Hypoxia-induced autophagy: a new player in cancer immunotherapy?

A major challenge in formulating an effective immunotherapy is to overcome the mechanisms of tumor escape from immunosurveillance. We showed that hypoxia-induced autophagy impairs cytotoxic T-lymphocyte (CTL)-mediated tumor cell lysis by regulating phospho-STAT3 in target cells. Autophagy inhibition in hypoxic cells decreases phospho-STAT3 and restores CTL-mediated tumor cell killing by a mechanism involving the ubiquitin proteasome system and SQSTM1/p62. Simultaneously boosting the CTL-response, using a TRP-peptide vaccination strategy, and targeting autophagy in hypoxic tumors, improves the efficacy of cancer vaccines and promotes tumor regression in vivo. Overall, in addition to its immunosuppressive effect, the hypoxic microenvironment also contributes to immunoresistance and can be detrimental to antitumor effector cell functions.     

Epigenetic Activity of Peroxisome Proliferator-Activated Receptor Gamma Agonists Increases the Anticancer Effect of Histone Deacetylase Inhibitors on Multiple Myeloma Cells

Epigenetic modifications play a major role in the development of multiple myeloma. We have previously reported that the PPARγ agonist pioglitazone (PIO) enhances, in-vitro, the cytotoxic effect of the Histone deacetylase inhibitor (HDACi), valproic acid (VPA), on multiple myeloma cells. Here, we described the development of a new multiple myeloma mouse model using MOLP8 cells, in order to evaluate the effect of VPA/PIO combination on the progression of myeloma cells, by analyzing the proliferation of bone marrow plasma cells. We showed that VPA/PIO delays the progression of the disease and the invasion of myeloma cells in the bone marrow. Mechanistically, we demonstrated that VPA/PIO increases the cleavage of caspase 3 and PARP, and induces the acetylation of Histone 3 (H3). Furthermore, we provided evidence that PPARγ agonist is able to enhance the action of other HDACi such as Vorinostat or Mocetinostat. Using PPARγ antagonist or siPPARγ, we strongly suggest that, as described during adipogenesis, PIO behaves as an epigenetic regulator by improving the activity of HDACi. This study highlights the therapeutic benefit of PIO/VPA combination, compared to VPA treatment as a single-arm therapy on multiple myeloma and further highlights that such combination may constitute a new promising treatment strategy which should be supported by clinical trials.     

Resistance of tumor cells to cytolytic T lymphocytes involves Rho-GTPases and focal adhesion kinase activation

Tumor cells evade adaptive immunity by a variety of mechanisms, including selection of variants that are resistant to specific cytotoxic T lymphocyte (CTL) pressure. Recently, we have reported that the reorganization of the actin cytoskeleton can be used by tumor cells as a strategy to promote their resistance to CTL-mediated lysis. In this study, we further examined the functional features of a CTL-resistant tumor variant and investigated the relationship between cytoskeleton alteration, the acquisition of tumor resistance to CTL-induced cell death, Rho-GTPases, and focal adhesion kinase (FAK) pathways. Our data indicate that although the resistant cells do not display an increased migratory potential, an alteration of adhesion to the extracellular matrix was observed. When Rho-GTPases were activated in cells by the bacterial CNF1 (cytotoxic necrotizing factor 1), striking changes in the cell morphology, including actin cytoskeleton, focal adhesions, and membrane extensions, were observed. More importantly, such activation also resulted in a significant attenuation of resistance to CTL-induced cell death. Furthermore, we demonstrate that FAK signaling pathways were constitutively defective in the resistant cells. Silencing of FAK in the sensitive target cells resulted in the inhibition of immune synapse formation with specific CTLs and their subsequent lysis. Expression of the FAK mutant (Y397F) resulted in an inhibition of IGR-Heu cell adhesion and of their susceptibility to specific lysis. These results suggest that FAK activation plays a role in the control of tumor cell susceptibility to CTL-mediated lysis.     

