biomarker candidates. Bias-free molecular biomarkers are a necessary progression towards personalized medicine and integral for advanced adaptive cancer radiotherapy and risk assessment.

Cytokine responses to LPS in reprogrammed monocytes are associated with the transcription factor PU.1.

Myeloid-derived suppressor cells (MDSCs) are functionally immunosuppressive cells that arise and expand during extensive inflammatory conditions by increased hematopoietic output or reprogramming of immune cells. In sepsis, an increase of circulating MDSCs is associated with adverse outcomes, but unique traits that can be used to identify increased activity of MDSCs are lacking. By using endotoxin tolerance as a model of sepsis-induced monocytic MDSCs (M-MDSC-like cells), this study aims to identify the mediator and transcriptional regulator profile associated with M-MDSC activity. After analyzing 180 inflammation-associated proteins, a profile of differentially expressed cytokines was found in M-MDSC-like cells versus normal monocytes stimulated with LPS. These cytokines were associated with 5 candidate transcription factors, where particularly PU.1 showed differential expression on both transcriptional and protein levels in M-MDSC-like cells. Furthermore, inhibition of PU.1 led to increased production of CXCL5 and CCL8 in M-MDSC-like cells indicating its role in regulating the ability of M-MDSC-like cells to recruit other immune cells. Taken together, the study identifies a unique profile in the pattern of immune mediators defining M-MDSC activity upon LPS stimulation, which offers a functional link to their contribution to immunosuppression.

Differential cytokine response in endotoxin induced M-MDSC-like cells and their associated regulators.

Changes in the Proteome in the Development of Chronic Human Papillomavirus Infection-A Prospective Study in HIV Positive and HIV Negative Rwandan Women.

BACKGROUND: Effects on the proteome when a high risk (HR)-HPV infection occurs, when it is cleared and when it becomes chronic were investigated. Moreover, biomarker panels that could identify cervical risk lesions were assessed. METHODS: Cytology, HPV screening and proteomics were performed on cervical samples from Rwandan HIV+ and HIV- women at baseline, at 9 months, at 18 months and at 24 months. Biological pathways were identified using the String database. RESULTS: The most significantly affected pathway when an incident HR-HPV infection occurred was neutrophil degranulation, and vesicle-mediated transport was the most significantly affected pathway when an HR-HPV infection was cleared; protein insertion into membrane in chronic HR-HPV lesions and in lesions where HR-HPVs were cleared were compared; and cellular catabolic process in high-grade lesions was compared to that in negative lesions. A four-biomarker panel (EIF1; BLOC1S5; LIMCH1; SGTA) was identified, which was able to distinguish chronic HR-HPV lesions from cleared HR-HPV/negative lesions (sensitivity 100% and specificity 91%). Another four-biomarker panel (ERH; IGKV2-30; TMEM97; DNAJA4) was identified, which was able to distinguish high-grade lesions from low-grade/negative lesions (sensitivity 100% and specificity 81%). CONCLUSIONS: We have identified the biological pathways triggered in HR-HPV infection, when HR-HPV becomes chronic and when cervical risk lesions develop. Moreover, we have identified potential biomarkers that may help to identify women with cervical risk lesions.

Proteoglycan profiling of human, rat and mouse insulin-secreting cells.

Proteoglycans (PGs) are proteins with glycosaminoglycan (GAG) chains, such as chondroitin sulfate (CS) or heparan sulfate (HS), attached to serine residues. We have earlier shown that prohormones can carry CS, constituting a novel class of PGs. The mapping of GAG modifications of proteins in endocrine cells may thus assist us in delineating possible roles of PGs in endocrine cellular physiology. With this aim, we applied a glycoproteomic approach to identify PGs, their GAG chains and their attachment sites in insulin-secreting cells. Glycopeptides carrying GAG chains were enriched from human pancreatic islets, rat (INS-1 832/13) and mouse (MIN6, NIT-1) insulinoma cell lines by exchange chromatography, depolymerized with GAG lyases, and analyzed by nanoflow liquid chromatography tandem mass spectrometry. We identified CS modifications of chromogranin-A (CgA), islet amyloid polypeptide, secretogranin-1 and secretogranin-2, immunoglobulin superfamily member 10, and protein AMBP. Additionally, we identified two HS-modified prohormones (CgA and secretogranin-1), which was surprising, as prohormones are not typically regarded as HSPGs. For CgA, the glycosylation site carried either CS or HS, making it a so-called hybrid site. Additional HS sites were found on syndecan-1, syndecan-4, nerurexin-2, protein NDNF and testican-1. These results demonstrate that several prohormones, and other constituents of the insulin-secreting cells are PGs. Cell-targeted mapping of the GAG glycoproteome forms an important basis for better understanding of endocrine cellular physiology, and the novel CS and HS sites presented here provide important knowledge for future studies.

