Reticulum Lumen Research Articles

Serine 663-dependent phosphorylation of SERCA2 in ischemic heart disease: Regulation of calcium homeostasis, cell death and myocardial infarct size

Ischemic heart disease is one of the leading cause of death and disability worldwide. Calcium handling in the post-ischemic myocardium is a prime target to treat patients with acute myocardial infarction. Although SERCA2 is a promising target for the treatment of Ca2+ dyshomeostasis and contractile dysfunction following ischemia-reperfusion, the mechanisms through which SERCA2 is regulated remains still unclear. Post-translational modifications of SERCA2 have been recently shown to modulate its activity, however specific phosphorylation sites of SERCA2 have not yet been identified. We hypothesized that a direct phosphorylation of SERCA2 controls SERCA2 activity, Ca2+ homeostasis and subsequent cell death in heart. We combined in silico analysis with in vitro and genetic manipulations to determine potential SERCA2 phosphorylation sites and their effect on Ca2+ homeostasis and cell death in heart. Importantly, we developed an in vivo AAV-myocardial rescue strategy to assess the effect of the SERCA2 phosphorylation event on infarct size and we investigated the potential application of our finding in patient hearts. Our results show for the first time that SERCA2 is a potential substrate for the enzyme GSK3β, whose interaction is improved during an ischemic stress, associated with an increase of the phosphorylation level of SERCA2 at S663 in human hearts. Using a CRISPR/Cas9 approaches, we demonstrated that when SERCA2 cannot be phosphorylated at serine 663, the activity of SERCA2 is significantly increased. As a consequence, we provided evidences that when SERCA2 is resistant to serine-663 phosphorylation, cells are protected cells against reperfusion injury, through a cellular detoxification of calcium into reticulum lumen. As in vivo proof of concept, using AAV-myocardial strategy, phosphoresistant S663 mutant showed an improved excitation-contraction coupling and developed significantly smaller infarct as compared to WT, whereas infarct size was drastically increased in phosphomimetic S663 rescued mice. We conclude that the increased phosphorylation of SERCA2 at serine 663 is a contemporary event of cardiovascular diseases in human hearts, and that avoiding the phosphorylation of SERCA2 at S663 offers a novel therapeutic strategy against acute myocardial infarction, through detoxification of cellular Ca2+ excess into reticulum lumen.

RNAi-Mediated Knockdown of Calreticulin3a Impairs Pollen Tube Growth in Petunia.

Pollen tube growth depends on several complex processes, including exo/endocytosis, cell wall biogenesis, intracellular transport, and cell signaling. Our previous results provided evidence that calreticulin (CRT)—a prominent calcium (Ca2+)-buffering molecular chaperone in the endoplasmic reticulum (ER) lumen—is involved in pollen tube formation and function. We previously cloned and characterized the CRT gene belonging to the CRT1/2 subgroup from Petunia hybrida (PhCRT1/2), and found that post-transcriptional silencing of PhCRT1/2 expression strongly impaired pollen tube growth in vitro. Here, we report cloning of a new PhCRT3a homolog; we identified the full-length cDNA sequence and described its molecular characteristics and phylogenetic relationships to other plant CRT3 genes. Using an RNA interference (RNAi) strategy, we found that knockdown of PhCRT3a gene expression caused numerous defects in the morphology and ultrastructure of cultivated pollen tubes, including disorganization of the actin cytoskeleton and loss of cytoplasmic zonation. Elongation of siPhCRT3a pollen tubes was disrupted, and some of them ruptured. Our present data provide the first evidence that PhCRT3a expression is required for normal pollen tube growth. Thus, we discuss relationships between diverse CRT isoforms in several interdependent processes driving the apical growth of the pollen tube, including actomyosin-dependent cytoplasmic streaming, organelle positioning, vesicle trafficking, and cell wall biogenesis.

Deciphering the Mechanism of Inhibition of SERCA1a by Sarcolipin Using Molecular Simulations.

