Use of an innovative system and nanotechnology-based strategy for therapeutic applications of Gla-rich protein (GRP)

Introduction: Gla-rich protein (GRP) is a vitamin K-dependent protein (VKDP) acting as a calcification inhibitor and anti-inflammatory agent in cardiovascular and articular systems, and THP1 monocyte/macrophage cells [1,2]. Calcification and inflammation processes are known to be involved in the etiology of several calcification-related chronic inflammatory diseases such as atherosclerosis, CKD and osteoarthritis, in a complex bi-directional interplay that drives disease progression. Here, we developed an innovative system to produce human c-carboxylated GRP (cGRP), and a nanotechnology strategy based on GRP loading into extracellular vesicles (EVs) as a gold standard delivery system for GRP in therapeutic applications. Materials and methods: Human GRP protein was co-expressed with c-carboxylase enzyme (GGCX), vitamin K oxidoreductase (GGCX) and furin, in the insect cell baculovirus system in the presence of vitamin K. GRP released in the cell culture media was characterized by mass spectrometry based techniques and Western blot analysis. EVs released by the insect cells overexpressing GRP were isolated by ultracentrifugation, and characterized for GRP content through TEM-immunogold staining, Western blot, ELISA, qPCR. Functional assays using isolated EVs containing GRP were performed in primary vascular smooth muscle cells (VSMCs) and THP1 monocyte/macrophage cells, for anti-mineralizing and anti-inflammatory screening.Results: GRP released in the cell culture media when co-expressed with GGCX, VKOR and furin in the presence of vitamin K, is processed at the pro-peptide and contain Gla residues. EVs released by the insect cells in this system were shown to be loaded with GRP protein and mRNA, and capable of reducing ECM calcium deposition of calcifying VSMCs and the production of TNFa in THP1 monocyte/macrophage cells stimulated with LPS. Discussion and conclusions: While the successful production of human cGRP constitutes a major achievement, this innovative methodology will open new opportunities for the production of other biological active VKDPs. Furthermore, EVs loaded with GRP were shown to have anti-mineralizing and anti-inflammatory properties, with promising therapeutic potentialities for calcification-related chronic inflammatory diseases.Portuguese Foundation for Science and Technology (EU/PID1003201)info:eu-repo/semantics/publishedVersio

Barnase as a New Therapeutic Agent Triggering Apoptosis in Human Cancer Cells

RNases are currently studied as non-mutagenic alternatives to the harmful DNA-damaging anticancer drugs commonly used in clinical practice. Many mammalian RNases are not potent toxins due to the strong inhibition by ribonuclease inhibitor (RI) presented in the cytoplasm of mammalian cells.In search of new effective anticancer RNases we studied the effects of barnase, a ribonuclease from Bacillus amyloliquefaciens, on human cancer cells. We found that barnase is resistant to RI. In MTT cell viability assay, barnase was cytotoxic to human carcinoma cell lines with half-inhibitory concentrations (IC(50)) ranging from 0.2 to 13 microM and to leukemia cell lines with IC(50) values ranging from 2.4 to 82 microM. Also, we characterized the cytotoxic effects of barnase-based immunoRNase scFv 4D5-dibarnase, which consists of two barnase molecules serially fused to the single-chain variable fragment (scFv) of humanized antibody 4D5 that recognizes the extracellular domain of cancer marker HER2. The scFv 4D5-dibarnase specifically bound to HER2-positive cells and was internalized via receptor-mediated endocytosis. The intracellular localization of internalized scFv 4D5-dibarnase was determined by electronic microscopy. The cytotoxic effect of scFv 4D5-dibarnase on HER2-positive human ovarian carcinoma SKOV-3 cells (IC(50) = 1.8 nM) was three orders of magnitude greater than that of barnase alone. Both barnase and scFv 4D5-dibarnase induced apoptosis in SKOV-3 cells accompanied by internucleosomal chromatin fragmentation, membrane blebbing, the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, and the activation of caspase-3.These results demonstrate that barnase is a potent toxic agent for targeting to cancer cells

Gene gymnastics:Synthetic biology for baculovirus expression vector system engineering

International audienceMost essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach

BIN1 modulation in vivo rescues dynamin-related myopathy

International audienceThe mechanoenzyme dynamin 2 (DNM2) is crucial for intracellular organization and trafficking. DNM2 is mutated in dominant centronuclear myopathy (DNM2-CNM), a muscle disease characterized by defects in organelle positioning in myofibers. It remains unclear how the in vivo functions of DNM2 are regulated in muscle. Moreover, there is no therapy for DNM2-CNM to date. Here, we overexpressed human amphiphysin 2 (BIN1), a membrane remodeling protein mutated in other CNM forms, in Dnm2 RW/+ and Dnm2 RW/RW mice modeling mild and severe DNM2-CNM, through transgenesis or with adeno-associated virus (AAV). Increasing BIN1 improved muscle atrophy and main histopathological features of Dnm2 RW/+ mice and rescued the perinatal lethality and survival of Dnm2 RW/RW mice. In vitro experiments showed that BIN1 binds and recruits DNM2 to membrane tubules, and that the BIN1-DNM2 complex regulates tubules fission. Overall, BIN1 is a potential therapeutic target for dominant centronuclear myopathy linked to DNM2 mutations

