Tetrameric Form Research Articles

Interaction of the Escherichia coli HU Protein with Various Topological Forms of DNA.

E. coli histone-like protein HU has been shown to interact with different topological forms of DNA. Using radiolabeled HU, we examine the effects of DNA supercoiling on HU–DNA interactions. We show that HU binds preferentially to negatively supercoiled DNA and that the affinity of HU for DNA increases with increases in the negative superhelical density of DNA. Binding of HU to DNA is most sensitively influenced by DNA supercoiling within a narrow but physiologically relevant range of superhelicity (σ = −0.06–0). Under stoichiometric binding conditions, the affinity of HU for negatively supercoiled DNA (σ = −0.06) is more than 10 times higher than that for relaxed DNA at physiologically relevant HU/DNA mass ratios (e.g., 1:10). This binding preference, however, becomes negligible at HU/DNA mass ratios higher than 1:2. At saturation, HU binds both negatively supercoiled and relaxed DNA with similar stoichiometries, i.e., 5–6 base pairs per HU dimer. In our chemical crosslinking studies, we demonstrate that HU molecules bound to negatively supercoiled DNA are more readily crosslinked than those bound to linear DNA. At in vivo HU/DNA ratios, HU appears to exist predominantly in a tetrameric form on negatively supercoiled DNA and in a dimeric form on linear DNA. Using a DNA ligase-mediated nick closure assay, we show that approximately 20 HU dimers are required to constrain one negative supercoil on relaxed DNA. Although fewer HU dimers may be needed to constrain one negative supercoil on negatively supercoiled DNA, our results and estimates of the cellular level of HU argue against a major role for HU in constraining supercoils in vivo. We discuss our data within the context of the dynamic distribution of the HU protein in cells, where temporal and local changes of DNA supercoiling are known to take place.

Antigen multimers: Specific, sensitive, precise, and multifunctional high-avidity CAR-staining reagents

Although chimeric antigen receptor (CAR) T-cell therapy has transformed cancer treatment, high-quality and universal CAR-staining reagents are urgently required to manufacture CAR T cells, predict therapy response, decipher CAR biology, and engineer new CARs. Here, we developed tetrameric and dodecameric forms of a multifunctional and extensible category of high-avidity CAR-staining reagents: antigen-multimers. Antigen-multimers detected CARs against CD19, HER2, and Tn-glycoside with significantly higher specificity, sensitivity, and precision than existing reagents. In addition to accurate CAR T-cell detection by flow cytometry, antigen-multimers also enabled ≥100-fold magnetic enrichment of rare CAR T cells, selective CAR T-cell stimulation, and high-dimensional CAR T-cell profiling by single-cell multi-omics analyses. Finally, antigen-multimers accurately captured clinical anti-CD19 CAR T cells from patients' cellular infusion products, post-infusion peripheral blood, and tumor biopsies. Antigen-multimers can be readily extended to other CAR systems by switching its antigen ligand. As such, antigen-multimers have broad clinical and research applications.

NMR-Based Analysis of Nanobodies to SARS-CoV-2 Nsp9 Reveals a Possible Antiviral Strategy Against COVID-19.

Following the entry into the host cell, SARS‐CoV‐2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B‐cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID‐19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild‐type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication.

Cryo-EM structure of a tetrameric photosystem I from Chroococcidiopsis TS-821, a thermophilic, unicellular, non-heterocyst-forming cyanobacterium

Photosystem I (PSI) is one of two photosystems involved in oxygenic photosynthesis. PSI of cyanobacteria exists in monomeric, trimeric, and tetrameric forms, in contrast to the strictly monomeric form of PSI in plants and algae. The tetrameric organization raises questions about its structural, physiological, and evolutionary significance. Here we report the ∼3.72 Å resolution cryo-electron microscopy structure of tetrameric PSI from the thermophilic, unicellular cyanobacterium Chroococcidiopsis sp. TS-821. The structure resolves 44 subunits and 448 cofactor molecules. We conclude that the tetramer is arranged via two different interfaces resulting from a dimer-of-dimers organization. The localization of chlorophyll molecules permits an excitation energy pathway within and between adjacent monomers. Bioinformatics analysis reveals conserved regions in the PsaL subunit that correlate with the oligomeric state. Tetrameric PSI may function as a key evolutionary step between the trimeric and monomeric forms of PSI organization in photosynthetic organisms.

