Gel Filtration Experiments Research Articles

Merlin tumor suppressor function is regulated by PIP2-mediated dimerization.

Neurofibromatosis Type 2 is an inherited disease characterized by Schwann cell tumors of cranial and peripheral nerves. The NF2 gene encodes Merlin, a member of the ERM family consisting of an N-terminal FERM domain, a central α-helical region, and a C-terminal domain. Changes in the intermolecular FERM-CTD interaction allow Merlin to transition between an open, FERM accessible conformation and a closed, FERM-inaccessible conformation, modulating Merlin activity. Merlin has been shown to dimerize, but the regulation and function Merlin dimerization is not clear. We used a nanobody based binding assay to show that Merlin dimerizes via a FERM-FERM interaction, orientated with each C-terminus close to each other. Patient derived and structural mutants show that dimerization controls interactions with specific binding partners, including HIPPO pathway components, and correlates with tumor suppressor activity. Gel filtration experiments showed that dimerization occurs after a PIP2 mediated transition from closed to open conformation monomers. This process requires the first 18 amino acids of the FERM domain and is inhibited by phosphorylation at serine 518. The discovery that active, open conformation Merlin is a dimer represents a new paradigm for Merlin function with implications for the development of therapies designed to compensate for Merlin loss.

Crystal structures reveal a novel dimer of the RWD domain of human general control nonderepressible 2

General control nonderepressible 2 (GCN2) is a serine/threonine protein kinase, detecting a variety of stresses including amino acid starvation, reactive oxygen species, etc. in eukaryotic cells. Activation of GCN2 requires the interaction of the N-terminal RWD domain with the upstream GCN1 protein and the dimerization by the kinase domain. In this study, we determined two crystal structures of the RWD domain of human GCN2 in two different crystal packing modes. These two different crystal structures reveal a same dimer of the RWD domain, which has not been reported in previous studies. We further confirmed this novel dimer interaction in solution using gel filtration experiments, and in human embryonic kidney (HEK) 293 cells using bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) assays. Together, this study discovers a potential protein-protein interface on the RWD domain of human GCN2, and suggests a possible regulation between the interaction of GCN1 and the formation of GCN2 dimer.

The tetrameric structure of the novel haloalkane dehalogenase DpaA from Paraglaciecola agarilytica NO2.

Haloalkane dehalogenases (EC 3.8.1.5) are microbial enzymes that catalyse the hydrolytic conversion of halogenated compounds, resulting in a halide ion, a proton and an alcohol. These enzymes are used in industrial biocatalysis, bioremediation and biosensing of environmental pollutants or for molecular tagging in cell biology. The novel haloalkane dehalogenase DpaA described here was isolated from the psychrophilic and halophilic bacterium Paraglaciecola agarilytica NO2, which was found in marine sediment collected from the East Sea near Korea. Gel-filtration experiments and size-exclusion chromatography provided information about the dimeric composition of the enzyme in solution. The DpaA enzyme was crystallized using the sitting-drop vapour-diffusion method, yielding rod-like crystals that diffracted X-rays to 2.0 Å resolution. Diffraction data analysis revealed a case of merohedral twinning, and subsequent structure modelling and refinement resulted in a tetrameric model of DpaA, highlighting an uncommon multimeric nature for a protein belonging to haloalkane dehalogenase subfamily I.

Tight binding of cytochrome b5 to cytochrome P450 17A1 is a critical feature of stimulation of C21 steroid lyase activity and androgen synthesis

It has been recognized for >50 years that cytochrome b5 (b5) stimulates some cytochrome P450 (P450)–catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b5 is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b5 function. One of the issues in studying b5–P450 interactions has been the limited solution assay methods. We constructed a fluorescently labeled variant of human b5 that can be used in titrations. The labeled b5 bound to WT P450 17A1 with a Kd of 2.5 nM and rapid kinetics, on the order of 1 s−1. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b5 binding. Kd values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b5 fluorescence at higher concentrations. The addition of NADPH–P450 reductase (POR) to an Alexa 488-T70C-b5:P450 17A1 complex resulted in a concentration-dependent partial restoration of b5 fluorescence, indicative of a ternary P450:b5:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b5 complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.

