Informatics and Computational Approaches for the Discovery and Optimization of Natural Product-Inspired Inhibitors of the SARS-CoV-2 2'-O-Methyltransferase.

The urgent need for new classes of orally available, safe, and effective antivirals─covering a breadth of emerging viruses─is evidenced by the loss of life and economic challenges created by the HIV-1 and SARS-CoV-2 pandemics. As frontline interventions, small-molecule antivirals can be deployed prophylactically or postinfection to control the initial spread of outbreaks by reducing transmissibility and symptom severity. Natural products have an impressive track record of success as prototypic antivirals and continue to provide new drugs through synthesis, medicinal chemistry, and optimization decades after discovery. Here, we demonstrate an approach using computational analysis typically used for rational drug design to identify and develop natural product-inspired antivirals. This was done with the goal of identifying natural product prototypes to aid the effort of progressing toward safe, effective, and affordable broad-spectrum inhibitors of Betacoronavirus replication by targeting the highly conserved RNA 2'-O-methyltransferase (2'-O-MTase). Machaeriols RS-1 (7) and RS-2 (8) were identified using a previously outlined informatics approach to first screen for natural product prototypes, followed by in silico-guided synthesis. Both molecules are based on a rare natural product group. The machaeriols (3-6), isolated from the genus Machaerium, endemic to Amazonia, inhibited the SARS-CoV-2 2'-O-MTase more potently than the positive control, Sinefungin (2), and in silico modeling suggests distinct molecular interactions. This report highlights the potential of computationally driven screening to leverage natural product libraries and improve the efficiency of isolation or synthetic analog development.

A highly versatile fungal glucosyltransferase for specific production of quercetin-7-O-ß-D-glucoside and quercetin-3-O-ß-D-glucoside in different hosts.

Glycosylation is an effective way to improve the water solubility of natural products. In this work, a novel glycosyltransferase gene (BbGT) was discovered from Beauveria bassiana ATCC 7159 and heterologously expressed in Escherichia coli. The purified enzyme was functionally characterized through in vitro enzymatic reactions as a UDP-glucosyltransferase, converting quercetin to five monoglucosylated and one diglucosylated products. The optimal pH and temperature for BbGT are 35 ℃ and 8.0, respectively. The activity of BbGT was stimulated by Ca2+, Mg2+, and Mn2+, but inhibited by Zn2+. BbGT enzyme is flexible and can glycosylate a variety of substrates such as curcumin, resveratrol, and zearalenone. The enzyme was also expressed in other microbial hosts including Saccharomyces cerevisiae, Pseudomonas putida, and Pichia pastoris. Interestingly, the major glycosylation product of quercetin in E. coli, P. putida, and P. pastoris was quercetin-7-O-ß-D-glucoside, while the enzyme dominantly produced quercetin-3-O-ß-D-glucoside in S. cerevisiae. The BbGT-harboring E. coli and S. cerevisiae strains were used as whole-cell biocatalysts to specifically produce the two valuable quercetin glucosides, respectively. The titer of quercetin-7-O-ß-D-glucosides was 0.34 ± 0.02 mM from 0.83 mM quercetin in 24 h by BbGT-harboring E. coli. The yield of quercetin-3-O-ß-D-glucoside was 0.22 ± 0.02 mM from 0.41 mM quercetin in 12 h by BbGT-harboring S. cerevisiae. This work thus provides an efficient way to produce two valuable quercetin glucosides through the expression of a versatile glucosyltransferase in different hosts. KEY POINTS: • A highly versatile glucosyltransferase was identified from B. bassiana ATCC 7159. • BbGT converts quercetin to five mono- and one di-glucosylated derivatives in vitro. • Different quercetin glucosides were produced by BbGT in E. coli and S. cerevisiae.

Naturally occurring cancer-associated mutations disrupt oligomerization and activity of protein arginine methyltransferase 1 (PRMT1).

