Types of Publications
Journal articles
Histone modification is well established as a fundamental mechanism driving the regulation of transcription, replication and DNA repair through the control of chromatin structure. Likewise, it is apparent that incorrect targeting of histone modifications contributes to misregulated gene expression and hence to developmental disorders and diseases of genomic instability such as cancer. The KMT2 family of SET domain methyltransferases, typified by MLL1, are responsible for histone H3 lysine-4 methylation, a marker of active genes. To ensure that this modification is correctly targeted, a multi-protein complex associates with the methyltransferase and directs activity. We have identified a novel interaction site on the core complex protein WDR5, and mapped the complementary site on its partner RbBP5. We have characterised this interaction by X-ray crystallography and show how it is fundamental to the assembly of the complex and to the regulation of methyltransferase activity. We show which region of RbBP5 contributes directly to MLL activation and combine our structural and biochemical data to produce a model to show how WDR5 and RbBP5 act cooperatively to stimulate activity.
Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific.
BACKGROUND: Emerging evidence implicates lysyl oxidase (LOX), an extracellular matrix-modifying enzyme, in promoting metastasis of solid tumors. We investigated whether LOX plays an important role in the metastasis of colorectal cancer (CRC). METHODS: We analyzed LOX expression in a patient CRC tissue microarray consisting of normal colon mucosa (n = 49), primary (n = 510), and metastatic (n = 198) tissues. LOX was overexpressed in CRC cell line SW480 (SW480+LOX), and the expression was knocked down in CRC cell line SW620 using LOX-specific short hairpin RNA (SW620+shLOX). Effect of LOX manipulation on three-dimensional cell proliferation and invasion was characterized in vitro. Effect of LOX manipulation on tumor proliferation and metastasis was investigated in a subcutaneous tumor mouse model (n = 3 mice per group) and in an intrasplenic metastatic mouse model (n = 3 mice per group). The mechanism of LOX-mediated effects via v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) was investigated using dasatinib, an inhibitor of SRC activation. All statistical tests were two-sided. RESULTS: Compared with normal colon tissue (n = 49), LOX expression was statistically significantly increased in tumor tissues (n = 510) of CRC patients (P < .001), and a greater increase was observed in metastatic tissue (n = 198). SW480+LOX cells showed a statistically significantly increased three-dimensional proliferation (P = .037) and invasion (P = .015), whereas SW620+shLOX cells showed reduced proliferation (P = .011) and invasion (P = .013) compared with controls. Subcutaneous tumor growth in mice was statistically significantly increased in SW480+LOX tumors (P = .036) and decreased in SW620+shLOX tumors (P = .048), and metastasis was statistically significantly increased in SW480+LOX tumors (P = .044) and decreased in SW620+shLOX tumors (SW620 control vs SW620+shLOX, mean = 1.0 luminescent signal, 95% confidence interval = 0.3 to 1.7 luminescent signal, vs mean = 0.3 luminescent signal, 95% confidence interval = 0.1 to 0.5 luminescent signal; P = .035) compared with controls. LOX-mediated effects on tumor progression were associated with SRC activation, and these effects were inhibited by dasatinib. CONCLUSIONS: LOX showed an important role in CRC cell proliferation and metastasis and was dependent on the activation of SRC. These results have the potential to identify patients with high SRC activity, who may benefit from dasatinib treatment.
Book chapters
Methylation also plays important roles in aging. This volume is intended to capture these recent developments concerning protein methyltransferases.
Types of Publications
Journal articles
Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34-318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34-318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34-318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34-318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection.
Histone modification is well established as a fundamental mechanism driving the regulation of transcription, replication and DNA repair through the control of chromatin structure. Likewise, it is apparent that incorrect targeting of histone modifications contributes to misregulated gene expression and hence to developmental disorders and diseases of genomic instability such as cancer. The KMT2 family of SET domain methyltransferases, typified by MLL1, are responsible for histone H3 lysine-4 methylation, a marker of active genes. To ensure that this modification is correctly targeted, a multi-protein complex associates with the methyltransferase and directs activity. We have identified a novel interaction site on the core complex protein WDR5, and mapped the complementary site on its partner RbBP5. We have characterised this interaction by X-ray crystallography and show how it is fundamental to the assembly of the complex and to the regulation of methyltransferase activity. We show which region of RbBP5 contributes directly to MLL activation and combine our structural and biochemical data to produce a model to show how WDR5 and RbBP5 act cooperatively to stimulate activity.
