Enzymes in the glutathione transferase (GST) superfamily catalyze the conjugation of glutathione (GSH) to electrophilic substrates. As a consequence they are involved in a number of key biological processes, including protection of cells against chemical damage, steroid and prostaglandin biosynthesis, tyrosine catabolism, and cell apoptosis. Although virtual screening has been used widely to discover substrates by docking potential noncovalent ligands into active site clefts of enzymes, docking has been rarely constrained by a covalent bond between the enzyme and ligand. In this study, we investigate the accuracy of docking poses and substrate discovery in the GST superfamily, by docking 6738 potential ligands from the KEGG and MetaCyc compound libraries into 14 representative GST enzymes with known structures and substrates using the PLOP program [Jacobson et al. Proteins 2004, SS, 351]. For X-ray structures as receptors, one of the top 3 ranked models is within 3 angstrom all-atom root mean square deviation (RMSD) of the native complex in 11 of the 14 cases; the enrichment LogAUC value is better than random in all cases, and better than 25 in 7 of 11 cases. For comparative models as receptors, near-native ligand-enzyme configurations are often sampled but difficult to rank highly. For models based on templates with the highest sequence identity, the enrichment LogAUC is better than 25 in 5 of 11 cases, not significantly different from the crystal structures. In conclusion, we show that covalent docking can be a useful tool for substrate discovery and point out specific challenges for future method improvement.

The cytosolic glutathione transferase (cytGST) superfamily comprises more than 13,000 nonredundant sequences found throughout the biosphere. Their key roles in metabolism and defense against oxidative damage have led to thousands of studies over several decades. Despite this attention, little is known about the physiological reactions they catalyze and most of the substrates used to assay cytGSTs are synthetic compounds. A deeper understanding of relationships across the superfamily could provide new clues about their functions. To establish a foundation for expanded classification of cytGSTs, we generated similarity-based subgroupings for the entire superfamily. Using the resulting sequence similarity networks, we chose targets that broadly covered unknown functions and report here experimental results confirming GST-like activity for 82 of them, along with 37 new 3D structures determined for 27 targets. These new data, along with experimentally known GST reactions and structures reported in the literature, were painted onto the networks to generate a global view of their sequence-structure-function relationships. The results show how proteins of both known and unknown function relate to each other across the entire superfamily and reveal that the great majority of cytGSTs have not been experimentally characterized or annotated by canonical class. A mapping of taxonomic classes across the superfamily indicates that many taxa are represented in each subgroup and highlights challenges for classification of superfamily sequences into functionally relevant classes. Experimental determination of disulfide bond reductase activity in many diverse subgroups illustrate a theme common for many reaction types. Finally, sequence comparison between an enzyme that catalyzes a reductive dechlorination reaction relevant to bioremediation efforts with some of its closest homologs reveals differences among them likely to be associated with evolution of this unusual reaction. Interactive versions of the networks, associated with functional and other types of information, can be downloaded from the Structure-Function Linkage Database (SFLD; http://sfld.rbvi.ucsf.edu).

Published Ahead of Print 23 September 2013. 10.1128/AAC.00734-13. 2013, 57(12):6063. DOI: Antimicrob. Agents Chemother. Michael J. Arrowood and Conor R. Caffrey Jacobson, Chakrapani Kalyanaraman, James H. McKerrow, Chow, Zachary Mackey, Kirkwood M. Land, Matthew P. Victoria Marcus, Susan T. Mashiyama, Judy Sakanari, Eric Momar Ndao, Milli Nath-Chowdhury, Mohammed Sajid, Infection from a Lethal Cryptosporidium parvum A Cysteine Protease Inhibitor Rescues Mice

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible to C. parvum, oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo, and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis.

