Table1_Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.XLSX

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.</p

Table3_Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.XLSX

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.</p

Table2_Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.XLSX

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.</p

DataSheet1_Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.PDF

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.</p

S100A9 is a Biliary Protein Marker of Disease Activity in Primary Sclerosing Cholangitis

<div><h3>Background and Aims</h3><p>Bile analysis has the potential to serve as a surrogate marker for inflammatory and neoplastic disorders of the biliary epithelium and may provide insight into biliary pathophysiology and possible diagnostic markers. We aimed to identify biliary protein markers of patients with primary sclerosing cholangitis (PSC) by a proteomic approach.</p> <h3>Methods</h3><p>Bile duct-derived bile samples were collected from PSC patients (n = 45) or patients with choledocholithiasis (n = 24, the control group). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to analyse the proteins, 2-D-gel patterns were compared by densitometry, and brush cytology specimens were analysed by RT-PCR.</p> <h3>Results</h3><p>A reference bile-duct bile proteome was established in the control group without signs of inflammation or maligancy comprising a total of 379 non-redundant biliary proteins; 21% were of unknown function and 24% had been previously described in serum. In PSC patients, the biliary S100A9 expression was elevated 95-fold (p<0.005), serum protein expression was decreased, and pancreatic enzyme expression was unchanged compared to controls. The S100A9 expression was 2-fold higher in PSC patients with high disease activity than in those with low activity (p<0.05). The brush cytology specimens from the PSC patients with high disease activity showed marked inflammatory activity and leukocyte infiltration compared to the patients with low activity, which correlated with S100A9 mRNA expression (p<0.05).</p> <h3>Conclusions</h3><p>The bile-duct bile proteome is complex and its analysis might enhance the understanding of cholestatic liver disease. Biliary S100A9 levels may be a useful marker for PSC activity, and its implication in inflammation and carcinogenesis warrants further investigation.</p> </div

Localization of O-mannosylated peptides of human RPTPζ.

<p>Schematic model of RPTPζ, illustrating the annotated domains. Plasma membrane (PM) is indicated as a lipid bilayer. Positions of peptides containing extended O-mannosyl glycans (indicated by green circles and -R) and O-linked N-Acetylgalactosamine mucin-type glycans (yellow squares) found by Trinidad and coworkers are shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref029" target="_blank">29</a>]. The position of O-hexosylated peptides identified in this study is indicated as a green circle with a horizontal black bar.</p

Mono-O-mannosyl glycans localize to distinct cell types throughout the murine brain.

<p>Sagittal section of WT murine brain counterstained with DAPI for nuclei labeling. A) Broad staining was achieved as shown in the overview (scale bar = 100 μm) and at higher magnifications in the cerebral cortex (1), the cerebellum (2), and the hippocampus (3). In more detail, staining in the cerebellum included single cells of the molecular layer, cells of the granular cell layer and cells of the Purkinje cell layer (indicated by asterisks). In the hippocampus, cells of the <i>cornu ammonis</i> region were labeled including their neuronal projections as indicated by arrowheads (picture 3 shows cells of the CA2 field). Individual cells of regions II to V of the cerebral cortex were stained by the α-O-Man antibody. Co-localization of mono-O-mannosyl glycans with neuronal cell marker (NeuN/Fox3) in the hippocampus (B) or with Purkinje cell marker (Calbindin) in the cerebellum (C) showing single channel signal and merged channels. NeuN-labeled hippocampal neurons of the <i>cornu ammonis</i> 2 (CA2) region were stained by the α-O-Man antibody. Purkinje cell localization of mono-O-mannosyl glycans was demonstrated by co-localization with Calbindin. Scale bar = 50 μm.</p

Validation of O-mannosylation on RPTPζ.

<p>Samples were dimethyl labeled in the light and medium form, respectively. After treating the light sample with α-mannosidase, both samples were mixed and analyzed by LC-MS/MS. Shown are four extracted ion chromatograms of the m/z values of light and medium labeled peptide LLLPSTATSK in (A) the deglycosylated form (543.8 amu and 547.9 amu) and in (B) the mannosylated form (624.9 amu and 628.9 amu, respectively). The glycopeptide was detected only in the untreated sample (medium labeled), whereas the deglycosylated peptide (light labeled) was only observed after mannosidase treatment. (C) HCD fragment spectrum of precursor mass 543.8 amu and (D) 628.9 amu confirmed the sequence of the deglycosylated and O-mannosylated peptide LLLPSTATSK of RPTPζ.</p

Schematic workflow for the enrichment of peptides bearing O-linked mannose.

<p>For the detailed protocol please refer to the Materials and Methods section. In brief, murine brains were extracted and pulverized before protein extraction. Protein suspensions were digested with trypsin and N-glycans removed by PNGase F treatment. Tryptic peptides were thereafter subjected to ConA LWAC to enrich for mono-O-mannosylated peptides. Elution fractions were either analyzed directly (a), or after dimethyl labeling (medium (M), light (L)) and α-mannosidase treatment (b) by LC-MS/MS and HCD fragmentation according to previously described protocols [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref031" target="_blank">31</a>].</p

Known O-mannosylated proteins are located to the cell types identified by the α-O-Man antibody.

<p>A) α-DG as indicated by the IIH6 antibody reactive to matriglycan, and its interacting partner laminin preferentially stained vasculature (arrowhead) and the <i>glia limitans</i>. Purkinje cell layer (asterisks) and granular cell layer (bracket) were stained by the anti-laminin, the IIH6 and α-O-Man antibodies. IIH6 staining was performed following the protocol of Beedle <i>et al</i>. 2012 to detect mouse antigens on mouse tissue [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref033" target="_blank">33</a>]. B) Plexin-B2 (Plxnb2) and RPTPζ showed comparable signal distribution as the α-O-Man antibody. Plxnb2 signals were located exclusively to the Purkinje cell layer (asterisks), whereas RPTPζ was primarily located to the granular cell layer in the cerebellum (abbreviated by C). In the cerebral cortex (CC), RPTPζ labeled single cells all of which were also stained for by the α-O-Man antibody. Nuclei were stained by DAPI, sagittal cryosections of WT mouse brains were used. Scale bars = 50 μm.</p