Biochemical characterization and cytotoxic evaluation of mutant isoforms of L-asparaginase II from Dickeya chrysanthemi (Erwinia chrysanthemi)

A leucemia linfoblástica aguda (LLA) é a neoplasia mais frequente em crianças e adolescentes. Esse tumor tem como característica a auxotrofia para o aminoácido Lasparagina, o que permite que o tratamento da doença seja feito com a L-asparaginase (ASNase), enzima obtida a partir das bactérias Escherichia coli e Dickeya chrysanthemi (também denominada Erwinia chrysanthemi). A ASNase hidrolisa a L-asparagina, e Lglutamina em menor quantidade, impedindo que as células tumorais obtenham L-asparagina da circulação sanguínea para síntese de proteínas, levando a morte celular por apoptose. No entanto, as formulações disponíveis para terapêutica estão associadas a um alto índice de efeitos adversos, como toxicidade e a resistência ao medicamento causada pela produção de anticorpos e pela ação de proteases. Desta maneira, o desenvolvimento de proteoformas mutantes a partir das enzimas já comercializadas e o estudo da ação de proteases durante o tratamento podem contribuir para o desenvolvimento de uma enzima com menos efeitos adversos. Por isso, o nosso grupo de pesquisa criou uma biblioteca de mutantes utilizando a ASNase de D. chrysanthemi, por PCR propenso a erro, e de dez clones mutantes estudados uma proteoforma dupla mutante (DM) apresentou melhores parâmetros cinéticos do que a enzima selvagem (WT), sendo altamente ativa em condições fisiológicas testadas in vitro. Além disso, a enzima DM apresentou um menor reconhecimento por anticorpos antiasparaginase e o mesmo potencial citotóxico do que a enzima WT, podendo ocasionar menos efeitos adversos. Além disso, criamos as células de LLA REH knockout para protease catepsina B (CTSB) por CRISPR/cas9, e avaliamos a viabilidade celular após o tratamento com as ASNases WT de E. coli e D. chrysanthemi, e com a enzima DM. Os nossos resultados sugerem que a expressão de CTSB pelas células REH é muito baixa e não alteram a resposta ao tratamento com ASNases quando avaliadas in vitro.Acute lymphoblastic leukemia (ALL) is the most frequent neoplasm in children and adolescents. This tumor is characterized by auxotrophy for the amino acid L-asparagine, which allows the treatment of the disease to be carried out with L-asparaginase (ASNase), an enzyme obtained from the bacteria Escherichia coli and Dickeya chrysanthemi (also called Erwinia chrysanthemi). ASNase hydrolyzes L-asparagine, and L-glutamine to a lesser extent, preventing tumor cells from obtaining L-asparagine from the bloodstream for protein synthesis, leading to cell death by apoptosis. However, the formulations available for therapy are associated with a high rate of adverse effects, such as toxicity and drug resistance caused by the production of antibodies and the action of proteases. In this way, the development of mutant proteoforms from enzymes already commercialized and the study of the action of proteases during treatment can contribute to the development of an enzyme with fewer adverse effects. Therefore, our research group created a library of mutants using the D. chrysanthemi ASNase, by error prone PCR, and from ten mutant clones studied a double mutant proteoform (DM) showed better kinetic parameters than the wild-type enzyme (WT), being highly active under physiological conditions tested in vitro. In addition, the DM enzyme showed less recognition by anti-asparaginase antibodies and the same cytotoxic potential as the WT enzyme, which may cause fewer adverse effects. In addition, we created REH ALL cells knockout for cathepsin B (CTSB) protease by CRISPR/cas9, and evaluated cell viability after treatment with E. coli and D. chrysanthemi WT ASNases, and with the DM enzyme. Our results suggest that the expression of CTSB by REH cells is very low and does not alter the response to treatment with ASNases when evaluated in vitro

Production, kinetic characterization and engineering of asparaginase 1 protein from Saccharomyces cerevisiae to evaluate its use as a biopharmaceutical.

