Cellular maintenance of nuclear protein homeostasis

The accumulation and aggregation of misfolded proteins is the primary hallmark for more than 45 human degenerative diseases. These devastating disorders include Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis. Over 15 degenerative diseases are associated with the aggregation of misfolded proteins specifically in the nucleus of cells. However, how the cell safeguards the nucleus from misfolded proteins is not entirely clear. In this review, we discuss what is currently known about the cellular mechanisms that maintain protein homeostasis in the nucleus and protect the nucleus from misfolded protein accumulation and aggregation. In particular, we focus on the chaperones found to localize to the nucleus during stress, the ubiquitin-proteasome components enriched in the nucleus, the signaling systems that might be present in the nucleus to coordinate folding and degradation, and the sites of misfolded protein deposition associated with the nucleus

Dynamic Sumoylation of a Conserved Transcription Corepressor Prevents Persistent Inclusion Formation during Hyperosmotic Stress

<div><p>Cells are often exposed to physical or chemical stresses that can damage the structures of essential biomolecules. Stress-induced cellular damage can become deleterious if not managed appropriately. Rapid and adaptive responses to stresses are therefore crucial for cell survival. In eukaryotic cells, different stresses trigger post-translational modification of proteins with the small ubiquitin-like modifier SUMO. However, the specific regulatory roles of sumoylation in each stress response are not well understood. Here, we examined the sumoylation events that occur in budding yeast after exposure to hyperosmotic stress. We discovered by proteomic and biochemical analyses that hyperosmotic stress incurs the rapid and transient sumoylation of Cyc8 and Tup1, which together form a conserved transcription corepressor complex that regulates hundreds of genes. Gene expression and cell biological analyses revealed that sumoylation of each protein directs distinct outcomes. In particular, we discovered that Cyc8 sumoylation prevents the persistence of hyperosmotic stress-induced Cyc8-Tup1 inclusions, which involves a glutamine-rich prion domain in Cyc8. We propose that sumoylation protects against persistent inclusion formation during hyperosmotic stress, allowing optimal transcriptional function of the Cyc8-Tup1 complex.</p></div

Net cholesterol efflux capacity of HDL enriched serum and coronary atherosclerosis in rheumatoid arthritis

Background/objectives: Cardiovascular (CV) risk is increased in patients with rheumatoid arthritis (RA), but not fully explained by traditional risk factors such as LDL and HDL cholesterol concentrations. The cholesterol efflux capacity of HDL may be a better CV risk predictor than HDL concentrations. We hypothesized that HDL's cholesterol efflux capacity is impaired and inversely associated with coronary atherosclerosis in patients with RA. Methods: We measured the net cholesterol efflux capacity of apolipoprotein B depleted serum and coronary artery calcium score in 134 patients with RA and 76 control subjects, frequency-matched for age, race and sex. The relationship between net cholesterol efflux capacity and coronary artery calcium score and other clinical variables of interest was assessed in patients with RA. Results: Net cholesterol efflux capacity was similar among RA (median [IQR]: 34% removal [28, 41%]) and control subjects (35% removal [27%, 39%]) (P = 0.73). In RA, increasing net cholesterol efflux capacity was not significantly associated with decreased coronary calcium score (OR = 0.78 (95% CI 0.51–1.19), P = 0.24, adjusted for age, race and sex, Framingham risk score and presence of diabetes). Net cholesterol efflux capacity was not significantly associated with RA disease activity score, C-reactive protein, urinary F2-isoprostanes, or degree of insulin resistance in RA. Conclusions: Net cholesterol efflux capacity is not significantly altered in patients with relatively well-controlled RA nor is it significantly associated with coronary artery calcium score

Tup1 and Cyc8 are sumoylated during hyperosmotic stress.

