Two phases of disulfide bond formation have differing requirements for oxygen

Most proteins destined for the extracellular space require disulfide bonds for folding and stability. Disulfide bonds are introduced co- and post-translationally in endoplasmic reticulum (ER) cargo in a redox relay that requires a terminal electron acceptor. Oxygen can serve as the electron acceptor in vitro, but its role in vivo remains unknown. Hypoxia causes ER stress, suggesting a role for oxygen in protein folding. Here we demonstrate the existence of two phases of disulfide bond formation in living mammalian cells, with differential requirements for oxygen. Disulfide. bonds introduced rapidly during protein synthesis can occur without oxygen, whereas those introduced during post-translational folding or isomerization are oxygen dependent. Other protein maturation processes in the secretory pathway, including ER-localized N-linked glycosylation, glycan trimming, Golgi-localized complex glycosylation, and protein transport, occur independently of oxygen availability. These results suggest that an alternative electron acceptor is available transiently during an initial phase of disulfide bond formation and that post-translational oxygen-dependent disulfide bond formation causes hypoxia-induced ER stress

Maintaining Golgi Homeostasis: A Balancing Act of Two Proteolytic Pathways

The Golgi apparatus is a central hub for cellular protein trafficking and signaling. Golgi structure and function is tightly coupled and undergoes dynamic changes in health and disease. A crucial requirement for maintaining Golgi homeostasis is the ability of the Golgi to target aberrant, misfolded, or otherwise unwanted proteins to degradation. Recent studies have revealed that the Golgi apparatus may degrade such proteins through autophagy, retrograde trafficking to the ER for ER-associated degradation (ERAD), and locally, through Golgi apparatus-related degradation (GARD). Here, we review recent discoveries in these mechanisms, highlighting the role of the Golgi in maintaining cellular homeostasis

Maintaining Golgi Homeostasis: A Balancing Act of Two Proteolytic Pathways

The Golgi apparatus is a central hub for cellular protein trafficking and signaling. Golgi structure and function is tightly coupled and undergoes dynamic changes in health and disease. A crucial requirement for maintaining Golgi homeostasis is the ability of the Golgi to target aberrant, misfolded, or otherwise unwanted proteins to degradation. Recent studies have revealed that the Golgi apparatus may degrade such proteins through autophagy, retrograde trafficking to the ER for ER-associated degradation (ERAD), and locally, through Golgi apparatus-related degradation (GARD). Here, we review recent discoveries in these mechanisms, highlighting the role of the Golgi in maintaining cellular homeostasis

Constant serum levels of secreted asialoglycoprotein receptor sH2a and decrease with cirrhosis

AIM: To investigate the existence and levels of sH2a, a soluble secreted form of the asialoglycoprotein receptor in human serum

Bur1 functions with TORC1 for vacuole‐mediated cell cycle progression

The vacuole/lysosome plays essential roles in the growth and proliferation of many eukaryotic cells via the activation of target of rapamycin complex 1 (TORC1). Moreover, the yeast vacuole/lysosome is necessary for progression of the cell division cycle, in part via signaling through the TORC1 pathway. Here, we show that an essential cyclin‐dependent kinase, Bur1, plays a critical role in cell cycle progression in cooperation with TORC1. A mutation in BUR1 combined with a defect in vacuole inheritance shows a synthetic growth defect. Importantly, the double mutant, as well as a bur1‐267 mutant on its own, has a severe defect in cell cycle progression from G1 phase. In further support that BUR1 functions with TORC1, mutation of bur1 alone results in high sensitivity to rapamycin, a TORC1 inhibitor. Mechanistic insight for Bur1 function comes from the findings that Bur1 directly phosphorylates Sch9, a target of TORC1, and that both Bur1 and TORC1 are required for the activation of Sch9. Together, these discoveries suggest that multiple signals converge on Sch9 to promote cell cycle progression.SynopsisThe yeast vacuole is required for cell cycle progression through early G1 phase via TORC1 signaling. This study reveals that Bur1/Cdk9, a cyclin‐dependent kinase, is also required for vacuole‐mediated cell cycle progression and acts in parallel with TORC1.Bur1/Cdk9 is required for cell cycle progression through G1 phase.Bur1 functions in parallel with TORC1 through direct phosphorylation of Sch9.TORC1 and Bur1 each phosphorylate unique sites on Sch9, and in addition phosphorylate sites in common.The yeast vacuole is required for cell cycle progression through early G1 phase via TORC1 signaling. This study reveals that Bur1/Cdk9, a cyclin‐dependent kinase, is also required for vacuole‐mediated cell cycle progression and acts in parallel with TORC1.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/172062/1/embr202153477.reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/172062/2/embr202153477.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/172062/3/embr202153477-sup-0001-EVFigs.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/172062/4/embr202153477_am.pd

Very low eIF2α-P levels in striatal cells, much increased by expression of Htt111Q.