Quantification of myocardial amyloid deposition in tafamidis-treated patients with transthyretin amyloid cardiomyopathy

Abstract Background Tafamidis kinetically stabilizes the tetrameric form of transthyretin (TTR) and thus may halt disease progression in patients with transthyretin amyloid cardiomyopathy (ATTR-CM). However, the effect of tafamidis treatment on the progression of myocardial amyloid deposition is still unclear. Methods In our explorative analysis, we aimed to investigate the treatment effect of tafamidis on myocardial amyloid deposition measured by myocardial standardized uptake value (SUV) peak and SUV retention index using quantitative single-photon emission computed tomography/computed tomography (SPECT/CT) of the thorax and to observe its association with clinical parameters. Therefore, clinical, laboratory, imaging, and SPECT/CT examinations were performed in twenty consecutive ATTR-CM patients who started treatment with tafamidis 61mg, both at baseline and at a median of 6 months. Results Main results are summarized in Table 1. In brief, we observed a significant reduction of mean myocardial SUV peak (baseline: 15.50 vs. follow-up: 11.61, p<0.001) and mean SUV retention index (5.64 vs. 3.58, p=0.001) after treatment with tafamidis (Figure 1A). Interestingly, a higher percentage decrease in the SUV retention index is more likely to be associated with clinical benefit, with a threshold of −30% distinguishing between patients who respond clinically (n=12) and those who do not (n=8, Figure 1B). Clinical response is demonstrated by improvement in exertional dyspnea (NYHA class III: 83.3% vs. 41.7%, p=0.047) and mean functional capacity as measured by 6-minute walk distance (349.5m vs. 356.7m, p=0.736). Cardiac biomarkers analysis showed a clear reduction in median NT-proBNP levels in the responder cohort (2765.0 pg/mL vs. 1904.0 pg/mL, p=0.041) compared to an increase in the non-responder cohort (1825.0 pg/mL vs. 1944.0 pg/mL, p=0.208; cohort comparison: p=0.026, Figure 1C). Echocardiographic findings revealed improvement in mean left ventricular (LV) strain (−12.0% vs. −13.5%, p=0.049) and mean LV ejection fraction (LVEF, 48.5% vs. 52.7%, p=0.287) in the responder cohort, while significant deterioration in mean LV function (LV strain: −13.9 vs. −10.5, p=0.035; LVEF: 53.2% vs. 46.5%, p=0.012) was observed in the non-responder cohort, with an additional substantial deterioration in right ventricular (RV) function as measured by tricuspid annular plane systolic excursion (TAPSE, mean, BL: 19.2mm vs. FU: 12.6mm, p=0.037) in those patients. These results are consistent with changes in the LV and RV function in cardiac magnetic resonance imaging parameters in each of the two cohorts. Conclusion Treatment with tafamidis in patients with ATTR-CM results in a significant reduction in myocardial amyloid deposition as measured by the SUV retention index, with a threshold of −30% distinguishing patients who respond clinically from those who do not. However, a larger patient sample is needed to verify these results. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Pfizer Inc.

Cardiac imaging in tafamidis-treatment patients with transthyretin amyloid cardiomyopathy

