Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates

The JmjC-domain-containing 2-oxoglutarate-dependent oxygenases catalyze protein hydroxylation and N^ε-methyllysine demethylation via hydroxylation. A subgroup of this family, the JmjC lysine demethylases (JmjC KDMs) are involved in histone modifications at multiple sites. There are conflicting reports as to the substrate selectivity of some JmjC oxygenases with respect to KDM activities. In this study, a panel of modified histone H3 peptides was tested for demethylation against 15 human JmjC-domain-containing proteins. The results largely confirmed known N^ε-methyllysine substrates. However, the purified KDM4 catalytic domains showed greater substrate promiscuity than previously reported (i.e., KDM4A was observed to catalyze demethylation at H3K27 as well as H3K9/K36). Crystallographic analyses revealed that the N^ε-methyllysine of an H3K27me3 peptide binds similarly to N^ε-methyllysines of H3K9me3/H3K36me3 with KDM4A. A subgroup of JmjC proteins known to catalyze hydroxylation did not display demethylation activity. Overall, the results reveal that the catalytic domains of the KDM4 enzymes may be less selective than previously identified. They also draw a distinction between the N^ε-methyllysine demethylation and hydroxylation activities within the JmjC subfamily. These results will be of use to those working on functional studies of the JmjC enzymes.     

The tRNA Recognition Mechanism of Folate/FAD-dependent tRNA Methyltransferase (TrmFO)

The conserved U54 in tRNA is often modified to 5-methyluridine (m⁵U) and forms a reverse Hoogsteen base pair with A58 that stabilizes the L-shaped tRNA structure. In Gram-positive and some Gram-negative eubacteria, m⁵U54 is produced by folate/FAD-dependent tRNA (m⁵U54) methyltransferase (TrmFO). TrmFO utilizes N⁵,N¹⁰-methylenetetrahydrofolate (CH₂THF) as a methyl donor. We previously reported an in vitro TrmFO assay system, in which unstable [¹⁴C]CH₂THF was supplied from [¹⁴C]serine and tetrahydrofolate by serine hydroxymethyltransferase. In the current study, we have improved the TrmFO assay system by optimization of enzyme and substrate concentrations and introduction of a filter assay system. Using this assay, we have focused on the tRNA recognition mechanism of TrmFO. 42 tRNA mutant variants were prepared, and experiments with truncated tRNA and microhelix RNAs revealed that the minimum requirement of TrmFO exists in the T-arm structure. The positive determinants for TrmFO were found to be the U54U55C56 sequence and G53-C61 base pair. The gel mobility shift assay and fluorescence quenching showed that the affinity of TrmFO for tRNA in the initial binding process is weak. The inhibition experiments showed that the methylated tRNA is released before the structural change process. Furthermore, we found that A38 prevents incorrect methylation of U32 in the anticodon loop. Moreover, the m¹A58 modification clearly accelerates the TrmFO reaction, suggesting a synergistic effect of the m⁵U54, m¹A58, and s²U54 modifications on m⁵s²U54 formation in Thermus thermophilus cells. The docking model of TrmFO and the T-arm showed that the G53-C61 base pair is not able to directly contact the enzyme.     

Structure,dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK

The enzyme m¹A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNA^Leu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.     

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro³¹⁴–Trp³¹⁶ turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro³¹⁴ of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.     

Mechanistic Diversity of Radical S-Adenosylmethionine (SAM)-dependent Methylation

Radical S-adenosylmethionine (SAM) enzymes use the oxidizing power of a 5′-deoxyadenosyl 5′-radical to initiate an amazing array of transformations, usually through the abstraction of a target substrate hydrogen atom. A common reaction of radical SAM (RS) enzymes is the methylation of unactivated carbon or phosphorous atoms found in numerous primary and secondary metabolites, as well as in proteins, sugars, lipids, and RNA. However, neither the chemical mechanisms by which these unactivated atoms obtain methyl groups nor the actual methyl donors are conserved. In fact, RS methylases have been grouped into three classes based on protein architecture, cofactor requirement, and predicted mechanism of catalysis. Class A methylases use two cysteine residues to methylate sp²-hybridized carbon centers. Class B methylases require a cobalamin cofactor to methylate both sp²-hybridized and sp³-hybridized carbon centers as well as phosphinate phosphorous atoms. Class C methylases share significant sequence homology with the RS enzyme, HemN, and may bind two SAM molecules simultaneously to methylate sp²-hybridized carbon centers. Lastly, we describe a new class of recently discovered RS methylases. These Class D methylases, unlike Class A, B, and C enzymes, which use SAM as the source of the donated methyl carbon, are proposed to methylate sp²-hybridized carbon centers using methylenetetrahydrofolate as the source of the appended methyl carbon.     

