Targeting defective sphingosine kinase 1 in Niemann-Pick type C disease with an activator mitigates cholesterol accumulation.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism.

A simple method for sphingolipid analysis of tissues embedded in optimal cutting temperature compound.

MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of OCT compound used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT compound also interferes with protein quantification assays, and that the presence of OCT compound impacts the quantification of extracted sphingolipids by LC-ESI-MS/MS. We developed and validated a simple and inexpensive method that removes OCT compound from OCT compound-embedded tissues. Our results indicate that removal of OCT compound from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC-ESI-MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer and to determine the long-term stability of sphingolipids in OCT compound-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT compound-cryopreserved normal lung tissues. This validated sphingolipidomic OCT compound-removal protocol should be a valuable addition to the lipid biologist's toolbox.

Design of new quinolin-2-one-pyrimidine hybrids as sphingosine kinases inhibitors.

Sphingosine-1-phosphate is now emerging as an important player in cancer, inflammation, autoimmune, neurological and cardiovascular disorders. Abundance evidence in animal and humans cancer models has shown that SphK1 is linked to cancer. Thus, there is a great interest in the development new SphK1 inhibitors as a potential new treatment for cancer. In a search for new SphK1 inhibitors we selected the well-known SKI-II inhibitor as the starting structure and we synthesized a new inhibitor structurally related to SKI-II with a significant but moderate inhibitory effect. In a second approach, based on our molecular modeling results, we designed new structures based on the structure of PF-543, the most potent known SphK1 inhibitor. Using this approach, we report the design, synthesis and biological evaluation of a new series of compounds with inhibitory activity against both SphK1 and SphK2. These new inhibitors were obtained incorporating new connecting chains between their polar heads and hydrophobic tails. On the other hand, the combined techniques of molecular dynamics simulations and QTAIM calculations provided complete and detailed information about the molecular interactions that stabilize the different complexes of these new inhibitors with the active sites of the SphK1. This information will be useful in the design of new SphK inhibitors.

Synthesis and biological evaluation of sphingosine kinase 2 inhibitors with anti-inflammatory activity.

The synthesis of inhibitors of SphK2 with novel structural scaffolds is reported. These compounds were designed from a molecular modeling study, in which the molecular interactions stabilizing the different complexes were taken into account. Particularly interesting is that 7-bromo-2-(2-phenylethyl)-2,3,4,5-tetrahydro-1,4-epoxynaphtho[1,2-b]azepine, which is a selective inhibitor of SphK2, does not exert any cytotoxic effects and has a potent anti-inflammatory effect. It was found to inhibit mononuclear cell adhesion to the dysfunctional endothelium with minimal impact on neutrophil-endothelial cell interactions. The information obtained from our theoretical and experimental study can be useful in the search for inhibitors of SphK2 that play a prominent role in different diseases, especially in inflammatory and cardiovascular disorders.

Sphingosine and Sphingosine Kinase 1 Involvement in Endocytic Membrane Trafficking.

The balance between cholesterol and sphingolipids within the plasma membrane has long been implicated in endocytic membrane trafficking. However, in contrast to cholesterol functions, little is still known about the roles of sphingolipids and their metabolites. Perturbing the cholesterol/sphingomyelin balance was shown to induce narrow tubular plasma membrane invaginations enriched with sphingosine kinase 1 (SphK1), the enzyme that converts the bioactive sphingolipid metabolite sphingosine to sphingosine-1-phosphate, and suggested a role for sphingosine phosphorylation in endocytic membrane trafficking. Here we show that sphingosine and sphingosine-like SphK1 inhibitors induced rapid and massive formation of vesicles in diverse cell types that accumulated as dilated late endosomes. However, much smaller vesicles were formed in SphK1-deficient cells. Moreover, inhibition or deletion of SphK1 prolonged the lifetime of sphingosine-induced vesicles. Perturbing the plasma membrane cholesterol/sphingomyelin balance abrogated vesicle formation. This massive endosomal influx was accompanied by dramatic recruitment of the intracellular SphK1 and Bin/Amphiphysin/Rvs domain-containing proteins endophilin-A2 and endophilin-B1 to enlarged endosomes and formation of highly dynamic filamentous networks containing endophilin-B1 and SphK1. Together, our results highlight the importance of sphingosine and its conversion to sphingosine-1-phosphate by SphK1 in endocytic membrane trafficking.

