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Zinc transporters take up/release zinc ions (Zn2+) across biological membranes and maintain intracellular and intra-organellar Zn2+ homeostasis. Since this process requires a series of conformational changes in the transporters, detailed information about the structures of different reaction intermediates is required for a comprehensive understanding of their Zn2+ transport mechanisms. Recently, various Zn2+ transport systems have been identified in bacteria, yeasts, plants, and humans. Based on structural analyses of human ZnT7, human ZnT8, and bacterial YiiP, we propose updated models explaining their mechanisms of action to ensure efficient Zn2+ transport. We place particular focus on the mechanistic roles of the histidine-rich loop shared by several zinc transporters, which facilitates Zn2+ recruitment to the transmembrane Zn2+-binding site. This review provides an extensive overview of the structures, mechanisms, and physiological functions of zinc transporters in different biological kingdoms.

Time-resolved direct observations of proteins in action provide essential mechanistic insights into biological processes. Here, we present mechanisms of action of protein disulfide isomerase (PDI)—the most versatile disulfide-introducing enzyme in the endoplasmic reticulum—during the catalysis of oxidative protein folding. Single-molecule analysis by high-speed atomic force microscopy revealed that oxidized PDI is in rapid equilibrium between open and closed conformations, whereas reduced PDI is maintained in the closed state. In the presence of unfolded substrates, oxidized PDI, but not reduced PDI, assembles to form a face-to-face dimer, creating a central hydrophobic cavity with multiple redox-active sites, where substrates are likely accommodated to undergo accelerated oxidative folding. Such PDI dimers are diverse in shape and have different lifetimes depending on substrates. To effectively guide proper oxidative protein folding, PDI regulates conformational dynamics and oligomeric states in accordance with its own redox state and the configurations or folding states of substrates. Single-molecule analysis by high-speed atomic force microscopy reveals that oxidized protein disulfide isomerase adopts a dynamic conformation in the absence of substrates and forms face-to-face dimers to accelerate oxidative folding in the presence of substrates.

Zinc ions (Zn 2+ ) are imported into the early secretory pathway by Golgi-resident transporters, but their handling and functions are not fully understood. Here, we show that Zn 2+ binds with high affinity to the pH-sensitive chaperone ERp44, modulating its localization and ability to retrieve clients like Ero1α and ERAP1 to the endoplasmic reticulum (ER). Silencing the Zn 2+ transporters that uptake Zn 2+ into the Golgi led to ERp44 dysfunction and increased secretion of Ero1α and ERAP1. High-resolution crystal structures of Zn 2+ -bound ERp44 reveal that Zn 2+ binds to a conserved histidine-cluster. The consequent large displacements of the regulatory C-terminal tail expose the substrate-binding surface and RDEL motif, ensuring client capture and retrieval. ERp44 also forms Zn 2+ -bridged homodimers, which dissociate upon client binding. Histidine mutations in the Zn 2+ -binding sites compromise ERp44 activity and localization. Our findings reveal a role of Zn 2+ as a key regulator of protein quality control at the ER-Golgi interface. Zinc ions (Zn 2+ ) are imported by Golgi-resident transporters but the function of zinc in the early secretory pathway has remained unknown. Here the authors find that Zn 2+ regulates protein quality control in the early secretory pathway by demonstrating that the pH-sensitive chaperone ERp44 binds Zn 2+ and solving the Zn 2+ -bound ERp44 structure.

Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.

Cysteine hydropersulfide (CysSSH) occurs in abundant quantities in various organisms, yet little is known about its biosynthesis and physiological functions. Extensive persulfide formation is apparent in cysteine-containing proteins in Escherichia coli and mammalian cells and is believed to result from post-translational processes involving hydrogen sulfide-related chemistry. Here we demonstrate effective CysSSH synthesis from the substrate l-cysteine, a reaction catalyzed by prokaryotic and mammalian cysteinyl-tRNA synthetases (CARSs). Targeted disruption of the genes encoding mitochondrial CARSs in mice and human cells shows that CARSs have a crucial role in endogenous CysSSH production and suggests that these enzymes serve as the principal cysteine persulfide synthases in vivo. CARSs also catalyze co-translational cysteine polysulfidation and are involved in the regulation of mitochondrial biogenesis and bioenergetics. Investigating CARS-dependent persulfide production may thus clarify aberrant redox signaling in physiological and pathophysiological conditions, and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

