A novel Golgi mannosidase inhibitor: Molecular design,synthesis, enzyme inhibition,and inhibition of spheroid formation

Effective chemotherapy for solid cancers is challenging due to a limitation in permeation that prevents anticancer drugs from reaching the center of the tumor, therefore unable to limit cancer cell growth. To circumvent this issue, we planned to apply the drugs directly at the center by first collapsing the outer structure. For this, we focused on cell–cell communication (CCC) between N-glycans and proteins at the tumor cell surface. Mature N-glycans establish CCC; however, CCC is hindered when numerous immature N-glycans are present at the cell surface. Inhibition of Golgi mannosidases (GMs) results in the transport of immature N-glycans to the cell surface. This can be employed to disrupt CCC. Here, we describe the molecular design and synthesis of an improved GM inhibitor with a non-sugar mimic scaffold that was screened from a compound library. The synthesized compounds were tested for enzyme inhibition ability and inhibition of spheroid formation using cell-based methods. Most of the compounds designed and synthesized exhibited GM inhibition at the cellular level. Of those, AR524 had higher inhibitory activity than a known GM inhibitor, kifunensine. Moreover, AR524 inhibited spheroid formation of human malignant cells at low concentration (10 µM), based on the disruption of CCC by GM inhibition.     

WFS1 and non-syndromic low-frequency sensorineural hearing loss: A novel mutation in a Portuguese case

Low-frequency sensorineural hearing loss (LFSNHL) is an unusual type of HL in which frequencies at 2000 Hz and below are predominantly affected. Most of the families with LFSNHL carry missense mutations in WFS1 gene, coding for wolframin.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Inhibition of palmitate‐induced GADD34 expression augments apoptosis in mouse insulinoma cells (MIN6)

Saturated fatty acids like palmitate induce endoplasmic reticulum (ER) stress in pancreatic beta‐cells, an event linked to apoptotic loss of β‐cells in type 2 diabetes. Sustained activation of the ER stress response leads to expression of growth arrest and DNA damage‐inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1. In the present study, we have used small interfering RNA in order to knockdown GADD34 expression in insulin‐producing MIN6 cells prior to induction of ER stress by palmitate and evaluated its consequences on RNA‐activated protein kinase‐like ER‐localized eIF2alpha kinase (PERK) signalling and apoptosis. Salubrinal, a specific inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, was used as a comparison. Salubrinal treatment augmented palmitate‐induced ER stress and increased GADD34 levels. Both GADD34 knockdown and salubrinal treatment potentiated the cytotoxic effects of palmitate as evidenced by increased DNA fragmentation and activation of caspase 3, with the fundamental difference that the former did not involve enhanced levels of GADD34. The data from this study suggest that sustained activation of PERK signalling and eIF2α phosphorylation sensitizes insulin‐producing MIN6 cells to lipoapoptosis independently of GADD34 expression levels. Copyright © 2014 John Wiley & Sons, Ltd.     

Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions.     

Three-dimensional organization of a transcellular tubulocisternal endoplasmic reticulum in epithelial cells of Reissner's membrane in the guinea-pig

Summary The ultrastructure of the epithelial cells of Reissner's membrane (membrana vestibularis) in the guinea-pig is described following vascular perfusion with glutaraldehyde of live, anaesthetised and artificially respirated animals. Postfixation in a solution containing O_sO₄ and potassium ferricyanide revealed a well-developed tubulocisternal endoplasmic reticulum, not previously described, the continuity of which has been mapped by serial sectioning and reconstruction. Large disc-shaped subsurface cisternae lining the cell membrane, but separated from it by a space approximately 10 nm wide, are in continuity with the smooth endoplasmic reticulum, forming an elaborated transcellular canalicular pathway. This structure is compared to that found in solute-transporting epithelia, e.g., renal proximal tubule, gall bladder, small intestine and choroid plexus. The fixation method used in the present study is compared to other techniques used for preservation of Reissner's membrane. Each epithelial cell of Reissner's membrane is endowed with one kinocilium, one to four multivesicular bodies, and a number of intercalated bodies. The functional significance of the canalicular pathway is discussed.     

Activation of the PDGF β Receptor by a Persistent Artificial Signal Peptide

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF β receptor (PDGFβR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFβR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFβR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFβR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFβR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFβR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.     

Temperature-induced transitions of function and structure in sarcoplasmic reticulum membranes

A transition in the temperature dependences of Ca²⁺ accumulation and ATPase activity occurs at 20 ° C in Sarcoplasmic reticulum membranes. The transition is characterized by an abrupt change in the activation energies for the cation transport process and the associated enzyme activities. The difference in activation energies below and above 20 °C appears to be due to changes in the entropy of activation rather than in the free energy of activation. Also, the temperature dependences of spectral parameters of lipophilic spin-labeled probes and protein-bound spin labels exhibit different behaviors on either side of this temperature. Above 20 °C the lipid matrix probed by the labels exhibits a large increase in molecular motion and a decrease in the apparent ordering of lipid alkyl chains. In addition, labels covalently bound to enzymic reactive sites indicate that the motion of protein side-chains is sensitive to this transition. The results are consistent with an order-disorder transition involving the lipid alkyl chains of the Sarcoplasmic membrane, and with a model in which molecular motion, Ca²⁺ transport and enzyme activity are limited by local viscosity of hydrophobic regions at temperatures below the transition.Another modification of the Sarcoplasmic reticulum membrane occurs between 37 and 40 °C. It appears that at this temperature the processes governing Ca²⁺ accumulation and ATPase activity are uncoupled, and Ca²⁺ accumulation is inhibited, while ATPase activity and passive Ca²⁺ efflux proceed at rapid rates. Parallel transitions of spectroscopic parameters originating from spin labels, covalently bound to the Sarcoplasmic reticulum ATPase, indicate that the uncoupling is due to a thermally-induced protein conformational change.     

M(en)TORship lessons on life and death by the integrated stress response

Cells employ pro-survival and pro-adaptive pathways to cope with different forms of environmental stress. When stress is excessive, and the damage caused by it is unsustainable, cells engage pro-death pathways, which are in place to protect the host from the deleterious effects of harmed cells. Two important pathways that determine the balance between survival and death of stressed cells are the integrated stress response (ISR) and the mammalian target of rapamycin (mTOR), both of which converge at the level of mRNA translation. The two pathways have established avenues of communication to control their activity and determine the fate of stressed cells in a context-dependent manner. The functional interplay between the ISR and mTOR may have significant ramifications in the development and treatment of human diseases such as diabetes, neurodegeneration and cancer.