Magnesium hydroxide-based dentifrice as an anti-erosive agent in an in situ intrinsic erosion model.

PURPOSE: To evaluate in situ a magnesium hydroxide-[Mg(OH)2] based dentifrice on enamel erosion. METHODS: Human dental enamel slabs were selected by surface microhardness and randomly assigned to one out of the following three groups (n=18): non-fluoride (control), NaF (1,450 ppm F), and Mg(OH)2 dentifrices. 18 volunteers were enrolled in a randomized, crossover and double-blind study, with three phases in 5 days. They wore acrylic palatal appliances containing two human enamel slabs, which were treated with one of the three dentifrices. During each experimental phase, the specimens were subjected to erosion by immersion in 0.01 M HCl for 60 seconds, 4x/day, followed by a 1-minute treatment with the correspondent slurry (saliva/dentifrice). Enamel changes were determined by the percentage of surface hardness loss (%SHL) and mechanical profilometry analysis. Data were analyzed by ANOVA, followed by Tukey's test (P< 0.05). RESULTS: The means (SD) for %SHL and surface wear (µm) were, respectively, as follows: control [50.67(17.48), 2.70(1.24) ], NaF [45.45(15.44), 1.95(0.70) ] and Mg(OH)2 [53.94(19.48), 1.95(0.67) ]. There was no statistically significant difference among the treated and control groups for %SHL (P= 0.349); however, for wear rates, a statistically significant difference was found between the groups treated with NaF and Mg(OH)2 and the control group (P= 0.04). CLINICAL SIGNIFICANCE: Dentifrices containing magnesium hydroxide or sodium fluoride might be an important strategy to minimize the effects of erosive challenges.

Nutritional Performance of Cattle Grazing during Rainy Season with Nitrogen and Starch Supplementation.

The objective of this work was to evaluate the effects of supplementation with nitrogen and starch on the nutritional performance of grazing cattle during the rainy season. Five rumen cannulated Nellore steers, averaging 211 kg of body weight (BW), were used. Animals grazed on five signal grass paddocks. Five treatments were evaluated: control (forage only), ruminal supplementation with nitrogen at 1 g of crude protein (CP)/kg BW, ruminal supplementation with starch at 2.5 g/kg BW, supplementation with nitrogen (1 g CP/kg BW) and starch (2.5 g/kg BW), and supplementation with nitrogen (1 g CP/kg BW) and a mixture of corn starch and nitrogenous compounds (2.5 g/kg BW), thereby resulting in an energy part of the supplement with 150 g CP/kg of dry matter (DM). This last treatment was considered an additional treatment. The experiment was carried out according to a 5 ×5 Latin square design following a 2×2+1 factorial arrangement (with or without nitrogen, with or without starch, and the additional treatment). Nitrogen supplementation did not affect (p>0.10) forage intake. Starch supplementation increased (p<0.10) total intake but did not affect (p<0.10) forage intake. There was an interaction between nitrogen and starch (p<0.10) for organic matter digestibility. Organic matter digestibility was increased only by supplying starch and nitrogen together. Nitrogen balance (NB) was increased (p<0.10) by the nitrogen supplementation as well as by starch supplementation. Despite this, even though a significant interaction was not observed (p>0.10), NB obtained with nitrogen plus starch supplementation was greater than NB obtained with either nitrogen or starch exclusive supplementation. Supplementation with starch and nitrogen to beef cattle grazing during the rainy season can possibly improve digestion and nitrogen retention in the animal..