Protein arginine methylation is a posttranslational modification catalyzed by the protein arginine methyltransferase (PRMT) enzyme family. Dysregulated protein arginine methylation is linked to cancer and a variety of other human diseases. PRMT1 is the predominant PRMT isoform in mammalian cells and acts in pathways regulating transcription, DNA repair, apoptosis, and cell proliferation. PRMT1 dimer formation, which is required for methyltransferase activity, is mediated by interactions between a structure called the dimerization arm on one monomer and a surface of the Rossman Fold of the other monomer. Given the link between PRMT1 dysregulation and disease and the link between PRMT1 dimerization and activity, we searched the Catalogue of Somatic Mutations in Cancer (COSMIC) database to identify potential inactivating mutations occurring in the PRMT1 dimerization arm. We identified three mutations that correspond to W215L, Y220N, and M224V substitutions in human PRMT1V2 (isoform 1) (W197L, Y202N, M206V in rat PRMT1V1). Using a combination of site-directed mutagenesis, analytical ultracentrifugation, native PAGE, and activity assays, we found that these conservative substitutions surprisingly disrupt oligomer formation and substantially impair both S-adenosyl-L-methionine (AdoMet) binding and methyltransferase activity. Molecular dynamics simulations suggest that these substitutions introduce novel interactions within the dimerization arm that lock it in a conformation not conducive to dimer formation. These findings provide a clear, if putative, rationale for the contribution of these mutations to impaired arginine methylation in cells and corresponding health consequences.

Toward Understanding Molecular Recognition between PRMTs and their Substrates.

Protein arginine methylation is a widespread eukaryotic posttranslational modification that occurs with as much frequency as ubiquitinylation. Yet, how the nine different human protein arginine methyltransferases (PRMTs) recognize their respective protein targets is not well understood. This review summarizes the progress that has been made over the last decade or more to resolve this significant biochemical question. A multipronged approach involving structural biology, substrate profiling, bioorthogonal chemistry and proteomics is discussed.

Rapid and direct measurement of methyltransferase activity in about 30 min.

Protein arginine methylation is a widespread eukaryotic posttranslational modification that occurs to both histone and non-histone proteins. The S-adenosyl-L-methionine (AdoMet or SAM)-dependent modification is catalyzed by the protein arginine methyltransferase (PRMT) family of enzymes. In the last several years a series of both direct and indirect assay formats have been described that allow the rate of methylation to be measured. Here we provide a detailed protocol to directly measure PRMT activity using radiolabeled AdoMet, reversed-phase resin-filled pipette tips (ZipTips®) and a liquid scintillation counter. Because the ZipTips® based quantitation relies only on the straightforward separation of unreacted AdoMet from a methylated substrate, this protocol should be readily adaptable to other methyltransferases. The method is fast, simple to employ with both peptide and protein substrates, and produces very little radioactive waste.

Modified substrate specificity of a methyltransferase domain by protein insertion into an adenylation domain of the bassianolide synthetase.

BACKGROUND: Creating designer molecules using a combination of select domains from polyketide synthases and/or nonribosomal peptide synthetases (NRPS) continues to be a synthetic goal. However, an incomplete understanding of how protein-protein interactions and dynamics affect each of the domain functions stands as a major obstacle in the field. Of particular interest is understanding the basis for a class of methyltransferase domains (MT) that are found embedded within the adenylation domain (A) of fungal NRPS systems instead of in an end-to-end architecture. RESULTS: The MT domain from bassianolide synthetase (BSLS) was removed and the truncated enzyme BSLS-ΔMT was recombinantly expressed. The biosynthesis of bassianolide was abolished and N-desmethylbassianolide was produced in low yields. Co-expression of BSLS-ΔMT with standalone MT did not recover bassianolide biosynthesis. In order to address the functional implications of the protein insertion, we characterized the N-methyltransferase activity of the MT domain as both the isolated domain (MTBSLS) and as part of the full NRPS megaenzyme. Surprisingly, the MTBSLS construct demonstrated a relaxed substrate specificity and preferentially methylated an amino acid (L-Phe-SNAC) that is rarely incorporated into the final product. By testing the preference of a series of MT constructs (BSLS, MTBSLS, cMT, XLcMT, and aMT) to L-Phe-SNAC and L-Leu-SNAC, we further showed that restricting and/or fixing the termini of the MTBSLS by crosslinking or embedding the MT within an A domain narrowed the substrate specificity of the methyltransferase toward L-Leu-SNAC, the preferred substrate for the BSLS megaenzyme. CONCLUSIONS: The embedding of MT into the A2 domain of BSLS is not required for the product assembly, but is critical for the overall yields of the final products. The substrate specificity of MT is significantly affected by the protein context within which it is present. While A domains are known to be responsible for selecting and activating the biosynthetic precursors for NRPS systems, our results suggest that embedding the MT acts as a secondary gatekeeper for the assembly line. This work thus provides new insights into the embedded MT domain in NRPSs, which will facilitate further engineering of this type of biosynthetic machinery to create structural diversity in natural products.