SERCA1a is an ATPase calcium pump that transports Ca2+ from the cytoplasm to the sarco/endoplasmic reticulum lumen. Sarcolipin (SLN), a transmembrane peptide, regulates the activity of SERCA1a by decreasing its Ca2+ transport rate, but its mechanism of action is still not well-understood. To decipher this mechanism, we have performed normal mode analysis in the all-atom model, with the SERCA1a-SLN complex, or the isolated SERCA1a, embedded in an explicit membrane. The comparison of the results allowed us to provide an explanation at the atomic level for the action of SLN that is in good agreement with experimental observations. In our analyses, the presence of SLN locally perturbs the TM6 transmembrane helix and as a consequence modifies the position of D800, one of the key metal-chelating residues. Additionally, it reduces the flexibility of the gating residues, V304, and E309 in TM4, at the entrance of the Ca2+ binding sites, which would decrease the affinity for Ca2+. Unexpectedly, SLN has also an effect on the ATP binding site more than 35 Å away, due to the straightening of TM5, a long helix considered as the spine of the protein. The straightening of TM5 modifies the structure of the P-N linker that sits above it, and which comprises the 351DKTG354 conserved motif, resulting in an increase of the distance between ATP and the phosphorylation site. As a consequence, the turn-over rate could be affected. All this gives SERCA1a the propensity to go toward a Ca2+ low-affinity E2-like state in the presence of SLN and toward a Ca2+ high-affinity E1-like state in the absence of SLN. In addition to a general mechanism of inhibition of SERCA1a regulatory peptides, this study also provides an insight into the conformational transition between the E2 and E1 states.

Defects in Signal Recognition Particle (SRP) Components Reveal an Essential and Non-Redundant Role for Granule Biogenesis and Differentiation of Neutrophil Granulocytes

Neutrophil granulocyte play pivotal roles in inflammatory responses, immune defence, tissue remodeling, and cancer control. Studying rare patients with defects in differentiation and/or function of neutrophil granulocytes highlights genes and pathways orchestrating these important cellular functions. A previously not appreciated role of the signal recognition particle (SRP) has emerged when monoallelic mutations in SRP54 were associated with congenital neutropenia and pancreatic insufficiency. The eukaryotic SRP is composed of six distinct polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68, SRP72) bound to an RNA molecule (the 7SL RNA). SRP and its receptor (SRPRA and SRPRB) cooperatively transport nascent proteins emerging from ribosome to the endoplasmic reticulum (ER) lumen via a protein-conducting channel. We hypothesized that other constituents of the SRP may be involved in the pathophysiology of congenital neutropenia and that disturbances of the SRP may result in defective proteostasis in neutrophil granulocytes. By virtue of our international Care-for-Rare Alliance we identified three kindred with molecularly unsolved congenital neutropenia. Exome-sequencing revealed a de novo monoallelic mutation in SRPRA in one individual in one family and a homozygous splice-site mutation in SRP19 in five individuals from two families. Clinically, all patients with mutations in SRPRA and SRP19 suffered from various degrees of neutropenia and susceptibility to bacterial infections. Pancreatic insufficiency was only seen in patients with mutation in SRPRA, whereas SRP19 deficiency was not associated with any non-hematopoietic symptoms. To functionally validate these novel genetic variants, we set up an in-vitro neutrophil differentiation system allowing us to model the genetic defects in human SRP complex subunits. We introduced patient-specific mutations (SRPRA Q464E and SRP19 c.189+5G>A) into wildtype iPS cells using CRISPR-Cas9 editing. The engineered cells were differentiated into bona fide neutrophil granulocytes. In vitro generated wildtype neutrophil granulocytes had a high degree of resemblance to primary peripheral blood neutrophil granulocytes, as evidenced by phenotypic (light microscopy, cell surface markers, gene expression) and functional (phagocytosis, bacterial killing, rolling and adhesion, chemotaxis) characteristics. In striking contrast to wildtype iPS cells, both SRPRA and SRP19 mutated iPS cells had a significantly reduced capacity to differentiate into neutrophil granulocytes, as shown by colony-forming unit assays and phenotypic analysis. Furthermore, in vitro generated neutrophil granulocytes had higher susceptibility to apoptosis and evidence of increased unfolded protein responses. We went on to characterize the proteome of primary neutrophil granulocytes from patients with SRP-defects by data-independent proteomics technology. Interestingly, neutrophil granulocytes with defects in SRP constituents shared a significantly reduced abundance of the proteins Pentraxin-3 (PTX3) and Glucuronidase Beta (GUSB). These findings were confirmed in both non-hematopoietic HELA cells and promyelocytic HL60 leukemia cells engineered to express the respective SRP mutants. Upon genetic reconstitution, the aberrant protein expression was re-established. In summary, our studies define novel genetic defects causing congenital neutropenia and highlight the relevance of coordinated protein trafficking pathways and homeostasis for physiological differentiation and function of human neutrophil granulocytes. Disclosures No relevant conflicts of interest to declare.