Domain Mapping of Chondroitin/Dermatan Sulfate Glycosaminoglycans Enables Structural Characterization of Proteoglycans.

Of all posttranslational modifications known, glycosaminoglycans (GAGs) remain one of the most challenging to study, and despite the recent years of advancement in MS technologies and bioinformatics, detailed knowledge about the complete structures of GAGs as part of proteoglycans (PGs) is limited. To address this issue, we have developed a protocol to study PG-derived GAGs. Chondroitin/dermatan sulfate conjugates from the rat insulinoma cell line, INS-1832/13, known to produce primarily the PG chromogranin-A, were enriched by anion-exchange chromatography after pronase digestion. Following benzonase and hyaluronidase digestions, included in the sample preparation due to the apparent interference from oligonucleotides and hyaluronic acid in the analysis, the GAGs were orthogonally depolymerized and analyzed using nano-flow reversed-phase LC-MS/MS in negative mode. To facilitate the data interpretation, we applied an automated LC-MS peak detection and intensity measurement via the Proteome Discoverer software. This approach effectively provided a detailed structural description of the nonreducing end, internal, and linkage region domains of the CS/DS of chromogranin-A. The copolymeric CS/DS GAGs constituted primarily consecutive glucuronic-acid-containing disaccharide units, or CS motifs, of which the N-acetylgalactosamine residues were 4-O-sulfated, interspersed by single iduronic-acid-containing disaccharide units. Our data suggest a certain heterogeneity of the GAGs due to the identification of not only CS/DS GAGs but also of GAGs entirely of CS character. The presented protocol allows for the detailed characterization of PG-derived GAGs, which may greatly increase the knowledge about GAG structures in general and eventually lead to better understanding of how GAG structures are related to biological functions.

Quantifying absolute gene expression profiles reveals distinct regulation of central carbon metabolism genes in yeast.

In addition to controlled expression of genes by specific regulatory circuits, the abundance of proteins and transcripts can also be influenced by physiological states of the cell such as growth rate and metabolism. Here we examine the control of gene expression by growth rate and metabolism, by analyzing a multi-omics dataset consisting of absolute-quantitative abundances of the transcriptome, proteome, and amino acids in 22 steady-state yeast cultures. We find that transcription and translation are coordinately controlled by the cell growth rate via RNA polymerase II and ribosome abundance, but they are independently controlled by nitrogen metabolism via amino acid and nucleotide availabilities. Genes in central carbon metabolism, however, are distinctly regulated and do not respond to the cell growth rate or nitrogen metabolism as all other genes. Understanding these effects allows the confounding factors of growth rate and metabolism to be accounted for in gene expression profiling studies.

Telomeric double-strand DNA-binding proteins DTN-1 and DTN-2 ensure germline immortality in Caenorhabditis elegans.

Telomeres are nucleoprotein complexes at the ends of chromosomes and are indispensable for the protection and lengthening of terminal DNA. Despite the evolutionarily conserved roles of telomeres, the telomeric double-strand DNA (dsDNA)-binding proteins have evolved rapidly. Here, we identified double-strand telomeric DNA-binding proteins (DTN-1 and DTN-2) in Caenorhabditis elegans as non-canonical telomeric dsDNA-binding proteins. DTN-1 and DTN-2 are paralogous proteins that have three putative MYB-like DNA-binding domains and bind to telomeric dsDNA in a sequence-specific manner. DTN-1 and DTN-2 form complexes with the single-strand telomeric DNA-binding proteins POT-1 and POT-2 and constitutively localize to telomeres. The dtn-1 and dtn-2 genes function redundantly, and their simultaneous deletion results in progressive germline mortality, which accompanies telomere hyper-elongation and chromosomal bridges. Our study suggests that DTN-1 and DTN-2 are core shelterin components in C. elegans telomeres that act as negative regulators of telomere length and are essential for germline immortality.

