Santiago Lima

A redox-active FKBP-type immunophilin functions in accumulation of the photosystem II supercomplex in Arabidopsis thaliana

Photosystem II (PSII) catalyzes the first of two photosynthetic reactions that convert sunlight into chemical energy. Native PSII is a supercomplex consisting of core and light-harvesting chlorophyll proteins. Although the structure of PSII has been resolved by x-ray crystallography, the mechanism underlying its assembly is poorly understood. Here, we report that an immunophilin of the chloroplast thylakoid lumen is required for accumulation of the PSII supercomplex in Arabidopsis thaliana. The immunophilin, FKBP20-2, belongs to the FK-506 binding protein (FKBP) subfamily that functions as peptidyl-prolyl isomerases (PPIases) in protein folding. FKBP20-2 has a unique pair of cysteines at the C terminus and was found to be reduced by thioredoxin (Trx) (itself reduced by NADPH by means of NADP-Trx reductase). The FKBP20-2 protein, which contains only two of the five amino acids required for catalysis, showed a low level of PPIase activity that was unaffected on reduction by Trx. Genetic disruption of the FKBP20-2 gene resulted in reduced plant growth, consistent with the observed lower rate of PSII activity determined by fluorescence (using leaves) and oxygen evolution (using isolated chloroplasts). Analysis of isolated thylakoid membranes with blue native gels and immunoblots showed that accumulation of the PSII supercomplex was compromised in mutant plants, whereas the levels of monomer and dimer building blocks were elevated compared with WT. The results provide evidence that FKBP20-2 participates specifically in the accumulation of the PSII supercomplex in the chloroplast thylakoid lumen by means of a mechanism that has yet to be determined.

Dual molecular signals mediate the bacterial response to outer-membrane stress.

Stress Inside Out In Gram-negative bacteria, the integrity of the outer membrane is crucial for survival and is an important aspect of resistance to antibiotics. The biogenesis of the major components lipopolysaccharide (LPS) and outer-membrane protein (OMP) of the outer membrane begins in the cytoplasmic compartment, involves export across the inner membrane and transport through the periplasm, and finally requires active insertion into the outer membrane by specialized assembly machines. Lima et al. (p. 837) supply results for a model in which serious defects in LPS biogenesis also create problems for OMP biogenesis, thereby producing the two signals needed to activate the σE stress response pathway. Gram-negative bacteria monitor lipopolysaccharide and outer-membrane protein status to detect and respond to problems. In Gram-negative bacteria, outer-membrane integrity is essential for survival and is monitored by the σE stress-response system, which initiates damage-repair pathways. One activating signal is unassembled outer-membrane proteins. Using biochemical and genetic experiments in Escherichia coli, we found that off-pathway intermediates in lipopolysaccharide transport and assembly provided an additional required signal. These distinct signals, arising from disruptions in the transport and assembly of the major outer-membrane components, jointly determined the rate of proteolytic destruction of a negative regulator of the σE transcription factor, thereby modulating the expression of stress-response genes. This dual-signal system permits a rapid response to dysfunction in outer-membrane biogenesis, while buffering responses to transient fluctuations in individual components, and may represent a broad strategy for bacteria to monitor their interface with the environment.

Revisiting the sphingolipid rheostat: Evolving concepts in cancer therapy.

Nearly two decades have passed since it was first proposed that regulation of the interconvertible sphingolipid metabolites, ceramide and sphingosine-1-phosphate (S1P), and their opposing signaling pathways are major determinants of cell fate, a concept referred to as the “sphingolipid rheostat”. Since then, many reports have substantiated the role of the sphingolipid rheostat in cell fate determination and in the initiation, progression, and drug sensitivity of cancer. Thus, modulation of the rheostat has emerged as a focus for treatment strategies to battle cancer. S1P regulates numerous processes important for cancer including proliferation, transformation, angiogenesis, metastasis, survival, and drug resistance. Ceramide on the other hand has been linked to cell growth arrest and cell death. With the increased understanding of sphingolipid metabolism and signaling, as well as the present focus on therapies designed to modulate the levels of sphingolipids in cancer, it is an appropriate time to re-examine the sphingolipid rheostat concept and determine how it fits within the current knowledge of sphingolipid signaling in cancer.