Fusion of agarase and neoagarobiose hydrolase for mono-sugar production from agar

In enzymatic saccharification of agar, endo- and exo-agarases together with neoagarobiose hydrolase (NABH) are important key enzymes for the sequential hydrolysis reactions. In this study, a bifunctional endo/exo-agarase was fused with NABH for production of mono-sugars (d-galactose and 3,6-anhydro-l-galactose) from agar using only one fusion enzyme. Two fusion enzymes with either bifunctional agarase (Sco3476) or NABH (Zg4663) at the N-terminus, Sco3476–Zg4663 (SZ) and Zg4663–Sco3476 (ZS), were constructed. Both fusion enzymes exhibited their optimal agarase and NABH activities at 40 and 35 °C, respectively. Fusions SZ and ZS enhanced the thermostability of the NABH activity, while only fusion SZ showed a slight enhancement in the NABH catalytic efficiency (K _cat/K _M) from 14.8 (mg/mL)⁻¹ s⁻¹ to 15.8 (mg/mL)⁻¹ s⁻¹. Saccharification of agar using fusion SZ resulted in 2-fold higher mono-sugar production and 3-fold lower neoagarobiose accumulation when compared to the physical mixture of Sco3476 and Zg4663. Therefore, this fusion has the potential to reduce enzyme production cost, decrease intermediate accumulation, and increase mono-sugar yield in agar saccharification.

     

Genetic Diversity of Fusarium oxysporum f.sp. lentis Population Affecting Lentil in Syria

Lentil (Lens culinaris Medik.) is an important food legume crop in Syria. Fusarium wilt (Fusarium oxysporum f.sp. lentis – Fol) is a key yield‐limiting factor in the country. The genetic diversity of Fol population was studied using 96 isolates collected from different parts of the country using molecular markers. A total of 16 markers, random amplified polymorphic DNA, simple sequence repeats and inter‐simple sequence repeats were used and 218 polymorphic markers (scorable bands) were obtained. Cluster and structure analyses grouped the isolates into three major groups and subgroups indicating high genetic diversity in the pathogen populations. The molecular variance within the population accounted 87% of the total variation indicating high diversity within population than among geographic locations. The result of this study showed that no alleles were linked to specific province, and therefore, screening for the Fusarium wilt in one location using virulent isolates could be enough to save time and resources.     

Synthesis of an iron(II)-braced proline-II tripod protein

Summary This paper describes the engineering of braced tripod proteins for use as molecular frameworks. Specifically, a 30-residue tripod-shaped protein with three proline-II helical legs braced by an iron(II)tris(bipyridine) complex was modularly designed, chemically synthesized, and biophysically characterized. Three copies of a 10-residue leg peptide were covalently linked through sulfide bonds to an N-terminal apex (1,3,5-tris(methylene)benzene) and by amide bonds to the brace (Fe^II (Mbc)₃: Mbc is 4′-methyl-2,2′-bipyridine-4-carbonyl). The leg peptide (H-Cys-Pro₅-Pra(Mbc)-Pro₃-NH₂: Pra iscis-4-amino-l-proline) was assembled by the solid-phase method using Boc-Pra(Mbc)-OH, which was synthesized in 75% overall yield by coupling Mbc-OH to the 4-amino group of Boc-Pra-OCH₃ and saponifying the methyl ester group. The iron(II)-braced tripod was assembled by S-alkylation of three copies of the leg peptide with 1,3,5-tris(bromomethyl)benzene followed by ligation of Fe²⁺ to the resulting unbraced tripod. The CD spectrum of the iron(II)-braced tripod showed a positive MLCT band at 570 nm and a negative π-π^* band at 312 nm, so its Fe^II(Mbc)₃ brace was predominantly in the Δ configuration. In a mostly acetonitrile solution at 25°C, the leg peptide and the unbraced tripod isomerized from the proline-II helical form into the proline-I helical form but the iron(II)-braced tripod remained in the proline-II helical form.     

Nitration of PECAM-1 ITIM tyrosines abrogates phosphorylation and SHP-2 binding

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a cell adhesion molecule with a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when phosphorylated, binds Src homology 2 domain-containing protein-tyrosine phosphatase (SHP-2). PECAM-1 is expressed at endothelial cell junctions where exposure to inflammatory intermediates may result in post-translational amino acid modifications that affect protein structure and function. Reactive nitrogen species (RNS), which are produced at sites of inflammation, nitrate tyrosine residues, and several proteins modified by tyrosine nitration have been found in diseased tissue. We show here that the RNS, peroxynitrite, induced nitration of both full-length cellular PECAM-1 and a purified recombinant PECAM-1 cytoplasmic domain. Mass spectrometric analysis of tryptic fragments revealed quantitative nitration of ITIM tyrosine 686. A synthetic peptide containing 3-nitrotyrosine at position 686 could not be phosphorylated nor bind SHP-2. These data suggest that ITIM tyrosine nitration may represent a mechanism for modulating phosphotyrosine-dependent signal transduction pathways.