Role of sphingosine kinase 1 and sphingosine-1-phosphate in CD40 signaling and IgE class switching.

The tumor necrosis factor (TNF) receptor family member CD40 plays an essential role in the activation of antigen-presenting cells, B cell maturation, and immunoglobulin (Ig) class switching critical for adaptive immunity. Although the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the kinase that produces it, sphingosine kinase 1 (SphK1), have long been implicated in the actions of TNF mediated by engagement of TNFR1, nothing is yet known of their role in CD40-mediated events. We have now found that ligation of CD40 activates and translocates SphK1 to the plasma membrane, leading to generation of S1P. SphK1 inhibition in human tonsil B cells, as well as inhibition or deletion of SphK1 in mouse splenic B cells, significantly reduced CD40-mediated Ig class switching and plasma cell differentiation ex vivo. Optimal activation of downstream CD40 signaling pathways, including NF-κB, p38, and JNK, also required SphK1. In mice treated with a SphK1 inhibitor or in SphK1(-/-) mice, isotype switching to antigen-specific IgE was decreased in vivo by 70 and 55%, respectively. Our results indicate that SphK1 is important for CD40-mediated B cell activation and regulation of humoral responses and suggest that targeting SphK1 might be a useful therapeutic approach to control antigen-specific IgE production.

A real-time high-throughput fluorescence assay for sphingosine kinases.

Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC50 values of inhibitors determined were consistent with those reported with other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format.

Serious games are more than just games. / Los serious games son más que juegos.

Serious games (SG) or educational games are complete games designed for a specific purpose that fulfill both their classic function of entertainment and promote the learning of specific concepts or skills and optimize health care in general. In the pediatric setting, these games combine strategies to educate about health issues, promote healthy behaviors, provide therapy or medical treatment. SG have been shown to promote adherence to treatment in children with chronic diseases, reduce anxiety in those undergoing invasive medical procedures, and stimulate the development of cognitive, emotional, or psychomotor skills. However, it is important to emphasize that the success of SG in pediatrics depends to a large extent on game quality, their design based on clear objectives, and their accurate adaptation to the individual needs and preferences of patients.

Los juegos serios, serious games (SG) o juegos formativos son juegos completos diseñados con un propósito determinado, que cumplen tanto su función clásica de entretenimiento como la de estimular el aprendizaje de conceptos o habilidades específicas y optimizar la atención médica en general. En el ámbito de la pediatría, estos juegos combinan estrategias para educar sobre temas de salud, promover comportamientos saludables, proporcionar terapia o tratamiento médico. Los SG han demostrado favorecer la adherencia al tratamiento en niños con enfermedades crónicas, reducir la ansiedad en aquellos que se enfrentan a procedimientos médicos invasivos y estimular el desarrollo de habilidades cognitivas, emocionales y/o psicomotoras. Sin embargo, es importante destacar que el éxito de los SG en pediatría depende en gran medida de la calidad de los juegos, del diseño basado en objetivos claros y de su adaptación precisa a las necesidades y preferencias individuales de los pacientes.

GBA Regulates EMT/MET and Chemoresistance in Squamous Cell Carcinoma Cells by Modulating the Cellular Glycosphingolipid Profile.

Glycosphingolipids (GSL) are plasma membrane components that influence molecular processes involved in cancer initiation, progression, and therapeutic responses. They also modulate receptor tyrosine kinases involved in EMT. Therefore, understanding the mechanisms that regulate GSLs in cancer has important therapeutic potential. One critical regulator of GSLs is the lysosomal glucosylceramidase ß1 (GBA) that catalyzes the last step in GSL degradation. We show that, in cancer, GBA copy number amplifications and increased expression are widespread. We show that depleting GBA in squamous cell carcinoma cell lines results in a mesenchymal-to-epithelial shift, decreased invasion and migration, increased chemotherapeutic sensitivity, and decreased activation of receptor tyrosine kinases that are involved in regulating EMT. Untargeted lipidomics shows that GBA depletion had significant effects on sphingolipids and GSLs, suggesting that increased GBA activity in cancer sustains EMT and chemoresistance by modulating receptor tyrosine kinase activity and signaling via effects on the cellular lipid profile.