Effect of heart failure on catecholamine granule morphology and storage in chromaffin cells.

One of the key mechanisms involved in sympathoexcitation in chronic heart failure (HF) is the activation of the adrenal glands. Impact of the elevated catecholamines on the hemodynamic parameters has been previously demonstrated. However, studies linking the structural effects of such overactivation with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not been previously reported. In this study, HF was induced in male Sprague-Dawley rats by ligation of the left coronary artery. Five weeks after surgery, cardiac function was assessed by ventricular hemodynamics. HF rats showed increased adrenal weight and adrenal catecholamine levels (norepinephrine, epinephrine and dopamine) compared with sham-operated rats. Rats with HF demonstrated increased small synaptic and dense core vesicle in splanchnic–adrenal synapses indicating trans-synaptic activation of catecholamine biosynthetic enzymes, increased endoplasmic reticulum and Golgi lumen width to meet the demand of increased catecholamine synthesis and release, and more mitochondria with dilated cristae and glycogen to accommodate for the increased energy demand for the increased biogenesis and exocytosis of catecholamines from the adrenal medulla. These findings suggest that increased trans-synaptic activation of the chromaffin cells within the adrenal medulla may lead to increased catecholamines in the circulation which in turn contributes to the enhanced neurohumoral drive, providing a unique mechanistic insight for enhanced catecholamine levels in plasma commonly observed in chronic HF condition.

Neuroprotection by salubrinal treatment in global cerebral ischemia.

Most common cerebrovascular accidents (CVAs) result from the occlusion of a blood vessel and are called ischemic stroke. The interruption of the blood flow reduces the oxygen and glucose levels in the neural parenchyma, which in turn decreases the energy of the cells and compromises their homeostasis. This condition has to be reverted for cell survival. One of the homeostatic processes impaired by cerebral ischemia is protein folding in the endoplasmic reticulum (ER) lumen where unfolded proteins are accumulated (Degracia and Monti, 2004). This situation, known as ER stress, elicits the so called unfolded protein response (UPR), whereby the cell tries to restore homeostasis to prevent its death. ER stress activates protein sensors that trigger the three different pathways described so far for UPR. Early UPR signaling events are initiated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK). This UPR pathway is characterized by the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α), which blocks most of protein translation and activates the transcription factor 4 (ATF4). This factor regulates the activation of genes that protect the cell against metabolic consequences of ER stress. PERK-ATF4 UPR pathway activation enhances the protein folding capacity of the ER and is essential for reducing ER stress. This pathway is turned off by protein phosphatase 1 (PP1) which dephosphorylates p-eIF2α (reviewed by Godin et al., 2016). The use of the PP1 blocker, salubrinal (Sal), which is able to penetrate into brain tissue in vivo (Sokka et al., 2007), provides a mechanism to maintain activated the PERK-UPR pathway (Ohri et al., 2013).

GW26-e2443 Changes in gene expression of calreticulin in the myocardium of streptozotocin-induced diabetic cardiomyopathy

Calreticulin (CRT) is major Ca2+ -binding protein mainly resident in the endoplasmic reticulum (ER) lumen. Evidences showed it was associated with many cardiovascular diseases. However, whether CRT is involved in the pathogenesis of diabetic cardiomyopathy (DCM) is still unknown. Our aim was to

Inhibition of autophagy promotes caspase-mediated apoptosis by tunicamycin in HepG2 cells