Background Hypocalcemia is cited as a complication of massive transfusion. However, this is not well studied as a primary outcome in trauma patients. Our primary outcome was to determine if transfusion of packed red blood cells (pRBC) was an independent predictor of severe hypocalcemia (ionized calcium ≤ 3.6 mg/dL). Methods Retrospective, single-center study (01/2004–12/2014) including all trauma patients ≥ 18 yo presenting to the ED with an ionized calcium (iCa) level drawn. Variables extracted included demographics, interventions, outcomes, and iCa. Regression models identified independent risk factors for severe hypocalcemia (SH). Results Seven thousand four hundred and thirty-one included subjects, 716 (9.8%) developed SH within 48 h of admission. Median age: 39 (Range: 18–102), systolic blood pressure: 131 (IQR: 114–150), median Glasgow Coma Scale (GCS): 15 (IQR: 10–15), Injury Severity Score (ISS): 14 (IQR: 9–24). SH patients were more likely to have depressed GCS (13 vs 15, p  < 0.0001), hypotension (23.2% vs 5.1%, p  < 0.0001) and tachycardia (57.0% vs 41.9%, p  < 0.0001) compared to non-SH patients. They also had higher emergency operative rate (71.8% vs 29%, p  < 0.0001) and higher blood administration prior to minimum iCa [pRBC: (8 vs 0, p  < 0.0001), FFP: (4 vs 0, p  < 0.0001), platelet: (1 vs 0, p  < 0.0001)]. Multivariable analysis revealed penetrating mechanism (AOR: 1.706), increased ISS (AOR: 1.029), and higher pRBC (AOR: 1.343) or FFP administered (AOR: 1.097) were independent predictors of SH. SH was an independent predictor of mortality (AOR: 2.658). Regression analysis identified a significantly higher risk of SH at pRBC + FFP administration of 4 units (AOR: 18.706, AUC:. 897 (0.884–0.909). Conclusion Transfusion of pRBC is an independent predictor of SH and is associated with increased mortality. The predicted probability of SH increases as pRBC + FFP administration increases.

AbstractObjectivesTo summarise and compare the accuracy of physical examination, computed tomography (CT), sonography of the optic nerve sheath diameter (ONSD), and transcranial Doppler pulsatility index (TCD-PI) for the diagnosis of elevated intracranial pressure (ICP) in critically ill patients.DesignSystematic review and meta-analysis.Data sourcesSix databases, including Medline, EMBASE, and PubMed, from inception to 1 September 2018.Study selection criteriaEnglish language studies investigating accuracy of physical examination, imaging, or non-invasive tests among critically ill patients. The reference standard was ICP of 20 mm Hg or more using invasive ICP monitoring, or intraoperative diagnosis of raised ICP.Data extractionTwo reviewers independently extracted data and assessed study quality using the quality assessment of diagnostic accuracy studies tool. Summary estimates were generated using a hierarchical summary receiver operating characteristic (ROC) model.Results40 studies (n=5123) were included. Of physical examination signs, pooled sensitivity and specificity for increased ICP were 28.2% (95% confidence interval 16.0% to 44.8%) and 85.9% (74.9% to 92.5%) for pupillary dilation, respectively; 54.3% (36.6% to 71.0%) and 63.6% (46.5% to 77.8%) for posturing; and 75.8% (62.4% to 85.5%) and 39.9% (26.9% to 54.5%) for Glasgow coma scale of 8 or less. Among CT findings, sensitivity and specificity were 85.9% (58.0% to 96.4%) and 61.0% (29.1% to 85.6%) for compression of basal cisterns, respectively; 80.9% (64.3% to 90.9%) and 42.7% (24.0% to 63.7%) for any midline shift; and 20.7% (13.0% to 31.3%) and 89.2% (77.5% to 95.2%) for midline shift of at least 10 mm. The pooled area under the ROC (AUROC) curve for ONSD sonography was 0.94 (0.91 to 0.96). Patient level data from studies using TCD-PI showed poor performance for detecting raised ICP (AUROC for individual studies ranging from 0.55 to 0.72).ConclusionsAbsence of any one physical examination feature is not sufficient to rule out elevated ICP. Substantial midline shift could suggest elevated ICP, but the absence of shift cannot rule it out. ONSD sonography might have use, but further studies are needed. Suspicion of elevated ICP could necessitate treatment and transfer, regardless of individual non-invasive tests.RegistrationPROSPERO CRD42018105642.