<p>(<b>A</b>) Scheme of the MS strategy to identify proteins sumoylated during hyperosmotic stress. (<b>B</b>) Total peptide counts identified for Tup1 and Cyc8 at 0 and 15 minutes of hyperosmotic stress. Total peptide counts for Smt3 are included to demonstrate equivalent levels of SUMO in the samples. (<b>C</b>) Total peptide counts identified for proteins where the significance of the changes between 0 and 15 minutes of hyperosmotic stress was <i>p</i>≤0.05. Gray areas represent ≥3 fold changes in the 15 minute samples compared with the 0 minute samples. Tup1 and Cyc8 are noted. (<b>D</b>) Cells expressing His₆-FLAG-Smt3 (HF-Smt3) and either a 3xHA epitope-tagged Tup1 (Tup1-3HA) or a 3xHSV epitope-tagged Cyc8 (Cyc8-3HSV) from their endogenous promoters were subject to hyperosmotic stress (1.2M sorbitol) over a 60-minute time course. Cell lysates (input) and purified sumoylated proteins (SUMO pulldown) were subject to western analyses using anti-HA, anti-HSV, or anti-Pgk1 antibodies to detect Tup1, Cyc8, or Pgk1 respectively. (<b>E</b>) Similar experiment as in (D) except cells were subject to hyperosmotic stress (1.2M sorbitol), heat shock (42°C), or high ethanol (10% v/v) for 0, 15, and 60 minutes.</p

Cyc8 inclusions form in the nucleus during hyperosmotic stress.

<p>(<b>A</b>) Structured Illumination Microscopy (SIM) images of cells subjected to 0 or 5 minutes of hyperosmotic stress (1.2M sorbitol). Wild-type or mutant Cyc8 is GFP-tagged and Nup53 is mCherry-tagged. (<b>B</b>) SIM images of cells during a 60 minute time course of hyperosmotic stress (1.2M sorbitol). Wild-type or mutant Cyc8 is GFP-tagged, and the cell wall is stained with Calcofluor White. Graph shows the percentage of cells with inclusions over time. (<b>C</b>) Confocal 3D images of cells subjected to 0 or 15 minutes of hyperosmotic stress (1.2M sorbitol). Wild-type Cyc8 is GFP-tagged and histone H2B (Htb1) is mCherry-tagged. Graph shows the intensity of Cyc8 or Htb1 across the white line drawn on the cells to the left. (<b>D</b>) Confocal 3D images of cells expressing Cyc8-GFP or Htb1-mCherry. Graph to the right shows the Mander's overlap coefficient [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005809#pgen.1005809.ref056" target="_blank">56</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005809#pgen.1005809.ref057" target="_blank">57</a>] for the colocalization of the proteins during 0 and 15 minutes of hyperosmotic stress (1.2M sorbitol).</p

Expression of a dominant-negative form of Hsp70 influences the persistence of Cyc8^4KtoR inclusions.

<p>(<b>A</b>) Cells expressing Cyc8^4KtoR-GFP from the endogenous <i>CYC8</i> locus and carrying either an empty 2 micron vector or a 2 micron vector with an dominant-negative form of Hsp70 (<i>SSA1</i>) were exposed to a time course of hyperosmotic stress (1.2M sorbitol), fixed at the times indicated, and imaged by fluorescence microscopy. (<b>B</b>) Analysis of data from (A). Six fields of cells for each condition, with at least 40 cells/field, were counted for the presence of cytoplasmic inclusions. Data represent the average percentage of cells that contained cytoplasmic inclusions within the six fields. Error bars are the standard deviation. A two-tailed, heteroscedastic student’s t-test was used to determine significance.</p

Identification of Tup1 and Cyc8 sumoylation sites.

