The activated endoplasmic reticulum stress sensor IRE1 oligomerizes into filaments contained in 30 nm membrane tubes of complex topology

The unfolded protein response (UPR) is an intracellular signaling network that adjusts the abundance and protein folding capacity of the endoplasmic reticulum (ER) according to need. The most conversed branch of the UPR is mediated by the ER‐resident transmembrane kinase/endoribonuclease IRE1. It senses unfolded protein accumulation within the ER and transduces the signal via a non‐conventional mRNA splicing mechanism. In response to direct binding of unfolded proteins in the ER lumen, IRE1 activates by oligomerization and accumulates in dynamic foci. IRE1 foci are not autophagosomes as they did not colocalize with the autophagosomal marker LC3. Fluorescence recovery after photobleaching (FRAP) experiments indicate that IRE1 molecules in the foci remain in equilibrium with IRE1 molecules in the surrounding ER network. We determined the structure of IRE1 foci in cells by whole cell correlative light – electron tomography. Our results show that IRE1 oligomers induce membrane deformations, leading to the protrusion of narrow 30 nm ribosome‐free tubes that remain connected to the ER and are twisted into glomeruli of complex topology. The tubes contain two parallel filaments in their lumen, likely representing oligomerized IRE1 ER‐lumenal domains. Taken together, our findings define a previously unrecognized subdomain of the ER membrane and shed new light on the structure and organization of active mammalian IRE1 inside the cell

Experimental study of VEGF immune expression dynamics in the retina using photoinduced BRVO model

Aim. To describe the dynamics of vascular endothelial growth factor (VEGF) immune expression in the retina using the model of photoinduced branch retinal vein occlusion (BRVO) and to establish the terms of neovascularization appearance.Materials and methods. BRVO was modelled on 21 chinchilla rabbits (21 eyes) weighing 1.5‑2 kg (fellow eyes served as controls). Photosensitizer «Fotoditazin» (2.5 mg / kg) was injected intravenously. 15 min later, transpupillary laser irradiation of branch retinal vein near the optic nerve head was performed. Irradiation energy density was 200 J / cm2. Histological analysis and immunohistochemistry of the retina was performed following 30 min, at days 1, 2, 3, 7, 14 and 30.Results. Maximum VEGF accumulation in photoinduced BRVO model was observed on day 2. From day 3, direct neovascularization was confirmed. VEGF levels were stably high throughout the follow-up to the day 30 inclusive.Conclusion. VEGF immune expression in the retina using the model of BRVO induced by photodynamic exposure was explored for the first time. These data can serve as the basis for future studies in order to define optimal anti-VEGF agent, its dosage and terms to manage this condition

The activated endoplasmic reticulum stress sensor IRE1 oligomerizes into filaments contained in 30 nm membrane tubes of complex topology

The unfolded protein response (UPR) is an intracellular signaling network that adjusts the abundance and protein folding capacity of the endoplasmic reticulum (ER) according to need. The most conversed branch of the UPR is mediated by the ER‐resident transmembrane kinase/endoribonuclease IRE1. It senses unfolded protein accumulation within the ER and transduces the signal via a non‐conventional mRNA splicing mechanism. In response to direct binding of unfolded proteins in the ER lumen, IRE1 activates by oligomerization and accumulates in dynamic foci. IRE1 foci are not autophagosomes as they did not colocalize with the autophagosomal marker LC3. Fluorescence recovery after photobleaching (FRAP) experiments indicate that IRE1 molecules in the foci remain in equilibrium with IRE1 molecules in the surrounding ER network. We determined the structure of IRE1 foci in cells by whole cell correlative light – electron tomography. Our results show that IRE1 oligomers induce membrane deformations, leading to the protrusion of narrow 30 nm ribosome‐free tubes that remain connected to the ER and are twisted into glomeruli of complex topology. The tubes contain two parallel filaments in their lumen, likely representing oligomerized IRE1 ER‐lumenal domains. Taken together, our findings define a previously unrecognized subdomain of the ER membrane and shed new light on the structure and organization of active mammalian IRE1 inside the cell

Long-Term Monitoring of Liver Fibrosis and Steatosis in Patients with Chronic Hepatitis C after Achieving a Sustained Virologic Response to Antiviral Therapy