A L-asparaginase (EC 3.5.1.1) é uma enzima importante para o tratamento da leucemia linfoblástica aguda (LLA), neoplasia mais frequente em crianças e adolescentes. A L-asparaginase hidrolisa a L-asparagina resultando em ácido aspártico e amônio, impedindo que as células tumorais utilizem esse aminoácido para síntese proteica, ocasionando a morte celular apoptótica. Atualmente a enzima é obtida a partir de Escherichia coli e Erwinia chrysanthemi; no entanto, ambas as formulações estão associadas a um alto índice de efeitos adversos que comprometem a evolução e eficácia do tratamento. A levedura Saccharomyces cerevisiae tem o gene ASP1 responsável pela produção de L-asparaginase 1 (Sc_ASPase1) que tem sido pouco estudada. Para elucidar as características de Sc_ASPase1 nós expressamos a proteína em E. coli BL21(DE3) e a purificamos por cromatografia de afinidade. Sc_ASPase1 tem uma atividade especifica de 195,4 U/mg para L-asparagina e de 0,36 U/mg para L-glutamina, e um comportamento alostérico com um K0.5 de 75 µM para L-asparagina. Por meio de mutação sitio dirigida demonstramos a importância dos resíduos Thr64-Thy78-Th141-Lys215 para a catálise. As isoformas mutantes da proteína A331D, K335E, Y243S, S301N e ΔG77 não apresentaram melhoria nos parâmetros cinéticos ou atividade específica. Construímos e clonamos Sc_ASPase1 com a deleção dos primeiros 52 aminoácidos, porém nas condições testadas a proteína foi expressa na forma insolúvel. Demonstramos que Sc_ASPase1 possui potencial antineoplásico, pois com 10 U/mL de enzima foi capaz causar a 85% de mortalidade da linhagem leucêmica MOLT-4. Na mesma concentração, a enzima de E. coli é capaz de matar 95% de células dessa mesma linhagem.L-Asparaginase (EC 3.5.1.1) is an important enzyme for the treatment of acute lymphoblastic leukemia (ALL), the most common malignancy in children and adolescents. L-asparaginase hydrolyzes L-asparagine resulting in ammonium and aspartic acid, preventing tumor cells of using such amino acid for protein synthesis, leading to apoptotic cell death. Currently, the enzyme is obtained from Escherichia coli and Erwinia chrysanthemi; however, both formulations are associated with a high incidence of side effects that compromise the progress and effectiveness of treatment. The yeast Saccharomyces cerevisiae has ASP1 gene responsible for the production of L-asparaginase 1 (Sc_ASPase1) that has been poor studied. To elucidate the characteristics of Sc_ASPase1, we expressed the protein in E. coli BL21 (DE3) cells and purified it by affinity chromatography. Sc_ASPase1 has a specific activity of 195.4 U/mg for L-asparagine and 0.36 U/mg for L-glutamine, and an allosteric behavior with a K0.5 of 75 µM for L-asparagine. Through site directed mutation, we demonstrated the importance of Thr64-Thy78-Th141-Lys215 residues for catalysis. The mutant protein isoforms A331D, K335E, Y243S, S301N and ΔG77 showed no improvement in kinetic parameters or specific activity. We build and cloned Sc_ASPase1 with the deletion of the first 52 amino acids, but under the conditions tested the protein was expressed in insoluble form. Sc_ASPase1 have demonstrated potential antineoplastic activityc, since 10 U/mL of enzyme lead to 85% of mortality in leukemia cell line MOLT-4. At the same concentration, the E. coli enzyme kills 95% of the cells of the same line

Engineered asparaginase from Erwinia chrysanthemi enhances asparagine hydrolase activity and diminishes enzyme immunoreactivity - a new promise to treat acute lymphoblastic leukemia

BACKGROUND: The treatment of acute lymphoblastic leukemia (ALL) uses the biopharmaceutical l-asparaginase (ASNase) as the main medication. This drug, from bacterial origin (Escherichia coli or Erwinia chrysanthemi), depletes l-asparagine (Asn) and secondarily l-glutamine (Gln – GLNase activity) from the bloodstream, leading leukemic cells to die by deprivation of Asn. The use of ASNase is limited by the high incidence of adverse effects, which collectively can specifically impair quality of life of patients. Its high toxicity caused by the product of the hydrolysis of amino acids and the formation of anti-ASNase antibodies often required treatment interruption, thus reducing the chances of cure and increasing the rates of disease relapse. RESULTS: In order to improve enzymatic activity, while reducing toxicity, we developed through directed evolution a double-mutant ASNase from Erwinia chrysanthemi (Erw_DM), which has specific activity for Asn 46% higher than the wild-type enzyme (Erw_WT). This makes it possible to reduce the amount of protein necessary for depletion of this amino acid and, consequently, the reduction of GLNase activity, considered toxic. In silico analysis showed that a lower number of epitopes was exposed, resulting in reduced recognition of the recombinant protein by antibody anti-ASNase observed in vitro assay. Furthermore, we observed the same cytotoxic profile for the MOLT-4 and REH ALL cell lines using 40% lower protein mass of Erw_DM to achieve the minimum enzyme activity required in the bloodstream during treatment. CONCLUSION: Altogether, our findings describe a potent and less immunogenic ASNase, an improvement that may alleviate treatment adverse effects developed in anti-ALL therapy. © 2021 Society of Chemical Industry (SCI)

