O-Glycomic and Proteomic Signatures of Spontaneous and Butyrate-Stimulated Colorectal Cancer Cell Line Differentiation.

Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano-liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell proteome. It was found that the fully differentiated butyrate-stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O-glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these patterns in homeostasis and dysbiosis at the gut-microbiota interface.

Retina-arrestin specific CD8+ T cells are not implicated in HLA-A29-positive birdshot chorioretinitis.

BACKGROUND: HLA-A29-positive birdshot chorioretinitis (BCR) is an inflammatory eye disorder that is generally assumed to be caused by an autoimmune response to HLA-A29-presented peptides from retinal arrestin (SAG), yet the epitopes recognized by CD8+ T cells from patients remain to be identified. OBJECTIVES: The identification of natural ligands of SAG presented by HLA-A29. To quantify CD8+ T cells reactive to antigenic SAG peptides presented by HLA-A29 in patients and controls. METHODS: We performed mass-spectrometry based immunopeptidomics of HLA-A29 of antigen-presenting cell lines from patients engineered to express SAG. MHC-I Dextramer technology was utilised to determine expansion of antigen-specific CD8+ T cells reactive to SAG peptides in complex with HLA-A29 in a cohort of BCR patients, HLA-A29-positive controls, and HLA-A29-negative controls. RESULTS: We report on the naturally presented antigenic SAG peptides identified by sequencing the HLA-A29 immunopeptidome of antigen-presenting cells of patients. We show that the N-terminally extended SAG peptide precursors can be trimmed in vitro by the antigen-processing aminopeptidases ERAP1 and ERAP2. Unexpectedly, no enhanced antigen engagement by CD8+ T cells upon stimulation with SAG peptides was observed in patients or HLA-A29-positive controls. Multiplexed HLA-A29-peptide dextramer profiling of a case-control cohort revealed that CD8+ T cells specific for these SAG peptides were neither detectable in peripheral blood nor in eye biopsies of patients. CONCLUSIONS: Collectively, these findings demonstrate that SAG is not a CD8+ T cell autoantigen and sharply contrast the paradigm in the pathogenesis of BCR. Therefore, the mechanism by which HLA-A29 is associated with BCR does not involve SAG.

WWP2 confers risk to osteoarthritis by affecting cartilage matrix deposition via hypoxia associated genes.

OBJECTIVE: To explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele rs1052429-A on WWP2 expression in a human 3D in vitro model of cartilage. METHOD: Co-expression behavior of WWP2 with genes expressed in lesioned OA articular cartilage (N = 35 samples) was explored. By applying lentiviral particle mediated WWP2 upregulation in 3D in vitro pellet cultures of human primary chondrocytes (N = 8 donors) the effects of upregulation on cartilage matrix deposition was evaluated. Finally, we transfected primary chondrocytes with miR-140 mimics to evaluate whether miR-140 and WWP2 are involved in similar pathways. RESULTS: Upon performing Spearman correlations in lesioned OA cartilage, 98 highly correlating genes (|ρ| > 0.7) were identified. Among these genes, we identified GJA1, GDF10, STC2, WDR1, and WNK4. Subsequent upregulation of WWP2 on 3D chondrocyte pellet cultures resulted in a decreased expression of COL2A1 and ACAN and an increase in EPAS1 expression. Additionally, we observed a decreased expression of GDF10, STC2, and GJA1. Proteomics analysis identified 42 proteins being differentially expressed with WWP2 upregulation, which were enriched for ubiquitin conjugating enzyme activity. Finally, upregulation of miR-140 in 2D chondrocytes resulted in significant upregulation of WWP2 and WDR1. CONCLUSIONS: Mimicking the effect of OA risk allele rs1052429-A on WWP2 expression initiates detrimental processes in the cartilage shown by a response in hypoxia associated genes EPAS1, GDF10, and GJA1 and a decrease in anabolic markers, COL2A1 and ACAN.

Carbamylation reduces the capacity of IgG for hexamerization and complement activation.

Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

Transient and constitutive repression of cytoplasmic translation signaling in cells with mtDNA mutation.

