The Structural Basis for Phospholamban Inhibition of the Calcium Pump in Sarcoplasmic Reticulum

P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca²⁺-ATPase), pumps contractile-dependent Ca²⁺ ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca²⁺ affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca²⁺-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca²⁺ in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca²⁺ binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca²⁺ affinity and depresses cardiac contractility.     

Insulin-induced Gene Protein (INSIG)-dependent Sterol Regulation of Hmg2 Endoplasmic Reticulum-associated Degradation (ERAD) in Yeast

Insulin-induced gene proteins (INSIGs) function in control of cellular cholesterol. Mammalian INSIGs exert control by directly interacting with proteins containing sterol-sensing domains (SSDs) when sterol levels are elevated. Mammalian 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR) undergoes sterol-dependent, endoplasmic-reticulum (ER)-associated degradation (ERAD) that is mediated by INSIG interaction with the HMGR SSD. The yeast HMGR isozyme Hmg2 also undergoes feedback-regulated ERAD in response to the early pathway-derived isoprene gernanylgeranyl pyrophosphate (GGPP). Hmg2 has an SSD, and its degradation is controlled by the INSIG homologue Nsg1. However, yeast Nsg1 promotes Hmg2 stabilization by inhibiting GGPP-stimulated ERAD. We have proposed that the seemingly disparate INSIG functions can be unified by viewing INSIGs as sterol-dependent chaperones of SSD clients. Accordingly, we tested the role of sterols in the Nsg1 regulation of Hmg2. We found that both Nsg1-mediated stabilization of Hmg2 and the Nsg1-Hmg2 interaction required the early sterol lanosterol. Lowering lanosterol in the cell allowed GGPP-stimulated Hmg2 ERAD. Thus, Hmg2-regulated degradation is controlled by a two-signal logic; GGPP promotes degradation, and lanosterol inhibits degradation. These data reveal that the sterol dependence of INSIG-client interaction has been preserved for over 1 billion years. We propose that the INSIGs are a class of sterol-dependent chaperones that bind to SSD clients, thus harnessing ER quality control in the homeostasis of sterols.     

Proprotein Convertases Process and Thereby Inactivate Formylglycine-generating Enzyme

Formylglycine-generating enzyme (FGE) post-translationally converts a specific cysteine in newly synthesized sulfatases to formylglycine (FGly). FGly is the key catalytic residue of the sulfatase family, comprising 17 nonredundant enzymes in human that play essential roles in development and homeostasis. FGE, a resident protein of the endoplasmic reticulum, is also secreted. A major fraction of secreted FGE is N-terminally truncated, lacking residues 34–72. Here we demonstrate that this truncated form is generated intracellularly by limited proteolysis mediated by proprotein convertase(s) (PCs) along the secretory pathway. The cleavage site is represented by the sequence RYSR⁷²↓, a motif that is conserved in higher eukaryotic FGEs, implying important functionality. Residues Arg-69 and Arg-72 are critical because their mutation abolishes FGE processing. Furthermore, residues Tyr-70 and Ser-71 confer an unusual property to the cleavage motif such that endogenous as well as overexpressed FGE is only partially processed. FGE is cleaved by furin, PACE4, and PC5a. Processing is disabled in furin-deficient cells but fully restored upon transient furin expression, indicating that furin is the major protease cleaving FGE. Processing by endogenous furin occurs mostly intracellularly, although also extracellular processing is observed in HEK293 cells. Interestingly, the truncated form of secreted FGE no longer possesses FGly-generating activity, whereas the unprocessed form of secreted FGE is active. As always both forms are secreted, we postulate that furin-mediated processing of FGE during secretion is a physiological means of higher eukaryotic cells to regulate FGE activity upon exit from the endoplasmic reticulum.     

General Control Nonderepressible 2 (GCN2) Kinase Protects Oligodendrocytes and White Matter during Branched-chain Amino Acid Deficiency in Mice

Branched-chain amino acid (BCAA) catabolism is regulated by branched-chain α-keto acid dehydrogenase, an enzyme complex that is inhibited when phosphorylated by its kinase (BDK). Loss of BDK function in mice and humans causes BCAA deficiency and epilepsy with autistic features. In response to amino acid deficiency, phosphorylation of eukaryotic initiation factor 2α (eIF2∼P) by general control nonderepressible 2 (GCN2) activates the amino acid stress response. We hypothesized that GCN2 functions to protect the brain during chronic BCAA deficiency. To test this idea, we generated mice lacking both Gcn2 and Bdk (GBDK) and examined the development of progeny. GBDK mice appeared normal at birth, but they soon stopped growing, developed severe ataxia, tremor, and anorexia, and died by postnatal day 15. BCAA levels in brain were diminished in both Bdk^−/− and GBDK pups. Brains from Bdk^−/− pups exhibited robust eIF2∼P and amino acid stress response induction, whereas these responses were absent in GBDK mouse brains. Instead, myelin deficiency and diminished expression of myelin basic protein were noted in GBDK brains. Genetic markers of oligodendrocytes and astrocytes were also reduced in GBDK brains in association with apoptotic cell death in white matter regions of the brain. GBDK brains further demonstrated reduced Sod2 and Cat mRNA and increased Tnfα mRNA expression. The data are consistent with the idea that loss of GCN2 during BCAA deficiency compromises glial cell defenses to oxidative and inflammatory stress. We conclude that GCN2 protects the brain from developing a lethal leukodystrophy in response to amino acid deficiencies.     