Factor XII and kininogen asymmetric assembly with gC1qR/C1QBP/P32 is governed by allostery

AbstractThe contact system is composed of factor XII (FXII), prekallikrein (PK), and cofactor high-molecular-weight kininogen (HK). The globular C1q receptor (gC1qR) has been shown to interact with FXII and HK. We reveal the FXII fibronectin type II domain (FnII) binds gC1qR in a Zn2+-dependent fashion and determined the complex crystal structure. FXIIFnII binds the gC1qR trimer in an asymmetric fashion, with residues Arg36 and Arg65 forming contacts with 2 distinct negatively charged pockets. gC1qR residues Asp185 and His187 coordinate a Zn2+ adjacent to the FXII-binding site, and a comparison with the ligand-free gC1qR crystal structure reveals the anionic G1-loop becomes ordered upon FXIIFnII binding. Additional conformational changes in the region of the Zn2+-binding site reveal an allosteric basis for Zn2+ modulation of FXII binding. Mutagenesis coupled with surface plasmon resonance demonstrate the gC1qR Zn2+ site contributes to FXII binding, and plasma-based assays reveal gC1qR stimulates coagulation in a FXII-dependent manner. Analysis of the binding of HK domain 5 (HKD5) to gC1qR shows only 1 high-affinity binding site per trimer. Mutagenesis studies identify a critical G3-loop located at the center of the gC1qR trimer, suggesting steric occlusion as the mechanism for HKD5 asymmetric binding. Gel filtration experiments reveal that gC1qR clusters FXII and HK into a higher-order 500-kDa ternary complex. These results support the conclusion that extracellular gC1qR can act as a chaperone to cluster contact factors, which may be a prelude for initiating the cascades that drive bradykinin generation and the intrinsic pathway of coagulation.

Humic Acid Transformation by the Fungus Cerrena unicolor Growing on Cellulose and Glucose

Microbial degradation of lignocellulose and soil organic matter is an important process controlling CO2 flow into the atmosphere, which is mostly carried out by fungi. The alkali-soluble fraction, which is represented mostly by the so-called humic acids (HA), dark-colored compounds of phenolic nature, constitute ~30‒70% of soil Corg. This is the first report on efficient degradation of soil HA under cellulolytic conditions; cellobiose dehydrogenase (CDH), an oxidative enzyme of the cellulolytic complex, was shown to play the key role in this process. Growth of a wood-decomposing fungus Cerrena unicolor on cellulose (CDH and laccase production) and glucose (laccase production) resulted in HA decolorization by 60 and 20%, respectively. HA depolymerization in the presence of CDH and its polymerization in the presence of laccase were shown by gel filtration experiments with the culture liquid and pure enzyme preparations. HA degradation in the presence of CDH was inhibited by radical scavengers, indicating the radical nature of this process (probably the Fenton’s reaction). HA depolymerization by CDH decreased in the presence of laccase, while HA polymerization by laccase decreased in the presence of CDH. Thus, the interaction of these enzymes affects HA transformation under cellulolytic conditions. These results improve our understanding of the mechanisms of microbial degradation of the aromatic components of soil humus and of the ecological functions of wood-decomposing fungi grown under cellulolytic conditions.

Biochemical and structural insights into how amino acids regulate pyruvate kinase muscle isoform 2

