Novel targets of a fungal specific protein methyltransferase

Protein methylation involves the transfer of a methyl group from S-adenosylmethionine to acceptor groups on substrate proteins. Proteins can be methylated on different sites such as lysine, arginine, histidine, or carboxyl residues. Arginine residues of proteins are modified by members of the protein arginine methyltransferase (PRMT) family. PRMTs are classified either as type I, II, or III based on the nature of the methylgroup introduced. Arginine methylation has been observed on a variety of proteins associated with gene regulation, including DNA-binding transcriptional activators, transcriptional coactivators, and many RNA-binding proteins involved in RNA processing, transport, and stability. In the filamentous fungus Aspergillus nidulans three distinct PRMTs are present which all possess in vitro and in vivo methyltransferase activity. Two of these proteins, termed RmtA and RmtC, exhibit significant sequence homology to the well characterized proteins PRMT1 and PRMT5, respectively. The third enzyme, termed RmtB, has an exceptional position because it displays both enzymatic and structural properties that are different from other known PRMTs. Extended investigations of substrate specificities revealed that recombinant Aspergillus RmtB can methylate several yet unknown cellular proteins. Our long term objective is to clarify the functional role of the fungal specific RmtB protein in A. nidulans. We therefore started to isolate and characterize enzymes involved in protein arginine methylation and to generate mutants of the corresponding PRMT genes by targeted gene replacement. A central aim of this project will be to apply these mutants for the isolation and identification of novel substrate proteins of Aspergillus RmtB. To reach this goal, we will start with the in vitro labeling of substrate proteins of Aspergillus wild type as well as rmtB mutant strains for the screening of selective and non-selective targets. Substrate proteins will be separated and identified by two dimensional gel electrophoresis and mass spectrometry. Finally, verification of identified proteins as in vivo targets of Aspergillus RmtB will be performed by immunological approaches and/or in vivo arginine methylation assays. The outcomes of our investigations will provide the basis for the long term analysis of the functional implication of this modification. Future projects will allow the specific mutation of substrate genes and/or methylation sites with subsequent analysis of effects on gene expression and physiology, as well as to explore the role of protein methylation for fungal specific processes such as the regulation of secondary metabolism. Secondary metabolites have tremendous importance to humankind in that they display a broad range of useful antibiotic and pharmaceutical activities as well as less desirable immunosuppressant and toxic activities.

Our long-term objective is to clarify the functional role of protein arginine methylation in the filamentous fungus Aspergillus nidulans. Arginine residues of proteins are modified by members of the protein arginine methyltransferase (PRMT) family. Arginine methylation has been observed on a variety of proteins associated with gene regulation, RNA processing, or DNA repair. However, little is known on the role of this modification in filamentous fungi which are of particular interest as model organisms for biotechnological (e.g. antibiotics) as well as medical (e.g. production of toxins like aflatoxin; aspergillosis) applications. In A. nidulans four genes encoding for PRMTs have been identified by our lab. To study the functional role of these PRMTs in Aspergillus we initially have deleted the coding sequences of rmtA, rmtB, and rmtC (rmtD was generated during this project) and analysed molecular and physiological consequences of the specific gene deletions. Deletion strains served as tool for the identification of novel targets of fungal PRMTs described in this project. We applied a proteomic approach (protein purification 2D gelelectrophoresis mass spectrometry) to identify more than 50 mostly yet unknown substrate proteins of Aspergillus PRMTs. Among the identified proteins we focused our investigations on the RmtB specific protein VipC that has sequence similarity to a regulator of secondary metabolism (LaeA), and the 14-3-3 homolog ArtB. 14-3-3 proteins are multiregulatory proteins of diverse cellular processes. Neither VipC nor ArtB have yet been reported as substrates of methyltransferases. For the verification of identified proteins as in vivo targets of Aspergillus PRMTs, VipC and ArtB proteins were expressed with an affinity tag (mark) in Aspergillus nidulans. The subsequent biochemical purification and analysis by mass spectrometry (MS) identified methylation sites on distinct arginines in both proteins. Moreover, the isolation and subsequent MS analysis of the endogenous form of ArtB in different mutant strains confirmed the finding of in vivo methylation of this substrate. However, in vitro methylation of recombinant substrates with isolated Aspergillus PRMTs was not successful which might be due to the lack of posttranslational modifications and/or missing complex partners in the recombinant proteins. Polyclonal antibodies will be used to study the localization of these proteins and for the identification of interacting proteins by immunoprecipitation. Further characterization of these substrates and their properties will finally help to elucidate the putative link between protein methylation and secondary metabolism in case of VipC, and the role of this modification in ArtB mediated alteration/coordination of protein interactions in Aspergillus.