Abstract Background Tafamidis kinetically stabilizes the tetrameric form of transthyretin (TTR) and thus may halt disease progression in patients with transthyretin amyloid cardiomyopathy (ATTR-CM). In our explorative analysis, we aimed to investigate the treatment effect on functional capacity and cardiac biomarkers as well as cardiac function and structure using echocardiography and cardiac magnetic resonance imaging (CMR), and to compare patients treated with tafamidis with an untreated control cohort. Methods Consecutive ATTR-CM patients either received tafamidis 61mg (n=64) or tafamidis 20mg (n=23) or were assigned to an untreated control cohort (n=54) reflecting the natural history of the disease. Subsequently, we performed clinical, laboratory, echocardiography and CMR follow-up at a median of 9 to 12.5 months. Results Main results are summarized in Table 1. In brief, we observed evidence of improvement in functional capacity as measured by the 6-minute walk distance (6MWD) in tafamidis 61mg treated patients (baseline: 377.1m vs. follow-up: 383.2m, p=0.678) compared to a significant decline in mean 6MWD in untreated patients (388.1m vs. 336.4m, p=0.002; cohort comparison: p=0.005). Analysis of cardiac biomarkers revealed evidence of therapeutic response by a decrease in median NT-proBNP levels in patients treated with tafamidis 61mg (2633.0pg/mL vs. 2244.0pg/mL, p=0.366), whereas a significant increase was observed in untreated patients (2798.0pg/mL vs. 3422.0pg/mL, p<0.001; cohort comparison: p<0.001). Echocardiographic findings revealed evidence of approximate stabilization in mean left ventricular (LV) strain (−11.75% vs. −11.58%, p=0.534) and mean right ventricular (RV) strain (−14.18% vs. −13.72, p=0.377) in the tafamidis 61mg treatment cohort compared to significant deterioration of mean LV longitudinal function (−11.71% vs. −10.59%, p=0.001) and mean RV longitudinal function (−14.36% vs. −12.99%, p=0.038) in the untreated cohort (cohort comparison: p=0.030 and p=0.269). Furthermore, cardiac structural assessment by CMR showed a significant increase in mean LV mass (199.1g vs. 214.3g, p=0.040) and mean extracellular volume (50.52% vs. 55.96%, p=0.026) in untreated patients, suggesting increased progression of myocardial amyloid deposition. Conclusion Treatment with tafamidis in patients with ATTR-CM results in significant improvements in functional capacity and cardiac biomarkers, and shows marked benefits in functional as well as structural imaging parameters compared with an untreated control cohort. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Pfizer Inc.

Cloning, Expression, Purification, and Oligomeric Characterization of the AopB-C-terminus Domain in T3SS Major Translocator Protein of Aeromonas hydrophila

Type three secretion system (T3SS) is found exclusively in gram-negative pathogens such as Yersinia spp., Escherichia coli, Salmonella spp., Shigella spp., Pseudomonas spp., Vibrio parahaemolyticus, and Aeromonas hydrophila. The translocon pore of T3SS comprises major and minor translocator proteins that assemble to provide passage of effectors through the host cell membrane. Major translocator protein AopB from Aeromonas hydrophila plays an important role in translocon pore formation. Despite tremendous efforts, structural information regarding the C-terminus domain of major translocator AopB remains elusive. In this study, the DNA fragment encoding for the C-terminus domain of the AopB major translocator from Aeromonas hydrophila AH-1 was cloned in to pET-M expression vector and expressed in BL21 (DE3) host cells. The recombinant AopB-C-terminus domain was successfully purified using immobilized nickel affinity chromatography as a soluble form. Crosslinking analysis among AopB-C-terminus molecules in solution showed that this domain exists as a mixture of tetramer, trimer, dimer and monomer forms. The three-dimensional structure model of AopB-C-terminus oligomerization was built by SWISS-MODEL and PyMol. The oligomeric model of AopB-C-terminus can be used for structural studies of the AopB-C-terminus domain which can contribute to the elucidation of the structure of the type III secretion system.

Abstract 606: Pkm2 activation modulates the tumor-immune microenvironment and enhances response to checkpoint inhibitors in preclinical solid tumor models

