Cell Secretory Machinery Research Articles

SARS-CoV-2 integral membrane proteins shape the serological responses of patients with COVID-19

SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has elicited a unique mobilization of the scientific community to develop efficient tools to understand and combat the infection. Like other coronavirae, SARS-CoV-2 hijacks host cell secretory machinery to produce viral proteins that compose the nascent virions; including spike (S), envelope (E), and membrane (M) proteins, the most exposed transmembrane proteins to the host immune system. As antibody response is part of the anti-viral immune arsenal, we investigate the immunogenic potential of S, E, and M using a human cell-based system to mimic membrane insertion and N-glycosylation. Both S and M elicit specific Ig production in patients with SARS-CoV-2. Patients with moderate and severe diseases exhibit elevated Ig responses. Finally, reduced Ig binding was observed with spike G614 compared to D614 variant. Altogether, our assay points toward an unexpected immune response against M and represents a powerful tool to test humoral responses against actively evolving SARS-CoV-2 variants and vaccine effectiveness.

Phytonematode peptide effectors exploit a host post-translational trafficking mechanism to the ER using a novel translocation signal.

Cyst nematodes induce a multicellular feeding site within roots called a syncytium. It remains unknown how root cells are primed for incorporation into the developing syncytium. Furthermore, it is unclear how CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptide effectors secreted into the cytoplasm of the initial feeding cell could have an effect on plant cells so distant from where the nematode is feeding as the syncytium expands. Here we describe a novel translocation signal within nematode CLE effectors that is recognized by plant cell secretory machinery to redirect these peptides from the cytoplasm to the apoplast of plant cells. We show that the translocation signal is functionally conserved across CLE effectors identified in nematode species spanning three genera and multiple plant species, operative across plant cell types, and can traffic other unrelated small peptides from the cytoplasm to the apoplast of host cells via a previously unknown post-translational mechanism of endoplasmic reticulum (ER) translocation. Our results uncover a mechanism of effector trafficking that is unprecedented in any plant pathogen to date, andthey illustrate how phytonematodes can deliver effector proteins into host cells and then hijack plant cellular processes for their export back out of the cell to function as external signaling molecules to distant cells.

New Insights on KCa3.1 Channel Modulation.

The human intermediate conductance calcium-activated potassium channel, KCa3.1, is involved in several pathophysiological conditions playing a critical role in cell secretory machinery and calcium signalling. The recent cryo-EM analysis provides new insights for understanding the modulation by both endogenous and pharmacological agents. A typical feature of this channel is the low open probability in saturating calcium concentrations and its modulation by potassium channel openers (KCOs), such as benzo imidazolone 1-EBIO, without changing calcium-dependent activation. In this paper, we proposed a model of KCOs action in the modulation of channel activity. The KCa3.1 channel has a very rich pharmacological profile with several classes of molecules that selectively interact with different binding sites of the channel. Among them, benzo imidazolones can be openers (positive modulators such as 1-EBIO, DC-EBIO) or blockers (negative modulators such as NS1619). Through computation modelling techniques, we identified the 1,4-benzothiazin-3-one as a promising scaffold to develop new KCa3.1 channel modulators. Further studies are needed to explore the potential use of 1-4 benzothiazine- 3-one in KCa3.1 modulation and its pharmacological application.

Exploitation of Drosophila Choriogenesis Process as a Model Cellular System for Assessment of Compound Toxicity: the Phloroglucinol Paradigm