A Ubiquitin-like Domain Recruits an Oligomeric Chaperone to a Retrotranslocation Complex in Endoplasmic Reticulum-associated Degradation

The Bag6-Ubl4A-Trc35 complex is a multifunctional chaperone that regulates various cellular processes. The diverse functions of Bag6 are supported by its ubiquitous localization to the cytoplasm, the nucleus, and membranes of the endoplasmic reticulum (ER) in cells. In ER-associated degradation (ERAD) pathways, Bag6 can interact with the membrane-associated ubiquitin ligase gp78 via its ubiquitin-like (UBL) domain, but the relative low affinity of this interaction does not reconcile with the fact that a fraction of Bag6 is tightly bound to the membranes. Here, we demonstrate that the UBL domain of Bag6 is required for interaction with the ER membranes. We find that in addition to gp78, the Bag6 UBL domain also binds a UBL-binding motif in UbxD8, an essential component of the gp78 ubiquitinating machinery. Importantly, Bag6 contains a proline-rich (PR) domain termed PDP (Proline rich-DUF3587-Proline rich) that forms homo-oligomer, allowing the UBL domain to form multivalent interactions with gp78 and UbxD8, which are essential for recruitment of Bag6 to the ER membrane. Furthermore, the PR domain comprises largely intrinsically disordered segments, which are sufficient for interaction with an unfolded substrate. We propose that simultaneous association with multiple ERAD factors helps to anchor a disordered chaperone oligomer to the site of retrotranslocation to prevent protein aggregation in ERAD.     

C/EBP Homologous Protein-induced Macrophage Apoptosis Protects Mice from Steatohepatitis

Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop^+/+) and knock-out (Chop^−/−) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop^−/− mice developed greater liver injury, inflammation, and fibrosis than Chop^+/+ mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop^+/+ and Chop^−/− were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop^+/+ and Chop^−/− macrophages, Chop^−/− macrophages were resistant to its lipotoxicity. Chop^−/− mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop^−/− and Chop^+/+ mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity.     

Mutations in the Polybasic Juxtamembrane Sequence of Both Plasma Membrane- and Endoplasmic Reticulum-localized Epidermal Growth Factor Receptors Confer Ligand-independent Cell Transformation

Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling.     

Altered Cortical GABAA Receptor Composition,Physiology, and Endocytosis in a Mouse Model of a Human Genetic Absence Epilepsy Syndrome

Patients with generalized epilepsy exhibit cerebral cortical disinhibition. Likewise, mutations in the inhibitory ligand-gated ion channels, GABA_A receptors (GABA_ARs), cause generalized epilepsy syndromes in humans. Recently, we demonstrated that heterozygous knock-out (Het_α1KO) of the human epilepsy gene, the GABA_AR α1 subunit, produced absence epilepsy in mice. Here, we determined the effects of Het_α1KO on the expression and physiology of GABA_ARs in the mouse cortex. We found that Het_α1KO caused modest reductions in the total and surface expression of the β2 subunit but did not alter β1 or β3 subunit expression, results consistent with a small reduction of GABA_ARs. Cortices partially compensated for Het_α1KO by increasing the fraction of residual α1 subunit on the cell surface and by increasing total and surface expression of α3, but not α2, subunits. Co-immunoprecipitation experiments revealed that Het_α1KO increased the fraction of α1 subunits, and decreased the fraction of α3 subunits, that associated in hybrid α1α3βγ receptors. Patch clamp electrophysiology studies showed that Het_α1KO layer VI cortical neurons exhibited reduced inhibitory postsynaptic current peak amplitudes, prolonged current rise and decay times, and altered responses to benzodiazepine agonists. Finally, application of inhibitors of dynamin-mediated endocytosis revealed that Het_α1KO reduced base-line GABA_AR endocytosis, an effect that probably contributes to the observed changes in GABA_AR expression. These findings demonstrate that Het_α1KO exerts two principle disinhibitory effects on cortical GABA_AR-mediated inhibitory neurotransmission: 1) a modest reduction of GABA_AR number and 2) a partial compensation with GABA_AR isoforms that possess physiological properties different from those of the otherwise predominant α1βγ GABA_ARs.     

Maturation of thyroglobulin protein region I

In vertebrates, the thyroglobulin (Tg) gene product must be exported to the lumen of thyroid follicles for thyroid hormone synthesis. In toto, Tg is composed of multiple type-1 repeats connected by linker and hinge (altogether considered as "region I," nearly 1,200 residues); regions II-III (~720 residues); and cholinesterase-like (ChEL) domain (~570 residues). Regions II-III and ChEL rapidly acquire competence for secretion, yet regions I-II-III require 20 min to become a partially mature disulfide isomer; stabilization of a fully oxidized form requires ChEL. Transition from partially mature to mature Tg occurs as a discrete "jump" in mobility by nonreducing SDS-PAGE, suggesting formation of at most a few final pairings of Cys residues that may be separated by significant intervening primary sequence. Using two independent approaches, we have investigated which portion of Tg is engaged in this late stage of its maturation. First, we demonstrate that this event is linked to oxidation involving region I. Introduction of the Tg-C1245R mutation in the hinge (identical to that causing human goitrous hypothyroidism) inhibits this maturation, although the Cys-1245 partner remains unidentified. Second, we find that Tg truncated after its fourth type-1 repeat is a fully independent secretory protein. Together, the data indicate that final acquisition of secretory competence includes conformational maturation in the interval between linker and hinge segments of region I.