Abstract Pyruvate kinase is a crucial enzyme responsible for the last step of glycolysis. Cancer cells can use the M2 isoform of pyruvate kinase (PKM2), to better balance respiration and biosynthesis due to allosteric switching between the less active dimeric and fully active tetrameric forms. Additionally, the dimeric form of PKM2 can translocate to the nucleus, altering transcription to enhance cancer cells' ability to grow and evade immune detection. Inducing tetramerization presents an opportunity to target PKM2 resulting in the metabolic reprogramming of tumor-immune microenvironment (TME). TP-1454 is a potent PKM2 activator with low nanomolar PKM2 activation in biochemical assays (AC50 = 10 nM) and multiple cell types (AC < 50 nM), tolerated in mice, rats and dogs after repeat doses as high as 1000 mg/kg/day and has recently entered a Phase I clinical trial (NCT04328740). We hypothesize that PKM2 activation may reverse the immune-suppressive TME. To test this hypothesis, we examined the activity of TP-1454 combination with immunotherapy (I/O) in multiple mouse syngeneic tumor models. TP-1454 and anti-PD-1 combination therapy in colorectal cancer models resulted in tumor growth inhibition versus vehicle (53% in CT26; 99% in MC38, P < 0.001). We observed decreases in multiple glycolytic intermediates in TP-1454-treated tumors versus vehicle. We conducted immunophenotyping of the TME in multiple models to identify targets of PKM2 activation. TP-1454 treatment reduced the CD4+ Foxp3+ T-regulatory (Treg) population in MC38, 4T1, RENCA models. Further, we assayed TP-1454 induced PKM2 activation in different immune cell types. To confirm the effect of PKM2 activation on Treg cells we conducted an in vitro assay to explore TP-1454 treatment response on polarization of Tregs and/or toxicity and proliferation. We further utilized LCMS to explore metabolic intermediates that play a critical role in Treg regulation, including regulation of the O-linked β-N-acetylglucosamine (O-GlcNac) post-translational modification, which is reported to stabilize Foxp3 in CD4+ cells. We are currently exploring the effect of TP-1454 treatment on O-GlcNac of Foxp3 and its stability in HEK293 cells, to support the link between PKM2 activation and stabilization of Foxp3. TP-1454 effects on tumor-specific immunity were validated using tumor rechallenge studies. The results of a tumor rechallenge study will be presented using murine MC38 or RENCA xenograft models that are treated with TP-1454 and I/O combination therapies that exhibited a complete response (CR) and were re-implanted. These preclinical studies indicate a unique mechanism modulating tumor metabolism and the TME to improve the response of cancer patients to immunotherapy. Citation Format: Salah Sommakia, Satya Pathi, Yuta Matsumura, Curtis Allred, Ethika Tyagi, Matthew Lalonde, Jason Foulks, Adam Siddiqui, Clifford Whatcott, David Bearss, Steven Warner. Pkm2 activation modulates the tumor-immune microenvironment and enhances response to checkpoint inhibitors in preclinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 606.

Stability of p53 oligomers: Tetramerization of p53 impinges on its stability

The p53 protein has been known to exist structurally in three different forms inside the cells. Earlier studies have reported the predominance of the lower oligomeric forms of p53 over its tetrameric form inside the cells, although only the tetrameric p53 contributes to its transcriptional activity. However, it remains unclear the functional relevance of the existence of other p53 oligomers inside the cells. In this study, we characterize the stability and conformational state of tetrameric, dimeric and monomeric p53 that spans both DNA Binding Domain (DBD) and Tetramerization Domain (TD) of human p53 (94–360 amino acid residues). Intriguingly, our studies reveal an unexpected drastic reduction in tetrameric p53 thermal stability in comparison to its dimeric and monomeric form with a higher propensity to aggregate at physiological temperature. Our EMSA study suggests that tetrameric p53, not their lower oligomeric counterpart, exhibit rapid loss of binding to their consensus DNA elements at the physiological temperature. This detrimental effect of destabilization is imparted due to the tetramerization of p53 that drives the DBDs to misfold at a faster pace when compared to its lower oligomeric form. This crosstalk between DBDs is achieved when it exists as a tetramer but not as dimer or monomer. Our findings throw light on the plausible reason for the predominant existence of p53 in dimer and monomer forms inside the cells with a lesser population of tetramer form. Therefore, the transient disruption of tetramerization between TDs could be a potential cue for the stabilization of p53 inside the cells.

Characterization of a Novel Family IV Esterase Containing a Predicted CzcO Domain and a Family V Esterase with Broad Substrate Specificity from an Oil-Polluted Mud Flat Metagenomic Library

Two novel esterase genes, est2L and est4L, were identified from a previously constructed metagenomic library derived from an oil-polluted mud flat sample. The encoded Est2L and Est4L were composed of 839 and 267 amino acids, respectively, without signal peptides. Est2L was a unique fusion type of protein composed of two domains: a domain of the CzcO superfamily, associated with a cationic diffusion promoter with CzcD, and a domain of the acetylesterase superfamily, belonging to family IV with conserved motifs, such as HGG, GXSAG, and GXPP. Est2L was the first fused esterase with a CzcO domain. Est4L belonged to family V with GXS, GXSMGG, and PTL motifs. Native Est2L and Est4L were found to be in dimeric and tetrameric forms, respectively. Est2L and Est4L showed the highest activities at 60 °C and 50 °C, respectively, and at a pH of 10.0. Est2L preferred short length substrates, especially p-nitrophenyl (pNP)-acetate, with moderate butyrylcholinesterase activity, whereas Est4L showed the highest activity with pNP-decanoate and had broad specificity. Significant effects were not observed in Est2L from Co2+ and Zn2+, although Est2L contains the domain CzcD. Est2L and Est4L showed high stabilities in 30% methanol and 1% Triton X-100. These enzymes could be used for a variety of applications, such as detergent and mining processing under alkaline conditions.