Phloroglucinol (1,3,5 tri-hydroxy-benzene) (PGL), a natural phenolic substance, is a peroxidase inhibitor and has anti-oxidant, anti-diabetic, anti-inflammatory, anti-thrombotic, radio-protective, spasmolytic and anti-cancer activities. PGL, as a medicine, is administered to patients to control the symptoms of irritable bowel syndrome and acute renal colic, in clinical trials. PGL, as a phenolic substance, can cause cytotoxic effects. Administration of PGL up to 300 mg/kg (bw) is well tolerated by animals, while in cell lines its toxicity is developed at concentrations above the dose of 10 μg/ml. Furthermore, it seems that tumor or immortalized cells are more susceptible to the toxic power of PGL, than normal cells. However, studies of its cytotoxic potency, at the cellular level, in complex, differentiated and meta-mitotic biological systems, are still missing. In the present work, we have investigated the toxic activity of PGL in somatic epithelial cells, constituting the follicular compartment of a developing egg-chamber (or, follicle), which directs the choriogenesis (i.e. chorion assembly) process, during late oogenesis of Drosophila melanogaster. Our results reveal that treatment of in vitro growing Drosophila follicles with PGL, at a concentration of 0.2 mM (or, 25.2 μg/ml), does not lead to follicle-cell toxicity, since the protein-synthesis program and developmental pattern of choriogenesis are normally completed. Likewise, the 1 mM dose of PGL was also characterized by lack of toxicity, since the chorionic proteins were physiologically synthesized and the chorion structure appeared unaffected, except for a short developmental delay, being observed. In contrast, concentrations of 10, 20 or 40 mM of PGL unveiled a dose-dependent, increasing, toxic effect, being initiated by interruption of protein synthesis and disassembly of cell-secretory machinery, and, next, followed by fragmentation of the granular endoplasmic reticulum (ER) into vesicles, and formation of autophagic vacuoles. Follicle cells enter into an apoptotic process, with autophagosomes and large vacuoles being formed in the cytoplasm, and nucleus showing protrusions, granular nucleolus and condensed chromatin. PGL, also, proved able to induce disruption of nuclear envelope, activation of nucleus autophagy (nucleophagy) and formation of a syncytium-like pattern being produced by fusion of plasma membranes of two or more individual follicle cells. Altogether, follicle cell-dependent choriogenesis in Drosophila has been herein presented as an excellent, powerful and reliable multi-cellular, differentiated, model biological (animal) system for drug-cytotoxicity assessment, with the versatile compound PGL serving as a characteristic paradigm. In conclusion, PGL is a substance that may act beneficially for a variety of pathological conditions and can be safely used for differentiated somatic -epithelial- cells at clinically low concentrations. At relatively high doses, it could potentially induce apoptotic and autophagic cell death, thus being likely exploited as a therapeutic agent against a number of pathologies, including human malignancies.

Extensive membrane systems at the host-arbuscular mycorrhizal fungus interface.

During arbuscular mycorrhizal (AM) symbiosis, cells within the root cortex develop a matrix-filled apoplastic compartment in which differentiated AM fungal hyphae called arbuscules reside. Development of the compartment occurs rapidly, coincident with intracellular penetration and rapid branching of the fungal hypha, and it requires much of the plant cell's secretory machinery to generate the periarbuscular membrane that delimits the compartment. Despite recent advances, our understanding of the development of the periarbuscular membrane and the transfer of molecules across the symbiotic interface is limited. Here, using electron microscopy and tomography, we reveal that the periarbuscular matrix contains two types of membrane-bound compartments. We propose that one of these arises as a consequence of biogenesis of the periarbuscular membrane and may facilitate movement of molecules between symbiotic partners. Additionally, we show that the arbuscule contains massive arrays of membrane tubules located between the protoplast and the cell wall. We speculate that these tubules may provide the absorptive capacity needed for nutrient assimilation and possibly water absorption to enable rapid hyphal expansion.

Role of Host Cell Secretory Machinery in Zika Virus Life Cycle.

The high human cost of Zika virus infections and the rapid establishment of virus circulation in novel areas, including the United States, present an urgent need for countermeasures against this emerging threat. The development of an effective vaccine against Zika virus may be problematic because of the cross reactivity of the antibodies with other flaviviruses leading to antibody-dependent enhancement of infection. Moreover, rapidly replicating positive strand RNA viruses, including Zika virus, generate large spectrum of mutant genomes (quasi species) every replication round, allowing rapid selection of variants resistant to drugs targeting virus-specific proteins. On the other hand, viruses are ultimate cellular parasites and rely on the host metabolism for every step of their life cycle, thus presenting an opportunity to manipulate host processes as an alternative approach to suppress virus replication and spread. Zika and other flaviviruses critically depend on the cellular secretory pathway, which transfers proteins and membranes from the ER through the Golgi to the plasma membrane, for virion assembly, maturation and release. In this review, we summarize the current knowledge of interactions of Zika and similar arthropod-borne flaviviruses with the cellular secretory machinery with a special emphasis on virus-specific changes of the secretory pathway. Identification of the regulatory networks and effector proteins required to accommodate the trafficking of virions, which represent a highly unusual cargo for the secretory pathway, may open an attractive and virtually untapped reservoir of alternative targets for the development of superior anti-viral drugs.