Pseudorapidity densities of charged particles with transverse momentum thresholds in pp collisions at √ s = 5.02 and 13 TeV

The pseudorapidity density of charged particles with minimum transverse momentum (pT) thresholds of 0.15, 0.5, 1, and 2 GeV/c is measured in pp collisions at the center of mass energies of √s=5.02 and 13 TeV with the ALICE detector. The study is carried out for inelastic collisions with at least one primary charged particle having a pseudorapidity (η) within 0.8pT larger than the corresponding threshold. In addition, measurements without pT-thresholds are performed for inelastic and nonsingle-diffractive events as well as for inelastic events with at least one charged particle having |η|2GeV/c), highlighting the importance of such measurements for tuning event generators. The new measurements agree within uncertainties with results from the ATLAS and CMS experiments obtained at √s=13TeV.

Study of the p−-p−-K+^+ and p−-p−-K−^- dynamics using the femtoscopy technique

International audienceThe interactions of kaons (K) and antikaons ($\mathrm{\overline{K}}$) with few nucleons (N) were studied so far using kaonic atom data and measurements of kaon production and interaction yields in nuclei. Some details of the three-body KNN and $\mathrm{\overline{K}}$NN dynamics are still not well understood, mainly due to the overlap with multi-nucleon interactions in nuclei. An alternative method to probe the dynamics of three-body systems with kaons is to study the final state interaction within triplet of particles emitted in pp collisions at the Large Hadron Collider, which are free from effects due to the presence of bound nucleons. This Letter reports the first femtoscopic study of p$-$p$-$K$^+$ and p$-$p$-$K$^-$ correlations measured in high-multiplicity pp collisions at $\sqrt{s}$ = 13 TeV by the ALICE Collaboration. The analysis shows that the measured p$-$p$-$K$^+$ and p$-$p$-$K$^-$ correlation functions can be interpreted in terms of pairwise interactions in the triplets, indicating that the dynamics of such systems is dominated by the two-body interactions without significant contributions from three-body effects or bound states

Studying strangeness and baryon production mechanisms through angular correlations between charged Ξ\Xi baryons and identified hadrons in pp collisions at s\sqrt{s} = 13 TeV

International audienceThe angular correlations between charged $\Xi$ baryons and associated identified hadrons (pions, kaons, protons, $\Lambda$ baryons, and $\Xi$ baryons) are measured in pp collisions at $\sqrt{s} = 13$ TeV with the ALICE detector to give insight into the particle production mechanisms and balancing of quantum numbers on the microscopic level. In particular, the distribution of strangeness is investigated in the correlations between the doubly-strange $\Xi$ baryon and mesons and baryons that contain a single strange quark, K and $\Lambda$. As a reference, the results are compared to $\Xi\pi$ and $\Xi\mathrm{p}$ correlations, where the associated mesons and baryons do not contain a strange valence quark. These measurements are expected to be sensitive to whether strangeness is produced through string breaking or in a thermal production scenario. Furthermore, the multiplicity dependence of the correlation functions is measured to look for the turn-on of additional particle production mechanisms with event activity. The results are compared to predictions from the string-breaking model PYTHIA 8, including tunes with baryon junctions and rope hadronisation enabled, the cluster hadronisation model HERWIG 7, and the core-corona model EPOS-LHC. While some aspects of the experimental data are described quantitatively or qualitatively by the Monte Carlo models, no one model can match all features of the data. These results provide stringent constraints on the strangeness and baryon number production mechanisms in pp collisions

Observation of medium-induced yield enhancement and acoplanarity broadening of low-pTp_\mathrm{T} jets from measurements in pp and central Pb−-Pb collisions at sNN=5.02\sqrt{s_{\rm NN}}=5.02 TeV

