Designer Adaptor Proteins for Functional Conversion of Peptides to Small-Molecule Ligands toward In-Cell Catalytic Protein Modification.

Peptides are privileged ligands for diverse biomacromolecules, including proteins; however, their utility is often limited due to low membrane permeability and in-cell instability. Here, we report peptide ligand-inserted eDHFR (PLIED) fusion protein as a universal adaptor for targeting proteins of interest (POI) with cell-permeable and stable synthetic functional small molecules (SFSM). PLIED binds to POI through the peptide moiety, properly orienting its eDHFR moiety, which then recruits trimethoprim (TMP)-conjugated SFSM to POI. Using a lysine-acylating BAHA catalyst as SFSM, we demonstrate that POI (MDM2 and chromatin histone) are post-translationally and synthetically acetylated at specific lysine residues. The residue-selectivity is predictable in an atomic resolution from molecular dynamics simulations of the POI/PLIED/TMP-BAHA (MTX was used as a TMP model) ternary complex. This designer adaptor approach universally enables functional conversion of impermeable peptide ligands to permeable small-molecule ligands, thus expanding the in-cell toolbox of chemical biology.

Author Correction: Meikin is a conserved regulator of meiosis-I-specific kinetochore function.

An Amendment to this Article has been published and is linked from the HTML version of this paper.

Malonylation of histone H2A at lysine 119 inhibits Bub1-dependent H2A phosphorylation and chromosomal localization of shugoshin proteins.

Post-translational modifications of histones, such as acetylation and phosphorylation, are highly conserved in eukaryotes and their combination enables precise regulation of many cellular functions. Recent studies using mass spectrometry have revealed various non-acetyl acylations in histones, including malonylation and succinylation, which change the positive charge of lysine into a negative one. However, the molecular function of histone malonylation or succinylation is poorly understood. Here, we discovered the functions of malonylation in histone H2A at lysine 119 (H2A-K119) in chromosome segregation during mitosis and meiosis. Analyses of H2A-K119 mutants in Saccharomyces cerevisiae and Schizosaccharomyces pombe showed that anionic mutations, specifically to aspartate (K119D) and glutamate (K119E), showed mis-segregation of the chromosomes and sensitivity to microtubule-destabilizing reagents in mitosis and meiosis. We found that the chromosomal localization of shugoshin proteins, which depends on Bub1-catalyzed phosphorylation of H2A at serine 121 (H2A-S121), was significantly reduced in the H2A-K119D and the H2A-K119E mutants. Biochemical analyses using K119-unmodified or -malonylated H2A-C-tail peptides showed that H2A-K119 malonylation inhibited the interaction between Bub1 and H2A, leading to a decrease in Bub1-dependent H2A-S121 phosphorylation. Our results indicate a novel crosstalk between lysine malonylation and serine/threonine phosphorylation, which may be important for fine-tuning chromatin functions such as chromosome segregation.

Meikin-associated polo-like kinase specifies Bub1 distribution in meiosis I.

In meiosis I, sister chromatids are captured by microtubules emanating from the same pole (mono-orientation), and centromeric cohesion is protected throughout anaphase. Shugoshin, which is localized to centromeres depending on the phosphorylation of histone H2A by Bub1 kinase, plays a central role in protecting meiotic cohesin Rec8 from separase cleavage. Another key meiotic kinetochore factor, meikin, may regulate cohesion protection, although the underlying molecular mechanisms remain elusive. Here, we show that fission yeast Moa1 (meikin), which associates stably with CENP-C during meiosis I, recruits Plo1 (polo-like kinase) to the kinetochores and phosphorylates Spc7 (KNL1) to accumulate Bub1. Consequently, in contrast to the transient kinetochore localization of mitotic Bub1, meiotic Bub1 persists at kinetochores until anaphase I. The meiotic Bub1 pool ensures robust Sgo1 (shugoshin) localization and cohesion protection at centromeres by cooperating with heterochromatin protein Swi6, which binds and stabilizes Sgo1. Furthermore, molecular genetic analyses show a hierarchical regulation of centromeric cohesion protection by meikin and shugoshin that is important for establishing meiosis-specific chromosome segregation. We provide evidence that the meiosis-specific Bub1 regulation is conserved in mouse.

The medaka dhc2 mutant reveals conserved and distinct mechanisms of Hedgehog signaling in teleosts.