Heme ligands were introduced in the hydrophobic core of an engineered monomeric ColE1 repressor of primer (rop-S55) in two different layers of the heptad repeat. Mutants rop-L63M/F121H (layer 1) and rop-L56H/L113H (layer 3) were found to bind heme with a K (D) of 1.1 +/- 0.2 and 0.47 +/- 0.07 microM, respectively. The unfolding of heme-bound and heme-free mutants, in the presence of guanidinium hydrochloride, was monitored by both circular dichroism and fluorescence spectroscopy. For the heme-bound rop mutants, the total free energy change was 0.5 kcal/mol higher in the layer 3 mutant compared with that in the layer1 mutant. Heme binding also stabilized these mutants by increasing the [DGobsH2O] by 1.4 and 1.8 kcal/mol in rop-L63M/F121H and rop-L56H/L113H, respectively. The reduction potentials measured by spectroelectrochemical titrations were calculated to be -154 +/- 2 mV for rop-56H/113H and -87.5 +/- 1.2 mV for rop-L63M/F121H. The mutant designed to bind heme in a more buried environment (layer 3) showed tighter heme binding, a higher stability, and a different reduction potential compared with the mutant designed to bind heme in layer 1.
Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific.
p53 is a tumour suppressor that regulates the cellular response to genotoxic stresses. p53 is a short-lived protein and its activity is regulated mostly by stabilization via different post-translational modifications. Here we report a novel mechanism of p53 regulation through lysine methylation by Set9 methyltransferase. Set9 specifically methylates p53 at one residue within the carboxyl-terminus regulatory region. Methylated p53 is restricted to the nucleus and the modification positively affects its stability. Set9 regulates the expression of p53 target genes in a manner dependent on the p53-methylation site. The crystal structure of a ternary complex of Set9 with a p53 peptide and the cofactor product S-adenosyl-l-homocysteine (AdoHcy) provides the molecular basis for recognition of p53 by this lysine methyltransferase.
The realisation that SET domains, which are found in numerous proteins involved in chromatin regulation, catalyse the methylation of lysine residues has led to intense interest in their cellular, biochemical and structural properties. The structures of five SET domain proteins have been reported over the past year. SET domains possess a novel fold, and use adjacent domains for both structural stabilisation and the completion of their active sites. The cofactor S-adenosyl-L-methionine and peptide substrates bind on opposite faces of the SET domain. Remarkably, the sidechain of the target lysine approaches the transferred methyl group through a narrow channel that passes through the middle of the domain.
Acetylation, phosphorylation and methylation of the amino-terminal tails of histones are thought to be involved in the regulation of chromatin structure and function. With just one exception, the enzymes identified in the methylation of specific lysine residues on histones (histone methyltransferases) belong to the SET family. The high-resolution crystal structure of a ternary complex of human SET7/9 with a histone peptide and cofactor reveals that the peptide substrate and cofactor bind on opposite surfaces of the enzyme. The target lysine accesses the active site of the enzyme and the S-adenosyl-l-methionine (AdoMet) cofactor by inserting its side chain into a narrow channel that runs through the enzyme, connecting the two surfaces. Here we show from the structure and from solution studies that SET7/9, unlike most other SET proteins, is exclusively a mono-methylase. The structure indicates the molecular basis of the specificity of the enzyme for the histone target, and allows us to propose a model for the methylation reaction that accounts for the role of many of the residues that are invariant across the SET family.
A nucleic acid binding protein, rop, has conserved topology with a number of redox proteins; this is exploited to engineer haem binding, expanding its function as a redox protein.
Biological methylation is a subject that has fascinated mechanistically minded chemists for over 50 years. While early studies were usually directed at C-methylation in natural products, more recent work on N-methylation in DNA and proteins is being supported by the results of X-ray crystallography. From this source, significant mechanistic detail can be gleaned and powerful insights gained into the nature of enzyme catalysis and selectivity in methyl-transfer processes. The case of the human histone H3 transmethylase SET7/9 is considered in detail and compared to cognate histone lysine methylases. It provides an analysis of Nature's solution to the task of avoiding over-methylation.
Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.
Resistance to mercuric ions in bacteria is conferred by mercuric reductase, which reduces Hg(II) to Hg(0) in the cytoplasmic compartment. Specific mercuric ion transport systems exist to take up Hg(II) salts and deliver them to the active site of the reductase. This short review discusses the role of transport proteins in resistance and the mechanism of transfer of Hg(II) between the mercury-resistance proteins.
