Accurate prediction of protein structures and interactions using a three-track neural network.

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.

Ceramide synthases: Reflections on the impact of Dr. Lina M. Obeid.

Sphingolipids are a family of lipids that are critical to cell function and survival. Much of the recent work done on sphingolipids has been performed by a closely-knit family of sphingolipid researchers, which including our colleague, Dr. Lina Obeid, who recently passed away. We now briefly review where the sphingolipid field stands today, focusing in particular on areas of sphingolipid research to which Dr. Obeid made valued contributions. These include the 'many-worlds' view of ceramides and the role of a key enzyme in the sphingolipid biosynthetic pathway, namely the ceramide synthases (CerS). The CerS contain a number of functional domains and also interact with a number of other proteins in lipid metabolic pathways, fulfilling Dr. Obeid's prophecy that ceramides, and the enzymes that generate ceramides, form the critical hub of the sphingolipid metabolic pathway.

Ceramide Synthase 6: Comparative Analysis, Phylogeny and Evolution.

Ceramide synthase 6 (CerS6, also known as LASS6) is one of the six members of ceramide synthase gene family in humans. Comparisons of CerS6 amino acid sequences and structures as well as of CerS6 gene structures/locations were conducted using data from several vertebrate genome projects. A specific role for the CerS6 gene and protein has been identified as the endoplasmic reticulum C14- and C16-ceramide synthase. Mammalian CerS6 proteins share 90⁻100% similarity among different species, but are only 22⁻63% similar to other CerS family members, suggesting that CerS6 is a distinct gene family. Sequence alignments, predicted transmembrane, lumenal and cytoplasmic segments and N-glycosylation sites were also investigated, resulting in identification of the key conserved residues, including the active site as well as C-terminus acidic and serine residues. Mammalian CerS6 genes contain ten exons, are primarily located on the positive strands and transcribed as two major isoforms. The human CERS6 gene promoter harbors a large CpG island (94 CpGs) and multiple transcription factor binding sites (TFBS), which support precise transcriptional regulation and signaling functions. Additional regulation is conferred by 15 microRNA (miRNA) target sites identified in the CERS6 3'-UTR region. Phylogenetic analysis of the vertebrate CerS1⁻6 gene families relationships supports a major role for the CerS6 enzyme that is strongly conserved throughout vertebrate evolution.

The revelation of selective sphingolipid pathway inhibition mechanism on fumonisin toxin binding to ceramide synthases in susceptible organisms and survival mechanism in resistant species.

Fumonisin B1 toxin (FB1) is a well-known competitive inhibitor of ceramide synthase (CS) in yeast. However, FB1 is unable to obstruct CS from Trichoderma spp., which are well-known biocontrol agents. To explore the contrasting binding modes, a comparative structural analysis of complexes of FB1 with these two CS proteins was carried out. Formation of activation loop on the binding of substrates with the CS from yeast was observed but when inhibitor interacted with the activation loop, it transformed into helix leading to the potentially inactivated state of the enzyme. In yeast homologue of the enzyme, the inhibitor and substrates compete for the same binding site. Whereas, in the CS protein from Trichoderma guizhouense, no such competition for substrate binding site was observed and the binding pocket of the enzyme could easily accommodate FB1 molecule along with the two interacting native substrates, which may lead to the successful catalysis.

Adoptive Transfer of Ceramide Synthase 6 Deficient Splenocytes Reduces the Development of Colitis.

Sphingolipids regulate critical cellular processes including inflammation. Ceramide, which serves a central role in sphingolipid metabolism, is generated by six ceramide synthases (CerS) that differ in substrate specificity. CerS6 preferentially generates C16-ceramide and its mRNA is highly expressed in immune tissues. In this study we analyzed how deficiency of CerS6 impacts on the development of colitis using an adoptive transfer model. Adoptive transfer of CerS6-deficient splenocytes, which have significantly decreased levels of C16-ceramide, showed that CerS6-deficiency protected against the development of colitis. However, adoptively transferred cells isolated from the lamina propria of the large intestine from wild type or CerS6-deficient groups showed no differences in the percentages of immune-suppressive regulatory T cells, pro-inflammatory Th17 cells, or their ability to express IL-17. In vitro polarization of wild type or CerS6-deficient splenocytes also revealed no defects in the development of T cell subsets. Our data suggest that protection from colitis following adoptive transfer of CerS6-deficient splenocytes maybe related to their ability to migrate and proliferate in vivo rather than subset development or cytokine expression.

Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.

Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with ß-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity.

Nuclear Drosophila CerS Schlank regulates lipid homeostasis via the homeodomain, independent of the lag1p motif.