Tunicamycin (TM) causes accumulation of unfolded protein in endoplasmic reticulum (ER) lumen and introduces from elsewhere ER stress. This study was to assess the apoptosis and autophagy effect induced by TM on HepG2 cells and the role of autophagy in the system. The viability of HepG2 cells was significantly inhibited by TM in a dose-dependent manner detected by MTT assay. Then, the apoptotic morphology change, increasing apoptotic cell rate suggested that apoptosis was induced by TM in a time- and dose-dependent manner. To further determine the involvement of caspase-dependent pathway in TM-induced apoptosis, we discover that the activity of caspase-3/7, 8, 9 and cleavage of PARP markedly increased after TM treatment and the apoptosis was effectively attenuated by using caspase-9 and pan caspase inhibitor. Moreover, provided the rising stained acidic vacuoles and an increased level of LC3II and activation of Beclin1, we concluded that autophagy could be triggered by TM in a time- and dose-dependent manner. In addition, the inhibition of autophagy efficiently promoted TM-induced cell death identified by MTT assay. Meanwhile, the apoptotic cell rate and caspase-3 activation increased significantly after autophagy blockage. In conclusion, we found that TM initiated apoptosis and autophagy both in a time- and dose-dependent manner in HepG2 cells; and inhibition of autophagy may promote TM-induced cell death through enhancing apoptosis.

Endoplasmic reticulum stress response in osteogenesis

Various cellular conditions such as synthesis of abundant proteins, expressions of mutant proteins and oxidative stress lead to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen. This type of stress is called ER stress. The excessive ER stress causes cellular damages followed by apoptosis. When ER stress occurs, cells are activated ER stress response (unfolded protein response) to avoid cellular damages. Recently, it has been clear that ER stress response plays crucial roles not only in cell survival after ER stress but also in regulating various cellular functions and tissue formations. In particular, ER stress and ER stress response regulate protein quality control, secretory protein production, and smooth secretion of proteins in the cells such as osteoblasts which synthesize and secrete enormous matrix proteins.

Ultrastructural and antioxidative changes in lupine embryo axes in response to salt stress

Embryo axes of lupine (<em>Lupinus luteus</em> L. ‘Mister’) were subjected to 0.1 M NaCl salt stress for 24 and 48 h. The ultrastructure modification and adjustment of antioxidant enzymes activities and izoenzymes profiles were observed. In cells of lupine embryo axes grown for 48 hours in medium with 0.1 M NaCl mitochondria took the forked shape and bulges of the outer mitochondrial membranes appeared. Moreover, the inflating and swelling of rough endoplasmic reticulum (RER) lumen and fragmentation of RER were noticed. The level of H<sub>2</sub>O<sub>2</sub> was higher in salt treated embryo axes after 24 hours and increase of thiobarbituric acid reactive substances was observed after both 24 and 48 h of salt treatment. Native gel electrophoresis showed increased intensities of bands for catalase isozymes in response to salt stress, whereas activity of catalase was higher only in embryo axes grown for 48 h in control conditions. Appearance of two new isoforms of ascorbate peroxidase was observed after 48 h only under control condition, however increased activities were stated for both control and salt-stress condition after 48 h. No changes in isozymes pattern for superoxide dismutase were observed, but significant decrease in superoxide dismutase activity was noticed in relation to time and salt stress. Possible role of these enzymes in salt stress tolerance is discussed. The 0.1 M salt stress is regarded as a middle stress for lupine embryo axes and the efficiency of stress prevention mechanisms is proposed.

Dislocation of Ricin Toxin A Chains in Human Cells Utilizes Selective Cellular Factors

Ricin is a potent A-B toxin that is transported from the cell surface to the cytosol, where it inactivates ribosomes, leading to cell death. Ricin enters cells via endocytosis, where only a minute number of ricin molecules reach the endoplasmic reticulum (ER) lumen. Subsequently, the ricin A chain traverses the ER bilayer by a process referred to as dislocation or retrograde translocation to gain access to the cytosol. To study the molecular processes of ricin A chain dislocation, we have established, for the first time, a human cell system in which enzymatically attenuated ricin toxin A chains (RTA(E177D) and RTA(Δ177-181)) are expressed in the cell and directed to the ER. Using this human cell-based system, we found that ricin A chains underwent a rapid dislocation event that was quite distinct from the dislocation of a canonical ER soluble misfolded protein, null Hong Kong variant of α(1)-antitrypsin. Remarkably, ricin A chain dislocation occurred via a membrane-integrated intermediate and utilized the ER protein SEL1L for transport across the ER bilayer to inhibit protein synthesis. The data support a model in which ricin A chain dislocation occurs via a novel strategy of utilizing the hydrophobic nature of the ER membrane and selective ER components to gain access to the cytosol.