Mercury resistance determinants are widespread in Gram-negative bacteria, but vary in the number and identity of genes present. We have shown that the merF gene from plasmid pMER327/419 encodes a 8.7 kDa mercury transport protein, by determining in vivo mercury volatilisation when MerF is expressed in the presence of mercuric reductase. We have confirmed that MerC of Tn21 is also a mercuric ion transporter. We have been able to detect interaction of the periplasmic protein MerP only with the MerT transporter, and not with MerF or MerC. Hydropathy analysis led to the prediction of models for MerT, MerC and MerF having three, four and two transmembrane regions respectively. In all three cases one pair of cysteine residues is predicted to be within the inner membrane with a second pair of cysteine residues on the cytoplasmic face, and the second helix contains a proline and at least one charged residue. The mechanisms of mercuric ion transport may be similar in these transporters even though their structures in the membrane differ.
BACKGROUND: Emerging evidence implicates lysyl oxidase (LOX), an extracellular matrix-modifying enzyme, in promoting metastasis of solid tumors. We investigated whether LOX plays an important role in the metastasis of colorectal cancer (CRC). METHODS: We analyzed LOX expression in a patient CRC tissue microarray consisting of normal colon mucosa (n = 49), primary (n = 510), and metastatic (n = 198) tissues. LOX was overexpressed in CRC cell line SW480 (SW480+LOX), and the expression was knocked down in CRC cell line SW620 using LOX-specific short hairpin RNA (SW620+shLOX). Effect of LOX manipulation on three-dimensional cell proliferation and invasion was characterized in vitro. Effect of LOX manipulation on tumor proliferation and metastasis was investigated in a subcutaneous tumor mouse model (n = 3 mice per group) and in an intrasplenic metastatic mouse model (n = 3 mice per group). The mechanism of LOX-mediated effects via v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) was investigated using dasatinib, an inhibitor of SRC activation. All statistical tests were two-sided. RESULTS: Compared with normal colon tissue (n = 49), LOX expression was statistically significantly increased in tumor tissues (n = 510) of CRC patients (P < .001), and a greater increase was observed in metastatic tissue (n = 198). SW480+LOX cells showed a statistically significantly increased three-dimensional proliferation (P = .037) and invasion (P = .015), whereas SW620+shLOX cells showed reduced proliferation (P = .011) and invasion (P = .013) compared with controls. Subcutaneous tumor growth in mice was statistically significantly increased in SW480+LOX tumors (P = .036) and decreased in SW620+shLOX tumors (P = .048), and metastasis was statistically significantly increased in SW480+LOX tumors (P = .044) and decreased in SW620+shLOX tumors (SW620 control vs SW620+shLOX, mean = 1.0 luminescent signal, 95% confidence interval = 0.3 to 1.7 luminescent signal, vs mean = 0.3 luminescent signal, 95% confidence interval = 0.1 to 0.5 luminescent signal; P = .035) compared with controls. LOX-mediated effects on tumor progression were associated with SRC activation, and these effects were inhibited by dasatinib. CONCLUSIONS: LOX showed an important role in CRC cell proliferation and metastasis and was dependent on the activation of SRC. These results have the potential to identify patients with high SRC activity, who may benefit from dasatinib treatment.
The delivery of site-specific post-translational modifications to histones generates an epigenetic regulatory network that directs fundamental DNA-mediated processes and governs key stages in development. Methylation of histone H4 lysine-20 has been implicated in DNA repair, transcriptional silencing, genomic stability and regulation of replication. We present the structure of the histone H4K20 methyltransferase Suv4-20h2 in complex with its histone H4 peptide substrate and S-adenosyl methionine cofactor. Analysis of the structure reveals that the Suv4-20h2 active site diverges from the canonical SET domain configuration and generates a high degree of both substrate and product specificity. Together with supporting biochemical data comparing Suv4-20h1 and Suv4-20h2, we demonstrate that the Suv4-20 family enzymes take a previously mono-methylated H4K20 substrate and generate an exclusively di-methylated product. We therefore predict that other enzymes are responsible for the tri-methylation of histone H4K20 that marks silenced heterochromatin.
Book chapters
Methylation also plays important roles in aging. This volume is intended to capture these recent developments concerning protein methyltransferases.