Search

Lysine methylation is an important post-translational modification that is also present on mitochondrial proteins, but the mitochondrial lysine-specific methyltransferases (KMTs) responsible for modification are in most cases unknown. Here, we set out to determine the function of human family with sequence similarity 173 member B (FAM173B), a mitochondrial methyltransferase (MTase) reported to promote chronic pain. Using bioinformatics analyses and biochemical assays, we found that FAM173B contains an atypical, noncleavable mitochondrial targeting sequence responsible for its localization to mitochondria. Interestingly, CRISPR/Cas9-mediated KO of FAM173B in mammalian cells abrogated trimethylation of Lys-43 in ATP synthase c-subunit (ATPSc), a modification previously reported as ubiquitous among metazoans. ATPSc methylation was restored by complementing the KO cells with enzymatically active human FAM173B or with a putative FAM173B orthologue from the nematode Caenorhabditis elegans. Interestingly, lack of Lys-43 methylation caused aberrant incorporation of ATPSc into the ATP synthase complex and resulted in decreased ATP-generating ability of the complex, as well as decreased mitochondrial respiration. In summary, we have identified FAM173B as the long-sought KMT responsible for methylation of ATPSc, a key protein in cellular ATP production, and have demonstrated functional significance of ATPSc methylation. We suggest renaming FAM173B to ATPSc-KMT (gene name ATPSCKMT).

Toll-like receptors (TLR)s are innate immune receptors for microbial molecules and also sense damage-associated molecular patterns released from host cells. Double-stranded RNA and the synthetic analog polyinosinic:polycytidylic acid (Poly(I:C)) bind and activate TLR3. This leads to recruitment of the adaptor molecule TRIF (Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing interferon β (IFNβ)), and downstream activation of the transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3), classically inducing IFNβ production. In this study we report that metastatic intestinal epithelial cells (IEC)s express TLR3, in contrast to non-metastatic IECs, and that TLR3 activation promotes the invasive properties of these cells. The metastatic IECs also induced CXCL10 in a TLR3- TRIF- IRF3- dependent manner in response to Poly(I:C) addition, but failed to produce IFNβ. This was in contrast to healthy and non-metastatic IECs which did not respond to Poly(I:C) stimulation. The endosomal transporter protein UNC93B1 and endolysosomal acidification was required for Poly(I:C) induced CXCL10 production, underscoring the role of endosomes in TLR3 activation. TLR3- induced CXCL10 was, however, triggered by immobilized ligand, was only modestly affected by inhibition of endocytosis, and could be blocked with an anti-TLR3 antibody, indicating that TLR3 still can signal from the cell surface of these cells. Plasma membrane fractions of metastatic IECs furthermore contained both full-length and cleaved TLR3. Our results imply that metastatic IECs express surface TLR3, allowing it to sense extracellular stimuli, which triggers chemokine responses, and promotes invasiveness in these cells. Altered TLR3 expression and localization may have implications for disease progression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)