Nitrogen limitation reveals large reserves in metabolic and translational capacities of yeast.

Cells maintain reserves in their metabolic and translational capacities as a strategy to quickly respond to changing environments. Here we quantify these reserves by stepwise reducing nitrogen availability in yeast steady-state chemostat cultures, imposing severe restrictions on total cellular protein and transcript content. Combining multi-omics analysis with metabolic modeling, we find that seven metabolic superpathways maintain >50% metabolic capacity in reserve, with glucose metabolism maintaining >80% reserve capacity. Cells maintain >50% reserve in translational capacity for 2490 out of 3361 expressed genes (74%), with a disproportionately large reserve dedicated to translating metabolic proteins. Finally, ribosome reserves contain up to 30% sub-stoichiometric ribosomal proteins, with activation of reserve translational capacity associated with selective upregulation of 17 ribosomal proteins. Together, our dataset provides a quantitative link between yeast physiology and cellular economics, which could be leveraged in future cell engineering through targeted proteome streamlining.

The BRCA2-MEILB2-BRME1 complex governs meiotic recombination and impairs the mitotic BRCA2-RAD51 function in cancer cells.

Breast cancer susceptibility gene II (BRCA2) is central in homologous recombination (HR). In meiosis, BRCA2 binds to MEILB2 to localize to DNA double-strand breaks (DSBs). Here, we identify BRCA2 and MEILB2-associating protein 1 (BRME1), which functions as a stabilizer of MEILB2 by binding to an α-helical N-terminus of MEILB2 and preventing MEILB2 self-association. BRCA2 binds to the C-terminus of MEILB2, resulting in the formation of the BRCA2-MEILB2-BRME1 ternary complex. In Brme1 knockout (Brme1-/-) mice, the BRCA2-MEILB2 complex is destabilized, leading to defects in DSB repair, homolog synapsis, and crossover formation. Persistent DSBs in Brme1-/- reactivate the somatic-like DNA-damage response, which repairs DSBs but cannot complement the crossover formation defects. Further, MEILB2-BRME1 is activated in many human cancers, and somatically expressed MEILB2-BRME1 impairs mitotic HR. Thus, the meiotic BRCA2 complex is central in meiotic HR, and its misregulation is implicated in cancer development.

Absolute yeast mitochondrial proteome quantification reveals trade-off between biosynthesis and energy generation during diauxic shift.

Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.

Glycosaminoglycan Domain Mapping of Cellular Chondroitin/Dermatan Sulfates.

Glycosaminoglycans (GAGs) are polysaccharides produced by most mammalian cells and involved in a variety of biological processes. However, due to the size and complexity of GAGs, detailed knowledge about the structure and expression of GAGs by cells, the glycosaminoglycome, is lacking. Here we report a straightforward and versatile approach for structural domain mapping of complex mixtures of GAGs, GAGDoMa. The approach is based on orthogonal enzymatic depolymerization of the GAGs to generate internal, terminating, and initiating domains, and nanoflow reversed-phase ion-pairing chromatography with negative mode higher-energy collision dissociation (HCD) tandem mass spectrometry (MS/MS) for structural characterization of the individual domains. GAGDoMa provides a detailed structural insight into the glycosaminoglycome, and offers an important tool for deciphering the complexity of GAGs in cellular physiology and pathology.