Cytoplasmic translation is under sophisticated control but how cells adapt its rate to constitutive loss of mitochondrial oxidative phosphorylation is unknown. Here we show that translation is repressed in cells with the pathogenic A3243G mtDNA mutation or in mtDNA-less rho(0) cells by at least two distinct pathways, one transiently targeting elongation factor eEF-2 and the other initiation factor eIF-2alpha constitutively. Under conditions of exponential cell growth and mammalian target of rapamycin (mTOR) activation, eEF-2 becomes transiently phosphorylated by an AMP-activated protein kinase (AMPK)-dependent pathway, especially high in mutant cells. Independent of AMPK and mTOR, eIF-2alpha is constitutively phosphorylated in mutant cells, likely a signature of endoplasmic reticulum (ER)-stress response induced by the loss of oxidative phosphorylation. While the AMPK/eEF-2K/eEF-2 pathway appears to function in adaptation to physiological fluctuations in ATP levels in the mutant cells, the ER stress signified by constitutive protein synthesis inhibition through eIF-2alpha-mediated repression of translation initiation may have pathobiochemical consequences.

Pitfalls in the detection of citrullination and carbamylation.

Carbamylation and citrullination are both post-translational modifications against which (auto)antibodies can be detected in sera of rheumatoid arthritis (RA) patients. Carbamylation is the chemical modification of a lysine into a homocitrulline, whereas citrullination is an enzymatic conversion of an arginine into a citrulline. It is difficult to distinguish between the two resulting amino acids due to similarities in structure. However, differentiation between citrulline and homocitrulline is important to understand the antigens that induce antibody production and to determine which modified antigens are present in target tissues. We have observed in literature that conclusions are frequently drawn regarding the citrullination or carbamylation of proteins based on reagents that are not able to distinguish between these two modifications. Therefore, we have analyzed a wide spectrum of methods and describe here which method we consider most optimal to distinguish between citrulline and homocitrulline. We have produced several carbamylated and citrullinated proteins and investigated the specificity of (commercial) antibodies by both ELISA and western blot. Furthermore, detection methods based on chemical modifications, such as the anti-modified citrulline-"Senshu" method and also mass spectrometry were investigated for their capacity to distinguish between carbamylation and citrullination. We observed that some antibodies are able to distinguish between carbamylation and citrullination, but an overlap in reactivity is often present in the commercially available anti-citrulline antibodies. Finally, we conclude that the use of mass spectrometry is currently essential to differentiate between citrullinated and carbamylated proteins present in complex biological samples.

Novel mitochondrial DNA length variants and genetic instability in a family with diabetes and deafness.

We have identified two locations with novel multiplasmic length variants in the mitochondrial DNA of a family with diabetes and deafness. At nt568 in the D-loop, the 6-bp polycytidine tract was found to be variable in length up to a total of 12 residues. A second region with length variants was found at nt8281 in the intergenic COII-tRNA(Lys) region, which consists of two copies of the 9-bp repeat CCCCCTCTA. Only the second repeat occurs in a heteroplasmic C(9-14)A form with both T residues largely deleted. In addition, the mtDNA contained a number of new homoplasmic point mutations. Both length variants are stably inherited in a maternal way with no major changes in their length distribution. In contrast, during culture of fibroblasts from the proband the average length of the polycytidine tracts is increased at both locations indicating a fibroblast-specific genetic instability. Cybrid cells containing mtDNA from the proband proliferate less efficient than cybrids with wild-type mtDNA in co-culture experiments, suggesting functional consequences of the mtDNA length variants or the additional homoplasmic point mutations. Since oxygen consumption was not severely affected, these mutation seem less detrimental for mitochondrial function than the A3243G diabetogenic mutation and most other pathogenic mtDNA mutations. The contribution of mtDNA length variants to the phenotype of members of this family is discussed.

Polyubiquitination and proteasomal turnover controls the anti-apoptotic activity of Bcl-B.

Anti-apoptotic Bcl-2 family members can contribute to tumorigenesis and may convey resistance to anti-cancer regimens. Therefore, they are important targets for novel therapeutics, particularly Bcl-2 homology (BH)3 mimetics. Bcl-B (BCL-2-like protein-10) is a relatively understudied member of the Bcl-2 protein family. Its physiological function is unknown, but it has been proven to have an anti-apoptotic activity and to act as a tumor promoter in mice. In human, high Bcl-B protein expression levels correlate with poor prognosis in various carcinomas and predict treatment resistance in acute myeloid leukemia. We here report that protein expression level and anti-apoptotic activity of Bcl-B are dictated by its ubiquitination. We demonstrate that Bcl-B is polyubiquitinated at steady state, in a unique loop between the BH1 and BH2 domains. Mutagenesis identified lysine (K)128 as an acceptor site for polyubiquitin chains, and K119 and K120, but not K181, as potential ubiquitination sites. Mass spectrometry confirmed K128 as a ubiquitination site and defined the polyubiquitin chains as K48-linked, which was confirmed by linkage-specific antibodies. Accordingly, Bcl-B proved to be an instable protein that is subject to ubiquitin-dependent proteasomal degradation at steady state. At equal mRNA expression, protein expression of a lysineless, nonubiquitinated Bcl-B mutant was fivefold higher than that of wild-type Bcl-B, demonstrating that ubiquitination is a key determinant for Bcl-B protein expression levels. Ubiquitination controlled the anti-apoptotic capacity of Bcl-B, in response to a variety of conventional and novel anti-cancer drugs. Certain anti-cancer drugs, known to reduce Mcl-1 protein levels, likewise downregulated Bcl-B. Together, these data demonstrate that polyubiquitination and proteasomal turnover dictate the expression level and anti-apoptotic capacity of Bcl-B.

