Tyrosine phosphorylation of S1PR1 leads to chaperone BiP-mediated import to the endoplasmic reticulum

Cell surface G protein–coupled receptors (GPCRs), upon agonist binding, undergo serine–threonine phosphorylation, leading to either receptor recycling or degradation. Here, we show a new fate of GPCRs, exemplified by ER retention of sphingosine-1-phosphate receptor 1 (S1PR1). We show that S1P phosphorylates S1PR1 on tyrosine residue Y143, which is associated with recruitment of activated BiP from the ER into the cytosol. BiP then interacts with endocytosed Y143-S1PR1 and delivers it into the ER. In contrast to WT-S1PR1, which is recycled and stabilizes the endothelial barrier, phosphomimicking S1PR1 (Y143D-S1PR1) is retained by BiP in the ER and increases cytosolic Ca2+ and disrupts barrier function. Intriguingly, a proinflammatory, but non-GPCR agonist, TNF-α, also triggered barrier-disruptive signaling by promoting S1PR1 phosphorylation on Y143 and its import into ER via BiP. BiP depletion restored Y143D-S1PR1 expression on the endothelial cell surface and rescued canonical receptor functions. Findings identify Y143-phosphorylated S1PR1 as a potential target for prevention of endothelial barrier breakdown under inflammatory conditions.

Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain

Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc N-glycan, and eliminating the N-glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated N-glycans that are unsuitable for therapeutics. However, Fc with a specifically truncated N-glycan still engages receptors with considerable affinity. Here we describe the creation and applications of a novel Saccharomyces cerevisiae strain that specifically modifies the IgG1 Fc domain with an N-glycan consisting of a single N-acetylglucosamine residue. This strain displayed glycoengineered Fc on its surface for screening yeast surface display libraries and also served as an alternative platform to produce glycoengineered Rituximab. An IgG-specific endoglycosidase (EndoS2) truncates the IgG1 Fc N-glycan. EndoS2 was targeted to the yeast ER using the signal peptide from the yeast protein disulfide isomerase (PDI) and a yeast ER retention signal (HDEL). Furthermore, >99% of the yeast expressed Rituximab displayed the truncated glycoform as determined by SDS-PAGE and ESI-MS analyses. Lastly, the yeast expressed Rituximab engaged the FcγRIIIa with the expected affinity (KD = 2.0 ± 0.5 μM) and bound CD20 on Raji B cells.

Aβ toxicity rescued by protein retention in the ER

Accumulation of Aβ in the brain is one of the hallmarks of Alzheimer's disease (AD). In the adult Drosophila brain, human Aβ over-expression is toxic and leads to deterioration of climbing ability and shortened lifespan. However, it remains unknown if Aβ is inherently toxic or if it triggers toxic downstream pathways that lead to neurodegeneration. Here, we describe a novel, and previously unidentified, protective role of intracellular laminin chain accumulation. Despite high Aβ levels, over-expression of the extracellular matrix protein subunit Laminin B1 (LanB1) resulted in a robust rescue of toxicity, highlighting a potential protective mechanism of resistance to Aβ. Over-expression of other Laminin subunits and a Collagen IV subunit also significantly rescued Aβ toxicity, while combining LanB1 with these subunits led to an even larger rescue. Imaging revealed that LanB1 was retained in the ER but had no effect on the secretion of Aβ into the extracellular milieu. LanB1 rescued toxicity independently of the IRE1α/XBP1-mediated branch of the ER stress response. Interestingly, over-expression of ER-targeted GFP also rescued Aβ toxicity, indicating a potentially broader benefit of ER protein retention. Finally, in proof-of-principle lentiviral transduction experiments using murine organotypic hippocampal slice cultures, over-expression of mouse Lamb1 resulted in ER-retention in transduced cells, highlighting a conserved mechanism. Typically, retention of proteins in the ER is detrimental to cellular health, but in the context of neuronal Aβ toxicity it may prove to be beneficial and a new therapeutic avenue for AD.

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.

Calmodulin-dependent KCNE4 dimerization controls membrane targeting

The voltage-dependent potassium channel Kv1.3 participates in the immune response. Kv1.3 is essential in different cellular functions, such as proliferation, activation and apoptosis. Because aberrant expression of Kv1.3 is linked to autoimmune diseases, fine-tuning its function is crucial for leukocyte physiology. Regulatory KCNE subunits are expressed in the immune system, and KCNE4 specifically tightly regulates Kv1.3. KCNE4 modulates Kv1.3 currents slowing activation, accelerating inactivation and retaining the channel at the endoplasmic reticulum (ER), thereby altering its membrane localization. In addition, KCNE4 genomic variants are associated with immune pathologies. Therefore, an in-depth knowledge of KCNE4 function is extremely relevant for understanding immune system physiology. We demonstrate that KCNE4 dimerizes, which is unique among KCNE regulatory peptide family members. Furthermore, the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4 is a structural platform in which Kv1.3, Ca2+/calmodulin (CaM) and dimerizing KCNE4 compete for multiple interaction partners. CaM-dependent KCNE4 dimerization controls KCNE4 membrane targeting and modulates its interaction with Kv1.3. KCNE4, which is highly retained at the ER, contains an important ER retention motif near the tetraleucine motif. Upon escaping the ER in a CaM-dependent pattern, KCNE4 follows a COP-II-dependent forward trafficking mechanism. Therefore, CaM, an essential signaling molecule that controls the dimerization and membrane targeting of KCNE4, modulates the KCNE4-dependent regulation of Kv1.3, which in turn fine-tunes leukocyte physiology.