Examining Product Specificity in Protein Arginine Methyltransferase 7 (PRMT7) Using Quantum and Molecular Mechanical Simulations.

Protein arginine methyltransferase 7 (PRMT7) catalyzes the formation of monomethylarginine (MMA) but is incapable of performing a dimethylation. Given that PRMT7 performs vital functions in mammalian cells and has been implicated in a variety of diseases, including breast cancer and age-related obesity, elucidating the origin of its strict monomethylation activity is of considerable interest. Three active site residues, Glu172, Phe71, and Gln329, have been reported as particularly important for product specificity and enzymatic activity. To better understand their roles, mixed quantum and molecular mechanical (QM/MM) calculations coupled to molecular dynamics and free energy perturbation theory were carried out for the WT, F71I, and Q329S trypanosomal PRMT7 (TbPRMT7) enzymes bound with S-adenosyl- L-methionine (AdoMet) and an arginine substrate in an unmethylated or methylated form. The Q329S mutation, which experimentally abolished enzymatic activity, was appropriately computed to give an outsized Δ G‡ of 30.1 kcal/mol for MMA formation compared to 16.9 kcal/mol for WT. The F71I mutation, which has been experimentally shown to convert the enzyme from a type III PRMT into a mixed type I/II capable of forming dimethylated arginine products, yielded a reasonable Δ G‡ of 21.9 kcal/mol for the second turnover compared to 28.8 kcal/mol in the WT enzyme. Similar active site orientations for both WT and F71I TbPRMT7 allowed Glu172 and Gln329 to better orient the substrate for SN2 methylation, enhanced the nucleophilicity of the attacking guanidino group by reducing positive charge, and facilitated the binding of the subsequent methylated products.

Phe71 in Type III Trypanosomal Protein Arginine Methyltransferase 7 (TbPRMT7) Restricts the Enzyme to Monomethylation.

Protein arginine methyltransferase 7 (PRMT7) is unique within the PRMT family as it is the only isoform known to exclusively make monomethylarginine (MMA). Given its role in epigenetics, the mechanistic basis for the strict monomethylation activity is under investigation. It is thought that PRMT7 enzymes are unable to add a second methyl group because of steric hindrance in the active site that restricts them to monomethylation. To test this, we probed the active site of trypanosomal PRMT7 (TbPRMT7) using accelerated molecular dynamics, site-directed mutagenesis, kinetic, binding, and product analyses. Both the dynamics simulations and experimental results show that the mutation of Phe71 to Ile converts the enzyme from a type III methyltransferase into a mixed type I/II, that is, an enzyme that can now perform dimethylation. In contrast, the serine and alanine mutants of Phe71 preserve the type III behavior of the native enzyme. These results are inconsistent with a sterics-only model to explain product specificity. Instead, molecular dynamics simulations of these variants bound to peptides show hydrogen bonding between would-be substrates and Glu172 of TbPRMT7. Only in the case of the Phe71 to Ile mutation is this interaction between MMA and the enzyme maintained, and the geometry for optimal SN2 methyl transfer is obtained. The results of these studies highlight the benefit of combined computational and experimental methods in providing a better understanding for how product specificity is dictated by PRMTs.

Understanding protein arginine methyltransferase 1 (PRMT1) product specificity from molecular dynamics.

Protein arginine methyltransferases (PRMTs) catalyze the post-translational methylation of specific arginyl groups within targeted proteins to regulate fundamental biological responses in eukaryotic cells. The major Type I PRMT enzyme, PRMT1, strictly generates monomethyl arginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA). Multiple diseases can arise from the dysregulation of PRMT1, including heart disease and cancer, which underscores the need to elucidate the origin of product specificity. Molecular dynamics (MD) simulations were carried out for WT PRMT1 and its M48F, H293A, H293S, and H293S-M48F mutants bound with S-adenosylmethionine (AdoMet) and the arginine substrate in an unmethylated or methylated form. Experimental site-directed mutagenesis and analysis of the resultant products were also performed. Two specific PRMT1 active site residues, Met48 and His293, have been determined to play a key role in dictating product specificity, as: (1) the single mutation of Met48 to Phe enabled PRMT1 to generate MMA, ADMA, and a limited amount of SDMA; (2) the single mutation of His293 to Ser formed the expected MMA and ADMA products only; whereas (3) the double mutant H293S-M48F-PRMT1 produced SMDA as the major product with limited amounts of MMA and ADMA. Calculating the formation of near-attack conformers resembling SN2 transition states leading to either the ADMA or SDMA products finds that Met48 and His293 may enable WT PRMT1 to yield ADMA exclusively by precluding MMA from binding in an orientation more conducive to SDMA formation, i.e., the methyl group bound at the arginine Nη2 position.

