Cysteine Methyltransferase Research Articles

Comparative Genomics of the Ectomycorrhizal Sister Species Rhizopogon vinicolor and Rhizopogon vesiculosus (Basidiomycota: Boletales) Reveals a Divergence of the Mating Type B Locus.

Divergence of breeding system plays an important role in fungal speciation. Ectomycorrhizal fungi, however, pose a challenge for the study of reproductive biology because most cannot be mated under laboratory conditions. To overcome this barrier, we sequenced the draft genomes of the ectomycorrhizal sister species Rhizopogon vinicolor Smith and Zeller and R. vesiculosus Smith and Zeller (Basidiomycota, Boletales)—the first genomes available for Basidiomycota truffles—and characterized gene content and organization surrounding their mating type loci. Both species possess a pair of homeodomain transcription factor homologs at the mating type A-locus as well as pheromone receptor and pheromone precursor homologs at the mating type B-locus. Comparison of Rhizopogon genomes with genomes from Boletales, Agaricales, and Polyporales revealed synteny of the A-locus region within Boletales, but several genomic rearrangements across orders. Our findings suggest correlation between gene content at the B-locus region and breeding system in Boletales with tetrapolar species possessing more diverse gene content than bipolar species. Rhizopogon vinicolor possesses a greater number of B-locus pheromone receptor and precursor genes than R. vesiculosus, as well as a pair of isoprenyl cysteine methyltransferase genes flanking the B-locus compared to a single copy in R. vesiculosus. Examination of dikaryotic single nucleotide polymorphisms within genomes revealed greater heterozygosity in R. vinicolor, consistent with increased rates of outcrossing. Both species possess the components of a heterothallic breeding system with R. vinicolor possessing a B-locus region structure consistent with tetrapolar Boletales and R. vesiculosus possessing a B-locus region structure intermediate between bipolar and tetrapolar Boletales.

Identification of a Distinct Substrate-binding Domain in the Bacterial Cysteine Methyltransferase Effectors NleE and OspZ

The type III secretion system effector protein NleE from enteropathogenic Escherichia coli plays a key role in the inhibition of NF-κB activation during infection. NleE inactivates the ubiquitin chain binding activity of host proteins TAK1-binding proteins 2 and 3 (TAB2 and TAB3) by modifying the Npl4 zinc finger domain through S-adenosyl methionine-dependent cysteine methylation. Using yeast two-hybrid protein interaction studies, we found that a conserved region between amino acids 34 and 52 of NleE, in particular the motif (49)GITR(52), was critical for TAB2 and TAB3 binding. NleE mutants lacking (49)GITR(52) were unable to methylate TAB3, and wild type NleE but not NleE(49AAAA52) where each of GITR was replaced with alanine restored the ability of an nleE mutant to inhibit IL-8 production during infection. Another NleE target, ZRANB3, also associated with NleE through the (49)GITR(52) motif. Ectopic expression of an N-terminal fragment of NleE (NleE(34-52)) in HeLa cells showed competitive inhibition of wild type NleE in the suppression of IL-8 secretion during enteropathogenic E. coli infection. Similar results were observed for the NleE homologue OspZ from Shigella flexneri 6 that also bound TAB3 through the (49)GITR(52) motif and decreased IL-8 transcription through modification of TAB3. In summary, we have identified a unique substrate-binding motif in NleE and OspZ that is required for the ability to inhibit the host inflammatory response.

Structure and specificity of the bacterial cysteine methyltransferase effector NleE suggests a novel substrate in human DNA repair pathway.

Enteropathogenic E. coli (EPEC) and related enterobacteria rely on a type III secretion system (T3SS) effector NleE to block host NF-κB signaling. NleE is a first in class, novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase that methylates a zinc-coordinating cysteine in the Npl4-like Zinc Finger (NZF) domains in TAB2/3 adaptors in the NF-κB pathway, but its mechanism of action and other human substrates are unknown. Here we solve crystal structure of NleE-SAM complex, which reveals a methyltransferase fold different from those of known ones. The SAM, cradled snugly at the bottom of a deep and narrow cavity, adopts a unique conformation ready for nucleophilic attack by the methyl acceptor. The substrate NZF domain can be well docked into the cavity, and molecular dynamic simulation indicates that Cys673 in TAB2-NZF is spatially and energetically favorable for attacking the SAM. We further identify a new NleE substrate, ZRANB3, that functions in PCNA binding and remodeling of stalled replication forks at the DNA damage sites. Specific inactivation of the NZF domain in ZRANB3 by NleE offers a unique opportunity to suggest that ZRANB3-NZF domain functions in DNA repair processes other than ZRANB3 recruitment to DNA damage sites. Our analyses suggest a novel and unexpected link between EPEC infection, virulence proteins and genome integrity.