A novel form of T cell antagonism by trapping of immunological synapse (106.49)

Abstract T cell activation results from productive T cell receptor (TCR) engagement by a cognate peptide-MHC (pMHC) complex on the antigen presenting cell (APC) surface, a process leading to the polarization of the T cell secretory machinery towards the interface of APC. We have previously shown that the half-life of the TCR/pMHC interaction and the density of pMHC on the APC are two parameters determining T cell activation. Here, by using APLs conferring different half-lives to the TCR/pMHC interaction we have tested how this parameter can control T cell polarization. We observed that only TCR/pMHC interactions with intermediate half-lives can promote the assembly of synapses that lead to T cell activation. Strikingly, intermediate half-life interactions can be competed out by short half-life interactions, which can efficiently promote T cell polarization and antagonize T cell activation that was induced by activating intermediate half-life interactions. However, short TCR/pMHC interactions fail at promoting phosphorylation of signaling molecules at the T cell-APC contact interface, which are needed for T cell activation. Our data suggest that while intermediate half-life pMHC ligands promote assembly of activating synapses, this process can be inhibited by short half-life antagonistic pMHC ligands, which promote the assembly of non activating synapses.

Hepatitis C virus targets the T cell secretory machinery as a mechanism of immune evasion

T cell activation and the resultant production of interleukin (IL-2) is a central response of the adaptive immune system to pathogens, such as hepatitis C virus (HCV). HCV uses several mechanisms to evade both the innate and adaptive arms of the immune response. Here we demonstrate that liver biopsy specimens from individuals infected with HCV had significantly lower levels of IL-2 compared with those with other inflammatory liver diseases. Cell culture-grown HCV particles inhibited the production of IL-2 by normal peripheral blood mononuclear cells, as did serum from HCV-infected patients. This process was mediated by the interaction of HCV envelope protein E2 with tetraspanin CD81 coreceptor. HCV E2 attenuated IL-2 production at the level of secretion and not transcription by targeting the translocation of protein kinase C beta (PKCβ), which is essential for IL-2 secretion, to lipid raft microdomains. The lipid raft disruptor methyl-β-cyclodextrin reversed HCV E2-mediated inhibition of IL-2 secretion, but not in the presence of a PKCβ-selective inhibitor. HCV E2 further inhibited the secretion of other cytokines, including interferon-γ. These data suggest that HCV E2-mediated disruption of the association of PKCβ with the cellular secretory machinery represents a novel mechanism for HCV to evade the human immune response and to establish persistent infection.

Activation of the Ancestral Polarity Regulator Protein Kinase Cζ at the Immunological Synapse Drives Polarization of Th Cell Secretory Machinery toward APCs

A key feature in T lymphocyte biology is that Th cells rapidly polarize their secretory machinery toward cognate APCs. The molecular mechanisms of these dynamic Th cell responses and their impact on APC biology remain to be elucidated. In this study, we demonstrate that protein kinase Czeta (PKCzeta) is rapidly activated at the immunological synapse (IS) in human Th cells interacting with cognate dendritic cells (DCs) and that a functional PKCzeta is required for the polarization of Th cell secretory machinery toward DCs. We also show that PKCzeta-dependent Th cell polarization allows dedicated delivery of IFN-gamma and CD40L at the IS and is required for the activation of cognate DCs to IL-12 production. PKCzeta synaptic activation is a low-threshold phenomenon and, in Th cells interacting with multiple DCs, selectively occurs at the IS formed with the DCs offering the strongest stimulus leading to dedicated Th cell polarization. Our results identify the PKCzeta signaling pathway as a key component of the Th cell polarization machinery and provide a molecular basis for T cell-dedicated activation of cognate DCs.

ATP-gated P2X 3 receptors constitute a positive autocrine signal for insulin release in the human pancreatic β cell

Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human beta cell's sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i), and hormone release in vitro, we show that human beta cells express ionotropic ATP receptors of the P2X(3) type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X(3) receptors in the beta-cell plasma membrane, resulting in increased [Ca(2+)](i) and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the beta cell's secretory machinery. This may explain how the human pancreatic beta cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.

Force Microscopy: Applications in Biology and Medicine. Edited by Bhanu P. Jena and J.K. Heinrich Hörber

Force Microscopy: Applications in Biology and Medicine. Edited by Bhanu P. Jena and J.K. Heinrich Hörber. John Wiley & Sons, Inc., Hoboken, NJ; 2006, 300 pages. ISBN 978-0-471-39628-4The excitement of man's imagination and discovery by the scope of imaging tools, whether tele- or micro-, from 1015 to 10−15 m, ranges from the limits of the known universe to the atomic level. Light and electron microscopy opened vistas of biology and medicine with discovery of the unit of life, the cell and viruses. In the mid-1980s development of scanning probe microscopy allowed studies on live cells at the near-nanometer level. Thus, results from studies using the atomic force microscope (AFM) have given birth to a new field, “NanoCellBiology”, by providing investigators a three-dimensional view of the structure and dynamics of live cells and biomolecules at near-angstrom resolution. Applications of force microscopy to the study of physiological processes of living cells combined with biochemical and molecular biology are yielding rapid advances on how normal and diseased cells function. This exciting book contains 15 chapters by invited experts that examine cell secretory machinery and the discovery of a new cellular structure—the porosome—involved in cell secretion, properties of microbial and cell plant surfaces, cellular interactions of nanodrug delivery systems, intermolecular forces and avidity modulation of leukocyte adhesion molecules, micromechanical properties of lipid bilayers and vesicles, imaging of soft surfaces, high-speed action of biomolecules in motion, cytogenetic applications, and the study of macromolecular interactions in hemostasis and thrombosis.