Drosophila Ceramide Synthase (CerS) Schlank regulates both ceramide synthesis and fat metabolism. Schlank contains a catalytic lag1p motif and, like many CerS in other species, a homeodomain of unknown function. Here, we show that the Drosophila CerS Schlank is imported into the nucleus and requires two nuclear localization signals (NLSs) within its homeodomain and functional Importin-ß import machinery. Expression of Schlank variants containing the homeodomain without functional lag1p motif rescued the fat metabolism phenotype of schlank mutants whereas a variant with a mutated NLS site did not rescue. Thus, the homeodomain of Schlank is involved in the regulation of lipid metabolism independent of the catalytic lag1p motif.

Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis.

Ceramide makes up the acyl-backbone of sphingolipids and plays a central role in determining the function of these essential membrane lipids. In Arabidopsis, the varied chemical composition of ceramide is determined by the specificity of three different isoforms of ceramide synthase, denoted LAG one homologue 1, -2 and -3 (LOH1, LOH2 and LOH3), for a range of long-chain base (LCB) and acyl-CoA substrates. The contribution of each of these isoforms to the synthesis of ceramide was investigated by in vitro ceramide synthase assays. The plant LCB phytosphingosine was efficiently used by the LOH1 and LOH3 isoforms, with LOH1 having the lowest Km for the LCB substrate of the three isoforms. In contrast, sphinganine was used efficiently only by the LOH2 isoform. Acyl-CoA specificity was also distinguished between the three isoforms with LOH2 almost completely specific for palmitoyl-CoA whereas the LOH1 isoform showed greatest activity with lignoceroyl- and hexacosanoyl-CoAs. Interestingly, unsaturated acyl-CoAs were not used efficiently by any isoform whereas unsaturated LCB substrates were preferred by LOH2 and 3. Fumonisin B1 (FB1) is a general inhibitor of ceramide synthases but LOH1 was found to have a much lower Ki than the other isoforms pointing towards the origin of FB1 sensitivity in plants. Overall, the data suggest distinct roles and modes of regulation for each of the ceramide synthases in Arabidopsis sphingolipid metabolism.

A gene variant in CERS2 is associated with rate of increase in albuminuria in patients with diabetes from ONTARGET and TRANSCEND.

Although albuminuria and subsequent advanced stage chronic kidney disease are common among patients with diabetes, the rate of increase in albuminuria varies among patients. Since genetic variants associated with estimated glomerular filtration rate (eGFR) were identified in cross sectional studies, we asked whether these variants were also associated with rate of increase in albuminuria among patients with diabetes from ONTARGET and TRANSCEND-randomized controlled trials of ramipril, telmisartan, both, or placebo. For 16 genetic variants associated with eGFR at a genome-wide level, we evaluated the association with annual rate of increase in albuminuria estimated from urine albumin:creatinine ratio (uACR). One of the variants (rs267734) was associated with rate of increase in albuminuria. The annual rate of increase in albuminuria among risk homozygotes (69% of the study population) was 11.3% (95%CI; 7.5% to 15.3%), compared with 5.0% (95%CI; 3.3% to 6.8%) for heterozygotes (27% of the population), and 1.7% (95%CI; -1.7% to 5.3%) for non-risk homozygotes (4% of the population); P = 0.0015 for the difference between annual rates in the three genotype groups. These estimates were adjusted for age, sex, ethnicity, and principal component of genetic heterogeneity. Among patients without albuminuria at baseline (uACR<30 mg/g), each risk allele was associated with 50% increased risk of incident albuminuria (OR = 1.50; 95%CI 1.15 to 1.95; P = 0.003) after further adjustment for traditional risk factors including baseline uACR and eGFR. The rs267734 variant is in almost perfect linkage-disequilibrium (r2 = 0.94) with rs267738, a single nucleotide polymorphism encoding a glutamic acid to alanine change at position 115 of the ceramide synthase 2 (CERS2) encoded protein. However, it is unknown whether CERS2 function influences albuminuria. In conclusion, we found that rs267734 in CERS2 is associated with rate of increase in albuminuria among patients with diabetes and elevated risk of cardiovascular disease.

A novel tumor metastasis suppressor gene LASS2/TMSG1 interacts with vacuolar ATPase through its homeodomain.