Phosphorylation of VACM-1/Cul5 by Protein Kinase A Regulates Its Neddylation and Antiproliferative Effect

Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific site in VACM-1/Cul5 (^S730AVACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if PKA-dependent phosphorylation of VACM-1/Cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a PKA-specific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with ^S730AVACM-1 cDNA when compared with the cytomegalovirus-transfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA activity with forskolin reduced modification of VACM-1 protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with ^S730AVACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nm) to induce PKC activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.     

Trm112p Is a 15-kDa Zinc Finger Protein Essential for the Activity of Two tRNA and One Protein Methyltransferases in Yeast

The degenerate base at position 34 of the tRNA anticodon is the target of numerous modification enzymes. In Saccharomyces cerevisiae, five tRNAs exhibit a complex modification of uridine 34 (mcm⁵U₃₄ and mcm⁵s²U₃₄), the formation of which requires at least 25 different proteins. The addition of the last methyl group is catalyzed by the methyltransferase Trm9p. Trm9p interacts with Trm112p, a 15-kDa protein with a zinc finger domain. Trm112p is essential for the activity of Trm11p, another tRNA methyltransferase, and for Mtq2p, an enzyme that methylates the translation termination factor eRF1/Sup45. Here, we report that Trm112p is required in vivo for the formation of mcm⁵U₃₄ and mcm⁵s²U₃₄. When produced in Escherichia coli, Trm112p forms a complex with Trm9p, which renders the latter soluble. This recombinant complex catalyzes the formation of mcm⁵U₃₄ on tRNA in vitro but not mcm⁵s²U₃₄. An mtq2-0 trm9-0 strain exhibits a synthetic growth defect, thus revealing the existence of an unexpected link between tRNA anticodon modification and termination of translation. Trm112p is associated with other partners involved in ribosome biogenesis and chromatin remodeling, suggesting that it has additional roles in the cell.     

Neuronal expression of ndst3 in early zebrafish development is responsive to Wnt signaling manipulation

Heparan sulfate proteoglycans (HSPGs) are constituents of the cell surface and extracellular matrix and are vital for various activities within the cell. The N-deacetylase/N-sulfotransferase (heparin glucosaminyl) family of enzymes, or NDST, modifies heparan sulfate (HS) by catalyzing both the N-deacetylation and the N-sulfation of N-acetylglucosamine residues. In zebrafish, a single ndst3 gene is an orthologue of both mammalian NDST3 and NDST4 genes. The role of ndst3 in zebrafish development has not been investigated and such study may provide insight into the role(s) of both mammalian orthologues. Here, we characterized expression of ndst3 during early development in zebrafish and found it to be predominately neuronal. We found that expression of ndst3 is sensitive to Wnt signaling manipulation, with stimulation of the Wnt pathway resulting in robust expansion of ndst3 expression domains. Finally, using CRISPR/Cas9 genome editing, we mutagenized the ndst3 gene and isolated an allele, ndst3^nu20, resulting in a frameshift and premature protein truncation. We discovered Ndst3 is not essential for zebrafish survival as ndst3^nu20 homozygous mutants are viable and fertile.     

A Nondiscriminating Glutamyl-tRNA Synthetase in the Plasmodium Apicoplast: THE FIRST ENZYME IN AN INDIRECT AMINOACYLATION PATHWAY*

The malaria parasite Plasmodium falciparum and related organisms possess a relict plastid known as the apicoplast. Apicoplast protein synthesis is a validated drug target in malaria because antibiotics that inhibit translation in prokaryotes also inhibit apicoplast protein synthesis and are sometimes used for malaria prophylaxis or treatment. We identified components of an indirect aminoacylation pathway for Gln-tRNA^Gln biosynthesis in Plasmodium that we hypothesized would be essential for apicoplast protein synthesis. Here, we report our characterization of the first enzyme in this pathway, the apicoplast glutamyl-tRNA synthetase (GluRS). We expressed the recombinant P. falciparum enzyme in Escherichia coli, showed that it is nondiscriminating because it glutamylates both apicoplast tRNA^Glu and tRNA^Gln, determined its kinetic parameters, and demonstrated its inhibition by a known bacterial GluRS inhibitor. We also localized the Plasmodium berghei ortholog to the apicoplast in blood stage parasites but could not delete the PbGluRS gene. These data show that Gln-tRNA^Gln biosynthesis in the Plasmodium apicoplast proceeds via an essential indirect aminoacylation pathway that is reminiscent of bacteria and plastids.     

