Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids (Review)

Glycosylphosphatidylinositols (GPIs) represent an abundant and ubiquitous class of eukaryotic glycolipids. Although these structures were originally discovered in the form of GPI-anchored cell surface glycoproteins, it is becoming increasingly clear that a significant proportion of the GPI synthetic output of a cell is not directed to protein anchoring. Indeed, pools of nonprotein-linked GPIs can approach 107 molecules per cell in some cell types, especially the protozoa, with a large proportion of these molecules being displayed at the cell surface. Recent studies which form the subject of this review indicate that there is (a) considerable diversity in the range of structural modifications found on GPI glycolipids within and between species and cell types, (b) complexity in the topological arrangement of the GPI biosynthetic pathway in the endoplasmic reticulum, and (c) spatial restriction of the biosynthetic pathway within the endoplasmic reticulum. Furthermore, consistent with additional functional roles for these lipids beyond serving as protein anchor precursors, products of the GPI biosynthetic pathway appear to be widely distributed in the cellular endomembrane system. These studies indicate that there is still much to learn about the organization of glycolipid biosynthetic pathways in eukaryotic cells, the nature and subcellular distribution of the lipid products of these pathways, and the function of these lipids within cells.     

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca²⁺ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca²⁺ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca²⁺ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca²⁺ indicator and a hydrophilic fluorescent dye/Ca²⁺ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F₀.     

Proteomic analysis reveals tanshinone IIA enhances apoptosis of advanced cervix carcinoma CaSki cells through mitochondria intrinsic and endoplasmic reticulum stress pathways

Cervix cancer is the second most common cancer among women worldwide, whereas paclitaxel, the first line chemotherapeutic drug used to treat cervical cancer, shows low chemosensitivity on the advanced cervical cancer cell line. Tanshinone IIA (Tan IIA) exhibited strong growth inhibitory effect on CaSki cells (IC₅₀ = 5.51 μM) through promoting caspase cascades with concomitant upregulating the phosphorylation of p38 and JNK signaling. Comprehensive proteomics revealed the global protein changes and the network analysis implied that Tan IIA treatment would activate ER stress pathways that finally lead to apoptotic cell death. Moreover, ER stress inhibitor could alleviate Tan IIA caused cell growth inhibition and ameliorate C/EBP‐homologous protein as well as apoptosis signal‐regulating kinase 1 mediated cell death. The therapeutic interventions targeting the mitochondrial‐related apoptosis and ER stress responses might be promising strategies to conquer paclitaxel resistance.     

An inside job: Annexin 1A-Inositol 1,4,5-trisphosphate receptor interaction conveys endoplasmic reticulum luminal Ca2+ sensitivity

Cytoplasmic Ca²⁺ is a pivotal regulator of IP₃R activity. It is however controversial whether the [Ca²⁺] in the Endoplasmic Reticulum lumen also directly regulates channel function. We highlight a recent paper that demonstrates that luminal [Ca²⁺] potently inhibits IP₃R activity. This regulation occurs indirectly by an interaction mediated through a binding partner, likely Annexin 1A.     

An Additional Function of the Rough Endoplasmic Reticulum Protein Complex Prolyl 3-Hydroxylase 1·Cartilage-associated Protein·Cyclophilin B: THE CXXXC MOTIF REVEALS DISULFIDE ISOMERASE ACTIVITY IN VITRO*

Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.     

Switching the Clientele: A LYSINE RESIDING IN THE C TERMINUS OF THE SEROTONIN TRANSPORTER SPECIFIES ITS PREFERENCE FOR THE COAT PROTEIN COMPLEX II COMPONENT SEC24C*

The serotonin transporter (SERT) maintains serotonergic neurotransmission via rapid reuptake of serotonin from the synaptic cleft. SERT relies exclusively on the coat protein complex II component SEC24C for endoplasmic reticulum (ER) export. The closely related transporters for noradrenaline and dopamine depend on SEC24D. Here, we show that discrimination between SEC24C and SEC24D is specified by the residue at position +2 downstream from the ER export motif (⁶⁰⁷RI⁶⁰⁸ in SERT). Substituting Lys⁶¹⁰ with tyrosine, the corresponding residue found in the noradrenaline and dopamine transporters, switched the SEC24 isoform preference: SERT-K610Y relied solely on SEC24D to reach the cell surface. This analysis was extended to other SLC6 (solute carrier 6) transporter family members: siRNA-dependent depletion of SEC24C, but not of SEC24D, reduced surface levels of the glycine transporter-1a, the betaine/GABA transporter and the GABA transporter-4. Experiments with dominant negative versions of SEC24C and SEC24D recapitulated these findings. We also verified that the presence of two ER export motifs (in concatemers of SERT and GABA transporter-1) supported recruitment of both SEC24C and SEC24D. To the best of our knowledge, this is the first report to document a change in SEC24 specificity by mutation of a single residue in the client protein. Our observations allowed for deducing a rule for SLC6 family members: a hydrophobic residue (Tyr or Val) in the +2 position specifies interaction with SEC24D, and a hydrophilic residue (Lys, Asn, or Gln) recruits SEC24C. Variations in SEC24C are linked to neuropsychiatric disorders. The present findings provide a mechanistic explanation. Variations in SEC24C may translate into distinct surface levels of neurotransmitter transporters.     

Isoform-selective Oligomer Formation of Saccharomyces cerevisiae p24 Family Proteins

p24 family proteins are evolutionarily conserved transmembrane proteins involved in the early secretory pathway. Saccharomyces cerevisiae has 8 known p24 proteins that are classified into four subfamilies (p24α, -β, -γ, and -δ). Emp24 and Erv25 are the sole members of p24β and -δ, respectively, and deletion of either destabilizes the remaining p24 proteins, resulting in p24 null phenotype (p24Δ). We studied genetic and physical interactions of p24α (Erp1, -5, and -6) and γ (Erp2, -3, and -4). Deletion of the major p24α (Erp1) partially inhibited p24 activity as reported previously. A second mutation in either Erp5 or Erp6 aggravated the erp1Δ phenotype, and the triple mutation gave a full p24Δ phenotype. Similar genetic interactions were observed among the major p24γ (Erp2) and the other two γ members. All the p24α/γ isoforms interacted with both p24β and -δ. Interaction between p24β and -δ was isoform-selective, and five major α/γ pairs were detected. These results suggest that the yeast p24 proteins form functionally redundant αβγδ complexes. We also identified Rrt6 as a novel p24δ isoform. Rrt6 shows only limited sequence identity (∼15%) to known p24 proteins but was found to have structural properties characteristic of p24. Rrt6 was induced when cells were grown on glycerol and form an additional αβγδ complex with Erp3, Erp5, and Emp24. This complex was mainly localized to the Golgi, whereas the p24 complex containing Erv25, instead of Rrt6 but otherwise with the same isoform composition, was found mostly in the ER.     

Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K⁺ channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1–4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12–17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19–21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.     

Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)

Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N_in-C_out (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.     

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca²⁺ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1^−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) responses in NOS1^−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca²⁺]_i across the range of pacing frequencies and increased myofilament Ca²⁺ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca²⁺ reuptake, and restored SR Ca²⁺ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca²⁺ leak, HYD improves Ca²⁺ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.