Abstract SY06-02: Activation of the UPR by c-Myc promotes transformation and tumorigenesis via cytoprotective autophagy

Abstract The Unfolded Protein Response (UPR) is a cellular homeostatic program initiated by an excess of unfolded/misfolded client proteins in the Endoplasmic Reticulum (ER) lumen, with a primarily cytoprotective effect. We have previously shown that tumor cell survival under hypoxic and nutrient deprivation stress is dependent on the ER resident protein and UPR effector PERK since the growth of PERK-null tumors is significantly inhibited in vivo. PERK deficient tumors are unable to tolerate ER stress induced by microenvironmental challenges such as hypoxia, and therefore, the tumors are unable to grow as ER-stress-induced apoptosis is induced. Similar findings have been confirmed for tumors lacking XBP1 (Romero-Ramirez et al). In addition to microenvironmental stresses such as hypoxia, oncogenes are known to activate cellular stress responses, including metabolic stress, apoptosis, DNA damage responses, and cellular senescence. MYC is the target of chromosomal translocation or gene amplification during the development of many human cancers. Most notably, almost every case of Burkitt's lymphoma involves the translocation of the MYC proto-oncogene to the enhancer region of the heavy or light chain immunoglobulin loci resulting in the constitutive overexpression of c-Myc in B lymphocytes. c-Myc expression has been associated with a two-fold increase in both total cellular proteins and the rates of protein synthesis in the Eµ-myc mouse model. This finding raises the possibility that c-Myc-transformed cells are exposed to a higher than normal level of ER stress. Therefore, we hypothesized that the increase in protein burden in cells overexpressing c-Myc corresponds to increased ER stress and activation of the UPR. Using multiple genetic models of regulated c-Myc and N-Myc activation, we demonstrate that Myc activates the PERK/eIF2α/Atf4 arm of the UPR. Activation of UPR leads to increased cell survival via the induction of cytoprotective autophagy as evidenced by upregulated LC3 processing, p62 degradation and increased double-membraned autophagosomes when analyzed by electron microscopy. Ablation of PERK significantly reduced Myc-induced autophagy, cell transformation and tumor formation in nude mice. Samples from Eμ-Myc mice and human lymphomas demonstrate higher levels of UPR activation (eIF2α phosphorylation and XBP1 splicing) compared to corresponding normal tissues and B-cells. Mechanistically, we genetically demonstrate an important link between Myc-dependent increases in protein synthesis and UPR activation. Specifically, by employing a mouse minute, which results in wild-type levels of protein synthesis and attenuation of Myc-induced lymphomagenesis, we show that Myc-induced UPR activation is reversed. Our findings establish a novel role of UPR activation as an enhancer of c-Myc-induced transformation and suggest that inhibition of the UPR may be particularly effective against malignancies characterized by c-Myc overexpression. The identification of cellular stress response pathways activated by c-Myc represents an opportunity to exploit such pathways in the development of novel therapeutics targeting oncogene-dependent malignancies. Supported by NCI grants CA94214 and CA 139362 to C.K. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY06-02. doi:10.1158/1538-7445.AM2011-SY06-02

Abstract 2078: PERK-mediated autophagy promotes Myc-induced transformation and tumorigenesis

Abstract The Unfolded Protein Response (UPR) is a coordinated cellular homeostatic program initiated by the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen, with the ultimate fate of the cell depending on both the intensity and persistence of the stress. We have previously shown that tumor cell survival under hypoxic stress is dependent on the UPR kinase PERK, as the growth of PERK-null tumors is significantly inhibited in vivo. The transcription factor c-Myc, the target of chromosomal translocation and gene amplification during the development of many human cancers, upregulates genes involved in ribosome biogenesis and increases the rate of protein synthesis, therefore, we hypothesized the UPR is activated by c-Myc overexpression. Using in vitro and in vivo models of c-Myc expression, we demonstrate that c-Myc activates PERK-dependent phosphorylation of eIF2α and upregulates the downstream effectors, ATF4 and XBP1s. UPR activation contributes to cell survival during c-Myc induced transformation via the induction of autophagy, as measured by LC3 processing and p62 degradation. Inhibition of PERK dramatically reduces c-Myc induced transformation and tumor growth in nude mice. We are currently developing a lymphoma mouse model to test the strategy of PERK inhibition orthotopically. We have established a role for c-Myc in the cellular response to ER-stress and our results highlight a role for PERK inhibition in the treatment of malignancies characterized by c-Myc overexpression, such as lymphomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2078. doi:10.1158/1538-7445.AM2011-2078