Biochemistry and regulation of the protein arginine methyltransferases (PRMTs).

Many key cellular processes can be regulated by the seemingly simple addition of one, or two, methyl groups to arginine residues by the nine known mammalian protein arginine methyltransferases (PRMTs). The impact that arginine methylation has on cellular well-being is highlighted by the ever growing evidence linking PRMT dysregulation to disease states, which has marked the PRMTs as prominent pharmacological targets. This review is meant to orient the reader with respect to the structural features of the PRMTs that account for catalytic activity, as well as provide a framework for understanding how these enzymes are regulated. An overview of what we understand about substrate recognition and binding is provided. Control of product specificity and enzyme processivity are introduced as necessary but flexible features of the PRMTs. Precise control of PRMT activity is a critical component to eukaryotic cell health, especially given that an arginine demethylase has not been identified. We therefore conclude the review with a comprehensive discussion of how protein arginine methylation is regulated.

Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity.

Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs) and is degraded by dimethylarginine dimethylaminohydrolase. Reports have shown that dimethylarginine dimethylaminohydrolase activity is down-regulated and PRMT1 protein expression is up-regulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. This oxidation event involves one or more cysteine residues that become oxidized to sulfenic acid (-SOH). We demonstrate a hydrogen peroxide concentration-dependent inhibition of PRMT1 activity that is readily reversed under physiological H2O2 concentrations. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis and demonstrate that enzymatic activity can be regulated in a redox-sensitive manner.

Structural determinants for the strict monomethylation activity by trypanosoma brucei protein arginine methyltransferase 7.

Trypanosoma brucei protein arginine methyltransferase 7 (TbPRMT7) exclusively generates monomethylarginine (MMA), which directs biological consequences distinct from that of symmetric dimethylarginine (SDMA) and asymmetric dimethylarginine (ADMA). However, determinants controlling the strict monomethylation activity are unknown. We present the crystal structure of the TbPRMT7 active core in complex with S-adenosyl-L-homocysteine (AdoHcy) and a histone H4 peptide substrate. In the active site, residues E172, E181, and Q329 hydrogen bond the guanidino group of the target arginine and align the terminal guanidino nitrogen in a position suitable for nucleophilic attack on the methyl group of S-adenosyl-L-methionine (AdoMet). Structural comparisons and isothermal titration calorimetry data suggest that the TbPRMT7 active site is narrower than those of protein arginine dimethyltransferases, making it unsuitable to bind MMA in a manner that would support a second turnover, thus abolishing the production of SDMA and ADMA. Our results present the structural interpretations for the monomethylation activity of TbPRMT7.

A remodeled protein arginine methyltransferase 1 (PRMT1) generates symmetric dimethylarginine.

Protein arginine methylation is emerging as a significant post-translational modification involved in various cell processes and human diseases. As the major arginine methylation enzyme, protein arginine methyltransferase 1 (PRMT1) strictly generates monomethylarginine and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA). The two types of dimethylarginines can lead to distinct biological outputs, as highlighted in the PRMT-dependent epigenetic control of transcription. However, it remains unclear how PRMT1 product specificity is regulated. We discovered that a single amino acid mutation (Met-48 to Phe) in the PRMT1 active site enables PRMT1 to generate both ADMA and SDMA. Due to the limited amount of SDMA formed, we carried out quantum mechanical calculations to determine the free energies of activation of ADMA and SDMA synthesis. Our results indicate that the higher energy barrier of SDMA formation (ΔΔG(‡) = 3.2 kcal/mol as compared with ADMA) may explain the small amount of SDMA generated by M48F-PRMT1. Our study reveals unique energetic challenges for SDMA-forming methyltransferases and highlights the exquisite control of product formation by active site residues in the PRMTs.

Substrate-induced control of product formation by protein arginine methyltransferase 1.