Lipid droplet-associated kinase STK25 regulates peroxisomal activity and metabolic stress response in steatotic liver.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide and have been recognized as one of the major unmet medical needs of the 21st century. Our recent translational studies in mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine kinase (STK)25 as a protein that coats intrahepatocellular lipid droplets (LDs) and critically regulates liver lipid homeostasis and progression of NAFLD/NASH. Here, we studied the mechanism-of-action of STK25 in steatotic liver by relative quantification of the hepatic LD-associated phosphoproteome from high-fat diet-fed Stk25 knockout mice compared with their wild-type littermates. We observed a total of 131 proteins and 60 phosphoproteins that were differentially represented in STK25-deficient livers. Most notably, a number of proteins involved in peroxisomal function, ubiquitination-mediated proteolysis, and antioxidant defense were coordinately regulated in Stk25-/- versus wild-type livers. We confirmed attenuated peroxisomal biogenesis and protection against oxidative and ER stress in STK25-deficient human liver cells, demonstrating the hepatocyte-autonomous manner of STK25's action. In summary, our results suggest that regulation of peroxisomal function and metabolic stress response may be important molecular mechanisms by which STK25 controls the development and progression of NAFLD/NASH.

Identification of a non-canonical chondroitin sulfate linkage region trisaccharide.

It is generally accepted that the biosynthesis of chondroitin sulfate and heparan sulfate is proceeding from a common linkage region tetrasaccharide comprising GlcA-Gal-Gal-Xyl-O-. The linkage region can undergo various modifications such as sulfation, phosphorylation and sialylation, and as the methods for studying glycosaminoglycan structure have been developed and refined, the number of discovered modifications has increased. Previous studies on the linkage region and the glycosyltransferases involved in the biosynthesis suggest that variants of the linkage region tetrasaccharide may also be possible. Here, using LC-MS/MS, we describe a non-canonical linkage region trisaccharide comprising GlcA-Gal-Xyl-O-. The trisaccharide was identified as a minor constituent in the proteoglycan bikunin from urine of human healthy donors present as a disulfated pentasaccharide, ΔHexA-GalNAc(S)-GlcA-Gal(S)-Xyl-O-, after chondroitinase ABC degradation. Furthermore, it was present as the corresponding disulfated pentasaccharide after chondroitinase ABC degradation in chondroitin sulfate primed on xylosides isolated from human cell lines. This linkage region trisaccharide may serve as an alternative point of entry for glycosaminoglycan biosynthesis.

Mice exposed to maternal androgen excess and diet-induced obesity have altered phosphorylation of catechol-O-methyltransferase in the placenta and fetal liver.

BACKGROUND/OBJECTIVES: Maternal obesity together with androgen excess in mice negatively affects placental function and maternal and fetal liver function as demonstrated by increased triglyceride content with dysfunctional expression of enzymes and transcription factors involved in de novo lipogenesis and fat storage. To identify changes in molecular pathways that might promote diseases in adulthood, we performed a global proteomic analysis using a liquid-chromatography/mass-spectrometry system to investigate total and phosphorylated proteins in the placenta and fetal liver in a mouse model that combines maternal obesity with maternal androgen excess. METHODS: After ten weeks on a control diet (CD) or high fat/high sugar-diet, dams were mated with males fed the CD. Between gestational day (GD) 16.5 and GD 18.5, mice were injected with vehicle or dihydrotestosterone (DHT) and sacrificed at GD 18.5 prior to dissection of the placentas and fetal livers. Four pools of female placentas and fetal livers were subjected to a global proteomic analysis. Total and phosphorylated proteins were filtered by ANOVA q < 0.05, and this was followed by two-way ANOVA to determine the effect of maternal obesity and/or androgen exposure. RESULTS: In placenta, phosphorylated ATP-citrate synthase was decreased due to maternal obesity, and phosphorylated catechol-O-methyltransferase (COMT) was differentially expressed due to the interaction between maternal diet and DHT exposure. In fetal liver, five total proteins and 48 proteins phosphorylated in one or more sites, were differentially expressed due to maternal obesity or androgen excess. In fetal liver, phosphorylated COMT expression was higher in fetus exposed to maternal obesity. CONCLUSION: These results suggest a common regulatory mechanism of catecholamine metabolism in the placenta and the fetal liver as demonstrated by higher phosphorylated COMT expression in the placenta and fetal liver from animals exposed to diet-induced maternal obesity and lower expression of phosphorylated COMT in animals exposed to maternal androgen excess.