Lipidomic Profiling Reveals Biological Differences between Tumors of Self-Identified African Americans and Non-Hispanic Whites with Cancer.

In the US, the incidence and mortality of many cancers are disproportionately higher in African Americans (AA). Yet, AA remain poorly represented in molecular studies investigating the roles that biological factors might play in the development, progression, and outcomes of many cancers. Given that sphingolipids, key components of mammalian cellular membranes, have well-established roles in the etiology of cancer progression, malignancy, and responses to therapy, we conducted a robust mass spectrometry analysis of sphingolipids in normal adjacent uninvolved tissues and tumors of self-identified AA and non-Hispanic White (NHW) males with cancers of the lung, colon, liver, and head and neck and of self-identified AA and NHW females with endometrial cancer. In these cancers, AA have worse outcomes than NHW. The goal of our study was to identify biological candidates to be evaluated in future preclinical studies targeting race-specific alterations in the cancers of AA. We have identified that various sphingolipids are altered in race-specific patterns, but more importantly, the ratios of 24- to 16-carbon fatty acyl chain-length ceramides and glucosylceramides are higher in the tumors of AA. As there is evidence that ceramides with 24-carbon fatty acid chain length promote cellular survival and proliferation, whereas 16-carbon chain length promote apoptosis, these results provide important support for future studies tailored to evaluate the potential roles these differences may play in the outcomes of AA with cancer.

Designing an Atopic Dermatitis Community that Integrates Patient Self-Uploaded Information into the EHR to Optimize Follow-up.

Atopic dermatitis is a common chronic dermatological disease in childhood that can affect people's quality of life. The aim of this study was to inquire about the difficulties, needs and interests related to the disease that people with eczema and their caregivers have; in order to develop a tool that is useful for the follow-up of the illness. Electronic surveys were sent to potential users and interviews were conducted with professionals who are specialized on the subject. The main findings allowed us to understand the challenges and situations they face on a daily basis, such as the difficulties related to the family support, the queries on the eczema flare-ups, the struggles with the adherence to treatment and the needs of optimizing their quality of life. These results helped us design a tool that allows patients and their companions to better monitor their disease while optimizing communication with their health professionals.

Properties of tryptophan indole-lyase from a piezophilic bacterium, Photobacterium profundum SS9.

Tryptophan indole-lyase (Trpase), PBPRA2532, from Photobacterium profundum SS9, a piezophilic marine bacterium, has been cloned, expressed in Escherichia coli, and purified. The P. profundum Trpase (PpTrpase) exhibits similar substrate specificity as the enzyme from E. coli (EcTrpase). PpTrpase has an optimum temperature for activity at about 30°C, compared with 53°C for EcTrpase, and loses activity rapidly (t(1/2)∼30min) when incubated at 50°C, while EcTrpase is stable up to 65°C. PpTrpase retains complete activity when incubated more than 3h at 0°C, while EcTrpase has only about 20% remaining activity. Under hydrostatic pressure, PpTrpase remains fully active up to 100MPa (986atm), while EcTrpase exhibits only about 10% activity at 100MPa. PpTrpase forms external aldimine and quinonoid intermediates in stopped-flow experiments with l-Trp, S-Et-l-Cys, S-benzyl-l-Cys, oxindolyl-l-Ala, l-Ala and l-Met, similar to EcTrpase. However, with l-Trp a gem-diamine is observed that decays to a quinonoid complex. An aminoacrylate is observed with l-Trp in the presence of benzimidazole, as was seen previously with EcTrpase [28] but not with S-Et-l-Cys. The results show that PpTrpase is adapted for optimal activity in the low temperature, high pressure marine environment.

