Golgi Alpha1,2-Mannosidase IA Promotes Efficient Endoplasmic Reticulum-Associated Degradation of NKCC2

Mutations in the apically located kidney Na-K-2Cl cotransporter NKCC2 cause type I Bartter syndrome, a life-threatening kidney disorder. We previously showed that transport from the ER represents the limiting phase in NKCC2 journey to the cell surface. Yet very little is known about the ER quality control components specific to NKCC2 and its disease-causing mutants. Here, we report the identification of Golgi alpha1, 2-mannosidase IA (ManIA) as a novel binding partner of the immature form of NKCC2. ManIA interaction with NKCC2 takes place mainly at the cis-Golgi network. ManIA coexpression decreased total NKCC2 protein abundance whereas ManIA knock-down produced the opposite effect. Importantly, ManIA coexpression had a more profound effect on NKCC2 folding mutants. Cycloheximide chase assay showed that in cells overexpressing ManIA, NKCC2 stability and maturation are heavily hampered. Deleting the cytoplasmic region of ManIA attenuated its interaction with NKCC2 and inhibited its effect on the maturation of the cotransporter. ManIA-induced reductions in NKCC2 expression were offset by the proteasome inhibitor MG132. Likewise, kifunensine treatment greatly reduced ManIA effect, strongly suggesting that mannose trimming is involved in the enhanced ERAD of the cotransporter. Moreover, depriving ManIA of its catalytic domain fully abolished its effect on NKCC2. In summary, our data demonstrate the presence of a ManIA-mediated ERAD pathway in renal cells promoting retention and degradation of misfolded NKCC2 proteins. They suggest a model whereby Golgi ManIA contributes to ERAD of NKCC2, by promoting the retention, recycling, and ERAD of misfolded proteins that initially escape protein quality control surveillance within the ER.

Cellular Functions of ER Chaperones in Regulating Protein Misfolding and Aggregation: An Emerging Therapeutic Approach for Preeclampsia

Proteinuria is one of the hallmarks of preeclampsia (PE) that differentiates other hypertensive disorders of pregnancy. Protein misfolding and aggregation is an emerging pathological condition underlying many chronic metabolic diseases and neurodegenerative diseases. Recent studies indicate protein aggregation as an emerging biomarker of preeclampsia, wherein several proteins are aggregated and dysregulated in the body fluids of preeclamptic women, provoking the multi-systemic clinical manifestations of the disease. At the cellular level, these misfolded and aggregated proteins are potentially toxic interfering with the normal physiological process, eliciting the unfolded protein response (UPR) pathway activators in the endoplasmic reticulum (ER) that subsequently augments the ER quality control systems to remove these aberrant proteins. ER resident chaperones, folding enzymes and other proteins serve as part of the ER quality control machinery in restoring nascent protein folding. These ER chaperones are crucial for ER function aiding in native protein folding, maintaining calcium homeostasis, as sensors of ER stress and also as immune modulators. Consequently, ER chaperones seems to be involved in many cellular processes, yet the association is expanding to be explored. Understanding the role and mechanism of ER chaperones in regulating protein misfolding and aggregation would provide new avenues for therapeutic intervention as well as for the development of new diagnostic approaches.

ER-Phagy and Microbial Infection

The endoplasmic reticulum (ER) is an essential organelle in cells that synthesizes, folds and modifies membrane and secretory proteins. It has a crucial role in cell survival and growth, thus requiring strict control of its quality and homeostasis. Autophagy of the ER fragments, termed ER-phagy or reticulophagy, is an essential mechanism responsible for ER quality control. It transports stress-damaged ER fragments as cargo into the lysosome for degradation to eliminate unfolded or misfolded protein aggregates and membrane lipids. ER-phagy can also function as a host defense mechanism when pathogens infect cells, and its deficiency facilitates viral infection. This review briefly describes the process and regulatory mechanisms of ER-phagy, and its function in host anti-microbial defense during infection.

Protein Aggregation in the ER: Calm behind the Storm

As one of the largest organelles in eukaryotic cells, the endoplasmic reticulum (ER) plays a vital role in the synthesis, folding, and assembly of secretory and membrane proteins. To maintain its homeostasis, the ER is equipped with an elaborate network of protein folding chaperones and multiple quality control pathways whose cooperative actions safeguard the fidelity of protein biogenesis. However, due to genetic abnormalities, the error-prone nature of protein folding and assembly, and/or defects or limited capacities of the protein quality control systems, nascent proteins may become misfolded and fail to exit the ER. If not cleared efficiently, the progressive accumulation of misfolded proteins within the ER may result in the formation of toxic protein aggregates, leading to the so-called “ER storage diseases”. In this review, we first summarize our current understanding of the protein folding and quality control networks in the ER, including chaperones, unfolded protein response (UPR), ER-associated protein degradation (ERAD), and ER-selective autophagy (ER-phagy). We then survey recent research progress on a few ER storage diseases, with a focus on the role of ER quality control in the disease etiology, followed by a discussion on outstanding questions and emerging concepts in the field.