International audienceThe ALICE Collaboration reports the measurement of semi-inclusive distributions of charged-particle jets recoiling from a high transverse momentum (high $p_{\rm T}$) hadron trigger in proton$-$proton and central Pb$-$Pb collisions at $\sqrt{s_{\rm NN}} = 5.02$ TeV. A data-driven statistical method is used to mitigate the large uncorrelated background in central Pb$-$Pb collisions. Recoil jet distributions are reported for jet resolution parameter $R=0.2$, 0.4, and 0.5 in the range $7 < p_{\rm T,jet} < 140$ GeV$/c$ and trigger$-$recoil jet azimuthal separation $\pi/2 < \Delta\varphi < \pi$. The measurements exhibit a marked medium-induced jet yield enhancement at low $p_{\rm T}$ and at large azimuthal deviation from $\Delta\varphi\sim\pi$. The enhancement is characterized by its dependence on $\Delta\varphi$, which has a slope that differs from zero by 4.7$\sigma$. Comparisons to model calculations incorporating different formulations of jet quenching are reported. These comparisons indicate that the observed yield enhancement arises from the response of the QGP medium to jet propagation

Skewness and kurtosis of mean transverse momentum fluctuations at the LHC energies

International audienceThe first measurements of skewness and kurtosis of mean transverse momentum ($\langle p_\mathrm{T}\rangle$) fluctuations are reported in Pb$-$Pb collisions at $\sqrt{s_\mathrm{NN}}$ = 5.02 TeV, Xe$-$Xe collisions at $\sqrt{s_\mathrm{NN}}$$=$ 5.44 TeV and pp collisions at $\sqrt{s} = 5.02$ TeV using the ALICE detector. The measurements are carried out as a function of system size $\langle \mathrm{d}N_\mathrm{ch}/\mathrm{d}\eta\rangle_{|\eta|<0.5}^{1/3}$, using charged particles with transverse momentum ($p_\mathrm{T}$) and pseudorapidity ($\eta$), in the range $0.2 < p_\mathrm{T} < 3.0$ GeV/$c$ and $|\eta| < 0.8$, respectively. In Pb$-$Pb and Xe$-$Xe collisions, positive skewness is observed in the fluctuations of $\langle p_\mathrm{T}\rangle$ for all centralities, which is significantly larger than what would be expected in the scenario of independent particle emission. This positive skewness is considered a crucial consequence of the hydrodynamic evolution of the hot and dense nuclear matter created in heavy-ion collisions. Furthermore, similar observations of positive skewness for minimum bias pp collisions are also reported here. Kurtosis of $\langle p_\mathrm{T}\rangle$ fluctuations is found to be in good agreement with the kurtosis of Gaussian distribution, for most central Pb$-$Pb collisions. Hydrodynamic model calculations with MUSIC using Monte Carlo Glauber initial conditions are able to explain the measurements of both skewness and kurtosis qualitatively from semicentral to central collisions in Pb--Pb system. Color reconnection mechanism in PYTHIA8 model seems to play a pivotal role in capturing the qualitative behavior of the same measurements in pp collisions

Light-flavor particle production in high-multiplicity pp collisions at s=13\mathbf{\sqrt{\textit{s}} = 13} TeV as a function of transverse spherocity

International audienceResults on the transverse spherocity dependence of light-flavor particle production ($\pi$, K, p, $\phi$, ${\rm K^{*0}}$, ${\rm K}^{0}_{\rm{S}}$, $\Lambda$, $\Xi$) at midrapidity in high-multiplicity pp collisions at $\sqrt{s} = 13$ TeV were obtained with the ALICE apparatus. The transverse spherocity estimator ($S_{{\rm O}}^{{\it p}_{\rm T}=1}$) categorizes events by their azimuthal topology. Utilizing narrow selections on $S_{\text{O}}^{{\it p}_{\rm T}=1}$, it is possible to contrast particle production in collisions dominated by many soft initial interactions with that observed in collisions dominated by one or more hard scatterings. Results are reported for two multiplicity estimators covering different pseudorapidity regions. The $S_{{\rm O}}^{{\it p}_{\rm T}=1}$ estimator is found to effectively constrain the hardness of the events when the midrapidity ($\left | \eta \right |< 0.8$) estimator is used. The production rates of strange particles are found to be slightly higher for soft isotropic topologies, and severely suppressed in hard jet-like topologies. These effects are more pronounced for hadrons with larger mass and strangeness content, and observed when the topological selection is done within a narrow multiplicity interval. This demonstrates that an important aspect of the universal scaling of strangeness enhancement with final-state multiplicity is that high-multiplicity collisions are dominated by soft, isotropic processes. On the contrary, strangeness production in events with jet-like processes is significantly reduced. The results presented in this article are compared with several QCD-inspired Monte Carlo event generators. Models that incorporate a two-component phenomenology, either through mechanisms accounting for string density, or thermal production, are able to describe the observed strangeness enhancement as a function of $S_{{\rm O}}^{{\it p}_{\rm T}=1}$