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme involved in ATP generation and critical for cancer metabolism. PKM2 is expressed in many human cancers and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various stimuli allosterically regulate PKM2 by cycling it between highly active and less active states. Several small molecules activate PKM2 by binding to its intersubunit interface. Serine and cysteine serve as an activator and inhibitor of PKM2, respectively, by binding to its amino acid (AA)-binding pocket, which therefore represents a potential druggable site. Despite binding similarly to PKM2, how cysteine and serine differentially regulate this enzyme remains elusive. Using kinetic analyses, fluorescence binding, X-ray crystallography, and gel filtration experiments with asparagine, aspartate, and valine as PKM2 ligands, we examined whether the differences in the side-chain polarity of these AAs trigger distinct allosteric responses in PKM2. We found that Asn (polar) and Asp (charged) activate PKM2 and that Val (hydrophobic) inhibits it. The results also indicate that both Asn and Asp can restore the activity of Val-inhibited PKM2. AA-bound crystal structures of PKM2 displayed distinctive interactions within the binding pocket, causing unique allosteric effects in the enzyme. These structure-function analyses of AA-mediated PKM2 regulation shed light on the chemical requirements in the development of mechanism-based small-molecule modulators targeting the AA-binding pocket of PKM2 and provide broader insights into the regulatory mechanisms of complex allosteric enzymes.

High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1.

Plants employ a number of phosphorylation cascades in response to a wide range of environmental stimuli. Previous studies in Arabidopsis and yeast indicate that histidine kinase AHK1 is a positive regulator of drought and osmotic stress responses. Based on these studies AHK1 was proposed a plant osmosensor, although the molecular basis of plant osmosensing still remains unknown. To understand the molecular role and signaling mechanism of AHK1 in osmotic stress, we have expressed and purified full-length AHK1 from Arabidopsis in a bacterial host to allow for studies on the isolated transmembrane receptor. Purification of the recombinant protein solubilized from the host membranes was achieved in a single step by metal-affinity chromatography. Analysis of the purified AHK1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting show a single band indicating that the preparation is highly pure and devoid of contaminants or degradation products. In addition, gel filtration experiments indicate that the preparation is homogenous and monodisperse. Finally, CD-spectroscopy, phosphorylation activity, dimerization studies, and protein–protein interaction with plant phosphorylation targeting AHP2 demonstrate that the purified protein is functionally folded and acts as phospho-His or phospho-Asp phosphatase. Hence, the expression and purification of recombinant AHK1 reported here provide a basis for further detailed functional and structural studies of the receptor, which might help to understand plant osmosensing and osmosignaling on the molecular level.

439 Melanoma cells export functional proteasomes by way of microvesiculation: a possible mechanism for generation of plasmatic proteasomes

Plasmatic proteasomes (p-proteasomes) are a reliable marker for metastatic dissemination in melanoma patients. Recent works have suggested that p-proteasome partially originated from tumour cell secretion via exosomes, which are also considered as a new biomarker for metastatic melanoma. Our objectives were to investigate the origin, mechanism of secretion and functionality of the melanoma p-proteasomes. We show by proteasome purification either from culture supernatant of melanoma cell lines (A-375, SK-MEL-28) or from plasma of melanoma patients, that the extracellular proteasome is released essentially as an assembled 20S core particle, contrarily to proteasome analyzed from cell lysate, which presents additional 26S capped proteasome. Using specific fluorogenic substrates we demonstrate that these extracellular proteasomes are functional and possess all the peptidase activities associated with b1, b2 and b5 subunits. There is no correlation between exosome and proteasome concentrations determined by ELISA in the plasma of melanoma patients. Immunoisolation of secreted proteasome using a7 antibody adsorbed on magnetic beads and pull-down experiments show that secreted proteasomes are not enwrapped in exosomes. To our point of view, previous reports of 20S proteasome subunits in exosomes are rather due to a co-purification along the exosome preparation. Extracellular proteasome concentration is increased in the secretome of melanoma cell lines after stimulation of membrane blebbing with the ionophore A23187. Moreover, proteasome is associated with microvesicles as shown by gel filtration experiments and immobilization of microvesicles on lectin-sepharose. Altogether our results show that melanoma cells secrete functional 20S proteasomes at least in part by membrane blebbing and microvesiculation and not through exosomal pathway.