BACKGROUND: Primary cilia are essential for Hedgehog (Hh) signal transduction in vertebrates. Although the core components of the Hh pathway are highly conserved, the dependency on cilia in Hh signaling is considered to be lower in fish than in mice, suggesting the presence of species-specific mechanisms for Hh signal transduction. RESULTS: To precisely understand the role of cilia in Hh signaling in fish and explore the evolution of Hh signaling, we have generated a maternal-zygotic medaka (Oryzias latipes) mutant that lacks cytoplasmic dynein heavy chain 2 (dhc2; MZdhc2), a component required for retrograde intraflagellar transport. We found that MZdhc2 exhibited the shortened cilia and partial defects in Hh signaling, although the Hh defects were milder than zebrafish mutants which completely lack cilia. This result suggests that Hh activity in fish depends on the length of cilium. However, the activity of Hh signaling in MZdhc2 appeared to be higher than that in mouse Dnchc2 mutants, suggesting a lower requirement for cilia in Hh signaling in fish. We confirmed that Ptch1 receptor is exclusively localized on the cilium in fish as in mammals. Subsequent analyses revealed that Fused, an essential mediator for Hh signaling in Drosophila and fish but not in mammals, augments the activity of Hh signaling in fish as a transcriptional target of Hh signaling. CONCLUSIONS: Ciliary requirement for Hh signaling in fish is lower than that in mammals, possibly due to fused-mediated positive feedback in Hh signaling. The finding of this fish-specific augmentation provides a novel insight into the evolution of Hh signaling.

Meikin is a conserved regulator of meiosis-I-specific kinetochore function.

The kinetochore is the crucial apparatus regulating chromosome segregation in mitosis and meiosis. Particularly in meiosis I, unlike in mitosis, sister kinetochores are captured by microtubules emanating from the same spindle pole (mono-orientation) and centromeric cohesion mediated by cohesin is protected in the following anaphase. Although meiotic kinetochore factors have been identified only in budding and fission yeasts, these molecules and their functions are thought to have diverged earlier. Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive. Here we have identified in mouse a meiosis-specific kinetochore factor that we termed MEIKIN, which functions in meiosis I but not in meiosis II or mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated mainly by the activity of Polo-like kinase PLK1, which is enriched to kinetochores in a MEIKIN-dependent manner. Our integrative analysis indicates that the long-awaited key regulator of meiotic kinetochore function is Meikin, which is conserved from yeasts to humans.

Acetylation regulates monopolar attachment at multiple levels during meiosis I in fission yeast.

In fission yeast, meiotic mono-orientation of sister kinetochores is established by cohesion at the core centromere, which is established by a meiotic cohesin complex and the kinetochore protein Moa1. The cohesin subunit Psm3 is acetylated by Eso1 and deacetylated by Clr6. We show that in meiosis, Eso1 is required for establishing core centromere cohesion during S phase, whereas Moa1 is required for maintaining this cohesion after S phase. The clr6-1 mutation suppresses the mono-orientation defect of moa1Δ cells, although the Clr6 target for this suppression is not Psm3. Thus, several acetylations are crucial for establishing and maintaining core centromere cohesion.

Shugoshin-PP2A counteracts casein-kinase-1-dependent cleavage of Rec8 by separase.

During meiosis, the cohesin complexes that maintain sister chromatid cohesion are lost in a stepwise manner. At meiosis I the cohesin subunit Rec8 is cleaved only along the chromosome arms; until meiosis II it is protected at centromeres by the action of shugoshin (Sgo1)-protein phosphatase 2A (PP2A). Although this regulation hypothetically involves phosphorylation that is antagonized by Sgo1-PP2A, the kinase and substrate that are responsible are as yet unknown. Using a genetic screen for 'anti-shugoshin', we identify Hhp2, an orthologue of casein kinase 1delta/epsilon (CK1), as a factor required for Rec8 cleavage in fission yeast. We show that CK1, rather than a Polo-like kinase that is widely believed to do so, acts as the cohesin kinase to promote this cleavage during meiosis. Crucially, forced localization of excess Hhp2 at the pericentromeric region abrogates the ability of Sgo1-PP2A to protect centromeric Rec8. Thus, our studies prove the key notion that the balance between Rec8 phosphorylation and its dephosphorylation by Sgo1-PP2A regulates the step-wise loss of chromosomal cohesion in meiosis.