Protein arginine methyltransferases (PRMTs) aid in the regulation of many biological processes. Accurate control of PRMT activity includes recognition of specific arginyl groups within targeted proteins and the generation of the correct level of methylation, none of which are fully understood. The predominant PRMT in vivo, PRMT1, has wide substrate specificity and is capable of both mono- and dimethylation, which can induce distinct biological outputs. What regulates the specific methylation pattern of PRMT1 in vivo is unclear. We report that PRMT1 methylates a multisite peptide substrate in a nonstochastic manner, with less C-terminal preference, consistent with the methylation patterns observed in vivo. With a single targeted arginine, PRMT1 catalyzed the dimethylation in a semiprocessive manner. The degree of processivity is regulated by substrate sequences. Our results identify a novel substrate-induced mechanism for modulating PRMT1 product specificity. Considering the numerous physiological PRMT1 substrates, as well as the distinct biological outputs of mono- and dimethylation products, such fine-tuned regulation would significantly contribute to the accurate product specificity of PRMT1 in vivo and the proper transmission of biochemical information.

Investigation of the molecular origins of protein-arginine methyltransferase I (PRMT1) product specificity reveals a role for two conserved methionine residues.

Protein-arginine methyltransferases aid in the regulation of many biological processes by methylating specific arginyl groups within targeted proteins. The varied nature of the response to methylation is due in part to the diverse product specificity displayed by the protein-arginine methyltransferases. In addition to site location within a protein, biological response is also determined by the degree (mono-/dimethylation) and type of arginine dimethylation (asymmetric/symmetric). Here, we have identified two strictly conserved methionine residues in the PRMT1 active site that are not only important for activity but also control substrate specificity. Mutation of Met-155 or Met-48 results in a loss in activity and a change in distribution of mono- and dimethylated products. The altered substrate specificity of M155A and M48L mutants is also evidenced by automethylation. Investigation into the mechanistic basis of altered substrate recognition led us to consider each methyl transfer step separately. Single turnover experiments reveal that the rate of transfer of the second methyl group is much slower than transfer of the first methyl group in M48L, especially for arginine residues located in the center of the peptide substrate where turnover of the monomethylated species is negligible. Thus, altered product specificity in M48L originates from the differential effect of the mutation on the two rates. Characterization of the two active-site methionines provides the first insight into how the PRMT1 active site is engineered to control product specificity.

A fast and efficient method for quantitative measurement of S-adenosyl-L-methionine-dependent methyltransferase activity with protein substrates.

Modification of protein residues by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases impacts an array of cellular processes. Here we describe a new approach to quantitatively measure the rate of methyl transfer that is compatible with using protein substrates. The method relies on the ability of reverse-phase resin packed at the end of a pipette tip to quickly separate unreacted AdoMet from radiolabeled protein products. Bound radiolabeled protein products are eluted directly into scintillation vials and counted. In addition to decreasing analysis time, the sensitivity of this protocol allows the determination of initial rate data. The utility of this protocol was shown by generating a Michaelis-Menten curve for the methylation of heterogeneous nuclear ribonucleoprotein K (hnRNP K) protein by human protein arginine methyltransferase 1, variant 1 (hPRMT1v1), in just over 1h. An additional advantage of this assay is the more than 3000-fold reduction in radioactive waste over existing protocols.

Efficient cleavage of problematic tobacco etch virus (TEV)-protein arginine methyltransferase constructs.

Protein arginine methyltransferases (PRMTs) are enzymes that are involved in many biological processes. Several studies have shown that the identity of the N-terminal fusion tag affects the substrate selectivity of PRMTs. Therefore, to accurately study substrate recognition, it is imperative that a tagless PRMT be used. However, cleavage of tagged PRMTs has been problematic. We have developed a successful method by which untagged PRMTs can be made using a tobacco etch virus (TEV) cleavage site at the N-terminal domain. This method may be useful for cleaving other challenging target proteins that have the TEV protease recognition site.

Substrate profiling of PRMT1 reveals amino acid sequences that extend beyond the "RGG" paradigm.