Abstract LB-323: Control of DNA repair and genome stability by the colon microbiome: The EPEC bacterially encoded NLEE virulence effector protein methylates and inactivates the human ZRANB3 DNA repair helicase

Abstract Colorectal cancer develops in the context of probably the most complex microenvironment in the human body: the colon microbiota comprised of >10¹³ bacteria, most of which are uncharacterized. Recently there has been great interest in defining the normal colon microbiome and how it is remodeled during disease states. Both commensal and infective enteropathogenic bacteria utilize a vast array of mechanisms to reduce inflammation in the gut, many of which are directed at the innate immune system, though undoubtedly, other cellular homeostasis systems are targeted. To limit inflammation bacterially encoded virulence proteins are injected into colon epithelial cells: many function by disrupting NFκB signaling. We have characterized the NLEE virulence protein which is encoded by EPEC bacteria and following injection by the T3SS, targets the TAB2 scaffold protein resulting in inactivation of the TAK1 kinase in the NFκB pathway. The Shao laboratory discovered that NLEE is an S-adenosyl-methionine (SAM)-dependent cysteine methyltransferase that methylates a single cysteine in the C4 zinc finger of TAB2 thereby unfolding and abolishing its function. The discovery of this first-in-class, Zinc Finger (ZF)-directed cysteine methytransferase, establishes the paradigm that eukaryotic ZFs can be subject to PTMs at the chelation residues that abolish finger function. We hypothesize that cysteine methylation of ZF domains is widespread and as yet, under appreciated PTM which regulates cellular signal transduction. The crystal structure of NLEE was solved and shows a novel SAM binding motif positioned in a deep cleft which confers a very high specificity for ZF binding and methylation. We searched for novel NLEE ZF-containing substrates in EPEC infected colon cells and discovered that the human DNA repair protein ZRANB3, a helicase which participates in the translesion synthesis repair pathway directly at replication forks, is a robust substrate for NLEE. Methylation of the C4 ZF of ZRANB3 is predicted to abolish its binding to K63-linked ubiquitinated-PCNA at stalled replication forks leading to fork collapse and replication stress. Additional NLEE substrates discovered in this DNA repair pathway include a PCNA-directed OTU class deubiquitinase and the NPL4 segregase complex, both of which limit repair functions. Together with recent work showing that bacterial virulence proteins can also down regulate the DNA mismatch repair system, these results suggest the remarkable conclusion that infectious colon bacteria have the capacity to abolish specific DNA repair systems in the colon epithelium. This work provides a unique and unexpected mechanistic link between the colon microbiome and genome stability in the colon. Whether a remodeled colon cancer specific microbiome contributes to tumor progression via induction of genome instability by inactivating DNA repair pathways is interesting to contemplate. Citation Format: Jayashree Karar, Hongzhuang Peng, Li Zhang, Qing Yao, Ilan Rosenshine, Feng Shao, Frank J. Rauscher. Control of DNA repair and genome stability by the colon microbiome: The EPEC bacterially encoded NLEE virulence effector protein methylates and inactivates the human ZRANB3 DNA repair helicase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-323. doi:10.1158/1538-7445.AM2014-LB-323

Purification, characterization, sequencing and molecular cloning of a novel cysteine methyltransferase that regulates trehalose-6-phosphate synthase from Saccharomyces cerevisiae

BackgroundIn Saccharomyces cerevisiae methylation at cysteine residue displayed enhanced activity of trehalose-6-phosphate synthase (TPS). MethodsThe cysteine methyltransferase (CMT) responsible for methylating TPS was purified and characterized. The amino acid sequence of the enzyme protein was determined by a combination of N-terminal sequencing and MALDI-TOF/TOF analysis. The nucleotide sequence of the CMT gene was determined, isolated from S. cerevisiae and expressed in E. coli. Targeted disruption of the CMT gene by PCR based homologous recombination in S. cerevisiae was followed by metabolite characterization in the mutant. ResultsThe purified enzyme was observed to enhance the activity of TPS by a factor of 1.76. The 14kDa enzyme was found to be cysteine specific. The optimum temperature and pH of enzyme activity was calculated as 30°C and 7.0 respectively. The Km Vmax and Kcat against S-adenosyl-l-methionine (AdoMet) were 4.95μM, 3.2U/mg and 6.4s−1 respectively. Competitive inhibitor S-Adenosyl-l-homocysteine achieved a Ki as 10.9μM against AdoMet. The protein sequence contained three putative AdoMet binding motifs. The purified recombinant CMT activity exhibited similar physicochemical characteristics with the native counterpart. The mutant, Mataα, cmt:: kanr exhibited almost 50% reduction in intracellular trehalose concentration. ConclusionA novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae. General significanceThis is the first report about a cysteine methyltransferase which performs S methylation at cysteine residue regulating TPS activity by 50%, which resulted in an increase of the intercellular stress sugar, trehalose.

