Opposing regulation of the Nα-trimethylase METTL11A by its family members METTL11B and METTL13.

N-terminal protein methylation (Nα-methylation) is a posttranslational modification that influences numerous biological processes by regulating protein stability, protein-DNA interactions, and protein-protein interactions. Although significant progress has been made in understanding the biological roles of Nα-methylation, we still do not completely understand how the modifying methyltransferases are regulated. A common mode of methyltransferase regulation is through complex formation with close family members, and we have previously shown that the Nα-trimethylase METTL11A (NRMT1/NTMT1) is activated through binding of its close homolog METTL11B (NRMT2/NTMT2). Other recent reports indicate that METTL11A co-fractionates with a third METTL family member METTL13, which methylates both the N-terminus and lysine 55 (K55) of eukaryotic elongation factor 1 alpha. Here, using co-immunoprecipitations, mass spectrometry, and in vitro methylation assays, we confirm a regulatory interaction between METTL11A and METTL13 and show that while METTL11B is an activator of METTL11A, METTL13 inhibits METTL11A activity. This is the first example of a methyltransferase being opposingly regulated by different family members. Similarly, we find that METTL11A promotes the K55 methylation activity of METTL13 but inhibits its Nα-methylation activity. We also find that catalytic activity is not needed for these regulatory effects, demonstrating new, noncatalytic functions for METTL11A and METTL13. Finally, we show METTL11A, METTL11B, and METTL13 can complex together, and when all three are present, the regulatory effects of METTL13 take precedence over those of METTL11B. These findings provide a better understanding of Nα-methylation regulation and suggest a model where these methyltransferases can serve in both catalytic and noncatalytic roles.

Fluorescence resonance energy transfer links membrane ferroportin, hephaestin but not ferroportin, amyloid precursor protein complex with iron efflux.

Iron efflux from mammalian cells is supported by the synergistic actions of the ferrous iron efflux transporter, ferroportin (Fpn) and a multicopper ferroxidase, that is, hephaestin (Heph), ceruloplasmin (Cp) or both. The two proteins stabilize Fpn in the plasma membrane and catalyze extracellular Fe3+ release. The membrane stabilization of Fpn is also stimulated by its interaction with a 22-amino acid synthetic peptide based on a short sequence in the extracellular E2 domain of the amyloid precursor protein (APP). However, whether APP family members interact with Fpn in vivo is unclear. Here, using cyan fluorescent protein (CFP)-tagged Fpn in conjunction with yellow fluorescent protein (YFP) fusions of Heph and APP family members APP, APLP1, and APLP2 in HEK293T cells we used fluorescence and surface biotinylation to quantify Fpn membrane occupancy and also measured 59Fe efflux. We demonstrate that Fpn and Heph co-localize, and FRET analysis indicated that the two proteins form an iron-efflux complex. In contrast, none of the full-length, cellular APP proteins exhibited Fpn co-localization or FRET. Moreover, iron supplementation increased surface expression of the iron-efflux complex, and copper depletion knocked down Heph activity and decreased Fpn membrane localization. Whereas cellular APP species had no effects on Fpn and Heph localization, addition of soluble E2 elements derived from APP and APLP2, but not APLP1, increased Fpn membrane occupancy. We conclude that a ferroportin-targeting sequence, (K/R)EWEE, present in APP and APLP2, but not APLP1, helps modulate Fpn-dependent iron efflux in the presence of an active multicopper ferroxidase.

Optimizing purification and activity assays of N-terminal methyltransferase complexes.

In vitro methyltransferase assays have traditionally been carried out with tritiated S-adenosyl-methionine (SAM) as the methyl donor, as site-specific methylation antibodies are not always available for Western or dot blots and structural requirements of many methyltransferases prohibit the use of peptide substrates in luminescent or colorimetric assays. The discovery of the first N-terminal methyltransferase, METTL11A, has allowed for a second look at non-radioactive in vitro methyltransferase assays, as N-terminal methylation is amenable to antibody production and the limited structural requirements of METTL11A allow for its methylation of peptide substrates. We have used a combination of Western blots and luminescent assays to verify substrates of METTL11A and the two other known N-terminal methyltransferases, METTL11B and METTL13. We have also developed these assays for use beyond substrate identification, showing that METTL11A activity is opposingly regulated by METTL11B and METTL13. Here we provide two methods for non-radioactive characterization of N-terminal methylation, Western blots with full-length recombinant protein substrates and luminescent assays with peptide substrates, and describe how each can be additionally adapted to look at regulatory complexes. We will review the advantages and disadvantages of each method in context with the other types of in vitro methyltransferase assays and discuss why these types of assays could be of general use to the N-terminal modification field.

Novel regulation of the transcription factor ZHX2 by N-terminal methylation.

N-terminal methylation (Nα-methylation) by the methyltransferase NRMT1 is an important post-translational modification that regulates protein-DNA interactions. Accordingly, its loss impairs functions that are reliant on such interactions, including DNA repair and transcriptional regulation. The global loss of Nα-methylation results in severe developmental and premature aging phenotypes, but given over 300 predicted substrates, it is hard to discern which physiological substrates contribute to each phenotype. One of the most striking phenotypes in NRMT1 knockout (Nrmt1-/-) mice is early liver degeneration. To identify the disrupted signaling pathways leading to this phenotype and the NRMT1 substrates involved, we performed RNA-sequencing analysis of control and Nrmt1-/- adult mouse livers. We found both a significant upregulation of transcripts in the cytochrome P450 (CYP) family and downregulation of transcripts in the major urinary protein (MUP) family. Interestingly, transcription of both families is inversely regulated by the transcription factor zinc fingers and homeoboxes 2 (ZHX2). ZHX2 contains a non-canonical NRMT1 consensus sequence, indicating that its function could be directly regulated by Nα-methylation. We confirmed misregulation of CYP and MUP mRNA and protein levels in Nrmt1-/- livers and verified NRMT1 can methylate ZHX2 in vitro. In addition, we used a mutant of ZHX2 that cannot be methylated to directly demonstrate Nα-methylation promotes ZHX2 transcription factor activity and target promoter occupancy. Finally, we show Nrmt1-/- mice also exhibit early postnatal de-repression of ZHX2 targets involved in fetal liver development. Taken together, these data implicate ZHX2 misregulation as a driving force behind the liver phenotype seen in Nrmt1-/- mice.