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BackgroundAmong sarcomas, which are rare cancers with an incidence of Cancers 2021; ClinicalTrials.gov Identifier: NCT02793050).MethodsA post-hoc analysis of case series to evaluate the efficacy and safety of trabectedin on patients with ultra-rare and other rare translocation-related sarcomas included in TrObs study was performed. Main outcomes comprised investigator-assessed overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and safety.ResultsThirty-six patients (18 women) with ultra-rare and other rare sarcoma and a median age of 53.0 years (range: 22-81) were included. Most patients had solitary fibrous tumor (SFT; n=11) followed by epithelioid sarcoma (n=5), malignant peripheral nerve sheath tumor (MPNST; n=4), extraskeletal myxoid chondrosarcoma (EMC; n=3), desmoplastic small round cell tumor (DSRCT; n=3), and alveolar soft part sarcoma (ASPS), rhabdomyosarcoma and clear cell sarcoma (n=2 each). Thirty-five patients had metastatic disease and 23 patients received trabectedin as a second-line treatment. Among 35 patients evaluable for response, two patients with SFT and ASPS had a partial response and one patient with DSRCT obtained a complete response, reaching an ORR of 8.6% (95% CI: 2.8-23.4%). Among patients with an ORR, 6-months PFS was 100% in patients with ASPS, 45.7% in patients with SFT and 33.3% in those with DSRCT. Two patients with epithelioid sarcoma and myoepithelioma had disease stabilization lasting >24 months. Nine patients had at least one grade 3/4 adverse event, mostly being bone marrow toxicity (n=6).ConclusionsTrabectedin has some anti-tumor activity in some ultra-rare and other rare sarcomas, particularly translocation-related sarcomas, with the well-known manageable safety profile.

International audience; Thioredoxins (TRXs) are ubiquitous disulfide oxidoreductases structured according to a highly conserved fold. TRXs are involved in a myriad of different processes through a common chemical mechanism. Plant TRXs evolved into seven types with diverse subcellular localization and distinct protein target selectivity. Five TRX types coexist in the chloroplast, with yet scarcely described specificities. We solved the crystal structure of a chloroplastic z-type TRX, revealing a conserved TRX fold with an original electrostatic surface potential surrounding the redox site. This recognition surface is distinct from all other known TRX types from plant and non-plant sources and is exclusively conserved in plant z-type TRXs. We show that this electronegative surface endows thioredoxin z (TRXz) with a capacity to activate the photosynthetic Calvin–Benson cycle enzyme phosphoribulokinase. The distinct electronegative surface of TRXz thereby extends the repertoire of TRX–target recognitions.

Oxygenic phototrophs use the Calvin–Benson cycle to fix CO2 during photosynthesis. In the dark, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), two enzymes of the Calvin–Benson cycle, form an inactive complex with the regulatory protein CP12, mainly under the control of thioredoxins and pyridine nucleotides. In the light, complex dissociation allows GAPDH and PRK reactivation. The GAPDH/CP12/PRK complex is conserved from cyanobacteria to angiosperms and coexists in land plants with an autoassembling GAPDH that is analogously regulated. With the recently described 3D structures of PRK and GAPDH/CP12/PRK, the structural proteome of this ubiquitous regulatory system has been completed. This outcome opens a new avenue for understanding the regulatory potential of photosynthetic carbon fixation by laying the foundation for its knowledge-based manipulation.

Background Soft tissue sarcomas (STS) are a rare group of solid neoplasm including among others liposarcoma, leiomyosarcoma (L-sarcoma) and undifferentiated pleomorphic sarcoma (UPS) entities. The current first-line treatment is represented by anthracycline based- regimens, second-line may include trabectedin. Currently the activity of trabectedin and its mechanism of action is not completely elucidated. Methods Taking the advantages of our 3D patient-derived primary culture translational model we performed genomic-, chemobiogram, proteomic- and in vivo analysis in a UPS culture (S1). Furthermore pharmacological profiling of a UPS and L-sarcoma patient-derived case series and in silico analysis were carried out. Results Trabectedin exhibited an increased activity in 3D respect to 2D cultures suggesting an extracellular matrix (ECM) and timp1 involvement in its mechanism of action. Moreover 3D S1 xenotranspanted zebrafish model showed an increased sensitivity to trabectedin. Finally the results were further validated in a UPS and L-sarcoma case series. Conclusions Taken together these results confirmed the activity of trabectedin in these STS histotypes. Moreover the data underline the ECM involvement in the cytotoxic effect mediated by trabectedin and could open the door for researches aimed to focus on the patient setting that could benefit from this agent.

Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. Its kinetic properties were determined and the three-dimensional structures of the apo-, NAD+- and NAD+/GSNO-forms were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity and folding, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed., Highlights • Chlamydomonas protein extracts catalyze NAD(P)H-dependent GSNO degradation. • Chlamydomonas GSNOR1 is a zinc-containing protein strictly relying on GSNO and NADH. • The 3D-structure of CrGSNOR1 revealed a conserved folding with other plant GSNORs. • CrGSNOR1 contains only two solvent-exposed/reactive cysteines. • Oxidizing and nitrosylating treatments have limited effects on CrGSNOR1 activity.

Culturing microalgae using dairy-wastes offers the opportunity of producing valuable biomass for different industrial applications. The capability of four Chlorella species and a recombinant Chlamydomonas reinhardtii strain to mixotrophically grow in wastewaters from an Italian dairy factory was investigated. A robust algal growth could be efficiently sustained in these wastes, despite the abundance of d -Lactose (~4% w/v), that could not be metabolized by any microalgal species. Non axenic cocultivation of microalgae together with microbial communities from the dairy wastes resulted in a marked decrease of their pollution load, thus reducing the necessity of expensive treatments before their discharge in the municipal sewage system. Microalgae cultivated using dairy-wastes were characterized by a lipid content ranging from 12% to 21% (w/w), with Auxenochlorella protothecoides reaching the highest lipid productivity (~0.16 g/L/d) whereas the transplastomic C. reinhardtii strain expressing a thermostable β-glucosidase reached a recombinant enzyme productivity of 0.18 mg/L/d.

Reactive oxygen species (ROS) are produced in cells as normal cellular metabolic by-products. ROS concentration is normally low, but it increases under stress conditions. To stand ROS exposure, organisms evolved series of responsive mechanisms. One such mechanism is protein S-glutathionylation. S-glutathionylation is a post-translational modification typically occurring in response to oxidative stress, in which a glutathione reacts with cysteinyl residues, protecting them from overoxidation. α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. The Arabidopsis genome contains three genes encoding α-amylases. The sole chloroplastic member, AtAMY3, is involved in osmotic stress response and stomatal opening and is redox-regulated by thioredoxins. Here we show that AtAMY3 activity was sensitive to ROS, such as H2O2. Treatments with H2O2 inhibited enzyme activity and part of the inhibition was irreversible. However, in the presence of glutathione this irreversible inhibition was prevented through S-glutathionylation. The activity of oxidized AtAMY3 was completely restored by simultaneous reduction by both glutaredoxin (specific for the removal of glutathione-mixed disulfide) and thioredoxin (specific for the reduction of protein disulfide), supporting a possible liaison between both redox modifications. By comparing free cysteine residues between reduced and GSSG-treated AtAMY3 and performing oxidation experiments of Cys-to-Ser variants of AtAMY3 using biotin-conjugated GSSG, we could demonstrate that at least three distinct cysteinyl residues can be oxidized/glutathionylated, among those the two previously identified catalytic cysteines, Cys499 and Cys587. Measuring the pKa values of the catalytic cysteines by alkylation at different pHs and enzyme activity measurement (pKa1 = 5.70 ± 0.28; pKa2 = 7.83 ± 0.12) showed the tendency of one of the two catalytic cysteines to deprotonation, even at physiological pHs, supporting its propensity to undergo redox post-translational modifications. Taking into account previous and present findings, a functional model for redox regulation of AtAMY3 is proposed., Frontiers in Plant Science, 10, ISSN:1664-462X