Distinct role of ERp57 and ERdj5 as a disulfide isomerase and reductase during ER protein folding.

Proteins entering the secretory pathway need to attain native disulfide pairings to fold correctly. For proteins with complex disulfides, this process requires the reduction and isomerisation of non-native disulfides. Two key members of the protein disulfide isomerase (PDI) family, ERp57 and ERdj5 (also known as PDIA3 and DNAJC10, respectively), are thought to be required for correct disulfide formation but it is unknown whether they act as a reductase, an isomerase or both. In addition, it is unclear how reducing equivalents are channelled through PDI family members to substrate proteins. Here, we show that neither enzyme is required for disulfide formation, but ERp57 is required for isomerisation of non-native disulfides within glycoproteins. In addition, alternative PDIs compensate for the absence of ERp57 to isomerise glycoprotein disulfides, but only in the presence of a robust reductive pathway. ERdj5 is required for this alternative pathway to function efficiently indicating its role as a reductase. Our results define the essential cellular functions of two PDIs, highlighting a distinction between formation, reduction and isomerisation of disulfide bonds.

Activation of the UPR sensor ATF6α is regulated by its redox-dependent dimerization and ER retention by ERp18.

SignificanceMembrane and secretory proteins are synthesized in the endoplasmic reticulum (ER). Perturbations to ER function disrupts protein folding, causing misfolded proteins to accumulate, a condition known as ER stress. Cells adapt to stress by activating the unfolded protein response (UPR), which ultimately restores proteostasis. A key player in the UPR response is ATF6α, which requires release from ER retention and modulation of its redox status during activation. Here, we report that ER stress promotes formation of a specific ATF6α dimer, which is preferentially trafficked to the Golgi for processing. We show that ERp18 regulates ATF6α by mitigating its dimerization and trafficking to the Golgi and identify redox-dependent oligomerization of ATF6α as a key mechanism regulating its function during the UPR.

A cytosolic reductase pathway is required for efficient N-glycosylation of an STT3B-dependent acceptor site.

N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here, we used an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could be mimicked by the addition of a membrane-impermeable reducing agent. We identified a hypoglycosylated acceptor site that is adjacent to a cysteine involved in a short-range disulfide. We show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A- and STT3B-dependent glycosylation. Our results provide further insight into the important role of the endoplasmic reticulum redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

Carbonate compensation depth drives abyssal biogeography in the northeast Pacific.

Abyssal seafloor communities cover more than 60% of Earth's surface. Despite their great size, abyssal plains extend across modest environmental gradients compared to other marine ecosystems. However, little is known about the patterns and processes regulating biodiversity or potentially delimiting biogeographical boundaries at regional scales in the abyss. Improved macroecological understanding of remote abyssal environments is urgent as threats of widespread anthropogenic disturbance grow in the deep ocean. Here, we use a new, basin-scale dataset to show the existence of clear regional zonation in abyssal communities across the 5,000 km span of the Clarion-Clipperton Zone (northeast Pacific), an area targeted for deep-sea mining. We found two pronounced biogeographic provinces, deep and shallow-abyssal, separated by a transition zone between 4,300 and 4,800 m depth. Surprisingly, species richness was maintained across this boundary by phylum-level taxonomic replacements. These regional transitions are probably related to calcium carbonate saturation boundaries as taxa dependent on calcium carbonate structures, such as shelled molluscs, appear restricted to the shallower province. Our results suggest geochemical and climatic forcing on distributions of abyssal populations over large spatial scales and provide a potential paradigm for deep-sea macroecology, opening a new basis for regional-scale biodiversity research and conservation strategies in Earth's largest biome.

Risk stratifying emergency department patients with acute pulmonary embolism: Does the simplified Pulmonary Embolism Severity Index perform as well as the original?

INTRODUCTION: The Pulmonary Embolism Severity Index (PESI) is a validated prognostic score to estimate the 30-day mortality of emergency department (ED) patients with acute pulmonary embolism (PE). A simplified version (sPESI) was derived but has not been as well studied in the U.S. We sought to validate both indices in a community hospital setting in the U.S. and compare their performance in predicting 30-day all-cause mortality and classification of cases into low-risk and higher-risk categories. MATERIALS AND METHODS: This retrospective cohort study included adults with acute objectively confirmed PE from 1/2013 to 4/2015 across 21 community EDs. We evaluated the misclassification rate of the sPESI compared with the PESI. We assessed accuracy of both indices with regard to 30-day mortality. RESULTS: Among 3006 cases of acute PE, the 30-day all-cause mortality rate was 4.4%. The sPESI performed as well as the PESI in identifying low-risk patients: both had similar sensitivities, negative predictive values, and negative likelihood ratios. The sPESI, however, classified a smaller proportion of patients as low risk than the PESI (27.5% vs. 41.0%), but with similar low-risk mortality rates (<1%). Compared with the PESI, the sPESI overclassified 443 low-risk patients (14.7%) as higher risk, yet their 30-day mortality was 0.7%. The sPESI underclassified 100 higher-risk patients (3.3%) as low risk who also had a low mortality rate (1.0%). CONCLUSIONS: Both indices identified patients with PE who were at low risk for 30-day mortality. The sPESI, however, misclassified a significant number of low-mortality patients as higher risk, which could lead to unnecessary hospitalizations.

Myth: Atropine should be administered before succinylcholine for neonatal and pediatric intubation.

Succinylcholine is often used to facilitate neonatal and pediatric rapid sequence intubation in the emergency department, and most relevant literature recommends administering atropine prior to succinylcholine to reduce the risk of bradycardia. Given the potential complications associated with combining these medications, we searched the published literature for evidence supporting this practice. Most studies recommending atropine premedication were undertaken in the operating room setting and pertained to repeated succinylcholine dosing. Furthermore, there is little published evidence to indicate that succinylcholine-related bradycardia is a clinically important side effect. Several authors have called for the practice to cease, but, to date, these calls have gone unheeded. We found no evidence supporting atropine's use in pediatric patients prior to single-dose succinylcholine. Atropine premedication for emergency department rapid sequence intubation is unnecessary and should not be viewed as a "standard of care."