The Endoplasmic Reticulum Role in the Plant Response to Abiotic Stress

The endoplasmic reticulum (ER) is the organelle where one third of the proteins of a cell are synthetized. Several of these proteins participate in the signaling and response of cells, tissues, or from the organism to the environment. To secure the proper synthesis and folding of these proteins, or the disposal of unfolded or misfolded proteins, the ER has different mechanisms that interact and regulate each other. These mechanisms are known as the ER quality control (ERQC), ER-associated degradation (ERAD) and the unfolded protein response (UPR), all three participants of the maintenance of ER protein homeostasis or proteostasis. Given the importance of the client proteins of these ER mechanisms in the plant response to the environment, it is expected that changes or alterations on their components have an impact on the plant response to environmental cues or stresses. In this mini review, we focus on the impact of the alteration of components of ERQC, ERAD and UPR in the plant response to abiotic stresses such as drought, heat, osmotic, salt and irradiation. Also, we summarize findings from recent publications looking for a connection between these processes and their possible client(s) proteins. From this, we observed that a clear connection has been established between the ERAD and UPR mechanisms, but evidence that connects ERQC components to these both processes or their possible client(s) proteins is still lacking. As a proposal, we suggest the use of proteomics approaches to uncover the identity of these proteins and their connection with ER proteostasis.

Multi-level inhibition of coronavirus replication by chemical ER stress

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. The ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types including primary differentiated human bronchial epithelial cells, (partially) reverses the virus-induced translational shut-down, improves viability of infected cells and counteracts the CoV-mediated downregulation of IRE1α and the ER chaperone BiP. Proteome-wide analyses revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including essential (HERPUD1) or novel (UBA6 and ZNF622) factors of ER quality control, and ER-associated protein degradation complexes. Additionally, thapsigargin blocks the CoV-induced selective autophagic flux involving p62/SQSTM1. The data show that thapsigargin hits several central mechanisms required for CoV replication, suggesting that this compound (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs.

Ladostigil attenuates the oxidative and ER stress in human neuroblast-like SH-SY5Y cells

A hallmark of the aging brain is the robust inflammation mediated by microglial activation. Neuroinflammation resulting from the induction of oxidative stress in neurodegenerative diseases and following brain injury. Chronic treatment of aging rats by ladostigil, a compound with antioxidant and anti-inflammatory function, prevented microglial activation and learning deficits. In this study, we investigate the effect of ladostigil on neuronal-like SH-SY5Y cells. We show that SH-SY5Y cells exposed to acute (by H2O2) or chronic oxidative stress (by Sin1, 3-morpholinosydnonimine) induced apoptotic cell death. However, in the presence of ladostigil, the decline in cell viability and the oxidative levels were partially reversed. RNA-seq analysis showed that chronic oxidation by Sin1 resulted in coordinated suppression of endoplasmic reticulum (ER) quality control and ER stress response gene sets. Chronic oxidative stress impacted ER proteostasis and induced the expression of numerous lncRNAs. Pre-incubation with ladostigil before exposing SH-SY5Y cells to Sin1 induced Clk1 (Cdc2-like kinase 1) which was implicated in psychophysiological stress in mice and Alzheimer disease. Ladostigil also suppressed the expression of Ccpg1 (Cell cycle progression 1) and Synj1 (Synaptojanin 1) that function in ER-autophagy and endocytic pathways. We postulate that ladostigil alleviated cell damage by oxidation and ER stress. Therefore, it may attenuate neurotoxicity and cell death that accompany chronic stress conditions in the aging brain.

The Brucella effector BspL targets the ER-associated degradation (ERAD) pathway and delays bacterial egress from infected cells

Perturbation of the endoplasmic reticulum (ER), a central organelle of the cell, can have critical consequences for cellular homeostasis. An elaborate surveillance system known as ER quality control ensures that cells can respond and adapt to stress via the unfolded protein response (UPR) and that only correctly assembled proteins reach their destination. Interestingly, several bacterial pathogens hijack the ER to establish an infection. However, it remains poorly understood how bacterial pathogens exploit ER quality-control functions to complete their intracellular cycle. Brucella spp. replicate extensively within an ER-derived niche, which evolves into specialized vacuoles suited for exit from infected cells. Here we present Brucella-secreted protein L (BspL), a Brucella abortus effector that interacts with Herp, a central component of the ER-associated degradation (ERAD) machinery. We found that BspL enhances ERAD at the late stages of the infection. BspL targeting of Herp and ERAD allows tight control of the kinetics of autophagic Brucella-containing vacuole formation, delaying the last step of its intracellular cycle and cell-to-cell spread. This study highlights a mechanism by which a bacterial pathogen hijacks ERAD components for fine regulation of its intracellular trafficking.

Distinct states of proinsulin misfolding in MIDY

A precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)–Cys(A20) “interchain” disulfide bond, facilitating formation of the Cys(B7)–Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)–Cys(A11), is not required for anterograde trafficking, i.e., a “lose-A6/A11” mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)–Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.