Enteropathogenic and enterohemorrhagic E. coli: ecology, pathogenesis, and evolution

EDITORIAL article Front. Cell. Infect. Microbiol., 30 April 2013Sec. Molecular Bacterial Pathogenesis https://doi.org/10.3389/fcimb.2013.00015

Modulation of k-Ras Signaling by Natural Products

Ras proteins regulate diverse cellular pathways that are important in the growth and spread of malignancies, including cell proliferation, cell cycle regulation, cell survival, angiogenesis and cell migration. These proteins lack the conventional transmembrane or hydrophobic domain typical of membrane associated proteins. Being small and hydrophilic in nature, these proteins undergo four-stage post-translational lipid modifications viz. prenylation, AAX proteolysis, carboxymethylation and palmitoylation for membrane localization which is important for their function. Therefore, enzymes involved in these modifications viz. farnesyl transferase (FTase), geranylgeranyl transferase-I (GGTase-I), geranylgeranyl transferase-II (GGTase-II), Ras converting enzyme-1 (Rce-1) and isoprenyl cysteine methyl transferase (ICMT) are emerging as potential therapeutic targets for the discovery of newer anticancer therapeutics. Several natural products have shown modulation of these post-translational enzymes. In the present review, natural products isolated from terrestrial as well as marine sources showing ability to modulate these k-Ras post-translational targets and their promise as potential anticancer agents have been discussed. A total of 157 natural products with 141 corresponding references have been covered.

Functional Analysis of Arabidopsis Postprenylation CaaX Processing Enzymes and Their Function in Subcellular Protein Targeting

Prenylation is a posttranslational protein modification essential for developmental processes and response to abscisic acid. Following prenylation, the three C-terminal residues are proteoliticaly removed and in turn the free carboxyl group of the isoprenyl cysteine is methylated. The proteolysis and methylation, collectively referred to as CaaX processing, are catalyzed by Ste24 endoprotease or Rce1 endoprotease and by an isoprenyl cysteine methyltransferase (ICMT). Arabidopsis (Arabidopsis thaliana) contains single STE24 and RCE1 and two ICMT homologs. Here we show that in yeast (Saccharomyces cerevisiae) AtRCE1 promoted a-mating factor secretion and membrane localization of a ROP GTPase. Furthermore, green fluorescent protein fusion proteins of AtSTE24, AtRCE1, AtICMTA, and AtICMTB are colocalized in the endoplasmic reticulum, indicating that prenylated proteins reach this compartment and that CaaX processing is likely required for subcellular targeting. AtICMTB can process yeast a-factor more efficiently than AtICMTA. Sequence and mutational analyses revealed that the higher activity AtICMTB is conferred by five residues, which are conserved between yeast Ste14p, human ICMT, and AtICMTB but not in AtICMTA. Quantitative real-time reverse transcription-polymerase chain reaction and microarray data show that AtICMTA expression is significantly lower compared to AtICMTB. AtICMTA null mutants have a wild-type phenotype, indicating that its function is redundant. However, AtICMT RNAi lines had fasciated inflorescence stems, altered phylotaxis, and developed multiple buds without stem elongation. The phenotype of the ICMT RNAi lines is similar to farnesyltransferase beta-subunit mutant enhanced response to abscisic acid2 but is more subtle. Collectively, the data suggest that AtICMTB is likely the major ICMT and that methylation modulates activity of prenylated proteins.

5043268 Substrates and inhibitors for prenyl cysteine methyltransferase enzymes: Jeffry Stock assigned to The Trustees of Princeton University

5043268 Substrates and inhibitors for prenyl cysteine methyltransferase enzymes: Jeffry Stock assigned to The Trustees of Princeton University