<p>(<b>A</b>) <i>His</i>_<i>6</i><i>-FLAG-SMT3</i> cells expressing either wild-type Tup1-3HA or Tup1^K270R-3HA from the endogenous <i>TUP1</i> promoter were examined for Tup1 sumoylation at 0 or 15 minutes of hyperosmotic stress (1.2M sorbitol) as in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005809#pgen.1005809.g002" target="_blank">Fig 2D</a>). (<b>B</b>) <i>His</i>_<i>6</i><i>-FLAG-SMT3</i> cells expressing the indicated Cyc8-3HSV deletion mutant were subject to hyperosmotic stress (1.2M sorbitol) for 15 minutes. Cell lysates were generated and subject to the same analysis as in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005809#pgen.1005809.g002" target="_blank">Fig 2D</a>). (<b>C</b>) <i>His</i>_<i>6</i><i>-FLAG-SMT3</i> cells expressing either wild-type Cyc8-3HSV or Cyc8^4KtoR-3HSV from the endogenous <i>CYC8</i> promoter were examined for Cyc8 sumoylation as in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005809#pgen.1005809.g002" target="_blank">Fig 2D</a>). (<b>D</b>) <i>His</i>_<i>6</i><i>-FLAG-SMT3</i> cells expressing wild-type Tup1-3HA from the endogenous <i>TUP1</i> promoter and either wild-type Cyc8-3HSV or Cyc8^4KtoR-3HSV from the endogenous <i>CYC8</i> promoter were examined for Tup1 sumoylation as in (A). (<b>E</b>) <i>His</i>_<i>6</i><i>-FLAG-SMT3</i> cells expressing wild-type Cyc8-3HSV from the endogenous <i>CYC8</i> promoter and either wild-type Tup1-3HA or Tup1^K270R-3HA from the endogenous <i>TUP1</i> promoter were examined for Cyc8 sumoylation as in (C). (<b>F</b>) <i>6His-FLAG-SMT3</i> wild-type or <i>tup1</i>^<i>K270R</i><i>cyc8</i>^<i>4KtoR</i> cells were subjected to hyperosmotic stress (1.2M sorbitol) for 0 or 15 minutes, and global sumoylation patterns examined by western analysis using an anti-FLAG antibody.</p

Cyc8 inclusions are not SDS-resistant, Hsp104-dependent amyloids.

<p>(<b>A</b>) Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) of lysates from the strains used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005809#pgen.1005809.g006" target="_blank">Fig 6A</a>. Cells were exposed to 0 or 15 minutes of hyperosmotic stress (1.2M sorbitol), and lysates were mixed with semi-denaturing buffer before loading into the agarose gel. Lysates from strains overexpressing either Rnq1-YFP or Cyc8^441-677-YFP are also included as positive controls for SDS-resistant amyloid formation. Western analysis was performed with anti-GFP antibody. The two images are different exposures of the same blot. (<b>B</b>) Fluorescence microscopy of cells expressing GFP-tagged wild-type or sumoylation-deficient Cyc8 after 3 passages on either YPD or YPD+4mM guanidinium hydrochloride (GuHCl). Cells were exposed to 0 or 15 minutes of hyperosmotic stress (1.2M sorbitol), fixed, and imaged by fluorescence microscopy. All constructs were integrated at the gene’s endogenous locus and expressed from the endogenous promoter. (<b>C</b>) Confocal 3D images of cells expressing mutant Cyc8^4KtoR tagged with GFP and Hsp104 tagged with mCherry were subject to a 15 minute 42°C heat shock to induce Hsp104 inclusions, 15 minute hyperosmotic stress (1.2M sorbitol) to induce Cyc8^4KtoR inclusions, or both. Cell wall was visualized by staining with Calcofluor White.</p

Loss of Cyc8 sumoylation leads to cytoplasmic inclusion formation of both Cyc8 and Tup1.

<p>(<b>A, B</b>) Strains expressing combinations of wild-type and/or sumoylation-deficient mutant versions of Cyc8 and Tup1, tagged with GFP or untagged, were exposed to a time course of hyperosmotic stress (1.2M sorbitol), fixed at the times indicated, and imaged by fluorescence microscopy. All constructs were integrated at the gene’s endogenous locus and expressed from the endogenous promoter. (A) GFP fluorescence images of cells in which wild-type or mutant Cyc8 is GFP-tagged and wild-type or mutant Tup1 is untagged. (B) GFP fluorescence images of cells in which wild-type or mutant Cyc8 is untagged and wild-type or mutant Tup1 is GFP-tagged. (<b>C</b>) GFP and mCherry images in which Cyc8^4KtoR is tagged with GFP and wild-type Tup1 is tagged with mCherry at their endogenous genomic loci. Cells were exposed to 0 or 15 minutes of hyperosmotic stress (1.2M sorbitol), fixed, and imaged by fluorescence microscopy. (<b>D</b>) GFP and mCherry images in which one copy of Cyc8^4KtoR is tagged with GFP at the endogenous genomic locus and another CEN plasmid-expressed copy is tagged with mCherry. Cells were exposed to 0 or 15 minutes of hyperosmotic stress (1.2M sorbitol), fixed, and imaged by fluorescence microscopy.</p