Aim: to analyze the dynamics of fibrosis and steatosis of the liver according to fibroelastometry in patients with chronic hep-atitis C (CHC) after ≥ 6 months from transient elastometry (TE) achieving a sustained virologic response (SVR) to antiviral therapy.Materials and methods. At baseline, a prospective observational study included 628 CHC patients with known stage of liver fibrosis (F) before AVT, some of whom were phased out due to non-compliance with the inclusion criteria. The final analysis included 297 patients who had transient elastometry (TE) data with CAP™ technology on the severity of liver fibrosis (± steatosis) before treatment and after ≥ 6 months after reaching SVR (67 % – interferonfree regimens of therapy). Median follow-up from the moment SVR was confirmed was 3 years [2; 6].Results. At the end of the study, the average age of patients was 49 ± 12 years, of which 53 % were men. In the long-term period after reaching SVR, regression of liver fibrosis was diagnosed in 80 % of cases (including in patients with cirrhosis), and the progression of fibrosis was in 3 % of patient. At the same time, regression of liver steatosis was detected only in 31 % of the patient, worsening of the results was in 23 % (26 % of them had the appearance of steatosis (S) of the liver of 1–3 degrees in persons with no fatty liver before the start of AVT). In the group of patients with liver steatosis, the proportion of men was significantly higher (p = 0.004). Clinically significant stages of fibrosis F3–F4 were significantly more often recorded in patients with hepatic steatosis, both before treatment (46 % S1–S3 and 22 % S0, p < 0.001) and after ≥ 6 months after reaching SVR (19 % S1–S3 and 9 % S0, p = 0.023).Conclusion. In patients with chronic hepatitis C with SVR achieved in the long term, despite a significant regression of liver fibrosis, a high prevalence of hepatic steatosis remains. The data obtained indicate the feasibility of routine diagnosis of both fibrosis and steatosis of the liver in the management of patients with chronic HCV infection before and after successful antiviral therapy

Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs

© 2016, Springer Science+Business Media Dordrecht.Objective: Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment. Results: Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT. Conclusion: Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD50 = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS

Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs

© 2016 Springer Science+Business Media DordrechtObjective: Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment. Results: Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT. Conclusion: Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD50 = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS

Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs

OBJECTIVE: Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment.RESULTS: Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT.CONCLUSION: Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD50 = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS

Direct observation shows superposition and large scale flexibility within cytoplasmic dynein motors moving along microtubules

Cytoplasmic dynein is a dimeric AAA+ motor protein that performs critical roles in eukaryotic cells by moving along microtubules using ATP. Here using cryo-electron microscopy we directly observe the structure of Dictyostelium discoideum dynein dimers on microtubules at near-physiological ATP concentrations. They display remarkable flexibility at a hinge close to the microtubule binding domain (the stalkhead) producing a wide range of head positions. About half the molecules have the two heads separated from one another, with both leading and trailing motors attached to the microtubule. The other half have the two heads and stalks closely superposed in a front-to-back arrangement of the AAA+ rings, suggesting specific contact between the heads. All stalks point towards the microtubule minus end. Mean stalk angles depend on the separation between their stalkheads, which allows estimation of inter-head tension. These findings provide a structural framework for understanding dynein’s directionality and unusual stepping behaviour

MscS-like mechanosensitive channels in plants and microbes

The challenge of osmotic stress is something all living organisms must face as a result of environmental dynamics. Over the past three decades, innovative research and cooperation across disciplines have irrefutably established that cells utilize mechanically gated ion channels to release osmolytes and prevent cell lysis during hypoosmotic stress. Early electrophysiological analysis of the inner membrane of Escherichia coli identified the presence of three distinct mechanosensitive activities. The subsequent discoveries of the genes responsible for two of these activities, the mechanosensitive channels of large (MscL) and small (MscS) conductance, led to the identification of two diverse families of mechanosensitive channels. The latter of these two families, the MscS family, consists of members from bacteria, archaea, fungi, and plants. Genetic and electrophysiological analysis of these family members has provided insight into how organisms use mechanosensitive channels for osmotic regulation in response to changing environmental and developmental circumstances. Furthermore, determining the crystal structure of E. coli MscS and several homologues in several conformational states has contributed to our understanding of the gating mechanisms of these channels. Here we summarize our current knowledge of MscS homologues from all three domains of life and address their structure, proposed physiological functions, electrophysiological behaviors, and topological diversity

Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements

Cilia are multi-functional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multi-motor assemblies with kinesin-II. Combining these data with insights from single-particle electron microscopy, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2’s mechanical “linker” and “stalk” domains within a novel motor-motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2’s recycling activity during IFT