This study describes new yeast expression systems for each subunit of the heterotrimeric epithelial sodium channel (ENaC). We found that a significant amount of each subunit resides in the ER and is destroyed via ERAD. We also found that the chaperone requirements for ENaC subunit degradation were unlike any other ERAD substrate examined.

Aquaporin (AQP) folding in the endoplasmic reticulum is characterized by two distinct pathways of membrane insertion that arise from divergent residues within the second transmembrane segment. We now show that in AQP1 these residues (Asn49 and Lys51) interact with Asp185 at the C terminus of TM5 to form a polar, quaternary structural motif that influences multiple stages of folding. Asn49 and Asp185 form an intramolecular hydrogen bond needed for proper helical packing, monomer formation and function. In contrast, Lys51 interacts with Asp185 on an adjacent monomer to stabilize the AQP1 tetramer. Although these residues are unique to AQP1, they share a highly conserved architecture whose functional properties can be transferred to other family members. These findings suggest a general mechanism by which evolutionary divergence of membrane proteins can confer new functional properties via alternative folding pathways that give rise to a common final structure.

Background:The epithelial sodium channel (ENaC) is a substrate for the endoplasmic reticulum associated degradation (ERAD) quality control system. Results: The chaperone Lhs1/GRP170 selects the nonglycosylated form of the ␣ subunit for ERAD. Conclusion: This study is the first to show a role for Lhs1/GRP170 in ERAD substrate selection. Significance: Mutations in ENaC are associated with human disease; therefore, Lhs1/GRP170, as a modulator of ENaC expression, may be a target for new therapeutic agents.

Most proteins in the secretory pathway are translated, folded, and subjected to quality control at the endoplasmic reticulum (ER). These processes must be flexible enough to process diverse protein conformations, yet specific enough to recognize when a protein should be degraded. Molecular chaperones are responsible for this decision making process. ER associated chaperones assist in polypeptide translocation, protein folding, and ER associated degradation (ERAD). Nevertheless, we are only beginning to understand how chaperones function, how they are recruited to specific substrates and assist in folding/degradation, and how unique chaperone classes make quality control "decisions".

Purpose Severe community-acquired pneumonia (CAP) requiring intensive care unit admission is associated with significant acute and long-term morbidity and mortality. We hypothesized that downregulation of systemic and pulmonary inflammation with prolonged low-dose methylprednisolone treatment would accelerate pneumonia resolution and improve clinical outcomes. Methods This double-blind, randomized, placebo-controlled clinical trial recruited adult patients within 72–96 h of hospital presentation. Patients were randomized in 1:1 ratio; an intravenous 40 mg loading bolus was followed by 40 mg/day through day 7 and progressive tapering during the 20-day treatment course. Randomization was stratified by site and need for mechanical ventilation (MV) at the time of randomization. Outcomes included a primary endpoint of 60-day all-cause mortality and secondary endpoints of morbidity and mortality up to 1 year of follow-up. Results Between January 2012 and April 2016, 586 patients from 42 Veterans Affairs Medical Centers were randomized, short of the 1420 target sample size because of low recruitment. 584 patients were included in the analysis. There was no significant difference in 60-day mortality between the methylprednisolone and placebo arms (16% vs. 18%; adjusted odds ratio 0.90, 95% CI 0.57–1.40). There were no significant differences in secondary outcomes or complications. Conclusions In patients with severe CAP, prolonged low-dose methylprednisolone treatment did not significantly reduce 60-day mortality. Treatment was not associated with increased complications. Supplementary Information The online version contains supplementary material available at 10.1007/s00134-022-06684-3.