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis.

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Insights into the mechanism of Pseudomonas dacunhae aspartate beta-decarboxylase from rapid-scanning stopped-flow kinetics.

The mechanism of wild-type and R37A mutant Pseudomonas dacunhae aspartate beta-decarboxylase (ABDC) was studied by rapid-scanning stopped-flow spectrophotometry. Mixing wild-type ABDC with 50 mM disodium l-Asp resulted in the formation of a 325 nm absorption peak within the dead time of the stopped-flow instrument, likely the ketimine of pyridoxamine 5'-phosphate and oxaloacetate or pyruvate. After consumption of the l-Asp, the 360 nm feature of the resting enzyme was restored. Thus, the 325 nm species is a catalytically competent intermediate. In contrast, mixing wild-type ABDC with the disodium salt of either threo- or erythro-beta-hydroxy-dl-Asp at 50 mM resulted in a much slower formation of the 325 nm complex, with an apparent rate constant of approximately 1 or 0.006 s(-1), respectively. When wild-type ABDC is mixed with disodium succinate, a nonreactive analogue of l-Asp, formation of a new peak at 425 nm is observed. The apparent rate constant for formation of the 425 nm band exhibits a hyperbolic dependence on succinate concentration, showing that there is a rapid binding equilibrium, followed by a slower reaction in which the internal aldimine is protonated on the Schiff base N. Hydrostatic pressure shifts the spectrum from the 425 nm form to the 360 nm form, consistent with a conformational change. It is likely that the binding of substrate or analogues induces a conformational change that releases strain in the Lys pyridoxal 5'-phosphate Schiff base and increases the pK(a), resulting in protonation of the Schiff base to initiate transaldimination. Mixing of R37A mutant ABDC with 50 mM l-Asp also results in the formation of the 325 nm complex, but with an apparent rate constant of 0.2 s(-1), at least 5000-fold slower than the rate of wild-type ABDC. In contrast to wild-type ABDC, R37A ABDC shows no change in the cofactor spectrum when mixed with disodium succinate. These results suggest that Arg-37, a conserved active site residue in ABDC, plays a role in modulating the pK(a) of the pyridoxal 5'-phosphate complexes during catalysis.

Influence of nonprotective autophagy and the autophagic switch on sensitivity to cisplatin in non-small cell lung cancer cells.

While therapy-induced autophagy is conventionally conceived to be cytoprotective in nature, previous studies have identified multiple functions of autophagy, including a nonprotective form, as well as the existence of a switch between the different forms of autophagy. The current work provides further evidence of an autophagic switch, in this case in response to the antitumor drug, cisplatin, in non-small cell lung cancer cells that are either wild-type (p53wt) or functionally null in p53 (crp53), the latter generated using CRISPR/Cas9 technology. Pharmacological and genetic inhibition of autophagy identified nonprotective autophagy in p53wt cells and cytoprotective autophagy in crp53 cells. Furthermore, differences in cisplatin sensitivity between the two cell lines proved to be largely a function of the nature of the autophagy. Specifically, autophagy inhibition in the crp53 cells converts the temporal profile for the loss of cell viability in response to cisplatin to essentially parallel that observed in the p53wt cells. This enhanced sensitivity is due to cisplatin-induced apoptosis that occurs without necessitating the restoration of functional p53. In contrast, inhibition of autophagy has no observable impact on the temporal response profile exhibited in response to cisplatin in the p53wt cells, or the extent of cisplatin-induced apoptosis in the p53wt cells, consistent with the functional definition of nonprotective autophagy. Taken together, our current studies provide evidence that nonprotective autophagy in p53wt non-small cell lung cancer cells can be "switched" to protective autophagy in isogenic crp53 cells, and furthermore that inhibition of cytoprotective autophagy is sufficient to restore cisplatin sensitivity in the crp53 cells, largely through the increased promotion of apoptosis, despite the absence of functional p53.