646 Characterization of platelets-derived compounds that induce antimicrobial peptides in human keratinocytes

The use of formulations based on thrombocyte concentrate lysates (platelet-released growth factors, PRGF) revealed promising effects in the treatment of chronic wounds. The underlying mechanisms are not well understood. We could show that PRGF induced the expression of antimicrobial peptides (AMPs) such as the human beta-defensin-2 and -3 (hBD-2, hBD-3) in keratinocytes which may contribute to the observed beneficial effects of PRGF-based formulations in wound treatment. Given that it is unclear which components of platelets induce AMPs in keratinocytes we aimed to biochemically identify PRGF-derived factors that induce hBD-2 in human keratinocytes. As PRGF contain a wide variety of different molecules we first investigated if larger (>10 kDa), medium (10 to 3kDa) or smaller (<3 kDa) molecules induced hBD-2. Using molecular weight filtration we found that the fraction >10 kDa was most active although induction levels were lower compared to native PRGF. Notably, reassembly of all three fractions (>10 kDa, 10 to 3 kDa and <3 kDa) restored the effect of the native PRGF indicating a synergistic action of different PRGF-components to induce hBD-2 in keratinocytes. Gel filtration experiments combined by mass spectrometric analyses revealed the presence of several proteins between 30-40 kDa in the hBD-2 inducing fraction. Further purification of the hBD-2 inducing protein(s) by conventional reversed-phase (RP)-HPLC failed due to acid labile properties of the hBD-2-inducing molecules. Optimized RP-HPLC separation strategies using low acidic conditions revealed a distinct hydrophobic fraction containing hBD-2- but no hBD-3-inducing activity. Interestingly, a less hydrophobic fraction contained hBD-3-, but no hBD-2-inducing activity suggesting that PRGF contains distinct proteins that selectively induce hBD-2 and hBD-3 in keratinocytes. These experiments are the basis for the final identification of PRGF-derived mediators that support healing of chronic wounds by their capacity to induce the expression of AMPs in keratinocytes.

Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices

Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.

A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

A predominant gelatinolytic enzyme with approximately 26 kDa was observed in gelatin zymogram of immature mice uterine luminal fluid (ULF). Size exclusion analysis revealed that the native size of this enzyme was close to that of human α2-macroglobulin (α2-MG), a 725 kDa protein. This large protease was isolated by a series of chromatographic steps on the Sephacryl S-400 and DEAE-Sepharose columns. The results from gelatin zymography and SDS-PAGE analysis supported that this large protease consists of gelatinolytic enzyme and a 360 kDa protein. Through tandem mass spectrometry analysis followed by MASCOT database search, the 360 kDa protein was identified as ovostatin homolog (accession: NP_001001179.2) assigned as a homolog of chicken ovostatin, a protease inhibitor. The co-fractionation analysis by gel filtration and mouse ovostatin homolog (mOH) co-immunoprecipitation experiments demonstrated that the mOH formed a complex with three gelatinolytic enzymes in immature mice ULF. Substrate zymography analysis revealed that the mOH-associated gelatinolytic enzymes were suitable to digest type I collagen rather than type IV collagen. In addition, the refolded mOH-associated 26 kDa gelatinolytic enzyme displayed the type I collagen-digesting activity in the assay, but the other two enzymes did not have this function. RT-PCR analysis showed that mOH gene was abundantly expressed in brain, spinal cord, lung, uterus, and in 17-day embryo. Taken together, our data suggest that mOH/cognate protease system may play a potential role in regulation of tissue remodeling and fetal development.

Physical and thermodynamic characterization of the rice gibberellin receptor/gibberellin/DELLA protein complex

Gibberellins (GAs) are phytohormones that regulate various developmental processes in plants. The initial GA signalling events involve the binding of a GA to the soluble GA receptor protein GID1, followed by the binding of the complex to the negative transcriptional regulator of GA signaling, the DELLA protein. Although X-ray structures for certain Arabidopsis GID1/GA/DELLA protein complexes have previously been determined, examination of these complexes did not fully clarify how a DELLA protein recognizes and binds to a GID1/GA complex. Herein, we present a study aimed at physically defining, via a combination of gel chromatography, isothermal titration calorimetry (ITC), small-angle X-ray scattering experiments (SAXS), NMR spectroscopy and mutagenesis, how the rice DELLA protein (SLR1) binds to the rice GID1/GA complex. We have identified the shortest SLR1 sequence (M28-A112) that binds the rice GID/GA complex tightly. The binding constant for the ternary complex that includes SLR1(M28-A112) is 2.9 × 107 M−1; the binding is enthalpically driven and does not depend on the chemical nature of the bound GA. Furthermore, the results of SAXS, ITC, and gel filtration experiments indicate that when free in solution, SLR1(M28-A112) is a natively unfolded protein. The NMR experiments expand this observation to show that the unfolded mutant also contains a small amount of marginally stable secondary structure. Conversely, the protein has a highly ordered structure when bound one-to-one to GID1/GA.