Pyruvate Kinase, Inflammation and Periodontal Disease.

Pyruvate kinase (PK) is the final and rate-limiting enzyme in glycolysis. It has four isoforms PKM1, PKM2, PKL and PKR. PK can form homo tetramers, dimers or monomers. The tetrameric form has the most catalytic activity; however, the dimeric form has non-canonical functions that contribute to the inflammatory response, wound healing and cellular crosstalk. This brief review explores these functions and speculates on their role in periodontal disease.

NAD(H)-mediated tetramerization controls the activity of Legionella pneumophila phospholipase PlaB

The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal α/β-hydrolase domain expanded by two noncanonical two-stranded β-sheets, β-6/β-7 and β-9/β-10. The C-terminal domain reveals a fold displaying a bilobed β-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. Δβ-9/β-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.

Expanding the Use of Tyrosine Phenol Lyase in the Enzymatic Synthesis of Unnatural Amino Acids

The use of enzymes in the synthesis of pharmaceuticals and biochemical tools offers an attractive approach to generating complex structures, as enzymatic reactions often lead to the formation of specific products in high yields. Tyrosine phenol lyase (TPL), an enzyme biologically involved in the degradation of tyrosine, has been used to synthesize a variety of substituted tyrosines for biochemical studies. Expanding the scope of substrates to naphthol-based compounds or substituted tyrosine-based structures offers an attractive target for enzymatic synthesis of complex molecules which can then be used for in potential pharmaceuticals. Using Citrobacter freundii TPL, we enzymatically synthesized the naphthol-analog of tyrosine, but product was in low yield. The simplest model for the low conversion is that this result suggests the larger substrate is poorly accommodated by the enzyme active site and this poor binding leads to the low yield in the enzymatic reaction. To test this hypothesis, we mutated several bulky residues near the substrate-binding site (M288, M397, F448) to open space for substrate binding and facilitate synthesis of the unnatural amino acid. A series of mutant plasmids were generated using site-directed mutagenesis and sequenced to confirm the mutation. The wild type and mutant enzymes were recombinantly expressed in E. coli and subjected to affinity and ion-exchange chromatography. Although gel electrophoresis of the samples showed a single band and initial enzyme assays with tyrosine showed that the wild type and mutants are active, subsequent size-exclusion chromatography assays suggested the samples contain potential different multimeric forms of the enzyme. As previous studies suggested TPL is tetrameric enzyme, we are currently developing additional purification steps to isolate the tetrameric form of the wild type and mutants. The purified enzyme will then be used to evaluate the effects of the mutations on enzyme activity and substrate specificity. While a goal of this study is aimed at the more efficient production of tyrosine derivatives, a larger goal is to better understand the features important for substrate binding in enzymatic reactions. This understanding may aid in the development of enzymes with a broad range of substrates efficiently converted to products in the biochemical generation of complex molecules.

Synthesis of dimeric and tetrameric trithiomannoside clusters through convenient photoinitiated thiol-ene click protocol for efficient inhibition of gram-negative bacteria

Glycoclusters with high glycan density accessible through grouping of monosaccharide units in multimeric forms are important due to their high affinity and capability in recognition with protein receptors as a result of multivalent effect. In this report, we have achieved the assembly of glycoclusters in dimeric to tetrameric forms using efficient light initiated free radical mediated thiol-ene click reaction. Bacterium (UTI89) inhibition study by yeast agglutination assay demonstrated that these multivalent glycoclusters showed more efficient inhibition of bacteria when compared to monovalent sugar units.

NMDA GluN2C/2D receptors contribute to synaptic regulation and plasticity in the anterior cingulate cortex of adult mice