We performed a genome-level computational study of sequence and structure similarity, the latter using crystal structures and models, of the proteases of Homo sapiens and the human parasite Trypanosoma brucei. Using sequence and structure similarity networks to summarize the results, we constructed global views that show visually the relative abundance and variety of proteases in the degradome landscapes of these two species, and provide insights into evolutionary relationships between proteases. The results also indicate how broadly these sequence sets are covered by three-dimensional structures. These views facilitate cross-species comparisons and offer clues for drug design from knowledge about the sequences and structures of potential drug targets and their homologs. Two protease groups ("M32" and "C51") that are very different in sequence from human proteases are examined in structural detail, illustrating the application of this global approach in mining new pathogen genomes for potential drug targets. Based on our analyses, a human ACE2 inhibitor was selected for experimental testing on one of these parasite proteases, TbM32, and was shown to inhibit it. These sequence and structure data, along with interactive versions of the protein similarity networks generated in this study, are available at http://babbittlab.ucsf.edu/resources.html.

Background Blood flukes of the genus Schistosoma are platyhelminth parasites that infect 200 million people worldwide. Digestion of nutrients from the host bloodstream is essential for parasite development and reproduction. A network of proteolytic enzymes (proteases) facilitates hydrolysis of host hemoglobin and serum proteins. Methodology/Principal Findings We identified a new cathepsin L termed SmCL3 using PCR strategies based on S. mansoni EST sequence data. An ortholog is present in Schistosoma japonicum. SmCL3 was heterologously expressed as an active enzyme in the yeast, Pichia pastoris. Recombinant SmCL3 has a broad pH activity range against peptidyl substrates and is inhibited by Clan CA protease inhibitors. Consistent with a function in degrading host proteins, SmCL3 hydrolyzes serum albumin and hemoglobin, is localized to the adult gastrodermis, and is expressed mainly in those life stages infecting the mammalian host. The predominant form of SmCL3 in the parasite exists as a zymogen, which is unusual for proteases. This zymogen includes an unusually long prodomain with alpha helical secondary structure motifs. The striking specificity of SmCL3 for amino acids with large aromatic side chains (Trp and Tyr) at the P2 substrate position, as determined with positional scanning-synthetic combinatorial library, is consistent with a molecular model that shows a large and deep S2 pocket. A sequence similarity network (SSN) view clusters SmCL3 and other cathepsins L in accordance with previous large-scale phylogenetic analyses that identify six super kingdoms. Conclusions/Significance SmCL3 is a gut-associated cathepsin L that may contribute to the network of proteases involved in degrading host blood proteins as nutrients. Furthermore, this enzyme exhibits some unusual sequence and biophysical features that may result in additional functions. The visualization of network inter-relationships among cathepsins L suggests that these enzymes are suitable 'marker sequences' for inclusion in future phylogenetic analyses.

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

Improvements are made to our gas-chromatography–mass-spectrometry-based assay for quantifying low levels of DNA-uracil. Folate deficiency leads to increased deoxyuridine monophosphate/thymidylate (dUMP/dTMP) ratios and uracil misincorporation into DNA, which may increase cancer risk. Vitamin B6 (B6) deficiency might also result in increased DNA-uracil because B6 is a cofactor for serine hydroxymethyltransferase, which catalyzes the methylation of tetrahydrofolate (THF) to methylene-THF, the folate form that is required to convert dUMP to dTMP. However, the low baseline levels of DNA-uracil in healthy human lymphocytes are difficult to measure accurately. This version of the assay (Uracil assay V3) has an approximately 10-fold increase in signal strength over the previous method and a 10-fold lower detection limit (0.2 pg uracil). Five micrograms of DNA, the amount in about 1ml of human blood, is a suitable amount for this assay. Using this improved assay, DNA-uracil was measured in lymphocytes from 12 healthy smoking or nonsmoking young men and women who consumed a B6-restricted diet (0.7mg B6/day, or ≈half the recommended dietary allowance) for 28 days. DNA-uracil concentration was not significantly related to B6 status or smoking. More severe and/or prolonged B6 deficiency may be necessary to detect significant changes in DNA-uracil in humans. The average concentration of DNA-uracil in these subjects was found to be approximately 3000 uracils per diploid lymphocyte, which is comparable to steady state levels of one of the oxidative adducts of DNA, 8-oxoguanine.