Protein arginine methyltransferase 1 (PRMT1) catalyzes the mono- and dimethylation of certain protein arginine residues. Although this posttranslational modification has been implicated in many physiological processes, the molecular basis for PRMT1 substrate recognition is poorly understood. Most modified arginine residues in known PRMT1 substrates reside in repeating "RGG" sequences. However, PRMT1 also specifically methylates Arg3 of histone H4 in a region that is not glycine-arginine rich, suggesting that PRMT1 substrates are not limited to proteins bearing "RGG" sequences. Because a systematic evaluation of PRMT1 substrate specificity has not been performed, it is unclear if the "RGG" sequence accurately represents the consensus target for PRMT1. Using a focused peptide library based on a sequence derived from the in vivo substrate fibrillarin we observed that PRMT1 methylated substrates that had amino acid residues other than glycine in the "RX (1)" and "RX (1)X (2)" positions. Importantly, eleven additional PRMT1 substrate sequences were identified. Our results also illustrate that the two residues on the N-terminal side of the modification site are important and need not both be glycine. PRMT1 methylated the eukaryotic initiation factor 4A1 (eIF4A1) protein, which has a single "RGG" sequence. Methylation of eIF4A1 and the similar eIF4A3 could be affected using single site mutations adjacent to the modification site, demonstrating the importance of amino acid sequence in PRMT1 protein substrates. Dimethylation of the parent library peptide was shown to occur through a dissociative mechanism. In summary, PRMT1 selectively recognizes a set of amino acid sequences in substrates that extend beyond the "RGG" paradigm.

Novel functional view of the crocidolite asbestos-treated A549 human lung epithelial transcriptome reveals an intricate network of pathways with opposing functions.

BACKGROUND: Although exposure to asbestos is now regulated, patients continue to be diagnosed with mesothelioma, asbestosis, fibrosis and lung carcinoma because of the long latent period between exposure and clinical disease. Asbestosis is observed in approximately 200,000 patients annually and asbestos-related deaths are estimated at 4,000 annually. Although advances have been made using single gene/gene product or pathway studies, the complexity of the response to asbestos and the many unanswered questions suggested the need for a systems biology approach. The objective of this study was to generate a comprehensive view of the transcriptional changes induced by crocidolite asbestos in A549 human lung epithelial cells. RESULTS: A statistically robust, comprehensive data set documenting the crocidolite-induced changes in the A549 transcriptome was collected. A systems biology approach involving global observations from gene ontological analyses coupled with functional network analyses was used to explore the effects of crocidolite in the context of known molecular interactions. The analyses uniquely document a transcriptome with function-based networks in cell death, cancer, cell cycle, cellular growth, proliferation, and gene expression. These functional modules show signs of a complex interplay between signaling pathways consisting of both novel and previously described asbestos-related genes/gene products. These networks allowed for the identification of novel, putative crocidolite-related genes, leading to several new hypotheses regarding genes that are important for the asbestos response. The global analysis revealed a transcriptome that bears signatures of both apoptosis/cell death and cell survival/proliferation. CONCLUSION: Our analyses demonstrate the power of combining a statistically robust, comprehensive dataset and a functional network genomics approach to 1) identify and explore relationships between genes of known importance 2) identify novel candidate genes, and 3) observe the complex interplay between genes/gene products that function in seemingly different processes. This study represents the first function-based global approach toward understanding the response of human lung epithelial cells to the carcinogen crocidolite. Importantly, our investigation paints a much broader landscape for the crocidolite response than was previously appreciated and reveals novel paths to study. Our graphical representations of the function-based global network will be a valuable resource to model new research findings.

Determinants of oligomerization of the bifunctional protein DCoHalpha and the effect on its enzymatic and transcriptional coactivator activities.

DCoH and DCoHalpha are bifunctional proteins that function as 4a-hydroxytetrahydrobiopterin dehydratases and as coactivators of HNF1alpha-dependent transcription. Although these isoforms share sequence and structural similarity and equivalent enzyme activities, DCoH is a hyperstable tetramer whereas DCoHalpha readily forms dimers. Differences in quaternary structure affect the formation of the DCoH(alpha):HNF1alpha complex. Because the interface used to bind HNF1alpha is masked in tetrameric DCoH, the DCoH:HNF1alpha complex is only formed in vivo, presumably by co-translational folding. Conversely, the DCoHalpha:HNF1alpha complex readily forms in vitro. We identified residues in DCoHalpha that differed from those in the dimer-dimer interface of tetrameric DCoH. Mutating these residues altered the quaternary state and concomitantly the ability of the mutated proteins to affect HNF1alpha-dependent DNA binding. Our results indicate that three residues, Asn61, Gln45, and Lys98 in DCoHalpha play a role in oligomeric flexibility, which enables DCoHalpha to more readily interact with HNF1alpha and increase DNA binding.