<p><b>A</b>) Basal level of eIF2α-P in murine cell lines normalized by total eIF2α. Graph: average of 3 experiments ± SE<b>.</b> **P = 0.004, ***P  = 0.001. <b>B</b>) Immunofluorescence images of cells fixed, permeabilized and stained with rabbit anti-eIF2α-P and mouse anti-eIF2α followed by secondary antibodies. Bar = 10 µm. Image exposure time was kept constant to be able to compare protein levels in the different cell types. Levels relative to ST<i>Hdh</i>^Q7/7 levels were quantified from images from 3 experiments ± SE (>20 cells, ***P<0.001).</p

High sensitivity of striatal neurons to ER stress, further aggravated by expression of pathogenic huntingtin.

<p><b>A-C</b>) Strong induction of GADD34 and CHOP upon prolonged ER stress in ST<i>Hdh</i>^Q7/7 cells and even stronger in ST<i>Hdh</i>^Q111/111 cells; (3 independent experiments ±SE). *P = 0.02, <b>*</b>*P = 0.01, ***P = 0.0002. Immunoblots of a representative experiment are shown in A. GAPDH levels served here as a loading control. <b>D</b>) Prolonged ER stress induced with Tun or MG-132 leads to extensive death of ST<i>Hdh</i>^Q7/7 cells, further aggravated in ST<i>Hdh</i>^Q111/111 cells, as measured by FACS analysis of cell cycle progression with propidium iodide (PI) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090803#pone.0090803.s002" target="_blank">Fig. S2</a>); (6 independent experiments ± SE). *P<0.05, **P = 0.01, ***P = 0.001.</p

Regulation of phosphorylated eIF2α levels by inhibition of its dephosphorylation.

<p><b>A</b>) Guanabenz (Gz), at a relatively high concentration (100 µM), inhibits eIF2α dephosphorylation in untreated ST<i>Hdh</i>^Q7/7 cells and also in those treated with Tun (5 µg/ml) up to 7h; this is also true in ST<i>Hdh</i>^Q111/111 cells but only after very short treatments. *P = 0.02, **P = 0.01. EIF2α-P levels were normalized by total eIF2α. <b>B</b>) Similar to (A), but for cells treated for 24 h. After these long treatments Gz did not inhibit ER stress-induced eIF2α dephosphorylation, it increased CHOP levels. The values in the graphs are averages from 3-4 independent experiments±SE. *P<0.05, **P = 0.002. <b>C</b>) Gz showed a minor effect in rescuing ST<i>Hdh</i>^Q111/111 cells from UPR-induced cell death (Tun for 48 h). ***P =  0.0001.</p

Striatal neurons show a low induction of early UPR markers, whereas later ER stress responses are upregulated.

<p>Early responses and in some cases late ones are increased by expression of Htt111Q. A,B) Levels of UPR markers after short term ER stress. ST<i>Hdh</i>^Q7/7 were compared to ST<i>Hdh</i>^Q111/111 and NIH 3T3 cells. Immunoblots show results of a representative experiment of 3. Vertical lines indicate removal of irrelevant lanes. C-H) Quantification of A,B. ST<i>Hdh</i>^Q7/7 cells do not activate properly an early stress response, mediated by PERK-P and its target eIF2α-P, induced by Tun (C, 10 µg/ml) or by MG-132 (D, 40 µM). In ST<i>Hdh</i>^Q111/111 cells, PERK-P and eIF2α-P are induced (C,D). Later ER stress responses are increased in ST<i>Hdh</i>^Q7/7 compared to NIH 3T3 cells (E-H). Htt111Q expression causes even more enhanced upregulation of the UPR markers in some cases. Values were normalized to β-actin levels as a loading control.</p

Regulation of phosphorylated eIF2α levels by inhibition of its phosphorylation and rescue of ST<i>Hdh</i>^Q111/111 cells.

<p><b>A</b>) EIF2α phosphorylation in ST<i>Hdh</i>^Q111/111 cells is PERK-mediated. ST<i>Hdh</i>^Q111/111 cells left untreated or treated with the PERK inhibitor A4 (50 µM) or the PKR inhibitor PKRi (1 µM) for the indicated times. **P = 0.009. <b>B</b>) ER stress-mediated eIF2α phosphorylation is inhibited by A4 and not by PKRi. As in (A), but with ST<i>Hdh</i>^Q7/7 cells treated for different times with Tun. <b>C</b>) PKR-mediated eIF2α phosphorylation is inhibited by PKRi and not by A4. As in (B), but with cells treated for 7h with the PKR inducer poly-I:C (200 µg/ml). <b>D-E</b>) A4 rescued ST<i>Hdh</i>^Q111/111 cells from UPR-induced cell death (Tun for 48 h, D), whereas PKRi had no effect (E). ***P =  0.0001. <b>F</b>) Total protein synthesis levels are much increased in ST<i>Hdh</i>^Q111/111 cells after prolonged ER stress (Tun for 24h) and reduced by A4 (50 µM). **P<0.002 (3 repeat experiments).</p