Folate deficiency increases dUMP/dTMP ratios and uracil misincorporation into DNA, which may increase cancer risk. Because vitamin B-6 (B6) is a required cofactor for serine hydroxymethyltransferase, catalyzing the methylation of tetrahydrofolate (THF) to 5,10-methylene THF, B6 deficiency may have similar consequences. Improvements made to our gas chromatography-mass spectrometry based assay for quantifying uracil in DNA resulted in an approximately 10-fold increase in signal strength over the previous method, and a 10-fold lower detection limit (0.2 pg uracil). Five micrograms of DNA, the amount in about 1 mL of human blood, is sufficient for this assay. DNA-uracil was measured in lymphocytes from 12 healthy young men and women who consumed a B6 restricted diet (0.7 mg B6/day, or half the RDA) for 28 days. No significant increases in DNA-uracil concentration were found with marginal B6 deficiency. The average concentration of DNA-uracil was found to be approximately 3,000 uracils per diploid lymphocyte, comparable to steady state levels of an oxidative adduct of DNA, 8-oxoguanine. More severe and/or prolonged B6 deficiency may be necessary to detect significant changes in DNA-uracil in humans. Support: NIH grants NCCAM K05 AT001323, R21 AT001918, NCMHD P60 MD00222, NFCR grant M2661 (BNA); R03 CA89722 (TDS)

Metagenomics projects based on shotgun sequencing of populations of micro-organisms yield insight into protein families. We used sequence similarity clustering to explore proteins with a comprehensive dataset consisting of sequences from available databases together with 6.12 million proteins predicted from an assembly of 7.7 million Global Ocean Sampling (GOS) sequences. The GOS dataset covers nearly all known prokaryotic protein families. A total of 3,995 medium- and large-sized clusters consisting of only GOS sequences are identified, out of which 1,700 have no detectable homology to known families. The GOS-only clusters contain a higher than expected proportion of sequences of viral origin, thus reflecting a poor sampling of viral diversity until now. Protein domain distributions in the GOS dataset and current protein databases show distinct biases. Several protein domains that were previously categorized as kingdom specific are shown to have GOS examples in other kingdoms. About 6,000 sequences (ORFans) from the literature that heretofore lacked similarity to known proteins have matches in the GOS data. The GOS dataset is also used to improve remote homology detection. Overall, besides nearly doubling the number of current proteins, the predicted GOS proteins also add a great deal of diversity to known protein families and shed light on their evolution. These observations are illustrated using several protein families, including phosphatases, proteases, ultraviolet-irradiation DNA damage repair enzymes, glutamine synthetase, and RuBisCO. The diversity added by GOS data has implications for choosing targets for experimental structure characterization as part of structural genomics efforts. Our analysis indicates that new families are being discovered at a rate that is linear or almost linear with the addition of new sequences, implying that we are still far from discovering all protein families in nature.

The Structure-Function Linkage Database (SFLD, http://sfld.rbvi.ucsf.edu/) is a manually curated classification resource describing structure-function relationships for functionally diverse enzyme superfamilies. Members of such superfamilies are diverse in their overall reactions yet share a common ancestor and some conserved active site features associated with conserved functional attributes such as a partial reaction. Thus, despite their different functions, members of these superfamilies 'look alike', making them easy to misannotate. To address this complexity and enable rational transfer of functional features to unknowns only for those members for which we have sufficient functional information, we subdivide superfamily members into subgroups using sequence information, and lastly into families, sets of enzymes known to catalyze the same reaction using the same mechanistic strategy. Browsing and searching options in the SFLD provide access to all of these levels. The SFLD offers manually curated as well as automatically classified superfamily sets, both accompanied by search and download options for all hierarchical levels. Additional information includes multiple sequence alignments, tab-separated files of functional and other attributes, and sequence similarity networks. The latter provide a new and intuitively powerful way to visualize functional trends mapped to the context of sequence similarity.

Folate is required for one-carbon transfer reactions and the formation of purines and pyrimidines for DNA and RNA synthesis. Deficiency of folate can lead to many clinical abnormalities, including macrocytic anemia, cardiovascular diseases, birth defects, and carcinogenesis. The nucleotide imbalance due to folate deficiency causes cell cycle arrest in the S phase and uracil misincorporation into DNA, which may result in DNA double-strand breaks during repair. The role of folate in the immune system has not been fully characterized. We cultured PHA-activated human T lymphocytes in varying concentrations of folate, and measured proliferation, cell cycle, apoptosis, uracil misincorporation, and proportions of Th cells (CD4+) and cytotoxic T (CD8+) cells. Folate deficiency reduced proliferation of T lymphocytes, induced cell cycle arrest in the S phase, induced apoptosis, and increased the level of uracil in DNA. Folate deficiency also increased the CD4+ to CD8+ ratio due to a marked reduction of CD8+ cell proliferation. Folate or nucleoside repletion of folate-deficient cells rapidly restored T lymphocyte proliferation and normal cell cycle, reduced the DNA uracil content, and lowered the CD4+ to CD8+ ratio. These data suggest that folate status may affect the immune system by reducing the capacity of CD8+ cells to proliferate in response to activation.