Examination of disproportionate permeability reduction mechanism on rupture of hydrogels performance

In this paper, the overall performance of a disproportionate permeability reduction mechanism for a sulfonated polyacrylamide copolymer and the chromium triacetate crosslinker was investigated. A home-built gel filtration system was used for visual inspections and evaluations of the hydrogel performance. The experiments were conducted based on rheological test plans, energy-dispersive X-ray spectroscopy, and scanning electron microscope. Accordingly, quadratic equations based on the polymer and crosslinker concentrations were presented predicting the rupture pressure gradient of the hydrogel and the hydrogel output due to the oil and water injection through the hydrogel. It was indicated that the polymer concentration was the main effect on the rupture pressure gradient of the hydrogel and hydrogel output. Moreover, under constant concentration of crosslinker, increasing polymer concentration showed an increase in rupture pressure gradient and output of the hydrogel due to the increase of elastic modulus of hydrogel network and its strength as a viscoelastic material. As the hydrogel structure illustrated no rupture during the gel filtration experiments, a mechanism presented justifying the disproportionate permeability reduction phenomenon which in that under constant condition, the oil permeability through hydrogel was greater than the water permeability.

Differential biochemical properties of three canonical Dps proteins from the cyanobacterium Nostoc punctiforme suggest distinct cellular functions

DNA-binding proteins from starved cells (Dps, EC: 1.16.3.1) have a variety of different biochemical activities such as DNA-binding, iron sequestration, and H2O2 detoxification. Most bacteria commonly feature one or two Dps enzymes, whereas the cyanobacterium Nostoc punctiforme displays an unusually high number of five Dps proteins (NpDps1-5). Our previous studies have indicated physiological differences, as well as cell-specific expression, among these five proteins. Three of the five NpDps proteins, NpDps1, -2, and -3, were classified as canonical Dps proteins. To further investigate their properties and possible importance for physiological function, here we characterized and compared them in vitro Nondenaturing PAGE, gel filtration, and dynamic light-scattering experiments disclosed that the three NpDps proteins exist as multimeric protein species in the bacterial cell. We also demonstrate Dps-mediated iron oxidation catalysis in the presence of H2O2 However, no iron oxidation with O2 as the electron acceptor was detected under our experimental conditions. In modeled structures of NpDps1, -2, and -3, protein channels were identified that could serve as the entrance for ferrous iron into the dodecameric structures. Furthermore, we could demonstrate pH-dependent DNA-binding properties for NpDps2 and -3. This study adds critical insights into the functions and stabilities of the three canonical Dps proteins from N. punctiforme and suggests that each of the Dps proteins within this bacterium has a specific biochemical property and function.

Bonsai Gelsolin Survives Heat Induced Denaturation by Forming \u03b2-Amyloids which Leach Out Functional Monomer

Here, we report that minimal functional gelsolin i.e. fragment 28–161 can display F-actin depolymerizing property even after heating the protein to 80 °C. Small angle X-ray scattering (SAXS) data analysis confirmed that under Ca2+-free conditions, 28–161 associates into monomer to dimer and tetramer, which later forms β-amyloids, but in presence of Ca2+, it forms dimers which proceed to non-characterizable aggregates. The dimeric association also explained the observed decrease in ellipticity in circular dichroism experiments with increase in temperature. Importantly, SAXS data based models correlated well with our crystal structure of dimeric state of 28–161. Characterization of higher order association by electron microscopy, Congo red and ThioflavinT staining assays further confirmed that only in absence of Ca2+ ions, heating transforms 28–161 into β-amyloids. Gel filtration and other experiments showed that β-amyloids keep leaching out the monomer, and the release rates could be enhanced by addition of L-Arg to the amyloids. F-actin depolymerization showed that addition of Ca2+ ions to released monomer initiated the depolymerization activity. Overall, we propose a way to compose a supramolecular assembly which releases functional protein in sustained manner which can be applied for varied potentially therapeutic interventions.