LASS2/TMSG1 was a novel tumor metastasis suppressor gene, which was first cloned by our laboratory from non-metastatic and metastatic cancer cell variants of human prostate carcinoma PC-3M using mRNA differential display in 1999. LASS2/TMSG1 could interact with the C subunit of vacuolar ATPase (V-ATPase, ATP6V0C) and regulate V-ATPase activity. In an attempt to provide molecular mechanism of the interaction between LASS2/TMSG1 and V-ATPase, we constructed four variant transfectants containing different functional domain of LASS2/TMSG1 and stably transfected the variants to human prostate cancer cell line PC-3M-1E8 cell with high metastatic potential. Results showed that there were no obvious differences of V-ATPase expression among different transfected cells and the control. However, V-ATPase activity and intracellular pH was significantly higher in the variant transfectants with Homeodomain of LASS2/TMSG1 than that in the control using the pH-dependent fluorescence probe BECEF/AM. Immunoprecipitation, immunofluorescence and immuno-electron microscope alone or in combination demonstrated the direct interaction of Homeodomain of LASS2/TMSG1 and ATP6V0C. Loss of Homeodomain markedly enhanced the proliferation ability but weakened the apoptotic effect of LASS2/TMSG1 in PC-3M-1E8 cells. These lines of results for the first time contribute to the conclusion that LASS2/TMSG1 could regulate V-ATPase activity and intracellular pH through the direct interaction of its Homeodomain and the C subunit of V-ATPase. Their interaction could play important roles in the apoptosis of tumor cells.

CLN5 and CLN8 protein association with ceramide synthase: biochemical and proteomic approaches.

Four patients with juvenile neuronal ceroid lipofuscinoses, a childhood neurodegenerative disorder that was previously described as CLN9 variant, are reclassified as CLN5 disease. CLN5-deficient (CLN5(-/-) ) fibroblasts demonstrate adhesion defects, increased growth, apoptosis, and decreased levels of ceramide, sphingomyelin, and glycosphingolipids. The CLN8 protein (CLN8p) corrects growth and apoptosis in CLN5(-/-) cells. Related proteins containing a Lag1 motif (CerS1/2/4/5/6) partially corrected these deficits, with CerS1, which is primarily expressed in brain, providing the best complementation, suggesting CLN5p activates CerS1 and may co-immunoprecipitate with it. CLN8p complements CLN5-deficient cells, consolidating the interrelationship of CLN5p/CLN8p, whose potential roles are explored as activators of (dihydro)ceramide synthases. Homozygosity mapping using microarray technology led to identification of CLN5 as the culprit gene in previously classified CLN9-defective cases. Similar to CLN5(-/-) cells, ceramide synthase activity, C16/C18:0/C24:0/C24:1 ceramide species, measured by MS is decreased in CLN8(-/-) cells. Comparison of normal versus CLN5(-/-) cell CerS1-bound proteins by immunoprecipitation, differential gel electrophoresis, and MS revealed absence of γ-actin in CLN5(-/-) cells. The γ-actin gene sequence is normal in CLN5(-/-) derived DNA. The γ-actin-bound proteins, vimentin and histones H2Afz/H3F3A/Hist1H4, were absent from the γ-actin protein complex in CLN5(-/-) cells. The function of CLN5p may require vimentin and the histone proteins to bind γ-actin. Defective binding could explain the CLN5(-/-) cellular phenotype. We explore the role of the CLN5/CLN8 proteins in ceramide species specific sphingolipid de novo synthesis, and suggest that CLN5/CLN8 proteins are more closely related than previously believed.

2-Hydroxy-ceramide synthesis by ceramide synthase family: enzymatic basis for the preference of FA chain length.

Ceramide is unusually abundant in epidermal stratum corneum and is important for permeability barrier function. Ceramides in epidermis also comprise an unusual variety, including 2-hydroxy (alpha-hydroxy)-ceramide. Six mammalian ceramide synthase/longevity assurance homologue (CerS/LASS) family members have been identified as synthases responsible for ceramide (CER) production. We reveal here that of the six, CerS3/LASS3 mRNA is the most predominantly expressed in keratinocytes. Moreover, its expression is increased upon differentiation. CerS family members have known substrate specificities for fatty acyl-CoA chain length and saturation, yet their abilities to produce 2-hydroxy-CER have not been examined. In the present study, we demonstrate that all CerS members can produce 2-hydroxy-CER when overproduced in HEK 293T cells. Each produced a 2-hydroxy-CER with a chain length similar to that of the respective nonhydroxy-CER produced. In HeLa cells overproducing the FA 2-hydroxylase FA2H, knock-down of CerS2 resulted in a reduction in total long-chain 2-hydroxy-CERs, confirming enzyme substrate specificity for chain length. In vitro CerS assays confirmed the ability of CerS1 to utilize 2-hydroxy-stearoyl-CoA as a substrate. These results suggest that all CerS members can synthesize 2-hydroxy-CER with specificity for 2-hydroxy-fatty acyl-CoA chain length and that CerS3 may be important in CER and 2-hydroxy-CER synthesis in epidermis.