The roles of Ser-36, Asp-132 and Asp-201 in the reaction of Pseudomonas fluorescens Kynureninase.

Kynureninase from Pseudomonas fluorescens (Pfkynase) catalyzes the pyridoxal-5'-phosphate (PLP) dependent hydrolytic cleavage of L-kynurenine to give anthranilate and L-alanine. Asp-132 and Asp-201 are located in the structure near the pyridine NH of the PLP, with Asp-201 forming a hydrogen bond. Mutation of Asp-132 to alanine and glutamate and Asp-201 to glutamate results in reduced catalytic activity with L-kynurenine and ß-benzoyl-L-alanine, but not O-benzoyl-l-serine. D132A, D132E D201E and S36A mutant Pfkynases all can form quinonoid and vinylogous amide intermediates with ß-benzoyl-L-alanine, similar to wild-type enzyme. D132A, D132E, and D201E Pfkynase react more slowly with ß-benzoyl-L-alanine and benzaldehyde to form an aldol product absorbing at 490 nm than wild-type, with D132E reacting the slowest. The 1H NMR spectra of wild-type and D201E Pfkynase are very similar in the low field region from 10 to 18 ppm, but that of D132A Pfkynase is missing a resonance at 13.1 ppm. These results show that these residues modulate the reactivity of the PLP at different stages during the reaction cycle. Ser-36 is located near the expected location of the carbonyl oxygen of the substrate. Mutation of Ser-36 to alanine results in a 230-fold reduction of kcat and 30-fold reduction in kcat/Km with L-kynurenine, but very little effect on the reaction of O-benzoyl-l-serine. Thus, the rate-determining step in the reaction of S36A Pfkynase is the Cß-Cγ bond cleavage. These results support the hypothesis that Ser-36 together with Tyr-226 is part of an oxyanion hole that polarizes the carbonyl of the substrate in the catalytic mechanism of Pfkynase.

Differential Radiation Sensitivity in p53 Wild-Type and p53-Deficient Tumor Cells Associated with Senescence but not Apoptosis or (Nonprotective) Autophagy.

Studies of radiation interaction with tumor cells often focus on apoptosis as an end point; however, clinically relevant doses of radiation also promote autophagy and senescence. Moreover, functional p53 has frequently been implicated in contributing to radiation sensitivity through the facilitation of apoptosis. To address the involvement of apoptosis, autophagy, senescence and p53 status in the response to radiation, the current studies utilized isogenic H460 non-small cell lung cancer cells that were either p53-wild type (H460wt) or null (H460crp53). As anticipated, radiosensitivity was higher in the H460wt cells than in the H460crp53 cell line; however, this differential radiation sensitivity did not appear to be a consequence of apoptosis. Furthermore, radiosensitivity did not appear to be reduced in association with the promotion of autophagy, as autophagy was markedly higher in the H460wt cells. Despite radiosensitization by chloroquine in the H460wt cells, the radiation-induced autophagy proved to be essentially nonprotective, as inhibition of autophagy via 3-methyl adenine (3-MA), bafilomycin A1 or ATG5 silencing failed to alter radiation sensitivity or promote apoptosis in either the H460wt or H460crp53 cells. Radiosensitivity appeared to be most closely associated with senescence, which occurred earlier and to a greater extent in the H460wt cells. This finding is consistent with the in-depth proteomics analysis on the secretomes from the H460wt and H460crp53 cells (with or without radiation exposure) that showed no significant association with radioresistance-related proteins, whereas several senescence-associated secretory phenotype (SASP) factors were upregulated in H460wt cells relative to H460crp53 cells. Taken together, these findings indicate that senescence, rather than apoptosis, plays a central role in determination of radiosensitivity; furthermore, autophagy is likely to have minimal influence on radiosensitivity under conditions where autophagy takes the nonprotective form.