PO-061 BCL-2 family of proteins, BCL-XL and MCL-1, regulate apoptosis and cancer cell survival by different mechanisms

IntroductionAbnormal cell survival through resistance to apoptosis is a cardinal feature of most malignancies and plays a key role in chemoresistance. As the major regulators of the intrinsic mitochondrial pathway of apoptosis, the pro-survival BCL-2 family proteins (BCL-2, BCL-XL and MCL-1) are attractive targets for novel cancer therapeutics. The concept of employing BH3 mimetics (small molecule inhibitors of the BCL-2 family) as anticancer agents has been substantiated by the efficacy of selective drugs, such as navitoclax and venetoclax, in treating BCL-2-dependent haematological malignancies. However, most solid tumours depend for survival on both BCL-XL and MCL-1, which are highly amplified in multiple cancers. Several MCL-1 inhibitors that have been generated so far and demonstrate early promise in vitro but most fail to exhibit specificity and potency in a cellular context.Material and methodsAll cell lines and primary cells, BH3 mimetics and antibodies, unless received as gift from our collaborators, were purchased from commercial vendors. Differential scanning fluorimetry (DSF) was performed using a StepOnePlus Real-Time PCR machine. The extent of cytochrome c release was quantitated using immunofluorescence and apoptosis assessed using an Attune NxT flow cytometer. Gel filtration and immunoprecipitation experiments were performed according to standard protocols. Statistical analysis was performed using a two-way ANOVA with p values: * for p≤0.05, ** for p≤0.005 and *** for p≤0.001.Results and discussionsEmploying a rapid DSF-based assay, we screened a panel of BH3 mimetics to identify that only S63845 and to a smaller extent, A-1210477, demonstrated enhanced binding to MCL-1 that correlated with a rapid, concentration-dependent apoptosis in relevant cell lines. At higher concentrations, S63845 also appeared to weakly bind BCL-2. Furthermore, S63845 synergized with other BH3 mimetics to induce apoptosis in several cancer cell lines. However, in the colorectal HCT-116 cells, BCL-XL-regulated apoptosis required all known BH3-only members, whereas apoptosis and cellular proliferation regulated by MCL-1 appeared to occur independently of all known BH3-only proteins.ConclusionThe anti-apoptotic and cell survival roles of BCL-XL and MCL-1 could be distinct, as antagonising BCL-XL induced BH3-dependent apoptosis, whereas MCL-1 appeared to regulate apoptosis even in the absence of all known BH3-only proteins.

PO-064 Tubulin-binding anti-cancer polysulfides induce cell death via mitotic arrest and autophagic interference in colon cancer

IntroductionAbnormal cell survival through resistance to apoptosis is a cardinal feature of most malignancies and plays a key role in chemoresistance. As the major regulators of the intrinsic mitochondrial pathway of apoptosis, the pro-survival BCL-2 family proteins (BCL-2, BCL-XL and MCL-1) are attractive targets for novel cancer therapeutics. The concept of employing BH3 mimetics (small molecule inhibitors of the BCL-2 family) as anticancer agents has been substantiated by the efficacy of selective drugs, such as navitoclax and venetoclax, in treating BCL-2-dependent haematological malignancies. However, most solid tumours depend for survival on both BCL-XL and MCL-1, which are highly amplified in multiple cancers. Several MCL-1 inhibitors that have been generated so far and demonstrate early promise in vitro but most fail to exhibit specificity and potency in a cellular context.Material and methodsAll cell lines and primary cells, BH3 mimetics and antibodies, unless received as gift from our collaborators, were purchased from commercial vendors. Differential scanning fluorimetry (DSF) was performed using a StepOnePlus Real-Time PCR machine. The extent of cytochrome c release was quantitated using immunofluorescence and apoptosis assessed using an Attune NxT flow cytometer. Gel filtration and immunoprecipitation experiments were performed according to standard protocols. Statistical analysis was performed using a two-way ANOVA with p values: * for p≤0.05, ** for p≤0.005 and *** for p≤0.001.Results and discussionsEmploying a rapid DSF-based assay, we screened a panel of BH3 mimetics to identify that only S63845 and to a smaller extent, A-1210477, demonstrated enhanced binding to MCL-1 that correlated with a rapid, concentration-dependent apoptosis in relevant cell lines. At higher concentrations, S63845 also appeared to weakly bind BCL-2. Furthermore, S63845 synergized with other BH3 mimetics to induce apoptosis in several cancer cell lines. However, in the colorectal HCT-116 cells, BCL-XL-regulated apoptosis required all known BH3-only members, whereas apoptosis and cellular proliferation regulated by MCL-1 appeared to occur independently of all known BH3-only proteins.ConclusionThe anti-apoptotic and cell survival roles of BCL-XL and MCL-1 could be distinct, as antagonising BCL-XL induced BH3-dependent apoptosis, whereas MCL-1 appeared to regulate apoptosis even in the absence of all known BH3-only proteins.

Purification and characterization of Stenotrophomonas maltophilia-derived l-amino acid ester hydrolase for synthesizing dipeptide, isoleucyl-tryptophan

In the present study, we purified α-amino acid ester hydrolase (AEH) from cell-free extracts of the Stenotrophomonas maltophilia strain HS1. The approximately 70-kDa AEH from S. maltophilia HS1 (SmAEH) was homogeneous in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and was present as a tetramer in gel-filtration experiments. The activity of the SmAEH enzyme was then determined by monitoring the synthesis of the antihypertensive agent dipeptide isoleucyl-tryptophan (Ile-Trp) from isoleucyl methyl ester (Ile-OMe) and tryptophan (Trp). In these experiments, SmAEH had wide substrate specificity for acyl donors, such as Gly-OMe, β-Ala-OMe, Pro-OMe and Trp-OMe and Ile-OMe, and maximal activity were observed under conditions of pH 9.0 and 30°C. SmAEH also showed the greatest stability at pH 9.0, whereas its activity was reduced by 40% after 10-min incubation at approximately 50°C. In subsequent activity assays in the presence of various metal ions, Ag+ strongly inhibited enzyme activity. Finally, SmAEH activity was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), suggesting that the protein is a serine protease.

Biochemical Characterization of Two Clinically-Relevant Human Fumarase Variants Defective for Oligomerization.

Background:Fumarase, a significant enzyme of energy metabolism, catalyzes the reversible hydration of fumarate to L-malate. Mutations in the FH gene, encoding human fumarase, are associated with fumarate hydratase deficiency (FHD) and hereditary leiomyomatosis and renal cell cancer (HLRCC). Fumarase assembles into a homotetramer, with four active sites. Interestingly, residues from three of the four subunits within the homotetramer comprise each active site. Hence, any mutation affecting oligomerization is predicted to disrupt enzyme activity.Methods:We constructed two variants of hexahistidine-tagged human recombinant fumarase, A308T and H318Y, associated with FHD and HLRCC, respectively. Both Ala308 and His318 lie within the fumarase intersubunit interface. We purified unmodified human fumarase and the two variants, and analyzed their enzymatic activities and oligomerization states in vitro.Results:Both variants showed severely diminished fumarase activity. Steady-state kinetic analysis demonstrated that the variants were largely defective due to decreased turnover rate, while displaying Km values for L-malate similar to unmodified human recombinant fumarase. Blue native polyacrylamide gel electrophoresis and gel filtration experiments revealed that each variant had an altered oligomerization state, largely forming homodimers rather than homotetramers.Conclusion:We conclude that A308T and H318Y render human fumarase enzymatically inactive via defective oligomerization. Therefore, some forms of FHD and HLRCC can be linked to improperly folded quaternary structure.