Nuclear inner membrane fusion facilitated by yeast Jem1p is required for spindle pole body fusion but not for the first mitotic nuclear division during yeast mating

During mating of budding yeast, Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. The process of nuclear fusion requires two J proteins, Jem1p in the endoplasmic reticulum (ER) lumen and Sec63p, which forms a complex with Sec71p and Sec72p, in the ER membrane. Zygotes of mutants defective in the functions of Jem1p or Sec63p contain two haploid nuclei that were closely apposed but failed to fuse. Here we analyzed the ultrastructure of nuclei in jem1 Delta and sec71 Delta mutant zygotes using electron microscope with the freeze-substituted fixation method. Three-dimensional reconstitution of nuclear structures from electron microscope serial sections revealed that Jem1p facilitates nuclear inner-membrane fusion and spindle pole body (SPB) fusion while Sec71p facilitates nuclear outer-membrane fusion. Two haploid SPBs that failed to fuse could duplicate, and mitotic nuclear division of the unfused haploid nuclei started in jem1 Delta and sec71 Delta mutant zygotes. This observation suggests that nuclear inner-membrane fusion is required for SPB fusion, but not for SPB duplication in the first mitotic cell division.

A cloned prokaryotic Cd 2+ P-type ATPase increases yeast sensitivity to Cd 2+

CadA, the P1-type ATPase involved in Listeria monocytogenes resistance to Cd 2+, was expressed in Saccharomyces cerevisiae and did just the opposite to what was expected, as it strikingly decreased the Cd 2+ tolerance of these cells. Yeast cells expressing the non-functional mutant Asp 398Ala could grow on selective medium containing up to 100 μM Cd 2+, whereas those expressing the functional protein could not grow in the presence of 1 μM Cd 2+. The CadA–GFP fusion protein was localized in the endoplasmic reticulum membrane, suggesting that yeast hyper-sensitivity was due to Cd 2+ accumulation in the reticulum lumen. CadA is also known to transport Zn 2+, but Zn 2+ did not protect the cells against Cd 2+ poisoning. In the presence of 10 μM Cd 2+, transformed yeasts survived by rapid loss of their expression vector.

Reconstitution of glycopeptide export in mixed detergent-solubilised and resealed microsomes depleted of lumenal components

Export of macromolecules from the endoplasmic reticulum (ER) lumen into the cytosol is a major aspect of the quality control systems operating within the early secretory system. Glycopeptides are exported from the ER by an ATP- and GTP-dependent pathway, which shares many similarities to the protein export system. Significantly, for glycopeptides, there is no requirement for cytosolic factors, biochemically distinguishing the glycopeptide and protein paths and probably reflecting the lower conformational complexity of the former substrate. Genetic studies in yeast, and biochemical data from higher eukaryotes, indicate that glycopeptides utilise the Sec61 translocon. Here, we report a new system allowing access to lumenal ER components, facilitating assessment of their importance in glycopeptide retrotranslocation and potentially other processes. Saponin, in combination with CHAPS, but not saponin alone, facilitated removal of >95% of lumenal protein disulphide isomerase (PDI) and BiP. Upon resealing, these microsomes retained glycopeptide export competence. These data suggest that the majority of lumenal components of the ER are most likely nonessential for glycopeptide export. In addition, export competence was highly sensitive to the addition of external protease, indicating a role for protein factors with cytoplasmically exposed determinants.

Effect of carticaine on the sarcoplasmic reticulum Ca2+-dependent adenosine triphosphatase.