A New Glycogen Storage Disease Caused by a Dominant PYGM Mutation

International audienceObjective: Glycogen storage diseases (GSDs) are severe human disorders resulting from abnormal glucose metabolism, and all previously described GSDs segregate as autosomal recessive or X-linked traits. In this study, we aimed to molecularly characterize the first family with a dominant GSD. Methods: We describe a dominant GSD family with 13 affected members presenting with adult-onset muscle weakness, and we provide clinical, metabolic, histological, and ultrastructural data. We performed exome sequencing to uncover the causative gene, and functional experiments in the cell model and on recombinant proteins to investigate the pathogenic effect of the identified mutation. Results: We identified a heterozygous missense mutation in PYGM segregating with the disease in the family. PYGM codes for myophosphorylase, the enzyme catalyzing the initial step of glycogen breakdown. Enzymatic tests revealed that the PYGM mutation impairs the AMP-independent myophosphorylase activity, whereas the AMP-dependent activity was preserved. Further functional investigations demonstrated an altered conformation and aggregation of mutant myophosphorylase, and the concurrent accumulation of the intermediate filament desmin in the myofibers of the patients. Interpretation: Overall, this study describes the first example of a dominant glycogen storage disease in humans, and elucidates the underlying pathomechanisms by deciphering the sequence of events from the PYGM mutation to the accumulation of glycogen in the muscle fibers. ANN NEUROL 2020;88:274–282

Binding and internalization of scFv 4D5-dibarnase in BT-474 cells visualized by confocal microscopy.

<p>(A) Cells were incubated with scFv 4D5-dibarnase at 4°C or (B) at 37°C. The scFv 4D5-dibarnase was detected with rabbit anti-barnase antiserum followed by GAR-PE. Fluorescence was observed predominantly on the surface of cells incubated at 4°C and inside the cells incubated at 37°C. This difference in the localization of the fluorescent label suggests internalization of scFv 4D5-dibarnase at 37°C in BT-474 cells.</p

Effects of recombinant proteins on cell viability as determined by MTT assay.

<p>(A) The effects of barnase and scFv 4D5-dibarnase on the viability of human cancer and normal cells. SKOV-3 cells were treated for 72 h with barnase (long dashed line) or scFv 4D5-dibarnase (solid line), and hPBMCs were treated with barnase (short dashed line) or scFv 4D5-dibarnase (dashed-dotted line). (B) The competitive inhibition of scFv 4D5-dibarnase cytotoxicity by scFv 4D5. SKOV-3 cells were treated for 72 h with scFv 4D5-dibarnase in the absence (black circles) or presence (white triangles) of 300 nM scFv 4D5 or with scFv 4D5 alone (white squares). (C) The inhibition of barnase cytotoxicity and scFv 4D5-dibarnase cytotoxicity by barstar. SKOV-3 cells were treated for 72 h with barnase (white circles), barnase and equimolar amounts of barstar (white triangles), scFv 4D5-dibarnase (black circles), scFv 4D5-dibarnase with three-fold molar excess of barstar (black triangles), or barstar alone (black squares). (D) The effects of hRI on the cytotoxicity of scFv 4D5-dibarnase. SKOV-3 cells were treated for 72 h with either scFv 4D5-dibarnase in the absence of hRI (black circles), scFv 4D5-dibarnase in the presence of hRI (white diamonds), or hRI alone (black diamonds). Cell viability is expressed as the percentage of the metabolic activity of treated cells with respect to untreated cells (crosshair). Each regression curve in panel A (with 95% confidence intervals indicated by dotted lines) represents at least three independent experiments. Sigmoid regression was performed with SigmaPlot software. Curves in B–D represent typical experiments. Error bars (B–D) were obtained from triplicate measurements.</p

Ribonuclease activity assay.

<p>(A) The ribonuclease activities of barnase (dashed line and diamonds) and scFv 4D5-dibarnase (dotted line and circles) were determined according to the method of Rushizky et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#pone.0002434-Rushizky1" target="_blank">[58]</a>. The x-axis represents the concentration of barnase alone or the half-concentration of scFv 4D5-dibarnase. The absorbance of 0.5 AU₂₆₀ corresponds to the activity of 2 nM native barnase as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#pone.0002434-Hartley2" target="_blank">[27]</a>. (B) Susceptibility of barnase to hRI (solid line and circles) and of scFv 4D5-dibarnase to barstar (dashed line and triangles). Data are means±SD of triplicate determinations; the curves are the results of sigmoid regression performed with SigmaPlot software.</p

Cellular RNA undergoes degradation in SKOV-3 cells treated with barnase.

<p>SKOV-3 cells were exposed to 50 µM barnase for 24 h (lane 3) or 48 h (lane 4). Total RNA was isolated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002434#s4" target="_blank">Materials and Methods</a> and analyzed on a 9% polyacrylamide gel containing 7.5 M urea. Each sample lane was loaded with RNA from 2×10⁵ treated (+) or untreated (−) cells. Lane 2 corresponds to mock-treated control. The positions of the RNA molecular weight standards (lane 1) are shown as the number of bases to the left of panel. Asterisks indicate the most prominent bands that appear as a result of enzymatic cleavage of high molecular weight rRNA by barnase (lane 3).</p