A novel granular cell type of locust Malpighian tubules: ultrastructural and immunocytochemical study

A novel secretory cell type in the initial segment of the Malpighian tubules of the locusts Schistocerca gregaria and Locusta migratoria is described ultrastructurally and studied by means of immunocytochemical techniques. The cells show abundant rough endoplasmic reticulum with interspersed Golgi zones. The richness of the cell secretory machinery and the presence of apical dense plemorphic granules suggest a role in secretion of proteinaceous material to the tubule lumen. The surprising finding of ACTH (1–24)-, α-MSH-, and 7B2-like immunoreactivity for this cell is discussed.

One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coli secretion machinery.

The E. coli secE (prlG) gene codes for an integral cytoplasmic membrane protein which is part of the cell's secretory machinery. A deletion of nearly the entire gene renders the cell dependent on the presence of a complementing secE+ plasmid, indicating that the SecE protein is essential for growth. Deletions which remove carboxy-terminal sequences or substantial amounts near the amino-terminus of SecE can still complement the lethal deletion. This deletion analysis suggests that the essential domain of the SecE protein includes only a single one of its three hydrophobic membrane-spanning segments. Two of three dominant prlG signal sequence suppressors map to this segment. Consistent with the insensitivity of SecE to major structural changes, several cold-sensitive mutations cause lethality not because of any change in the protein, but because of a reduction in its level of expression. Our results suggest that higher levels of the protein are needed at the lower temperature. These findings are discussed in terms of the interactions between various components of the secretory machinery.

Rapid and reversible secretion changes during uncoupling of rat insulin-producing cells.

To determine whether insulin secretion is affected by a blockage of gap junctions between B cells, we have studied the secretion of rat pancreatic islets of Langerhans, primary dispersed islet cells, and cells of the RINm5F line, during short-term exposure to heptanol. Within minutes, this alkanol blocked gap junctions between the B cells of intact islets and abolished their normal secretory response to glucose. These two changes were rapidly and fully reversible after return of the islets to control medium. We further found that heptanol had no significant effect on the glucose-stimulated secretion of single B cells but inhibited that of B cell pairs. In the clone of RINm5F cells, whose junctional coupling and D-glyceraldehyde-induced stimulation of insulin release by aggregated cells were also inhibited by heptanol, this alkanol did not perturb intracellular pH and Ca2+ and the most distal steps of the secretion pathway. In summary, a gap junction blocker affected the secretion of insulin-producing cells by a mechanism which is dependent on cell contact and is not associated with detectable pleiotropic perturbations of the cell secretory machinery. The data provide evidence for the involvement of junctional coupling in the control of insulin secretion.

Dynamics of foreign protein secretion from Saccharomyces cerevisiae

A mathematical model describing the dynamics of foreign protein secretion from yeast cells is developed. The secretion events, which are a series of complicated enzymatic reactions and carrier-involved transport, are lumped to a practically applicable model structure, based on the major interactions between the heterologous polypeptides and the host cell's secretory machinery through the pathway. The developed model structure predicts that the secretion rate constant is represented as a saturated form with respect to the host cell's specific growth rate. The validity of the proposed model structure is tested by generating dynamic response data to a step input of cycloheximide. The model system used in the experiment is SEY2102-s21, which has an integrated copy of a yeast secretion-mutant invertase that simulates well typical gene cassettes designed to secrete mature foreign proteins utilizing the yeast cell's secretion signals. Protein quantification is done by gel electrophoresis followed by immuno-blot on nitrocellulose filters and subsequent scanning with a reflectance densitometer. Experimental data confirm the proposed model structure.

The role of hydroxylation in the secretion of collagen by mouse fibroblasts in culture

The secretion of collagen may be specifically and quantitatively measured in a cell culture system using mouse fibroblasts, line 3T6. The results of such a study show that inhibition of the hydroxylation of collagen with α,α′-dipyridyl reduces the rate of secretion to 27% of the rate of normally hydroxylated collagen. Dipyridyl does not hinder passage of the normally hydroxylated collagen species to the medium. Therefore its effect on the secretion of under-hydroxylated collagen (protocollagen) is due to its inhibition of hydroxylation and not to an inhibition of the cell's secretory machinery.