Folate deficiency leads to increased dUMP/dTMP ratios and uracil misincorporation into DNA, which may increase cancer risk. We improved a previously described gas chromatography-mass spectrometry (GC-MS) assay for uracil in DNA and validated the assay by analyzing the DNA-uracil content of normal, primary human lymphocytes that were cultured in 0–3000nM folic acid. In addition, the effects of nucleoside mixtures T or TdCA (T, thymidine; A, adenosine; dC, deoxycytidine) were investigated. Over 4 consecutive days, the inter- and intraassay coefficients of variation (CVs) were 2.3–3.9 and 0.6–2.2%. Mean recovery was 99.4%. Oligonucleotides containing 100pg of uracil yielded a mean uracil measurement of 110.1pg (CV=2.7%). Cells grown in different concentrations of folate showed a bimodal response, with maximum DNA-uracil at 12nM, and minima at 0 and 3000nM folate. Extremely folate-deficient cells may incorporate less uracil because DNA synthesis is reduced. A wide response to folate deficiency was seen in cells from different donors, suggesting that genetic background plays a critical role in individual susceptibility to DNA damage and cancer risk. Unexpectedly, TdCA supplementation caused increased DNA-uracil (vs 3000nM folate for 10 days, P<0.05), probably due to the conversion of deoxycytidine to deoxyuridine by cytidine deaminase, leading to elevated dUMP/dTMP ratios. This improved uracil assay could serve as a useful tool in the study of the mechanism of uracil misincorporation into DNA. The assay requires 3μg of DNA per folate-deficient sample, but more may be required for baseline DNA-uracil detection in healthy humans.

Folate is required for one-carbon transfer reactions and the formation of purines and pyrimidines for DNA and RNA synthesis. Deficiency of folate can lead to many clinical abnormalities, including macrocytic anemia, cardiovascular diseases, birth defects, and carcinogenesis. The nucleotide imbalance due to folate deficiency causes cell cycle arrest in the S phase and uracil misincorporation into DNA, which may result in DNA double-strand breaks during repair. The role of folate in the immune system has not been fully characterized. We cultured PHA-activated human T lymphocytes in varying concentrations of folate, and measured proliferation, cell cycle, apoptosis, uracil misincorporation, and proportions of Th cells (CD4(+)) and cytotoxic T (CD8(+)) cells. Folate deficiency reduced proliferation of T lymphocytes, induced cell cycle arrest in the S phase, induced apoptosis, and increased the level of uracil in DNA. Folate deficiency also increased the CD4(+) to CD8(+) ratio due to a marked reduction of CD8(+) cell proliferation. Folate or nucleoside repletion of folate-deficient cells rapidly restored T lymphocyte proliferation and normal cell cycle, reduced the DNA uracil content, and lowered the CD4(+) to CD8(+) ratio. These data suggest that folate status may affect the immune system by reducing the capacity of CD8(+) cells to proliferate in response to activation.