A region within the third extracellular loop of rat Aquaporin 6 precludes trafficking to plasma membrane in a heterologous cell line

The inability to over-express Aquaporin 6 (AQP6) in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail using crystallographic approaches. Using an Aquaporin 3-tGFP Reporter (AGR) system we have identified a region within loop C of AQP6 that is responsible for severely hampering plasma membrane expression. Serine substitution corroborated that amino acids present within AQP6194–213 of AQP6 loop C contribute to intracellular endoplasmic reticulum (ER) retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous expression systems.

Computational Modeling of C-Terminal Tails to Predict the Calcium-Dependent Secretion of Endoplasmic Reticulum Resident Proteins

The lumen of the endoplasmic reticulum (ER) has resident proteins that are critical to perform the various tasks of the ER such as protein maturation and lipid metabolism. These ER resident proteins typically have a carboxy-terminal ER retention/retrieval sequence (ERS). The canonical ERS that promotes ER retrieval is Lys-Asp-Glu-Leu (KDEL) and when an ER resident protein moves from the ER to the Golgi, KDEL receptors (KDELRs) in the Golgi recognize the ERS and return the protein to the ER lumen. Depletion of ER calcium leads to the mass departure of ER resident proteins in a process termed exodosis, which is regulated by KDELRs. Here, by combining computational prediction with machine learning-based models and experimental validation, we identify carboxy tail sequences of ER resident proteins divergent from the canonical “KDEL” ERS. Using molecular modeling and simulations, we demonstrated that two representative non-canonical ERS can stably bind to the KDELR. Collectively, we developed a method to predict whether a carboxy-terminal sequence acts as a putative ERS that would undergo secretion in response to ER calcium depletion and interacts with the KDELRs. The interaction between the ERS and the KDELR extends beyond the final four carboxy terminal residues of the ERS. Identification of proteins that undergo exodosis will further our understanding of changes in ER proteostasis under physiological and pathological conditions where ER calcium is depleted.

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation

Lignocellulosic biomass from plants has been used as a biofuel source and the potent acidic endoglucanase GtCel12A has been isolated from Gloeophyllum trabeum, a filamentous fungus. In this study, we established a plant-based platform for the production of active GtCel12A fused to family 3 cellulose-binding module (CBM3). We used the signal sequence of binding immunoglobulin protein (BiP) and the endoplasmic reticulum (ER) retention signal for the accumulation of the produced GtCel12A in the ER. To achieve enhanced enzyme expression, we incorporated the M-domain of the human receptor-type tyrosine-protein phosphatase C into the construct. In addition, to enable the removal of N-terminal domains that are not necessary after protein expression, we further incorporated the cleavage site of Brachypodium distachyon small ubiquitin-like modifier. The GtCel12A-CBM3 fusion protein produced in the leaves of Nicotiana benthamiana exhibited not only high solubility but also efficient endoglucanase activity on the carboxymethyl cellulose substrate as determined by 3,5-dinitrosalicylic acid assay. The endoglucanase activity of GtCel12A-CBM3 was maintained even when immobilized on microcrystalline cellulose beads. Taken together, these results indicate that GtCel12A endoglucanase produced in plants might be used to provide monomeric sugars from lignocellulosic biomass for bioethanol production.

Deletion of ER-retention motif on SARS-CoV-2 spike protein reduces cell hybrid during cell–cell fusion

Background The novel SARS-CoV-2 has quickly become a global pandemic since the first reported case in December 2019, with the virus infecting millions of people to date. The spike (S) protein of the SARS-CoV-2 virus plays a key role in binding to angiotensin-converting enzyme 2 (ACE2), a host cell receptor for SARS-CoV-2. S proteins that are expressed on the cell membrane can initiate receptor-dependent syncytia formation that is associated with extensive tissue damage. Formation of syncytia have been previously observed in cells infected with various other viruses (e.g., HIV, Ebola, Influenza, and Herpesviruses). However, this phenomenon is not well documented and the mechanisms regulating the formation of the syncytia by SARS-CoV-2 are not fully understood. Results In this study, we investigated the possibility that cell fusion events mediated by the S protein of SARS-CoV-2 and ACE2 interaction can occur in different human cell lines that mimic different tissue origins. These cell lines were transduced with either wild-type (WT-S) S protein or a mutated variant where the ER-retention motif was removed (Δ19-S), as well as human ACE2 expression vectors. Different co-culture combinations of spike-expressing 293T, A549, K562, and SK-Hep1 cells with hACE2-expressing cells revealed cell hybrid fusion. However, only certain cells expressing S protein can form syncytial structures as this phenomenon cannot be observed in all co-culture combinations. Thus, SARS-CoV-2 mediated cell–cell fusion represents a cell type-dependent process which might rely on a different set of parameters. Recently, the Δ19-S variant is being widely used to increase SARS-CoV-2 pseudovirus production for in vitro assays. Comparison of cell fusion occurring via Δ19-S expressing cells shows defective nuclear fusion and syncytia formation compared to WT-S. Conclusions This distinction between the Δ19-S variant and WT-S protein may have downstream implications for studies that utilize pseudovirus-based entry assays. Additionally, this study suggest that spike protein expressed by vaccines may affect different ACE2-expressing host cells after SARS-CoV-2 vaccine administration. The long-term effects of these vaccines should be monitored carefully. Δ19-S mRNA may represent a safer mRNA vaccine design in the future.

Characterization of membrane topology and retention signal of pestiviral glycoprotein E1

Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1 and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface, but localizes predominantly in the ER. Using engineered chimeric TM domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labelling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C-terminus whereas it adopts a hairpin-like structure with the C-terminus located in the ER lumen when the pre-cleavage situation is mimicked by blocking the cleavage site between E1 and E2. Importance The shortage of specific antibodies against E1 making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the TM domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy-terminus located on the luminal side of the ER in the pre-cleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.