Polytopic protein topology is established in the endoplasmic reticulum (ER) by sequence determinants encoded throughout the nascent polypeptide. Here we characterize 12 topogenic determinants in the cystic fibrosis transmembrane conductance regulator, and identify a novel mechanism by which a charged residue is positioned within the plane of the lipid bilayer. During cystic fibrosis transmembrane conductance regulator biogenesis, topology of the C-terminal transmembrane domain (TMs 7-12) is directed by alternating signal (TMs 7, 9, and 11) and stop transfer (TMs 8, 10, and 12) sequences. Unlike conventional stop transfer sequences, however, TM8 is unable to independently terminate translocation due to the presence of a single charged residue, Asp 924 , within the TM segment. Instead, TM8 stop transfer activity is specifically dependent on TM7, which functions both to initiate translocation and to compensate for the charged residue within TM8. Moreover, even in the presence of TM7, the N terminus of TM8 extends significantly into the ER lumen, suggesting a high degree of flexibility in establishing TM8 transmembrane boundaries. These studies demonstrate that signal sequences can markedly influence stop transfer behavior and indicate that ER translocation machinery simultaneously integrates information from multiple topogenic determinants as they are presented in rapid succession during polytopic protein biogenesis.The topology of most eukaryotic polytopic proteins is generated in the endoplasmic reticulum (ER) 1 through the collective action of sequence determinants encoded within the nascent polypeptide. These determinants encompass hydrophobic transmembrane (TM) segments that, together with their flanking residues, interact with cytosolic and ER translocation machinery to initiate and terminate translocation and integrate the polypeptide into the lipid bilayer (reviewed in Refs. 1-3). In the simplest model, topology can be established cotranslationally by alternating topogenic determinants that function as signal (anchor) and stop transfer sequences (4 -7). As the first signal sequence emerges from the ribosome, it targets the ribosome nascent-chain complex (RNC) to the ER and gates open an aqueous channel in the membrane (the Sec61 translocon) (8). Because the ribosome exit site is directly aligned with the axial pore of the translocon, newly synthesized polypeptide is cotranslationally directed into the aqueous environment of the translocon as it emerges from the ribosome (9 -11). Subsequent synthesis of a stop transfer sequence gates the translocon closed to the ER lumen, terminates translocation, and provides the growing nascent polypeptide access to the cytosol (12, 13). Through sequential iterations of these events, signal and stop transfer sequences can alternately direct the polypeptide into the ER lumen or the cytosol and thus establish topology of transmembrane segments and lumenal and cytosolic peptide loops.Not all native polytopic proteins utilize a simple cotranslational biogenesis pathway. For ex...

Here we re-examine AQP1 biogenesis and show that irrespective of the reporter or fusion site used, oocytes and mammalian cells yielded similar topologic results. AQP1 intermediates containing the first three TM segments generated two distinct cohorts of polypeptides in which TM3 spanned the ER membrane in either an Ncyto/Cexo (mature) or Nexo/Ccyto (immature) topology. Pulse-chase analyses revealed that the immature form was predominant immediately after synthesis but that it was rapidly degraded via the proteasome-mediated endoplasmic reticulum associated degradation (ERAD) pathway with a half-life of less than 25 min in HEK cells. As a result, the mature topology predominated at later time points. We conclude that (i) differential stability of biogenesis intermediates is an important factor for in vivo topological analysis of truncated chimeric proteins and (ii) cotranslational events of AQP1 biogenesis reflect a common AQP1 folding pathway in diverse expression systems.Polytopic membrane proteins are synthesized and oriented in the endoplasmic reticulum (ER) 1 by the ribosome and Sec61 translocon complex (1-3). In the simplest model, topology of each transmembrane (TM) segment is established in a vectoral and sequential manner (N to C termini) (4) as independent signal anchor and stop transfer sequences alternately gate the translocon and the ribosome-translocon junction and direct TM segment integration into the lipid bilayer (cotranslational model) (5-7). However, a growing body of evidence has demonstrated that the final topology of many native proteins is not necessarily established cotranslationally but rather through cooperative interactions between topogenic determinants (TM segments) located within different regions of the polypeptide (post-translational model) (8 -16).One example of the post-translational model occurs during the biogenesis of aquaporin-1 (AQP1), a hydrophobic membrane protein of ϳ29 kDa that exists as a homo-tetramer in cell membranes. AQP1 is a member of the MIP (major intrinsic protein) family (17, 18). In its mature form it exhibits a characteristic topology with six TM segments and two additional short helical regions flanked by conserved NPA motifs that fold inward within the plane of the membrane to form a monomeric, water-selective pore (19,20). AQP1 is expressed in diverse cell types and is localized in the kidney to the proximal tubule and descending limb of the loop of Henle where it plays a major role in renal water reabsorption (18,21).Early biogenesis studies of AQP1 in cell-free systems and microinjected Xenopus oocytes revealed a novel mechanism in which only four of its six transmembrane segments cotranslationally acquired a membrane spanning topology (22). This four-spanning intermediate later matures in the ER membrane to form the final six-spanning structure (23,24). AQP1 biogenesis differs from the cotranslational pathway utilized by a close homolog, AQP4 (25), in part because hydrophilic residues within the N terminus of TM2 (Asn 49 and Lys 51 ) disrupt stop tran...

Molecular chaperones reside in nearly every organelle within a eukaryotic cell, and in each of these compartments, they ensure that protein homeostasis (or proteostasis) is maintained. In this issue, Wiseman and colleagues find that an ER lumenal chaperone escapes this compartment when a specific stress pathway is activated. The chaperone, an Hsp40 homolog known as ERdj3, transits through the secretory pathway to the extracellular space. During this journey, ERdj3 can escort an aggregation‐prone protein or it can identify aggregation‐prone proteins extracellularly, thereby functioning outside of its normal environment.