We performed a comprehensive study on the genotoxic and cytotoxic effects of in vitro folic acid deficiency on primary human lymphocytes. Lymphocytes were cultured in medium containing 12-120 nM folic acid for 9 days in a novel cytokinesis-block micronucleus (CBMN) assay system (n = 20). Besides identifying optimal folic acid concentrations for in vitro genomic stability, we tested the hypothesis that lymphocytes from individuals homozygous for the C677T methylenetetrahydrofolate reductase (MTHFR) polymorphism (TTs, n = 10) are protected against chromosome damage relative to controls (CCs, n = 10) under conditions of folic acid deficiency. This hypothesis is based on the assumption that reduced :MTHFR activity in TT lymphocytes causes a diversion of 5,10-methylene tetrahydrofolate toward thymidine synthesis, which minimizes uracil-induced double-stranded DNA breakage. Cells were scored for micronuclei, apoptosis, necrosis, nucleoplasmic bridges, and nuclear budding. The latter two endpoints are indicative of chromosome rearrangements and gene amplification, respectively, and to the best of our knowledge, this is the first report of their association with folic acid concentration. Folic acid concentration correlated significantly (P < 0.0001) and negatively (r, -0.63 to -0.74) with all markers of chromosome damage, which were minimized at 60-120 nM folic acid, much greater than concentrations assumed "normal," but not necessarily optimal in plasma. Two-way ANOVA revealed no effect of the MTHFR genotype on any of the endpoints. Results show that the C677T polymorphism does not affect the ability of a cell to resist chromosome damage induced by folic acid deficiency in this in vitro system.

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme which converts 5,10-methylene tetrahydrofolate (5,10-MnTHF) to 5-methyl tetrahydrofolate. A common C to T transition (C677T) in the MTHFR gene is reported to reduce the risk for colorectal cancer and acute lymphocytic leukemia in homozygotes (TTs). It is hypothesized that because TTs have reduced MTHFR activity, more 5,10-MnTHF is available to provide methyl groups for the conversion of uracil to thymidine. Folic acid deficiency causes the intracellular accumulation of dUMP and the subsequent incorporation of uracil into DNA. The removal of uracil from DNA may result in double-stranded DNA breaks, the accumulation of which is a putative risk factor for cancer. We tested whether human lymphocytes taken from TTs (n = 10) were more able to resist uracil incorporation into DNA than controls (n = 14 CCs and 6 CTs) when cultured in medium containing 12-120 nM folic acid for 9 days. DNA uracil content of these lymphocytes was measured by CG-MS. TTs and controls showed a dose-dependent increase in DNA uracil content during folic acid deficiency (P < 0.0001, R2 = 0.23 for TTs and P < 0.0001, R2 = 0.19 for controls). DNA uracil content was not different between the two groups at any of the folic acid concentrations (two-way ANOVA: media [folic acid], P < 0.0001; genotype, P = 0.4). The results show that, in this in vitro system, the MTHFR C677T polymorphism does not affect the cell's ability to resist uracil incorporation into DNA. Chromosome breakage, as measured by micronuclei, was also shown to correlate with folic acid concentration in a preliminary experiment (P < 0.0001). Although the results appear not to support the hypothesis that a reduced risk for certain cancers in TTs is due to diversion of folic acid to thymidine synthesis, differences between the in vivo and in vitro situation make this conclusion not definitive.

The oxidized DNA base 8-oxoguanine has been commonly measured by enzymatic digestion of DNA to nucleosides followed by high-performance liquid chromatography (HPLC) separation of the adduct 8-oxodeoxyguanosine. There has recently been an enormous debate surrounding the validity of this approach, from which it has become clear that artifactual oxidation of the native base to 8-oxoguanine can occur at numerous stages in sample preparation. Hence, we have designed an alternative protocol to traditional enzymatic digestion of DNA which (i) limits the potential for artifactual oxidation, (ii) speeds up the assay markedly, (iii) increases the assay's sensitivity moderately, and (iv) addresses criticisms that have been raised concerning the efficiency of DNA digestion by nucleases. In short, we use the Escherichia coli repair enzyme formamidopyrimidine (Fapy) glycosylase to release the base 8-oxoguanine from full-length DNA, then separate 8-oxoguanine from high molecular weight molecules by ultrafiltration (10,000 Da exclusion) and analyze the base adduct by reverse-phase HPLC. Benefits of this approach include (i) rapid removal of the roughly million-fold molar excess of unaltered bases from the sample, (ii) reduction in the length of enzymatic incubations and the number of steps, (iii) elimination of high temperature incubation, (iv) a very clean chromatographic separation, and (v) rapid elution of the analyte and correspondingly greater throughput. Using this improved method, we have followed the induction of 8-oxoguanine in the DNA of peroxide-treated HeLa cells, an experiment that had proved cumbersome with traditional methods.