Structure of the First Sphingosine 1-Phosphate Receptor

The lipid sphingosine 1-phosphate may laterally diffuse through the membrane to bind a receptor. The sphingosine 1-phosphate receptor 1 (S1P1) and its ligand, sphingosine 1-phosphate (S1P), have now emerged as critical regulators of lymphocyte trafficking, vascular development and integrity, and immunity. S1P1 is targeted by the phosphorylation product of fingolimod, which has been approved for the treatment of multiple sclerosis. The recent progress in the structural biology of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors has now enabled the elucidation of the structure of S1P1. Analysis of the structure, along with structure activity and mutagenesis analysis, highlighted key interactions associated with the binding of S1P and agonists and suggested that the ligand may gain access to the binding pocket by lateral diffusion within the plasma membrane. The S1P1 crystal structure will be helpful for designing ligands that specifically target S1P1.

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.

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.

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.—Kim, E. Y., Sturgill, J. L., Hait, N. C., Avni, D., Valencia, E. C., Maceyka, M., Lima, S., Allegood, J., Huang, W.‐C., Zhang, S., Milstien, S., Conrad, D., Spiegel, S., Role of sphingosine kinase 1 and sphingosine‐1‐phosphate in CD40 signaling and IgE class switching. FASEB J. 28, 4347–4358 (2014). www.fasebj.org

An integrative study to identify novel scaffolds for sphingosine kinase 1 inhibitors.

Sphingosine kinase 1 (SphK1), the enzyme that produces the bioactive sphingolipid metabolite, sphingosine-1-phosphate, is a promising new molecular target for therapeutic intervention in cancer and inflammatory diseases. In view of its importance, the main objective of this work was to find new and more potent inhibitors for this enzyme possessing different structural scaffolds than those of the known inhibitors. Our theoretical and experimental study has allowed us to identify two new structural scaffolds (three new compounds), which could be used as starting structures for the design and then the development of new inhibitors of SphK1. Our study was carried out in different steps: virtual screening, synthesis, bioassays and molecular modelling. From our results, we propose a new dihydrobenzo[b]pyrimido[5,4-f]azepine and two alkyl{3-/4-[1-hydroxy-2-(4-arylpiperazin-1-yl)ethyl]phenyl}carbamates as initial structures for the development of new inhibitors. In addition, our molecular modelling study using QTAIM calculations, allowed us to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analyses indicate that the cationic head of the different compounds must be refined in order to obtain an increase in the binding affinity of these ligands.

TP53 is required for BECN1- and ATG5-dependent cell death induced by sphingosine kinase 1 inhibition

ABSTRACT The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the enzyme that produces it, SPHK1 (sphingosine kinase 1), regulate many processes important for the etiology of cancer. It has been suggested that SPHK1 levels are regulated by the tumor suppressor protein TP53, a key regulator of cell cycle arrest, apoptosis, and macroautophagy/autophagy. However, little is still known of the relationship between TP53 and SPHK1 activity in the regulation of these processes. To explore this link, we examined the effects of inhibiting SPHK1 in wild-type and TP53 null cancer cell lines. SK1-I, an analog of sphingosine and isozyme-specific SPHK1 inhibitor, suppressed cancer cell growth and clonogenic survival in a TP53-dependent manner. It also more strongly enhanced intrinsic apoptosis in wild-type TP53 cells than in isogenic TP53 null cells. Intriguingly, SK1-I induced phosphorylation of TP53 on Ser15, which increases its transcriptional activity. Consequently, levels of TP53 downstream targets such as pro-apoptotic members of the BCL2 family, including BAX, BAK1, and BID were increased in wild-type but not in TP53 null cells. Inhibition of SPHK1 also increased the formation of autophagic and multivesicular bodies, and increased processing of LC3 and its localization within acidic compartments in a TP53-dependent manner. SK1-I also induced massive accumulation of vacuoles, enhanced autophagy, and increased cell death in an SPHK1-dependent manner that also required TP53 expression. Importantly, downregulation of the key regulators of autophagic flux, BECN1 and ATG5, dramatically decreased the cytotoxicity of SK1-I only in cells with TP53 expression. Hence, our results reveal that TP53 plays an important role in vacuole-associated cell death induced by SPHK1 inhibition in cancer cells.

Sphingosine Kinase: A Closer Look at Last

Sphingosine-1-phosphate is a potent sphingolipid mediator, and the kinase that produces it, sphingosine kinase 1 (SphK1), has been implicated in cancer progression, inflammation, and cardiovascular diseases. In this issue of Structure, Wang and colleagues provide the scientific community with the long awaited structure of SphK1.