ERGIC-53 transports certain subsets of newly synthesized secretory proteins and membrane proteins from the endoplasmic reticulum to the Golgi apparatus. Despite numerous structural and functional studies since its identification, the overall architecture and mechanism of action of ERGIC-53 remain unclear. Here we present cryo-EM structures of full-length ERGIC-53 in complex with its functional partner MCFD2. These structures reveal that ERGIC-53 exists as a homotetramer, not a homohexamer as previously suggested, and comprises a four-leaf clover-like head and a long stalk composed of three sets of four-helix coiled-coil followed by a transmembrane domain. 3D variability analysis visualizes the flexible motion of the long stalk and local plasticity of the head region. Notably, MCFD2 is shown to possess a Zn 2+ -binding site in its N-terminal lid, which appears to modulate cargo binding. Altogether, distinct mechanisms of cargo capture and release by ERGIC- 53 via the stalk bending and metal binding are proposed. ERGIC-53 engages in the ER-to-Golgi transport of secretory and membrane proteins by unknown mechanisms. Here authors report a long flexible tetrameric structure of full-length ERGIC-53 complexed with its functional partner MCFD2 by cryo-EM.

Many secretory enzymes acquire essential zinc ions (Zn 2+ ) in the Golgi complex. ERp44, a chaperone operating in the early secretory pathway, also binds Zn 2+ to regulate its client binding and release for the control of protein traffic and homeostasis. Notably, three membrane transporter complexes, ZnT4, ZnT5/ZnT6 and ZnT7, import Zn 2+ into the Golgi lumen in exchange with protons. To identify their specific roles, we here perform quantitative Zn 2+ imaging using super-resolution microscopy and Zn 2+ -probes targeted in specific Golgi subregions. Systematic ZnT-knockdowns reveal that ZnT4, ZnT5/ZnT6 and ZnT7 regulate labile Zn 2+ concentration at the distal, medial, and proximal Golgi, respectively, consistent with their localization. Time-course imaging of cells undergoing synchronized secretory protein traffic and functional assays demonstrates that ZnT-mediated Zn 2+ fluxes tune the localization, trafficking, and client-retrieval activity of ERp44. Altogether, this study provides deep mechanistic insights into how ZnTs control Zn 2+ homeostasis and ERp44-mediated proteostasis along the early secretory pathway. Zinc is vital to protein quality control in the early secretory pathway. Here, authors demonstrate distinct roles of the Golgi-resident zinc transporters for the maintenance of zinc homeostasis and the regulation of a zinc-dependent chaperone, ERp44.

BackgroundSince 2007, clinical practice guidelines by the Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) recommend early surgical management with laparoscopic cholecystectomy for pregnant women with symptomatic gallbladder disease regardless of trimester. However, little is known about practice patterns in the management of pregnant patients with acute cholecystitis. This study aims to examine nationwide trends in the surgical management of acute cholecystitis, as well as their impact on clinical outcomes during pregnancy.MethodsThe National Inpatient Sample was queried for all pregnant women diagnosed with acute cholecystitis between January 2003 and September 2015. After applying appropriate weights, multivariate regression analysis adjusted for patient- and hospital-level characteristics and quantified the impact of discharge year (2003–2007 versus 2008–2015) on cholecystectomy rates and timing of surgery. Multivariate regression analysis was also used to examine the impact of same admission cholecystectomy and its timing on maternal and fetal outcomes.ResultsA total of 23,939 pregnant women with acute cholecystitis satisfied our inclusion criteria. The median age was 26 years (interquartile range: 22–30). During the study period, 36.3% were managed non-operatively while 59.6% and 4.1% underwent laparoscopic and open cholecystectomy, respectively. After adjusting for covariates, laparoscopic cholecystectomy was more commonly performed after 2007 (odds ratio [OR] 1.333, p < 0.001). Furthermore, time from admission to surgery was significantly shorter in the latter study period (regression coefficient -0.013, p < 0.001). Compared to non-operative management, laparoscopic cholecystectomy for acute cholecystitis was significantly associated with lower rates of preterm delivery, labor, or abortion (OR 0.410, p < 0.001). Each day that laparoscopic cholecystectomy was delayed significantly associated with an increased risk of fetal complications (OR 1.173, p < 0.001).ConclusionsThis nationwide study exhibits significant trends favoring surgical management of acute cholecystitis during pregnancy. Although further studies are still warranted, early laparoscopic cholecystectomy should be considered in pregnant patients with acute cholecystitis.