IntroductionN-Methyl-D-aspartate receptors (NMDARs) play a critical role in different forms of plasticity in the central nervous system. NMDARs are always assembled in tetrameric form, in which two GluN1 subunits and two GluN2 and/or GluN3 subunits combine together. Previous studies focused mainly on the hippocampus. The anterior cingulate cortex (ACC) is a key cortical region for sensory and emotional functions. NMDAR GluN2A and GluN2B subunits have been previously investigated, however much less is known about the GluN2C/2D subunits.ResultsIn the present study, we found that the GluN2C/2D subunits are expressed in the pyramidal cells of ACC of adult mice. Application of a selective antagonist of GluN2C/2D, (2R*,3S*)-1-(9-bromophenanthrene-3-carbonyl) piperazine-2,3-dicarboxylic acid (UBP145), significantly reduced NMDAR-mediated currents, while synaptically evoked EPSCs were not affected. UBP145 affected neither the postsynaptic long-term potentiation (post-LTP) nor the presynaptic LTP (pre-LTP). Furthermore, the long-term depression (LTD) was also not affected by UBP145. Finally, both UBP145 decreased the frequency of the miniature EPSCs (mEPSCs) while the amplitude remained intact, suggesting that the GluN2C/2D may be involved in presynaptic regulation of spontaneous glutamate release.ConclusionsOur results provide direct evidence that the GluN2C/2D contributes to evoked NMDAR mediated currents and mEPSCs in the ACC, which may have significant physiological implications.

Involvement of different hemoprotein thiol groups of Oncorhynchus mykiss in cadmium toxicity

BackgroundCadmium is considered the seventh most toxic heavy metal as per ATSDR ranking but its mechanism of toxicity is debated. Recently, we evaluated the effects of this metal on the erythrocyte of teleost fish (Oncorhynchus mykiss) leading us to hypothesize that the pro-oxidant activity of cadmium is not linked to mitochondria but more likely to haemoglobin. In this context, the main aim of this work was to detect the ability of Cd to induce structural perturbation in haemoproteins that present different structures and thus different functional properties and to identify what sites of interaction are mainly involved. MethodsThe effect of Cd on the structural destabilization of the different haemoproteins was followed spectrophometrically through their precipitation. In addition, the sites of interaction between the different haemoproteins and bivalent cadmium ions were identified by MIB server followed by molecular docking/molecular dynamics simulations both in the dimeric and tetrameric associations. ResultsCadmium does not influence the autoxidation rate of Mb, HbA and trout HbI. However, the presence of this metal accelerates the precipitation process in trout HbIV in a dose-dependent manner. Moreover, the presence of 1−10-50−250-500−1000 μM GSH, a chelating agent, reduces the ability of cadmium to accelerate the denaturation process although it is not able to completely prevent it. In order to explain the experimental results, a computational investigations was carried out to identify the cadmium cation affinity for the studied haemoglobins and myoglobin, both in their dimeric and tetrameric forms. As a result, the highest affinity cadmium binding sites for fish HbIV are located at the interface between tetramer-tetramer association, indicating that the cation can assist supramolecular protein aggregations and induce complex precipitation. For mammalian Hb, Mb and fish HbI computational investigation did not detect any site where Cd could to induce such aggregation, in line with the experimental results. ConclusionThe present study provides new information on the mechanisms of toxicity of cadmium by specific interaction with trout O. mykiss haemoglobin component.

Structure and activation mechanism of the human liver-type glutaminase GLS2

Cancer cells exhibit an altered metabolic phenotype, consuming higher levels of the amino acid glutamine. This metabolic reprogramming depends on increased mitochondrial glutaminase activity to convert glutamine to glutamate, an essential precursor for bioenergetic and biosynthetic processes in cells. Mammals encode the kidney-type (GLS) and liver-type (GLS2) glutaminase isozymes. GLS is overexpressed in cancer and associated with enhanced malignancy. On the other hand, GLS2 is either a tumor suppressor or an oncogene, depending on the tumor type. The GLS structure and activation mechanism are well known, while the structural determinants for GLS2 activation remain elusive. Here, we describe the structure of the human glutaminase domain of GLS2, followed by the functional characterization of the residues critical for its activity. Increasing concentrations of GLS2 lead to tetramer stabilization, a process enhanced by phosphate. In GLS2, the so-called “lid loop” is in a rigid open conformation, which may be related to its higher affinity for phosphate and lower affinity for glutamine; hence, it has lower glutaminase activity than GLS. The lower affinity of GLS2 for glutamine is also related to its less electropositive catalytic site than GLS, as indicated by a Thr225Lys substitution within the catalytic site decreasing the GLS2 glutamine concentration corresponding to half-maximal velocity (K0.5). Finally, we show that the Lys253Ala substitution (corresponding to the Lys320Ala in the GLS “activation” loop, formerly known as the “gating” loop) renders a highly active protein in stable tetrameric form. We conclude that the “activation” loop, a known target for GLS inhibition, may also be a drug target for GLS2.