Mitochondrial diabetes and its lessons for common Type 2 diabetes.

Multiple pathogenic pathways are able to deregulate glucose homoeostasis leading to diabetes. The 3243A>G mutation in the mtDNA (mitochondrial DNA)-encoded tRNALeu,UUR gene was found by us to be associated with a particular diabetic subtype, designated MIDD (maternally inherited diabetes and deafness). This mutation causes an imbalance in the mitochondrion between proteins encoded by the nuclear and mitochondrial genomes, resulting in a gradual deterioration of glucose homoeostasis during life. Remarkably, carriers of the 3243A>G mutation are generally not obese. The mutation also results in enhanced radical production by mitochondria. We propose that this mutation leads to the development of diabetes due to an inappropriate storage of triacylglycerols within adipocytes. The result is a fatty acid-induced deterioration of pancreatic beta-cell function. In combination with an enhanced radical production in the beta-cell due to the mutation, this leads to an age-dependent, accelerated decline in insulin production. In common Type 2 (non-insulin-dependent) diabetes, which is generally associated with obesity, a decline in mitochondrial function in adipose cells seems to result in an inappropriate scavenging of fatty acids by beta-oxidation. As a consequence, a systemic overload with fatty acids occurs, leading to an enhanced decline in beta-cell function due to lipotoxicity.

In situ metal precipitation in a zinc-contaminated, aerobic sandy aquifer by means of biological sulfate reduction.

The applicability of in situ metal precipitation (ISMP) based on bacterial sulfate reduction (BSR) with molasses as carbon source was tested for the immobilization of a zinc plume in an aquifer with highly unsuitable initial conditions (high Eh, low pH, low organic matter content, and low sulfate concentrations), using deep wells for substrate injection. Batch experiments revealed an optimal molasses concentration range of 1-5 g/L and demonstrated the necessity of adding a specific growth medium to the groundwater. Without this growth medium, even sulfate, nitrogen, phosphorus, and potassium addition combined with pH optimization could not trigger biological sulfate reduction. In column experiments, precipitation of ZnS(s) was induced biologically as well as chemically (by adding Na2S). In both systems, zinc concentrations dropped from about 30 mg/L to below 0.02 mg/L. After termination of substrate addition the biological system showed continuation of BSR for at least 2 months, suggesting the insensitivity of the sulfate reducing system for short stagnations of nutrient supply, whereas in the chemical system an immediate increase of Zn concentrations was observed. A pilot experiment conducted in situ at the zinc-contaminated site showed a reduction of zinc concentrations from around 40 mg/L to below 0.01 mg/L. Termination of substrate supply did not result in an immediate stagnation of the BSR process, but continuation of BSR was observed for at least 5 weeks.

Mitochondrial diabetes mellitus.

This review discusses the current insight by which mutations in mitochondrial DNA (mtDNA) contribute to the development of particular disease states with emphasis on diabetes mellitus. Mitochondria are the power factories of the cells and produce ATP by oxidizing reducing equivalents via the respiratory chain. These reducing equivalents originate mainly from the citric acid cycle that also occurs within the mitochondria. Human mitochondria contain their own genetic material in the form of circular DNA that encodes for only a fraction of the mitochondrial components. The other mitochondrial components are nuclear encoded. Pathogenic mutations in mtDNA can affect the activity of the respiratory chain, thereby leading to the reduced generation of ATP. However, mitochondria not only produce ATP but they also regulate cytosolic concentrations of signaling molecules such as calcium and iron ions. The metabolic processes within mitochondria such as the citric acid cycle determine the concentration of metabolites that can also act as signalling molecules. Furthermore, the respiratory chain and mitochondrion-associated monoamine oxidase are major producers of reactive oxygen radicals. As a result, mutations in mtDNA can deregulate multiple processes within cells and the balance of this deregulation may contribute to the clinical phenotype.