The sarcoplasmic reticulum Ca2+-ATPase (calcium-dependent adenosine triphosphatase) transports Ca2+ from the myoplasm to the reticulum lumen at the expense of free energy from ATP hydrolysis. Carticaine is a local anesthetic of frequent use in dentistry which is now entering other clinical fields. We studied the action of carticaine on the sarcoplasmic reticulum (SR) skeletal muscle Ca2+-ATPase. SR vesicles from rabbit fast skeletal muscle were used. Carticaine inhibits the enzymatic activity. The inhibition of the enzymatic activity depends on pH, [Ca2+] and the presence of calcimycin. Half-maximal carticaine concentration that inhibits the ATPase activity tends to a maximal value upon increasing [Ca2+]. Carticaine concentrations required to inhibit the enzymatic activity at myoplasmic calcium concentration are lower than usual clinical doses: Ki=6.0+/-1.4 mM carticaine (n=5) for 0.1 microM [Ca2+]. ATP-dependent calcium uptake is also inhibited by the local anesthetic: Ki=30.5+/-3.4 mM (n=4). Besides, carticaine inhibits the phosphorylation of the enzyme by inorganic phosphate (Pi): Ki=20.0+/-3.4 (n=5) - 33.2+/-4.6 (n=4) mM, for [Pi] 1-4 mM. Carticaine increases the membrane permeability to Ca2+. Ca2+ efflux from preloaded vesicles is prevented by Ca2+ and Mg2+. Our results suggest that the diffusion of the local anesthetic into muscle fibers might trigger undesired effects such as sustained contraction of the masticatory muscles.

Down-regulation of MHC class I antigen presentation by HCMV; lessons for tumor immunology

MHC class I antigen presentation is a tightly regulated process that begins in the cell cytoplasm with the generation of peptide ligands by the proteasome (1, 2). Peptides are translocated from the cytosol into the endoplasmic reticulum (ER) lumen through the MHC-encoded TAP complex. These peptides are loaded onto the class I heterodimer that is comprised of a 43 kDa type I membrane glycoprotein heavy chain and a 12 kDa soluble light chain (p2m). Shortly after synthesis of the class I heavy chain and &m, the complex is properly folded with assistance from ER resident chaperones such as calnexin, calreticulin and the thiol-reductase, ERp57(3). Tapasin, an ER-resident protein that associates with TAP, can interact with peptide-free class I heterodimerkhaperone complex and guide it to the TAP, where it is subsequently loaded with peptide. The generation of the trimeric class I complex allows its exit out of the ER and through the Golgi to the cell surface. The major histocompatibility complex (MHC) class I molecules present peptides derived from microbial and cellular proteins on the cell surface (4). The interaction of the MHC class I molecules with cytotoxic CD8'T lymphocytes (CTL) can activate an immune response and target the host cell for lysis, thereby eliminating the infected or abnormal cell. Several viruses and tumor cells can down-regulate the surface expression of class I molecuies at the transcriptional and post-translational levels(5, 6). The prevention of class I surface expression allows these cells to escape CTL detection. The mechanism by which tumors down-regulate class I molecules is not clearly understood. However, viral proteins have been identified which interfere with class I antigen presentation and may provide insight into how class I down-regulation is achieved in tumor cells as well.

Getting in is easy, but how to get out?

In yeast, the heat-shock proteins Kar2p (BiP) and Ssa1p play essential roles in the import of nascent proteins into the ER by working together as ratchets or motors in the endoplasmic reticulum (ER) lumen and the cytosol, respectively. Defective imported proteins are scouted out by other ER chaperones such as calnexin (Cne1p) and prepared for export back into the cytosol, where they are degraded in proteasomes. Both the import of nascent proteins into the ER as well as the export of misfolded proteins from the ER require Kar2p and the Sec61p translocation complex, suggesting a mechanistic link between these two processes.To investigate this hypothesis, Brodsky et al.1xThe requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct. Brodsky, J., Werner, E.D., Dubas, M.E., Goeckeler, J.L., Kruse, K.B., and McCracken, A.A. J. Biol. Chem. 1999; 274: 3453–3460Crossref | PubMed | Scopus (195)See all References1 generated mutant alleles of some of the major players involved, namely Cne1p, Kar2p and Ssa1p. By following the degradation of pro-α factor in vitro and A1PiZ in vivo, they could show that mutations in Kar2p inhibit this degradation and that Kar2p is required for export and for the recognition of degradation substrates. By contrast, mutations in Ssa1p had no effect on export or degradation, although they prevented protein import into the ER both in vivo and in vitro, showing that the essential nature of the role for Ssa1p for protein import does not extend to protein export. Finally, they observed a synthetic interaction between kar2 and cne1 at elevated temperatures.The authors suggest that misfolded proteins are captured by Cne1p in the ER lumen but must be handed over to Kar2p for proper re-translocation. Mutations in either chaperone might compromise the release of the substrate and therefore abolish this handover. Taken together, the mechanisms for both the import of nascent polypeptides and for the export of misfolded proteins are distinct, although at least one player (Kar2p) is involved in both processes.