MTT based L-aminoacid oxidase activity test for determination of antivenom potency against Vipera ammodytes envenomation

The MTT assay is routinely used to detect the activity of living cells. While working with Vipera ammodytes venom we detected the reduction of MTT without the presence of cells, in a concentration-dependent manner. By combining non-reducing PAGE, L-amino acid oxidase (LAAO) assays, and standard MTT assays, we established and confirmed that venom MTT reduction is catalyzed by only one enzyme, the LAAO. Even though it was previously known that the dimeric and tetrameric forms of LAAO are active, we conclude that the enzyme is also active in the monomeric form. Our results have led to the definition of a new MTT assay in a microtiter plate for in vitro testing of svLAAO activity i.e. from the venom of the V. ammodytes snake. Potentially, this method can be used for testing hemorrhagic venoms of other snakes as well as the LAAO neutralization capability of appropriate antivenoms.

AK104 (PD-1/CTLA-4 bispecific) combined with chemotherapy as first-line therapy for advanced gastric (G) or gastroesophageal junction (GEJ) cancer: Updated results from a phase Ib study.

232 Background: OS and PFS benfits have been observed in combination of anti-PD-1 agent plus chemotherapy compared to chemotherapy alone as first-line advanced G/GEJ cancer (Checkmate-649). It uncovers the significant treatment prospects of immune checkpoint inhibitors combination therapies. The PD-1/CTLA-4 dual blockade has consistently demonstrated higher response rate compared to PD-1 monotherapy but higher toxicity. AK104, a PD-1/CTLA-4 bispecific antibody, is designed as a novel tetrameric form. It could preferentially binds to tumor-infiltrating lymphocytes (TILs) co-expressing PD-1 and CTLA-4 with higher avidity in the tumor micro-environment than peripheral sites. Therefore, AK104 is designed to retain the efficacy benefit derived from the combination of anti-PD-1 and anti-CTLA-4 while conferring superior safety compared to the co-administration of these individual agents. This Phase Ib study evaluates the safety and efficacy of AK104 and mXELOX in the first-setting of G/GEJ cancer cohorts (NCT03852251). Methods: Pts with untreated, inoperable advanced G/GEJ adenocarcinoma regardless of PD-L1 status were enrolled to cohorts of 3-6 pts at AK104 doses of 4, 6 and 10 mg/kg q2w + mXELOX [oxaliplatin 85 mg/m2 and capecitabine 1000 mg/m2] during dose escalation. Selected cohorts were expanded up to 18 pts to further establish the recommended Phase II dose. Tumor tissue for determination of PD-L1 status with combined positive score (CPS) must be provided from ≤ 6 months before study treatment. Antitumor activity was assessed by RECIST v1.1. Results: As of Sep 9, 2020, 34 pts (73.5% male, median age 63.1 yrs [29-75], 76.5% G and 23.5% GEJ ) have received AK104 at doses of 4 mg/kg (n = 18), 6 mg/kg (n = 14) and 10 mg/kg (n = 2) + mXELOX. AK104-related adverse events (TRAEs) occurred in 79.4% of pts. G3 TRAEs occurred in 29.4% (10/34) and no G4 or G5 TRAE was reported. Most frequent TRAEs (incidence ≥ 15%) were neutrophil count decreased (26.5%), platelet count decreased (20.6%), white blood cell count decreased (17.6%), anaemia (17.6%) and infusion related reaction(17.6%). Grade ≥3 TRAEs reported in ≥2 pts were neutrophil count decreased (8.8%). Grade ≥3 immune-related AEs were reported in 8.8% of pts (hepatitis, pneumonitis, hyponatraemia). Of 24 pts evaluable for antitumor activity, ORR was 66.7% (95% CI 44.7, 84.4 ) including 2 CRs and 14 PRs . The disease control rate (DCR) was 95.8% (95% CI 78.9, 99.9). Response was seen regardless of PD-L1 status. At a median follow-up of 8.6 mons for the 4mg/kg cohort, 6-mons PFS rate was 69.5% (95%CI 41.3, 86.1). Conclusions: AK104 in combination with mXELOX had a manageable safety profile and encouraging antitumor activities in pts with advanced G/GEJ adenocarcinoma regardless of PD-L1 status. Enrollment is currently ongoing for 6 mg/kg and 10 mg/kg cohort. Clinical trial information: NCT03852251.

Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure.

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in Escherichia coli. We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/β hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. Km values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.