The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth

N⁶-Threonylcarbamoyl-adenosine (t⁶A) is a universal modification occurring at position 37 in nearly all tRNAs that decode A-starting codons, including the eukaryotic initiator tRNA (tRNA_i^Met). Yeast lacking central components of the t⁶A synthesis machinery, such as Tcs3p (Kae1p) or Tcs5p (Bud32p), show slow-growth phenotypes. In the present work, we show that loss of the Drosophila tcs3 homolog also leads to a severe reduction in size and demonstrate, for the first time in a non-microbe, that Tcs3 is required for t⁶A synthesis. In Drosophila and in mammals, tRNA_i^Met is a limiting factor for cell and animal growth. We report that the t⁶A-modified form of tRNA_i^Met is the actual limiting factor. We show that changing the proportion of t⁶A-modified tRNA_i^Met, by expression of an un-modifiable tRNA_i^Met or changing the levels of Tcs3, regulate target of rapamycin (TOR) kinase activity and influences cell and animal growth in vivo. These findings reveal an unprecedented relationship between the translation machinery and TOR, where translation efficiency, limited by the availability of t⁶A-modified tRNA, determines growth potential in eukaryotic cells.     

Human NAT10 Is an ATP-dependent RNA Acetyltransferase Responsible for N4-Acetylcytidine Formation in 18 S Ribosomal RNA (rRNA)

Human N-acetyltransferase 10 (NAT10) is known to be a lysine acetyltransferase that targets microtubules and histones and plays an important role in cell division. NAT10 is highly expressed in malignant tumors, and is also a promising target for therapies against laminopathies and premature aging. Here we report that NAT10 is an ATP-dependent RNA acetyltransferase responsible for formation of N⁴-acetylcytidine (ac⁴C) at position 1842 in the terminal helix of mammalian 18 S rRNA. RNAi-mediated knockdown of NAT10 resulted in growth retardation of human cells, and this was accompanied by high-level accumulation of the 30 S precursor of 18 S rRNA, suggesting that ac⁴C1842 formation catalyzed by NAT10 is involved in rRNA processing and ribosome biogenesis.     

Double-stranded RNA-stimulated enzyme activities isolated from human placental extracts

A (2′–5′)A_n synthetase activity was isolated from human placental extracts by affinity chromatography on poly(rI)·poly(rC)-agarose. The oligonucleotide (2′–5′)A_n was identified by (1) chromatography on PEI-cellulose and DEAE-cellulose, (2) inhibition of polypeptide synthesis in lysed rabbit reticulocytes (3) competition of the binding of pppA(pA)3,3′-[³²P]pCp to rabbit reticulocyte lysates, and (4) alkaline phosphatase digestion. The synthetase activity in most placental preparations is activated by natural or synthetic dsRNA. However, in a few placental synthetase preparations, dsRNA is only marginally stimulatory and only becomes effective by prior treatment of the enzyme preparations with the calcium-dependent micrococal nuclease. This suggeststhat there is an endogenous placental dsRNA contaminant in the enzyme preparations. In some synthetase preparations, a second dsRNA-stimulated product, tentatively identified as the nucleotide 5′-IMP, is also observed. Because the specific AMP deaminase inhibitor coformycin (10 μM) blocks the formation of IMP from ATP and causes a quantitative accumulation of AMP, and because the formation of IMp becomes independent of dsRNA when ADP or AMP is used in plase of ATP, the presence of a dsRNA-stimulated ATP phosphohydrolase (ATPase) activity in human placenta is suggested.     

PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast

The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His⁹⁹ and Cys¹³⁶), and an Asp (Asp¹³⁴) in the potential S₁ binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.     

Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates

The JmjC-domain-containing 2-oxoglutarate-dependent oxygenases catalyze protein hydroxylation and N^?-methyllysine demethylation via hydroxylation. A subgroup of this family, the JmjC lysine demethylases (JmjC KDMs) are involved in histone modifications at multiple sites. There are conflicting reports as to the substrate selectivity of some JmjC oxygenases with respect to KDM activities. In this study, a panel of modified histone H3 peptides was tested for demethylation against 15 human JmjC-domain-containing proteins. The results largely confirmed known N^?-methyllysine substrates. However, the purified KDM4 catalytic domains showed greater substrate promiscuity than previously reported (i.e., KDM4A was observed to catalyze demethylation at H3K27 as well as H3K9/K36). Crystallographic analyses revealed that the N^?-methyllysine of an H3K27me3 peptide binds similarly to N^?-methyllysines of H3K9me3/H3K36me3 with KDM4A. A subgroup of JmjC proteins known to catalyze hydroxylation did not display demethylation activity. Overall, the results reveal that the catalytic domains of the KDM4 enzymes may be less selective than previously identified. They also draw a distinction between the N^?-methyllysine demethylation and hydroxylation activities within the JmjC subfamily. These results will be of use to those working on functional studies of the JmjC enzymes.     

Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog

Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs.